Principles of Plant-Microbe Interactions

Ben Lugtenberg
Editor

Principles of Plant-Microbe
Interactions
Microbes for Sustainable Agriculture

2123
Editor
Ben Lugtenberg
Molecular Microbiology and Biotechnology
Leiden University, Sylvius Laboratory
Leiden
The Netherlands

ISBN 978-3-319-08574-6 ISBN 978-3-319-08575-3 (eBook)

DOI 10.1007/978-3-319-08575-3
Springer Cham Heidelberg New York Dordrecht London

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I dedicate this book to my wife Faina and my
children Annelieke, Martijn and Marjolein
Preface

The field of Plant Microbe Interactions is very broad. It covers all topics in which mi-
crobes influence or even determine plant activities. Plant enemies can be pathogenic
viruses, microbes or insects which cause pests. Fortunately, these enemies in turn
have natural enemies in the form of beneficial microbes, which can protect plants
against pathogens and pests. As is rather common in this field, we included nema-
todes and insects in the book. Although they are not microbes, they have in common
with microbes that some can cause harm to, and others help protect, the plant. An-
other group of microbes is beneficial for plant growth. Some microbes promote plant
growth, for example by producing “plant” hormones or by making nutrients avail-
able to the plant. Other beneficial microbes can alleviate plant stress or can inactivate
environmental pollutants, thereby cleaning the environment and allowing plants to
grow without toxic residues. The present market share of biologicals is estimated
at 1.6 billion USDs and is growing fast. In the past years the trend is that major
chemical companies buy smaller biotech companies.
For this book I have invited the world’s top scientists to summarize the basic
principles of all these topics in brief chapters which give a helicopter view on the
subjects. The book also contains important techniques, success stories and future
prospects. The topics include basic as well as applied aspects. Hereby we make an
attempt to close the gap that still exists between fundamental and applied research.
In my opinion the two fields need each other and cooperation will create a win-win
situation for both parties. Since space is limited, the authors have often referred to
reviews. For more detailed information, the reader can consult primary articles listed
as references in these reviews.
This book is meant for everybody who is interested in plant-microbe interactions
and in the roles microbes can play in making agriculture and horticulture more
sustainable. These include academic scientists, industrial professionals working in
agriculture, horticulture, biotech and food industry, students, teachers, as well as
government officials and decision makers who quickly want to make themselves
familiar with particular aspects of this broad field. Using this information as a basis,
also a non-specialist reader should be able to understand more complicated articles
and to discuss selected topics with colleagues. To read the book, basic knowledge of
plant science, microbiology, biochemistry, and molecular biology is helpful.
Ben Lugtenberg, editor
vii
Acknowledgement

I am very much indebted to all authors for their contributions. I am particularly

thankful to the following people who have contributed by useful advice and discus-
sions: Gabriele Berg, Rainer Borriss, Frans de Bruijn, Faina Kamilova, Christoph
Keel, Corné Pieterse, and Clara Pliego. I am greatly obliged to Izabela Witkowska
and Melanie van Overbeek of Springer Dordrecht for their help and patience during
the preparation of the manuscript.
The following sponsors made the editing of the book more pleasant. Their con-
tributions will go to a foundation which supports the promotion of knowledge about
plant-microbe interactions and their applications.

DIAMOND SPONSORS

ix
x Acknowledgement

GOLD SPONSORS
Contents

1 Introduction to Plant-Microbe Interactions . . . . . . . . . . . . . . . . . . . . . . 1

Ben Lugtenberg

Part I Introductory Chapters

2 The Importance of Microbiology in Sustainable Agriculture . . . . . . . . 5

Thomas Schäfer and Tom Adams

3 Life of Microbes in the Rhizosphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Ben Lugtenberg

4 Life of Microbes on Aerial Plant Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Johan H. J. Leveau

5 Life of Microbes Inside the Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Jesús Mercado-Blanco

6 Microbial Cell Surfaces and Secretion Systems . . . . . . . . . . . . . . . . . . . 33

Jan Tommassen and Han A. B. Wösten

7 Microbial Biofilms and Quorum Sensing . . . . . . . . . . . . . . . . . . . . . . . . . 45

Aurelien Carlier, Gabriella Pessi and Leo Eberl

8 Bacterial Volatiles as Airborne Signals for Plants and Bacteria . . . . . 53

Choong-Min Ryu

Part II Phytopathogens and Pest Insects

9 Phytopathogenic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Jan van der Wolf and Solke H. De Boer

10 Plant Pathogenic Fungi and Oomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Pierre J. G. M. de Wit

xi
xii Contents

11 Phytopathogenic Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Johannes Helder, Mariëtte Vervoort, Hanny van Megen, Katarzyna
Rybarczyk-Mydłowska, Casper Quist, Geert Smant and Jaap Bakker

12 Herbivorous Insects—A Threat for Crop Production . . . . . . . . . . . . . . 103

Eddy van der Meijden

13 Phytopathogenic Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Carmen Büttner, Susanne von Bargen and Martina Bandte

14 Induced Disease Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Corné M. J. Pieterse and Saskia C. M. Van Wees

15 Apologies to the Planet—Can We Restore the Damage? . . . . . . . . . . . . 135

Dulce Eleonora de Oliveira and Marc Van Montagu

16 Will the Public Ever Accept Genetically Engineered Plants? . . . . . . . 145

Inge Broer

Part III Control of Plant Diseases and Pests using Beneficial Microbes

17 Microbial Control of Phytopathogenic Nematodes . . . . . . . . . . . . . . . . 155

Xiaowei Huang, Keqin Zhang, Zefen Yu and Guohong Li

18 Microbial Control of Root-Pathogenic Fungi and Oomycetes . . . . . . . 165

Linda Thomashow and Peter A. H. M. Bakker

19 Control of Insect Pests by Entomopathogenic Nematodes . . . . . . . . . . 175

Vladimír Pu◦ ža

20 Bacillus thuringiensis-Based Products for Insect Pest Control . . . . . . 185

Ruud A. de Maagd

21 Post Harvest Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Emilio Montesinos, Jesús Francés, Esther Badosa
and Anna Bonaterra

Part IV Plant Growth Promotion by Microbes

22 The Nitrogen Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Martine A. R. Kox and Mike S. M. Jetten

23 Biological Nitrogen Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Frans J. de Bruijn

24 Phosphate Mobilisation by Soil Microorganisms . . . . . . . . . . . . . . . . . . 225

José-Miguel Barea and Alan E. Richardson
Contents xiii

25 Arbuscular Mycorrhizas: The Lives of Beneficial Fungi and Their

Plant Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Paola Bonfante and Alessandro Desirò

26 Plant Hormones Produced by Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Stijn Spaepen

27 Stress Control and ACC Deaminase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Bernard R. Glick

28 Plant-Microbe Interactions and Water Management in Arid

and Saline Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Daniele Daffonchio, Heribert Hirt and Gabriele Berg

29 Rhizoremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Sofie Thijs and Jaco Vangronsveld

Part V Important Technologies

30 Microbial Communities in the Rhizosphere Analyzed

by Cultivation-Independent DNA-Based Methods . . . . . . . . . . . . . . . . . 289
Susanne Schreiter, Namis Eltlbany and Kornelia Smalla

31 Visualization of Plant-Microbe Interactions . . . . . . . . . . . . . . . . . . . . . . 299

Massimiliano Cardinale and Gabriele Berg

Part VI Products for Plant Growth-promotion and Disease Suppression

32 Commercialisation of Microbes: Present Situation

and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Willem J. Ravensberg

33 Commercialization of Microbes: Manufacturing, Inoculation,

Best Practice for Objective Field Testing, and Registration . . . . . . . . . 319
Faina Kamilova, Yaacov Okon, Sandra de Weert and Katja Hora

34 Towards a New Generation of Commercial Microbial Disease

Control and Plant Growth Promotion Products . . . . . . . . . . . . . . . . . . . 329
Rainer Borriss

35 Important Organizations and Companies . . . . . . . . . . . . . . . . . . . . . . . . 339

Ben Lugtenberg

Part VII Paradigms in Plant-Microbe Interactions

36 Trichoderma: A Multi-Purpose Tool for Integrated Pest

Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Matteo Lorito and Sheridan L. Woo
xiv Contents

37 Agrobacterium, The Genetic Engineer . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Paul J. J. Hooykaas

38 Take-All Decline and Beneficial Pseudomonads . . . . . . . . . . . . . . . . . . . 363

David M. Weller

39 The Oomycete Phytophthora infestans, the Irish Potato

Famine Pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Charikleia Schoina and Francine Govers

40 Bacillus, A Plant-Beneficial Bacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

Rainer Borriss

41 Soybean Production in the Americas . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

Woo-Suk Chang, Hae-In Lee and Mariangela Hungria

Part VIII Future Prospects and Dreams

42 Exploring the Feasibility of Transferring Nitrogen Fixation

to Cereal Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Muthusubramanian Venkateshwaran

43 The Minimal Rhizosphere Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . 411

Jos M. Raaijmakers

44 The Edible Plant Microbiome: Importance and Health Issues . . . . . . 419

Gabriele Berg, Armin Erlacher and Martin Grube

45 From Nodulation to Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

Eva Kondorosi

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
Contributors

Tom Adams Monsanto Company, St. Louis, MO, USA

Carmen Büttner Division Phytomedicine, Faculty of Life Sciences, Humboldt-
Universität zu Berlin, Berlin, Germany
Esther Badosa Laboratory of Plant Pathology, Institute of Food and Agricultural
Technology, University of Girona, Girona, Spain
Jaap Bakker Laboratory of Nematology, Wageningen University, Wageningen,
The Netherlands
Peter A. H. M. Bakker Plant-Microbe Interactions, Institute of Environmental
Biology, Utrecht University, Utrecht, The Netherlands
Martina Bandte Division Phytomedicine, Faculty of Life Sciences, Humboldt-
Universität zu Berlin, Berlin, Germany
José-Miguel Barea Soil Microbiology and Symbiotic Systems Department,
Estación Experimental del Zaidín, CSIC, Granada, Spain
Gabriele Berg Institute of Environmental Biotechnology, Graz University of
Technology, Graz, Austria
Anna Bonaterra Laboratory of Plant Pathology, Institute of Food and Agricultural
Technology, University of Girona, Girona, Spain
Paola Bonfante Department of Life Sciences and Systems Biology, University of
Torino, Torino, Italy
Rainer Borriss ABiTEP GmbH, Berlin, Germany
Inge Broer Agricultural and Environmental Faculty, University of Rostock,
Rostock, Germany
Massimiliano Cardinale Institute of Applied Microbiology, Justus-Liebig-
University Giessen, Giessen, Germany
Aurelien Carlier Institute of Plant Biology, University of Zurich, Zurich,
Switzerland
xv
xvi Contributors

Woo-Suk Chang Department of Biology, University of Texas-Arlington, Arlington,

Texas, USA
Daniele Daffonchio DeFENS, Department of Food, Environmental and Nutritional
Sciences, University of Milan, Milan, Italy
BESE Division, King Abdullah University of Science and Technology, Thuwal,
Kingdom of Saudi Arabia
Solke H. De Boer Emeritus Scientist, Charlottetown, PE, Canada
Frans J. de Bruijn INRA/CNRS Laboratory of Plant-Microbe Interactions,
Castanet-Tolosan Cedex, France
Ruud A. de Maagd Plant Research International, Wageningen UR, Wageningen,
The Netherlands
Dulce Eleonora de Oliveira VIB–Institute of Plant Biotechnology Outreach,
Department of Biotechnology and Bioinformatics, Ghent University, Gent-
Zwijnaarde, Belgium
Sandra de Weert Koppert Biological Systems, Berkel en Rodenrijs, The
Netherlands
Pierre J. G. M. de Wit Laboratory of Phytopathology, Wageningen University,
Wageningen, The Netherlands
Alessandro Desirò Department of Life Sciences and Systems Biology, University
of Torino, Torino, Italy
Leo Eberl Institute of Plant Biology, University of Zurich, Zurich, Switzerland
Namis Eltlbany Julius Kühn-Institut, Federal Research Centre for Cultivated Plants
(JKI), Braunschweig, Germany
Armin Erlacher Institute of Environmental Biotechnology, Graz University of
Technology, Graz, Austria
Institute of Plant Sciences, University of Graz, Graz, Austria
Jesús Francés Laboratory of Plant Pathology, Institute of Food and Agricultural
Technology, University of Girona, Girona, Spain
Bernard R. Glick Department of Biology, University of Waterloo, Waterloo, ON,
Canada
Francine Govers Laboratory of Phytopathology, Wageningen University,
Wageningen, The Netherlands
Martin Grube Institute of Plant Sciences, University of Graz, Graz, Austria
Johannes Helder Laboratory of Nematology, Wageningen University,
Wageningen, The Netherlands
Contributors xvii

Heribert Hirt BESE Division, King Abdullah University of Science and

Technology, Thuwal, Kingdom of Saudi Arabia
Paul J. J. Hooykaas Institute of Biology, Sylvius Laboratory, Leiden University,
Leiden, The Netherlands
Katja Hora Koppert Biological Systems, Berkel en Rodenrijs, The Netherlands
Xiaowei Huang Yunnan University, Kunming, People’s Republic of China
Mariangela Hungria Embrapa Soja, Londrina, Paraná, Brazil
Mike S. M. Jetten Department of Microbiology, Institute of Water and Wet-
land Research, Faculty of Science, Radboud University Nijmegen, Nijmegen, The
Netherlands
Faina Kamilova Koppert Biological Systems, Berkel en Rodenrijs, The
Netherlands
Eva Kondorosi Institute of Biochemistry, Biological Research Centre of the
Hungarian Academy of Sciences, Szeged, Hungary
Martine A. R. Kox Department of Microbiology, Institute of Water and
Wetland Research, Faculty of Science, Radboud University Nijmegen, Nijmegen,
The Netherlands
Hae-In Lee Department of Biology, University of Texas-Arlington, Arlington,
Texas, USA
Johan H. J. Leveau Department of Plant Pathology, University of California, Davis,
CA, USA
Guohong Li Yunnan University, Kunming, People’s Republic of China
Matteo Lorito Department of Agriculture, University of Naples Federico II, and
Institute of Plant Protection IPP–CNR, Portici (NA), Italy
Ben Lugtenberg Institute of Biology, Sylvius Laboratory, Leiden University,
Leiden, The Netherlands
Jesús Mercado-Blanco Department of Crop Protection, Institute for Sustainable
Agriculture, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC),
Córdoba, Spain
Emilio Montesinos Laboratory of Plant Pathology, Institute of Food and
Agricultural Technology, University of Girona, Girona, Spain
Yaacov Okon Department of Plant Pathology and Microbiology, Faculty of
Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot,
Israel
Vladimír Pu◦ ža Institute of Entomology, Biology Centre of the AS CR, České
Budějovice, Czech Republic
xviii Contributors

Gabriella Pessi Institute of Plant Biology, University of Zurich, Zurich,

Switzerland
Corné M. J. Pieterse Plant-Microbe Interactions, Department of Biology, Faculty
of Science, Utrecht University, Utrecht, The Netherlands
Casper Quist Laboratory of Nematology, Wageningen University, Wageningen,
The Netherlands
Jos M. Raaijmakers Department of Microbial Ecology, Netherlands Institute of
Ecology, Wageningen, The Netherlands
Willem J. Ravensberg International Biocontrol Manufacturers Association
(IBMA), Brussels, Belgium
Koppert Biological Systems, Berkel en Rodenrijs, The Netherlands
Alan E. Richardson CSIRO Plant Industry, Canberra, Australia
Katarzyna Rybarczyk-Mydłowska Museum and Institute of Zoology PAS,
Warsaw, Poland
Choong-Min Ryu Molecular Phytobacteriology Laboratory, KRIBB, Daejeon,
Republic of Korea
Thomas Schäfer Novozymes A/S, Bagsvaerd, Denmark
Charikleia Schoina Laboratory of Phytopathology, Wageningen University,
Wageningen, The Netherlands
Susanne Schreiter Julius Kühn-Institut, Federal Research Centre for Cultivated
Plants (JKI), Braunschweig, Germany
Kornelia Smalla Julius Kühn-Institut, Federal Research Centre for Cultivated
Plants (JKI), Braunschweig, Germany
Geert Smant Laboratory of Nematology, Wageningen University, Wageningen,
The Netherlands
Stijn Spaepen Centre of Microbial and Plant Genetics, KU Leuven, Heverlee,
Belgium
Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding
Research, Köln, Germany
Sofie Thijs Centre for Environmental Sciences, Hasselt University, Diepenbeek,
Belgium
Linda Thomashow USDA-ARS, Root Disease and Biological Control Research
Unit, Washington State University, Pullman, WA, USA
Jan Tommassen Section Molecular Microbiology, Department of Biology, Utrecht
University, Utrecht, The Netherlands
Contributors xix

Eddy van der Meijden Institute of Biology, Leiden University, Leiden, The
Netherlands
Jan van der Wolf Plant Research International, Wageningen, The Netherlands
Hanny van Megen Laboratory of Nematology, Wageningen University,
Wageningen, The Netherlands
Marc Van Montagu VIB–Institute of Plant Biotechnology Outreach, Department
of Biotechnology and Bioinformatics, Ghent University, Gent-Zwijnaarde, Belgium
Saskia C. M. Van Wees Plant-Microbe Interactions, Department of Biology,
Faculty of Science, Utrecht University, Utrecht, The Netherlands
Jaco Vangronsveld Centre for Environmental Sciences, Hasselt University,
Diepenbeek, Belgium
Muthusubramanian Venkateshwaran School of Agriculture, University of
Wisconsin-Platteville, Platteville, WI, USA
Mariëtte Vervoort Laboratory of Nematology, Wageningen University,
Wageningen, The Netherlands
Susanne von Bargen Division Phytomedicine, Faculty of Life Sciences, Humboldt-
Universität zu Berlin, Berlin, Germany
Han A. B. Wösten Section Molecular Microbiology, Department of Biology,
Utrecht University, Utrecht, The Netherlands
David M. Weller United States Department of Agriculture-Agricultural
Research Service, Root Disease and Biological Control Research Service,
Pullman, Washington, USA
Sheridan L. Woo Department of Agriculture, University of Naples Federico II, and
Institute of Plant Protection IPP–CNR, Portici (NA), Italy
Zefen Yu Yunnan University, Kunming, People’s Republic of China
Keqin Zhang Yunnan University, Kunming, People’s Republic of China
Abbreviations

ABA abscisic acid

ACC 1-aminocyclopropane-1-carboxylate
AFM atomic force microscopy
AHL N-acyl homoserine lactone
AMF arbuscular mycorrhizal fungus
ARISA Automated rRNA Intergenic Spacer Analysis
BC biocontrol
BCA biocontrol agent
BCPC British Crop Production Council
BNF Biological nitrogen fixation
BPSG BioPesticide Steering Group
Bt Bacillus thuringiensis
CFU colony forming units
CIPAC Collaborative International Pesticides Analytical Council
CK cytokinin
c-LP cyclic lipopeptide
CLSM confocal laser scanning microscopy
CMV Cassava mosaic virus
CNN competition for nutrients and niches
CRAfT Cre Reporter Assay for Translocation
CSP Common Symbiotic Pathway
DAMP damage-associated molecular patterns
DAPG 2,4-diacetylphloroglucinol
DGGE denaturing gradient gel electrophoresis
DIC Differential Interference Contrast
DSF diffusible signal factor
EBIC European Biostimulant Industry Council
ECM Ectomycorrhizal fungi
EPA Environmental Protection Agency
EPN Entomopathogenic nematode
EPPO European and Mediterranean Plant Protection Organization
ET Ethylene
xxi
xxii Abbreviations

ETI effector-triggered immunity

ETS effector-triggered susceptibility
FAME Fatty Acid Methyl Esters
FAO Food and Agriculture Organization
FeMoCo Iron-molybdenum cofactor
GA gibberellin
GAP Good agricultural practice
GAs gibberellins
GFP Green fluorescent protein
GM genetically modified
GMO genetically modified organism
GOGAT Glutamine oxoglutarate aminotransferase
GS Glutamine Synthase
HR Homologous recombination
HR hypersensitive response
HSL homoserine lactone
IAA indole-3-acetic acid
IAM indole-3-acetamide
IBCA invertebrate biocontrol agent
IJ infective juvenile
IncP-1 incompatibility groups of plasmids
InsP5 1D-myo-inositol 1,2,4,5,6 pentakisphosphate
IPM Integrated pest management
IPyA indole-3-pyruvate
ISO international organization for standardization
ISR induced systemic resistance
ITS fragments internal transcribed spacer ribosomal DNA (used as the universal
barcode for fungi)
JA Jasmonic Acid
LCO lipochitooligosaccharide
LPS lipopolysacchide
LysM lysine-motif
MALDI MSI Matrix-Assisted Laser Desorption/Ionization Mass Spectrome-
try Imaging
MAMP microbe associated molecular pattern
MGE mobile genetic element (DNA fragments, which can be trans-
ferred from cell to cell)
MHB mycorrhiza helper bacteria
MRL maximum residue limit
Myc factor mycorrhization factor
N2 H 4 hydrazine
N2 O nitrous oxide
NB-LRR nucleotide binding-leucine-rich repeat
NH2 OH hydroxylamine
NHEJ Non-homologous end-joining
Abbreviations xxiii

nif gene nitrogen fixation gene

NO nitric oxide
Nod factor nodulation factor
NOx nitrogen oxides
OECD organisation for economic co-operation and development
OMZ oxygen minimum zone
OTU operational taxonomic unit (used as species equivalent)
PAHs polycyclic aromatic hydrocarbons
PAMP pathogen-associated molecular pattern
PCA phenazine-1-carboxylic acid
PCB poly-chlorinated biphenyls
PCN phenazine-1-carboxamide
PCR polymerase chain reaction
PCWDE plant cell wall degrading enzyme
PGP plant growth-promotion
PGPB plant growth-promoting bacteria
PGPF plant growth promoting fungi
PGPM plant growth promoting microbe
PGPR plant growth-promoting rhizobacteria
PhyloChip microarray for the comprehensive identification of microbial
organisms
Pi inorganic phosphate
PIP plant-incorporated protein
PIR protein with internal repeat
PIT pre-infection thread
PKS polyketide synthase
PMRA pest management regulatory agency
PPA pre-penetration apparatus
PPP plant protection product
PP-transferase phospho-pantetheinyl-transferase
PQQ pyrroloquinoline quinone
PR pathogenesis related
PRR pattern recognition receptor
PSM phosphate solubilising microorganism
PTI pathogen-triggered immunity
PZN plantazolicin
qPCR quantitative real-time PCR (provides copy-number of the target
organism)
QS quorum-sensing
rDNA ribosomal DNA
RLP receptor-like protein
RNAi ribonucleic acid interference
ROL radial oxygen loss
ROS reactive oxygen species
RP rock phosphate
xxiv Abbreviations

SA salicylic acid
SAM S-adenosylmethionine
SAR systemic acquired resistance
SIMS secondary ion mass spectrometry
SIP stable-isotope probing
SL strigolactone
SRP signal recognition particle
SSCP single strand conformation polymorphism (method to analyze
composition of microbial communities)
SWOT strengths, weaknesses, opportunities and threats
SYM genes common symbiosis genes
SYM pathway symbiosis pathway
T1-6SS type 1-6 secretion system
T4SS type 4 secretion system
TAD take-all decline
TC-DNA DNA obtained from the total community present in one sample
T-RFLP terminal restriction fragment length polymorphism (method to
analyze the composition of microbial communities)
TTSS type III secretion system
US EPA OCSPP United States Environmental Protection Agency Office of
Chemical Safety and Pollution Prevention
USDA US Department of Agriculture
VBNC viable but non-culturable
VOC volatile organic compound
Chapter 1
Introduction to Plant-Microbe Interactions

Ben Lugtenberg

Abstract Pathogenic microbes and pest organisms as well as unfavorable growth

conditions can be a threat for plant growth. Other beneficial microbes and small
organisms can be used to protect plants against these attackers or to assist the plant
to overcome the unfavorable conditions. These plant-beneficial organisms can be
divided into classes which (i) reduce plant diseases, (ii) which regulate plant growth,
(iii) help plants to overcome stresses, and (iv) inactivate soil pollutants which inhibit
plant growth or make (parts of) the plant unsuitable for consumption.

Plant Pathogens and Pest Organisms are a Threat for Plant Growth In this book
we discuss pathogens and pest organisms which are a threat for plant growth. We
highlight the roles which microbes can play in making agriculture and horticulture
more sustainable. Selected microbes are able to (partly) replace most chemicals
which are presently used in agriculture. In addition, microbes can often be used
against diseases for which no chemicals are available. In this book, the following
activities and applications of microbes will be discussed.
Biological Control of Plant Diseases Approximately 25 % of the world’s crop yield
is lost every year, mainly due to diseases caused by fungi, by other pathogens, and by
pests. Plant protection products are on the market to fight these diseases. Presently
these are mainly chemicals. Their use can be threatening the health of people and
polluting the environment. Disease control with beneficial microbes is an alternative
which allows sustainable crop production. The use of microbial plant protection
products is growing and their importance will strongly increase because of political
and public pressure.
Regulation of Plant Growth The world population is growing and the amount of
food needed by 2050 will be the double of what is being produced now, whereas the
area of agricultural land is decreasing. We have to increase crop yield in a sustainable
way, i.e. chemical plant growth regulators have to be replaced by microbiological

B. Lugtenberg ()
Institute of Biology, Sylvius Laboratory, Leiden University,
Sylviusweg 72, 2333 BE Leiden, The Netherlands
Tel.: +31629021472
e-mail: Ben.Lugtenberg@gmail.com

© Springer International Publishing Switzerland 2015 1

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_1
2 B. Lugtenberg

products. Also here, the use of microbial products is growing and their importance
will strongly increase.
Control of Plant Stress by Microbes An increasing area of agricultural land is arid
and/or salinated. Global warming will increase this area. Plant growth is inhibited, or
even made impossible, by drought and salt. It has been proven already that microbes
can be used successfully to alleviate such stresses.
Microbial Cleaning of Polluted Land Chemical pollution of land can make plant
growth difficult or even impossible. But even when crop plants grow on such lands,
their products are often polluted and not suitable for consumption. Selected microbes
have been already been used successfully to detoxify chemical pollutants in soil and
to remove heavy metals, thereby allowing the growth of healthy plants.
The field of Plant-Microbe Interactions has made important progress thanks to
the development of new technologies. Attention to state-of-the-art DNA and visual-
ization techniques is paid in two separate chapters. Moreover, successful examples
of progress are presented under Paradigms of Plant-Microbe Interactions. The book
ends with the presentation of a number of real innovative research projects of which
the future will show whether these are dreams or big steps forwards.
 Part I
Introductory Chapters
Chapter 2
The Importance of Microbiology in Sustainable
Agriculture

Thomas Schäfer and Tom Adams

Abstract Deriving from various naturally-occurring microorganisms such as bac-

teria and fungi, microbial technologies can protect crops from pests and diseases
and enhance plant productivity and fertility. They enable farmers to increase yield
and productivity in a sustainable way and are expected to play a significant role in
agriculture.

As the global population’s rapid growth is set to continue, the need to significantly in-
crease agricultural output without increasing pressure on the environment also grows.
Microbial solutions enable farmers to drive yield and productivity in a sustainable
way. Deriving from various naturally-occurring microorganisms such as bacteria and
fungi, these solutions can protect crops from pests and diseases and enhance plant
productivity and fertility.
Microbial solutions make up approximately two thirds of the agricultural bio-
logicals industry. Representing roughly US$ 2.3 billion in annual sales, agricultural
biologicals have posted double-digit sales growth each of the last several years. There
are numerous biological products currently on the market that contain microorgan-
isms as active ingredients, including seed treatment and foliar applied products.
Microbial technologies can help improve nutrient acquisition, promote growth and
yield, control insects and protect against disease. These emerging agricultural bio-
logical technologies complement the integrated systems approach that is necessary
in modern agriculture, bringing together breeding, biotechnology and agronomic
practices to improve and protect crop yields.
There has been significant interest in agricultural biologicals in the past few
years from major crop chemical manufacturers, including Bayer’s acquisition of
Agraquest, BASF’s acquisition of Becker Underwood, and Syngenta’s acquisitions
of Pasteuria and Devgen. Most recently, Novozymes and Monsanto established The

T. Schäfer ()
Novozymes A/S, Brudelysvej 32, 2880 Bagsvaerd, Denmark
Tel.: + 45 44460000
e-mail: TSch@novozymes.com
T. Adams
Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA
Tel.: + 1 (314) 258-1547
e-mail: tom.h.adams@monsanto.com

© Springer International Publishing Switzerland 2015 5

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_2
6 T. Schäfer and T. Adams

BioAg Alliance in December 2013, with a goal to discover, develop and sell mi-
crobial solutions that enable farmers worldwide to increase crop yields with less
chemical input. Novozymes brought an established product portfolio and strengths
within microbial discovery, application development and fermentation to this part-
nership. Combined with Monsanto’s highly-developed seeds and traits discovery,
field-testing and extensive commercial network, the aim is to deliver a comprehen-
sive research, development and commercial collaboration from which agriculture,
consumers, the environment and society at large can benefit.
Microbial solutions provide more choice for farmers and help meet the demand
for more sustainable agricultural practices. Such solutions can increase crop yields
and develop a more sustainable industry impact profile, ultimately resulting in more
food to feed the growing world and new opportunities to protect the planet.
Chapter 3
Life of Microbes in the Rhizosphere

Ben Lugtenberg

Abstract Life of microbes in the rhizosphere is best characterized as starvation for

nutrients and attempts to survive. All microbes are hunting for food of which a sub-
stantial amount is supplied by the root in the form of exudate. The most successful
microbes are attracted to food sources, such as to the root and to each other, by
chemotaxis to specific exuded compounds. Subsequently they colonize the target
organism. Specific exudate compounds can also initiate communication between
organisms as the start of more specialized interactions, such as nodulation, patho-
genesis, DNA transfer and the production of antibiotics. Some target organisms have
developed defense reactions against such attacks which allows them to survive. All
these processes are described in this chapter.

3.1 The Rhizosphere

The rhizosphere was defined by Lorentz Hiltner as “the soil compartment influenced
by the roots of growing plants”. The rhizosphere is supposed to be no more than a
few mm thick. It is 10- to 100-fold richer in microbes than the surrounding “bulk”
soil because 6–21 % of the carbon fixed by the plant is secreted by the root. This phe-
nomenon is called the rhizosphere effect. It is good to realize that the concentration
of nutrients in the rhizosphere is still 100-fold lower than that in the usual laboratory
media. The life style of microbes in the rhizosphere is therefore best characterized as
starvation. Recent reviews on the rhizosphere are those of Haas and Défago (2005),
Lugtenberg and Bloemberg (2004), Lugtenberg and Kamilova (2009), and Pinton
et al. (2007).
Roots of many plants are colonized by mycorrhizal fungi which can function
as fine extensions of the root and allow the plant to reach nutrients which can-
not be reached by the thicker roots. The combination of root surface and attached
mycorrhizal fungi is designated as the mycorrhizosphere (Chap. 25).

B. Lugtenberg ()
Institute of Biology, Sylvius Laboratory, Leiden University, Sylviusweg 72,
2333 BE Leiden, The Netherlands
Tel.: + 31629021472
e-mail: Ben.Lugtenberg@gmail.com

© Springer International Publishing Switzerland 2015 7

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_3
8 B. Lugtenberg

Plant life is affected by both abiotic and biotic conditions. For the estimation of
local conditions, bioreporters have been developed. These are bacterial derivatives
harboring a promoter that reacts on the compound or condition of choice, and that is
fused to a reporter gene encoding a protein which can easily be detected and quantified
(for example gfp, lux, lacZ or inaZ). Reporter constructs respond for example to
the presence of certain sugars, amino acids, or to conditions such as pH or the
bioavailability of carbon, phosphate and oxygen (Chap. 10 in ref. Pinton et al. 2007).
Abiotic Conditions Abiotic conditions affecting plant growth include temperature,
pH, soil type, water potential, and concentrations of bioavailable essential nutrients
and salts. Soils can be rich or poor for plant growth. Rich soils contain sufficient water
and nutrients. Drought is a major and increasing problem for plant growth (Chap. 28),
and so is salination (Chap. 28). Nitrogen and phosphorous are the major nutrients
whereas ions of potassium, iron and micronutrients are also required for plant life.
Poor soils can be fertilized chemically, for example by adding N-P-K fertilizer. This
can increase plant growth enormously but is not sustainable. Therefore the trend is
to replace such chemicals by other means, for example by bacteria which generate
nutrients in forms that can be used by the plant (Chap. 23 and 24). A neutral or
high pH makes ferric iron ions insoluble and will therefore reduce plant growth. In
contrast, at acid pH these ions will be soluble.
Biotic Conditions The rhizosphere contains microbes (bacteria and fungi) as well
as small animals, such as amoebae, insect larvae, mites, nematodes and protozoa
(Bonkowski et al. 2009). The microbes are collectively called the rhizosphere mi-
crobiome (Mendes et al. 2013) (Chap. 30 and 43). The main factors shaping the
rhizosphere microbiome are the soil type and the plant genotype. The microbes can
have a beneficial, neutral, or negative effect on plant growth. Some of the benefi-
cial microbes have been cultured and formulated and are being sold as commercial
products (Chaps. 32–34) and are applied as biopesticides or plant protection prod-
ucts (Chap. 18), biofertilizers (Chaps. 23 and 24), rhizoremediators (Chap. 29),
phytostimulators (Chaps. 25 and 26), or stress controllers (Chap. 27). Beneficial
microbes include strains of the bacteria Bacillus and Pseudomonas, of Arbuscular
Mycorrhizal Fungi (AMF) (Chap. 25), and of the fungus Trichoderma (Chap. 36).
Plant pathogens can be viruses (Chap. 13), bacteria (Chap. 9), fungi and oomycetes
(Chap. 10), or nematodes (Chap. 11). Some insects cause pests (Chap. 12).
A healthy soil hardly contains pathogens. Soils harboring pathogens are called
disease-conducive soils. However, in some cases, soils harbor pathogens but plants
growing in this soil remain healthy. These disease-suppressive soils contain beneficial
microbes which suppress the action of the pathogens (Chap. 38). Some soils contain
human pathogens (Berg et al. 2005) which can be risky for agricultural workers as
well as for consumers. It has been suggested that treatment of seeds of crop plants
with products containing enhanced colonizing bacteria will reduce the number of
pathogens on the root and, therefore, reduce this risk for humans (Egamberdieva
et al. 2008).
3 Life of Microbes in the Rhizosphere 9

Nutrition of Rhizosphere Microbes Between 6 and 21 % of the carbon fixed by the

plant can be released by the plant root. This material consists of sloughed-off cells,
macromolecules and small molecules. The latter are the favorite food sources of the
rhizosphere microbes. Based on mutational studies it was concluded that the most
important food sources exuded by tomato roots are organic acids, such as citric,
malic, lactic, succinic, oxalic, and pyruvic acids. Other root exudate compounds
include sugars (such as glucose, xylose, fructose, maltose, sucrose and ribose),
amino acids, fatty acids, nucleotides, putrescine, and vitamins. A special group of
exudate compounds are the signal molecules which are used for communication
between the plant and microbes (see Sect. 3.4).
The qualitative and quantitative composition of root exudate is heavily influenced
by the presence of microbes in the rhizosphere in a microbe species-dependant way.
For example, the addition of the pathogenic fungus Fusarium oxysporum f. sp.
radicis-lycopersici to a mono-axenic tomato system strongly decreased the amount
of citric acid and increased the amount of succinic acid but did not influence the total
amount of organic acids in exudate. In contrast, when the Pseudomonas biocontrol
strain WCS365 was added in amounts sufficient for biocontrol, the total amount
of organic acids—especially of citric acid—strongly increased whereas the level of
succinic acid decreased dramatically (Kamilova et al. 2006).

3.2 Interactions Between Organisms in the Rhizosphere

Using sophisticated techniques (Chap. 31), interactions occurring between organisms

in the rhizosphere can be visualized.
Fungus—Plant Interactions A fungal spore in the soil will germinate when it
senses the presence of a plant root through certain chemicals secreted by the root
(Lugtenberg and Kamilova 2009). Subsequently, the formed mycelium will grow into
the direction of the root, attach to the root, colonize the root surface, and penetrate
the root. This process is shown for the fungus Fusarium oxysporum f. sp. radicis-
lycopersici in Fig. 3.1a–3.1d.
Bacterium—Plant Interactions During treatment of seeds with microbes, use is
made of the fact that microbes which are present on the seed are in the best position to
colonize the roots of the emerging seedling. Therefore, the best way to colonize the
root with a beneficial bacterium is to surface-sterilize the seed and coat it subsequently
with cells of the beneficial bacterium of choice. After growth, the result is that the
top of the seedling root is best colonized (with approx. 106 bacteria per cm root).
A good colonizing bacterium can even reach the growing root tip. Coming from
the seed, a bacterium colonizes the root, initially as individual cells (Fig. 3.1e)
which subsequently multiply and grow out to micro-colonies (Fig. 3.1f), presently
designated as biofilms (Chap. 7). Only approximately 15 % of the plant root surface
can be covered by bacteria (Fig. 3.1f). Most bacteria are found on the junctions
between epidermal cells (Fig. 3.1e). Mature biofilms usually consist of multiple
layers of cells (Fig. 3.1f) and are covered with a mucous layer (Fig. 3.1g,h).
10 B. Lugtenberg

Fig. 3.1 Visualization of plant-microbe and microbe-microbe interactions during biocontrol. Con-
focal Laser Scanning Microscopy (a–g and i) and scanning electron microscopy (h) were used to
visualize control of tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici
by Pseudomonas biocontrol bacteria. For explanation, see text. Panels a, c, and d were reproduced
from Lagopodi et al. (2002), panel b from Bolwerk et al. (2003), and panel h from Chin-A-Woeng
et al. 1997. Panel e is from Bolwerk, Lagopodi and Bloemberg, unpublished. Panels f and g are
from Bloemberg et al. 1997; Copyright ©American Society for Microbiology. Reproduced from
Lugtenberg and Girard (2013) by permission of the publisher

Bacterium—Fungus Interactions Some bacteria attach to fungal hyphae in order

to use them as a food source (Fig. 3.1i).

3.3 Competitive Root Tip Colonization

In order to be successful in their beneficial action, cells of applied microbes have

to reach the root in high numbers. Therefore, they have to win the competition with
indigenous microbes for nutrients secreted by the root and for niches on the root. In
order to identify bacterial traits important for competitive root colonization, Simons
et al. (1996) screened individual random mutants of the efficient P. fluorescens root
colonizing strain WCS365 in a mono-axenic sand system. Surface-sterilized tomato
seeds were coated with a 1:1 mixture of one of the mutants and the wild type strain.
After the root system had developed, the root tip was isolated and the ratio of wild
type to mutant cells was determined. Competitive colonization mutants loose this
competition and were found in lower numbers than the wild type. The mutated genes
were characterized and the traits lost in the mutants were identified. It turned out
that the number of traits involved in competitive root colonization is high. The best
understandable traits can be grouped as follows. An excellent root tip colonizer
should (i) be able to synthesize amino acids, vitamin B1, uracil, as well as the O-
antigenic side chain of lipopolysaccharide; (ii) grow fast in root exudate, show a
3 Life of Microbes in the Rhizosphere 11

chemotactic response towards the root, and adhere to the root; (iii) have a number
of properties related to secretion (see Chap. 6), such as the secB gene involved in a
protein secretion pathway, the type three secretion system, and an intact ColR/ColS
two-component system. The latter property is supposed to be related to keeping
protein pores in the outer membrane open. The most likely explanation for the need
of the type three secretion system is that the needle of this system is required for
tapping nutrients from the plant cell. For better understanding of these traits, see
Chap. 6.
Good tomato root colonizers have also been tested on other crop plants. In contrast
to general predictions, pseudomonads colonize roots of many other plants—such as
cucumber and wheat—well, so there is hardly any host plant specificity involved in
colonization. Moreover, mutants which are not colonizing tomato roots well, are also
impaired in the colonization of wheat and cucumber roots, indicating that bacterial
colonization traits are common for several plants.
Interestingly, it is possible to increase the colonization ability of bacteria or to
select strongly enhanced root tip colonizers. (i) When cells of a transposon mutant
library of P. fluorescens strain WCS365 were put on a seedling and selected for
enhanced root tip colonizers, the best colonizing mutant was characterized as a
strain carrying a mutation in the gene encoding MutY, an enzyme which repairs
mutations in the DNA. We assume that this enhanced colonizing mutY derivative
has collected a combination of mutations which have fine-tuned the strain in such a
way that it is better adapted to the conditions on the root (De Weert et al. 2004a). It
would be interesting to compare the nucleotide sequences of these strains in order to
evaluate which traits contribute to enhanced colonization. (ii) Using the same mono-
axenic system and a mixture of numerous rhizosphere strains, it appeared possible
to select strains which are enhanced colonizers (Fig. 3.2). Half of these strains are
even able to control the disease tomato foot and root rot by a mechanism designated
as Competition for Nutrients and Niches (CNN) (Kamilova et al. 2005; Fig. 3.2;
Chap. 18).

3.4 Communication Between Organisms by Chemical Signaling

Many compounds secreted by plant roots and microbes function as signals that play
a role in the communication between plant and microbe or between microbes. A
selection of examples is given below.
Chemoattraction Bacteria in the rhizosphere usually live under conditions of star-
vation for nutrients and, consequently, are hunting for food. They have developed
sensing systems which guide them towards compounds secreted by plant roots and
by fungal hyphae. Pseudomonas cells from soil find the tomato root because they
are attracted by root exudate. In order to identify the exudate component which is
most active in the tomato rhizosphere, the chemo attractant activity of the individ-
ual representatives of the major groups of exudate components of tomato, namely
organic acids, sugars, and amino acids, was analyzed. Experiments showed that
12 B. Lugtenberg

Inoculate seed or seedlings

Growth in quartz
sand/nutrient solution

Suspension of rhizosphere
bacteria

Collect all colonies

Judge colony diversity

Isolation root tip
Growth o/n

After 3 Cycles
Vortex and dilute on KB plate
Strains acting through CNN

Fig. 3.2 Enrichment of bacteria which compete efficiently for nutrients and niches. Starting from
a seed on which a crude mixture of rhizosphere bacteria is applied, enhanced competing bacteria
are enriched for by repeatedly selecting for those cells which—after application to a sterile seed—
reach the root tip first. Reproduced from Pliego et al. (2011 by permission of the publisher), after
modification by Clara Pliego

sugars are inactive, that dicarboxylic acids are active, and that amino acids (espe-
cially L-leucine) are the most active chemoattractants. However, when this data is
corrected for the levels of the individual components estimated to be present in the
tomato rhizosphere, it was concluded that malic acid and citric acid are the major
chemoattractants for Pseudomonas cells in the rhizosphere (De Weert et al. 2002).
After reaching the root surface, the bacteria colonize it (Fig. 3.1e,f).
After reaching the fungal hyphae, the bacteria colonize the surface (Fig. 3.1i).
Bacterial cells also try to find fungal hyphae because they can be used as food sources.
They have developed a system to detect specific fungal products. This process has
been extensively studied in the case of Pseudomonas cells trying to find hyphae of
the fungus Fusarium oxysporum fsp. radicis-lycopersici. Using a series of Fusarium
oxysporum f. sp. radicis-lycopersici strains which differ in the levels of secreted
fusaric acid, it was found that the more fusaric acid is secreted, the stronger bacteria
are attracted to the fungus. Finally, using synthesized chemically pure fusaric acid,
the notion that fusaric acid is the major chemo attractant for Pseudomonas cells, was
confirmed (De Weert et al. 2004b).
3 Life of Microbes in the Rhizosphere 13

Initiation of the Nodulation Process Certain flavonoids secreted by roots of legu-

minous plants signal to (Brady)rhizobium bacteria that a root of their leguminous
host plant is close by and that the process of nodule formation can be initiated. These
flavonoids induce the nodulation (nod) genes in the bacterium which encode the en-
zymes for the synthesis of Nod-metabolites or lipochitin oligosaccharides (LCOs).
These LCOs initiate the formation of nodules in which bacteria, in the form of
bacteroides, fix atmospheric nitrogen for the plant.
Induction of vir gene Expression in Agrobacterium Phenolic compounds released
from plant wound sites, such as 3’,5’ dimethoxy-4’-hydroxy acetophenone (com-
monly known as acetosyringone) and α-hydroxy acetosyringone, are the key inducers
of bacterial virulence genes. A pH of around 5.5 and a temperature below 30 ◦ C are
also required. Particular sugars enhance the level of induction (Chap. 37).
N-Acyl Homoserine Lactones (AHLs) and Quorum Sensing (Chap. 7) AHLs are
signal molecules secreted by many Gram-negative bacteria. They are used to rec-
ognize bacteria of their own kind. These molecules are supposed to diffuse more or
less freely over the membranes. If the bacterial cell concentration reaches a certain
density, the quorum, the intracellular AHL concentration reaches a level sufficiently
high to initiate the synthesis of increased amounts of AHL, which in turn initiates the
production of several proteins, including those involved in the synthesis of many an-
tibiotics (Chap. 18) as well as virulence factors. AHLs also play a role in exchanging
DNA by inducing F-pilus-mediated conjugation .
AMF-Plant Interaction Flavonoids present in root exudates can initiate interac-
tions of plant roots with AMF such as stimulation of spore germination and hyphal
branching. More recently, it was reported that secreted representatives of a group of
plant hormones, the strigolactones, can also cause branching of neighboring AMF
spores, thereby increasing their chance to encounter a plant root. In addition, theAMF
releases signal molecules, identified as lipochito-oligosaccharides or Myc factors
which stimulate root growth and branching (Chap. 25).
Volatiles Both plants and microbes can produce a range of volatile organic com-
pounds, briefly designated as volatiles. Whereas volatile plant hormones such as
ethylene, methyl jasmonate, and methyl salicylate function as airborne signals in
mediating plant communication, several bacterial volatiles play a role in biocon-
trol (HCN) or induce systemic resistance in plants (such as 2,3-butanediol and its
precursor acetoin) (Chap. 8).
War in the Rhizosphere Microbes in the rhizosphere can be attacked by their
colleagues but they are not always simple victims. Like bacteria, some fungi have
developed resistance mechanisms against antibiotics. The following mechanisms of
fungal and bacterial tolerance or resistance have been shown. (i) Detoxification of the
antibiotic. Some biocontrol strains produce the antibiotic 2,4-diacetyl phlorogluci-
nol, for example to kill Fusarium strains. Some Fusarium strains produce an enzyme
that deacetylates the antibiotic to mono-acetyl phloroglucinol which is a lot less fun-
gitoxic. (ii) Efflux of the antibiotic. Upon exposure to phenazine antibiotics, some
14 B. Lugtenberg

strains of the fungus Botrytis cinerea induces an efflux pump for the antibiotic. (iii)
Repression of the synthesis of an antibiotic. The biocontrol fungus Trichoderma
atroviride P1 produces chitinase enzymes to attack the cell wall of fungi (Chap. 6).
The Fusarium mycotoxin deoxynivalenol inhibits the expression of the chitinase
genes ech42 and nag1 which contribute to the biocontrol activity. Another example
of repression of antibiotic synthesis is the Fusarium metabolite fusaric acid which
inhibits the syntheses of the antibiotics 2,4-diacetyl phloroglucinol and phenazine-1-
carboxamide produced by some Pseudomonas biocontrol strains. Finally, AHLs are
often required for the synthesis of antibiotics and virulence factors. Some bacteria
protect themselves by enzymatic inactivation of AHL.
Gene Transfer in the Rhizosphere A well known form of gene transfer between
bacteria, namely F-pilus mediated conjugation, requires AHL. In biofilms, bacteria
are very close to each other and covered by a mucous layer (Fig. 3.1f–h). These
conditions seem ideal for keeping the intracellular AHL concentration high and
therefore for stimulating conjugation. Indeed, it has been reported that gene transfer
between bacteria in the rhizosphere is very efficient (van Elsas et al. 1988).

Acknowledgements This research was supported by Leiden University as well as by numer-

ous grants, especially from the European Commission, EET, INTAS as well as from the NWO
departments of ALW, CW, and STW.

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Chapter 4
Life of Microbes on Aerial Plant Parts

Johan H. J. Leveau

Abstract The phyllosphere, or leaf environment, is a temporally erratic, spatially

heterogeneous, and inherently transient habitat that supports a large and diverse
population of microorganisms. This chapter offers an introductory exploration of the
leaf surface as a microbial biome and of the genes and gene functions that underlie the
unique adaptations for epiphytic survival in this inhospitable milieu. Also reviewed
are the various ways in which host plant and environmental conditions affect the
assembly, structure, and function of microbial communities on plant foliage. Special
emphasis is placed on the challenges of studying microbial life on leaf surfaces,
on the impact of leaf-associated microbiota on the ecosystem services provided by
plant leaves, and on the interactions of epiphytic microorganisms with their host,
each other, and plant and human pathogens in the context of food security and food
safety.

4.1 The Phyllosphere

As an ecosystem or ‘biome’for microorganisms, leaves and other above-ground plant

parts are referred to as the ‘phyllosphere’, a term coined in the 1950s and inspired
by Hiltner’s ‘rhizosphere’ (Chap. 3). The prefix ‘phyllo’ is derived from the Greek
ϕ υλλo
’ for ‘leaf’ and shares the same base as ‘filo’, i.e. the dough used for flaky
pastries such as baklava and apfelstrudl. Many use the term ‘phyllosphere’ (some-
times ‘phylloplane’) exclusively for plant foliage, and refer to other above-ground
plant parts with alternative language, for example the anthosphere (flower), carpo- or
fructosphere (fruit), caulosphere (bark/stem), calusphere (bud), and spermosphere
(seed). This chapter will stick to the original definition of phyllosphere and only cover
microbial life on leaves (Leveau 2006; Meyer and Leveau 2012; Vorholt 2012), not

J. H. J. Leveau ()
Department of Plant Pathology, University of California, One Shields Avenue,
476 Hutchison Hall, Davis, CA 95616, USA
Tel.: +1 530 752-5046
e-mail: jleveau@ucdavis.edu

© Springer International Publishing Switzerland 2015 17

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_4
18 J. H. J. Leveau

other above-ground plant parts. Also, its narrative will be limited to epiphytic mi-
croorganisms (i.e. surface-associated, in contrast to endophytes, see also Chap. 5)
and focus on land-based plants only, not submerged plants or macro-algae.
Based on satellite imagery of Earth’s terrestrial foliage, the phyllosphere micro-
biome is estimated to encompass about half a billion square kilometers. However,
the ‘coastline paradox’ stipulates that this value is many times larger if one considers
that the leaf surface represents an intricate three-dimensional topography at the scale
at which it is occupied and experienced by its microscopic inhabitants, i.e. the phyl-
losphere microbiota, which include bacteria, filamentous fungi, yeast, oomycetes,
algae, lichens, protists, and protozoa. The extraordinary structural heterogeneity
of the leaf surface is a key driver of the nonrandom, micrometer-scale spatial dis-
tribution of epiphyllous microorganisms. For example, the cuticle that covers the
leaf surface varies laterally in composition, thickness, and permeability, which con-
tributes to differential leaching of plant substances (including photosynthates) to the
surface where they become available for exploitation by microorganisms (Leveau
and Lindow 2001). Spatial variation in the topography-driven ‘waterscape’ on the
leaf surface also impacts the ability of microorganisms to avoid drought, to laterally
disperse, and to sense, respond to and interact with other colonizers on the same leaf
surface.
Microbial Colonization Patterns on Leaves Bacteria are by far the most abundant
microbial colonizers of leaf surfaces, an estimated 1026 cells on the foliage of all
terrestrial plants combined. Characteristic of bacterial life in the phyllosphere is
that the majority of bacteria occur not as single cells but in large clusters (often
referred to as ‘aggregates’) of up to thousands of cells. The term ‘aggregate’ is
somewhat misleading, as the formation of these clusters is not just a function of cells
aggregating (in the literal sense, i.e. bacteria coming together, actively or passively,
to form a cluster), but also of cells staying together, i.e. the formation of offspring by
a single immigrant to the leaf surface into a colony or cluster. The observation that
trichomes (leaf hairs) often support large clusters of bacteria at their base is probably
a result of both mechanisms: increased leaching at these sites favors the replication
and staying together of cells, while the process of evaporation forces accumulation
of cells in areas where water is retained longest, including the base of trichomes. The
distribution curve of bacterial cluster sizes on leaves is typically right-hand skewed,
with many small clusters and few very large clusters. Underlying this pattern is the
ability of individual bacteria to leave larger clusters and start a new one elsewhere
on the leaf (Perez-Velazquez et al. 2012; van der Wal et al. 2013). In this, bacterial
life in aggregates on leaf surfaces resembles that in biofilms on variably-saturated
surfaces (Chap. 7).
Processes of Leaf Inoculation There are several processes by which leaves get
colonized. Some microorganisms are already present in buds and therefore among
the first to explore the developing leaf. Most microorganisms will arrive after leaf
emergence, by various mechanisms and from different sources, including wind, rain,
dust, soil splash, and deposition from the air and by insects. As leaves get older, sur-
face topography, cuticle waxiness, availability of water and nutrients, and microbial
4 Life of Microbes on Aerial Plant Parts 19

colonization patterns all change and affect the ability of newly arriving immigrants
to the leaf to settle, disperse or start reproducing. An underappreciated factor in the
assembly of microbial communities on leaf surfaces is chance. The leaf represents
a patchy environment in terms of availability of nutrients, presence or absence of
water, and microbial colonization, so an incoming microorganism may just have to
be lucky to land in a spot that isn’t already taken by other microorganisms and that
(still) offers food and shelter. For bacterial immigrants to bean leaves, such luck has
been quantified (Remus-Emsermann et al. 2012): there is a 3 % chance that they
land in a location that allows five or more doublings, compared to an approximately
50 % chance of landing somewhere where they double once or less. Precolonization
significantly reduces immigrant ability to produce offspring, indicating that early
arrival by phyllosphere-fit microorganisms offers greatest likelihood to benefit from
available nutrients and to dominate the community structure. The stochastic nature of
arrival probably underlies the often observed variation in bacterial community com-
position on individual leaves of plants in the same field or greenhouse (Maignien
et al. 2014).

4.2 Structure and Function of Phyllosphere Microbiota

The leaf surface is an extreme habitat. In the course of a single day, leaves may go
from wet to dry, dark to light, and cold to hot. On a longer time scale, plant leaves
will eventually senesce and shed, so epiphyllous microorganisms also need to avoid,
anticipate, or survive life outside of the leaf environment, for example in the soil
or air. This picture of the leaf surface as a harsh, quickly changing, and ephemeral
environment predicts that phyllosphere microorganisms must be well adapted to
deal with phyllosphere stresses. Indeed, many possess genes for the production
of pigments that protect against ultraviolet radiation, or DNA repair systems that
deal with the damage caused by it. The ability to accumulate solutes to withstand
drought is a common property among bacteria and fungi isolated from foliage. Many
phyllosphere-related adaptations don’t convey mere tolerance to leaf stresses, but
contribute to habitat modification in favor of microbial survival and growth: biofilm
formation, ice nucleation (see Box 4.1), and surfactant production are all examples
of ecosystem engineering by epiphytic microorganisms, designed to provide shelter,
release nutrients, and/or facilitate dispersal.
20 J. H. J. Leveau

Box 4.1
Bacterial ice nucleation was discovered as a property of plant pathogens of
the species Pseudomonas syringae which cause frost damage on crop foliage.
Pioneering work by Dr. Steve Lindow showed that this activity was due to a
single gene and that mutants lacking this gene (so-called ice-minus bacteria)
not only lost the ability to cause frost, but in the first deliberate release of a
genetically modified organism also protected plants against ice-positive P. sy-
ringae. Bacterial ice nucleation has been commercially exploited; for example,
artificial snow at the 2014 Sochi Olympic Games was generated with the use
of devitalized ice-nucleating bacteria.

Phyllosphere Functional Genomics Whole-genome sequencing, transcriptional

profiling, and proteomic analysis of phyllosphere-competent microorganisms such
as bacteria from the genera Pseudomonas, Erwinia, Pantoea, Methylobacterium,
and Arthrobacter provide valuable insights into leaf surface-specific adaptations of
these organisms: they confirm the contribution or involvement of gene functions
hypothesized to contribute to phyllosphere fitness (‘epiphitness’), or they reveal
new ones. For example, Arthrobacter chlorophenolicus upregulates genes for the
utilization of hydroquinone, a compound not previously detected (looked for) on
leaf surfaces but now confirmed to be present in low concentrations (Scheublin
et al. 2013). The phyllosphere-induced expression of genes for phenylalanine degra-
dation by Pseudomonas syringae is presumed to counter phenylpropanoid-based
plant defenses (Yu et al. 2013). Proteomics-identified PhyR is a master regulator
for phyllosphere-survival of Methylobacterium extorquens, with a pleiotropic role
in resistance to desiccation, UV, and heat (Gourion et al. 2008). Metagenomic ap-
proaches to understanding life on leaf surfaces are on the rise (Delmotte et al. 2009)
and beginning to uncover the functional diversity contained within entire epiphytic
microbial communities, not just individual phyllosphere isolates.
Plant and Human Pathogens Much of the interest in studying leaf microorganisms
comes from the realization that plant foliage provides many ecosystem services, be
it directly, for example as food, shade, or drugs, or indirectly as a harvester of solar
energy to produce flowers, fruits, fibers, seeds, roots, and other plant parts. Histor-
ically, the field of phyllosphere microbiology has seen overwhelming emphasis on
understanding foliar pathogens: their ability to cause chlorosis, wilt, browning, and
other symptoms that interfere with proper leaf function represents a great threat to
many of the above services. An emerging concern is the foliar contamination of leafy
greens with human pathogens (Brandl and Sundin 2013). Research in this area has
contributed significantly to the latest phyllosphere-related scientific literature, offer-
ing new insights into the sources of disease outbreaks on raw-consumed leafy greens
such as spinach and lettuce and the factors that impact the foliar establishment of
such pathogens as E. coli and Salmonella. Little is known still about the interactions
that plant and human pathogens have with other members of leaf-surface micro-
bial communities, and how such interactions affect the abundance and activity of
4 Life of Microbes on Aerial Plant Parts 21

said pathogens. Positive as well as negative correlations between pathogen presence

and community composition have been reported (Rastogi et al. 2012), and experi-
mental evidence exists for facilitative effects of natural leaf associates on pathogen
establishment (Cooley et al. 2006), or antagonistic effects, be it direct, for exam-
ple through resource competition (Innerebner et al. 2011) or indirect, for example
through induction of plant defense genes (Kurkcuoglu et al. 2007).
DNA-Based Community Profiling Early efforts to describe microbial diversity
on plant foliage relied on differences in the number and appearance of microbial
colonies that formed after pushing a leaf or plating leaf washes onto an agar sur-
face. This culture-dependent approach has dominated the field for a long time,
although additional techniques allowed the differentiation and naming of isolates
based on properties other than colony appearance, including cell composition (for
example, fatty acid methyl esters), phenotype (for example, substrate utilization),
or genotype (for example, small-subunit rRNA genes or internal transcribed spacer
sequences). Not until the availability and embrace of techniques such as Denatur-
ing Gradient Gel Electrophoresis (DGGE), Terminal Restriction Fragment Length
Polymorphism (T-RFLP), Automated rRNA Intergenic Spacer Analysis (ARISA),
phylochips, clone library sequencing, and most recently massively parallel amplicon
sequencing, transcriptional profiling, and community proteogenomics, did questions
about size, structure, and activity of microbial communities on leaves become an-
swerable without having to depend on ability or willingness of microbes to grow in
the lab (Rastogi et al. 2013).
Culture-Independent versus Culture-Dependent Data As do other microbiomes,
the phyllosphere reveals greater population sizes and a richer biodiversity when an-
alyzed by culture-independent (i.e. DNA-based) approaches than culture-dependent
ones (Rastogi et al. 2010). One explanation for this is that some fraction of the
bacterial population is truly unculturable, i.e. these bacteria simply cannot form a
colony on the media that are used in the lab. A relevant example are bacteria from
the genus Alkanindiges which have been detected on the leaves of several plant
species, but which have been described as obligate alkane degraders, so they cannot
be expected to form colonies on standard media. Another explanation for the culture-
dependent/independent discrepancy is that the harsh conditions on the leaf surface
forces some bacteria into a state of nonculturability. Evidence in support of this no-
tion is that DNA-based community profiles often reveal abundant representation of
bacteria from genera such as Pseudomonas, Erwinia, and Pantoea, which one would
expect to form colonies if they were alive and well. Another reason for culturable-
based underestimation is the incomplete breakup of bacterial aggregates from the
leaf surface, as one such aggregate will be counted as one colony-forming unit, al-
though it might consist of hundreds or even thousands of individual bacterial cells.
Culture-independent counts may be overestimated because most culture-independent
approaches are based on relative abundances of small-subunit rRNA gene amplicons,
and most bacteria possess more than one copy of this gene. A DNA-based challenge
that is unique to the phyllosphere involves the fact that plant leaf cells contain chloro-
plasts that have their own genome and small-subunit rRNA genes. A single plant cell
22 J. H. J. Leveau

may harbor as many as 105 copies of the chloroplast 16S rRNA gene, which is a
huge source of potential contamination considering that many leaves carry the same
number of bacterial 16S rRNA genes per gram of tissue (Rastogi et al. 2010).

4.3 Drivers of Microbial Community Structure on Plant Leaves

Two major questions in phyllosphere microbiology that DNA-based profiling of leaf-

associated microbial communities is finding answers to are: (1) to what extent does
plant genotype drive community structure, and (2) how is microbial community
structure impacted by the environment in which the plant is growing?
Plant Genotype The first question can be addressed in several ways. One is the
‘common garden’ experiment, where plants from different species are grown or al-
ready available under similar environmental conditions. Such studies have indeed
demonstrated that different plant species and even plant cultivars vary in the types
and abundances of microorganisms that they carry on their foliage. Many labs focus
on finding the genetic factors or loci that contribute to these differences. Quantitative
trait loci mapping is used to identify genes that contribute to differences in bacterial
diversity on leaves. Another approach is the use of mutant plants to evaluate the effect
of the mutation on leaf surface microbiology. For example, bacterial communities
on the leaves of Arabidopsis mutants with an altered cuticular wax biosynthesis are
demonstrably distinct from those on wildtype plants. The notion that one or more
plant genes can alter the diversity of leaf-associated microbiota feeds the idea that
the structure and function of microbial communities have potential as a breeding
target. To realize this potential, there is a need not only for knowing the genes that
underlie foliar differences in colonization, but also for a definition of what consti-
tutes desirable microbiota. For example, are there combinations of bacterial taxa that
consistently protect plant leaves from infection by a plant pathogen? The answers
will come from a continued investment in correlative surveying of phyllosphere mi-
crobiota on different types of plants, in different states of health, and under different
environmental conditions.
Environmental Impacts The fact that demonstration of a plant genotype impact
on epiphytic microbiota often requires tight control of environmental conditions
suggests that the environment can have a significant impact also. Three such impacts
can be distinguished. One is that of the environment as a source of microorganisms
(inoculum): different locations harbor different microbial communities in the air, soil
and on nearby plants, and represent different sink-source dynamics. The environment
may also play a facilitating role in the delivery of new immigrants (inoculation): rain
splash, overhead irrigation, wind, and insects all may deposit microorganisms to
the leaf surface, and geographical variation in these factors may drive variation
in microbiota structure. A third type of environmental impact is that as a driver
of microbial activity on the leaf surface (incubation); this includes factors such as
temperature and relative humidity. Distance-decay relationships among phyllosphere
4 Life of Microbes on Aerial Plant Parts 23

microbiota (Finkel et al. 2012; Rastogi et al. 2012) are probably best explained by
the existence of environmental gradients along different geographical scales in terms
of inoculum, inoculation, and incubation, as defined above.

4.4 Future Aspects

The future of phyllosphere microbiology will hopefully bring an integration of many

of the approaches and insights described in this chapter. One of the field’s future
challenges will be to connect knowledge about microbial community structure to
what we have come to understand about the heterogeneous nature of the biotic and
abiotic leaf environment. In other words, it is time to start asking questions about
the spatial distribution of all this recorded microbial diversity on leaves, in order
to get basic answers about who is interacting how with whom and where in the
phyllosphere, and how these interactions scale up to matters of food security and
food safety.

References

Brandl MT, Sundin GW (2013) Focus on food safety: human pathogens on plants. Phytopathology
103:304–305
Cooley MB, Chao D, Mandrell RE (2006) Escherichia coli O157: H7 survival and growth on lettuce
is altered by the presence of epiphytic bacteria. J Food Prot 69:2329–2335
Delmotte N, Knief C, Chaffron S et al (2009) Community proteogenomics reveals insights into the
physiology of phyllosphere bacteria. Proc Natl Acad Sci U S A 106:16428–16433
Finkel OM, Burch AY, Elad T et al (2012) Distance-decay relationships partially determine diversity
patterns of phyllosphere bacteria on Tamarix trees across the Sonoran desert. Appl Environ
Microbiol 78:6187–6193
Gourion B, Francez-Charlot A, Vorholt JA (2008) PhyR is involved in the general stress response
of Methylobacterium extorquens AM1. J Bacteriol 190:1027–1035
Innerebner G, Knief C, Vorholt JA (2011) Protection of Arabidopsis thaliana against leaf-pathogenic
Pseudomonas syringae by Sphingomonas strains in a controlled model system. Appl Environ
Microbiol 77:3202–3210
Kurkcuoglu S, Degenhardt J, Lensing J et al (2007) Identification of differentially expressed genes
in Malus domestica after application of the non-pathogenic bacterium Pseudomonas fluorescens
Bk3 to the phyllospere. J Exp Bot 58:733–741
Leveau JHJ (2006) Microbial communities in the phyllosphere. In: Riederer M, Mueller C (eds)
Biology of the plant cuticle. Blackwell, Oxford, pp 334–367
Leveau JHJ, Lindow SE (2001) Appetite of an epiphyte: quantitative monitoring of bacterial sugar
consumption in the phyllosphere. Proc Natl Acad Sci U S A 98:3446–3453
Maignien L, DeForce EA, Chafee ME et al (2014) Ecological succession and stochastic variation
in the assembly of Arabidopsis thaliana phyllosphere communities. MBio 5:e00682–e00613
Meyer KM, Leveau JHJ (2012) Microbiology of the phyllosphere: a playground for testing
ecological concepts. Oecologia 168:621–629
Perez-Velazquez J, Schlicht R, Dulla G et al (2012) Stochastic modeling of Pseudomonas syringae
growth in the phyllosphere. Math Biosci 239:106–116
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Rastogi G, Tech JJ, Coaker GL et al (2010) A PCR-based toolbox for the culture-independent
quantification of total bacterial abundances in plant environments. J Microbiol Methods 83:
127–132
Rastogi G, Sbodio A, Tech JJ et al (2012) Leaf microbiota in an agroecosystem: spatiotemporal
variation in bacterial community composition on field-grown lettuce. ISME J 6:1812–1822
Rastogi G, Coaker GL, Leveau JHJ (2013) New insights into the structure and function of phyl-
losphere microbiota through high-throughput molecular approaches. FEMS Microbiol Lett
348:1–10
Remus-Emsermann MNP, Tecon R, Kowalchuk GA et al (2012) Variation in local carrying capacity
and the individual fate of bacterial colonizers in the phyllosphere. ISME J 6:756–765
Scheublin TR, Deusch S, Moreno-Forero SK et al (2013) Transcriptional profiling of gram-positive
Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant
phenolic compounds. Environ Microbiol 16:2212–2225
van der Wal A, Tecon R, Kreft J-U et al (2013) Explaining bacterial dispersion on leaf surfaces with
an individual-based model (PHYLLOSIM). PLoS One 8:e75633
Vorholt JA (2012) Microbial life in the phyllosphere. Nat Rev Microbiol 10:828–840
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growth in epiphytic versus apoplastic leaf sites. Proc Natl Acad Sci U S A 110:E425–E434
Chapter 5
Life of Microbes Inside the Plant

Jesús Mercado-Blanco

Abstract A hidden microbial world is present in the interior of all plants. Myriads
of bacteria and fungi live inside them without causing apparent deleterious effects to
their hosts. They are designated as endophytes. Endophytic communities are variable
and diverse. Their structure and composition are shaped by a number of (a)biotic fac-
tors. Endophytes have found evolutionary solutions to cope with defensive responses
deployed by host plants to face colonization by microbes. In return, they live within
an ecological niche that provides better protection against a number of stresses and
a reliable and constant source of nutrients. Endophytes seem to contribute to plant
fitness and development, displaying beneficial traits that can be exploited in agri-
cultural biotechnology. However, many questions related to the endophytic lifestyle
remain to be answered. This chapter summarizes present knowledge on how en-
dophytes are able to establish and endure within plants. Potential biotechnological
applications are also briefly presented.

5.1 Introduction: Beneficial Endophytes Defined

Plants live in close association with a huge diversity of microorganisms. In fact, the
composite genome of these accompanying microbial communities is far larger than
that of the host plant, and thus is also referred to as the plant’s second genome. Most
of the components of the plant-associated ‘microbiome’ (Chap. 30) are only able
to colonize and persist on plant tissue surfaces or in the soil rhizosphere (Chap. 3).
However, some can also establish themselves as non-deleterious endophytes. It is
likely that all plants carry endophytes, which play an important role in plant fitness
and development. Plants can thus be considered as super organisms of which both
the plant and its endophytic microbiome work coordinately to shape and sustain
an extraordinary ecosystem. It is generally recognized that endophytes represent
just a minor fraction of the microbiota inhabiting plant surfaces or living in close

J. Mercado-Blanco ()
Department of Crop Protection, Institute for Sustainable Agriculture,
Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC),
Campus ‘Alameda del Obispo’ s/n, Apartado 4084, 14080 Córdoba, Spain
Tel.: +34957499261
e-mail: jesus.mercado@ias.csic.es
© Springer International Publishing Switzerland 2015 25
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_5
26 J. Mercado-Blanco

proximity to them, and comprise mainly microbes originating from the soil region
associated with roots (i.e. the rhizosphere, Chap. 3). In fact only small subpopulations
of rhizosphere and phyllosphere microorganisms are able to enter and live inside
the plant. Relevant reviews suggested are: Bacon and Hinton (2006); Schulz et al.
(2006); Rosenblueth and Martínez-Romero (2006); Hardoim et al. (2008); Ryan
et al. (2008); Reinhold-Hurek and Hurek (2011); VV.AA. (2013).
The word endophyte means ‘in the plant’ and is derived of the Greek words
endon (within) and phyton (plant). Endophytes have been defined by various authors
in somewhat different ways (Bacon and Hinton 2006; Rosenblueth and Martínez-
Romero 2006; Schulz et al. 2006; Mercado-Blanco and Lugtenberg 2014). It is
generally agreed that they are bacteria and fungi that can be detected at any moment
within the tissues of healthy plants, and that do not produce disease symptoms.
Microbial phytopathogens, nodule-producing microbes, and mycorrhizal fungi may
display endophytic lifestyles during part of their lives but they are not considered
here as endophytes. Moreover, this chapter is only focused on bacterial endophytes
(for endophytic fungi see, for instance, VV.AA. 2013). Mere isolation from surface-
disinfected tissues is not enough to claim a true endophyte. Plant surface sterilization
protocols must be sufficiently stringent to eradicate the external microbiota without
killing bacteria with the tissue. Moreover, the ‘candidate endophyte’ must be shown
to be a true endophyte by both its ability to re-infect disinfected seedlings, and by
microscopic evidence (Reinhold-Hurek and Hurek 1998).
The vast majority of bacterial endophytes are non-culturable or VBNC. This may
represent a survival strategy to persist hostile conditions within the plant. By imple-
menting culture-independent and metagenomics approaches our understanding of
endophytes is being steadily enriched. These methodologies will undoubtedly con-
tinue to reveal a much wider diversity and abundance of endophytic communities
than that uncovered by traditional culture-dependent methods.

5.2 How Do Endophytes Get into the Plant and Spread

to Distant Tissues?

Endophytes gain entrance into plants predominantly through the roots but also
through leaves, flowers, stems or cotyledons. Indeed, the vast majority of endo-
phytes are soil-inhabitants and plant colonization seems to mainly originate from the
rhizosphere. Evidence confirming this possibility has been obtained by combining
biotechnological and microscopy tools. Some endophytes have thus been shown to
spread systemically from the original penetration site(s), and be found in distant
plant tissues and organs. Consequently, the population density of endophytes is usu-
ally higher in roots than in any other plant organ. It is important to stress that an
amazing variety of endophytic bacteria and fungi (hundreds of different taxa) can be
found in diverse organs/tissues of any individual herbaceous, woody or moss species
(Hallmann and Berg 2006; Schulz et al. 2006; Mercado-Blanco and Lugtenberg
2014).
5 Life of Microbes Inside the Plant 27

Overall, our knowledge about the specific sites at which endophytic bacteria
attach and penetrate into root tissues is scant. Nevertheless, it is generally assumed
that bacteria invade roots passively using cracks or wounds located, for instance,
at the emergence points of lateral roots. Such root cracks can also be produced
by microbial, nematode or arthropod activities. Preferential sites for rhizosphere
bacteria attachment and subsequent entry can also be the thin-walled surface layers
located in the apical root region, including the root differentiation, elongation and root
hair zones as well as the intercellular spaces of the root epidermis. Specific bacterial
components that are known to be involved in endophyte attachment to plant tissue
include Type IV pili, lipopolysaccharides, and exopolysaccharides (Hardoim et al.
2008; Reinhold-Hurek and Hurek 2011; Mercado-Blanco and Lugtenberg 2014).
Besides being an important attachment structure, root hairs play also a role in root
endophytic colonization. Using fluorescently-tagged bacteria and CLSM allowed
demonstration that endophytic Pseudomonas spp. strains can internally colonize
olive root hairs (Prieto et al. 2011) prior to becoming established within the intercel-
lular spaces of the root cortex (Fig. 5.1). Despite root hairs seeming to play a role
in bacterial entrance into the roots, several questions remain to be elucidated: (i) the
exact timing of and site(s) used for bacterial penetration of the root hair cell; (ii)
how the bacteria move to the intercellular spaces of the root cortex; and (iii) whether
these bacteria enter root hairs via active or passive mechanisms (Mercado-Blanco
and Prieto 2012).
Mechanisms used by endophytic bacteria to enter the plant are largely unknown,
although several bacterial traits have been proposed to participate in endophytic
colonization of plant roots. Once bacteria overcome the exodermal barrier, they may
remain either at the site of entry or move towards the intercellular space of the cortex
and even to distant parts (Compant et al. 2005; Hardoim et al. 2008; Reinhold-Hurek
and Hurek 2011).

5.3 How Do Endophytes Cope with and Adapt to the Inner

Plant Environment?

Living inside plant tissues requires adaptation to an environment that provides food
and low exposure to (a)biotic stresses. Therein, competition among endophytic
microorganisms can be expected, although nothing is yet known about trophic in-
teractions within such microbial communities. However, compared to the highly
competitive/predatory environment found outside the plant, its interior is a “safe
heaven” for endophytes since it is a reliable and constant source of nutrients. En-
dophytes have thus evolved to adapt themselves to nutrients available inside plant
tissues (Bacon and Hinton 2006; Mercado-Blanco and Lugtenberg 2014).
Data on the ability of endophytes to utilize nutrients found in the plant interior
are not abundant. Comparing the abilities of endophytic and highly-related non-
endophytic strains to utilize nutrient sources is a strategy to unravel the feeding
capabilities of the endophytes. For instance, utilization of l-arabinose has been
28 J. Mercado-Blanco

Fig. 5.1 CLSM images of in vitro micropropagated olive roots (cv. Manzanilla) colonized by
enhanced green fluorescent protein (EGFP)-tagged Pseudomonas fluorescens PICF7. a Surface
colonization of root hairs by PICF7 cells; b Detection of EGFP-tagged PICF7 cells inside two root
hairs; c Intercellular colonization of the root cortical tissue by PICF7 cells. Scale bar represents 50
μm in panel a and 20 μm in panels b and c. For details on olive roots-PICF7 colonization bioassays,
tissue sectioning and CLSM imagery, see Prieto et al (2011). These CLSM microphotographs are
reproduced from Prieto et al. (2011), doi:10.1007/s00248-011-9827-6

suggested to be an important trait contributing to the endophytic lifestyle of several

Pseudomonas spp. strains in cucumber (Malfanova et al. 2013). It is likely that other
carbon sources may play similar roles in other plant-endophyte associations, and
evidence/suggestions for such a role are available (Mercado-Blanco and Lugtenberg
2014). Because of the ability of endophytes to preferentially metabolize specific
carbon sources, competition for these nutrients can be a determining factor in shaping
the endophytic community in a given host plant or tissue. Indeed, adaptation to
specific nutrients’ availability may limit the number of endophytic microbes that can
be taken up and survive inside plant tissues either under natural conditions or upon
artificial inoculation. Comparative genomics approaches aimed to unravel specific
traits linked to endophytic lifestyles can provide relevant information on this issue
(Mitter et al. 2013; Brader et al. 2014; Ali et al. 2014).
As a toll to be paid for their in planta protection, endophytes must develop strate-
gies to silence or evade the various responses that plants use to confront ‘non-hostile’
endophyte colonization, or attacks by phytopathogens. The strategies that endophytes
have evolved to elude such responses and be recognized as innocuous invaders occur
by an as yet poorly-understood modulation of the plant-deployed immune response
(Zamioudis and Pieterse 2012). It is conceivable that the prevalence of non-culturable
5 Life of Microbes Inside the Plant 29

and/or VBNC states encountered among endophytes could be a survival strategy to

prevail over stresses operating within plant tissues.
With respect to how endophytes cope with plant defense responses our knowledge
is also scant (Reinhold-Hurek and Hurek 2011). Inner colonization and persistence
of endophytes within plant tissues likely entail broad changes at the transcriptomic
level for both partners. However, little information is available about the genetics
changes that an endophyte provokes in the host plant and how colonization modifies
the behavior of the new resident. Likewise, the effects that introduced endophytes
may provoke on a pre-existing endophytic microbiome and vice versa are largely
unknown. However, some studies are available and results indicate that colonization
of plant tissues by an endophyte triggers, among other changes, a broad range of
defensive responses (Conn et al. 2008; Schilirò et al. 2012).
Finally, it is worth mentioning that the composition, abundance, distribution and
functionality of the endophytic microbiome found in a given host plant, plant tissue or
organ is not static. On the contrary, an endophytic microbiome can be modified over
time by the plant growth phase and physiological state, and/or diverse environmental,
biological and physical-chemical factors (see, for instance, Van Overbeek and van
Elsas 2008; Ardanov et al. 2012).

5.4 The Search for Endophytic-Specific Traits

An important question still to be solved is whether endophytes have specific traits that
define their lifestyle. An environment providing specific nutrients, but hostile because
of active defense responses, should be a driving force selecting adapted pheno-
types (see previous section). Similarly, the presence of microbe-specific machineries
for plant tissue penetration could be necessary for establishment as beneficial en-
dophytes, although it is likely that some of the mechanisms could be shared by
pathogens. It is also possible that, in contrast to facultative endophytes, obligate
endophytes might carry genetic and metabolic determinants more essential to the
endophytic lifestyle. Comparative genomics and bioinformatics approaches could
definitively help in unraveling specific traits linked to endophytism. Even though
studies like these show that some characteristics seem to be shared by different en-
dophytes, the emerging picture is of a broad genetic diversity (Mitter et al. 2013; Ali
et al. 2014).
Quorum-sensing (QS) (Chap. 8) and the ability to overcome plant defenses seem
to be commonly found in the genomes of the endophytic bacteria analyzed so far.
QS systems allow bacteria to ‘sense’ their own concentration/abundance, thereby
triggering the expression of specific target genes only at a high cell density. Con-
sidering that bacterial endophytes can reach high populations densities in defined
sites, production of QS signals within plant tissues and how those signals oper-
ate in endophytic-mediated processes deserve investigation. In contrast, Type III
(Chap. 7) secretion systems, which mediate, in gram-negative pathogens and rhi-
zobial symbionts, the delivery to the host plant of effector proteins that suppress
30 J. Mercado-Blanco

the host defense response, seem rare among endophytes. Instead, other types of se-
cretion systems such as Type VI seem to be more frequent. Such findings can thus
shed light on the potential differences between pathogenic and endophytic lifestyles
displayed by plant-associated bacteria. Currently, however, the number of genomes
and genomic information available from claimed endophytes is still too low to draw
sound conclusions (Reinhold-Hurek and Hurek 2011; Mitter et al. 2013; Ali et al.
2014).

5.5 What Endophytes Do for the Plant and Their Potential

to be Exploited in Agro-Ecosystems

The presence of non-deleterious endophytes can benefit the host plant in differ-
ent ways, a scenario of utmost interest once these microbial communities and
their multitrophic interactions are properly characterized, understood and harnessed.
Endophytes are of increasing interest because of their potential biotechnological ap-
plications (see, for instance, Ryan et al. 2008; VV.AA. 2013; Brader et al. 2014;
Mercado-Blanco and Lugtenberg 2014). Among the beneficial traits with potential
to be exploited in agro-ecosystems, promotion of plant growth, and control of plant
diseases are of particular significance. In most cases the mechanism(s) involved,
particularly those related to biocontrol, remain to be elucidated. Nevertheless, it has
been suggested that beneficial effects deployed by bacterial endophytes might operate
through mechanisms similar to those described for rhizosphere bacteria (Kloepper
and Ryu 2006). Additional applications of endophytes beyond agricultural biotech-
nology rely on the ability of the organisms to produce a broad range of bioactive
metabolites which are relevant for other purposes, including human health (i.e. an-
tibiotics, antitumor compounds, anti inflammatory agents, etc.) (see, for instance,
Christina et al. 2013; Brader et al. 2014).
Plant growth promotion can be achieved either directly or indirectly. There is little
knowledge available about mechanisms of growth promotion exerted by endophytes,
and operating in planta (Hardoim et al. 2008). Nevertheless, considering that many
endophytes are also free-living rhizosphere microorganisms, it is plausible to assume
that mechanisms to stimulate plant growth deployed by the latter may also operate
once endophytic growth is established. However, this assumption still needs to be
confirmed.
Direct promotion of plant growth by endophytic bacteria and fungi can be achieved
by the microbe providing (micro)nutrients (biofertilization) and/or phytohormones
(phytostimulation) to the plant. Indirect plant growth promotion is a consequence
of the suppression of plant diseases exerted by pathogenic microorganisms, and can
be mediated by direct antagonism/antibiosis against the pathogens, advantageous
out-competing for nutrients and/or space, or by triggering in the host plant enhanced
defense capacities against pathogen attack. Growth can also be stimulated indirectly
by alleviation of stress caused by environmental pollutants (rhizoremediation, see
5 Life of Microbes Inside the Plant 31

Chap. 29) or other stressful abiotic (heavy metals, drought, salinated soils) condi-
tions. For instance, synthesis of the enzyme ACC deaminase reduces the level of
the stress hormome ethylene by converting ACC into α-ketobutyrate and ammonia.
Production of this enzyme by plant-growth promoting bacteria, including those dis-
playing endophytic lifestyles, can make host plants tolerant to a number of stresses
(Chap. 27; Hardoim et al. 2008).

5.6 Concluding Remarks

Many questions on how, why and when any given endophyte(s)-plant consortium
is established remain to be elucidated. A list of questions to be answered on this
topic has been recently outlined by Mercado-Blanco and Lugtenberg (2014). For
instance, little is known on how an ‘endophytic candidate’ is able to overcome or
modulate defensive barriers/responses to successfully penetrate and establish within
plant tissues. The identification of traits involved in the colonization and persistence
within the plant is still incomplete. Similarly, understanding the influence of environ-
mental, physiological, developmental stages and/or genetic factors on endophytes
is essential if these organisms are expected to be further developed as biocontrol
or (phyto)rhizoremediation tools. In summary, two main questions that should now
be put forth are: (i) which driving forces are operating to build up an endophytic
community, and (ii) what does the endophytic microbiome do for the plant. The
implementation of currently available and powerful ‘-omics’ and microscopy tech-
nologies (see Chap. 31) will undoubtedly provide some answer to these questions,
as well as those aimed at unraveling the molecular processes that define endophytes
(Mitter et al. 2013; Ali et al. 2014).
From a practical perspective, more studies are needed to understand whether the
performance of any artificially-introduced endophyte can be affected by the native
microbiome of the host plant; and vice versa, i.e. how the indigenous endophytic
microbiota can be influenced by the introduction of a ‘newcomer’ into this delicately
balanced microenvironment, and whether such introductions may alter the plant’s
development and fitness.

Acknowledgments Supported by grants P07-CVI-02624 from Junta de Andalucía (Spain) and

AGL2009-07275 from Spanish MICINN/MINECO, both co-financed by ERDF of the EU. Thanks
are due to Katherine Dobinson and Pilar Prieto for their critical reading and interesting suggestions.

References

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genes involved in endophytic behavior in Burkholderia spp. J Theor Biol 343:193–198
Ardanov P, Sessitsch A, Haggman H et al (2012) Methylobacterium-induced endophyte community
changes correspond with protection of plants against pathogen attack. PLoS One 7(10):e46802
32 J. Mercado-Blanco

Bacon CW, Hinton DM (2006) Bacterial endophytes: the endophytic niche, its occupants, and its
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Brader G, Compant S, Mitter B et al (2014) Metabolic potential of endophytic bacteria. Curr Opin
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Compant S, Duffy B, Nowak J et al (2005) Use of plant growth promoting bacteria for biocontrol of
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Conn VM, Walker AR, Franco CMM (2008) Endophytic actinobacteria induce defense pathways
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Hardoim PR, van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and their
proposed role in plant growth. Trends Microbiol 16:463–471
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Heidelberg, pp 33–52
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of Pseudomonas spp.? Arch Microbiol 195:9–17
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Mercado-Blanco J, Prieto P (2012) Bacterial endophytes and root hairs. Plant Soil 361:301–306
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mans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with
host plants. Front Plant Sci 4:120
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colonization of olive roots by Pseudomonas spp. with biocontrol activity. Microb Ecol 62:435–
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Reinhold-Hurek B, Hurek T (1998) Interactions of gramineous plants with Azoarcus spp. and other
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Sci 17:29–54
Reinhold-Hurek B, Hurek T (2011) Living inside plants: bacterial endophytes. Curr Opin Plant
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Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts.
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applications. FEMS Microbiol Lett 278:1–9
Schilirò E, Ferrara M, Nigro F et al (2012) Genetic responses induced in olive roots upon colonization
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Schulz B, Boyle C, Sieber T (2006) Microbial root endophytes. Springer, Berlin
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Microbe Interact 25:139–150
Chapter 6
Microbial Cell Surfaces and Secretion Systems

Jan Tommassen and Han A. B. Wösten

Abstract Microbial cell surfaces, surface-exposed organelles, and secreted proteins

are important for the interaction with the environment, including adhesion to hosts,
protection against host defense mechanisms, nutrient acquisition, and intermicrobial
competition. Here, we describe the structures of the cell envelopes of bacteria, fungi,
and oomycetes, and the mechanisms they have evolved for the transport of proteins
across these envelopes to the cell surface and into the extracellular milieu.

6.1 Basic Structure of Bacterial Cell Envelopes

Based on the Gram-staining method, bacteria are classically divided into two groups,
Gram-positives and Gram-negatives, which have different cell envelope architecture.
Gram-positives are enveloped by a cytoplasmic membrane (CM) and a thick cell wall.
In Gram-negatives, the cell wall is thinner, but an additional membrane is present,
the outer membrane (OM), which is located peripheral to the cell wall. The space in
between the membranes is called the periplasm and because of the presence of the
OM, the CM is also called the inner membrane (Fig. 6.1).
The Cytoplasmic Membrane The CM is a phospholipid bilayer with inserted pro-
teins. The membrane-spanning segments of the integral membrane proteins are
α-helical and consist of ∼20 amino acids, the vast majority of them containing
hydrophobic side chains (Fig. 6.1).
The Cell Wall The rigid cell wall consists of peptidoglycan and provides strength
and shape to the bacteria. For example, it protects the bacteria against osmotic

J. Tommassen () · H. A. B. Wösten

Section Molecular Microbiology, Department of Biology,
Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Tel.: +31-30-2532999
e-mail: j.p.m.tommassen@uu.nl
H. A. B. Wösten
Tel.: +31-30-2533448
e-mail: h.a.b.wosten@uu.nl

© Springer International Publishing Switzerland 2015 33

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_6
34 J. Tommassen and H. A.B. Wösten

Fig. 6.1 Structure of the Gram-negative bacterial cell envelope. OM outer membrane containing
LPS in its outer leaflet, PP periplasm containing a layer of peptidoglycan (PG), IM inner membrane.
Examples of a typical β-barrel OMP and a typical α-helical inner-membrane protein are shown at the
left and the right, respectively. Reproduced from Tommassen (2010) by permission of the publisher

pressure. Peptidoglycan consists of oligomers of a disaccharide composed of N-

acetylglucosamine (GlcNAc) and N-acetylmuramic acid, which are covalently
interconnected by small peptides, thus creating a network that enwraps the entire
bacterial cell (Vollmer and Seligman 2010). In Gram-positive bacteria, the cell wall
also contains large amounts of polymers known as (lipo)teichoic acids, which provide
a net negative surface charge to the bacteria.
The Outer Membrane The OM protects Gram-negative bacteria from harmful
compounds in the environment, including many antibiotics. This asymmetric bilayer
contains phospholipids and lipopolysaccharides (LPS) in the inner and outer leaflets,
respectively. LPS (Raetz and Whitfield 2002) consists of two or three moieties: lipid
A, a core oligosaccharide, and a polysaccharide, the O-antigen, which is absent in
several bacterial species. Lipid A is a signaling molecule for the innate immune sys-
tem. It consists of a phosphorylated glucosamine disaccharide substituted with four
3-OH hydroxylated fatty acids, which can be esterified with secondary fatty acids.
Repulsive forces between the negatively charged phosphate groups are compensated
by divalent cations. The resulting network, together with the dense packing of the
acyl chains, generates a barrier that is barely permeable to hydrophobic substances.
Variations in the lipid A structure, affecting the acyl chains or the phosphate groups,
are induced by environmental conditions and help bacteria to escape from the host’s
innate immune responses.
The core moiety of LPS is divided in an inner and an outer core. The inner
core usually contains L-glycero-d-manno-heptose and 2-keto-3-deoxyoctonate with
various substituents and is conserved among different strains of the same species.
The outer core is more variable and the O-antigen, which is a polymer of repeating
6 Microbial Cell Surfaces and Secretion Systems 35

mono- or oligosaccharides, is highly variable. The O-antigen can form an effective

barrier for bacteriophages, bacteriocins, and antibodies, targeting the underlying
conserved parts of the OM.
Integral OM proteins (OMPs) structurally deviate from other membrane proteins.
The membrane-spanning segments are not α-helices but β-strands, which form a β-
barrel (Fig. 6.1). These β-strands are amphipathic with hydrophobic residues facing
the lipids and hydrophilic ones directed toward the interior of the barrel. Some of these
β-barrels form open channels through which small hydrophilic solutes, including
nutrients, such as amino acids and small sugars, can pass by diffusion. Such pore-
forming proteins are called porins. Thus, the OM functions as a molecular sieve,
allowing the passage of small hydrophilic molecules but holding larger hydrophilic
molecules and hydrophobic ones.
Besides integral OMPs, the OM also contains lipoproteins, which are bound to the
membrane via an N-terminal lipid moiety. These lipoproteins can be very abundant,
e.g. Braun’s lipoprotein is, with ∼106 copies per cell, the most abundant protein in
Escherichia coli. This lipoprotein is also covalently bound to the peptidoglycan and
functions to anchor the OM to the cell wall. Lipoproteins are also found in the CM.
Deviant Cell Envelope Architectures The general architecture of bacterial en-
velopes as described above was derived from studies on model organisms such as E.
coli and Bacillus subtilis. However, deviations are now well known. For example,
although not Gram-negative, Mycobacteria are covered with an OM, however, with a
composition entirely different from the Gram-negative OM (Niederweis et al. 2010).
It contains a large variety of lipids, including mycolic acids, which are covalently
attached to the peptidoglycan via an arabinogalactan polymer. Also, bacteria that
lack a cell wall have been described, such as mycoplasmas.

6.2 Additional Layers and Surface Appendices

Peripheral to the cell envelope, bacteria can be covered with additional layers, such
as capsules and S-layers, and they can contain organelles, such as flagella, pili and
fimbriae, which extend into the extracellular milieu.
Additional Layers Capsules (Bazaka et al. 2011) consist of polysaccharides with a
highly variable composition. They have various functions, e.g. in protection against
desiccation or against phagocytosis and other defense mechanisms of the host. In
addition, they can have a role in the attachment of bacteria to biotic or abiotic
surfaces. Bacteria can also produce extracellular polysaccharides (EPS), which do
not form a capsule but are released in the environment (Bazaka et al. 2011). EPS
can be important components of the extracellular matrix (ECM) of biofilms, which
are surface-attached microbial communities embedded in a self-produced ECM (see
also Chap. 7). S-layers are paracrystalline arrays of identical protein subunits with a
large variety of functions (Fagan and Fairweather 2014). Amongst others, they may
function as a molecular sieve, like the OM, or in determining the cell shape, and
they may protect against bacteriophages, host defense mechanisms, or osmotic and
mechanical stresses.
36 J. Tommassen and H. A.B. Wösten

Flagella Flagella (Van Gerven et al. 2011) are long surface appendages that are
used by bacteria to move towards favorable conditions or away from repellents in
a process called chemotaxis. A flagellum consists of a long filament composed of
multiple copies of a protein called flagellin. The filament is connected via a hook
structure to a basal body that anchors the flagellum into the cell envelope and also
forms the channel for the export of flagellin from the cytoplasm to the cell surface.
The flagellum can rotate like a propeller to move the bacterium. Energy for this
process is derived from the proton gradient across the CM. Flagella are also used for
initial attachment of bacteria to a substratum during biofilm formation and, like LPS,
flagellin is an important signaling molecule for defense mechanisms of animals and
plants.
Pili and Fimbriae The names pili and fimbriae are often used interchangeably.
These structures are built of subunits called pilins (Van Gerven et al. 2011). An
abundant major pilin forms the filament, which usually exposes several minor pilins.
Many pili/fimbriae have a role in adhesion, where one of the minor pilins functions
as the adhesin that binds, for example, a eukaryotic target cell. The type IV pili form
a special class of pili with multiple functions. These pili, which are based in the
CM and cross the OM via a large oligomeric protein called secretin, are retractile.
Extension and retraction of these pili, both at the expense of ATP, can be used by
bacteria such as Pseudomonas aeruginosa to move over surfaces, a process called
twitching motility. Some bacteria that are naturally transformable use type IV pili to
take up DNA from the environment. Type IV pili can also function as nanowires that
transfer electrons from the respiratory chain to extracellular electron acceptors. Also
sex pili are retractile. They are produced by donor cells in the process of conjugation
to establish contact with a recipient cell. Retraction of the pilus then results in the
formation of a stable mating pair that allows for the transfer of DNA from donor
to recipient. DNA transfer from Agrobacterium tumefaciens to plant cells requires
similar machinery as in bacterial conjugation (see Chap. 37). A final class of pili
is constituted by curli. Curli form amyloid fibers similar to the amyloids that cause
neurodegenerative diseases in humans. Curli fibers are involved in adhesion, bacterial
aggregation and biofilm formation.

6.3 Protein Export

Translocation Across the CM Proteins destined for export are synthesized as pre-
cursors with an N-terminal signal peptide of ∼25 amino-acid residues. Different
signal peptides share a similar organization with three domains: an N-domain con-
taining positively charged residues, an H-domain of ∼10–12 hydrophobic residues,
and a C-domain containing the motif recognized by the enzyme that cleaves off the
signal peptide after export. The signal peptide directs the precursor to the Sec (se-
cretion) machinery, which mediates its transport across the CM (Kudva et al. 2013).
The central component of this machinery is a complex of three integral CM proteins,
the SecYEG translocon, which forms the protein-conducting channel. It is widely
6 Microbial Cell Surfaces and Secretion Systems 37

conserved in nature and corresponds to the Sec61 complex in the endoplasmic retic-
ulum of eukaryotes. Energy for export is provided by the motor protein SecA, which
hydrolyzes ATP, and the proton-motive force. The Sec machinery also inserts pro-
teins into the CM. CM proteins are generally not produced with a cleavable signal
peptide, but the most N-terminal membrane-spanning α-helix is recognized by the
signal-recognition particle (SRP) and targeted via an SRP receptor (FtsY) to the Sec
translocon. When such a long hydrophobic α-helix enters the translocon, the sub-
strate is not released in the periplasm, but the translocon opens laterally to insert it
into the CM. This is the reason why OMPs have a deviant structure: if OMPs would
also consist of hydrophobic α-helices, they would never reach their destination but
be inserted by the translocon into the CM. Besides the Sec translocon, YidC protein
constitutes an alternate insertase for CM proteins.
The Sec translocon contains a narrow channel and exports delineated proteins.
Some proteins need to be exported in a folded conformation, for example because
they bind a co-factor in the cytoplasm. These proteins are exported via the Tat system
(Kudva et al. 2013). Tat stands for twin-arginine translocation and refers to a char-
acteristic twin-arginine motif in the signal peptides of the substrate proteins. The Tat
system consists of three CM proteins, TatA, TatB, and TatC. Energy for transport is
provided by the proton gradient.
OMP Assembly The Sec machinery releases OMPs into the periplasm, where they
are bound by the chaperones Skp and/or SurA, which prevent their aggregation.
Subsequently, they are folded and inserted into the OM by the Bam (β-barrel assembly
machinery) complex. The central component of this complex, known as BamA or
Omp85, is highly conserved and found in the OM of all Gram-negatives and even
in mitochondria and chloroplasts. These eukaryotic cell organelles also contain β-
barrel proteins in their OM, probably reflecting their endosymbiont origin. The Bam
complex contains a variable number of accessory components, usually lipoproteins,
which are less conserved (Tommassen 2010).

6.4 Protein Secretion

In Gram-positive bacteria, exported proteins are bound to the peptidoglycan or re-

leased in the environment. In Gram-negatives, secreted proteins have to pass another
hurdle, the OM. These bacteria have developed six widely disseminated protein-
secretion mechanisms, designated type 1–6 secretion systems (T1-6SS) (Chang et al.
2014). Many of these systems can be present in a single cell, often in multiple copies,
each one dedicated to the secretion of a specific protein or set of proteins. For example,
P. aeruginosa possesses five of the six secretion systems (Fig. 6.2).
Two-Step Mechanisms In two-step mechanisms, a periplasmic intermediate is
translocated across the OM. The T2SS is dedicated to the secretion of folded proteins.
The substrates, usually hydrolytic enzymes or toxins, are either folded in the cyto-
plasm and exported via the Tat system, or exported via the Sec system and folded in
38 J. Tommassen and H. A.B. Wösten

Fig. 6.2 Protein secretions systems in P. aeruginosa. Details are described in the text. Reproduced
from Bleves et al. (2010) by permission of the publisher

the periplasm (Fig. 6.2). The T2SS consists of 12–16 proteins and resembles the ma-
chinery that builds type IV pili. It includes several pilin-like proteins and a secretin in
the OM, which forms the protein-conducting channel. The model is that substrates
bind the secretin, and a pilus-like structure that grows from the CM provides the
mechanical force to push them through the secretin into the milieu.
The T5SS consists of five subtypes (a–e), including four variants of an autotrans-
porter mechanism. Autotransporters consist of a signal peptide for export via the Sec
system, a passenger, which is the secreted part, and a translocator domain, which
forms a β-barrel that inserts into the OM via the Bam complex. During insertion, the
connected passenger is translocated across the OM. Protein folding starts when the
first part of the passenger appears at the external side and presumably provides
the energy to thread the rest of the protein through the translocation channel. The
passenger may stay associated with the translocator and function, for example, as
an adhesin, or it may be released into the milieu often via autocatalytic proteolysis.
In the fifth T5SS, known as two-partner secretion system or T5bSS (Fig. 6.2), the
translocator is not connected to the secreted protein but is a separate protein. The
secreted proteins are very large (up to > 6000 residues) β-helical proteins. Many
of them contain a small toxic domain at the C terminus that inhibits the growth of
related bacteria competing for the same niche (Ruhe et al. 2013).
One-Step Mechanisms The T1SS consists of three proteins, a CM-based ATPase,
an OM-based tunnel protein, and a membrane-fusion protein that connects the other
two. Together, they form a channel that translocates substrates directly from the
6 Microbial Cell Surfaces and Secretion Systems 39

cytoplasm into the external milieu. The substrates are very large proteins, belonging
to the RTX (repeat-in-toxin) family, which refers to a glycine/aspartate-rich Ca2+ -
binding nonapeptide repeat near the C terminus. Many substrates are toxins, but the
family also includes adhesins, enzymes, and S-layer proteins.
The T3SS and T4SS translocate substrates directly from the cytoplasm into a
eukaryotic target cell (Fig. 6.2), where they interfere with signal transduction and
metabolism. The T4SS is very similar to the conjugation apparatus, which translo-
cates DNA with associated proteins into other bacterial or eukaryotic cells. The T3SS
contains a basal body resembling the structure that anchors the flagellum in the cell
envelope and functions as the translocon for flagellin. In T3SS, the basal body is
connected to an extracellular needle or pilus in animal or plant pathogens, respec-
tively. These structures reach through any additional surface layers of the bacteria
and the eukaryotic cell wall, if present. The T3SS first inserts a translocon into the
eukaryotic membrane that serves to deliver effector proteins into these cells.
The T6SS delivers toxic proteins into competing bacteria or into eukaryotic cells.
Several components of the T6SS resemble phage tail proteins, which serve to inject
phage DNA into the bacterial cytoplasm. Thus, the T6SS appears to function as an
inverted phage driving proteins out of the bacterial cell and delivering them straight
into target cells.

6.5 Basic Structure of Cell Envelopes of Fungi and Oomycetes

The Fungal Cell Envelope The fungal cell envelope is the target of microbial
control agents. Ergosterol is a main anti-fungal plasma-membrane target, while
chitinases, glucanases and proteases attack the cell wall. These enzymes often work
synergistically, thereby weakening or even killing the pathogen. The cell envelope
has been best studied in Saccharomyces cerevisiae. This yeast functions as a model
for the plasma membrane and cell walls of plant-pathogenic and plant-beneficial
fungi such as Fusarium and Trichoderma, respectively.
The Fungal Plasma Membrane The plasma membrane of S. cerevisiae consists
of the phospholipids phosphatidylcholine, phosphatidylethanolamine, phosph-
atidylinositol, phosphatidylserine as well as inositol sphingolipid and the sterol er-
gosterol (van Meer et al. 2008). The molar ratio between ergosterol and phospholipids
is 0.5. Mechanical stress resistance is acquired by the relative dense packing of sph-
ingolipids and sterols. Plasma membrane proteins are encoded by only ∼4 % of the
S. cerevisiae genome (i.e. ∼250 proteins). Szopinska et al. (2011) identified > 100
of them and 68 % were integral membrane proteins. About one third were classi-
fied as transporters including, for example, seven glucose transporters. The plasma
membrane also contains signaling proteins, e.g. of the cell-wall integrity pathway,
and proteins involved in cell-wall synthesis, including two chitin synthases, one
1,3-β-d-glucan synthase, and three glucan elongases.
40 J. Tommassen and H. A.B. Wösten

The Fungal Cell Wall The cell wall stabilizes internal osmotic conditions and tur-
gor pressure and provides physical protection and shape to the cells. It represents a
considerable metabolic investment; in S. cerevisiae, it accounts for ∼10–25 % of the
total cell mass (Klis et al. 2006). Cell walls of fungi generally consist of one or more
fibrillar components, one or more matrix components, and may have an outer pro-
tein layer. Often, the fibrillar components include both chitin (a 1,4-β-linked GlcNAc
polymer) and β-glucans, but occasionally mainly chitin (e.g. Encephalitozoon cuni-
culi) or 1,3-β-glucan (e.g. Schizosaccharomyces pombe) is present (Xie and Lipke
2010). Mannoproteins often form the matrix of cell walls, e.g. in S. cerevisiae, but
also galactomannoproteins and α-glucan are used, e.g. in S. pombe. The composi-
tion, molecular organization and thickness of the cell wall can vary depending on
environmental conditions. This adaption may be functional in an environment where
fungi are exposed to antibiotics, lytic enzymes secreted by other microorganisms or
to immune systems.
The cell walls of filamentous ascomycetes and even basidiomycetes have been
proposed to be very similar to the well-studied cell wall of S. cerevisiae, which
consists of an inner and outer layer (De Groot et al. 2005). The inner layer consists
of 1,3-β-glucan, 1,6-β-glucan and chitin (Fig. 6.3). The moderately branched 1,3-β-
glucan is the main polysaccharide. Its side-chains allow only local mutual association
by hydrogen bonding. Consequently, a three-dimensional network is formed that is
highly elastic and extended under normal osmotic conditions (Klis et al. 2006). Cells
of S. cerevisiae shrink when exposed to hypertonic conditions. The cell wall also
reduces in size then resulting in a surface loss of up to 50 % and a cell-wall porosity
of less than 1000 Da, while even medium-sized proteins can pass under normal
osmotic conditions. Chitin and 1,6-β-glucan are linked to the inner and outer parts
of the 1,3-β-glucan network, respectively. Growing buds have not yet formed chitin;
hence this polymer is not essential for cell-wall assembly and function. The ‘alkali-
sensitive linkage’ cell-wall proteins (ASL-CWPs) are linked to the 1,3-β-glucan in
the inner layer of the cell wall. Among the ASL-CWPs are the protein with internal
repeats (PIR)-CWPs, which interconnect two or even more 1,3-β-glucan chains,
thereby strengthening the cell wall (De Groot et al. 2005). Increased presence of
PIR-CWPs has been proposed to produce a less elastic cell wall as occurs during the
G1 phase of the cell cycle and during cell-wall stress (Klis et al. 2006).
The outer cell-wall layer is formed by mannoproteins, which are heavily glyco-
sylated; each glycochain may contain hundreds of mannose residues. At least 20
different proteins make up this layer. Their composition varies depending on the cul-
ture conditions (Klis et al. 2006). The glycosylphosphatidylinositol (GPI)-modified
CWPs form the largest group of CWPs within this outer layer. They are covalently
linked to 1,6-β-glucan through a truncated form of their original GPI-anchor (De
Groot et al. 2005). The genome of S. cerevisiae contains 66 GPI-CWP genes (De
Groot et al. 2003).
The cell wall of S. cerevisiae also contains proteins that are not covalently linked
to β-glucans. Collectively, the covalently linked and the non-covalently linked CWPs
have a wide range of functions. Both classes are involved in cell-wall synthesis and
remodeling. The GPI-CWPs also have a structural role by making the cell wall
6 Microbial Cell Surfaces and Secretion Systems 41

Fig. 6.3 Representation of the cell wall of S. cerevisae. Mannoproteins are delivered to the cell wall
via secretory vesicles that fuse with the plasma membrane. The polysaccharides β-1,3-glucan and
chitin are synthesized by synthases in the plasma membrane. The mechanism by which β-1,6-glucan
is formed is not known. The cell wall polysaccharides and mannoproteins form a complex in the cell
wall. TheASL-CWPs and proteins that are not covalently linked are not shown. Et ethanolamine, Glc
Glucose, P phosphate. Reproduced from Cabib and Arroyo (2013) by permission of the publisher

less porous (Klis et al. 2006). Reduced porosity has been proposed to retain high-
molecular-weight soluble proteins in the cell-wall matrix and may also protect the cell
against lytic enzymes of competing microbes or plants or animals. In the latter case,
it thus contributes to virulence. CWPs can also be involved in virulence by acquiring
iron in the host or by inactivation of oxygen radicals released by the immune system
(De Groot et al. 2005). Adhesins also contribute to the infection process. Adhesion of
S. cerevisiae to foreign surfaces depends on the GPI-CWP Flo11 (Bojsen et al. 2012).
This protein, which can also mediate mutual binding of yeast cells, is characterized
by an A, B, and C domain. The A domain mediates cell-surface or cell-cell adherence,
while the C domain contains the GPI anchor. Flo1, Flo5, Flo9 and Flo10 show high
42 J. Tommassen and H. A.B. Wösten

mutual homology and similarity to Flo11. These proteins also have A, B, and C
domains. The A domain is a β-barrel involved in mutual binding of yeast cells.
Binding to a cell surface or to each other is often accompanied by the formation
of an ECM, which creates a micro-environment that may prevent dehydration or
access of antibiotics to the cells. In the case of S. cerevisiae, glucose and mannose
polysaccharides and proteins constitute the ECM (Beauvais et al. 2009).
As in bacteria, some surface-exposed fungal proteins can form amyloid-like struc-
tures (Gebbink et al. 2005). Amyloids are filamentous protein structures of ∼10 nm
wide and 0.1–10 μm long that share a structural motif, the cross-beta structure,
and have been associated with neurodegenerative diseases. One of the best studied
amyloid-forming microbial proteins is the hydrophobin SC3 of Schizophyllum com-
mune. The water-soluble form of this protein affects the polysaccharide cell-wall
composition. When confronted with the interface between the cell wall and the air
or a hydrophobic surface, such as that of a plant, the structure of SC3 changes. It
self-assembles into an amphipathic two-dimensional mosaic film of parallel amyloid
fibrils. In this conformation, SC3 has different functions. It allows fungi to escape the
aqueous substrate to grow into the air, it confers hydrophobicity to aerial hyphae and
mediates attachment of the fungus to a hydrophobic support (Wösten 2001). Adhesins
in the yeast cell wall (see above) have also been proposed to adopt the amyloid struc-
ture. This would explain the paradox that the adhesins often show weak binding to
ligands, yet mediate remarkably strong adherence. Experimental evidence indicates
that the strength of adhesion results partly from amyloid-like clustering of hundreds
of adhesin molecules to form an array of ordered binding sites (Lipke et al. 2012).
The Cell Wall of Oomycetes Oomycetes are more related to brown algae and di-
atoms than to fungi and include some of the most devastating plant and animal
pathogens. Their cell envelopes represent an excellent target to control disease but
little is known about their composition. The cell wall is classically described to con-
sist of 1,3-β- and 1,6-β-glucans and 4–20 % of cellulose (Aronson et al. 1967). A
recent detailed cell wall analysis of 10 species from two oomycete orders revealed
high heterogeneity (Mélida et al. 2013). Three different cell wall types were dis-
tinguished primarily based on GlcNAc content. Types I, II and III contain 0 %, up
to 5 %, and > 5 % of this sugar, respectively. Each type is also characterized by
additional compositional features. For example, the type I cell walls of Phytophtora
spp. contain glucuronic acid and mannose and have a cellulose content of 32–35 %.
Saprolegnia has a type II cell wall. Its GlcNAc residues are contained in chitin. A
unique feature of this type is the 1,3,4-linked glucosyl residues, which are indicative
of cross-links between cellulose and 1,3-β-glucans. Aphanomyces euteiches has a
type III cell wall and contains nearly 10 % GlcNAc including 1,6-linked polymers.

6.6 Protein Export in Fungi

Most fungal proteins are transported to the cell wall or beyond via the ER
(Conesa et al. 2001). S. cerevisiae translocates proteins over the ER membrane
via SRP-dependent and -independent pathways, in which translocation occurs co-
6 Microbial Cell Surfaces and Secretion Systems 43

or post-translationally, respectively. Proteins with a less hydrophobic signal peptide

are targeted through the SRP-independent route, which involves the ER chaperone
BiP, whereas proteins with a more hydrophobic signal can take both routes. Both
pathways make use of the same translocon, the Sec61 complex. Also in other fungi,
both pathways are probably operational. In the ER lumen, proteins are modified
and folded and then transported to the Golgi, where they are further processed, e.g.
by proteolytic modification and/or by modifications of their glycan-chains. Subse-
quently, the proteins are packed in vesicles which fuse with the plasma membrane to
release the proteins into the cell wall. Vesicle fusion does not occur uniformly along
the plasma membrane but mainly at the growing bud of the daughter cell and later
re-localizes at the mother-bud neck just prior to cytokinesis. This implies that pro-
teins are mainly incorporated in the cell wall or released into the medium when the
cell is formed and not once cell division has occurred (Sietsma and Wessels 2006).
In filamentous fungi, which form hyphae that extend at their tips, vesicles also fuse
at the growth site. Once extruded at the hyphal tip, proteins migrate together with the
newly synthesized cell-wall polymers to the outer part of the wall. This migration is
driven by the turgor pressure in the hyphae and the apposition of newly synthesized
polymers at the inner part of the wall. At the outside, the proteins diffuse into the
medium (Sietsma and de Vries 2006). The newly synthesized cell-wall polymers
are initially not cross-linked, resulting in a deformable cell wall at the tip. Once
deposited, the polymers start to crosslink, and the cell wall becomes more and more
rigid and less porous in subapical direction. This implies that proteins that are se-
creted more subapically will be captured in the cell wall. Yeast buds grow over their
whole surface and do not have an “ever” extending tip that creates a continuous flow
of proteins to the medium. Therefore, protein release in this case depends more on
pores in the cell wall.

References

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335
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matrix in FLO1-expressing Saccharomyces cerevisiae cells. FEMS Yeast Res 9:411–419
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Bojsen RK, Andersen KS, Regenberg B (2012) Saccharomyces cerevisiae–a model to uncover
molecular mechanisms for yeast biofilm biology. FEMS Immunol Med Microbiol 65:169–182
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plant pathogenesis. Annu Rev Phytopathol. doi:10.1146/annurev-phyto-011014-015624
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biotechnological view. Fungal Genet Biol 33:155–171
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De Groot PW, Hellingwerf KJ, Klis FM (2003) Genome-wide identification of fungal GPI proteins.
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cell walls. Fungal Genet Biol 42:657–675
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12:211–222
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Nat Rev Microbiol 3:333–341
Klis FM, Boorsma A, De Groot PW (2006) Cell wall construction in Saccharomyces cerevisiae.
Yeast 23:185–202
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negative bacteria: the Sec and Tat dependent protein transport pathways. Res Microbiol 164:505–
534
Lipke PN, Garcia MC, Alsteens D et al (2012) Strengthening relationships: amyloids create
adhesion nanodomains in yeasts. Trends Microbiol 20:59–65
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bohydrates in oomycetes unveil the existence of three different cell wall types. Eukaryot Cell
12:194–203
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proteins. Trends Microbiol 18:109–116
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Chapter 7
Microbial Biofilms and Quorum Sensing

Aurelien Carlier, Gabriella Pessi and Leo Eberl

Abstract Many bacteria that form biofilms on various plant surfaces use small
signal molecules for intra- and interspecies communication. In this chapter we will
review the current knowledge on bacterial cell-to-cell signaling, referred to as quorum
sensing (QS), in biofilms on the surfaces of plant roots and leaves. Particular focus
will be laid on the role of QS in the formation of nitrogen-fixing root nodules and the
expression of virulence factors in biofilms formed by plant pathogens. We will also
discuss how plants can interfere with bacterial QS and thus manipulate microbial
activity and persistence.

7.1 Quorum Sensing in Plant-Associated Biofilms

The term quorum sensing (QS) describes the phenomenon that bacteria are capa-
ble of perceiving and responding to self-generated signal molecules to coordinate
their behavior in response to their population size (Fuqua et al. 1994). The general
consensus is that bacteria trigger QS only when their cell density has reached a cer-
tain threshold (the “quorum”), upon which the expression of target genes is either
activated or repressed. Among the various QS signal molecules identified to date,
N-acyl-homoserine lactones (AHL) have been investigated to the greatest extent and
have been shown to control the expression of various traits, including virulence,
symbiosis, motility, biofilm formation, the production of antibiotics and toxins, and
conjugation. Various AHL molecules have been described that all have a homoserine

L. Eberl () · G. Pessi · A. Carlier

Institute of Plant Biology, University of Zurich, Zollikerstrasse 107,
CH-8008 Zurich, Switzerland
Tel.: + 41 44 634 8220
e-mail: leberl@botinst.uzh.ch
A. Carlier
Tel.: + 41 44 63 48227
e-mail: aurelien.carlier@gmail.com
G. Pessi
Tel.: + 41 44 63 52904
e-mail: gabriella.pessi@botinst.uzh.ch

© Springer International Publishing Switzerland 2015 45

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_7
46 A. Carlier et al.

lactone (HSL) moiety but differ in the length and structure of the acyl side chain.
Cha et al. (1998) showed that the majority of plant-associated bacteria produce AHL
signal molecules. All isolates of the genera Agrobacterium, Erwinia, Pantoea, and
Rhizobium, and about half of the erwinias and pseudomonads tested, synthesize de-
tectable levels of AHLs while only few AHL producers could be identified among
Xanthomonas isolates. It is worthwhile to note that members of the latter genus
are known to use another type of signal molecule, namely DSF (Diffusible Signal
Factor; cis-11-methyl-2-dodecenoic acid). A structurally related molecule, BDSF
(Burkholderia Diffusible Signal Factor; cis-2-dodecenoic acid), is also produced by
many plant-associated Burkholderia species, which additionally produce AHL sig-
nal molecules (Suppiger et al. 2013). Elasri et al. (2001) screened 137 soil-borne
and plant-associated Pseudomonas sp. strains using biosensors and identified 54 that
were positive for AHL production. The authors of this study concluded that plant-
associated and plant-pathogenic bacteria produce AHLs more frequently than soil
borne strains and hypothesized that the more intimate the relationship of the bacteria
with the host plant, the higher the probability that it produces AHLs.
QS is a particularly valuable regulatory mechanism when bacteria are living in
close contact to each other. This is the case in biofilms, where the cells are embedded
in a self-produced extracellular matrix, which consists of polysaccharides, proteins
and DNA and acts as a diffusion barrier for signal molecules, thus creating an ideal
environment for the induction of QS. Moreover, a direct role for AHL-mediated QS
in biofilm formation has been demonstrated for many bacteria that are usually as-
sociated with plants, including members of the genera Burkholderia, Pseudomonas
and Serratia (Aguilar et al. 2009). Employing a quorum quenching approach (i.e. the
enzymatic degradation of AHL signal molecules), it was shown that AHL signaling
regulates biofilm formation in the large majority of Burkholderia species. In several
Pseudomonas putida strains AHL-dependent production of powerful cyclic lipopep-
tide biosurfactants (putisolvins) strongly affects biofilm formation. Putisolvins were
found to inhibit biofilm formation and were shown to even break down existing
biofilms. As a consequence it was observed that QS mutants produce denser biofilms
than the wild-type strains. For Serratia sp. it has been shown that QS plays an impor-
tant role in biofilm structural development, ultimately resulting in a highly porous
biofilm composed of cell chains, filaments, and cell clusters.
Plants are known to support the growth of bacterial biofilms on and within their
tissues, including aerial portions of the plant, the vascular network, and root tissues
below ground (Fig. 7.1). These plant-associated biofilms may establish commensal,
mutualistic and pathogenic interactions with plants, or simply grow saprophytically
on the nutrients released.
QS in the Rhizosphere The rhizosphere is the narrow soil compartment that is di-
rectly influenced by root secretions (see Chap. 3). Hence this niche is relatively rich
in nutrients compared with the bulk soil and allows bacteria to form microcolonies
on the root surface, consisting of multiple layers of cells that are embedded in a self-
produced matrix. Steidle et al. (2001) screened over 300 bacterial strains isolated
from the rhizosphere of tomato on standard laboratory media, and found that approxi-
mately 12 % of the isolates produced detectable AHLs. In this study, GFP-based AHL
7 Microbial Biofilms and Quorum Sensing 47

lactonase acylase

AHLs

BDSF
Phyllosphere

DSF

Vascular Pathogens 3-OH PAME

QS
QS

Rhizosphere / Nodules

EPS

adhesion aggregation maturation Plant surface

Fig. 7.1 Microbial biofilms on and within plants. In many plant-associated bacteria biofilm forma-
tion is controlled by QS. These bacterial communication systems utilize chemically diverse signal
molecules. The site of action of two classes of enzymes that inactivate AHL signal molecules is
indicated by blue arrows

monitor strains were used to visualize interspecies communication between bacteria

colonizing the rhizosphere of tomato plants. These experiments provided evidence
that AHL signal molecules serve as a universal language for communication be-
tween root-associated Gram-negative bacteria. Several subsequent studies provided
further evidence that AHL-mediated QS is common among root-associated bacteria.
In situ quantitative data on the spatial scale of AHL-mediated cell-to-cell commu-
nication of P. putida on tomato and wheat root surfaces were generated employing
computer-assisted microscopy in combination with geostatistical modeling (Gant-
ner et al. 2006). This analysis suggested that the effective “calling distance” on root
surfaces was most frequent at 4–5 μm, extended to 37 μm in the root tip/elongation
zone and further out to 78 μm in the root hair zone. This quantitative analysis also
lent support to the hypothesis that the bacteria are able to use AHL gradients for
sensing their positions relative to each other in the rhizosphere, an ability that may
be particularly important for the formation of biofilms. An interesting link between
AHL-dependent signaling and nitrogen cycling was noted for the bacterial consor-
tium colonizing the Avena (wild oat) rhizosphere. In many rhizosphere isolates the
expression of chitinolytic and proteolytic activity was found to be AHL-regulated,
suggesting that QS could be a control point in the complex process of rhizosphere
nitrogen mineralization (DeAngelis et al. 2008) .
QS in Aggregates and Biofilms of the Phyllosphere The leaf surface is a stressful
environment for colonizing bacteria, due to light, heat and desiccation (see Chap. 4).
48 A. Carlier et al.

Multicellular aggregates preferentially form around stomates and trichomes, or along

leaf veins, responding to moisture and nutrient concentration. Such aggregates gen-
erally grow from one or a few cells that proliferate at the site of deposition to form
a microcolony. Among the best studied epiphytes is Pseudomonas syringae, which
causes brown spot disease on bean. Although single P. syringae cells can be observed
on the leaf surface, the bulk of the adherent biomass that results from colonization
is contained in large aggregates comprised of thousands of cells, which not only
survive desiccation stress better than solitary cells but are also ideally suited for QS.
In fact, the fitness of adherent P. syringae populations was shown to be dependent on
a functional AHL-dependent QS system, as it controls various functions important
for epiphytic persistance, including extracellular polysaccharide (EPS) production,
oxidative stress tolerance, and motility (Quinones et al. 2005). QS of P. syringae on
leaves was found to be strongly influenced both by the size of the aggregates and by
the availability of water, as AHL transport on leaves appears to be a relatively local
phenomenon, in contrast to that in the rhizosphere (see above). Moreover, a high
level of AHL-mediated cross-talk as well as of QS quenching has been demonstrated
for epiphytic communities and it has been suggested that these processes can be
exploited for disease control (Dulla et al. 2009).

7.2 QS and Root Nodule Symbiosis

Rhizobia enter a symbiotic relationship with leguminous plants, resulting in differ-

entiated bacteria that live enclosed in intracellular organelle-like structure within
nodules on the root, called symbiosomes (see Chap. 23). Rhizobia are taxonomi-
cally diverse and polyphyletic; they include members of both the α-proteobacteria
group (Azorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium, and Sinorhi-
zobium) as well as Burkholderia and Cupriavidus strains, which belong to the
β-proteobacteria. The Rhizobia-legume interaction begins with an initial attraction
step involving various signal molecules, attachment to root hairs via formation of
micro-colonies and biofilms, followed by root colonization and infection, and sub-
sequent growth within legume roots, both in infection threads and in nodules where
the bacteria fix atmospheric nitrogen (see Downie 2010).
The interaction between rhizobia and their leguminous host plants requires an
exchange of molecular signals. In addition to well characterized flavonoids and Nod
factor-mediated signalling, rhizobia also use EPS and QS molecules. The cell density
of Rhizobium species is important for biofilm formation around the plant roots and for
an efficient symbiotic life inside the root nodules. Most of the QS systems identified
so far are based on AHLs. Compared to other genera, rhizobia produce the greatest
diversity of AHLs, with strains producing from as few as one to as many as seven
detectable AHL signals which range from short (C4) to very long acyl side chains.
More recently, the soybean symbiont B. japonicum has been shown to produce low
concentrations of isovaleryl-HSL that belongs to a new class of HSL signals, namely
the aryl-HSLs. All the identified rhizobial QS regulation systems so far are based
7 Microbial Biofilms and Quorum Sensing 49

on acyl- or aryl-HSL synthesis and perception. Within a single rhizobial species,

different isolates may have different AHL-dependent QS systems. The production
of diverse, partially overlapping sets of AHLs by different strains suggests that QS
regulation in each strain is likely to be complex and distinct.
QS affects many aspects of rhizobial physiology including EPS production,
biofilm formation, legume nodulation and nitrogen fixation. Moreover, QS has been
shown to induce the transfer of plasmids in several rhizobia and integrated symbiosis
islands in Mesorhizobium loti as well as motility, growth inhibition and adaptation
to abiotic stresses (Sanchez-Contreras et al. 2007). A conserved QS system is im-
portant for nitrogen fixation as well as growth and development of nitrogen-fixing
symbiosomes in the pea symbiont Rhizobium etli. QS mutant bacteroids are always
individually packed in the symbiosome membrane and are devoid of a large sym-
biosome space whereas wild-type symbiosomes usually contain multiple bacteroids.
The R. etli QS system was shown to be expressed in infection threads and in dif-
ferentiated bacteroids and AHLs could be extracted from R. etli (pea) bacteroids. In
Rhizobium leguminosarum bv. viciae four QS systems have been identified which
are hierarchically arranged. Interestingly, one of the AHL molecules produced by
R. leguminosarum, N-(3-hydroxy-7-cis-tetradecenoyl) homoserine lactone, has been
shown to be a small bacteriocin that inhibits growth of susceptible strains. This AHL-
dependent inhibition of growth of R. leguminosarum is unusual and is mediated by
two QS regulators that also regulate plasmid transfer. In S. meliloti and in several
Mesorhizobium isolates, EPS production has been shown to be under the control of
QS and is important for infection, attachment and biofilm formation on root hairs.
EPS mutant strains form empty nodules that lack bacteroids, suggesting that EPS is
essential for the development of nitrogen-fixing nodules.
Rhizobia belonging to the β-proteobacteria group, such as Burkholderia phy-
matum, use an AHL QS system that is highly conserved among plant-beneficial-
environmental Burkholderia species. The B. phymatum QS system is responsible
for the production and perception of several different AHL molecules. Although
this system is not essential for legume nodulation, a QS mutant is impaired in EPS
production (Coutinho et al. 2013).

7.3 QS in Plant Pathogens

The fact that QS can affect plant colonization at various stages is also reflected in the
diversity of strategies adopted by phytopathogens for biofilm formation and dispersal
as well as for infection.
Pathogens of the Plant Surface Biofilms are a high-cell density environment and
thus not only a natural site for QS regulation but also for conjugative plasmid trans-
fer. Agrobacterium tumefaciens, the agent that causes crown gall disease, regulates
conjugation of the Ti virulence plasmid via an AHL-based QS system. The biofilm
that A. tumefaciens forms on plant surfaces provides an ideal site for QS-dependent
plasmid transfer (Danhorn and Fuqua 2007) .
50 A. Carlier et al.

Vascular Pathogens The role of biofilms in the later stages of infection is best
studied in vascular pathogens, probably because of the well-established link between
EPS production and virulence. Pantoea stewartii subsp. stewartii forms biofilms that
clog the xylem vessels of susceptible varieties of sweet corn, causing water stress and
the typical symptoms associated with Stewart’s wilt. Dense bacterial mats are formed
upon initial attachment to the protoxylem annular rings, structures that may provide
nourishment to P. stewartii. Maturation of these biofilms is an essential process,
as mutants blocked in EPS synthesis produce immature, flat biofilms and fail to
colonize tissues beyond the infection site. An AHL-dependent QS system regulates
the biosynthesis of EPS and biofilm maturation. At low cell density P. stewartii does
not produce the EPS virulence factor, while at high cell density, the QS system
activates EPS biosynthesis (Von Bodman et al. 2003). The importance of proper
timing of the different stages of biofilm formation by QS is illustrated by the fact
that mutants that express EPS constitutively form loose biofilms but display reduced
virulence (Koutsoudis et al. 2006). Ralstonia solanacearum is the causative agent
of wilts affecting over 200 plant species and can persist in soil or in water systems.
When it encounters a susceptible host, R. solanacearum breaches the root cortex
and spreads through the host’s vasculature. Similar to P. stewartii, R. solanacearum
biofilms lethally hinder the flow of nutrients through the xylem. Most of the traits
required for infection, including EPS production and biofilm formation, are regulated
by the Phc (phenotype conversion) regulatory system in a cell-density dependent
manner. The Phc system is an atypical QS system that relies on the synthesis of the
volatile 3-hydroxy-palmitic acid methyl ester (3-OH PAME) by a methyltransferase
and its perception by a two-component sensor histidine kinase-response regulator
pair. This QS system provides a regulatory switch from a free-living to a pathogenic
lifestyle. This is illustrated by the fact that QS mutants produce low levels of EPS
and plant cell wall degrading enzymes, while they display enhanced traits associated
with the free-living stage in soil, e.g. motility and siderophore biosynthesis (Von
Bodman et al. 2003) .
Xanthomonas campestris is a xylem-dwelling pathogen that is causing econom-
ically significant diseases. Pathovars of X. campestris cause black rot disease of
virtually all cultivated brassicas, bacterial spot of pepper and tomato and angular
leaf spot of cotton. The rpf genes of X. campestris are responsible for the synthesis
and perception of the signal molecule DSF. DSF-dependent QS in X. campestris
regulates the synthesis of extracellular hydrolytic enzymes and the polysaccharide
xanthan, a structural component of biofilms. Mutations inactivating any of the rpf
genes in X. campestris pv. campestris lead to reduced synthesis of xanthan EPS,
extracellular enzymes and decreased virulence. Moreover, the DSF system posi-
tively controls the expression of an endo-β-(1,4)-mannase, involved in virulence and
biofilm dispersal in vitro (Dow et al. 2003) .
7 Microbial Biofilms and Quorum Sensing 51

7.4 Cross-Talk Between Bacteria and Plants

Interkingdom Signaling via Small Molecules Evidence has accumulated that

plants can perceive and respond to bacterial signal molecules. Both Medicago trun-
catula and tomato have been shown to respond to exogenous AHLs by altering the
global patterns of gene expression, including the activation of pathogenesis-related
proteins that might contribute to disease resistance (Hartmann and Schikora 2012).
AHLs with acyl side chain lengths smaller than C8 have been found to enter the
roots readily and are transported up to the shoots whereas AHLs with side chains
longer than C10 tend to stick to the root surface and are not transported substantially.
The presence of AHL-producing bacteria in the tomato rhizosphere was shown to in-
crease the salicylic acid levels in the leaves and enhance systemic resistance against
the fungal leaf pathogen Alternaria alternata. Macro-array analyses showed that
synthetic AHLs systemically induce salicylic acid- and ethylene-dependent defense
genes, suggesting that AHLs can play an important role in the biocontrol activity
of rhizobacteria. Several reports have demonstrated that AHLs can also influence
root development of plants. Depending on the structure of the AHL molecule, (par-
ticularly the length of the acyl side chain), the thickness and length of the root but
also root hair development can be affected. Recent research has also shown that a
sub-family of bacterial AHL receptors responds specifically to yet unidentified small
molecules produced by plants (Patel et al. 2013).
QS Inhibition and Quenching Plants also produce compounds that mimic or inhibit
AHL-dependent QS of plant-associated bacteria (Kalia 2012). Signal antagonists, so-
called QS inhibitors, have been isolated from a large number of plants and some of
these compounds are currently considered for exploitation as antibacterial and an-
tibiofilm agents. Many studies have demonstrated that AHL signals can be degraded
by various bacteria by the production of lactonases or acylases and thereby “quench”
QS (Fig. 7.1). Bacteria that are capable of quenching QS have been isolated from the
rhizosphere as well as from the phyllosphere of various plants and have been shown
to play an important role in the ecology of plant-associated consortia. Finally, some
plants have also the capability to enzymatically inactivate AHL signal molecules.

References

Aguilar C, Carlier A, Riedel K et al (2009) Cell-to-cell communication in biofilms of gram-negative

bacteria. In: Krämer R, Jung K (eds) Bacterial signaling. Wiley-VCH, Weinheim, pp 23–40
Cha C, Gao P, Chen Y et al (1998) Production of acyl-homoserine lactone quorum-sensing signals
by gram-negative plant-associated bacteria. Mol Plant-Microbe Interact 11:1119–1129
Coutinho B, Mitter B, Talbi C et al (2013) Regulon studies and in planta role of the BraI/R
quorum-sensing system in the plant-beneficial Burkholderia cluster. Appl Environ Microbiol
79:4421–4432
Danhorn T, Fuqua C (2007) Biofilm formation by plant-associated bacteria. Annu Rev Microbiol
61:401–422
52 A. Carlier et al.

DeAngelis KM, Lindow SE, Firestone MK (2008) Bacterial quorum sensing and nitrogen cycling
in rhizosphere soil. FEMS Microbiol Ecol 66:197–207
Dow JM, Crossman L, Findlay K et al (2003) Biofilm dispersal in Xanthomonas campestris is
controlled by cell-cell signaling and is required for full virulence to plants. Proc Natl Acad Sci
U S A 100:10995–11000
Downie JA (2010) The roles of extracellular proteins, polysaccharides and signals in the interactions
of rhizobia with legume roots. FEMS Microbiol Rev 34:150–170
Dulla G, Lindow S (2009) Acyl-homoserine lactone mediated cross talk among epiphytic bacteria
modulates behavior of Pseudomonas syringae on leaves. ISME J 3:825–834
Elasri M, Delorme S, Lemanceau P et al (2001) Acyl-homoserine lactone production is more
common among plant-associated Pseudomonas spp. than among soilborne Pseudomonas spp.
Appl Environ Microbiol 67:1198–1209
Fuqua WC, Winans SC, Greenberg EP (1994) Quorum sensing in bacteria: the LuxR-LuxI family
of cell density-responsive transcriptional regulators. J Bacteriol 176:269–275
Gantner S, Schmid M, Dürr C et al (2006) In situ quantitation of the spatial scale of calling distances
and population density-independent N-acylhomoserine lactone-mediated communication by
rhizobacteria colonized on plant roots. FEMS Microbiol Ecol 56:188–194
Hartmann A, Schikora A (2012) Quorum sensing of bacteria and trans-kingdom interactions of
N-acyl homoserine lactones with eukaryotes. J Chem Ecol 38:704–713
Kalia VC (2012) Quorum sensing inhibitors: an overview. Biotechnol Adv 31:224–245
Patel HK, Suárez-Moreno ZR, Degrassi G et al (2013) Bacterial LuxR solos have evolved to respond
to different molecules including signals from plants. Front Plant Sci 4:447
Quinones B, Dulla G, Lindow SE (2005) Quorum sensing regulates exopolysaccharide production,
motility, and virulence in Pseudomonas syringae. Mol Plant Microbe Interact 18:682–693
Sanchez-Contreras M, Bauer WD, Gao M et al (2007) Quorum sensing regulation in rhizobia and its
role in symbiotic interactions with legumes. Philos Trans R Soc Lond B Biol Sci 362:1149–1163
Steidle A, Siegl K, Schuhegger R, Ihring A et al (2001) Visualization of N-Acylhomoserine (AHL)-
mediated cell-cell communication between bacteria colonizing the tomato rhizosphere. Appl
Environ Microbiol 67:5761–5770
SuppigerA, Schmid N,Aguilar C et al (2013) Two quorum sensing systems control biofilm formation
and virulence in members of the Burkholderia cepacia complex. Virulence 4:400–409
Von Bodman SB, Bauer WD, Coplin DL (2003) Quorum sensing in plant-pathogenic bacteria. Annu
Rev Phytopathol 41:455–482
Chapter 8
Bacterial Volatiles as Airborne Signals for Plants
and Bacteria

Choong-Min Ryu

Abstract Volatile compounds are found in all organisms. Bacteria communicate

with their surrounding ecosystem using a diverse array of volatile metabolites. Some
rhizosphere bacteria (rhizobacteria) emit volatile organic compounds (VOCs) that
promote plant growth and elicit induced systemic resistance (ISR) and induced sys-
temic tolerance (IST). This chapter reviews recent progress in understanding how
VOCs mediate interactions between rhizobacteria and plants and among bacteria.
Recent proteomics analysis of plant ISR and IST induced by rhizobacterial VOCs as
well as the potential of VOCs for field applications will be discussed. These studies
provide novel insights into the biological and ecological potential of rhizobacterial
VOCs for modulating biotic and abiotic stress tolerance in modern agriculture.

8.1 Airborne Bacterial Signals

Bacterial volatile organic compounds (VOCs) are signaling molecules that are per-
ceived by other bacteria, animals, insects, plants, and microorganisms (Farag et al.
2013). The first report of microbial VOCs was in 1921 (Zoller and Clark 1921).
Bacterial volatiles are recognized as insect semiochemicals and inhibitors of fungal
and plant growth (Baily and Weisskopf 2012; Davis et al. 2013). The study of bac-
terial volatiles traditionally focused on soil ecology and interactions with plants. In
2003, Ryu et al. identified bacterial VOCs that positively promote growth of Ara-
bidopsis thaliana seedlings. These VOCs were emitted from a rhizobacterial class
called plant-growth promoting rhizobacteria (PGPR). A year later, the same research
group reported that PGPR trigger ISR (Ryu et al. 2004), which is a form of plant
systemic defense. These two reports provided new information about novel bacterial
compounds that elicit plant growth and ISR. There has been considerable progress
in recent years in the understanding of bacterial VOC-mediated plant responses, in-
cluding abiotic stress tolerance, and new evidence for the role of bacterial VOC in

C.-M. Ryu ()

Molecular Phytobacteriology Laboratory, KRIBB, Daejeon 305-806,
Republic of Korea
Tel.: + 82 42 879 8229
e-mail: cmryu@kribb.re.kr

© Springer International Publishing Switzerland 2015 53

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_8
54 C.-M. Ryu

c
Fig. 8.1 Inter-kingdom and intra-kingdom communication-mediated by bacterial volatile organic
compounds. a Bacterial volatiles are captured in fiber membranes, extracted with organic solvents,
and identified using gas chromatography-mass spectrometry (GC-MS). The bacterial volatiles de-
coded by GC-MS analysis helps us to understand the roles of bacterial airborne signals in their
interactions with plants and bacteria. b Plant protection by bacterial volatiles against biotic and abi-
otic stresses in in vitro as well as even under field conditions. c Bacterial interactions mediated by
volatile compounds. The two plates indicate inhibition of the motility of a gram-negative bacterium
(E. coli) by bacterial VOCs from the gram-positive bacterium Bacillus subtilis on the agar media

plants, animals, and bacteria. This chapter will focus on the role of bacterial VOCs
in plants, bacteria, and other microbes (Fig. 8.1).
The major obstacle for early studies of the effect of bacterial VOCs on plants
was the lack of suitable culture systems to perform the experiments, because natural
culture conditions also contain VOCs produced by plants and microbes. It was nec-
essary to develop a gnotobiotic system, in which plants and bacteria were spatially
separated into a closed culture system that controls both biotic and abiotic variables.
This problem was solved by using an I-plate system, which is a Petri dish that con-
tains two physically separated compartments for growth of seedlings and bacteria
and allows the free exchange of air. The I-plate system did not perform well in
studies on fungal VOCs, due to fungal spore contamination of the seedlings. There-
fore, microtiter plates (24- or 48-well) were used to study fungal VOCs, because
the airspace between the plate and the lid prevented spore transmission but allowed
VOC transmission.
The identification of VOCs from PGPR strains was necessary to isolate bioac-
tive VOCs and study their effects on plants. Several analytical methods have been
developed to capture, analyze, identify, and quantify airborne volatiles released from
8 Bacterial Volatiles as Airborne Signals for Plants and Bacteria 55

bacteria. Most of these techniques were adapted from those used to study plant VOCs.
The most common method captures gasses in the headspace of culture systems and
then employs gas chromatography (GC) for analysis of the extracted compounds. The
volatile compounds contained in the dynamic air flow over a bacterial culture grown
on solid Murashige-Skoog (MS) medium can be trapped by an absorbent filter from
the headspace and then released by rinsing the filter with specific organic solvents.
MS medium appeared to yield lower background signals compared to those pro-
duced by other microbial media (Ryu et al. 2003, 2004). Recently, a more advanced
solid-phase micro extraction technique, coupled with software-driven extraction of
compounds from overlapping peaks in GC analysis, was developed. Using this pro-
cedure, compounds are rapidly and directly released from bacterial cultures into a
heated GC-injector in a no-flow, low-oxygen environment. The solid-phase micro
extraction method can collect and identify more than 30 different volatile compounds
and has been used to collect bacterial VOCs in several systems (Fig. 8.1a; Farag et al.
2006; Lee et al. 2012). A comparative review of different techniques used for volatile
compound analysis is provided by Tholl et al. (2006).

8.2 Aromatic Therapy for Plant Stresses: Beneficial Effects

of Bacterial VOCs on Plant Growth and Immunity

The I-plate enabled identification of specific airborne chemicals emitted from soil
bacteria as signals that trigger plant growth and ISR (Ryu et al. 2003, 2004). A recent
review by Bailly and Weisskopf (2012) describes the growth-promoting effects of
bacterial VOCs. In the present chapter, I will focus on ISR/induced systemic tolerance
(IST) triggered by bacterial VOCs (Fig. 8.1b).
Sweet Odor for Inducing Immunity Our early studies investigated the
necrotrophic pathogen Pectobacterium carotovorum subsp. carotovorum (syn. Er-
winia carotovora subsp. carotovora) because this pathogen causes visible soft-rot
symptoms in many plant species within 24 h. In later studies we investigated bacte-
rial speck disease caused by Pseudomonas syringae pv. tomato; however, the disease
symptoms caused by this bacterium become visible only after 7 days (Lee et al. 2012).
Maximum protection against necrotrophic disease was conferred by rhizobacterial
strains Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, which also
trigger ISR. Four other PGPR strains that trigger ISR when inoculated onto seeds
failed to induce resistance against P. carotovorum subsp. carotovorum in the I-plate
test. Continuous analysis for 24 h revealed that GB03 and IN937a consistently re-
leases 2,3-butanediol and its precursor 3-hydroxy-2-butanone, whereas E. coli strain
DH5α and Pseudomonas putida strain 89B61 did not release these VOCs and did
not trigger ISR. Most bacterial species of the Proteobacteria and Firmicute groups
produce 2,3-butanediol and acetoin under low-oxygen conditions; these VOCs pro-
vide an alternative electron sink for NAD+ regeneration when aerobic respiration
is limited (Ramos et al. 2000; Xiao and Xu 2007). Aerobic respiration may be
56 C.-M. Ryu

limited in the rhizosphere due to low-oxygen conditions, and this is where PGPR
naturally reside. The bioactivity of stereoisomers (2R, 3R and 2S, 3S) should be
compared to determine whether PGPR VOCs have stereoisomer specificity, and to
identify the stereoisomer that triggers plant ISR and/or growth. Treatment with a syn-
thetic 2,3-butanediol and a volatile extract collected from strain GB03 induce similar
disease-resistance responses, which are comparable to those induced by direct inoc-
ulation of PGPR on plants (Ryu et al. 2004). Treatments with 0.2 pg/ml–0.2 μg/ml of
the synthetic 2,3-butanediol (in increments of 1:100 dilutions) trigger similar levels
of ISR in plants.
A recent proteomics study investigated Arabidopsis tissue exposed to VOCs de-
rived from GB03. This study provided new insights into how plants perceive PGPR
VOCs (Kwon et al. 2010). The study identified 95 peptides that differentially respond
to GB03 VOCs, including 61 up-regulated and 34 down-regulated proteins. Of these,
20 spots correspond to twelve proteins involved in ethylene (ET) biosynthesis. An-
other proteomic study, exploring in planta effects of bacterial volatiles, confirmed
that ISR triggered by B. subtilis FB17 against P. syringae pv. tomato DC3000 was
mediated by the salicylic acid (SA) and ET signaling pathways, but was independent
of the jasmonic acid (JA) pathway (Rudrappa et al. 2010). Bacterial mutants in the
acetoin and 2,3-butanediol biosynthetic pathways fail to trigger ISR, which confirms
that acetoin and 2,3-butanediol function as VOCs in eliciting ISR. In addition to
these C4 volatile compounds, long-chain VOCs, such as tridecane released from P.
polymyxa E681, prime transcriptional expression of marker genes for the SA, JA,
and ethylene signaling pathways, including PR1, ChiB, and VSP2, respectively (Lee
et al. 2012).
Treatment of maize seeds with the endophytic bacterium Enterobacter aerogenes
led to the identification of 2,3-butanediol from soil-grown maize seedlings. Bacterial
production of 2,3-butanediol resulted in maize plants that had greater resistance to
the Northern corn leaf blight fungus Setosphaeria turcica. Treatment of seeds with E.
aerogenes decreased plant attractiveness for the parasitoid Cotesia marginiventris,
whereas treatment with the soil microbiome enhanced parasitoid recruitment. These
contrasting observations indicate an indirect effect of bacterial VOCs on parasitoids,
which depends on the microbiota composition (D’Alessandro et al. 2014).
Cool Scent for Thirsty Plants An increasing number of studies demonstrated that
PGPR VOCs trigger plant tolerance to abiotic stress, including drought stress, salt
stress, and nutrient deficiency. Our previous work proposed the term induced sys-
temic tolerance for “PGPR-induced physical and chemical changes in plants that
result in enhanced tolerance to abiotic stress”. “Biotic stress is excluded from
IST because conceptually it is part of biological control and induced resistance”
(Yang et al. 2009). Plants treated with GB03-derived VOCs had greater photosyn-
thetic capacity and increased iron uptake (Zhang et al. 2009). GB03-derived VOCs
also modulate AtHKT1 function, which confers shoot-to-root Na+ recirculation, pos-
sibly by loading Na+ into phloem vessels. This result supports the role of AtHKT1
in controlling shoot-to-root Na+ recirculation, and explains VOC-induced salt toler-
ance (Zhang et al. 2008). Arabidopsis plants grown with GB03 or with Pseudomonas
8 Bacterial Volatiles as Airborne Signals for Plants and Bacteria 57

chlororaphis O6 in the soil exhibit increased drought tolerance (Cho et al. 2008;
Zhang et al. 2010). The SA signaling pathway may be involved in P. chlororaphis
O6-mediated drought tolerance, because drought-stressed plants exposed to bacte-
rial volatiles or 2,3-butanediol accumulate higher levels of SA compared to those in
untreated plants (Cho et al. 2008).

8.3 Airborne Chemicals Mediate Communication

Between Bacteria

Many bacterial species coexist in dynamic communities where they compete or

cooperate with one another (see Chap. 43). Intercellular signaling systems enable
bacteria to adapt to biotic and abiotic stresses. The most well-known phenomenon
is quorum sensing (QS), which is mediated by a complex regulatory network (see
Chap. 7). Many bacteria release a wide variety of bioactive VOCs that diffuse through
heterogeneous mixtures of solids, liquids, and gasses (Kai et al. 2009). The mech-
anisms underlying VOC-mediated bacterial communication systems remain largely
unknown.
Let’s Speak with Odors Although many bacteria produce VOCs, little is known
about their physiological functions. Several recent reports indicate that bacteria are
responsive to ammonia, which is a volatile organic compound. Ammonia is a valuable
nutrient for the production of proteins and nucleic acids. The Burgess group recently
showed that the soil bacterium Bacillus licheniformis was sensitive to the presence of
ammonium sulfate (Nijland and Burgess 2010), which was metabolized, converted
to ammonia, and induced biofilm formation. The mechanisms underlying VOC-
mediated bacterial communication are not known. Bernier et al. (2011) showed that
ammonia released from E. coli influences antibiotic resistance in aerially-exposed
Gram-positive and -negative bacteria. These authors discovered a previously unchar-
acterized antibiotic resistance mechanism, and showed that this contributes to the
induced expression of genes involved in ammonia-polyamine metabolism during the
stationary growth phase. This was the first evidence for a mechanism underlying
VOC-modified gene expression in interacting bacteria. The same group recently re-
ported that exposure to trimethylamine, a volatile compound produced by E. coli
and other Gram-negative bacteria, altered antibiotic resistance in all tested bacteria.
The study revealed that exposure to trimethylamine elevated the growth-medium pH,
which modified antibiotic uptake and transiently altered antibiotic resistance.
E. coli and Bacillus subtilis were used as representative models to study the roles of
VOCs in Gram-negative and Gram-positive species, respectively (Kim et al. 2013).
Microarray analysis was used to examine gene expression in E. coli exposed to
B. subtilis VOCs. The results showed that VOCs dramatically affected bacterial
phenotypes in receiver cells and altered the expression of more than one hundred
genes. Changes in global gene expression related to motility and antibiotic resistance
phenotypes were observed after treatment with 2,3-butanedione and glyoxylic acid
58 C.-M. Ryu

from nanomolar to micromolar concentrations. VOC-mediated interactions were

modulated by the previously uncharacterized ypdB gene product and its downstream
transcription factors soxS, rpoS, and yjhU. These results strongly suggest that VOCs
represent an interspecific signal for rapid sensing between bacteria, which can trigger
variations in gene expression. VOC-mediated bacterial communication may induce
modifications that affect defense and locomotion responses of physically separated
bacteria (Fig. 8.1).

8.4 Volatile Compounds for Agricultural Applications:

From Lab to Field

Many microbially produced VOCs function as signals that mediate interactions be-
tween microbes and plants or between different microbes. There have been few
studies of field applications of VOCs. Treatment of aerial plant parts with the highly
bioactive and inexpensive 2,3-butanediol to promote growth, induce ISR, and con-
fer tolerance to drought and salinity offers a promising agricultural application. A
selection of novel strategies utilizing bacterial VOCs is discussed.
Working in the Open Field? Although volatile compounds rapidly dissipate un-
der natural conditions, VOCs may be useful for practical applications. Many VOCs
(including 2,3-butanediol) are water soluble, inexpensive (< US $/kg), function at
extremely low concentrations (ng/ml to pg/ml), and are not overtly toxic to animals
and humans. Under growth-chamber conditions, drenching roots with acetoin re-
duced the pathogen population on leaves (Rudrapa et al. 2010). Therefore, bacterial
VOCs are promising candidates for triggering plant systemic defense and improving
disease control. The primary requirement for field trials of bacterial VOCs is to de-
velop an appropriate treatment protocol. Under natural conditions, bacteria emit the
volatiles at a very low, steady level. This process is difficult to mimic, and the rapid
and nonuniform evaporation of bacterial VOCs after application in an open field can
cause inconsistent results.
Drench application of a bacterial volatile, 2-butanone, to pepper and cucumber
plants has been successfully performed for four consecutive years under field con-
ditions (Song and Ryu 2013). In another field trial, 4-week-old pepper plants were
dip-treated with 1 mM 2-butanone before they were transplanted into the field. This
successfully protected pepper crops against bacterial spot during a 2-year field trial
(Fig. 7.2). Similarly drench treatment of cucumber with 2-butanone up-regulated the
defense-related gene CsLOX, reduced the aphid (Myzus persicae) population, and
increased the ladybird beetle population (the natural enemy of aphid). These results
suggest that VOC-mediated induction of the oxylipin pathway, related to produce
airborne signal molecules methyl jasmonate and green leaf volatiles, can help recruit
a natural aphid enemy and may ultimately prevent plant disease and insect damage by
eliciting induced resistance, even under open-field conditions (Song and Ryu 2013).
Whether PGPR volatiles function to directly attract natural herbivore enemies, or
8 Bacterial Volatiles as Airborne Signals for Plants and Bacteria 59

Fig. 8.2 Bacterial VOC-elicited induced resistance against naturally occurring bacterial spot caused
by Xanthomoans vesicatoria and Cucumber mosaic virus in a pepper field. The picture was taken at
3 month after drench application of a bacterial volatile, 2-butanone when the pepper seedlings were
transplanted. The upper and bottom rows indicate 2-butanone and water treatments respectively.
The three plants in the first row show severe stunting by Cucumber mosaic virus infection. The
white arrows indicate the same height of the two rows. The left and right plants on the first row
demonstrate the beginning of chlorosis that is a typical symptom of bacterial spot caused by X.
vesicatoria. The red arrows indicate the symptoms of bacterial spot: necrosis and chlorosis. The
empty hole in the bottom row is caused by the removal of a plant because of infection by the late
blight pathogen, Phytophthora capsici

whether bacterial VOCs induce volatile emissions from plants (Farag and Pare 2002;
Arimura et al 2000) that function to attract herbivore enemies, has yet to be
determined (Fig. 8.2).

8.5 Perspectives

The experiments in 2003 (Ryu et al. 2003), 2004 (Ryu et al. 2004), and 2013
(Kim et al. 2013) presented a new paradigm for interactions between bacteria and
plants and among different bacteria. These studies revealed that bacterial VOCs can
act as signal molecules that trigger plant growth and immunity. A detailed analysis
of cellular and molecular mechanisms involved in plant and bacterial responses to
VOCs is required. Potential agricultural applications of bacterial VOCs for promot-
ing biotic and abiotic resistance in crop plants will be an important topic of future
studies. The following topics remain to be addressed: (i) The mechanism of plant
60 C.-M. Ryu

perception of bacterial VOCs is unknown. It is not known how plants differentiate

between similar C4-alcohols such as 2,3-butanediol and acetoin. (ii) It is unknown
whether plants possess a signaling pathway for bacterial VOCs that is distinct from
known plant defense signaling pathways. (iii) It is unknown whether plant sensors for
bacterial VOCs are tissue-specific or organ-specific. (iv) It is unknown how a single
bacterial volatile compound promotes plant growth and elicits ISR. Theoretically,
ISR occurs at the cost of plant fitness and may result in a growth penalty. (v) Future
work will determine if bacterial volatiles can trigger the emission of plant green leaf
volatiles.

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 Part II
Phytopathogens and Pest Insects
Chapter 9
Phytopathogenic Bacteria

Jan van der Wolf and Solke H. De Boer

Abstract A few hundred bacterial species, belonging to the Proteobacteria, Molle-

cutes and Actinomycetes cause a large number of different plant diseases, some of
which are devastating for agricultural crops. Symptoms of bacterial plant diseases are
diverse and include necrosis, tissue maceration, wilting, and hyperplasia. For suc-
cessful infection to occur, the pathogen must overcome plant defense mechanisms,
which it often does by injecting effector molecules directly into plant cells to suppress
a host response. Virulence may also involve production of plant cell wall-degrading
enzymes, toxins and/or plant hormones often under control of quorum sensing mech-
anisms. Some phytopathogenic bacteria actively move to their host via chemotaxis
and enter the plant through natural openings such as stomata and lenticels or wounds
caused by insect feeding, fungal infection, or mechanical plant damage. Host plants
are internally colonized locally through intercellular spaces and systemically via the
vascular system. Control of bacterial plant diseases is achieved mainly by prevention
and exclusion of the pathogen since there are few effective chemical control agents
and sources of resistance against bacterial diseases are limited.

9.1 Introduction

While estimates of the number of bacterial species on earth vary widely from tens of
thousands to billions (Schloss and Handelsman 2004), only a few hundred species
cause significant damage to agricultural crops (Kado 2010). Phytopathogenic bacte-
ria are a major threat to crop production due to (a) the lack of suitable agrochemicals
for their control, (b) the absence of resistance or immunity in host plants, and (c)
their inadvertent and undetected spread as contaminants or asymptomatic (latent)

J. van der Wolf ()

Plant Research International, PO BOX 69, 6700 AB Wageningen, The Netherlands
Tel.: +31.317.480598
e-mail: Jan.vanderWolf@wur.nl
S. H. De Boer
Emeritus Scientist, 29 Donald Drive, Charlottetown, PE C1E 1Z5, Canada
e-mail: Solkedb@gmail.com

© Springer International Publishing Switzerland 2015 65

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_9
66 J. van der Wolf and S. H. De Boer

infections in plant propagation materials. It is therefore not surprising that a rela-

tively large number of phytopathogenic bacteria are quarantine organisms. In this
chapter the taxonomy, host range, symptomatology and virulence factors are briefly
discussed after which the fate of bacterial pathogens is followed from the first con-
tact with their hosts to their dissemination and survival. The chapter closes with a
description of disease management strategies in which exclusion and prevention play
dominant roles.

9.2 Taxonomy, Host Range and Symptomatology

Plant disease-causing bacteria are a highly diverse group of prokaryotes. Although

they are classified into many different taxa, the majority of them are found in the
phylum Proteobacteria. A complete list of names of phytopathogenic bacteria has
been published recently (Bull et al. 2010) as well as a general reference book on
plant bacteriology (Kado 2010).
The current transition from phenotypic to genotypic methods for classification
is expected to stabilize the taxonomy of plant pathogenic bacteria, which has been
in flux for some time. In particular, the development of low-cost, next generation
sequencing techniques enabling whole genome analysis will result in more accurate
species delineation and the inclusion of virulence determinants as taxonomic criteria.
Some highly specialized bacterial species are pathogenic on only a single crop
as illustrated by Clavibacter michiganensis subsp. sepedonicus, the causal agent
of the potato bacterial ring rot disease. Other bacterial species are polyphagous and
cause disease on multiple host plants as exemplified by Ralstonia solanacearum÷ the
causal agent of wilting diseases in various monocot and dicot crop plants. There are
also a few phytopathogenic species that are so-called “kingdom crossers” which cause
diseases in both plants and animals. Dickeya dadantii causes soft rot of vegetables
and ornamental plants but is also an entomopathogen being highly virulent for pea
aphid; Serratia marcescens causes onion bulb rot and clinical sepsis in humans.
Bacterial plant diseases occur in all climatic zones, but tend to be less prevalent in arid
climates because moisture is important for disease development and dissemination
of the pathogens.
Infection of plants by phytopathogenic bacteria often, but not always, results in
multiple symptoms of disease (Fig. 9.1) (Kado 2010). When seed, bulbs, tubers or
rootstocks are infected, plants developing from them may succumb to disease before
even emerging as sometimes occurs with Clavibacter michiganensis subsp. michi-
ganensis in tomato and Pectobacterium spp. in potato. Many of the phytopathogenic
Xanthomonas and Pseudomonas spp. initially cause water-soaked lesions in in-
fected plant parts followed by necrosis and chlorosis of the tissues. Dieback of tree
fruit branches, usually preceded by necroses of leaves, is caused by such bacteria
as Erwinia amylovora and Pseudomonas syringae pv. morsprunorum. Rhizobium
rhizogenes, Rhodococcus fascians and Clavibacter michiganensis subsp. michiga-
nensis form cankers in their hosts, while Streptomyces spp. causes hyperplasia in the
9 Phytopathogenic Bacteria 67

Fig. 9.1 Examples of symptoms caused by plant pathogenic bacteria. a Chlorosis and necrosis
on geranium leaves caused by Xanthomonas hortorum pv. pelargonii, b blackrot caused by Xan-
thomonas campestris pv. campestris on cauliflower leaves, characterized by V-shaped chlorotic
and nectrotic lesions and black veins, c leaf cracking caused by Curtobacterium flaccumfaciens
pv. oortii in tulip, d tumors caused by Agrobacterium tumefaciens on chrysanthemum stems, e
wilting of potato plants caused by Pectobacterium atrosepticum, f potato tuber maceration caused
by Dickeya solani. Figures 9.1a, b, d and e are from the collection of Jan van der Wolf. Figure 9.1c
is a gift from Khanh Pham, PPO, Lisse, the Netherlands (unpublished). Figure 9.1f was reproduced
from John Elphinstone, FERA, UK Crown copyright, by permission of the publisher

periderm of underground plant structures. Dickeya, Pectobacterium, Burkholderia,

Serratia and Ralstonia spp. all cause soft rot in which plant tissue is macerated by the
activity of microbial pectinolytic enzymes. Disease symptoms caused by phytoplas-
mas and spiroplasmas include stunting, phyllody (retrograde development of floral
organs), witches’ broom (development of multiple stalks), virescence (greening) and
bolting (growth of elongated stalks) (Hogenhout and Loria 2008). Infection by some
plant pathogenic bacteria such as Erwinia amylovora and Ralstonia solanacearum
are characterized by oozing of bacterial slime from infection loci. Asymptomatic
infections, while usually not causing direct disease losses, can still result in crop loss
when detection of a quarantine pathogen dictates severe statutory measures including
crop destruction.

9.3 Virulence Factors and Plant Defense

To successfully invade host plants, phytopathogenic bacteria must cope with a num-
ber of plant defense mechanism and have a means for acquiring water and nutrients
for growth and colonization of plant tissues. Hence plant disease-causing bacteria
display a broad variety of virulence factors such as cell-wall degrading enzymes,
toxins, plant hormones, and effectors to overcome plant defense responses. Viru-
lence factors are often associated with transposable elements or genomic islands and
may be acquired from other microbes via lateral gene transfer which usually involve
68 J. van der Wolf and S. H. De Boer

Fig. 9.2 Schematic representation of the interaction between a bacterial pathogen and a plant cell.
Molecules are released from the pathogen into the host cell by different types of secretion systems
(T1SS—T6SS). These molecules may be effectors that suppress plant defense mechanisms, but
also cell wall degrading enzymes, toxins or phytohormones. Various bacterial components such as
lipopolysaccharides and flagellar proteins, act as pathogen associated molecular patterns (PAMPS)
that are recognized by cell surface pattern recognition receptors (PRRs) of the host, and elicit PAMP
triggered immunity (PTI), which involve such responses as the production of reactive oxygen
species (ROS), callose deposition, cell thickening etc. To suppress PTI, many plant pathogens
release effector molecules. However, in some pathosystems effectors are recognized by nucleotide-
binding leucine rich repeats (NB-LRR) of the host inducing effector triggered immunity (ETI). Some
pathogens can also detoxify ROS. Figure from author Jan van der Wolf

mobile genetic elements, such as plasmids, bacteriophages and other integrative and
conjugative elements. Major virulence factors will be discussed in the following and
are indicated in bold.
Secretion Systems Phytopathogenic bacteria deliver pathogenicity factors directly
into plant cells by various secretion systems (Chap. 6; Kado 2010) (Fig. 9.2). The
type 1 secretion system (T1SS) is found in many proteobacteria and utilizes an
ABC transporter cassette for ATP driven transmembrane delivery of biologically
active molecules. It is involved in the translocation of metalloproteases, e.g. in
Dickeya and Pectobacterium spp., and of lipases, sterols, toxins and drugs in other
phytopathogenic bacteria. The type 2 secretion system (T2SS) is also found in
9 Phytopathogenic Bacteria 69

many Gram-negative phytopathogenic bacteria. In T2SS, proteins sequentially pass

through the cytoplasmic membrane and, after modification, through a pore in the
outer membrane. This system is involved in the secretion of cell wall degrading en-
zymes by Pectobacterium sp., levansucrase by Erwinia amylovora, and a cysteine
protease by Xanthomonas oryzae pv. oryzae. A functional type III secretion system
(T3SS) is required for virulence of many important bacterial pathogens including
Pseudomonas syringae, Xanthomonas campestris, Ralstonia solanacearum and Er-
winia amylovora (Rosenberg et al. 2013). The expression of T3SS, which involves the
injection of biologically active effector molecules into host cells through a pilus-like
structure, is regulated by environmental factors such as pH and the balance between
concentrations of calcium and magnesium ions. Effectors transported by T3SS gen-
erally have host defense-suppressing proteolytic activity as found in Pseudomonas
syringae and Xanthomonas campestris, elicit water and nutrients from host cells into
apoplasts, and modulate plant hormone physiology as evidenced by auxin regulation
in Xanthomonas campestris. The chromosome or plasmid-encoded type 4 secretion
system (T4SS) in which a pilus also forms an integral part of the secretion system,
is yet another system by which various plant pathogenic bacteria transfer effectors,
nucleoproteins and nucleic acids into host cells. Agrobacterium tumefaciens, for ex-
ample, incites the crown gall disease by transferring its T-DNA into host cells via
T4SS (Chap. 37). The type 5 secretion system (T5SS) is a so-called autotransporter
protein system and is found in several plant pathogens including Xylella fastidiosa,
Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. vesicatoria, and Dick-
eya spp., where it plays a role in the adherence of the bacteria to surfaces of host
tissues. Finally, the type 6 secretion system (T6SS), reminiscent of the injection
machinery of phages, occurs in at least thirteen species of plant pathogenic bacteria.
It appears to be involved in virulence, immunity responses, and possibly host speci-
ficity of Pectobacterium atrosepticum, Pseudomonas syringae and Agrobacterium
tumefaciens (Records 2011).
PAMPS, DAMPS and Effectors Pathogen-associated molecular patterns (PAMPS)
describe the immune response of host plants to bacterial infections that result in a
basal level of resistance. PAMPS are triggered by conserved microbial molecules
such as lipopolysaccharides, flagellar proteins, peptidoglycans, elongation factor
peptides, activators of XA21-mediated immunity, double-stranded DNA, methylated
DNA, and cell wall pieces (Li et al. 2013; Zeng et al. 2010) (Fig. 9.1). PAMP triggers
are perceived by their cognate pattern-recognition receptors and their role in defence
against infection is regulated by specific signal molecules that include ethylene,
jasmonic acid and salicylic acid. Resulting defence mechanisms involve callose
deposition, cell-wall thickening, stomata closure, and production of antimicrobial
compounds and reactive oxygen species (ROS). Some pathogens are equipped with
mechanisms to detoxify antimicrobial compounds and ROS. For example catalase
and peroxidases are produced by Xanthomonas campestris pv. campestris and alkyl
hydroperoxidase reductases are produced by Xanthomonas campestris pv. phaseoli
to neutralize cell-damaging ROS. Damage-associated molecular patterns (DAMPS)
are similar to PAMPS and are triggered by small endogenous peptides or cell wall
70 J. van der Wolf and S. H. De Boer

fragments that are released from damaged or stressed cells and serve as signals to
activate the immune response in infected host plants (Li et al. 2013).
A second layer of defence against phytopathogenic bacteria is effector-triggered
immunity (ETI). All major groups of phytopathogenic bacteria studied to date pro-
duce effector proteins. Specific effector proteins, expressed by avirulence (Avr)
genes, are injected into host cells via T3SS to suppress plant defences. In incompat-
ible interactions, which are often host cultivar specific, the effectors are recognized
by the product of corresponding resistance (R) genes and induce defence responses
such as the hypersensitive response (HR). In some cases, however, the effector rec-
ognized by R gene products induces modifications to the effector mediated cascade
so that instead of activating ETI, it suppresses it and results in effector-triggered
susceptibility (ETS).
Quorum Sensing Many pathogenic bacteria use quorum sensing, a cell-to-cell com-
munication mechanism, which allow them to respond to population density and delay
production of virulence factors until a critical cell density has been achieved (Chap. 7;
Venturi and Fuqua 2013). This mechanism postpones production of virulence factors,
such as plant cell wall-degrading enzymes by soft rot Enterobacteriaceae and the
transfer of the Ti-plasmid by Agrobacterium tumefaciens, until bacterial cell numbers
are high enough to overwhelm the plant’s defense response. Typically, these systems
consists of a synthase enzyme belonging to the LuxI family and is responsible for the
production of acyl homoserine lactone (AHL) signal molecules and a transcription
factor of the LuxR family, which activates gene expression but can also be involved
in gene repression. The luxR protein consists of an autoinducer-binding and a DNA-
binding domain separated by a linker. Most LuxR proteins are inactive in the absence
of the AHLs but some are inactivated in the presence of AHLs. Also LuxR solo sys-
tems have been described that respond to AHLs produced by other bacteria or by
plant compounds. For instance the genes involved in the motility of Xanthomonas
oryzae pv. oryzae are regulated during infection by host plant molecular activators.
Conversely, AHLs can also influence expression of plant genes including those in-
volved in plant defense mechanisms, stress responses and hormones as was found
in Medicago truncatula and Arabidopsis thaliana. Moreover, these plants as well
as rice and pea, produce compounds mimicking AHLs and thereby stimulate bac-
terial quorum sensing gene expression. These observations are indeed indicative of
complex interkingdom communication.
Lipopolysaccharides The outer membrane of Gram-negative bacteria contains
lipopolysacchides (LPS) that play an important role in their interaction with the
environment and contact with the host (Newman et al. 2001). Some LPS mutants are
also more sensitive to microbial compounds such as antibiotics, detergents, and an-
timicrobial peptides, possibly because the ability of their outer membrane to exclude
them is impaired. An LPS mutant of Ralstonia solanacearum, for example, was un-
able to induce the hypersensitive response in its host and a mutant of Xanthomonas
oryzae pv. oryzicola, causal agent of bacterial leaf streak of rice, was affected in the
T3SS-mediated delivery of effector proteins.
9 Phytopathogenic Bacteria 71

Extracellular Polysaccharides (EPS) EPS can be either closely or loosely attached

to the bacterial cell and serve different functions (Rosenberg et al. 2013). In Ralsto-
nia solanacearum EPS is involved in the absorption of water, minerals, and nutrients
whereas in Xanthomonas campestris EPS plays a role in adhesion and biofilm forma-
tion, protecting the pathogen against biotic and abiotic stresses such as desiccation.
In Erwinia amylovora , EPS suppresses host recognition of bacterial colonization of
plant tissues.
Phytotoxins Phytotoxins that cause direct damage to plant cells or modulate the
metabolism and physiology of the host are produced by many plant pathogenic bac-
teria. One example is the production of taxtomin, a cyclic dipeptide that induces scab
lesions, by Streptomyces spp. (Hogenhout and Loria 2008). The two lipodepsipeptide
phytotoxins produced by Pseudomonas syringae pathovars, syringomycin and sy-
ringopeptin, induce necrosis by inserting into plant cell plasma membranes, thereby
disrupting the membrane electrical potential (Rosenberg et al. 2013). These toxins
also act as biosurfactants that enhance bacterial dispersion. The modified peptides
tabtoxin and phaseolotoxin produced by other pseudomonads cause severe yellowing,
possibly by inhibiting photosynthesis. Another pseudomonad phytoxin, coronatine,
is a polyketide that mimics the plant hormones jasmonic acid and ethylene, upset-
ting plant defense mechanisms. Coronatine also promotes lesion formation, induces
chlorosis, enhances pathogen multiplication, and inhibits root growth.
Plant Cell Wall Degrading Enzymes (PCWDE) PCDWE are important viru-
lence factors of both Gram-positive and Gram-negative bacteria. Plant cell walls
are composed of carbohydrate polymers in which pectins, hemicelluloses and cel-
luloses are most abundant, but they also contain proteins and lignins. Plant cell
wall degradation by invading pathogens allows the bacteria to spread in infected
tissues, and makes water and nutrients available. Soft rot Enterobacteriaceae secrete
multiple (iso)enzymes involved in pectin degradation as well as hemicellulases, cel-
lulases and proteases which in concerted action very rapidly macerate host tissues
(Charkowski et al. 2012). Plasmid encoded cellulases are also essential for virulence
of the Gram-positive pathogens Clavibacter michiganensis subsp. sepedonicus and
subsp. michiganensis.
Siderophores There is an important role for siderophores in plant infection for
species of Xanthomonas, Pseudomonas, Erwinia and soft rot Enterobacteriaceae.
Siderophores are ferric iron chelators essential for scavenging iron in iron-limited
environments. For example, Dickeya dadantii produces two siderophores, achro-
mobactin and chrysobactin, that are required for systemic progression of tissue
maceration. Interestingly, chrysobactin not only supports the pathogen in its compe-
tition for iron but also modulates the defence mechanism of its host by interfering
with salicylic and jasmonic acid signalling cascades (Dellagi et al. 2009).
Plant Hormones Several phytopathogenic bacteria produce hormones that enhance
virulence by interfering in the physiology of host plants. Rhodococcus fascians, for
example, produces a mixture of five cytokinins that directly affects plant development
and indirectly stimulates the host to produce auxins and polyamines resulting in gall
72 J. van der Wolf and S. H. De Boer

formation. Such galls serve as specific niches for survival of the bacterial pathogen.
Similarly, Pseudomonas syringae subsp. savastanoi causes hyperplasia in several
tree hosts by the production of indole-3-acetic acid inducing formation of knots that
are typical of the disease. Ethylene production is also a virulence factor for Ralstonia
solanacearum and Pseudomonas syringae.

9.4 Ecology and Epidemiology

Microbial population dynamics is a function of the ability to grow in an environment

where water and nutrients may be limiting, and the ability to compete within a
hostile environment. Competitive factors include such characteristics as possession
of protective pigments and capsular materials, ability to form biofilms, production of
antimicrobial metabolites, and ability to maintain cellular reserves. Plant pathogenic
bacteria only cause disease if a suitable host plant is present and environmental
conditions are conducive to infection.
The First Contact The first step in pathogenesis is host recognition. Some phy-
topathogenic bacteria are chemotactic in that they are attracted by specific compounds
released by host plants and move to suitable infection sites by swimming, swarm-
ing, or twitching types of motility. Dickeya dadantii, for example, is attracted by
the phytohormone jasmonate, synthesized in plant wounds (Antunez-Lamas et al.
2009). Mutants affected in motility have reduced virulence. Similarly Ralstonia
solanacearum is attracted to amino acids and organic acids in the root exudate of
host tomato plants (Yao and Allen 2006). Some pathogenic bacteria are epiphytic on
leaves and must often survive for considerable periods before conditions favor the
infection process. Survival during periods of dessication is enhanced by osmopro-
tectants such as choline or glycine betaine of either microbial or host origin (Beattie
2011). Xanthomonas species on plant surfaces are protected from lethal ultraviolet
light damage and host-produced photosensitizers by production of xanthomonadins,
membrane-bound yellow pigments (Kado 2010).
Penetration Plant pathogenic bacteria cannot penetrate plant cuticles directly and
require openings to enter into their hosts. Bacteria invade plant tissue through wounds
caused by cultural cropping practices and severe weather or naturally by lateral root
formation, as well as by wounds caused by feeding insects and fungal growth. Other
natural openings such as lenticels, hydathodes and in particular stomata are ma-
jor routes of entry (Zeng et al. 2010). As part of their defense mechanism, some
plants such as tomato, pea and grape close their stomata to prevent entry of bacte-
ria. In response, some phytopathogenic bacteria including Pseudomonas syringae
and Xanthomonas campestris produce compounds as virulence factors that reverse
stomatal closure. Non-motile bacteria such as Clavibacter michiganensis subsp.
sepedonicus, are introduced into susceptible vascular tissues of their potato hosts
through crop management practices. Candidatus Liberibacter spp. and Xylella fas-
tidiosa are among the few plant pathogenic bacteria that are specifically vectored by
9 Phytopathogenic Bacteria 73

insects; host specific psyllids and leafhoppers, respectively, introduce these bacteria
directly into the host. Both Ca. Liberibacter and Xylella spp. lack a T3SS appara-
tus, which is likely unnecessary for pathogenesis because the bacteria are directly
injected into the xylem vessels bypassing many of the plant defense mechanisms.
Some pathovars of Pseudomonas syringae possess unique ice-nucleating proteins
that cause rapid crystallization of water molecules when air temperature approaches
freezing. Frost damage to plants intensified by such ice crystals provides a means
for epiphytically dwelling pathogens to invade host plants and take advantage of
nutrients released from damaged tissue.
Colonization During colonization of host plants, many bacterial pathogens (e.g.
Xanthomonas fragariae and Clavibacter michiganensis subsp. michiganensis) form
biofilms in xylem or phloem vessels. For biofilm formation, attachment to internal
plant surfaces is enhanced by extrapolysaccharides, several types of pili (fimbriae),
non-fimbrial adhesions, and lipopolysaccharides. To move within the plant, bacterial
cells must be released again from biofilms and in some disease scenarios rhamnolipids
with biosurfactant proporties play a role in reestablishing planktonic cells. Within
plants, bacteria often lose their flagella and move passively or by a twitching process
through the xylem vessels.
Dissemination Phytopathogenic bacteria are disseminated via plant propagation
material, including seeds, irrigation and other surface water sources, wind-blown rain
and aerosols, and surface-contaminated harvesting, grading and cultivating equip-
ment and the walls of storages and transportation vehicles. Several phytopathogenic
bacteria are transmitted via insects (Nadarasah and Stavrinides 2011). Flying in-
sects in particular can spread bacterial inoculum over relatively long distances, e.g.
survival times of up to 12 days have been recorded for phytopathogenic bacteria
on aphids. Most phytobacterial pathogens do not multiply within insect carriers but
rather are dispersed by them as non-specific surface contaminants as found for Er-
winia amylovora, the causal agent of the fireblight disease, on honey bees. Fruit
flies and other insects transmit Pectobacterium and Dickeya spp. when attracted by
soft rotted plant tissues and move with contaminated surface parts to adjacent sus-
ceptible plant material. Other phytopathogenic bacteria do, however, multiply in
their insect vectors. For example Xylella fastidiosa and Ca. Liberibacter are plant
pathogenic bacteria that replicate, in the gut and salivary glands of their leafhopper
and psyllid vectors, respectively. In most cases of insect transmission of bacterial
plant pathogens, bacterial cells are introduced directly into wounds generated during
piercing, sucking or chewing activities of the insects.
Survival Generally plant pathogenic bacteria are hemi-biotrophs that do not survive
very well outside their plant hosts. They do survive for various lengths of time as
epiphytes, as root colonizers, in water streams, in plant rhizospheres, attached to soil
particles, or in organic plant debris. There are exceptions, however, because some
phytopathogens, such as Streptomyces spp., are classical soil-dwelling organisms
and do not need plant hosts to survive. For some pathogens, populations of bacteria
build up on perennial host plants or in the soil environment when the same crops
74 J. van der Wolf and S. H. De Boer

are replanted year after year without crop rotation. There are also bacterial plant
pathogens, such as Ralstonia solanacearum that survive well in aquatic environments
and others that survive in association with insect vectors as described above.

9.5 Control

Control of bacterial plant diseases is challenging because few options are available.
There are very few agrichemicals that are effective or practical to use, so control
strategies need to be largely based on avoiding contamination of crop plants with
pathogenic bacteria, or by growing disease-resistant plant cultivars. Initiation of crop
plants from pathogen-tested seed or vegetative plant propagules such as cuttings,
tubers, or bulbs is an effective strategy for avoiding bacterial pathogens. Elimination
of overt inoculum sources by roguing and destruction of diseased and suspect plant
material is another way to minimize disease spread. Cultural practices that minimize
plant damage prevent creating entry points for bacteria, and meticulous sanitation
on farm sites prevents the build-up of inoculum sources. To avoid pathogens, seed
production is done in arid and semi-arid regions which reduces the risk of air-borne
inoculum. Containment of seed production in glasshouses is another strategy that
minimizes airborne pathogens. Planting resistant crop cultivars is effective when
good varieties are available but care must be taken that disease tolerant plants do not
play a role in maintaining and dispersing inoculum sources as latent infections.
Testing of Plant Propagation Material Control strategies that are based on the
planting of material free from bacterial pathogens require a means to propagate such
material and tests to ensure absence from pathogens. Most bacteria can be eliminated
from plants by in vitro tissue culturing from growing tips and be maintained pathogen-
free by axenic culture. Further multiplication of plant propagation material in a
protected environment helps ensure that recontamination is kept to a minimum level.
To ensure that propagation material remains free from pathogens, robust, specific and
sensitive methods need to be applied to test for the possible presence of pathogens.
Reliable serological and DNA-based amplification methods have been developed for
detection of many bacterial plant pathogens. For some assays, sensitivity is enhanced
by sample incubation in selective growth media to increase the population of target
bacteria prior to application of the detection assay.
Cultivation based techniques provide the most definitive evidence for the pres-
ence of plant pathogenic bacteria but is challenging for many of them because
of unavailability of good selective media or poor growth characteristics in the
laboratory. Furthermore, some pathogens may persist in a viable but noncultur-
able (VBNC) state in response to environmental stress (Oliver 2010). The VBNC
state has been described for a number of phytopathogenic bacteria, including
Agrobacterium tumefaciens, Erwinia amylovora, Pseudomonas syringae, Ralstonia
solanacearum, Xanthomonas campestris and Xanthomonas axonopodis pv. citri. The
use of cultivation-independent detection methods for viable cells is therefore favored
if they possess adequate detection sensitivity.
9 Phytopathogenic Bacteria 75

Disease Detection and Diagnosis Many bacterial plant diseases can be identified
on the basis of symptomology although confirmation of the diagnosis by isolation of
the pathogen is usually recommended. Techniques for non-destructive monitoring of
bacterial plant diseases during crop production and post-harvest have been developed,
although so far they have not been used widely in practice (Sankaran et al. 2010).
Such methods include spectroscopic and imaging techniques, including fluorescence
and spectral imaging, infrared-, fluorescence-, multiband- and nuclear magnetic
resonance spectroscopy. Fluorescence spectroscopy has been used specifically to
detect Xanthomonas axonopodis pv. citri and GC-MS analysis has been used to
detect volatiles in onion bulbs infected with Pectobacterium carotovorum subsp.
carotovorum. Such indirect techniques detect the response of plants to infections
rather than the pathogen. Consequently, latent infections cannot be detected and in
particular the spectroscopic and imaging techniques cannot always distinguish the
response of plants to a pathogen from that caused by other biotic or abiotic stress
factors.
Chemical and Biocontrol Treatments In some cases, chemical treatments or bio-
control agents can decrease populations of phytopathogenic bacteria in and on plant
material to reduce damage and yield loss. The efficacy of chemical treatments, in-
cluding antibiotics, copper compounds and general biocides—such as peroxides and
chlorine—are limited. However in some pathosystems acceptable control levels can
be achieved with streptomycin or copper sprays, such as for control of fireblight
caused by Erwinia amylovora. However, with the high mutation rate of bacteria,
typically one per million bacterial cells, development and selection of resistant mu-
tants is a considerable problem for chemical control strategies. True seed, but also
bulbs and tubers, can be treated with warm water, aerated steam or hot air to reduce
populations of bacterial pathogens associated with such propagation materials. The
temperature window between control and plant damage however, is very small.
The first example of successful biocontrol of a phytobacterial disease is the use of
Rhizobium rhizogenes K84 against Agrobacterium tumefaciens, the causative agent
of crown gall. Selective control of Agrobacterium tumefaciens occurs because its
Ti plasmid codes for production of agrocinopine permease that is inserted into its
cell membrane for uptake of nutrients, as well as plasmid-induced agrocinopines
produced by the host plant. The agrocinopine permease also allows specific uptake
of agrocin produced by the biocontrol agent; agrocin, a nucleotide analogue, blocks
DNA synthesis in the pathogen. Another biocontrol agent is Pantoea herbicola used
to control fireblight caused by Erwinia amylovora. Phage therapy has been applied
commercially for control of the foliar tomato pathogens Xanthomonas vesicatoria
and Pseudomonas syringae pv. tomato but the rapid development of resistance against
the phages requires a constant selection of new hypervirulent strains.
Resistance Breeding Knowledge of molecular plant-pathogen interactions, such as
pattern and effector—triggered immune systems (PTI and ETI) is useful in resistance
breeding, often via marker-assisted selection. One typical example is how knowledge
of the receptor-like kinase involved in PTI signaling controlled by the rice disease
resistance gene Xa21 was exploited for developing broad spectrum resistance against
76 J. van der Wolf and S. H. De Boer

Xanthomonas oryzae pv. oryzae, causative agent of bacterial blight (Zhang et al.
2013). ETI, particularly the NBS-LRR R genes that contain a nucleotide binding
site and a leucine rich repeat region are also useful targets for resistance breeding.
To avoid pathogens overcoming ETI-based resistance, a strategy is used by which
several R genes, each recognizing a specific range of pathogen strains, are stacked.
Disease Resistant Transgenic Crops Development of transgenic disease resistant
crops is controversial because of consumers’ concern and skepticism (Chaps. 15 and
16; Collinge et al. 2010). Currently no GM crops with resistance to bacterial diseases
are grown commercially but release of such crops is imminent. Recently, in the US,
127 applications representing 12 % of those submitted for field testing, concerned
transgenic crops with resistance against bacterial pathogens. In most cases, resis-
tance was achieved by insertion of genes for antimicrobial proteins or metabolites.
For example, transgenic potatoes were developed with resistance to Pectobacterium
carotovorum using a synthetic antimicrobial magainin peptide originating from the
amphibian Xenopus. Other traits introduced to achieve bacterial disease resistance
include enzymes involved in plant resistance such as protein kinases, transcription
factor proteins, and disruptors of quorum sensing. The use of cisgenic crops in
which recipient plants are modified with homologous R-genes from the same or
related species may be more acceptable to consumers and opponents of GM crops.

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bacteria, 1980–2007. J Plant Pathol 92:551–592
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current trends and future prospects. Annu Rev Phytopathol 48:269–291
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Chapter 10
Plant Pathogenic Fungi and Oomycetes

Pierre J. G. M. de Wit

Abstract Fungi and Oomycetes are notorious plant pathogens and use similar strate-
gies to infect plants. The majority of plants, however, is not infected by pathogens as
they recognize pathogen-associated molecular patterns (PAMPs) by pattern recogni-
tion receptors that mediate PAMP-triggered immunity (PTI), a basal defense response
effective against potential pathogens. Successful pathogens secrete effectors to sup-
press PTI and alter host plant physiology. In turn, plants have evolved immune
receptors that recognize effectors, resulting in effector-triggered immunity (ETI).
ETI includes the hypersensitive response which is effective against biotrophic plant
pathogens that require living cells to feed on. Other pathogens are hemi-biotrophic,
which start infection as a biotroph, but after having colonized the host tissue can also
feed on death tissue. Necrotrophic pathogens kill host tissue before they start to feed
on it. Co-evolution between pathogens and their hosts had led to the development of
numerous effectors produced by pathogens and corresponding resistance proteins in
host plants, which has generated an arms race genetically described by the gene-for-
gene concept. Resistance genes can now successfully be transferred to crop plants
by classical breeding or as transgenes stapled into one cultivar.

10.1 Fungal and Oomycetous Pathogens and Their Life Styles

Fungi and Oomycetes are uni- or multicellular eukaryotic heterotrophic organisms

producing filamentous structures, designated hyphae, that show tip growth, except
for yeasts that multiply by budding. Hyphae are usually 1–2 μm in diameter, but may
reach more than 100 μm for some fungi. Cells of hyphae contain one, two or multiple
nuclei often divided by septa. Until the 1990s, Oomycetes were considered true
fungi, but based on genome analyses they are now classified among the Chromista
(Chap. 39). Pathogenic Oomycetes are still treated as fungi because they have many
properties in common, including their filamentous growth and their mode of plant

P. J. G. M. de Wit ()
Laboratory of Phytopathology, Wageningen University,
Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
Tel.: + 31 317 48 31 30
e-mail: Pierre.dewit@wur.nl

© Springer International Publishing Switzerland 2015 79

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_10
80 P. J. G. M. de Wit

infection. Fungal cell walls contain chitin, α- and β- glucans and (glyco)proteins, but
no cellulose, while those of Oomycetes contain cellulose and glucans but lack chitin.
Several fungi and Oomycetes are important for industrial production of enzymes,
bread, cheese, alcohol and organic acids. As producers of antibiotics they can have
important medical applications. On the other hand, many fungi and Oomycetes can
cause disease to humans, animals and plants. Around 150,000 fungal species have
now been described, which represent around 10 % all fungi estimated to be present
on earth (Hawksworth 1991). The most important fungal plant pathogens belong to
(i) Ascomycetes, producing sexual spores (ascospores) in a sac-like structure, the
ascus, and asexual spores (conidia), (ii) Basidiomycetes producing sexual spores
(basidiospores) on a basidium, dikaryotic vegetative mycelium and asexual spores,
(iii) Oomycetes producing sexual spores (oospores) and asexual spores (sporangia
that can germinate directly or produce zoospores) (Agrios 2005). In the remainder of
this chapter I will jointly discuss fungi and Oomycetes as fungi, unless phenomena
are specific to Oomycetes only. Fungi are one of the largest living organisms; the soil-
borne fungus Armillaria ostoyae is estimated to be 2400 years old, covering 8.4 km2
of soil in Oregon, USA (Burdsall and Volk 2008). Pathogenic fungi enter plants via
natural openings (e.g. stomata) or penetrate directly by a penetration peg produced
by an appressorium on a host cell (see Chap. 25). Many fungi produce haustoria
in plant cells, specialized feeding organs for retrieval of nutrients. Extracellular
pathogens grow as epiphytes on the outside of plants or in the apoplastic space
between cells without producing haustoria. Ascomytous fungi are haploid for the
major part of their life and produce haploid spores (1N). During the sexual stage
haploid hyphae ofAscomycetes may fuse to produce a dikaryon inside the ascogenous
hyphae where the nuclei soon fuse to produce a zygote (2N) that quickly divides
meiotically to produce eight haploid ascospores per ascus. Oomycetes are diploid
for the major part of their life and have a life cycle that is very similar to that of algae.
During the sexual stage Oomycetes produce gametangia in which meiosis occurs,
followed by fertilization and production of the diploid zygote, the oospore, that
produces a sporangium that geminates directly or indirectly by producing zoospores.
Basidiomycetes are dikaryotic (N + N) for the major part of their life. During the
sexual stage in the basidium the paired nuclei fuse and form a zygote (2N) that
quickly divides meiotically to produce four haploid basidiospores (Agrios 2005). In
most fungi the asexual cycle repeats multiple times during the growth season and
is most damaging to plants, whereas the sexual cycle usually occurs only once a
year at the end of the growth season when host plants senesce and nutrients become
limiting.
Diseases Caused by Oomycetes The most important oomycetous plant pathogens
comprising species belonging to the genera Pythium, Phytophthora, Peronospora and
Plasmopara. The late blight disease of potato is an important oomycetous pathogen
discussed in Chap. 39. Downy mildew of grape is another important disease caused
by Plasmopara viticola (Fig. 10.1a), that almost completely destroyed the grape and
wine industry in France soon after it was imported into Europe from the United States
around 1875; the first fungicide, Bordeaux mixture, effective against P. viticola was
10 Plant Pathogenic Fungi and Oomycetes 81

Fig. 10.1 Symptoms of economically important plant diseases caused by fungi and Oomycetes.
a Plasmopara viticola, downy mildew of grape (http://www.biolib.cz/cz/image/id100651/).
b Blumeria graminis f.sp. tritici, powdery mildew of wheat (http://www.apsnet.org/
publications/imageresources/Pages/fi00189.aspx). c Nectria galligena, apple canker (http://www.
downgardenservices.org.uk/cankerapp.htm). d Venturia inaequalis, apple scab (http://www.
nature.com/news/us-regulation-misses-some-gm-crops-1.13580). e Ophiostoma ulmi, Dutch elm
disease (http://commons.wikimedia.org/wiki/File:Ceratocystis_ulmi_1_beentree.jpg). f Melamp-
sora lini, flax rust (http://www.sciencearchive.org.au/events/frontiers/frontiers2008/dodds.html).
g Puccinia graminis f.sp. tritici, wheat leaf rust (http://www.mississippi-crops.com/2012/
03/02/wheat-leaf-rust-and-stripe-rust-update-march-2-2012/). h Ustilago tritici, loose smut of
wheat (http://www.bayercropscience.cl/soluciones/fichaproblema.asp?id=27). i Ustilago may-
dis, corn smut (http://commons.wikimedia.org/wiki/File:Maisbrand,_Maisbeulenbrand_(Usti-
lago_maydis)_-_hms(1).jpg)

discovered by accident in 1885. Downy mildews are obligate biotrophic pathogens

that overwinter as oospores. Many resistance genes against downy mildews have
been cloned (Takken and Goverse 2012). They belong to the cytoplasmic nucleotide
binding, leucine-rich repeat protein receptors (NB-LRRs) (discussed later).
Diseases Caused by Ascomycetes Powdery mildews can infect virtually all plant
species and are easy recognizable by the white powdery overlay on infected plant
leaves (Fig. 10.1b). Powdery mildews are obligate biotrophic pathogens that can
only infect epidermis cells from which they retrieve nutrients by multiple haustoria.
Short conidiophores are produced on the plant surface producing chains of conidia.
82 P. J. G. M. de Wit

At the end of the growth season the fungus may produce cleistothecia with asci and
ascospores. Many resistance genes against powdery mildews have been cloned. They
belong to the cytoplasmic nucleotide binding, NB-LRRs (Takken and Goverse 2012)
(discussed later).
Tree Cankers These are caused by ascomycetous fungi. Cankers generally begin
at a wound from which they expand in all directions, but the host may survive the
disease by producing callus tissue around the dead areas thereby limiting the canker.
In subsequent years the fungus invades additional healthy tissue, and new concentric
ridges of callus tissue are produced every year, resulting in a typical canker. Canker of
apple is caused by Nectria galligena (Fig. 10.1c), one of the most important diseases
of apple worldwide. Apple scab is caused by Venturia inaequalis (Fig. 10.1d), and
occurs in areas with cool, moist springs and summers. Infected fruits develop scab
lesions and the cuticle is ruptured at the margin of these lesions. The mycelium in
living tissues is located between the cuticle and epidermal cells, and produces short
conidiophores bearing conidia. The fungus is a hemi-biotroph starting infection as
a biotroph, but at the end of the growth season, the mycelium grows through dead
leaf tissues and produces pseudothecia with asci and ascospores. In spring, when
pseudothecia become thoroughly wet, the asci forcibly discharge the ascospores that
infect young apple leaves. Apple varieties resistant against apple scab exist, and the
resistance genes encode typical receptor-like proteins (RLPs) (discussed later).
Vascular Wilts These are widespread, very destructive plant diseases and are caused
by fungal pathogen residing in the xylem vessels of plants that may be clogged
with mycelium, spores, or polysaccharides produced by the fungus and gels and
gums produced by plant cells upon attack by the fungus. In some hosts, tyloses are
produced by parenchyma cells adjoining xylem vessels. The disease can sometimes
be controlled by using disease-resistant cultivars.
Dutch Elm Disease This disease is caused by Ophiostoma ulmi (Fig. 10.1e). It is a
very destructive wilt disease that affects all elm species. Usually trees that become
infected in spring or early summer die quickly, whereas those infected in late summer
are less seriously affected and may recover. Perithecia with asci and ascospores may
be produced. The spread of the Dutch elm disease fungus depends on bark beetles
belonging to the genus Scolytus that carry fungal spores from infected wood to
healthy elm trees. The fungus overwinters in the bark of dead elm trees as mycelium
or spores. Adult female beetles tunnel through the bark and lay eggs that, after
hatching, develop into adult beetles that carry thousands of spores. The beetles feed
on healthy elms and carry fungal spores that infect healthy xylem vessels. Control
of Dutch elm disease depends primarily on removal and destruction of diseased elm
trees. Inoculation of the xylem vessels with a spore suspension of the wilt fungus
Verticillium dahliae offers protection through induced resistance (see Chap. 14),
whereas protection can also be achieved by inoculating trees with particular strains
of Pseudomonas bacteria. Resistance genes against wilt diseases have been cloned;
they encoded either NB-LRRs or RLPs (discussed later).
10 Plant Pathogenic Fungi and Oomycetes 83

Diseases Caused by Basidiomycetes Basidiomycetes produce their sexual spores,

basidiospores, on a club shaped basidium. Most Basidiomycetes are either saprobes
or fungi causing wood decay including root and stem rots of trees. However, they
also include plant pathogens that cause rust and smut diseases.
Rusts Rusts are obligate biotrophic pathogens that have caused many famines in
the history of many countries by destroying cereal crops. Presently the rust strain
Ug99 causes a dramatic epidemic on wheat in eastern Africa from which it has spread
into Arabia (Ward 2007). There are about 5000 species of rusts, mostly belonging
to the genus Puccinia. They are very specialized pathogens attacking only particular
genera or only certain species or even only some varieties of particular cereals. In
the latter case they are called races that can be identified only by a set of differential
varieties carrying different resistance genes. The gene-for-gene system proposed by
Flor in the USA was based on research performed on the rust fungus of flax caused by
Melampsora lini (Fig. 10.1f), (Flor 1971). Rust fungi can produce up to five different
types of spores: uredospores, teliospores and basidiospores (on wheat), spermatia
and aeciospores (on barbary) (Agrios 2005).
Stem Rust of Wheat This disease is caused by Puccinia graminis f.sp. trititici
(Fig. 10.1g), and affects wheat wherever it is grown. It attacks all aboveground parts of
wheat and the alternate host barberry (Berberis vulgaris). Symptoms on wheat appear
as pustules and uredia. The latter produce red-colored uredospores. Later in the
season two-celled black teliospores are produced in telia. On barberry, spermagonia
with spermatia and receptive hyphae are produced on the upper side of leaves, and
on the lower side orange cup-like aecia are produced containing aeciospores. The
most effective means of control of the rust pathogen is by growing resistant wheat
varieties. The resistance proteins are typical NB-LRR receptor proteins (Takken and
Goverse 2012) (discussed later).
Smut Fungi More than 1200 species of smut fungi exist that occur throughout the
world. Most smut fungi attack the ovaries of grains and grasses and develop in the
kernels, which they destroy completely. Some smuts infect seeds or seedlings before
they emerge from the soil, in which they grow systemically until they reach the
inflorescence. Cells in affected tissues are either destroyed and replaced by black
smut spores, or they are first stimulated to divide and enlarge to produce a swelling
or gall that is subsequently destroyed and replaced by the black smut spores. Most
smut fungi produce only teliospores and basidiospores. When haploid basidiospores
germinate, the germ tubes fuse to produce dikaryotic infectious mycelium. Important
smut fungi are Ustilago tritici (Fig. 10.1h), causing smut on wheat and Ustilago
maydis (Fig. 10.1i), causing smut galls on corn. Together with Flor in the USA,
Oort in the Netherlands proposed the gene-for-gene hypothesis based on research
performed on Ustilago tritici and wheat (Oort 1944).
Root Rots Root rots of trees are often caused by species of Armillaria that occur
worldwide and affect hundreds of species of trees. The pathogen Armillaria mellea is
one of the most common fungi in forest soils. Diagnostic characteristics of Armillaria
root rot appear at decayed areas in the bark, at the root-stem junction, and on the roots.
84 P. J. G. M. de Wit

White mycelial mats are formed between the bark and wood. Another characteristic
sign of the disease is the formation of rhizomorphs or “shoe strings”, cordlike threads
of mycelium 1–3 mm in diameter that can grow long distances and infect healthy
trees (Agrios 2005).

10.2 Characteristcis of Fungal and Oomycetous

Plant Pathogens

These organisms can be obligate biotrophic, biotrophic, hemi-biotrophic or

necrotrophic pathogens. Biotrophic pathogens thrive on living host cells. Some
biotrophs cannot be cultured on synthetic media and are called obligate biotrophic
pathogens like rusts, downy mildews and powdery mildews. However, many
biotrophic pathogens grow on host plants under natural conditions but can still be
cultured on synthetic media like the tomato pathogen Cladosporium fulvum (Joosten
and De Wit 1999). Hemi-biotrophic pathogens start infection as a biotroph, but later
in the growth season they can live as a saprophyte on death host tissue. A defense
response that is very efficient against obligate biotrophic and biotrophic pathogens is
the hypersensitive response (HR), death of a few plant cells at the site of infection. In
contrast, necrotrophic fungal pathogens kill host tissue before they retrieve nutrients,
and the HR is not effective against them.
Extracellular and Intracellular Pathogens Many fungi live on plants as epiphytes
of which some have developed into pathogens. They usually enter plants through
stomata and thrive in the apoplastic space surrounding cells without producing haus-
toria. Most extracellular pathogens are slow growers and show long latent periods
as the apoplast often contains antimicrobial compounds and antimicrobial enzymes,
and is poor in nutrients. Intracellular pathogen often produce haustoria.

10.3 Fungal and Oomycetous Infection Strategies

and Host Defense Mechanisms

Infection Strategies Infection strategies employed by fungal pathogens depend

strongly on where they thrive in their host plants. The cell wall is a major obstacle for
plant pathogens which contains physical and chemical barriers like the plant waxy
cuticle, (lignified) cell walls and antimicrobial metabolites and proteins (Balmer
et al. 2013). Many fungal genomes have been sequenced showing that pathogenic
fungi contain many different classes of cell-wall degrading enzymes. The expres-
sion of these genes is highly up-regulated during infection by necrotrophic plant
pathogens (Zhao et al. 2013). Biotrophic pathogens often contain similar numbers
of genes encoding cell-wall degrading enzymes as necrotrophs, but they are often
lowly expressed by these pathogens or only at specific sites and phases of infection.
Necrotrophic fungi often also produce secondary metabolites that are toxic to plant
10 Plant Pathogenic Fungi and Oomycetes 85

cells thereby facilitating their necrotrophic lifestyle, while those metabolites are often
absent in (obligate) biotrophic pathogens. For example, the powdery mildew Blume-
ria graminis contains hardly any genes encoding secondary metabolites (Spanu 2012)
and in the tomato leaf pathogen C. fulvum these genes are down-regulated during
infection (De Wit et al. 2012). Apart from producing enzymes that enable pathogens
to degrade plant cell walls and to retrieve the released mono/oligosaccharides, fungi
also need to protect themselves against antifungal proteins that are present in plant-
cell walls and the apoplast by detoxifying enzymes like tomatinase produced by
Cladosporium fulvum that detoxifies the toxic saponin, α-tomatine occurring at high
concentrations in tomato (Okmen et al. 2013).
Basal Defense Strategies Plants have developed sophisticated defense strategies to
recognize pathogens and to defend themselves against fungal pathogens. All plants
can recognize pathogen-associated molecular patterns (PAMPs), like chitin from
fungi, by pattern recognition receptors (PRRs) that mediate PAMP-triggered im-
munity (PTI), a response that protects plants against potential microbial pathogens
(Jones and Dangl 2006; Liebrand et al. 2014). PRRs are extracellular LRR-containing
receptor-like transmembrane proteins with a cytoplasmic kinase signaling domain
known as RLKs. They mediate PAMP-triggered basal structural and chemical
defense responses including callose deposition, accumulation of reactive oxygen
species (ROS), cell wall enforcements and accumulation of pathogenesis-related
(PR) proteins including chitinases, proteases and glucanases.
Effector-Triggered Susceptibility Although plants have developed basal defense
strategies against microbes, successful pathogens have found ways to overcome basal
defense responses. They can suppress PTI by secreting different types of effectors that
target various components of PTI (Stergiopoulos and de Wit 2009). By suppressing
PTI they cause effector-triggered susceptibility (ETS). Various types of effectors have
been described in various pathogenic fungi (Stergiopoulos and De Wit 2009). Overall
they manipulate host defenses and host physiology to facilitate virulence in various
ways. Here I discuss the intrinsic functions of two Cladosporium fulvum effectors
that can serve as an example of many other fungal effectors. C. fulvum secretes
the Avr2 effector protein which inhibits plant cysteine proteases including Rcr3pim
(required for C. fulvum resistance 3) (Rooney et al. 2005). Heterologous expression
of Avr2 in tomato and Arabidopsis resulted in increased susceptibility to different
fungal pathogens including Botrytis cinerea and Verticillium dahliae (Van Esse et al.
2008). Mutation studies on Rcr3pim showed it also to be active against Phytophthora
infestans, which secretes EPIC1 and EPIC2B proteins that also bind and inhibit
Rcr3pim . Also the root parasitic nematode Globodera rostochiensis secretes a venom
allergen-like effector protein, Gr-VAP1, that targets and inhibits Rcr3 perturbing its
active site (Lozano-Torres et al. 2012).
C. fulvum also secretes the cysteine-rich Avr4 protein which is a chitin-binding
lectin that protects fungal cell walls against plant chitinases, providing a defensive
role during infection (Van den Burg et al. 2006). In addition, silencing of the Avr4
gene in C. fulvum reduces virulence on tomato plants (Van Esse et al. 2007). Protec-
tion against plant chitinases is important for virulence of most plant pathogenic fungi
86 P. J. G. M. de Wit

Fig. 10.2 Schematic overview of an evolutionary scenario for adaptation of the leaf mould pathogen
Cladosporium fulvum to tomato. a Chitin fragments (pattern-associated molecular pattern; PAMP)
are recognized by the tomato chitin receptor (SlCERK) that triggers PAMP-triggered immunity
10 Plant Pathogenic Fungi and Oomycetes 87

because chitin is a basic component of fungal cell walls. Indeed homologs of Avr4
have been identified in several dothideomycetous fungi, including Mycosphaerella fi-
jiensis and Dothistroma septosporum (Stergiopoulos et al. 2010). Interestingly many
fungi, including C. fulvum, also secrete proteins that contain LysM carbohydrate-
binding domain proteins (Kombrink and Thomma 2013). The LysM protein Ecp6
of C. fulvum also binds to chitin, but does not protect the fungus against basic plant
chitinases. Ecp6 mainly binds small chitin fragments (PAMPs) that are released
from the fungal cell wall in planta and prevents them from being recognized by
chitin receptors present in plants to induce PTI (De Jonge et al. 2010). Ecp6 is
a virulence factor because silencing of Ecp6 results in reduced virulence (Kom-
brink and Thomma 2013). Many homologs of Ecp6 are identified in various fungal
species (De Jonge and Thomma 2009; Kombrink and Thomma 2013) , suggesting
an important function in preventing chitin-triggered immunity in many plant-fungus
interactions. Recently, it was shown that Mycosphaerella graminicola secretes three
LysM effectors called Mg1LysM, Mg3LysM and MgxLysM (Marshall et al. 2011) of
which both Mg1LysM and Mg3LysM bind chitin, whereas only Mg3LysM prevents
chitin-triggered immunity. However, only the Mg3LysM deletion mutant showed
significantly reduced virulence. Magnaporthe oryzae LysM effector Slp1 also binds
and scavenges small chitin fragments. In rice, the chitin elicitor binding protein,
CEBiP, recognizes chitin and activates PTI. Indeed it was found that Slp1 prevents
chitin-triggered immunity by competing with the CEBiP receptor for chitin binding
(Mentlak et al. 2012).
Effector-Triggered Immunity In turn, plants have evolved sophisticated ways to
recognize and respond to effectors. In addition to PRRs that recognize PAMPs,
plants have developed immune receptors that recognize effectors or host plant tar-
gets manipulated by effectors resulting in effector-triggered immunity (ETI) (Jones
and Dangl 2006). One of the most typical characteristics of ETI is the HR. At the
host species and cultivar level, co-evolution between hosts and their pathogens has
caused an arms race that has led to the development of numerous novel effectors and
corresponding resistance proteins, which are described by the gene-for-gene concept
(De Wit et al. 2009) (Fig. 10.2a, 10.2b, 10.2c, 10.2d).

←
Fig. 10.2 (continued) (PTI) providing basal defense against the pathogen. b To become pathogenic,
C. fulvum secretes the Ecp6 effector that scavenges chitin fragments to prevent PTI. c C. fulvum
secretes additional effectors to increase virulence including Avr2 inhibiting apoplastic cysteine
protease Rcr3, and Avr4 protecting chitin present in the cell wall of the fungus against tomato
chitinases. d Tomato responds by developing RLP immune receptors Cf-Ecp6, Cf-2 and Cf-4 to
recognize Ecp6, Avr2 and Avr4, respectively, and to mediate effector-triggered immunity (ETI)
leading to the hypersensitive response (HR) and resistance against fungal strains secreting these
effectors. C. fulvum is supposed to produce numerous effectors and tomato a comparable number
of immune receptors
88 P. J. G. M. de Wit

10.4 Resistance Against Fungi and Oomycetes

The cloning of effector genes speeded up the cloning of the matching resistance
genes that encode either cell surface-localized receptor-like proteins known as
RLPs (Liebrand et al. 2014) or cytoplasmic nucleotide binding, leucine-rich repeat
(NB-LRR) or NLR proteins (Takken and Goverse 2012). RLPs are integral plant
membrane proteins containing an extracellular leucine-rich repeat or LRR domain, a
membrane spanning domain, and a short cytoplasmic tail without signaling domain.
RLPs recognize effectors of extracellular fungal pathogens and mediate ETI. How-
ever, cytoplasmic pathogens exploit the cytoplasm of plant cells by injecting effectors
into host cells that interact with cytoplasmic targets to suppress PTI. The cytoplasmic
effectors are usually recognized by cytoplasmic NLR immune receptors. Pathogens
can develop mutations in effectors or simply loose them to escape recognition by
RLP and NRL immune receptors or develop new effectors for compensation. Some
immune receptors can work in concert in receptor complexes active against more
than one pathogen (Macho and Zipfel 2014). Defense systems of plants are able to
respond to different types of PAMPs and effectors, but they cannot always be distin-
guished easily and downstream defense pathways in plants activated during PTI and
ETI often overlap and operate against a broad spectrum of pathogens (Thomma et al.
2011). These responses include the generation of reactive oxygen species (ROS), an-
timicrobial phytoalexins, chitinases, glucanases, proteases, and often the HR. Many
immune receptors encoded by resistance genes have been cloned in the last decade.
They can now be introduced in many copies by breeders in crops by classical breed-
ing or by cis/transgenesis (Zhu et al. 2012). It is important to introduce multiple
immune receptor genes in plants against multiple pathogen effectors in order to ob-
tain durable plant resistance (Brunner et al. 2010; Vleeshouwers and Oliver 2014)
. Overcoming multiple immune receptors by a pathogen by modification or loss of
effectors is expected to cause a fitness cost and decrease in virulence.

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Chapter 11
Phytopathogenic Nematodes

Johannes Helder, Mariëtte Vervoort, Hanny van Megen, Katarzyna

Rybarczyk-Mydłowska, Casper Quist, Geert Smant and Jaap Bakker

Abstract Soil is teeming with life, and rhizosphere soil is even more densely in-
habited than bulk soil. In terms of biomass, bacteria and fungi are dominant groups,
whereas nematodes (roundworms) are the most abundant Metazoans. Bulk soil, soil
not directly affected by living plant roots, typically harbours around 2000–4000 ne-
matodes per 100 g, while in the rhizosphere these numbers should be multiplied by
a factor 3–5. This difference is not only explained by a higher density of plant para-
sites, as also bacterivorous and fungivorous nematodes benefit from the local boost
of the bacterial and fungal community. Most nematodes feeding on higher plants
are obligatory parasites. In this chapter four independent lineages of plant-parasitic
nematodes are discussed. Facultative plant parasites often occupy basal positions

J. Helder () · M. Vervoort · H. van Megen · C. Quist · G. Smant · J. Bakker

Laboratory of Nematology, Wageningen University, Droevendaalsesteeg 1,
6708 PB Wageningen, The Netherlands
Tel.: +31 317 483 136
e-mail: Hans.Helder@wur.nl
K. Rybarczyk-Mydłowska
Museum and Institute of Zoology PAS, Wilcza 64, 00-679 Warsaw, Poland
Tel.: +48 888981281
e-mail: katarzynar@miiz.waw.pl
M. Vervoort
Tel.: +31 317 483136
e-mail: Jet.Vervoort@wur.nl
H. van Megen
Tel.: +31 33 2864283
e-mail: Hannyvanmegen@kpnmail.nl
C. Quist
Tel.: +31 317 483136
e-mail: Casper.Quist@wur.nl
G. Smant
Tel.: +31 317 485154
e-mail: Geert.Smant@wur.nl
J. Bakker
Tel.: +31 317 483136
e-mail: Jaap.Bakker@wur.nl

© Springer International Publishing Switzerland 2015 91

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_11
92 J. Helder et al.

Fig. 11.1 Schematic overview of a plant-parasitic nematode. Amphids and phasmids are chemosen-
sory organs. The stylet is a protrusible, hollow puncturing device, that is used to penetrate plant cell
walls. Figure courtesy of Shinya et al. (2013)

within a lineage. Most, but not all, economically high impact plant parasites such a
root knot, cyst and lesion nematodes belong to the most distal nematode clade (Clade
12; Holterman et al. Mol Biol Evol 23:1792–1800, 2006). In this chapter, some of
the latest insights on the evolution, the ecology and the biology of phytopathogenic
nematodes will be covered.

11.1 Nematodes

Nematodes are small (mostly between 0.2 and 2.5 mm in length), worm-shaped
animals that together constitute the phylum Nematoda. This phylum, which is thought
to have arisen during early phases of the Cambrian explosion (≈ 550 million years
ago) in marine habitats, belongs to the superphylum Ecdysozoa that encompasses
all moulting animals (Aguinaldo et al. 1997). Nematodes are not only present in
terrestrial systems, but also in freshwater and marine habitats (Bongers and Ferris
1999). Next to their ubiquity, nematodes are abundant and can reach densities of up to
millions per square meter in soil, residing in the water films attached to soil particles
or e.g. in or around plant roots. Due to their size and their mobility, nematodes
are easily extractable from soil as compared to protozoans, fungi and bacteria. The
phylum Nematoda show a high trophic diversity; nematodes may feed upon bacteria,
fungi, protozoa, algae, other nematodes, a combination of the aforementioned food
sources (omnivores), or they may be facultative or obligate parasites of plants or
animals (Yeates et al. 1993). Due to this diversity in feeding habits, nematodes can
be found at all three levels of the soil food web (Ferris et al. 2001). Plant parasites, in
fact herbivores, reside in the first trophic level as they feed directly on roots of higher
plants. Bacterivores and fungivores are found in the second trophic level as they feed
on primary decomposers. Carnivorous nematodes, also referred to as predators, feed
mainly on other nematodes and are therefore positioned at the third trophic level.
It should be noted that plant-parasites (see Fig. 11.1) usually constitute a minority
within the nematode community, both in terms of number of individuals as well as
of species.
11 Phytopathogenic Nematodes 93

11.2 Use of an SSU rDNA Framework to Reconstruct

the Evolution of Nematodes

Nematodes are morphologically highly conserved; even for experts, the ability to
identify certain species depends on the life stage or sex of the present individuals
(Floyd et al. 2002). Considering the age of this phylum, the saying ‘never change a
winning team’ is certainly applicable. As the number of informative morphological
characters is limited, identification of nematodes on the basis of such characters is
challenging and requires a considerable amount of time, experience and expertise.
Keeping the economical relevance of this animal phylum in mind, it is remarkable
to see that nematode systematics is far from established. It has a long history of con-
stant revision, and over a dozen general schemes for nematode classification have
been proposed. One of the first phylum-wide classifications was proposed by Chit-
wood and Chitwood (1933). They divided the phylum into two classes, the Phasmidia
and Aphasmidia, later renamed to Secernentea and Adenophorea respectively. This
was based mainly on the fact that the Secernentea share several characters, including
the presence of phasmids, small sensory organs on the tail. Although it was already
recognized at the time that the Adenophorea did not form a natural group, the division
of the Nematoda into these two groups persisted for a long time. The first person to
apply cladistic principles to nematode systematics was Lorenzen (Lorenzen 1981).
He also recognized that the Adenophorea were not a monophyletic group, but could
not provide an alternative. Also at lower taxonomic levels (order, family and genus
level), systematics were far from stable (De Ley and Blaxter 2002). Despite their
ecological and physiological diversity, their conserved morphology and small size
resulted in a paucity of observable, phylogenetically informative characters. Further-
more, many characters show a convergent evolution. In recent years DNA sequence
data have brought a revival to the field of systematics. The first major classifica-
tion to incorporate both morphological and molecular phylogenetic information was
presented by De Ley and Blaxter (2002).
Appreciating that nematodes arose early in animal evolution, it would be con-
ceivable that even relatively conserved genes such as the ribosomal RNA-encoding
genes could offer us insights in the evolution of plant parasitism within this phylum.
Especially among invertebrates, the small subunit ribosomal DNA (SSU rDNA) gene
(coding for SSU rRNA) is frequently used to deduce deep phylogenetic relationships.
Because of their vital role in the assembly of proteins in the ribosomes, there is a
strong selection on the SSU and LSU ribosomal DNA genes (LSU: large subunit).
As a consequence these genes—at least parts thereof—are very conserved. Among
the ribosomal RNA encoding genes, the SSU rDNA is most conserved. Riboso-
mal DNA genes are usually present in multiple copies (the Caenorhabditis elegans
genome harbours ≈ 55 copies; Ellis et al. 1986) and this implies that a relatively
small quantity of starting material (e.g. a single nematode corresponding to ≈ 0.2 ng
DNA) is sufficient for a polymerase chain reaction (PCR)-based amplification. Nor-
mally it would not be advisable to use a multicopy gene in phylogenetics because
there could be the risk of comparing paralogs instead of orthologous gene copies.
94 J. Helder et al.

However, intrachromosomal homogenization ensures that mutations in a copy of a

SSU rDNA gene are either removed or taken over in the other SSU rDNA copies
(Dover et al. 1993).
For some time, SSU rDNA in nematodes was suggested to have a substitution
rate 2–3 times higher than in most other Metazoa. In a study on the phylogenetic
position of the arthropods, Aguinaldo and co-workers carefully selected nema-
tode species with relatively low SSU rDNA substitution rates (Aguinaldo et al.
1997). The SSU rDNA substitution rates within the phylum Nematoda appeared
to be relatively variable. The model organism Caenorhabditis elegans and the
animal parasite Strongyloides showed remarkably high substitution rates; respec-
tively 0.192 and 0.187 substitutions per site, whereas another, more basal animal
parasite—Trichinella—was shown to have a significantly lower rate of change (0.110
substitutions per site). Related Arthropods have values ranging from 0.040 to 0.080
(Aguinaldo et al. 1997). Higher rDNA substitution rates as observed for nematodes
with a short life cycle and/or an advanced parasitic life style could result in unexpected
resolution at lower phylogenetic levels.
The small subunit ribosomal DNA gene (≈ 1700 bp) has been used successfully for
the generation of molecular frameworks throughout the animal kingdom. The SSU
rDNA harbours a few regions that are almost fully conserved among eukaryotes and
this allowed the design of so-called “universal primers” for the amplification of this
particular gene from a wide range of Metazoans. In fact SSU rDNA has—in terms
of diversity—a mosaic structure of very conserved regions interspersed by more
variable elements. Amplifiability from unknown samples in combination with the
presence of multiple regions with ample phylogenetic signals explains why this gene
became popular in nematode phylogenetics. A first key paper by Blaxter et al. (1998)
(53 nematode taxa), was followed by Holterman et al. (2006) (360 taxa), and Van
Megen et al. (2009) (1250 taxa). Currently, the SSU rDNA molecular framework
of nematodes at the Laboratory of Nematology, Wageningen University, includes
approximately 2800 taxa. Bayesian inference based analysis of 360 nematode taxa
by Holterman et al. (2006) gave rise to a phylogenetic framework characterized by
a backbone consisting of 12 consecutive bifurcations. Based on these bifurcations,
12 clades were defined within the phylum Nematoda. From this analysis it was clear
that trophic specialisations such as plant, mammal or insect parasitism all have arisen
multiple times during evolution. It should be noted that due to the absence of good
fossil records it is not possible to label nodes with a proper assessment of the era in
which such a hypothetical common ancestor has lived.

11.3 The Evolution of Plant Parasitism Among Nematodes

From molecular analyses of a wide diversity of representatives of the phylum Nema-

toda, it became clear that plant parasitism has arisen at least four times independently.
In all lineages plant parasites are equipped with a protrusible, hollow, injection
11 Phytopathogenic Nematodes 95

Fig. 11.2 Head regions of representatives of two distinct lineages of plant-parasitic nematodes.
Left: Trichodorus primitivus (Triplonchida, Clade 1) and right: Longidorus intermedius (Dory-
laimida, Clade 2). Pictures were taken at a 1000 times magnification. (Source: Hanny van Megen,
Wageningen University, Laboratory of Nematology, The Netherlands)

Fig. 11.3 Head regions of representatives of two distinct lineages of plant-parasitic nematodes.
Left: Aphelenchoides subtenuis (Aphelenchida, Clade 10) and right: Globodera rostochiensis (Ty-
lenchida, Clade 12). Pictures taken at a 1000 times magnification. (Source: Hanny van Megen,
Wageningen University, Laboratory of Nematology, The Netherlands)

needle-like device that is used to puncture the plant cell wall, to deliver pathogenicity-
related secretions in the plan root, and to take up nutrients from the plant (see
Figs. 11.1, 11.2, and 11.3). Another common denominator among plant parasitic
nematodes is the large size of the pharyngeal gland cells (usually a single dorsal
and multiple subventral glands (Fig. 11.1), and the high activity of these glands just
before and during plant parasitism.
Within Clade 1 (most basal clade of this phylum), obligatory plant parasites can
be found within the order Triplonchida, in the family Trichodoridae. Plant-parasitic
representatives within this family are generally known as ‘stubby root nematodes’.
These nematodes are ectoparasites, and they use a curved protrusible onchiostyle to
96 J. Helder et al.

puncture rhizodermis cells (Fig. 11.2). Nematode secretions that are thought to be
involved in root cell penetration and manipulation are produced in the pharyngeal
glands located in the basal bulb. Stubby root nematodes harbour five pharyngeal
glands cells: one dorsal gland, two posterior and two anterior ventrosublateral glands.
These glands open into the lumen of the nematode, and produce pathogenicity related-
proteins. It is so far unclear whether these five glands act during distinct phases of
plant feeding (Karanastasi et al. 2003).
The second lineage can be found in Clade 2, in a family embedded in the order
Dorylaimida called Longidoridae. The common name of this category of plant par-
asites is ‘dagger nematodes’. Dagger nematodes are equipped with an odontostyle,
a protrusible hollow spear (Fig. 11.2) that is used to puncture the plant cell wall,
to secrete pathogenicity-related proteins and to take up food from the plant cell. As
compared to stubby root nematodes, members of the family Longidoridae tend to
feed on deeper cell layers (e.g. cortical instead of rhizodermal cells). The terminal
bulb in the Dorylaimida contains five pharyncheal gland cells, but in case of the fam-
ily Longidoridae, two ventrosublateral glands seems to be degenerated (or fused)
as the nuclei of this second pair have disappeared (although the five gland orifices
are still intact) (Loof and Coomans 1972). Also in this respect, the plant parasitic
Longidoridae are distinct from the more basal plant-parasitic members of the order
Triplonchida. It might be worth noting that both a number of representatives of both
the Trichodoridae and the Longidoridae are transmitters of plant viruses.
The plant-parasitic members of the third lineage are mainly (or even exclusively)
facultative plant parasites; as an alternative food source they can feed on fungi as
well. They are members of the families Aphelenchoididae and Parasitaphelenchidae,
and the most notorious representatives of these groups are the causal agent of white
tip in rice, Aphelenchoides besseyi, and the pine wood nematode Bursaphelenchus
xylophilus. Currently these two families are positioned in Clade 10, but this clade
positioning is not robust, as the GC contents of SSU rDNAs of these families is
relatively low as compared to related families (Holterman et al. 2006). Members of
these two families are equipped with a stomatostylet (Fig. 11.3), a device functionally
comparable with the onchiostyle and the odontostyle mentioned above. However,
as suggested by their names, the origins of these injection needle-like devices are
fundamentally distinct.
The fourth, and economically most important group of plant parasitic nematodes
can be found in the most distal nematode clade (Clade 12). This clade roughly
corresponds to the order Tylenchida, with the exception of the suborder Hexatylina, a
branch that mainly comprises insect parasitic nematodes. The other three suborders,
the Hoplolaimina, the Criconematina, and the Tylenchina, harbour plant-parasitic
nematode species. Contrary to the first two suborders, numerous members of the
Tylenchina are facultative parasites of higher plants, being able to use lower plants,
fungi and oomycetes as alternative food sources. The most well-known and best-
studied plant parasites such as the root knot (Meloidogyne sp.), cyst (Heterodera and
Globodera sp.) and lesion (Pratylenchus sp.) nematodes all belong to the suborder
Hoplolaimina. Tylenchida are equipped with a stomatostylet (Fig. 11.3), and at least
of part of the secretory components involved in plant parasitism are produced in
11 Phytopathogenic Nematodes 97

the dorsal and in the two subventral pharyngeal glands. It should be noted that the
subventral glands are most active in pre-parasitic second stage juveniles, and their
activity slows down rapidly in parasitic second and third stage juveniles. The peak
of dorsal gland activity is observed in parasitic life stages, and thus lags behind the
peak of subventral gland activity.

11.4 Some Biological Characteristics of Migratory

and Endoparasitic Plant Parasites

Root-knot, lesion and cyst nematodes pose a serious threat to main agricultural
crops such as potato, sugar beet, and soybean. As such these distal representatives
of the order Tylenchida constitute the economically most detrimental group of plant
parasitic nematodes. Among the three genera mentioned above, root-knot nematodes
such as Meloidogyne incognita, M. hapla, and M. chitwoodi, are most polyphagous,
being able to infect almost all domesticated plants worldwide (Trudgill and Blok
2001). Lesion nematodes are members of a species-rich genus named Pratylenchus
(ca 70 valid species), and species such as P. penetrans, P. neglectus, and P. thornei
are notorious parasites in crops such as potato and tomat as well as a range of cereals
and legumes. Lesion nematodes are migratory endoparasites (see below), and as
such provide other opportunistic soil bacteria and fungi access to the plant root. The
invasion of plant roots by root-knot and cyst nematodes leads to the formation of
nematode feeding sites. In case of sedentary endoparasites, establishment of so called
‘giant cells’ or ‘syncytia’ (root-knot and cyst nematodes, respectively) is considered
as one of the most sophisticated adaptations of plant parasitism. The syncytium, a
conglomerate of plant cells fused by partial degradation of plant cell walls, functions
as a metabolic sink that transfers plant assimilates from the conductive tissues in
the vascular cylinder to the sedentary nematode. In case of rot knot nematodes, the
formation of five to seven giant-cells grouped around the head region is induced.
Upon the injection of nematode secretions, parasitized cells rapidly become larger,
hypertrophied and multinucleate as nuclear division occurs in the absence of cell
wall formation. Feeding sites, giant cells or syncytia, are used till the end of the
nematode life cycle and serve as sink tissues to which nutrients are imported in a
symplastic and/or apoplastic manner (Hoth et al. 2008).
On the other hand, migratory endoparasites, such as Pratylenchus species, exhibit
feeding strategies that may be considered as less refined, but they are no less success-
ful keeping their proliferation and host range in mind. In various life stages, lesion
nematodes move freely through the root to feed and reproduce, creating numerous
local tissue lesions, which are used as an entrance by the secondary pathogens such
as bacteria or fungi. The feeding takes place mostly in the root cortex, but root hair
feeding is observed for younger life stages as these are unable to perforate thicker
epidermal cell wall (Zunke 1990). The parasitic success of the mentioned groups
of nematodes is undoubtedly a result of their unusual ability to overcome the bar-
rier of the plant cell wall. This biological obstacle is mechanically and chemically
98 J. Helder et al.

weakened by the stylet, spear or onchiostyle-driven puncturing of the cell wall in

combination with the local release of cell wall-degrading and modifying proteins.

11.5 The Role of Lateral Gene Transfer in the Evolution

of Plant Parasitism

Contrary to vertical gene transfer, the transmission of genes from parents to their off-
spring, lateral gene transfer (LGT) is about the stable acquisition of (parts of) genes
in a manner other than traditional reproduction. Such non-conventional transfer of
genetic material requires, among other things, close physical contact between the
between donor and recipient. LGT could for instance occur between organisms with
a trophic relationship. As an example, Doolittle described the transfer of genes to
early eukaryotes from the bacteria taken by them as food (Doolittle 1998). In the
same year, Smant and co-workers (Smant et al. 1998) discovered that the potato
and soybean cyst nematodes (G. rostochiensis and H. glycines (order Tylenchida)
produce and secrete β-1,4-endoglucanase (cellulases). Till this discovery, animals
were thought to be incapable degrading plant cell walls; herbivores use microbial
endosymbionts for the degradation of these recalcitrant polymers.
This finding constituted the starting point of a series of papers reporting a range
of cell wall-degrading enzymes (CWDE) from plant-parasitic nematodes, including
pectate lyases (Popeijus et al. 2000), exo-polygalacturonase (Jaubert et al. 2002),
xylanases (Mitreva-Dautova et al. 2006) and expansins (Qin et al. 2004). It should be
noted that CWDEs are produced in the subventral glands of plant parasitic nematodes
during the early phases of infection. These enzymes are unlikely to be involved in
feeding site formation in the plant root.
The discovery of a set of cell wall-degrading enzymes (CWDE) is remarkable.
Nematodes are devoid of plant cell wall-like structures, and hence it seems safe to
state that plant cell wall penetration by parasitic nematodes is the result of mechanical
weakening and local depolymerization. Remarkably these plant cell wall-degrading
enzymes produced by nematodes were far more similar to their bacterial equivalents
than to orthologs from other eukaryotes such as higher plants, fungi or oomycetes.
This prompted Keen and Roberts (1998) in a commentary paper to the hypothesize
that ancestral bacterivorous nematodes could have acquired a pathogenicity island
with multiple plant parasitism-related genes by the ingestion of (plant-parasitic) soil
bacteria. Such an acquisition could have enabled them to penetrate a plant cell wall,
and exploit a food source that was till that time inaccessible.
If it is true that the evolution of plant parasitism among nematodes was facilitated
by the acquisition of CWDEs of bacterial origin, one might wonder whether the four
independent lineages as described in 10.3 harbour distinct or similar core sets of
cell wall-degrading enzymes. Here we will use cellulases as an example as this is
so far the best-characterized CWDE among plant parasitic nematodes. As indicated
above, β-1,4-endoglucanases (cellulases) were first discovered in two cyst nema-
tode species Globodera rostochiensis and Heterodera glycines (Smant et al. 1998).
11 Phytopathogenic Nematodes 99

They are encoded by a multi-copy gene family, which is expressed in the subventral
esophageal glands of the infective second stage juveniles (J2 ). The biochemical ac-
tivity of these enzymes is determined as a hydrolysis of β-1,4 glycosidic bonds of
cellulose microfibrils. According to their biochemical characteristics, cellulases are
represented in various glycoside hydrolase (GH) families. All members of the order
Tylenchida (lineage 4 in this chapter) investigated so far (even the insect parasite
Delandenus siridicola) harbour cellulases belonging to family 5 (GHF5; glutamic
acid (Glu) residues essential for catalysis).
The GHF5 genes in Tylenchida comprise at least a catalytic domain, and occasion-
ally this is connected to a linker and/or a type II cellulose-binding domain (CBDII).
Due to the slight differences in intron-exon composition and noticeable phyloge-
netic distance, those cellulases are thought to belong to at least two distinct lineages
(Kyndt et al. 2008). These lineages are referred to as CelI and CelII by (Rehman
et al. 2009a), and in a more recent study these are similar to catalytic domains
type B and type C respectively (Rybarczyk-Mydłowska et al. 2012). Phylogenetic
analysis of nematode GHF5 cellulases suggests for an early acquisition of this cate-
gory of plant cell wall-degrading enzymes, maybe even by the common ancestor of
the order Tylenchida and the family Aphelenchidae (subfamilies Aphelenchinae and
Paraphelenchinae) (Rybarczyk-Mydłowska et al. 2012).
A facultative plant parasite belonging to lineage 3, the pinewood nematode Bur-
saphelenchus xylophilus, was shown to produce cellulases from glycoside hydrolase
family 45 (GHF 45; aspartic acid (Asp) residues essential for catalysis) (Kikuchi et al.
2004). Bursaphelenchus belongs to the family Parasitaphelenchidae, and recently a
cellulase from its sister family, Aphelenchoididae, was isolated and characterized.
A small-scale analysis of the transcriptome of the foliar nematode Aphelenchoides
besseyi resulted in the discovery of another GHF45 cellulase that is produced and
presumably secreted (as the core protein is preceded by a predicted signal peptide for
secretion) by this nematode species (Kikuchi et al. 2014). So far each plant parasite
lineage was thought to be characterized by the production of cellulases from a single
GH family. Therefore the finding of a GHF5 cellulase from another facultative plant
parasite within this lineage, Aphelenchoides fragariae, by Fu et al. (2012) was re-
markable Notably, this finding was the result of a directed search as the authors used
degenerated GHF5 primers to screen for the presence of cellulases in A. fragariae.
The GHF5 cellulases described from A. fragariae seemed to have some unusual
features. For example, it was not possible to detect genomic copies of this sequence
in nematodes reared on fungi. Until the finding of this GHF5 cellulase in this foliar
nematodes species is confirmed by more detailed research, we tend to state that the
presence of GHF45 cellulases is a typical and unique characteristic of this lineage
of plant parasitic nematodes.
As compared to the two most distal lineages of plant parasites, the two remaining
families within the more basal clade 1 and 2 are poorly characterized. Within the
family Longidoridae, a cellulase was identified from Xiphinema index, and this
enzyme belonged to yet another GH family (Jones and Helder, unpublished results).
Based on this fragmentary information, we hypothesize that individual lineages of
100 J. Helder et al.

plant-parasitic nematodes are characterized by the presence of a single, lineage-

dependent kind of cellulase.

11.6 Plant Manipulation by Parasitic Nematodes

Even plant-parasitic nematode species residing in the most basal lineage, ectopara-
sites belonging the family Trichodoridae, do not show a hit-and-run strategy, i.e. a
strategy by which the nematode would just insert its puncturing device into a plant
cell, and take up the cytosol, and move on to the next plant cell. In case of the stubby
root nematode species Paratrichodorus anemones the delivery of nematode secretion
into the rhizodermis cell resulted in the redistribution of cytoplasm from all areas of
the cell towards the penetration site (Karanastasi et al. 2003). In the interaction of
Ficus carica seedlings with a representative of the second lineages of plant parasites,
the dagger nematode Xiphinema index, hypertrophied, multinucleate cells were in-
duced. Most likely the re-differentiation of plant cells is induced by saliva proteins
produced by this ectoparasite (Wyss et al. 1980). Hence, plant cell re-differentiation
as an essential process linked to parasitism is not a unique characteristic for the most
distal nematode taxa in Clade 12, and also this trait seems to have arisen multiple
times. Most likely proteins produced in de dorsal glands are responsible for plant
cell re-differentiation, but so far the underlying mechanism is unknown. Recently, a
number of effector proteins has been identified that are produced in these glands in
infectious life stages. It should be noted that the examples given below are all from
cyst and root knot nematodes, representatives of the most distal lineages. Indica-
tions for local auxin manipulation as an early step in feeding site formation (Goverse
et al. 2000) were recently confirmed by the identification of an effector protein from
the soybean cyst nematode Heterodera glycines, called 19C07, that was shown to
interact with an auxin influx transporter (LAX3) in Arabidopsis (Lee et al. 2011). An-
other category of dorsal gland proteins, the so-called SPRYSECs (secreted proteins
containing a SPRY domain) have been identified from the potato cyst nematodes Glo-
bodera rostochiensis and G. pallida, close relatives of the soybean cyst nematode. As
compared to e.g. Caenorhabditis elegans, the pinewood nematode Bursaphelenchus
xylophilus and the tropical root knot nematode Meloidogyne incognita, this protein
family has expanded enormously in cyst nematodes; both potato cyst nematodes were
shown to harbor dozens of secreted SPRY proteins (Rehman et al. 2009b, Cotton
et al. 2014). Although the function of these proteins in the interaction with the host
plant is unknown for most members of this family, one member, SPRYSEC-19, was
demonstrated to suppress the CC-NB-LRR disease resistance response in host plants
(Postma et al. 2012).
Hence, plant parasitism within the phylum Nematoda is characterized by ample
convergent evolution. As we have seen, all of the plant-parasitic lineages developed
similar morphological adaptations that allow them to overcome the plant cell wall,
a major physical barrier. A similar picture starts to arise for the non-morphological
11 Phytopathogenic Nematodes 101

characteristics of these lineages. Although the origin and the exact blend of cell wall-
degrading enzymes might be unique for individual taxa, a roughly similar pallet of
enzymes is likely to be produced by all obligatory plant parasites. It will probably
become clear in the next years whether or not convergent evolution can be observed in
the mechanisms underlying host cell manipulation and the suppression of resistance
responses.

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Chapter 12
Herbivorous Insects—A Threat for Crop
Production

Eddy van der Meijden

Abstract It is estimated that, in spite of plant breeding and pest control efforts, 15 %
of crop yield is worldwide lost to herbivory by insects. Examples demonstrate how
insect pests have developed in the past and why they will develop in the future. The
evolutionary potential of insects to become new pests is considered for traditionally
and genetically modified crop varieties. The immune system of plants is presented
step by step. Generalist herbivores can be effectively repelled, but specialist her-
bivores are much harder to repel. They use plant defenses as cues for host plant
recognition. Next to direct defense, indirect defense by attracting natural enemies
of (specialist) herbivores is explained. Finally, the interactions of plants and insect
herbivores with microbial symbionts—and their consequences—are discussed.

12.1 Introduction

Probably not a single wild plant will complete its life cycle without being victim of
herbivory by one or usually many more insect species during some stage of its life.
Without special treatment, like the application of insecticides, the release of natural
enemies and/or modification of their immune system, crop plants are even more
vulnerable than their wild relatives. The worldwide loss of crop yield to insects is
estimated to be 15 % (Maxmen 2013). Loss of crops has been familiar to man as long
as he has been growing them. Locust pests were already well known in ancient Egypt.
Especially the Migratory locust (Locusta migratoria) belongs to the most voracious
pests and at the same time to the most difficult pests to control. A high local juvenile
population density stimulates individuals to adapt their physiological development.
They grow into adults with effective wings and lightweight energy reserves. These
individuals start migrating (Fig. 12.1) in search for new food sources. During their
flight they mix with other groups of the same species and eventually these swarms
may become incredibly large. Swarms have been observed with an estimated number
of 70 billion individuals, ten times as many individuals as the total human world

E. van der Meijden ()

Institute of Biology, Leiden University, P.O. Box 9505, RA 2300 Leiden, The Netherlands
Tel.: +31715275119
e-mail: e.van.der.meijden@biology.leidenuniv.nl

© Springer International Publishing Switzerland 2015 103

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_12
104 E. van der Meijden

Fig. 12.1 A swarm of locusts is landing, an insect pest develops. (Reproduced with permission by
FAO (©FAO/Yasuyoshi Chibam))

population. Locusts feed on Sorghum, maize and wheat and other grass species. A
recent outbreak led to 42 % reduction in farmland grass production of 36,000 ha in
Northern China in 2003 (Tanaka and Zhu 2005).
Interestingly, already long ago (2500 BC) Sumerians used sulphur compounds
to control insects and mites (Dent 2000). Recent control methods include warning
systems with drones, insecticides, resistant plant varieties and biological control
with predators, parasitoids or insect pathogens. Despite immense scientific efforts,
plagues continue to affect plants and consequently human food supply. It may be
argued that we will probably never get completely rid of insect (and other) pests
(Chaps. 9, 10, 11, and 13). What makes insects such outstanding guerilleros? Their
short generation time (relative to that of their food sources) and their extremely high
fecundity (their potential number of offspring), make them evolutionarily extremely
successful. They can often adapt to insecticides and to new resistance genes within
a few generations. Moreover, for each plant species probably several hundreds of
insect herbivore species exist that may grow into effective pest species.
To demonstrate how insect pests work, how they develop and how some pests are
in the waiting room to develop, I will give some examples. This continuous battle
is probably best illustrated by one of the most important crop plants worldwide,
maize. Maize (Zea mays) originates from Central America and has been grown for
more than 6000 years. Today maize is grown all over the world. By far the largest
part of the maize crop is fed to livestock; smaller amounts serve as human food
or are turned into ethanol. Economically, maize is extremely important but it is
vulnerable to insect feeding. In the U.S. more than 100 different insect species were
reported to cause important pest damage. Some of these can be dealt with by using
insecticides. Others—like the European corn borer (Ostrinia nubilalis)—feed inside
12 Herbivorous Insects—A Threat for Crop Production 105

the stem, in immature kernels, or inside the roots. In these cases insecticides are less
effective. Alternative measures are biological control by insect parasitoids, viruses
or microorganisms. Another alternative is the development of resistant varieties by
genetic modification. Products containing the soil bacterium Bacillus thuringiensis
(Bt) have been known to be effective insecticides since the first half of the last
century. More recently, several toxic proteins were detected and the coding genes
were identified (Chap. 40). Maize is now one of the crop plants for which varieties
have been developed which harbour Bt-genes. They produce the Bt-proteins, which
make them resistant to several butterfly, moth and beetle pests such as the European
corn borer. These genetically modified varieties have become very popular in some
countries, like the USA and Spain. It must be stressed however, that similar to natural
varieties of wild plants and varieties of crop plants that have been developed with
traditional breeding techniques, these phenotypes are prone to loss of their resistance
by natural selection and evolution of their insect herbivores. Counter resistance is
especially expected to develop when host plants are grown in large monocultures
over longer periods of time. In 2012, Gassmann reported the first examples in which
the European corn borer broke through the resistance of Bt-maize in fields in Iowa
that had been grown with this variety for 3 to 6 years. Right now it has become
an urgent question how to deal with these economically extremely important plant
resistance characteristics. This makes future pest control one of the most urgent and
at the same time most exciting fields of the biological sciences.
A very ancient but yet illustrative example of loss of resistance comes from the
grape vine. Wine production has been of great economic importance for ages, prob-
ably even before the Greek and Roman cultures. In 1864 a disease was observed
among grape vines in Southern France that eventually affected most European vine-
yards. It took quite some time before it was realized that the disease was in fact
caused by sap sucking on leaves and roots by the Grape phylloxera. This small,
aphid-related, insect may even kill vines. In France the total production of wine
between 1875 and 1889 was reduced by three quarters. The insect, which is native
to North America, must have been accidentally introduced in France. Native vines
in North America, however, were not seriously affected by this herbivore. The so-
lution to control the Grape phylloxera in Europe has been to graft native varieties
onto phylloxera-resistant North American rootstocks (Campbell 2006). However, be
careful. Also the grape phylloxera is continuously evolving and is presently causing
disasters in Northern California.
Each year crop failures due to insect herbivores take place worldwide, sometimes
only locally, sometimes on a very large scale. All major and minor crops are vulner-
able. Rice, the second most important crop plant with its main areas of production in
China and Thailand, is frequently subject to crop losses of 30 % due to pest insects
like the Rice brown plant hopper. Cassava, an important crop of Africa, is frequently
subject to large-scale destruction by the Cassava mealy bug, the Cassava green mite,
the whitefly Bemisia tabaci, locusts and other insects. Bemisia is not only a serious
threat itself, it is also a vector for the Cassava mosaic virus (CMV). Yield losses in
Africa by CMV have been estimated up to 50 %.
106 E. van der Meijden

The climate of our planet will change in the future as it did in the past. There
are several indications that global warming takes place. Will warming affect the
influence of insects on human crops? Population dynamics of insects are usually
affected to a great extent by weather conditions. Weather can have direct and indirect
effects. Especially drought conditions may lead to insect pest development (a. o.
White 1969). Under drought conditions that are limited in duration, sap-feeders may
benefit from stress-induced increases in plant nitrogen. If our climate continues to
change towards higher temperatures and periods of drought, it is very likely that we
will be faced with more insect outbreaks all over the world in all kinds of crops from
timber to food crops like wheat, maize and potato (Maxmen 2013).

12.2 Pest and Beneficial Insects

The overwhelming variety of insect species that feed on plants may be challenging
for an entomologist, but is an absolute threat for plant growers. There are more than
a million different insect species and it is estimated that more than 360,000 species
are plant feeders.
Insects of some orders undergo complete metamorphosis. Larvae turn into pu-
pae, and pupae into adult insects. The larval stage is usually the most voracious
life stage. This is typical for beetles (Coleoptera), butterflies and moths (Lepi-
doptera) and flies (Diptera). In other groups, like the bugs, whiteflies, leafhoppers and
aphids (Hemiptera), locusts and grasshoppers (Orthoptera) and thrips (Thysanoptera)
there is so-called incomplete metamorphosis during which immature stages (called
nymphs) already resemble the mature stage.
Beetle larvae, with chewing mouthparts, often feed on or inside plant roots or
stems, or behave as leaf-miners. Adult beetles feed on leaves of trees, shrubs or
herbs. Some species are specialized on seeds. Lepidopteran larvae are mainly leaf
chewers. Leaf chewers can have very different feeding patterns; some start eating at
the leaf rim, some make feeding holes, and others skeletonise leaves. Hemiptera have
piercing mouthparts that enable them to feed from the vascular system of plants or
even from individual cells. In this way they can avoid feeding contact with particular
(less palatable) tissues. Locusts and grasshoppers are leaf chewers; thrips suck from
epidermis cells. Although all insects may be vectors of plant diseases by transmission
of bacteria, fungi or viruses from one plant to another, especially the groups with
piercing mouthparts bring along this extra threat for plants. Because they feed with
their mouthparts inside plants, they may be difficult to control with insecticides.
Beneficial insects are found in the orders of Diptera (flies) and Hymenoptera (bees,
wasps and ants). Both orders have pollinators and parasitoids of herbivorous insects.
These parasitoids may be extremely important in controlling pest species. Several
companies, (like Koppert: http://www.koppert.com/) are specialized in breeding par-
asitoids for crop protection and pollinators for glasshouse environments. Among the
12 Herbivorous Insects—A Threat for Crop Production 107

beetles, the bugs and the ants, several species are specialized as predator of her-
bivorous insects. Because they immediately kill their prey upon finding it, their
herbivory-reducing effect may be considerable.

12.3 Which Characteristics Make Insects Dangerous?

For each individual plant, the immune system provides protection against by far the
largest majority of 360,000 potential herbivore species. However, they are quite vul-
nerable to a much smaller group, the so-called specialists. These specialists (a few
to more than a hundred different insect species per plant species) have apparently
penetrated the plant’s immune system during their (co)evolution. These particular
plants have become their specific food plants. Specialist insects often use the defense
substances of their food plants to recognize and locate these plants. This phenomenon
will be illustrated with an example of Brassica species and their herbivores. Brassica
species like Cabbage and Oilseed rape (Fig. 12.2) contain a large group of specific
chemical substances, the glucosinolates. We are all familiar with these substances
because of their distinct “cabbage smell”. Specialist insect herbivores like the Di-
amondback moth (Plutella xylostella), the Cabbage white butterflies (Pieris spec.)
and the Crucifer flea beetle (Psylliodes chrysocephala), all important pest species of
Brassica on a worldwide scale, use these glucosinolates to find their food plants and
to start feeding. Generalist feeders (other insects, birds and slugs, etc) on the other
hand, are effectively repelled by the same substances (Fig. 12.2). Alkaloids constitute
another group of plant substances that is highly toxic to generalist herbivores (like
horses and cattle) but not to specialists. Small amounts of alkaloids spread on pieces
of filter paper are sufficient to attract individuals of the specialist Cinnabar moth
(Macel and Vrieling 2003). This leads to an awkward dilemma for plant breeders
and plant protection in general. The use of these plant substances as insecticides
may increase the level of defense of crop plants towards generalist herbivores. At the
same time it makes them more attractive to the specialists.
All insects of crop plants are potential pest species. If they can multiply fast,
they will soon cause damage. Under natural circumstances the low number of food
plants available and the presence of natural enemies, like parasitoids, will (often)
keep numbers low. However, under agricultural circumstances large monocultures
provide them with excess of food. Development of populations of natural enemies
large enough for control can only follow after a time lag of at least one generation.
That means after at least one season of crop growth. A special group of insects that
may cause crop pests are the so-called invaders. We have seen examples of the Grape
phylloxera (Daktulosphaira vitifoliae), colonizing Europe from the United States,
and the European corn borer colonizing the United States. These species enter a new
continent without their natural enemies which under natural circumstances might
control them. This clearly gives them a head start in their new environment.
108 E. van der Meijden

5
Adult flea beetle damage

0
0 5 10 15 20 25 30 35
a Total leaf glucosinolates (µmol/g DW)

60
Bird grazing (% leaf area)

50

40

30

20

10

0
0 5 10 15 20 25 30 35
b Total leaf glucosinolates (µmol/g DW)

Fig. 12.2 a Relationship between (specialist) Crucifer flea beetle damage and total leaf glucosi-
nolates in Oilseed rape (Brassica rapa). (Inserted photograph of Crucifer flea beetle by Richard
Mithen). b Relationship between (generalist) bird feeding and total leaf glucosinolates in Oilseed
rape. (Inserted photograph of oilseed rape by Eddy van der Meijden. Figures redrawn from
Giamoustaris and Mithen (1995) by permission of the publisher)
12 Herbivorous Insects—A Threat for Crop Production 109

12.4 The Plant’s Immune System, Step by Step

Firstly, each plant produces a blend of volatile chemicals. These substances pass
through the stomata and cuticle and surround the whole plant. Such a cloud may
contain a few dozens to several hundreds of different compounds. Undamaged plants
emit leaf volatiles. Upon damage the blend of compounds may change considerably.
The production of some new substances is induced by herbivore damage. They are
plant-species specific and their composition sometimes also depends on the particular
herbivore. Leaf volatiles constitute the outer layer of a plant’s immune system. They
provide information to the majority of herbivores that can smell that a particular plant
is not their host plant and is consequently unsuitable to feed or lay eggs upon. As was
mentioned earlier, each plant has also some specialist herbivores that are not repelled
by the plant’s immune system, and these use these volatiles to find their particular
host plant. Insects have advanced olfactory organs in their antennae that enable them
to sense particular volatile substances and blends in extremely low concentrations
(Schoonhoven et al. 2005).
The second component of the plant’s immune system that an insect has to deal
with is the outer surface, the epidermis. It provides protection by its toughness
caused by lignin and cellulose. Grasses in general, are three times tougher than herbs
(Schoonhoven et al. 2005). The epidermis is covered with a wax layer which contains
a great variety of molecules that play an essential role in plant defense. Insects can
sense these substances with their antennae and with the sense organs in their tarsae.
The maintenance of the chemical composition of these compounds in the wax layer
is an active process. Trichomes on the leaf surface are penetrating through this layer.
They may be glandular or non glandular. In the first case they may secrete repelling
or even toxic substances.
The insects that have not been stopped by leaf volatiles and other external defenses
are subsequently confronted by a world that is dominated by an incredible variety of
complicated chemical substances within the plant. These chemicals do not play an
important role in the primary activity of growth, and are therefore called secondary
metabolites. Up till now about 200,000 of these substances have been detected.
Many of them reduce herbivory by particular insect species. This is the constitutive
defense system of plants. These metabolites may be toxic or just repelling. Some
act as digestibility reducers. For instance, saponins inhibit enzymes in the gut of
insects that digest proteins. Some even act as attractors of pollinators. Some of
these compounds clearly have several different functions within a plant. Most of the
substances in the cloud of leaf volatiles are also secondary metabolites.
The glucosinolates or mustard oil glucosides are characteristic for the Brassi-
caceae, a plant family with more than 3500 species. All the cabbage varieties—Black
mustard, Indian mustard and Oilseed rape—belong to this family, but also the model
species for molecular research, Arabidopsis thaliana. About 120 different glucosi-
nolates have been identified and each different species usually contains twenty or
more of them, providing a specific fingerprint for that particular species. Typical is
that they contain at least one glucose residue, one sulphur and one nitrogen atom.
110 E. van der Meijden

Some other groups of secondary metabolites are the alkaloids (16,000) that occur in,
for instance, the Solanaceae (a. o. potato, tomato) and the sesquiterpenes (6500) of
the Asteraceae (a. o. sunflower and artichoke).
Not all plant parts contain the same concentration of secondary metabolites.
Hound’s tongue (Cynoglossum officinale) is a poisonous plant with a high concentra-
tion of pyrrolizidine alkaloids. However, young leaves may have a ten- to fifty-fold
higher concentration than old leaves. Specialist herbivores and generalist herbivores
that were fed on these leaves demonstrated a totally different preference. Specialists
fed predominantly on the younger leaves; generalists avoided these leaves and fed
on the older leaves with much lower concentrations (Van Dam et al. 1995; Fig. 12.3).
In general, much higher concentrations of secondary metabolites are found in
the reproductive organs of plants and in younger leaves that are important for future
photosynthesis, than in older leaves. The former plant parts are thus better protected
(against generalist herbivores). The Hound’s tongue example demonstrates that less
important plant parts (from the plant’s point of view) with low concentrations may
be attacked by generalist herbivores. Significantly different patterns in secondary
compounds were even detected among cell layers, like epidermis and mesophyll
(Nuringtyas et al. 2012).
Insect herbivore-challenged plants do not only act passively with their constitutive
defenses, but also respond to herbivory with the production of toxins and defensive
proteins that target physiological processes in insects (Howe and Jander 2008). This
is the extremely important inducible defense system. Contrary to the constitutive de-
fenses, induced responses follow upon particular damage cues and may thus be more
directed towards defense against the specific herbivore that is causing the damage.
To respond in this specific way, plants should be able to recognize herbivore species.
Such recognition has been earlier found in several plant-pathogen studies (Chap. 14).
During the past 20 years many experimental studies have demonstrated differences
in plant physiological responses to mechanical damage and insect herbivory. These
are the result of differences in induced gene expression patterns and transcriptional
responses.
There is strong evidence from experimental studies that plant defense is induced
by the oral secretions of insects (Howe and Jander 2008). The presence of fatty acid-
amino acid conjugates (FACs) in insect oral secretions was found to be an important
induction elicitor. These substances are derived from moieties from both insect and
host plant. There are indications that plants have specific FAC receptors. Other
insect and plant derived substances with similar functions are being studied. This
field of research is very actively developing right now. Signal transduction pathways
from insect signals to induced plant responses are still relatively unknown. What
we do know, is that especially the jasmonates play a crucial role in signalling and
regulating defense responses to insect herbivory. Many studies have demonstrated
that jasmonate mutants lack the ability to induce defenses against a wide variety of
insect species, whereas experimentally application of jasmonate to leaves increases
the level of defenses (Howe and Jander 2008).
12 Herbivorous Insects—A Threat for Crop Production 111

6
PA concentration (mg/g FW)
5

0
1 2 3 4 5 6 7 8 9 10
70

60
% of leaf area eaten

50

40

30

20

10

0
1 2 3 4 5 6 7 8 9 10
6
% of leaf area eaten

0
1 2 3 4 5 6 7 8
Leaf number

Fig. 12.3 a Pyrrolizidine alkaloid concentration in leaves of Hound’s tongue (1 is the youngest
leaf). b Fraction of leaves eaten by the generalist Helix Aspersa; (Inserted photograph of Helix
aspersa by Eddy van der Meijden.) c Fraction of leaves eaten by the specialist Mogulones cruciger.
(Inserted photograph of Mogulones cruciger by Henri Goulet, Agriculture and Agri-Food Canada.
Figures redrawn from Van Dam et al. (1995) by permission of the publisher)
112 E. van der Meijden

How Specific is the Plant Immunity System? The impression that one gets today
from a wide variety of studies on different species from feeding guilds like leaf
chewing caterpillars to phloem feeding aphids, is that the type of defense reaction
is more related to feeding guild than to individual insect species (Howe and Jander
2008).

12.5 Tritrophic Systems

Why would a plant change its production of leaf volatiles after herbivory? One
possibility is that it follows from damage without any particular function. Stud-
ies demonstrate an incredible variety of organisms that can perceive these volatile
signals. During the last few decades, evidence has been collected that natural ene-
mies of herbivores—parasitoids, predators and pathogens—use this induced blend
of compounds to locate their hosts and prey. Insect-eating birds (great tits) are being
attracted by volatiles of trees with damage caused by caterpillars. Predatory soil-
living nematodes are attracted by volatiles to feeding sites of larvae of the Western
corn borer in maize roots. It is tempting to believe that the induction process was the
next evolutionary step after the immune system of the plant was hacked by specialist
herbivores. Induction of plant volatiles provides information to parasitoids, predators
and pathogens that may lead to parasitization or predation of insect herbivores. If
this eventually would lead to a reduction of herbivore damage and a higher fitness
of individual plants, natural selection would favour this type of induced response.
Another function of the production of volatiles by herbivore-damaged leaves might
be that they signal other leaves of the same plant to become induced so that further
damage can be restricted. An alternative would be to signal through the phloem.
Future research will give the answer, but it is clear that rapid reactions are extremely
important for limiting herbivore damage.
How fast inducible responses through volatile emission work, is beautifully illus-
trated by a study of Allmann and Baldwin (2010). Coyote tobacco in the Western
U.S. produces nicotine as a constitutive defense substance. Nicotine is toxic to most
insects, but not to the Tobacco hornworm, a specialist insect herbivore of tobacco.
Mechanically damaged leaves produce a. o. the volatiles hexenal and hexenol. So
do Tobacco hornworm-damaged leaves. But there is an important difference. The
cloud of volatiles of mechanically damaged leaves is dominated by z-isomers of these
substances, whereas the tobacco hornworm-damaged leaves have more or less equal
concentrations of z- and e-isomers. The change from the production of e-isomer
dominated volatiles to z-e balanced blends takes place within a few minutes after
the onset of damage, and is induced by the oral secretion of the specialist herbivore.
Oral secretions of two generalist insect herbivores had no such effect. Blends with
predominantly e-isomers were much more attractive for an important predator of the
tobacco hornworm, the predatory bug Geocoris. By producing the induced blend
locally in an extremely fast way, predation may start immediately and the search
activity of the predator is guided by signals of the damaged leaf to its prey.
12 Herbivorous Insects—A Threat for Crop Production 113

12.6 Insect Symbiotic Microbes Hijack Plant Defense Signalling

There are relatively few studies on the role of microorganisms on insect-plant inter-
actions, but that is not a reliable indicator for their impact. Both insects and plants
carry microbial pathogens and symbionts. One of the most important functions of this
symbiosis is that microbial symbionts provide insects with (essential) amino acids
that are not available in their food plants. They also play a role in resistance against
natural enemies. Microbial symbionts of plants fulfil the same roles: the uptake of
nutrients and the protection against pathogens.
Recent studies suggest that microbial symbionts of herbivorous insects play a
crucial role in modifying food plant defenses. Larvae of the Colorado potato beetle
produce oral secretions that suppress the induced defenses in tomato and potato. To
unravel the mechanism, larvae were fed either antibiotic-treated leaves or non-treated
leaves. The first group was not able to suppress the jasmonate regulated defenses.
The second group did suppress these defenses. The bacteria in the insect’s oral secre-
tion (belonging to the genera Stenotrophomonas, Pseudomonas and Enterobacter)
elicit salicylic acid-regulated defenses. This negatively cross-talks with jasmonate
signalling, which in turn disables the plant to fully activate its jasmonate-mediated re-
sistance. Apparently the food plant does not recognize the insect herbivore any more,
but instead it defends itself against a microbial attack (Chung et al. 2013). Several
more or less similar experiments have been published now, which gives support to
the idea that we are dealing with an important phenomenon. Further study in this
particular field is expected to give information on why plant defense sometimes fails.

References

Allmann S, Baldwin IT (2010) Insets betray themselves in nature to predators by rapid isomeration
of green leaf volatiles. Science 329:1075–1077
Campbell C (2006) The Botanist and the Vintner: how wine was saved for the world. Algonquin
Books, Chapel Hill
Chung SH, Rosa C, Scully ED et al (2013) Herbivore exploits orally secreted bacteria to suppress
plant defenses. Proc Natl Acad Sci U S A 10:15278–15733
Dent D (2000) Insect pest management, 2nd edn. cabi Wallingford
Gassmann AJ (2012) Field-evolved resistance to Bt maize by western corn rootworm: predictions
from the laboratory and effects in the field. J Invertebr Pathol 110:287–293
Giamoustaris A, Mithen R (1995) The effect of modifying the glucosinolate content of leaves of
oilseed rape (Brassica napus ssp oleifera) on its interaction with specialist and generalist pests.
Ann Appl Biol 126:347–363
Howe GA, Jander G (2008) Plant immunity to insect herbivores. Annu Rev Plant Biol 59:41–66
Macel M, Vrieling K (2003) Pyrrolizidine alkaloids as oviposion stimulants for the cinnabar moth,
Tyria jacobaeae. J Chem Ecol 29:1435–1446
Maxmen A (2013) Under attack. The threat of insects to agriculture is set to increase as the planet
warms. What action can we take to safeguard our crops? Nature 501:15–17
Nuringtyas TR, Choi YH, Verpoorte R et al (2012) Differential tissue distribution of metabolites in
Jacobaea vulgaris, Jacobaea aquatica and their crosses. Phytochemistry 78:89–97
114 E. van der Meijden

Schoonhoven LM, van Loon JJA, Dicke M (2005) Insect-plant biology, 2nd edn. Oxford: Oxford
University Press
Tanaka S, Zhu DH (2005) Outbreaks of the migratory locust Locusta migratoria (Orthoptera:
Acrididae) and control in China. Appl Entomol Zool 40:257–263
Van Dam NM, Vuister LWM, Bergshoeff C et al (1995) The ‘raison d’ê tre’of pyrrolizidine alkaloids
in Cynoglossum officinale—Deterrent effects against generalist herbivores. J Chem Ecol 21:
507–523
White TCR (1969) An index to measure weather-induced stress of trees associated with outbreaks
of psyllids in Australia. Ecology 50:905–909
Chapter 13
Phytopathogenic Viruses

Carmen Büttner, Susanne von Bargen and Martina Bandte

Abstract Plant viruses are small sized plant pathogens. They are obligate parasites
and among the major limiting factors to modern agriculture. The incidence of plant
viruses has been shown in woody and herbaceous plants, soil and surface waters.
Many of them have a wide host range and are characterized by efficient virus trans-
mission. Since curative plant protection measures are lacking, it is important to
focus on preventive measures according to phytosanitary practices, interruption of
transmission pathways and vector control to combat plant viruses in practical crop
production. Viral diseases require a constant vigil. The suitability and efficacy of
different measures depend on the specific characteristics of the virus and the biology
of the plants, potential vectors, and the environment.

13.1 Characteristics of Plant Viruses

Viruses are responsible for severe losses in crop plants worldwide. Annual crop losses
caused by plant viruses were estimated in billions of US$. To date, more than 400
different plant virus species have been described and are summarized in the Ninth
Report of the International Committee on Taxonomy of Viruses (King et al. 2012).
Among plant viruses, Tomato spotted wilt virus (TSWV) is responsible for nu-
merous epidemics in different regions of the world and considered to have one of
the largest host ranges including at least 1090 plants species. It causes reductions in
yield of 1 billion USDs annually while losses due to African cassava mosaic virus

C. Büttner () · S. von Bargen · M. Bandte

Division Phytomedicine, Faculty of Life Sciences, Humboldt-Universität zu Berlin,
Lentzeallee 55/57, 14195 Berlin, Germany
Tel.: +49-30-2093-46445
e-mail: carmen.buettner@agrar.hu-berlin.de
S. von Bargen
Tel.: +49-30-2093-46447
e-mail: susanne.von.bargen@agrar.hu-berlin.de
M. Bandte
Tel.: +49-30-2093-46447
e-mail: martina.bandte@agrar.hu-berlin.de

© Springer International Publishing Switzerland 2015 115

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_13
116 C. Büttner et al.

Fig. 13.1 Electron micrographs of different particle morphologies of plant viruses and intercellular
movement via plasmodesmata. Rod shaped particles of Tobacco mosaic virus (TMV ) 300 nm in
length (a). Flexible particles of Potato virus Y (PVY ) with an estimated length of 800 nm (b).
Isometric particles with a diameter of approx. 28 nm of Cherry leaf roll virus (CLRV ) (c) and cell-
to-cell transport of the virus via tubules induced at plasmodesmata of Sambucus nigra cells (d).
(Figures 13.1a, b, c are from the authors. Figure 13.1d has not been published previously and was
kindly provided by J. Hamacher, Agro-Horti Testlabor, Bonn, Germany)

(ACMV) and related species, involved in the cassava disease pandemic in East and
Central Africa, are now estimated at US$ 1.9–2.7 billion (Scholthof et al. 2011).
Depending on the virus-host interaction and environmental conditions, the response
of the plant to virus infection may range from symptomless to severe disease or
even plant death. The induced symptoms include deformations in shape, undesirable
color, changes in taste or a reduction in keeping quality of the product.
Virus Properties Viruses are of small size, ranging within nanometers. They vary
from simple helical and icosahedral shapes to more complex structures (Fig. 13.1a,
b, c). Their morphology is only visible by electron microscopy. Viruses carry the
genetic information encoded in one type of nucleic acid, DNA or RNA. Transcription,
translation of viral proteins as well as replication of the viral genome is completely
dependent upon the cellular metabolism of the host. Therefore, plant viruses are often
considered replicators rather than forms of life. Some scientists classify viruses as
13 Phytopathogenic Viruses 117

microorganisms, but others consider them as nonliving. Rybicki (1990) described

viruses as "organisms at the edge of life". They replicate by creating multiple copies
of themselves through self-assembly processes, and evolve by natural selection but
do not metabolize.
Plant viruses have at least three genes encoding the capsid protein, the movement
protein and a protein accounting for nucleic acid replication.
The current state of taxonomy that follows a Linnaean-like classification of order,
family, subfamily, genus, and species is summarized by King et al. 2012. As more
sequence data have accumulated, more viruses have been placed in newly described
or existing taxa. The highest level of virus classification recognizes six major taxa,
based on the nature of the genome. In contrast to human and animal viruses the
majority of plant viruses have a positive sense single-stranded RNA (ssRNA (+))
which functions directly as messenger RNA after entering the host cell and release
from the virus particle. Only very few plant viruses have negative sense single-
stranded RNA (ssRNA (−)), single stranded DNA (ssDNA), or double-stranded
RNA (dsRNA).
The infection process of plants by viruses differs from that of other host’s such
as fungi, bacteria and animals. The plant has a robust cellulose cell wall covered
by a waxy epidermis and viruses cannot penetrate them unaided. The entry into
the cell succeeds in most cases through wounds by mechanical damage and/or vec-
tor assistance. In contrast entry of viruses infecting bacteria or animals is usually
mediated by cell surface receptors. There are also differences in the distribution of
viruses within the plant. Plasmodesmata enable virus movement by cytoplasmatic
connections between cells and vascular tissue throughout most parts of the plant
(Fig. 13.1d).
Transmission The mode of transmission is characteristic for each virus and often
serves as a starting-point for plant protection management. Few viruses, such as
tobamoviruses, rely on passive mechanical transmission from plant to plant. These
viruses belong to the stable ones which can be easily transmitted also through water
and soil, and they were detected even in clouds (Büttner and Koenig 2014). Other
viruses can be transmitted by pollen and seeds (Sastry 2013). The major types of
vector organisms of plant viruses are plant-feeding insects, nematodes and plant-
parasitic fungi (Bragard et al. 2013). The economically most important insect vectors
are restricted to a few hemipteran families such as aphids (Aphididae), whiteflies
(Aleyrodidae), leafhoppers (Cicadellidae) and delphacid planthoppers (Delphacidae)
(Hogenhout et al. 2008). For instance, aphids transmit about 200 plant virus species of
which 110 belong to the genus Potyvirus. Whiteflies transmit 128 virus species; 115
of them are solely transmitted by these vectors and belong to the genus Begomovirus.
Four different mechanisms of insect transmission of plant viruses have been described
to date. If the vector retains the virus for only a few seconds/minutes after acquisition
of the virus from plants and the virus is lost after molting (ecdysis), the transmission
mode is called non-persistent. In contrast for persistent viruses, the vector retains the
virus for long periods (days to weeks), often throughout the vector’s lifespan, and
keeping it even after molting. Semipersistent viruses display an intermediate category
118 C. Büttner et al.

whereupon the vector retains the virus for a few hours to a few days and vectors lose
it upon molting. Propagative viruses not only infect plants but also use the vectors
as hosts as they are able to invade and replicate in various tissues of their vectors.
Plant viruses can induce changes in vector behavior, survival and performance that
promotes transmission efficiency (Ingwell et al. 2012; de Oliveira et al. 2014).
To date, 14 different viruses are known to be transmitted by ectoparasitic nema-
todes. These viruses belong to two genera (ii) nepoviruses which are transmitted by
nematodes belonging to the family Longidoridae (genera Longidorus, Paralongi-
dorus, and Xiphinema) and (ii) tobraviruses which are transmitted by nematodes
from the family Trichodoridae (genera Trichodorus and Paratrichodorus). Ectopar-
asitic nematodes feed from the outside of the root, most often targeting the region at
or near the root tip.
Six fungal species vector plant viruses: all are soilborne zoosporic obligate en-
doparasites that belong either to the protists (plasmodiophorids) or to the chytrid
fungi (Olpidium). For instance, Polymyxa betae transmits four viruses to sugar beet,
Polymyxa graminis transmits 14 viruses to cereals and groundnut, and the chytrid
fungi Olpidium brassicae, Olpidium bornovanus, and Olpidium virulentus vector 15
viruses, all grouped in the Ophioviridae and Tombusviridae families as well as in the
unassigned genus Varicosavirus.

13.2 Virus Interference with Host Plants

The presence of plant viruses has been shown in herbaceous and woody plants. Many
of them have a wide host range and are characterized by efficient virus transmission,
the rapidity with which new variants arise, and difficulties in vector control. Some
hosts are more studied than others, e.g. investigations of viruses in forest and urban
trees are rare (Gonthier and Nicolotti 2013).
Symptoms of viral diseases vary according to the virus and its host. The effect
may range from severe and easily perceived to negligible depending on the plants’
predisposition. The most evident symptoms are caused by a systemic infection.
Deformation and coloring appear in fruits, leaves, stems, roots, or other parts of the
plant, leading directly to crop yield reductions or losses of quality and/or quantity.
One common symptom is hyperplasia: the abnormal proliferation of cells that causes
the appearance of plant tumors known as galls. Other viruses induce hypoplasia, or
decreased cell growth, in the leaves of plants, causing thin, yellow areas.
Virus-Host Interactions One of the most effective defense mechanisms of plants is
the presence of resistance genes. Each of these genes confers resistance to a particular
virus by triggering localized areas of cell death in the proximity to the infected cell,
which is often visible as necrotic lesions. This phenomenon prevents the viral infec-
tion from spreading within the plant. Local lesions are often induced under experi-
mental conditions when inoculating indicator plants within the scope of bioassays.
Latent viruses do not cause symptoms in one crop, but may do so in another crop.
Another central part of the plants resistance response to virus infection and
spread is called RNA silencing. It is a conserved intrinsic mechanism in eukaryotes,
13 Phytopathogenic Viruses 119

Virus entry

genome replicaon
uncoang

virus life cycle

virus transport
protein expression
DCL assembly

DCL systemic infecon and

silencing of symptom expression
vRNA suppression of
RNA silencing
primary
secondary
siRNA
siRNA
RDR
RISC
slicing DCL
siRNA amplificaon

siRNA induced anviral siRNA induced anviral

immunity without immunity without
symptoms symptoms

Fig. 13.2 Schematic representation of plant-virus interactions in host plant tissue. Infection process
of a plant virus (virus life cycle), RNA-based antiviral immune response of the host plant cell
(silencing of viral RNA, amplification and systemic spread of small interfering RNA-based signal),
and counter strategy of the virus (suppression of RNA silencing) leading to mosaic symptoms.
DCL dicer like protein, RISC RNA-induced silencing complex, RDR plant RNA-dependent RNA
polymerase, VSR viral suppressor of RNA silencing, CP coat protein, MP movement protein. (This
figure was created by the authors)

mediating antiviral defense via sequence-specific small RNA molecules. Virus-

induced RNA silencing and regulation of endogenous plant genes are the major
components of the mechanism called RNA interference (RNAi). In plants it is a key
regulator of gene expression and involved in many developmental processes. In the
RNA-based antiviral immunity, viral double-stranded RNAs, produced as replicative
intermediates or present in viral genomic RNAs with extensive hairpin structures, are
recognized by a host specific pathway as pathogen-associated molecules (Fig. 13.2).
This silencing pathway leads either to post-transcriptional gene silencing (PTGS) by
translational repression or to RNA degradation by endonucleolytic cleavage (slic-
ing) of targeted cognate vRNAs. The aberrant RNAs generated by slicing can be
amplified by host RNA-dependent RNA polymerases (RDRs) and may accumulate
to high levels of secondary vsiRNAs serving as a systemic signal which triggers
specific antiviral immunity throughout the plant.
Plant viruses are efficient pathogens having evolved various strategies to avoid
or overcome silencing. Most notably, many viruses encode one or more proteins
functioning as viral suppressors of RNA silencing (VSRs). The functions of VSRs
are very diverse as they target different sites of the silencing pathway (Burgyan and
120 C. Büttner et al.

Havelda 2011). Many VSRs are also responsible for virus-induced symptoms and
are important pathogenic determinants and effectors of virulence in virus-host inter-
actions. Furthermore, it has been demonstrated that manipulation of the host’s RNA
interference pathway by viral pathogens affects the regulation of plant resistance
genes (R genes). This indicates a host counter-counter defense mechanism account-
ing for a close co-evolution between viruses as successful obligate parasites with
their host plants.
In general, plant viruses must infect their host systemically to cause a negative
effect on crop yield and/or quality. This viral spread involves the utilization of in-
tracellular and intercellular pathways of the host tissue which is mediated by virus
encoded movement proteins (MPs). Plant viruses employ different MPs facilitating
distribution of the virus from the site of entry into new tissues. Systemic infection
in combination with the RNA interference-based-defense mechanisms and counter
strategies employed by plant viruses described above are considered to be the main
causes of symptom induction by these plant pathogens. For instance, mosaic is the
most characteristic symptom of plant virus infections (Fig. 13.2). Mosaic is com-
prised of light green or yellow cell areas interspersed with dark green islands (DGIs).
DGI formation can be affected by VSRs thus indicating the plant’s defense mech-
anism through RNA silencing which are active in the tissues which have not yet
been invaded by the virus. The formation of mosaic symptoms are therefore a good
example of the multiple sites a plant virus interferes within its host. The under-
lying molecular mechanisms of these fundamental processes involved in the RNA
interference-based immune response of plants to viral attack and its relevance to
symptom induction are thoroughly discussed in Pumplin and Voinnet (2013), Wang
et al. (2012), and Palukaitis (2011).
Beneficial Aspects of Plant Viruses Some plant viruses have been discovered to
be beneficial and used by horticulturists to enhance the aesthetics of ornamental
plants. Desirable aesthetic value is realized through flower color breaking, vein
discolorations and foliar or flower variegations. Often this is the product of
mutations that affect plastid development or transposable genetic elements that
result in anomalous production of pigments but plant viruses can cause symptoms
that mimic genetic variegations. One of the oldest virus diseases recorded is tulip
breaking disease. Tulip breaking virus (TBV) causes the petals to variegate due
to the irregular distribution of pigments, instead of being uniformly colored. Such
an appearance may be considered a benefit due to its economic value, but most
virus-infected plants do indicate less vitality in the long term.

13.3 Management of Viral Diseases

Introduction of viruses to new areas, often a result of human-aided transport, and

change in vector dynamics are the main factors underlying the global emergence of
viral diseases. Higher levels of human management result in higher disease and virus
infection risk, which is associated with decreased habitat species diversity and host
genetic diversity, and with increased host plant density (Rodelo-Urrego et al. 2013).
13 Phytopathogenic Viruses 121

Phytosanitary Measures Virus disease management begins with a precise diagno-

sis of the pathogen. The most important tools of integrated disease management are
the use of virus free planting or sowing material, the removal of vectors that transmit
the virus as well as of weed populations serving as secondary hosts and the inter-
ruption of transmission paths while being aware of sources of contamination. The
quality of water and soil is a condition precedent to virus-free crop production. If
crops have to be irrigated, all potentially contaminated nutrient solutions have to be
controlled. Such contamination often occurs in surface water collected from inten-
sively cropped landscapes and in nutrient solutions of recirculating systems (Hong
et al. 2014). In order to identify possible sources of contamination threatening the
production a risk assessment shall be carried out.
Several procedures have been developed to deliver water that does not pose a risk
to infect crop plants. Just recently, Hong et al. (2014) summarized the knowledge on
biology, detection and management of plant pathogens in irrigation water.
Development of Virus Resistant Plants The above described measures focus pri-
marily on avoiding the (i) introduction of plant viruses into an area and (ii) dispersion
of plant viruses by controlling the vector (iii) interruption of all possible paths of
virus transmission. Removal of weedy hosts and volunteer crops can reduce the virus
reservoir but may contribute to a more homogenous agricultural landscape, which
in turn decrease populations of predators and parasitoids that may regulate vector
populations. With low cost-benefit ratio, environmental concerns and concomitant
development of insecticide resistance, other strategies should be taken into consider-
ation. An alternative control strategy for viruses is the use of resistant crop cultivars
or varieties. These resistant genotypes carry heritable traits that are responsible for
the suppression of virus multiplication or/and spread even under environmental con-
ditions favoring infection. Thus no additional costs arise for the grower during the
growing season. Virus resistance traits are available so that cultivars with varying
degrees of resistance have been obtained by conventional breeding and gained com-
mercial status. Technical solutions exist to provide transgenic virus resistance even
for those crop species where virus resistance traits are lacking. For instance coat pro-
tein genes of many plant viruses have been transformed into a wide range of many
plant species to obtain virus protection. Today, papaya, potato, squash, bean and
plum have been introduced to the global market with transgenic virus resistance. For
instance in 1998, two transgenic papaya (Carica papaya L.) with resistance to Pa-
paya ringspot virus (PRSV) were released for commercial cultivation in Hawaii
(Gonsalves 2014). The development and commercialization of these transgenic
cultivars controlled the devastating aphid-vectored disease and saved the papaya
industry in Hawaii. Currently, the cultivar Rainbow accounts for about 70 % of
Hawaii’s papaya acreage.
General Strategies Several national programs have been developed for the man-
agement of viruses that affect vegetatively propagated perennial plants including
fruit trees, small fruits, grapevines and hop plants. These programs are based on
virus-free stock providing certified virus-free propagation or growing material to
be used in nurseries and vineyards. Prevention and control of vector transmission
122 C. Büttner et al.

of plant viruses is imperative to promote sustainable agricultural practices and to

reduce species invasion. Vector transmission of plant viruses is multi-scale and
driven by characteristics of the specific virus, crop, and field properties. Therefore
a good understanding of plant viruses, their ecology and vectors is required to de-
velop and implement reliable prediction models for timely intervention. Intervention
can be conducted using chemical or biological methods, particularly in greenhouse
production.

References

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interference with vector transmission. Annu Rev Phytopathol 51:1–25
Burgyan J, Havelda Z (2011) Viral suppressors of RNA silencing. Trends Plant Sci 16:265–272
Büttner C, Koenig R (2014) Viruses in water. In: Hong C, Moorman GW, Wohanka W, Büttner C
(eds) Biology, detection and management of plant pathogens in irrigation water. ISBN 978-0-
89054-426-6. St. Paul, Minnesota, USA
de Oliveira CF, Long EY, Finke DL (2014) A negative effect of a pathogen on its vector? A
plant pathogen increases the vulnerability of its vector to attack by natural enemies. Oecologia
174:1169–1177
Gonsalves D (2014) Hawaii’s transgenic papaya story 1978–2012: a personal account. In: Ming R,
Moore P (eds) Genetics and genomics of papaya. Springer, New York, pp 115–142
Gonthier P, Nicolotti G (2013) Infectious forest diseases. Oxfordshire, UK
Hogenhout SA, Ammar ED, Whitfield AE et al (2008) Insect vector interactions with persistently
transmitted viruses. Annu Rev Phytopathol 46:327–359
Hong C, Moorman GW, Wohanka W et al (2014) Biology, detection and management of plant
pathogens in irrigation water. St. Paul, Minnesota, USA
Ingwell LL, Eigenbrode SD, Bosque-Perez NA (2012) Plant viruses alter insect behavior to enhance
their spread. Sci Rep 2, Article number 578. doi:10.1038/srep00578
King AMQ, Adams MJ, Carstens EB et al (2012) Virus taxonomy: classification and nomenclature
of viruses. Ninth report of the International Committee on Taxonomy of viruses. London, UK
Palukaitis P (2011) The road to RNA silencing is paved with plant-virus interactions. Plant Pathol
J 27:197–206
Pumplin N, Voinnet O (2013) RNA silencing suppression by plant pathogens: defence, counter-
defence and counter-counter-defence. Nat Rev Microbiol 11:745–760
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parasite interactions and results in apparent plant virus codivergence. Mol Ecol 22:2325–2340
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Microbe Interact 25:1275–1285
Chapter 14
Induced Disease Resistance

Corné M. J. Pieterse and Saskia C. M. Van Wees

Abstract During the co-evolutionary arms race between plants and pathogens, plants
evolved a sophisticated defense system to ward off their enemies. In this plant im-
mune system, plant receptor proteins recognize non-self molecules of microbial
origin, which leads to the activation of a basal level of disease resistance. The onset
of these local plant immune reactions often triggers a systemic acquired resistance
(SAR) in tissues distal from the site of infection. Beneficial microbes in the rhizo-
sphere microbiome stimulate a phenotypically similar induced systemic resistance
(ISR) that, like SAR, is effective against a broad spectrum of pathogens. There are
differences and similarities in the SAR and ISR signaling pathways. The plant de-
fense hormone salicylic acid is a major regulator of SAR, whereas jasmonic acid
and ethylene play important roles in ISR. Priming of systemic tissue to express an
accelerated defense response upon attack by a pathogen is a common phenomenon
in both SAR and ISR. This chapter will outline the current concept of the plant im-
mune system, with special emphasis on mechanisms of systemically induced disease
resistance and priming for enhanced defense.

14.1 The Plant Immune System

In the past decade, ground-breaking conceptual advances have been made in the
understanding of the evolutionary development and functioning of the plant immune
system (Jones and Dangl 2006). In the current concept of the plant immune sys-
tem, pattern-recognition receptors (PRRs) have evolved to recognize pathogen- or
microbe-associated molecular patterns (PAMPs or MAMPs), such as bacterial flag-
ellin or fungal chitin (Boller and Felix 2009). MAMP recognition is translated into

C. M. J. Pieterse () · S. C. M. Van Wees

Plant-Microbe Interactions, Department of Biology, Faculty of Science,
Utrecht University, PO BOX 800.56, 3508 TB Utrecht, The Netherlands
Tel.: +31302536887
e-mail: C.M.J.Pieterse@uu.nl
S. C. M. Van Wees
Tel.: +31302536861
e-mail: S.VanWees@uu.nl

© Springer International Publishing Switzerland 2015 123

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_14
124 C. M. J. Pieterse and S. C. M. Van Wees

a basal defense called pattern-triggered immunity (PTI) (Dodds and Rathjen 2010).
Successful pathogens evolved virulence effector molecules to bypass this first line
of defense, either by preventing detection by the host, or by suppressing PTI sig-
naling (Dodds and Rathjen 2010; Pel and Pieterse 2013). To fight these successful
pathogens, plants developed a second line of defense in which resistance (R) pro-
teins mediate recognition of attacker-specific effectors (formerly known as avirulence
factors), resulting in highly powerful effector-triggered immunity (ETI) (Dodds and
Rathjen 2010). ETI is a manifestation of the classic gene-for-gene resistance that is
accompanied by a hypersensitive response that prevents biotrophic pathogens from
further entry (Chap. 10).
Activation of PTI and ETI in locally infected tissues often triggers an induced
resistance in tissues distal from the site of infection and involves one or more long-
distance signals that propagate an enhanced defensive capacity in still undamaged
plant parts. This pathogen-induced systemic resistance is known as systemic acquired
resistance (SAR) (Fu and Dong 2013). While PTI and ETI are activated rapidly and
act locally to limit growth of the specific invader at the site of infection, SAR takes
more time to develop but confers an enhanced defensive capacity that is typically
effective against a broad spectrum of pathogens (Walters et al. 2013; Fu and Dong
2013).
Besides pathogen infection, also colonization of plant roots by beneficial
microbes has been shown to stimulate the plant immune system, resulting in
a phenotypically similar type of broad-spectrum disease resistance, commonly
referred to as induced systemic resistance (ISR) (Pieterse et al. 2014). Moreover,
insect herbivory and specific chemicals can also induce resistance (Howe and
Jander 2008; Pastor et al. 2013). After more than three decades of research, the
picture is emerging that the different forms of induced resistance are regulated by
a complex network of interconnecting signaling pathways in which plant hormones
play an important regulatory role (Pieterse et al. 2012). Induced resistance signaling
pathways that are triggered by pathogens, beneficial microbes, and insects partly
overlap and share common signaling components (Pieterse et al. 2014). This
provides plants with an enormous regulatory potential to rapidly adapt to their
biotic environment and to utilize their limited resources for growth and survival in
a cost-efficient manner. Intriguingly, successful pathogens evolved mechanisms to
rewire the plant’s hormone signaling network to suppress or evade the host immune
system (Robert-Seilaniantz et al. 2011; Pieterse et al. 2012), highlighting the central
role of plant hormones in the regulation of immunity.
The concepts of PTI and ETI that act locally in the plant immune system will be
discussed in more depth elsewhere in this issue (see Chap. 10). In this chapter we will
focus on the important principles and recent findings of induced disease resistance
that acts systemically throughout the plant.
14 Induced Disease Resistance 125

14.2 Pathogen-Induced Systemic Acquired Resistance (SAR)

Hallmarks of SAR The term SAR was first coined by Ross for the phenomenon
that uninfected systemic plant parts become more resistant in response to a prior
infection elsewhere in the plant (Ross 1961). SAR is typically triggered upon local
activation of a PTI or ETI response (Shah and Zeier 2013). In systemic tissues, SAR
is characterized by increased levels of the hormone salicylic acid (SA), one of the
hallmarks of SAR (Vlot et al. 2009) (Fig. 14.1). Early genetic studies in tobacco
showed that SA accumulation and signaling is essential for the establishment of
SAR (Vernooij et al. 1994). Another hallmark of SAR is the coordinate activation of
PATHOGENESIS-RELATED (PR) genes, several of which encode PR proteins with
antimicrobial activity (Van Loon et al. 2006). PR −1 is amongst the best characterized
PR genes and is in many plant species used as a marker for SAR (Van Loon et al.
2006; Fu and Dong 2013).
Long-Distance Signals Because the expression of SAR occurs in plant parts that
are distant from the site of induction, a long-distance mobile signal is required that
is produced locally and is responsible for the systemic onset SAR in still healthy tis-
sues. The identity of the mobile SAR signal(s) has been a subject of controversy for
many years. The lipid-transfer protein DEFECTIVE IN INDUCED RESISTANCE1
(DIR1) was shown to act as a chaperone for an unknown mobile SAR signal in the vas-
cular tissue (Maldonado et al. 2002; Champigny et al. 2011). Despite the fact that SA
accumulates in the phloem sap of SAR-expressing plants, grafting experiments with
tobacco showed that SA itself is not the mobile SAR signal (Vernooij et al. 1994). Re-
cent genetic and biochemical studies uncovered several plant metabolites involved in
long-distance SAR signaling. These include the methyl ester of SA (MeSA), the diter-
penoid (DA), a glycerol -3-phosphate (G3P)-dependent factor, azelaic acid (AzA),
and pipecolic acid (Pip) (Fig. 14.1). From these findings a more comprehensive view
on the identity and functioning of the long-distance SAR signals started to emerge in
which different signals may be operative under different environmental conditions
(Shah and Zeier 2013; Dempsey and Klessig 2012; Kachroo and Robin 2013). In
systemic tissues, the onset of SAR requires the function of FLAVIN-DEPENDENT
MONOOXYGENASE 1 (FMO1) (Mishina and Zeier 2006), possibly to transduce
or amplify long-distance signals originating from primary leaves, which then results
in enhanced SA biosynthesis in still healthy tissues.
SAR Signaling Upon activation of SAR, the SA signal is transduced by the redox-
regulated protein NONEXPRESSOR OF PR GENES1 (NPR1), which functions as
a transcriptional co-activator of a large set of PR genes (Fu and Dong 2013). In
non-stimulated cells, NPR1 is sequestered in the cytoplasm as an oligomer through
intermolecular disulfide bonds. Upon SA accumulation, changes in the cellular redox
state mediate monomerization of NPR1, which allows translocation of NPR1 into
the nucleus. In the nucleus, NPR1 interacts with TGA transcription factors that
together with WRKY transcription factors activate SA-responsive PR genes. Proper
functioning of NPR1 requires that the protein is broken down by the proteasome,
126 C. M. J. Pieterse and S. C. M. Van Wees

MPKs priming
priming of
SA-dependent JA/ET-dependent
Ac Me defense genes defense genes

TGA WRKY

SYSTEMIC
NPR1 MYC2 TFs

ISR
SAR SA
FMO1 NPR1

Pathogen SAR DIR1 ISR

infec on
SA-dependent
defense genes

NPR1

PTI

LOCAL
AzA Fe-deficiency
response ??
SA MeSA
DA
G3P
PTI ETI Pip MYB72

vola les
PRRs R protein PRRs
Rhizosphere
MAMPs
PAMPs

microbiome effectors

a b Pathogen Beneficial microbe

Fig. 14.1 a Schematic representation of biologically induced disease resistance triggered by

pathogen infection (SAR; red arrow) and colonization of the roots by beneficial microbes (ISR;
purple arrow). Induced resistance involves long-distance signals that are transported through the vas-
culature or as airborne signals, and systemically propagate an enhanced defensive capacity against
a broad spectrum of attackers in still healthy plant parts. b Schematic representation of molecular
components and mechanisms involved in pathogen-induced SAR and rhizobacteria-mediated ISR.
Solid black lines indicate established interactions; dashed black lines indicate hypothetical inter-
actions. Colored arrows indicate systemic translocation of long-distance signals (indicated in the
same color at the base of the arrows). Ac acetylation, ET ethylene, ETI effector-triggered immunity,
Fe iron, ISR induced systemic resistance, JA jasmonic acid, MAMP microbe-associated molecular
pattern, Me methylation, PAMP pathogen-associated molecular pattern, PRR pattern-recognition
receptor, PTI PAMP-triggered immunity, R protein Resistance protein, SA salicylic acid, SAR
systemic acquired resistance, TF transcription factor

possibly to allow new NPR1 proteins to reinitiate the PR transcription cycle (Spoel
et al. 2009). Recently, NPR1 and its paralogues NPR3 and NPR4 were identified as
SA receptors that bind to SA with different affinity thereby influencing the stability
of NPR1 (Fu et al. 2012; Wu et al. 2012).
14 Induced Disease Resistance 127

14.3 Induced Systemic Resistance (ISR) by Beneficial Microbes

Besides microbial pathogens, also large communities of commensal and mutual-

istic microbes interact with plants providing them with essential services, such as
enhanced mineral uptake, nitrogen fixation, growth promotion, and protection from
pathogens (Chap. 20; Lugtenberg and Kamilova 2009; Zamioudis and Pieterse 2012;
Pieterse et al. 2014). This community of microbes is predominantly hosted by the
root system and is also referred to as the rhizosphere microbiome (Chap. 28; Berend-
sen et al. 2012; Mendes et al. 2011). In 1991, it was demonstrated that colonization
of plant roots by selected strains of plant growth-promoting rhizobacteria (PGPR)
can stimulate the plant immune system in above-ground plant parts, resulting in
a broad-spectrum disease resistance called rhizobacteria-ISR (Fig. 14.1) (Van Peer
et al. 1991; Wei et al. 1991; Alström 1991). Since then, hundreds of studies in di-
cots and monocots have reported on the ability of PGPR to promote plant health
via ISR. These studies mainly involved Bacillus , Pseudomonas, and Serratia PGPR
strains. In addition, non-pathogenic plant growth-promoting fungi (PGPF) strains
from species like Fusarium oxysporum, Trichoderma spp., and Piriformospora in-
dica strains, but also symbiotic arbuscular mycorrhizal fungi have been shown to
trigger ISR (Pieterse et al. 2014).
Microbial Elicitors of ISR In order to stimulate ISR, beneficial microbes must pro-
duce elicitors that are responsible for the onset of systemic immunity. Early research
on MAMPs and other elicitors of ISR-inducing Pseudomonas and Bacillus PGPR
focused on the involvement of lipopolysaccharides (LPS) and the iron-regulated
metabolites pyoverdin and SA (De Vleesschauwer and Höfte 2009). Other micro-
bial ISR elicitors include antibiotics, like 2,4-diacetylphloroglucinol (DAPG) and
pyocyanin, flagella, N-acyl homoserine lactones, siderophores, and biosurfactants
(De Vleesschauwer and Höfte 2009). Also specific volatile organic compounds pro-
duced by beneficial microbes were demonstrated to elicit ISR (Ryu et al. 2004; Lee
et al. 2012). Several of these ISR elicitors were shown to act redundantly, indicat-
ing that multiple microbial elicitors can trigger common signaling pathway leading
to systemic immunity (Bakker et al. 2003). This resembles PTI in plant-pathogen
interactions, where recognition of multiple PAMPs is channeled into the same PTI
signaling pathway (Boller and Felix 2009).
Rhizobacteria-ISR Signaling Pathways Because of its broad spectrum effec-
tiveness, rhizobacteria-ISR was initially thought to be mechanistically similar to
pathogen-induced SAR. However, in radish it was shown that Pseudomonas flu-
orescens WCS417r (hereafter called WCS417r) triggered ISR without stimulating
the accumulation of the PR proteins that are characteristic for SAR (Hoffland et al.
1995). Also in Arabidopsis thaliana (Arabidopsis) WCS417r-ISR developed without
the activation of PR genes (Pieterse et al. 1996). Transgenic SA-nonaccumulating
Arabidopsis NahG plants mounted wild-type levels of ISR upon colonization of the
roots by WCS417r, providing genetic evidence that ISR can be mediated via an SA-
independent signaling pathway (Pieterse et al. 1996). Hence, rhizobacteria-mediated
128 C. M. J. Pieterse and S. C. M. Van Wees

ISR and pathogen-induced SAR are regulated by distinct signaling pathways

(Fig. 14.1). Analysis of a large number of ISR-triggering plant-beneficial microbe
interactions in which a role for SA had been functionally tested, revealed that the
ability to activate an SA-independent ISR pathway is common for beneficial mi-
crobes and occurs in a broad range of plant species (Van Loon and Bakker 2005; Van
Wees et al. 2008).
Although ISR by beneficial microbes is often regulated through SA-independent
mechanisms, certain strains of beneficial microbes have been reported to trigger ISR
in an SA-dependent fashion, which resembles pathogen-induced SAR (De Vleess-
chauwer and Höfte 2009; Van de Mortel et al. 2012). In these cases, reactive oxygen
species that accumulate at the site of tissue colonization seem to act as important elic-
itors (De Vleesschauwer and Höfte 2009). Since SA-dependent signaling triggered
by beneficial microbes is likely to follow the SAR signaling pathway, we refer to the
above section on pathogen-induced SAR for information on mechanisms underlying
this phenomenon.
Role of Jasmonic Acid and Ethylene in ISR After the discovery of SA as an
important defense hormone, also the plant hormones jasmonic acid (JA) and ethylene
(ET) emerged as important regulators of plant immunity (Pieterse et al. 2012). By
using JA or ET signaling mutants of Arabidopsis, it was shown that not SA, but
JA and ET are central regulators of WCS417r-ISR (Pieterse et al. 1998). For many
other PGPR and PGPF genetic evidence pointed to a role for JA and/or ET in the
regulation of ISR (Pieterse et al. 2014), supporting the notion that JA and ET are
dominant players in the regulation of SA-independent systemic immunity conferred
by beneficial soil-borne microbes.
Master Regulators of ISR The first regulatory protein identified as being essential
for rhizobacteria-ISR was NPR1 (Pieterse et al. 1998). While in SAR, NPR1 func-
tions as a transcriptional co-activator of SA-responsive PR genes, JA/ET-dependent
ISR typically functions without PR gene activation. Hence, the role of NPR1 in ISR
seems to be different from that in SAR. In SA signaling, NPR1 is clearly connected to
a nuclear function (Fu and Dong 2013), while in JA/ET signaling and ISR evidence
is accumulating for a cytosolic function of NPR1 (Spoel et al. 2003; Stein et al.
2008; Pieterse et al. 2012). Interestingly, simultaneous activation of SAR and ISR
leads to an additively enhanced defensive capacity (Van Wees et al. 2000). Whether
this is based on the notion that SAR and ISR do not seem to compete for the same
subcellular pool of NPR1 is unknown, as the exact molecular mechanism by which
NPR1 functions in JA/ET-dependent ISR remains to be investigated.
Although ISR involves long-distance root-to-shoot signaling, only few studies
have investigated the signaling components of the plant root that are involved in
the onset of ISR. Analysis of the transcriptome of WCS417-colonized Arabidopsis
roots revealed the R2R3 type MYB transcription factor gene MYB72 as one of the
significantly induced genes (Verhagen et al. 2004). In non-stimulated plants, MYB72
is lowly expressed in the root vascular bundle, but becomes highly expressed in
root epidermis and cortical cells upon colonization by ISR-inducing PGPR or their
volatiles (Zamioudis et al. 2014b). Knockout myb72 mutants are impaired in their
14 Induced Disease Resistance 129

ability to express ISR, indicating that this root-specific transcription factor is es-
sential for the onset of ISR (Van der Ent et al. 2008). MYB72 is also induced in
Trichoderma-colonized Arabidopsis roots and shown to be crucial for Trichoderma-
ISR (Segarra et al. 2009), suggesting that MYB72 is a node of convergence in the ISR
signaling pathway triggered by different beneficial microbes. Being a transcriptional
regulator, it was postulated that MYB72 plays an important role in the generation
and/or translocation of a long-distance ISR signal. Besides its crucial role in the on-
set of ISR, MYB72 is also implicated in the iron-deficiency response of plant roots
(Zamioudis et al. 2014a; Zamioudis et al. 2014b). How ISR and the iron-deficiency
response are interconnected is currently unknown.

14.4 Induced Disease Resistance: Priming for Enhanced

Defense

While SA accumulation and PR gene expression are hallmarks of SAR, ISR triggered
by beneficial microbes is lacking such universal characteristics associated with the
onset of systemic immunity. In many cases, colonization of plant roots by beneficial
microbes does not lead to major changes in defense-related gene expression in the
above-ground plant parts. Instead, pathogen infection or insect herbivory on ISR-
expressing plants often leads to an accelerated expression of defense-related gene
expression in comparison to similarly attacked control plants (Van Wees et al. 1999;
Van Oosten et al. 2008). Large-scale analysis of the WCS417r-ISR transcriptome
of Arabidopsis before and after pathogen challenge showed that ISR is associated
with potentiated expression of a large set of JA/ET-regulated defense genes that are
induced upon pathogen challenge (Fig. 14.1) (Verhagen et al. 2004). This preparation
of the whole plant to better combat pathogen or insect attack is called ‘priming’ and is
characterized by a faster and/or stronger activation of cellular defenses upon invasion,
resulting in an enhanced level of resistance (Conrath 2011). To date, a large number
of studies with PGPR and PGPF have supported the notion that ISR by beneficial
microbes is commonly based on defense priming (Pieterse et al. 2014).
Priming for enhanced defense emerged as an important cellular process in many
types of biologically and chemically induced systemic immunity, including SAR,
ISR, and herbivore-induced resistance (Frost et al. 2008; Luna et al. 2014; Pastor
et al. 2013; Conrath 2011). For instance, low doses of SAR-inducing agents do
not directly activate PR gene expression, but prime systemic tissues for enhanced
PR gene expression after pathogen challenge, indicating that priming is also an
important component of this type of induced resistance (Conrath 2011). By studying
the costs and benefits of defense priming, it was shown that the fitness costs of
priming are lower than those of constitutively activated defenses (Van Hulten et al.
2006; Walters et al. 2008; Vos et al. 2013). The fitness benefit of priming was shown
to outweigh its cost when under pathogen pressure, suggesting that priming functions
as an ecological adaptation of the plant to respond faster to its hostile environment.
130 C. M. J. Pieterse and S. C. M. Van Wees

Priming: A Molecular Memory of Immunization Because defense priming is

clearly expressed at the transcriptional level, research on the mechanisms under-
lying the primed state has focused on the expression of signaling intermediates in
transcriptional networks. These factors are thought to remain inactive in the absence
of an attacker, but their accumulation can provide the plant with the capacity to react
with an accelerated defense response upon perception of a pathogen- or insect-derived
stress signal. In Arabidopsis, the ISR-primed state was shown to be associated with
elevated transcript levels of genes that encode transcription factors of the AP2/ERF
family and MYC2, both of which have been implicated in the regulation of JA- and/or
ET-dependent defenses (Van der Ent et al. 2009; Pozo et al. 2008). This is in agree-
ment with the observation that in particular JA/ET-regulated genes show a primed
expression pattern in challenged ISR-expressing plants (Verhagen et al. 2004).
Mitogen-activated protein kinases (MAPKs) have also been implicated in defense
priming. Inactive forms of the MPK3 and MPK6 were shown to accumulate after
treatment of plants with low concentrations of the SAR-inducing SA-analogue ben-
zothiadiazole (BTH) (Beckers et al. 2009). After pathogen challenge, these latent
signaling molecules were activated, resulting in accelerated PR -1 gene expression
and the development of enhanced disease resistance. Priming is also associated with
chromatin modifications in the promoters of WRKY transcription factor genes that
regulate SA-dependent defenses, thereby facilitating potentiated expression of these
regulatory genes upon pathogen attack (Jaskiewicz et al. 2011). Recently, epigenetic
regulation of pathogen- and chemically-induced priming for SA-dependent defenses
and herbivore-induced priming for JA-dependent defenses was shown to be inher-
ited to the offspring via chromatin remodeling (Slaughter et al. 2012; Rasmann et al.
2012; Luna et al. 2012). Hence, plants seem to have the capacity to “memorize” a
stressful situation and subsequently immunize not only themselves, but also their
next generation against future attacks (Pastor et al. 2013).

14.5 Induced Resistance: Shaping the Plant’s Social Network

Exciting developments in induced disease resistance research provided a wealth of

information on the molecular details of how this adaptive defense system functions.
In nature, plants are attacked by a multitude of pathogens and pests. However, bene-
ficial associations between plants and mutualistic microbes are abundant in nature as
well, improving plant growth and health. Hormone-regulated plant defense signal-
ing networks finely balance plant responses to beneficial microbes, pathogens, and
insects to maximize both profitable and protective functions. Defense signaling path-
ways that are recruited in response to parasitic and beneficial organisms can overlap,
indicating that the regulation of the plant’s adaptive response to its biotic environment
is finely balanced between protection against aggressors and acquisition of benefits.
Plant hormones play pivotal roles in the regulation of the defense signaling network.
Their signaling pathways interact in a synergistic or antagonistic manner, providing
the plant with the capacity to tailor its immune response to the attacker encountered
14 Induced Disease Resistance 131

(Pieterse et al. 2012). In agricultural and ecological settings, plants often interact
with a whole suite of other organisms that range from beneficial microbes on their
root system to foliar pathogens and insect herbivores. Detailed mechanistic knowl-
edge on how the plant immune signaling network functions during multi-organisms
interactions is fundamental to develop novel strategies for sustainable protection of
our future crops that need to produce more with less input of pesticides and fertilizers.

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Chapter 15
Apologies to the Planet—Can We Restore
the Damage?

Dulce Eleonora de Oliveira and Marc Van Montagu

Abstract Many of us believe that human ingenuity can promote the innovations
required to face the challenge of demographic pressure. But twentieth century ex-
perience has shown that pollution by human beings (through increased population),
by manufacturing industries and by agricultural practices have severely damaged
the environment, requiring continuous mitigation efforts. And still we are unable to
bring an acceptable standard of living to half of the world population.
How to render intensive agriculture and small farming more sustainable? Good
governance and innovative science are essential, but we can no longer delay apply-
ing the knowledge generated in the last decennia by the plant scientists. Intensive
cooperation among agronomists, agro-ecologists and biotechnologists is urgently
needed, together with communication to society on the value of applying science to
agriculture, to achieve global food security and improved environment.
Why not doubt about the ingenuity of this Homo sapiens sapiens, if he is not
able or willing to make birth control acceptable; unable or unwilling to develop
an economy with better profit sharing; unable or unwilling to apply science for
developing sustainable agriculture and industry.

15.1 Death in this Garden

Humanity’s path was not a wandering around a Garden of Eden. For most of our
history human beings survived as hunters and gatherers, depleting locally the wild
food resources. Extensive land areas were necessary to sustain small groups. Our
possessions were limited to what we could transport when moving to a new site.
Depending on the local environment this could be very hard and it is probably the

M. V. Montagu () · D. E. de Oliveira

VIB—Institute of Plant Biotechnology Outreach, Department of Biotechnology
and Bioinformatics, Ghent University, IIC/UGent Technologiepark 3,
B-9052 Gent-Zwijnaarde, Belgium
Tel.: + 32 9 473 78 47 79
e-mail: marc.vanmontagu@vib-ugent.be
D. E. de Oliveira
Tel.: + 32 9 264 87 27
e-mail: dulce.deoliveira@vib-ugent.be

© Springer International Publishing Switzerland 2015 135

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_15
136 D. E. de Oliveira and M. V. Montagu

reason why humans developed environmental engineering skills to alter terrestrial

habitats.
Clearing vegetation, tilling the soil and domesticating wild animals and plants
to meet specific requirements resulted in the advent of organized agriculture, which
played an enormous role in the development of humanity. The battle was not won at
the first round though. Early farmers have paid a price for their new way of life as
they traded quality for quantity. Paleophatology studies reveal that with the adoption
of agriculture the average height of individuals of post-agricultural communities in
Greece and Turkey reduced drastically. Skeletons from different archaeological sites
also indicated childhood malnutrition, anemia, and diseases such as tuberculosis and
leprosy, as well as significant decrease in life expectancy (Diamond 1987). Although
the hunter and gatherer diet was probably superior to that of new agricultural societies,
there is little question that the advent of the agriculture supported the increase of
populations.
Successive cultural revolutions have led to surges in population. Together with
medical advances, the increase in agricultural productivity is responsible for the
phenomenal population growth of the last century. Already in 1798 Thomas Malthus
studied the population growth in Europe and claimed that the population was increas-
ing faster than food production. He feared global starvation, which did not happen
thanks to the development of new technologies that expanded food production.
When the Club of Rome asked the world to pay attention to overpopulation
(Meadows et al. 1972), stressing that food security would become a problem, many
thoughtful scientists were wondering if Thomas Malthus had not been right with his
predictions. The majority however continued to deride these scientists who didn’t
trust human ingenuity. The successes of diligent innovative plant breeders such as
Norman Borlaug, the “Father of the Green Revolution”, brought overconfidence. It
took a period of time before it became accepted that intensive agriculture, with its
need for a high input of synthetic fertilisers, crop protection chemicals and irrigation
caused environmental problems. Today it is too late to argue against intensive agri-
culture; world food production cannot go on without this high yielding agriculture;
we are too numerous. We were 2 billion when Norman was a student. We are now
well over the 7.2 billion and demographers predict we will be about 10 or 11 billion
within 40 years. But at this very moment there are 1.2 billion people who are under-
fed amongst which 0.8 billion enter the FAO statistics of being underfed for a whole
year

15.2 From Enlightenment to Darwin and Back to Superstition

The Enlightenment displaced beliefs and revelation by the scientific method, includ-
ing empiricism and rational thought. Hand in hand with it came a new concept of
nature. The nineteenth century gave us Darwin’s Origin of Species with its revolu-
tionary theory of evolution and Gregor Mendel’s laws of genetic inheritance, two
wonderful outcomes of the newly-born biological science.
15 Apologies to the Planet—Can We Restore the Damage 137

Life Sciences evolved at an exponential rate in the twentieth century and became
one of the most relevant fields of research and innovation to the benefit of mankind,
and hopefully in the future to the entire ecosystem. In the last decade we have wit-
nessed an eruption of information generated by the ‘omics’, computational models,
genome engineering, and bio-imagining. These large datasets are progressively gen-
erating knowledge on the biological interactions behind the phenotype, allowing
biology to evolve from the classical reductionist mode to the more holistic approach
of systems biology. We can now appreciate how genomes within a metaorganism
interact and affect one another. It will soon be possible to unravel the complex net-
work of interactions that link a host with its associate microbiome and how this can
influence the host’s performance and even evolution (Guerrero et al. 2013)
Agrobacterium had already given us a hint on how it can be relevant to plants.
The holistic approach will bring us new insights on other types of plant-microbe
interactions such as symbiosis with endophytes. Although plant-growth promoting
endophytes have been identified for a long time, the predictive success at positively
influencing plant growth under field conditions has been limited. We can now tackle
this issue through the extensive examination of endophyte community dynamics and
how they are influenced by abiotic and biotic factors such as soil conditions, biogeog-
raphy, plant species, microbe–microbe interactions and plant–microbe interactions
(Gaiero et al. 2013)
High-throughput deep sequencing has shown that many non-coding sequences
produce RNA molecules that regulate gene expression. Several studies have indi-
cated that small RNAs participate in plant resistance to bacterial pathogens (Seo
et al. 2013). We are tantalized by the future discoveries on the role of small RNAs
in the intra-holobiont communication, leading to better understanding of the mecha-
nisms underlying the new Hologenome Theory of Evolution (Zilber-Rosenberg and
Rosenberg 2008),
All these advancements in our knowledge of how biology works are sources of ma-
jor innovations and bring us hope that we can improve agricultural productivity while
reducing its environmental footprint. And yet, ironically, despite all the scientific
progress, from the standpoint of public acceptability it seems that we have returned
to medieval times. We face now a kind of fundamentalism which deems all that is
“natural” to be “sacred”. The “natural” or “back to nature” viewpoint is opposed to
human intervention in the natural world, and therefore to the biotechnologies, as if
what is “natural” can only be good and science can only be bad.
To a certain extent this is nothing new. Human beings have always had mixed
feelings in their relationship to Nature. Sometimes nature is perceived as hostile to
the persons who try to transform it, such as farmers or engineers. Sometimes it is
perceived as a sacred “Mother Earth” by more contemplative people such as poets,
philosophers, naturalists and traditional cultures. From a Science point of view,
Nature is neither good nor bad. It is our living environment. Ecology shows the
interdependence between the natural world and human beings, who have shaped the
world and environment as we see it and experience it today over thousands of years.
Modern biology shows the way an organism works to interact with other organisms
in a habitat. It is also an invaluable tool to find strategies to improve human life while
138 D. E. de Oliveira and M. V. Montagu

preserving the environment. Again the example of how Agrobacterium survives is

an excellent illustration.
Our society rightfully wants to have a bottom-up voice on every aspect of social
life like education, technology, ecology, arts, kinship, or sexuality. Such laudable
search for self-management can only be successful in a well-informed society. We
have to cultivate discernment based on intellectual honesty and not on individual
passions. Human passions, especially self-esteem and fear, are at the origin of our
emotion-driven myth-making tendency (Russel 1943). These traits were useful to
our hunters and gatherers ancestors, but are a threat to modern society. Irrational fear
is nourished by ignorance and is an easy prey to intellectual dishonesty.

15.3 Agrobacterium has Opened Our Eyes: The Living World

is a Continuous Genetic Experiment

The TIP Story In the beginning of the twentieth century, several new scientific
disciplines such as virology, epidemiology, internal medicine and public health de-
veloped. Some discoveries, like the demonstration (in 1911 by Peyton Rous) that a
cancer on chicken wings could be caused by an infectious agent, were so revolution-
ary that the medical world could not be convinced. At the Rockefeller Institute, the
quest for the “Tumour Inducing Principle” (TIP) started. A Belgian scientist, Albert
Claude, joined the team in the late 1920s. He stressed that new enabling technologies
would be needed and introduced ultracentrifugation and electron microscopy to the
life sciences. This opened the field for “cell biology” and allowed the identifica-
tion of TIP as a virus, later called Rous Sarcoma Virus. It brought a Nobel Prize
to Peyton Rous (in 1966) and to Albert Claude (together with Christian de Duve
and George Palade in 1974). Another astonishing infectious agent was a bacterium
inducing tumorous growth (called crown galls) on a wide variety of plants. Amin
Braun (also at Rockefeller) initiated the quest for the TIP present in Agrobacterium
tumefaciens with the postulate that this “principle” was transferred by the bacterium
to the plant cell to induce transformation. Again it was ultracentrifugation and elec-
tron microscopy that showed that TIP was a product of a large plasmid, which was
then called Ti-plasmid (Tumour inducing plasmid) (Zaenen et al. 1974). Later in
the 1970s it was demonstrated that a particular DNA segment of the bacterial Ti
plasmid, called the transferred (T)-DNA, was integrated into the plant cell genome
upon Agrobacterium infection (Depicker et al. 1978; Chilton et al. 1980; Zambryski
et al. 1980).
The First Transgenic Plants As soon as it was established that gene transfer from
Agrobacterium to plant cells occurred, there was a rush to use Agrobacterium as a
vector for plant transformation. It was soon employed worldwide for a systematic
and refined analysis of the impact of single genes on all aspects of plant biology,
giving a new life to plant science.
15 Apologies to the Planet—Can We Restore the Damage 139

At Plant Genetic Systems (PGS), a spin-off company from the plant science lab at
Ghent University, this tool was employed to develop transgenic plants with different
useful agronomic traits: tolerance to the herbicide glufosinate ammonium (De Block
et al. 1987); insect resistance using the insecticidal protein genes from Bacillus
thuringiensis (Vaeck et al. 1987; see Chap. 20); and nuclear male sterility, a trait that
formed the basis for an efficient hybrid production system (Mariani et al. 1990, 1992).
Scientific Hit, Public Denial Since the first transgenic plants, scientists, mostly
from the public sector, have identified numerous genes for introducing new traits into
plants, including resistance to pests and disease-causing agents, enhanced stability or
shelf-life, increased yield, environmental tolerances including salt resistance, nutri-
tional enhancements (especially for vitamin A deficiency), and pharmaceutical and
industrial value-added traits. Sadly today, in contrast to the scientific achievements,
20 years after their first commercialization, new GM varieties with novel genes are
being introduced very slowly worldwide. The commercial development of transgenic
germplasm has advanced mostly in stacking the few traits commercially available in
essentially four crops (maize, soybean, canola and cotton). The cost of global reg-
ulation coupled with the lack of consumer acceptance is stopping the spread of the
technology beyond the most profitable seed crops previously mentioned, with R&D
(but no commercialization) occurring in rice and wheat. Specialty crops and traits
with high environmental or social value but low economic value for commercial seed
companies have not been introduced or considered or have been abandoned in the
face of NGO opposition. Today only a handful of companies with global experience
in deregulation have the capability of developing and launching a GM plant product.
Much of the frustrating delay on the development and use of GM crops can
be attributed to the significant and successful opposition deployed by NGOs such
as GreenPeace and Friends of the Earth through highly sophisticated marketing
campaigns. These campaigns raise the specter of adverse social implications, as well
as health and environmental risks. The latter have been debated at length and no
serious study has concluded that GMOs are unsafe for health or the environment
(EASAC report 2013). But fear persists at consumer level and is mainly linked to
the tool of genetic engineering with which these crops are constructed. The fact that
one can transfer a gene from a particular species into a completely different species
has triggered people’s irrational fears.
Scientific thinking is alien to irrational gut feelings. The scientific community
did not expect to face this challenge. The gene splicing technology used to gener-
ate a transgenic organism is a minute genetic alteration compared to the genomic
changes induced during all crosses and breeding events traditionally practiced in
agriculture and husbandry. Our planet is one large natural genetic laboratory, where
all the living organisms continuously activate and silence part of their genomes, as a
reaction to all the environmental stresses endured (Pigliucci 2005; Heger and Wiehe
2014; Bateson et al. 2014). The natural gene engineering of Agrobacterium is, as
indicated above, one example of very large phenomena. Biological evolution cannot
be achieved by single point mutations in a static genome. It is essential to adaptation
and survival that living organisms can alter their genomes through transposition of
140 D. E. de Oliveira and M. V. Montagu

movable elements, accumulation of deletions, insertions, gene amplifications, and

point mutations. Taxonomy is a useful tool to group and categorize organisms accord-
ing to important homologous attributes, but in reality species often have indistinct
boundaries. Genomic studies of the last decade have well documented that a genome
is a dynamic structure continuously refining its gene pool. We are convinced that,
when the metagenomic studies will be comprehensibly analysed, we will find that
horizontal gene transfer is a frequent and essential event for evolution (Fuentes et al.
2014). Once again the example of Agrobacterium will have shown us the way.

15.4 Give Science a Chance

According to calculations by the FAO, the world already produces enough food to
feed 12 billion people (da Silva 2012). But global estimates can be misleading be-
cause access to food varies enormously around the world (Vermeulen et al. 2012).
Many of the world’s poorest rural populations continue to rely on locally produced
food distributed in economies that are poorly integrated into global markets. There-
fore improvement of the quality and quantity of the agricultural production of the
smallholder farmer is central to eradicating hunger and poverty in the world. Some
3 billion poor people still live in rural areas of low-income countries and derive the
major part of their income from the agricultural sector and related activities (Dethier
and Effenberger 2012).
Several empirical studies support the highly positive impact of agricultural
progress on poverty alleviation. Agricultural growth generates income and employ-
ment in rural areas and provides cheaper food for urban areas. A labour-intensive
sector such as agriculture has a larger impact on poverty reduction than less labour-
intensive activities. The difference is greatest among the extremely poor, who live
on less than $ 1 a day (Christiaensen et al. 2010).
It is undisputed that solutions to improve the agriculture in low-income countries
pass through good governance, political will and concerted actions of different seg-
ments of society. But there is a broad consensus that agricultural technologies are at
the heart of long-run agricultural growth. The Green Revolution in Asia is an example
of how R&D for seeds, fertilizers, pesticides and irrigation was able to significantly
expand agricultural production. However, any New Green Revolution for Asia and
developing countries will have to be sustainable over time, economically, environ-
mentally and from a societal dimension, as explained above. Smallholder farmers in
low-income countries face this challenge now. Drought, low-yielding crop varieties,
pests and diseases, poor soils, low fertilizer use, limited irrigation and lack of modern
technologies are among the problems that plague tropical agriculture.
Modern biotechnology is part of the answer to achieve an agricultural revolution in
low-income countries. Already, the technology has brought significant improvement
to earned income, quality of life and per acre productivity. The crops produced by
biotechnology and molecular-assisted breeding currently on the market are helping
agriculture to achieve higher yields in a more sustainable way. One remarkable
15 Apologies to the Planet—Can We Restore the Damage 141

example is Bt cotton, which is grown by over 15 million smallholder farmers in

India, China, Pakistan, and a few other developing countries. This technology has
reduced food insecurity by 15–20 % among cotton-producing households (Qaim and
Kouser 2013). Another example from the Philippines shows that the adoption of Bt
corn has a positive yield impact especially to farmers at the lower end of the yield
distribution (Sanglestsawai et al. 2014). Other studies indicate that if restrictions on
the present GM-crops were lifted in all countries, including sub-Saharan Africa, the
region might have significant gains in income generation and improved public health
(Dethier and Effenberger 2012). These insights can help to develop policies that
encourage the use of GM technology among smallholder farmers as the high costs
restrict its adoption. Indeed, the capacity to adopt modern technologies is directly
related to the health of the economy of the developing country.
However, other constraints have prevented the adoption of GM technology where
is it needed most. One major obstacle is the fact that the other three major GM crops—
soybean, maize, and canola—do not match the interests of most of smallholder
farmers of the least developed countries. Although more than half of the global GM
crop area is located in developing countries, much of this relates to large farms
in South America. Relevant development country crops such as sorghum, millet,
groundnut, cowpea, common bean, chickpea, pigeon pea, cassava, yam and sweet
potato are grown in niche geographical areas by small farmers. These key staple
crops are often referred to as ‘orphan crops’. Because they are not extensively traded,
except for sorghum these crops present little economic interest for commercial seed
companies and therefore have been mostly ignored by GM agriculture.
Public research funded plant science has produced numerous breakthroughs that
can help to alleviate many of the entrenched problems of impoverished nations. But
most of these are proof-of-concept in model plants. For downstream applications
relevant to low-income countries, it is essential to develop efficient transformation
protocols for niche geographical crops and that breeding programs are funded for
each crop.
Plant transformation is a daring research topic because, although Agrobacterium
has a wide host range in plants, the external conditions that permit or prohibit
transformation are diverse and the balance needs to be determined empirically. De-
spite intensive research it is still not established why some plant species can be
transformed easily, while others are recalcitrant to Agrobacterium-mediated trans-
formation. There is a continuous striving for simpler, more robust and more efficient
transformation protocols for model plants and crop species for intensive agricul-
ture (Pitzschke 2013). The advances are driven by the demand for higher yields and
improved stress tolerance. Unfortunately major crops of marginal environments of
Africa, Asia and South America have not received the same attention; generating
stable transformed plants is still a challenge for most orphan crops.
Poor farmers might benefit especially from transgenic food crops that are more nu-
tritious or particularly tolerant to biotic and abiotic stress. Research is well advanced
in the development of more nutritious maize and rice (Pérez-Massot et al. 2013).
Still a lot has yet to be done for staple crops of geographical niches, many of them
142 D. E. de Oliveira and M. V. Montagu

requiring not only nutrient improvements but also the elimination of anti-nutritional
factors. Lathyrus sativa is one such example (Van Moorhem et al. 2011).
As for biotic stress, development of resistant phenotypes via classical breeding
is no longer an option. Pests and pathogens easily break down resistance based on
single genes. Crop breeding is limited by the time taken to move resistance traits
into elite crop genetic backgrounds and the limited gene pools in which to search for
novel resistance (Bruce 2011). The pyramiding of resistance genes from different
wild relatives using gene engineering is a strategy that deserves to be applied to
orphan crops
Systems biology is showing that biotic and abiotic regulatory pathways are inte-
grated in multidimensional ways that include genetic variation, abiotic environment
and plant life history (Kliebenstein 2014). The understanding on how a plant modu-
lates defence, symbiosis and growth as well as the signalling communication between
individual plants will help to find the best ways to develop agroecology. This approach
is widely recognized as a way to enhance the sustainability of intensive agricultural
systems.
Modern agroecology cannot ignore the needs of low-income countries. The
paradigm shift proposed in agroecology is not an option but a prerequisite to poor
farmers. There is no other choice but to adapt the plant genotypes to the environment,
since the inverse is not affordable. Actions must be taken to immediately apply the
discoveries of this research to orphan crops.
This is even more necessary in view of the predicted impacts of climate change,
particularly to tropical countries. Rain-fed crops account for nearly 60 % of cropland
area (Vermeulen et al. 2012). Climate instability will have large negative impacts
on the productivity of these agricultural systems, which do not have the required
adaptive capacity, with major implications for rural poverty and both rural and urban
food security.
Food aid can be of help in emergency situations but is quite questionable in a longer
term development strategy. It has been shown that this type of assistance can have
adverse effects such as price fluctuations, disincentives to agricultural production and
market development, and can cause a cycle of dependency in farming communities
(Dethier and Effenberger 2012). The solution will have to come from innovations in
agriculture. We have to develop better-adapted crops faster. It can be that, even using
GM technology, the development of new varieties will not fast enough respond to our
needs. It is predicted that new races of pathogens will evolve rapidly under elevated
temperature and CO2 . Another implication will be a changing pathogen geographic
distribution (Chakraborty 2013). The unpredictability and the speed of the events
will demand more flexible and speedier responses. Novel biotechnology tools will
have to come. We dare to bet that the rapid evolution of miRNA technology will soon
allow the production of these pathogen-specific molecules in quantities sufficient to
apply on the field at the moment of a disease outburst. More human solidarity will
be necessary for this tool to be used by the poor farmers.
15 Apologies to the Planet—Can We Restore the Damage 143

15.5 Ite Missa Est

It has been well documented that the unprecedented population growth and the in-
equity in resource availability have generated huge famines, massive deforestation
and intensive industrial pollution in the last century. To achieve global food secu-
rity both intensive and subsistence agriculture are necessary. We head straight for
disaster if we do not find ways to enhance land use productivity while preventing
worldwide ecosystem degradation and fragmentation. This can only happen when
the best technologies available are being used.
GM plant technology is a mature technology that has proved its social and eco-
nomic benefits as well as its safety. After 26 years of field trials and 18 years of
regular consumption, not a single health or environmental incident that could be at-
tributed to GMO has been reported. Academies of Sciences worldwide endorse GM
technology.
Science evolves with the development of new tools and all knowledge is tentative
and may be adapted in the light of future knowledge. The Scientific Method cannot
prove or disprove ideas and all scientific ideas are subject to change if warranted
by the evidence. This normal and healthy process of Science is often misused by
NGOs to serve political projects or simply to raise money. Mediatised “experts”
abuse people’s fears of the unknown by proclaiming that something new is more
dangerous than something old. More alarming is that certain governments embrace
this anti-science and anti-progress ideology.
It is time to learn not to surrender to fear and the images that fear engenders.
We will have to learn to share emotions without fooling ourselves by blurring the
real world with mythical dreams. Time is pressing, we have to act now and fear is a
breeding ground for inactivity.

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Chapter 16
Will the Public Ever Accept Genetically
Engineered Plants?

Inge Broer

Abstract Compared to transgenic herbicide- or insect-resistant plants, pathogen-

resistant ones are rarely found in the field. There are several economic reasons for
this, but an additional cause is the scientific and public debate about potential risks
and a low attractiveness for consumers to buy these plants and their products. This
paper gives a short overview of traits theoretically available or already on the market
and describes the concerns raised that reduce their market opportunity. Finally it
proposes solutions on how to proceed in the future to allow a rational dealing with
the technology.

16.1 Transgenic Pathogen Resistant Plants

Since the beginning of their existence plants and pathogens survive by ongoing de-
velopment of sophisticated strategies to either attack or defend. In plant breeding,
humans try to pyramid as many defense strategies as possible in crops in order to
combine a healthy growth with a minimal input of pesticides and with optimal yield.
Theoretically, this can be achieved using pathogen resistant plants. Nevertheless,
since in classical breeding complete genomes are mixed, stacking of many bene-
ficial genes without retaining deleterious ones is complicated and analysis of high
numbers of crossings and descendants is unavoidable. In spite of the development of
molecular markers, which reduce these numbers drastically, it is still an enormous
challenge. In addition, classical breeding is restricted to defense genes present in sex-
ually compatible cultivars. Hence several attempts have been made to enhance crop
resistance to pathogens via genetic engineering which enables scientists to exploit
the whole gene pool of the world.
Most of the defense genes transferred up to now originate from plants, but in
many cases donor and recipient are not sexually compatible like for instance rice
and maize. According to Collinge et al. (2008) out of twenty eight examples for
transgene encoded disease resistance only 8 of the donors were sexually compatible

I. Broer ()
Agricultural and Environmental Faculty, University of Rostock,
18059 Rostock, Germany
Tel.: + 49 381 498 3080
e-mail: Inge.Broer@uni-rostock.de
© Springer International Publishing Switzerland 2015 145
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_16
146 I. Broer

to the recipient. Only for these a transfer via conventional breeding would be possible.
All other donors were non compatible plants, viruses, insects, fungi or bacteria. The
establishment of plant resistance using these resources is therefore strictly dependent
on gene transfer.
So far forty five different cultivars have been successfully transformed to receive
pathogen resistance. However, only virus resistant squash and papaya are cultivated
in the USA and some other countries and virus resistant pepper and tomato in China
(James 2014). The transgenic papaya was developed and commercialized by the
University of Hawaii and is now approved for consumption in Japan (Dec 2011)
and China (Dangl et al. 2013). This seems surprisingly little considering that her-
bicide and insect resistant plants are grown worldwide with great success. In 2013,
transgenic plants, most of them herbicide or insect resistant, covered 175 million
ha worldwide (ISAAA 2014). In the USA, about half of the total land was used
for GM crops (USDA 2014). E.g. no pathogen resistant soybean variety is on the
market (Hudson et al. 2013) although more than 81 % of the worldwide production
is transgenic. So up till now, transgenic pathogen resistant plants play no visible role.
This is especially surprising considering that crop losses due to bacteria, fungi and
viruses account for about 16 % yield loss over all major crops (wheat, rice, maize,
potato and soybean for the period 2001–2003) (Oerke 2006). An overview of the sta-
tus of transgenic pathogen-resistant plants is shown in Table 16.1. Numbers of field
trials for pathogen resistant plants are also much lower than those of herbicide and
insecticide tolerance. 2616 total field releases of transgenic plants with virus/fungal
resistance have been conducted in the USA until September 2013. This has to be
compared to 6772 releases of plants with herbicide tolerance, 4809 with insect re-
sistance, 4896 with improved product quality and 5190 with agronomic properties
(USDA 2014). One interesting example of a commercially potent, virus resistant
plant is a virus resistant bean, created by the Brazilian Agricultural Research Corpo-
ration (EMBRAPA). Beans are very important crops in South and Middle America
and are mostly produced by small farmers. The golden mosaic virus causes severe
yield loses (40–100 %). Descendants of one single transformant (event) showed re-
sistance up to 93 %, hence this might be a real breakthrough for the small farmers
(Bonfim et al. 2007). The bean is approved in Brazil und expected to appear on the
market in 2014/2015.

16.2 Reasons of Why so Few Transgenic Pathogen Resistant

Plants Exist in Agriculture

There seem to be several reasons for the minimal use of transgenic pathogen resis-
tant plants in the world. In many countries, economic concerns might be of most
importance. In Europe, political and public concerns are dominant while scientific
concerns seem to matter less.
16 Will the Public Ever Accept Genetically Engineered Plants 147

Table 16.1 Approval and cultivation of transgenic, pathogen resistant plants worldwide. (Source:
ISAAA/TransGEN/Hudson et al. 2013)
Culture Trait Approval Cultivation
Apple Fungal/bacterial resistance – –
Apricot Virus resistance – –
Avocado Fungal resistance – –
Banana Fungal/bacterial/virus resistance – –
Barley Fungal resistance – –
Broad bean (Vicia Fungal resistance – –
faba)
Carrot Fungal resistance – –
Cassava Virus/bacterial resistance – –
Chestnut Fungal resistance – –
citrus fruits Fungal/virus/bacterial resistance – –
Cocoa Fungal/virus resistance – –
Coconut Virus resistance – –
cowpea (Vigna un- Fungal resistance – –
guiculata)
Eggplant Fungal resistance – –
Grape vine Fungal/virus/bacterial resistance – –
Grapefruit Fungal/virus/bacterial resistance – –
Hop plant Fungal resistance – –
Kidney bean Virus/fungal resistance Brazil Starting 2014
(Phaseolus
vulgaris)
Kiwi Fungal resistance – –
Lettuce Fungal/virus resistance – –
Lupine Virus resistance – –
Mango Fungal/bacterial resistance – –
Melon Virus resistance – –
Oat Virus resistance – –
Olive Fungal resistance – –
Papaya Virus resistance USA 2, China 1, USA, China
Canada 1, Japan 1
Pea Fungal/virus resistance – –
(Pisum sativum)
Peanut Fungal/virus resistance – –
Pear Bacterial resistance – –
Pepper Virus resistance China Cultivated In China
148 I. Broer

Table 16.1 (continued)

Culture Trait Approval Cultivation
Persimmon Fungal resistance – –
Pineapple Virus resistance – –
Plum Virus resistance USA 1 USA 1
Potato Fungal/virus/bacterial resistance USA/ Canada Not now
Raspberry Virus resistance – –
Soybean Virus/fungal resistance – –
Squash Virus resistance USA/ Canada USA
Strawberry Fungal resistance – –
Sugar beet Virus resistance – –
Sunflowers Fungal resistance – –
Sweet potato Virus/fungal resistance – –
Taro Fungal resistance – –
Tobacco Virus resistance – –
Tomato Virus resistance – Cultivated in China
Water melon Virus resistance – –
Wheat Fungal resistance – –

Economic Causes The approval process required to develop permitted pathogen

resistant plants is often too costly to make it viable for any business to pursue. Un-
fortunately, pathogen resistance is much more complex compared to herbicide or
insect resistance. Since there are several kinds of organisms causing disease (bac-
teria, fungi, viruses, oomycetes) no single gene is expected to protect against all of
them. In addition, the pathogen’s strategies to exploit the plants resources are quite di-
vergent. Hence the diversity of defense genes is huge, ranging from chitinases, virus
repressor genes and viral coat proteins, bacterial (Bt-toxin) and viral toxins (KP4) to
RNAi (Collinge et al. 2008). Unfortunately, most plants suffer from the constitutive
expression of defense genes that quite often causes cell death. Consequently, in order
to sustain yield, inducible expression is often necessary, complicating the process
further.
The high divergence of pathogens and defense mechanisms also complicates their
administrative approval. In Europe 10–50 million € are estimated as final costs for
approval of a single event. Compared to a herbicide or insect resistance, the risk
assessment of a pathogen resistant plant is much more complex and expensive. Due to
the high capability of pathogens to adapt to the plant defense, several different defense
mechanisms should be introduced. In addition to the location of these transgenes in
the plants genome, potential side effects of each transgene have to be analyzed.
Although from a scientific point of view it might be assumed that the analysis of
each combination of transgene and cultivar is sufficient (trait specific) the European
16 Will the Public Ever Accept Genetically Engineered Plants 149

legislation requests the analysis of each event for approval (event specific). Taking
into account that the market for pathogen resistant plants is much smaller compared
to insect- or herbicide resistant plants, the chance for a sufficient return of investment
seems for the time being too low.
Scientific Causes The most obvious scientific concern is the spread of the resistance
gene to wild relatives, giving these hybrids a better chance to survive. Although this
could also happen to the resistance genes in classical breeding, and is not regulated
there, in transgenic plants it is required to be analyzed when cultivars are used that
have sexual compatible wild relatives in the area where the transgenic plants are
likely to grow. This is for instance important for pathogen resistant maize in South
America, for transgenic canola at river beets where Brassica oleracea is found, or for
sugar beets at the coastline of Europe where wild beets are growing. Hence intensive
out crossing studies have been conducted (www.transgen.de).
Secondly, unwanted effects on non-target organisms have to be taken into account.
Stefani and Hamelin (2010) summarize eighty six studies investigating the impact
of transgenic fungal resistant crops on target and non-target fungi. No significant
changes in the populations of non-target fungi could be detected. Similar results were
obtained for fungal resistant trees. Nevertheless, this result cannot be generalized
since the studies were mostly carried out under controlled conditions; only eleven
were conducted in the field.
The uncontrolled spread of the transgenic plants is quite often named as one of
the biggest problems. Nevertheless, as long as crop plants are used that are not
able to survive without human support, this risk is restricted to the agricultural area.
Here, strict seed thresholds defining the maximum content of transgenic seeds in a
conventional seed lot and corresponding thresholds for the labeling of products that
contain small amounts of transgenic plants in combination with guidelines that define
the distance between fields where conventional or transgenic sexual compatible plants
are grown, is sufficient to secure the availability of non GMP products on the market,
as long as the demand is high enough to justify the costs of the separation. In case
of plants that are able to spread into the wild, more caution has to be taken. It has
to be assured that the expression of the transgene does not lead to any advantage
compared to the near isogenic lines.
Big advantages of certain varieties lead to an increase of these varieties in the
field. Hence it might be assumed that transgenic, pathogen resistant varieties will be
preferred by the farmers and thereby reduce the number of different varieties available
on the market. However, this holds true for any interesting variety independent on
the method with which it was created. Actually, it could be much easier to integrate
an interesting trait into several varieties via genetic engineering instead of crossings,
but this is only possible if the legislation changes from event specific to a trait specific
approval.
In contrast to insect or herbicide resistant plants, the creation of resistance is not
of great concern. Resistance of pathogens will occur at some point, no matter which
strategy is used to create the resistant plant; this is just a matter of time and evolution.
Nevertheless, the pyramiding of resistance genes, which is much easier and more
150 I. Broer

effective using genetic engineering, should prolong the utilization phase of specific
traits drastically.
Political and Social Concerns As shown in Brazil, pathogen resistant plants are
of importance for small farmers and could support a sustainable agriculture due to
the reduction of pesticides. This is of public interest and should therefore—as hap-
pened in Brazil—be supported by public money. For several years the development
as well as risk assessment of transgenic plants was funded by governments in the
whole world. Nevertheless, opposition against this policy arose quite early and with
increasing intensity. Several non-governmental organizations and even political par-
ties declared the opposition to genetically modified plants as a primary goal, leading
to increasing financial and political support by the public. Unjustified dispraise of
scientists conducting risk assessment analysis led to a loss of trust in the indepen-
dence of science. Politicians expressing their doubts on the governmental assessment
system and countless scandals in food and feed production undermined the public
faith in security systems. Contradictive scientific studies also caused confusion. For
example, a study by Séralini et al. concluded that a herbicide-tolerant maize caused
tumors and early death in rats (Séralini et al. 2012). This was widely dismissed by
scientists including EFSA (European Food Safety Authority 2012). In November
2013, this article was retracted by the journal concerned (Elsevier 2013). All this
supported attacks on field trials, scientists and watchmen by activists without dis-
approval from the public. Understandably, neither the business nor the governments
are now very keen to support the production and cultivation of GMP in Europe.
In addition to the scientific reasons, which are unfortunately less and less part of
the debate, ethical and moral concerns influence the public opinion. The integrity
of a being and of its genome is often mentioned as an important value, ignoring
the fact that genomes always undergo changes due to the mixing in sexual mating,
recombination, mutation through environmental factors and jumping genes. These
changes are the prerequisite of evolution. The potential dependence on multinational
companies is another, and very relevant, concern. Only very few big multinational
companies are able to finance and support the approval process necessary, especially
in Europe. All applications for approval in Europe have been made by only nine
companies. This is much different for instance in the United States, where approval
is cheaper and easier and therefore also smaller companies are able to bring their
events on the market. Due to the high cost in Europe, the companies join to apply
for the approval of one event that is then inbreed into the different varieties.
One last important concern is the fact that high licensing costs make it impossible
for small farmers to use transgenic, pathogen resistant plants. The example of the
golden rice, where license holders agreed to waive the fee if the income of the farmer
is below a certain level, shows that it is possible to solve this problem. In addition, the
support of governmental research independent of companies, like in Brazil, would
allow more innovations to come to the market with lower licensing fees.
16 Will the Public Ever Accept Genetically Engineered Plants 151

16.3 What has to Happen?

Pathogen resistant transgenic plants have the potential to make agriculture more cost-
effective and with less adverse impact upon the natural environment. The following
suggestions might contribute to use this potential and to ensure that all people, not
just multinationals, can benefit from the technology.
Worldwide, mainly plants with small market potential are problematic. Here, first
of all the costs for approval and licenses have to go down while keeping the relevant
safety level. In addition, the trait has to reduce the costs for the farmer in order to
justify a technology fee. Even then, it might also be necessary to refund the farmer
for the environmentally friendly cultivation in order to set enough incentives to saw
these seeds. In addition, public acceptance is a prerequisite for the cultivation. This
seems only achievable when there is an advantage for the consumer that is directly
obvious, which unfortunately seems to be not the case for ecological advantages.
Even then, taking the huge opposition into account, it is unrealistic to think that
there will be transgenic pathogen resistant plants on the market in Europe in 10
or even 20 years. Here, scientists should prepare for the time when cultivation of
transgenic plants will be needed and possible too, since it takes more than 20 years
from the first idea to a safety assessed transgenic variety ready for the market. This is
only possible if field trials can be conducted. For instance in 2014, not a single field
trial is planned in Germany. This is also due to the high costs of field protection (for
fences and watchmen) that exceed the actual research costs. The Swiss government is
funding a protected site where all field trails can be conducted (Romeis et al. 2013).
This is a good start; nevertheless the site is much too small. Similar attempts have to
be undertaken in other European countries or even by the EU.

References

Bonfim K et al (2007) RNAi-mediated resistance to Bean golden mosaic virus in genetically

engineered common bean (Phaseolus vulgaris). Mol Plant Microbe Interact 20:717–726
Collinge DB, Lund OS, Thordal-Christensen H (2008) What are the prospects for genetically
engineered, disease resistant plants? Eur J Plant Pathol 121:217–231
Dangl JL, Horvath DM, Staskawicz BJ (2013) Pivoting the plant immune system from dissection
to deployment. Science 341(2013):746–751
Elsevier (2013) Elsevier announces article retraction from Journal Food and Chemical Tox-
icology November 28, 2013. http://www.elsevier.com/about/press-releases/research-and-
journals/elsevier-announces-article-retraction-from-journal-food-and-chemical-toxicology
European FoodSafety Authority(2012) Final review of the Séralini et al. (2012) publication on a
2-year rodent feeding study with glyphosate formulations and GM maize NK603 as published
online on 19 September 2012 in Food and Chemical Toxicology, 1. EFSA J 10(11):1–10
Hudson LC et al (2013) Advancements in transgenic soy: from field to bedside. In: Board J (ed) A
comprehensive survey of international soybean research—genetics, physiology, agronomy and
nitrogen relationships. InTech. doi:10.5772/52467
James C (2014) Status of Commercialized Biotech/GM Crops: 2013 ISAAA Brief 47, Ithaca, NY
Oerke EC (2006) Crop losses to pests. J Agric Sci 144:31–43
152 I. Broer

Romeis J et al (2013) Plant biotechnology: research behind fences. Trends Biotechnol 31(4):
222–224
Séralini GE et al (2012) Long term toxicity of a Roundup herbicide and a Roundup-tolerant
genetically modified maize. Food Chem Toxicol 50(11):4221–4231
Stefani FOP, Hamelin RC (2010) Current state of genetically modified plant impact on target and
non-target fungi. Environ Rev 18:441–475
USDA (United States Department of Agriculture) (2014) Report Summary from the Economic
Research Service 2014
 Part III
Control of Plant Diseases and Pests using
Beneficial Microbes
Chapter 17
Microbial Control of Phytopathogenic
Nematodes

Xiaowei Huang, Keqin Zhang, Zefen Yu and Guohong Li

Abstract Phytopathogenic nematodes, mainly comprised of plant parasitic nema-

todes, cause serious losses in a variety of agricultural crops worldwide. However,
because traditional nematicides are associated with major environmental and health
concerns, developing safe and effective nematicides is urgently needed. Among the
recent developments, biocontrol measures using nematophagous microorganisms
have shown significant promise and attracted much attention. Nematophagous mi-
croorganisms are the most important natural enemies of phytopathogenic nematodes
and these microorganisms employ a variety of physical, chemical, and biochem-
ical mechanisms to attack nematodes. This chapter introduces nematophagous
microorganisms as well as their virulence factors against nematodes.

17.1 Introduction to Nematodiasis and Biocontrol

Nematodes are among the most abundant multi-cellular organisms on earth and can
reach densities of up to 10 million individuals/m3 in soil. Plant parasitic nematodes
(Chap. 11), a major group of worms that feed and reproduce on living plants, widely
exist as one of the main pathogens for global agriculture and cause crop damages
greater than those by bacteria and viruses (Cai et al. 1997). The significant impact
of specific nematodes on world agriculture is the result of their wide distribution,

X. Huang () · K. Zhang · Z. Yu · G. Li

Yunnan University, 650091 Kunming, People’s Republic of China
Tel.: +13888865821
e-mail: xwhuang@ynu.edu.cn
K. Zhang
Tel.: +86-871-6503487
e-mail: kqzhang@ynu.edu.cn
Z. Yu
Tel.: +86-871-65033805
e-mail: zfyuqm@hotmail.com
G. Li
Tel.: +86-871-65031092
e-mail: ligh@ynu.edu.cn

© Springer International Publishing Switzerland 2015 155

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_17
156 X. Huang et al.

their ability to attack a variety of cultivated plants, as well as the multiple types
of damages to individual plants. Take Meloidogyne. spp as an example. The root-
knot nematodes (i.e. Meloidogyne spp.) and the cyst nematodes (i.e. Heterodera and
Globodera spp.) are the two main groups of plant parasitic nematodes responsible
for the majority of crop losses (Molinari 2011). Meloidogyne. spp. infects about
3000 flowering plant species belonging to 114 different families, resulting in 10–
20 % crop failure per year (Wang et al. 2002). They preferentially invade the roots of
plants and cause the formation of giant cells and root knots in plants. Such damages
lead to the fracture in catheter, epidermis and cortical tissue, which consequently
endangers the plants by slowing their respiration, photosynthesis, and transpiration.
Furthermore, infection by Meloidogyne can also weaken the immunity or protective
enzyme system in plants, which makes the damaged plants easier to become infected
by pathogenic fungi or/and bacteria. Through the different modes described above,
plant parasitic nematodes cause damages to plants ranging from negligible injury
to serious destruction. It has been estimated that globally, plant parasitic nematodes
bring direct economic losses worth about US$ 157 billion per year (Abad et al. 2008).
The management of nematodes is more difficult than that for other pests be-
cause nematodes mostly inhabit the soil and usually attack the underground parts
of the plants. For a long time, due to its effectiveness, rapid effects, and ease of
use, the application of chemical products has been the most commonly used method
to control nematodes. However, public health and environmental concerns have
focused scientific interest on the development of environmentally acceptable alterna-
tives to chemical nematocides. Biological control, exploiting the interaction between
nematode-antagonistic microorganisms and their hosts, represents an alternative or
complementary approach to chemical controls.

17.2 Microbial Populations Antagonizing Phytopathogenic

Nematodes

A diversity of microorganisms exist in natural environments that function as an-

tagonists of nematodes. These microorganisms include fungi, bacteria, and viruses.
Those nematocidal microorganisms have evolved a variety of physical, chemical, and
biochemical mechanisms to kill nematodes, and they have been the most important
resources used in biocontrol of phytopathogenic nematodes. Among the nematocidal
microorganisms, nematophagous fungi and nematophagous bacteria have been the
most frequently investigated.
Nematophagous Fungi More than 700 species of nematophagous fungi have been
described. These fungi belong to widely divergent Orders and Families. The re-
ported fungal taxa of nematophagous fungi include Ascomycetes (e.g. asexual
Orbiliaceae and Clavicipitaceae), Basidiomycetes (e.g. Pleurotaceae), Chytrid-
iomycetes, Oomycetes and Zygomycetes (Zoopagales). They are broadly distributed
in terrestrial and aquatic ecosystems (Hao et al. 2005)
17 Microbial Control of Phytopathogenic Nematodes 157

According to the modes of nematicidal action, the nematophagous fungi can also
be divided into four groups, consisting of endoparasitic fungi, nematode-trapping
fungi, opportunistic fungi and toxic fungi respectively. Endoparasitic fungi infect
nematodes via special spores and subsequently mycelia grow from spores within the
nematodes. Endoparasitic fungi are typically regarded as obligate parasites since they
are poor saprotrophic competitors in soil. So far, about 120 nematode-endoparasitic
species have been reported, and the typical genera include Drechmeria W. Gams
& H.-B. Jansson, Myzocytium Schenk, Harposporium Lohde, Hirsutella Pat. and
Nematoctonus Drechsler (Zhang et al. 2011). The second group is the nematode-
trapping fungi and they capture nematodes by trapping devices produced from the
vegetative mycelia. The typical traps mainly include adhesive networks, adhesive
knobs, constricting rings, non-constricting rings, and adhesive branches (Fig. 17.1).
The majority of nematode-trapping fungi are asexual taxa, mostly known as hy-
phomycetes. Three hundred and forty seven nematode-trapping species have been
described, and they are mainly in Ascomycota, Basidiomycota and Zygomycota. The
representative genera include Arthrobotrys Corda, Cystopage Drechsler, Dactylella
Grove, Dactylellina M. Morelet, Drechslerella Subram, Hyphoderma Wallr., Ho-
henbuehelia Schulzer, Monacrosporium Oudem, Nematoctonus Drechsler, Orbilia
Fr., Stylopage Drechsler, Triposporina Höhnel, Tridentaria Preuss and Zoophagus
Sommerst (Zhang et al. 2011). The third group is the opportunistic nematophagous
fungi. These are also called egg and cyst parasitizing fungi because they can col-
onize nematode reproductive structures and affect their reproductive capabilities.
These fungi commonly use appressoria or zoospores to infect their hosts. This group
of fungi includes species in genera Pochonia, Paecilomyces, Lecanicillium and Ne-
matophthora (Siddiqui and Mahmood 1996). The fourth group is the toxic fungi and
they produce low molecular metabolites that are toxic to nematodes. Generally, these
fungi first immobilize nematodes by secreting toxins and then their hyphae pene-
trate the nematode cuticle. About 270 species of toxin-producing fungi have been
reported and they belong to widely divergent orders and families. Most of these fungi
are Basidiomycota (e.g., Pleurotus, Coprinus) although several ascomycete species
(e.g., Lecanicillium, Paecilomyces, Pochonia) also produce nematocidal compounds
(Lòpez-Llorca et al. 2008).
Nematophagous Bacteria Since nematophagous bacteria have several advantages
over fungi, such as their fast multiplication, ease of cultivation and mass production,
they have been more commonly applied as biocontrol agents in the field. Several bac-
terial agents have been shown similar effectiveness in controlling nematodes as chem-
ical pesticides (Zhou et al. 2002). Strains of the following bacterial genera have shown
the capabilities to infect nematodes: Actinomycetes, Agrobacterium, Arthrobacter,
Alcaligenes, Aureobacterium, Azotobacter, Bacillus, Beijerinckia, Chromobac-
terium, Clavibacter, Clostridium, Comamonas, Corynebacterium, Curtobacterium,
Desulforibtio, Enterobacter, Flavobacterium, Gluconobacter, Hydrogenophaga,
Klebsiella, Methylobacterium, Pasteuria, Pseudomonas, Phyllobacterium, Phingob-
acterium, Rhizobium, Stenotrotrophomonas, and Variovorax. Additionally, several
human bacterial pathogens, such as species of Burkholderia, Enterococcus, Staphy-
lococcus, Serratia and Streptococcus have also been reported to have antagonistic
effects against nematodes (Liu et al. 2013).
158 X. Huang et al.

Fig. 17.1 The typical trapping devices in nematode-trapping fungi and the formation process of the
adhesive networks. a adhesive networks, b constricting rings, c adhesive knob, d adhesive branch,
and e–h adhesive networks after adding nematode extracts. e 8 h, f 16 h, g 24 h, h 32 h. Bars are 20
μm in length
17 Microbial Control of Phytopathogenic Nematodes 159

Based on the modes of nematicidal action, nematophagous bacteria can be di-

vided into three groups. These groups are obligate parasitic bacteria, parasporal
crystal-forming bacteria, and opportunistic parasitic bacteria. Four Pasteuria species
(Pasteuria ramosa, P. penetrans, P. thornei, and P. nishizawae) are obligate, mycelial
and endospore-forming bacterial parasites of nematodes. They parasitize 323 nema-
tode species belonging to 116 genera, including main phytopathogenic nematode
genera Meloidogyne spp, Heterodera and Globodera (Atibalentja et al. 2000). During
their pathogenesis, the spores of Pasteuria attach to the cuticles of the second-stage
juveniles, and germinate after the worms enter plant roots and begin feeding. Paras-
poral crystal-forming bacteria, represented by Bacillus thuringiensis (Chap. 20), can
produce one or more parasporal crystal inclusions (Cry proteins) toxic to the larvae
of free-living or plant parasitic nematodes (Wei et al. 2003; Kotze et al. 2005). Most
of nematophagous bacteria, except for obligate parasitic bacteria, are opportunistic
parasites because they usually live a saprophytic life. But once the conditions be-
come suitable for parasitism, the bacteria will infect the nematode hosts as a possible
nutrient source. Most of the opportunistic parasitic bacteria are also rhizobacteria,
represented by Bacillus and Pseudomonas (Table 17.1). The rhizobacteria antago-
nize plant-parasitic nematodes by a variety of strategies to directly or indirectly kill
nematodes.

17.3 Virulence Factors as well as Molecular Mechanisms Used

by Nematophagous Microorganisms

Nematophagous microorganisms produce a series of virulence factors or employ

different strategies to reduce the populations of plant parasitic nematodes. They
antagonize nematodes mainly by competing for essential nutrients, promoting plant
growth, inducing systemic resistance, interfering with plant-nematode recognition,
regulating nematode behavior and/or directly killing the nematodes by means of
enzymes, small molecular metabolic products, protein toxins, and trapping devices.
In the next section, the microbial virulence factors that are directly used for killing
nematodes will be described in detail.
Hydrolytic Enzymes The cuticle of nematodes, which completely surrounds the
animal except for small openings, is a very rigid but flexible multilayered extracel-
lular exoskeleton structure and an effective barrel preventing nematodes from being
environmentally damaged. The cuticle of adult nematodes consists mainly of pro-
teins including keratin, collagen and fibers running diagonally in opposite directions
from each other. Therefore, penetration to the nematode cuticle requires the activi-
ties of hydrolytic enzymes. Indeed, both nematophagous fungi and nematophagous
bacteria have been reported to secrete different types of extracellular enzymes to help
the infectious process of pathogens (Huang et al. 2004; Tian et al. 2007).
Subtilisin-like serine protease is a virulence enzyme characterized by its catalytic
domain of aspartic acid-histidine-serine, and its roles in infection have been most
160 X. Huang et al.

Table 17.1 The main rhizobacterial species and their target nematodes
Genus Species Target nematodes
Bacillus B. subtilis, B. thuringiensis Meloidogyn javanica
B. cereus, B. circulans Meloidogyn incognita
B. mycoides, B. polymyxa Meloidogyn hapla
B. pumilus, B. sphaericus Heterodera glycines
B. stearothermophilus Trichodorus primitivus
Paratrichodorus pachydermus
Globodera pallida
Pseudomonas P. aeruginosa, P. putida Meloidogyn javanica
P. chlororaphis, P. cepacia Meloidogyn incognita
P. fluorescens, P. gladioli Criconemella xenoplax
P. mendocina, P. picketti Heterodera glycines
P. aureofaciens Pratylenchus penetrans
Radopholus similes
Tylenchulus semipenetrans
Globodera pallida
Rotylenchulus reniformis
Burkholderia B. cepacia, B. ambifaria Meloidogyn incognita
B. glathel Heterodera glycines
Streptomyces S. violaceus niger Meloidogyn incognita
S. lavendulae Heterodera glycines
Hydrogenophaga H. flava Meloidogyn incognita
H. pseudofalva Heterodera glycines

intensively studied in nematophagous microbes. Currently, more than 20 serine pro-

teases from nematophagous fungi and four from nematophagous bacteria have been
identified respectively. These serine proteases share several biochemical traits: most
of them are highly sensitive to the serine protease inhibitor phenylmethylsulfonyl
fluoride (PMSF); their maximal activities can generally be obtained under alkaline
conditions; the purified proteases can degrade the proteins from the nematode cuti-
cle and even destroy the adult nematode cuticle or nematode eggshells. Additionally,
comparisons of the deduced amino acid sequences showed relatively low similarities
between those from nematophagous fungi and nematophagous bacteria except for
the active center of catalytic domains and the substrate-binding pockets. However,
higher degrees of similarity can be found among members within either fungal ser-
ine proteases or bacterial serine proteases. Besides the alkaline serine proteases, a
few neutral metalloproteinase have also been identified as a virulence factor during
bacterial infection.
17 Microbial Control of Phytopathogenic Nematodes 161

A chitinous layer, the thickest component in the shell of nematode eggs, is also
the major barrier against infections by nematophagous microorganisms. Therefore
chitinases, a group of inducible enzymes capable of degrading chitin, have been as-
sumed to be required for egg infection. Since the first extracellular chitinase (CHI43)
was identified from Pochonia chlamydosporia and P. rubescens, several chintinases
were subsequently investigated in the opportunistic nematophagous fungi. The ex-
perimental data from microscopic observations demonstrated a series of changes
in the chintinase-treated eggs: large vacuoles in the chitin layer of nematode eggs;
swollen, deformed and even degraded eggs; abnormal development or hatch of eggs
(Gan et al. 2007; Mi et al. 2010).
Certainly, other extracellular enzymes, such as collagenase, lipases and elastases,
are also reported to be involved in the infection of worms or eggs. In conclusion, those
hydrolytic enzymes are believed to be involved in several steps of infection, such
as facilitating microbial penetration by degrading proteins of the cuticle, causing
nematode deaths by targeting intestinal epithelium, and releasing nutrients to further
support the growth of nematophagous microbes.
Cry Proteins Cry proteins, called δ-endotoxins, are generally produced by B.
thuringiensis during sporulation. Those Cry proteins are encoded by cry genes that
are commonly located on a plasmid in most strains of B. thuringiensis. Cry proteins
were first shown to be strictly insecticidal, but later some authors described them
as also toxic to other invertebrates, including nematodes. To date, six Cry proteins
(Cry5, Cry6, Cry12, Cry13, Cry14, and Cry21) have been described to have nema-
totoxic activities to free-living and phytopathogenic nematodes (Kotze et al. 2005).
Based on their amino acid sequences, the Cry proteins with nematocidal activity can
be divided into two clusters. Among them, Cry5, Cry12, Cry13, Cry14, Cry21 were
arranged together in a branch; but Cry6 was assigned to a separate cluster that had
relatively low similarity in either amino acid sequence or protein crystal structure
to the first cluster. The molecular mechanism underlying the Cry5 cluster has been
elucidated in the model nematode Caenorhabditis elegans. Once the Cry5 toxin has
been ingested by the target nematode larvae, the crystals dissolve within the gut and
then the toxin molecules bind to a receptor in the epithelial cell. This binding leads
to vacuole and pore formation, pitting, and eventual degradation of the intestine
(Crickmore 2005; Marroquin et al. 2000).
Small Toxic Metabolites Nematophagous microorganisms can produce a large
number of small molecular metabolites to reduce nematode reproduction, egg hatch-
ing and juvenile survival, and even kill nematodes directly. So far, more than 200
compounds with nematicidal activities have been reported from about 280 fun-
gal species in 150 genera of Ascomycota and Basidiomycota. These nematocidal
compounds are very diverse in structure and include alkaloids, quinones, pyrans,
benzofuran, furan, peptides, macrolides, lactones, terpenoids, fatty acids, dike-
topiperazines, simple aromatics, as well as alkynes etc (Li et al. 2007). Fewer
metabolites have been obtained from nematophagous bacteria, likely due to the
much simpler metabolic pathway present in bacteria than in fungi.
162 X. Huang et al.

It is particularly worth mentioning that avermectin compounds, which have potent

insecticidal and nematocidal properties, are extensively used in agriculture world-
wide. The avermectins consist of a series of 16-membered macrocyclic lactone
derivatives. These naturally occurring compounds are generated as fermentation
products by the soil actinomycete Streptomyces avermitilis (Omura and Shiomi 2007)
and have shown significant antagonistic effects to some plant parasitic nematodes
such as Ditylenchus dipsaei and Meloidogyne hapla. Experiments with the model
worm C. elegans suggested that avermectins mediate their nematocidal effects via
interacting with a common receptor molecule, glutamate-gated chloride channels
(Arena et al. 1995).
Trap Formation Mechanical activities, especially trapping devices in nematode-
trapping fungi mentioned above, is another important factor involved in the infection
against nematodes. Though the trapping devices are developed from vegetative hy-
phae, they show a large variation in morphology and a distinguishable ultrastructure
from regular hyphae. For example, the dense bodies of peroxisomes and the extra-
cellular polymers are much more prominent in traps than in regular hyhae. Because
the development of traps is a complex process involving many genes, a few high
throughput methods of genomics, microarray, proteomics, and transcriptomics have
been applied to investigate the mechanisms of trap formation. Recently, a model
of trap formation in A. oligospora (three-dimensional networks) was suggested to
include active cytoskeleton assembling, enhanced cell wall biosynthesis, increased
glycerol synthesis and accumulation, as well as peroxisome biogenesis (Yang et al.
2011). Additionally, a few novel mechanical structures with nematicidal activity
have been reported in our lab, including the spiny ball from Coprinus comatus and
acanthocytes from Stropharia rugosoannulata, which can damage the cuticle of the
nematode with the mechanical force provided by their sharp projections (Luo et al.
2006, 2007).

17.4 Commercial Biocontrol Nematicides

Despite its long history and recent developments, relatively few commercial bio-
control products based on nematophagous fungi and bacteria have been developed.
However, the pace of discovery and commercialization is accelerating. For instance,
several commercial products based on the bacteria P. penetrans, Bacillus firmus,
Burkholderia cepacia and Bacillus spp., as well as on the fungi Purpureocillium lilac-
inus, Pochonia chlamydosporia and Myrothecium verrucaria have been developed
to control the root-knot nematodes Meloidogyne spp. (LamovŠek et al. 2013). How-
ever, for more effective biocontrol, a more thorough understanding of the complexity
of soil ecology in agricultural fields is required.
17 Microbial Control of Phytopathogenic Nematodes 163

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Chapter 18
Microbial Control of Root-Pathogenic Fungi
and Oomycetes

Linda Thomashow and Peter A. H. M. Bakker

Abstract The rhizosphere is a complex and dynamic environment in which microbes

introduced to control root pathogens must establish and maintain populations of suf-
ficient size and activity to antagonize pathogens directly or by manipulating the host
plant’s own defenses. Genetic and physiological studies of rhizobacteria with the
capacity to control root pathogens have given considerable insight into the microbial
side of these interactions, but much remains to be learned about the physical condi-
tions and the chemical and biological activities that take place at the root-microbe
interface. This chapter focuses on advances in our understanding of the constraints
to the successful introduction of microbial agents for the control of soil-borne root
pathogens and the mechanisms involved in pathogen suppression. Chapters else-
where in this volume address related topics including plant growth promotion, stress
control, the activation of the plant’s own defense mechanisms by introduced mi-
crobes, and powerful new biotechnological advances available to gain insight into
rhizosphere processes.

18.1 Colonization: A Necessary Requisite for Biological Control

Root colonization, a challenge to both indigenous microbes and those introduced to

control root pathogens, is the process by which bacteria become distributed along
the root, propagate, and persist for weeks or longer in the presence of the indigenous
rhizosphere microflora. How many bacteria are needed to achieve pathogen control?
The minimum population size required will depend in part on the disease pressure,

L. Thomashow ()
USDA-ARS, Root Disease and Biological Control Research Unit,
Washington State University, PO Box 646430, Pullman, WA, USA
Tel.: +1-509-335-0930
e-mail: thomashow@wsu.edu
P. A. H. M. Bakker
Plant-Microbe Interactions, Institute of Environmental Biology,
Utrecht University, 3508 TB Utrecht, The Netherlands
Tel.: +31 30 253 6861
e-mail: P.A.H.M.Bakker@uu.nl

© Springer International Publishing Switzerland 2015 165

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_18
166 L. Thomashow and P. A. H. M. Bakker

the distribution of the bacteria along the root, and the mechanism of control, but
several lines of evidence suggest a minimum average population size of 105 per
gram of root, at least for antibiosis and the induction of systemic resistance (Pieterse
et al. 2014).
Bacteria on root surfaces are not uniformly distributed; they reside in discrete
mucigel-enclosed aggregates termed biofilms where nutrients are available. Such
sites include wounds along the root, root tips, the junctions between epidermal cells,
and regions where root hairs and lateral roots emerge. Exudates from these sites are
a dominant source of nutrients for rhizobacteria, and there is increasing evidence
that the sugars, organic and amino acids, phenolics, and other signal molecules in
exudates maintain a complex chemical dialog between the plant and its associated
microflora (Zolla et al. 2013). The quality, quantity, and composition of root exudates
vary widely with the plant species and the biotic and abiotic stresses acting upon it,
and these factors have a significant effect on the structure and composition of the
associated microbial communities. There has been considerable effort in recent years
to isolate and characterize exudates, but the best methods now available still identify
only a fraction of the compounds present (Zolla et al. 2013).
Biofilms The extracellular matrix in which bacterial cells on roots are embedded in
biofilms is composed mainly of proteins and exopolysaccharides, of which the latter
vary in composition among strains but are key structural components (Martínez-Gil
et al. 2013). In nature, biofilms provide microbes with a stable protective barrier
against chemical stresses and protozoal grazing. In the model strain Pseudomonas
putida KT2440, an efficient colonizer of seeds and roots, two very large extracellular
proteins, LapA and LapF, have sequential roles in biofilm development. LapA first
facilitates a cell-surface interaction resulting in irreversible bacterial attachment, and
LapF mediates subsequent cell-cell interactions, providing support for expansion and
maturation of the biofilm. Complex interactions modulated by the two-component
global regulators GacS/GacA and the bacterial universal second messenger cyclic
dimeric guanosine phosphate (c-di-GMP) have integral roles in the balance of protein
and polysaccharide constituents within the biofilm (Martínez-Gil et al. 2014) and
therefore its structural integrity and characteristics. c-di-GMP also has an important
role in the transition of cells in biofilms to the planktonic form associated with
motility. Biosurfactants such as cyclic lipopeptides (cLPs) and rhamnolipids also can
influence the formation, as well as the stability and dissolution, of biofilms, and it has
been postulated that the contrasting roles of this diverse family of compounds may
be due to differences in their chemical structures and physicochemical properties, as
well the ionic conditions and pH of their environment (Raaijmakers et al. 2010).
Motility Bacterial movement along roots may occur passively with root elongation
or redistribution with water. Alternatively, dispersal mediated by flagellar swimming
or swarming can be active. Numerous studies have demonstrated that bacterial mu-
tants defective in motility and chemotaxis, the ability to detect and move towards
nutrients, also are impaired in rhizosphere colonization (Lugtenberg and Kamilova
2009; Pliego et al. 2011). Recent studies suggest that these mechanisms are likely
to require relatively hydrated root surfaces (Dechesne et al. 2010; Dechesne and
18 Microbial Control of Root-Pathogenic Fungi and Oomycetes 167

Smets 2012) and/or the presence of biosurfactants that can modulate the viscosity
and surface tension of the thin water films that are thought to be present on roots
under common agricultural conditions. Biosurfactants clearly contribute to micro-
bial surface motility in vitro, but whether they have a similar role in the environment
remains uncertain (Raaijmakers et al. 1999). Collectively, however, it is apparent that
introduced bacteria can and do move along the root, that movement can be facilitated
by one or more different mechanisms, and that a greater understanding is needed of
the physicochemical and hydrodynamic forces that prevail in the rhizosphere.

18.2 Mechanisms of Biological Control

Biological control of plant root pathogens may be mediated indirectly, by compe-

tition for nutrients and niches on the root or by induction of resistance in the host
plant. Alternatively, biocontrol agents may directly antagonize the pathogen via the
production of biosurfactants, antibiotics, or enzymes that hydrolyze the pathogen
cell wall. Biocontrol agents often express more than one mechanism of action, and
metabolites such as the antibiotics and iron-sequestering siderophores produced by
many biocontrol agents may have multiple activities, affecting not only the target
pathogen but also the host plant and other members of the microbial community.
Competition for Niches and Nutrients (CNN) Kamilova et al. (2005) used a clas-
sic selective enrichment procedure to recover novel biocontrol agents enhanced in
competitive colonization of the root. A mixture of rhizosphere strains was introduced
onto surface-sterilized tomato seeds and the seeds were allowed to germinate and
grow for one week in a gnotobiotic (germ-free) system. Bacteria that had moved
along the root to the tip were then collected, introduced onto sterile seeds, and the
cycle was repeated three times. Many of the isolates recovered from root tips af-
ter this procedure were found to protect tomato against foot and root rot caused by
Fusarium oxysporum f. sp. radicis-lysopersici, and studies with mutants showed that
both motility and the ability to grow efficiently on nutrients in tomato root exudate
were important for biocontrol (Lugtenberg and Kamilova 2009; Pliego et al. 2011).
Not all of the recovered isolates were effective against the pathogen, however, and
results of another study (Pliego et al. 2011) involving the white root rot pathogen of
avocado indicated that not only aggressive colonization of the root, but also colo-
nization of specific target sites of pathogen attack on the root, or even colonization
of the surface of the pathogen itself, may be required to achieve disease control.
Induced Systemic Resistance (ISR) In induced systemic resistance, a plant’s first
line of defense against pathogens, selected beneficial microbe-associated inducers
on roots activate and sensitize (i.e., prime) the entire plant, including the above-
ground parts, for defense against diverse pathogens (Pieterse et al. 2014). Like
immunization in animals, priming sensitizes the immune system to an enhanced
state of readiness such that defenses can be mobilized rapidly and systemically upon
168 L. Thomashow and P. A. H. M. Bakker

subsequent pathogen attack. However, priming differs from immunization in that im-
munization targets specific pathogens whereas priming by beneficial root-associated
microbes enhances defenses against diverse pathogens and even herbivorous insects.
The exposure of roots to certain common microbial structural elements such as flag-
ella, lipopolysaccharides, and chitin, and microbially-produced compounds such as
siderophores, certain antibiotics, biosurfactants, and volatiles, can result in priming
in a wide variety of plant species.
Because the induction of defense and the enhanced defense reaction in ISR take
place in spatially separated locations, plants must employ a complex long-distance
signaling pathway that starts at the root-microbe interface. Signaling is modulated by
the hormones ethylene and jasmonic acid (and less commonly by salicylic acid). In all
cases, however, a complex array of signaling proteins and transcriptional regulators
is involved that accumulates after induction of the primed state. This pathway, and the
identity of ISR long-distance signals, are currently subjects of active investigation.
Siderophore-Mediated Competition for Iron and Induced Systemic Resistance
Under conditions of low iron availability bacteria produce siderophores, low molecu-
lar weight compounds that sequester iron in the environment and facilitate its uptake
by bacterial cells (Höfte 1993). Since basically all organisms require iron to grow
and function, and iron availability in soil is extremely low, competition for iron be-
tween microorganisms in the rhizosphere is expected to be a common phenomenon.
Siderophores produced by different microorganisms can belong to different classes
and they have different structures (Höfte 1993). Accordingly, specific receptors are
required to utilize ferric siderophore complexes (Hartney et al. 2011). Thus it was
postulated that bacteria that produce siderophores with a high affinity for iron and that
are specific, that is they can be utilized by the producer but not by other microbes,
could be effective biological control agents. Indeed competition for iron between
microorganisms in the rhizosphere has been demonstrated using reporter gene con-
structs that respond to bioavailability of iron (Loper and Henkels 1999). In several
studies on fluorescent pseudomonads it was demonstrated that mutants unable to pro-
duce their fluorescent siderophore also (partially) lost their ability to control disease
(see for example De Boer et al. 2003). Based on such studies, siderophore-mediated
competition for iron is considered an important mechanism in biological control of
soil-borne diseases.
Siderophores also have been implicated as effective elicitors of ISR in plants
(Meziane et al. 2005). Thus, when plants are protected from disease by siderophores
produced by biological control agents, it is difficult to assess if this is due to competi-
tion for iron, to ISR, or to both. In radish, Pseudomonas putida WCS358 effectively
controls fusarium wilt, caused by Fusarium oxysporum f. sp. raphani, through the
production of its pyoverdin siderophore (De Boer et al. 2003). Strain WCS358
cannot elicit ISR in radish and thus in this case the suppression of disease most
likely depends on competition for iron between the biological control agent and the
pathogen. Evidence that the fluorescent siderophore produced by strain WCS358
can elicit ISR comes from studies in Arabidopsis thaliana, bean and tomato. In all
three plant species, application of the purified pyoverdin of WCS358 did elicit ISR.
18 Microbial Control of Root-Pathogenic Fungi and Oomycetes 169

In A. thaliana and bean, a pyoverdin mutant was as effective as the wild-type, but
in tomato the pyoverdin mutant did not elicit ISR (Meziane et al. 2005). For tomato
the role of pyoverdin in ISR is straightforward but for A. thaliana and bean there
seems to be redundancy of bacterial elicitors of ISR and next to the pyoverdin, both
lipopolysaccharides and flagella play a role. The observed redundancy may give ro-
bustness to biological control, since if one of the factors involved in biocontrol is
not produced by the bacterium the additional determinants can still be effective. This
situation may however further complicate studies on siderophore-mediated compe-
tition for iron. In several studies it was suggested that siderophores do not play a
role in disease suppression since knock out mutants were as effective as the wild-
type. When we consider that there can be redundancy in biological control traits in a
single biological control agent, the mutant approach to study possible involvement
of siderophores is not waterproof and in several cases, rejecting their role may have
been unjustified. Nevertheless, siderophores have been demonstrated to play a sig-
nificant role in biocontrol and both competition for iron and triggering ISR can be
involved. If both mechanisms were active simultaneously this would first weaken
the pathogen in the rhizosphere by iron depletion and then confront it with an en-
hanced plant defense resulting in effective biocontrol. Such a scenario may explain
why siderophores were early on revealed as being important effectors of biological
control of soil-borne diseases.
Antibiosis Antibiotics are small organic molecules produced by microorganisms
that are deleterious to the growth or metabolic activities of other microorganisms.
Most bacteria involved in biocontrol have the capacity to produce antibiotics, and
many produce multiple such metabolites with broad-spectrum activity against a wide
range of plant pathogens (Raaijmakers and Mazzola 2012). Antibiotic-producing
strains typically are detected by their ability to inhibit the growth of target pathogens
in vitro and then are tested for biocontrol in soil, but this approach often fails due to
the producer strain’s inability to compete successfully in the rhizosphere or because
conditions there do not support the synthesis of inhibitory levels of the active agent in
the sites where pathogens attack. While the spatiotemporal and quantitative aspects of
antibiotic production in nature are only now beginning to be explored, it has become
apparent in recent years that antibiotics play multiple roles in natural systems. At
subinhibitory concentrations these molecules can function as molecular signals in
such diverse activities as biofilm formation and cellular differentiation, motility and
dispersal, and defense against predators and competitors (Raaijmakers and Mazzola
2012), all of which may impact upon biological control, and some antibiotics are
produced in the environment in sufficient quantities to inhibit pathogens (Mavrodi
et al. 2012). The availability since 2005 of genomic DNA sequences not only for
model biocontrol strains, but also for environmental isolates, has greatly facilitated
the identification of a repertoire of novel metabolites and gene clusters with the
potential to exhibit antibiotic activity. Pseudomonas and Bacillus spp. are the most
widely studied biocontrol agents to date, and Bacillus spp. are the most frequently
commercialized because they are more readily formulated.
170 L. Thomashow and P. A. H. M. Bakker

Biosurfactant Antibiotics Produced by Bacillus spp Cyclic lipopeptides (cLP)

produced by root-associated bacilli are composed of a lipid tail linked to a short cyclic
oligopeptide and include members of three main families, the surfactins, iturins and
fengycins, which differ in the type, number and sequence of amino acid residues and
the nature of peptide cyclization. Given their structural diversity, it is not surprising
that these antibiotics also differ in their modes of action and their contributions to the
biocontrol activity of the cells that produce them. For example, surfactins are power-
ful biosurfactants that can interfere with biological membrane integrity, but they have
no marked toxicity to fungi. In contrast, both iturins and fengycins exhibit antifungal
activity which is thought to be due to their ability to form ion-conducting pores in
target cell membranes, leading to an imbalance in transmembrane ion fluxes and
cell death. These latter classes of cLPs have been implicated directly in antagonism
of soil-borne and foliar fungal phytopathogens including Fusarium graminearum,
Rhizoctonia solani, Botrytis cinerea, Penicillium roqueforti, and Aspergillus flavus
(Raaijmakers et al. 2010).
Rhizosphere factors profoundly influence the production of cLPs and the induction
of systemic resistance by Bacillus spp. Members of the surfactin family, which
are inducers of ISR in a broad range of plants, are the main cLPs secreted by B.
amyloliquefaciens S499 in biofilms and on the roots of tomato, whereas iturins
were detected at much lower concentrations and fengycins were not produced in
measurable quantities. This is in marked contrast to production in vitro, where iturins
and fengycins are produced in similar quantities and each was more than half the
amount of surfactin present. A combination of elegant electrospray and imaging mass
spectrometry-based approaches showed that cLPs, mainly surfactin, were formed by
microcolonies directly on tomato roots and root hairs, and that surfactin diffused
into the surrounding medium, reaching biologically relevant concentrations within
the diffusion zone close to the colonized roots (Nihorimbere et al. 2012). Moreover,
a strong correlation was demonstrated between the amount of surfactin produced
and the ability to elicit ISR, suggesting that screening for strong producers among
members of the B. subtilis/amyloliquefaciens complex could facilitate the selection
of effective biocontrol agents (Cawoy et al. 2014).
Antibiotics produced by Pseudomonas spp Lipopeptide-producing strains of
Pseudomonas spp., like those of Bacillus, are widely distributed in nature. Many
have significant impacts on oomycetes such as Phytophthora and Pythium spp., with
members of the viscosin group of compounds including massetolide A of particu-
lar note for their effects (ranging with increasing concentration from inducement of
encystment to immobilization and lysis) on zoospoores. Effects of these cLPs on
mycelial morphology and physiology not only of oomycetes, but also of Rhizocto-
nia, typically are less pronounced than on zoospores and include increased hyphal
branching and swelling (Raaijmakers et al. 2010; D’aes et al. 2011). cLPs pro-
duced by Pseudomonas spp. have been implicated in the control of pathogens such
as Pythium ultimum on sugar beet, Phytophthora infestans on tomato, Pythium and
Rhizoctonia spp. on bean, and R. solani and Gaeumannomyces graminis var. tritici
on wheat (Yang et al. 2014).
18 Microbial Control of Root-Pathogenic Fungi and Oomycetes 171

Among the most commonly detected small metabolite antibiotics produced by

Pseudomonas spp. are phenazine compounds, 2, 4-diacetylphloroglucinol (DAPG),
pyrrolnitrin and pyoluteorin. The genetics, regulation of synthesis, and mode(s)
of action of these compounds have been studied intensively, and the compounds
themselves have, in most cases, been isolated from roots colonized by introduced
or indigenous strains of the producing bacteria (Raaijmakers and Mazzola 2012).
Of particular note are DAPG, produced by indigenous strains responsible for con-
trol of the wheat root disease take-all throughout much if not all of the USA and
Europe (Chap. 38) and phenazine-1-carboxylic acid (PCA), present on the roots of
wheat grown in dryland regions throughout theAmerican Pacific Northwest (Mavrodi
et al. 2012). The polyketide antibiotic DAPG is active against a wide range of phy-
topathogens, and also can elicit ISR in plants (Weller et al. 2012). The phenazines
encompass a large family of pigmented, heterocyclic redox-active compounds that
function not only in biological control, but also as microbial fitness determinants,
virulence factors in plant and animal disease, and in microbial community dynamics
and physiology. The phenazine-producing strain P. aureofaciens 1393 is marketed
as an active ingredient in the biopesticide “Pseudobacterin-2” in the Russian Federa-
tion for the control of a wide range of phytopathogens as well as for the induction of
resistance to plant diseases in organic and conventional crops (Thomashow 2013).
PCA is the major ingredient in Shenqinmeisu, a microbial pesticide produced in
China and applied commercially to protect rice and vegetables against important
phytopathogens (Xu 2013).

18.3 Conclusions

In this chapter we have touched briefly upon recent insights towards understanding
interactions among microorganisms and their plant hosts, as well as knowledge gaps
and needs for future research. Technological advances that enable sensitive detection
of metabolites including root exudate components produced in situ will continue to
be critical to unraveling the complex molecular and organismal interrelationships in
the rhizosphere habitat. Better knowledge of the microbe-plant dialogue is essential
given the need for increased agricultural productivity to provide food and biofuel
feedstocks in the face of climate change, the increasing world population and the
loss of arable lands.

Acknowledgements Parts of this work were supported by USDA-NRI Grant No. 2011-67019-
30212 from the USDA-NIFA Soil Processes program. Mention of trade names or commercial
products in this publication is solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal
opportunity provider and employer. We thank Dr. David Weller for review comments.
172 L. Thomashow and P. A. H. M. Bakker

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Chapter 19
Control of Insect Pests by Entomopathogenic
Nematodes
◦
Vladimír Puža

Abstract Entomopathogenic nematodes (EPNs) of the families Steinernematidae

and Heterorhabditidae (Rhabditida, Nematoda) are obligate pathogens of insects
and are associated with specific symbiotic bacteria of the genera Xenorhabdus and
Photorhabdus, respectively. Due to their ability to infect various insects, the possi-
bility of mass production by industrial techniques, and safety to non-target organisms
and the environment, these nematode-bacteria complexes are attractive agents for the
biological control of many insect pests. In this chapter, a brief characteristic of these
organisms is given, together with information on their use in the biological control
of insect pests.

19.1 Entomopathogenic Nematodes and their Bacterial

Symbionts

Entomopathogenic nematodes of the families Stenernematidae and Heterorhabditi-

dae do not form a monophyletic group, but evolved independently from a free-living
bacterivorous nematode in the Devonian period (Poinar 1993). Both Xenorhabdus
and Photorhabdus are gram-negative rods belonging to the family of Enterobac-
teriaceae, within the gamma subdivision of Proteobacteria. Unlike their nematode
associates, both genera form a monophyletic group and Proteus bacteria are the
closest sister taxon.
The family Steinernematidae comprises over 80 species, and the genus Xenorhab-
dus over 20 described species. The family Heterorhabditidae, probably a phyloge-
netically younger group of nematodes, has 18 described species, and four species of
associated Photorhabdus bacteria. Single species of Steinernema may be associated
with only one species of Xenorhabdus. The same applies to Heterorhabditis with
the exception of H. bacteriophora that is associated either with P. luminescens or

◦
V. Puža ()
Institute of Entomology, Biology Centre of the AS CR, Branišovská 31,
37005 České Budějovice, Czech Republic
Tel.: +420732561579
e-mail: vpuza@seznam.cz

© Springer International Publishing Switzerland 2015 175

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_19
 ◦
176 V. Puža

Fig. 19.1 Generalized life cycle of entomopathogenic nematodes

P. temperata. On the other hand, species of Photorhabdus and certain species of

Xenorhabdus are hosted by several species of Heterorhabditis and Steinernema.
Over 80 % of EPNs and bacteria have been described since the year 2000 thanks
to the use of molecular methods, and further descriptions of new EPN and bacterial
species are to be expected in the future. New EPN species present a unique combina-
tion of characteristics and may also have a great potential for the biological control
of particular insect pests.
Life Cycle (Fig. 19.1) The only free-living stage is a non-feeding and develop-
mentally arrested third stage larva, called infective juvenile (IJ). Infective juveniles
actively seek insect host, using various chemical cues. Once the host is found, they
penetrate into the haemocoel via natural openings. Upon entering an insect host, the
nematode expels its bacterial symbionts, which produce extracellular toxins and kill
the host within 1–3 days. The insect carcasses with Steinernema-Xenorhabdus turn
yellowish-brown in color, while those killed by Heterorhabditis-Photorhabdus turn
red. The bacteria produce degradative enzymes, and proliferate, providing a food
source that supports the reproduction of an increasing population of nematodes. Lar-
vae develop into amphimictic adults in the genus Steinernema or hermaphrodites in
19 Control of Insect Pests by Entomopathogenic Nematodes 177

Heterorhabditis. The first generation is followed by several amphimictic generations

in both genera. When the insect carcass is depleted of nutrients, the nematodes revert
back to the resting form, and their receptacle is colonized by bacterial cells. This
usually happens within 7–14 days after infection, depending on the host size, tem-
perature, and other factors. At the end of the cycle, thousands of infective juveniles
emerge from the carcass in search of a new insect host (Fig. 19.1).
Symbiosis The relationship between Steinernema and Xenorhabdus and Het-
erorhabditis and Photorhabdus is obligate in the natural environment (Akhurst and
Boemare 1990). For an excellent review on this subject, see Ciche et al. (2006).
Under experimental conditions, the nematodes may also consume other bacteria, but
the IJs can associate only with their own symbionts. Thus, under natural conditions,
the nematodes do not live without their symbiont, and, with the exception of P.
asymbiotica, the bacteria do not live freely without the nematodes.
The location of the symbiont within the IJ nematode differs between the two
genera. In the genus Steinernema the IJs carry dozens of symbionts in a specialized
structure called the receptacle. IJs of Heterorhabditis species transmit the symbionts
in the anterior part of the intestine. The bacteria persist for many weeks within the
free-living IJs in a quiescent state until becoming pathogenic within the insect host.
Besides killing the host and providing a food source to the nematodes, the bacteria
also protect the cadaver against other microorganisms and scavengers by the produc-
tion of bacteriocins, antibiotics, antimicrobials, and scavenger deterrent compounds.
On the other hand, the nematodes protect the bacteria in the external environment
and vector them into the insect hemocoel. In some associations, the nematode also
inhibits the insect immune response by producing proteases and immune suppressive
factors.
Typical for symbionts of both genera, Xenorhabdus and Photorhabdus, is the
phenomenon of phase variation. The primary phase is isolated from IJ nematodes
and in EPN-infected insects, while the secondary phase sometimes occurs during the
late stages of nematode development in insect cadavers and during the sub-culturing
of the primary form. Secondary stage cells are smaller, produce a lower number
of secondary metabolites, and have lower nutritional value. When the bacteria are
cultured under standard conditions, they are in the primary phase, however, stress
conditions can evoke a shift to the secondary stage. The shift to the secondary phase
has a detrimental effect on nematode development, particularly in liquid culture
(Ehlers et al. 1990). Thus all measures should be taken during production to avoid
the occurrence of phase variation.
Host Range The host range of EPNs is still not fully understood. In laboratory
experiments, they are capable of invading and killing a large number of insects and
even other arthropods, e.g. spiders, ticks and millipedes (Poinar 1979). However,
under natural condition, the host spectrum is much narrower and can include only
insects that spend at least some time within the soil environment. Insects naturally
infected with EPNs are rarely found. The available data show that the most frequently
infected larval stages of Diptera, Coleoptera, Lepidoptera, and Hymenoptera prevail
(Peters 1996). Peters (1996) concluded that some species like S. carpocapsae and S.
 ◦
178 V. Puža

feltiae are generalists with a broader host range, whereas specialists like S. glaseri and
S. scapterisci have a more restricted host range. On the other hand Pu̇ža and Mráček
(2010) found no difference in the natural host range of S. affine and S. kraussei.
Many insects, such as elaterid larvae (wireworms) possess various morphological
barriers that may protect them from invasion. Other insects, e.g. scarab larvae, display
aggressive and evasive behaviour, or other responses such as frequent defecation to
expel nematodes from the gut, as observed in some fly larvae. Alternatively, some
insects are able to detect EPNs, and move to an EPN-free place to prevent invasion,
as observed in fire-ant colonies treated with Steinernema carpocapsae. Once in the
host haemocoel, the nematodes may encounter a strong immune response. Often,
these defense strategies are powerful enough to make the insect resistant to infection
by EPNs.
Foraging Strategies From the biocontrol point of view, the foraging strategies of
entomopathogenic nematodes are of particular interest, because they make the ne-
matodes differentially suitable for the control of different insect pests. The strategies
range from sit and wait (ambusher) to actively foraging (cruiser) (Lewis et al. 1992).
Cruisers move through the soil and look for the sedentary host whereas ambushers
remain near the soil surface preferably attacking passing moving insects. Moreover,
the foraging strategy is not a two-class model, but it is a continuum with two poles
(Campbell and Gaugler 1997) and the majority of species possess an intermediate
strategy. Recently, Wilson et al. (2012) have demonstrated, that a typical ‘ambush’
forager, Steinernema carpocapsae, can use cruiser strategy in habitats other than
mineral soils and successfully control sedentary or cryptic pests in organic habitats.
The authors concluded that the classification of S. carpocapsae as an ambush forager
cannot be sustained.

19.2 Entomopathogenic Nematodes in Biocontrol

Collection For the use in biocontrol, EPN collection should be made in areas with
the occurrence of target insects, where EPN strains adapted to this pest are to be
expected. As was mentioned above, naturally infected insects are rarely found. Thus
the most frequently used technique used for EPN isolation is Galleria baiting (Bed-
ding and Akhurst 1975) using larvae of the greater wax moth, Galleria mellonella, as
bait. The insects caged in a wire mesh are placed in the soil (either in a soil sample,
or in situ). A few days later, the infected insect cadavers are removed, washed, and
placed on a White trap (White 1927) (top of a Petri dish with moist filter paper placed
in a larger Petri dish with water). One to two weeks later, thousands of fresh IJs are
collected for further use.
Mass Production EPNs can be mass produced using in vivo and in vitro methods.
In vivo production uses living insects, mostly the greater wax moth (Galleria mel-
lonella) larvae, or mealworms (Tenebrio molitor). The method is simple and cheap,
but is labor and cost effective only at a small scale. This method is thus appropriate
for laboratory use or small scale applications. It may also be used by small growers,
where large investments into in vitro culture technology cannot be made.
19 Control of Insect Pests by Entomopathogenic Nematodes 179

Solid or liquid fermentation in vitro technologies are used when large scale produc-
tion is needed at a reasonable quality and cost. In both these methods the nematodes
are cultured monoxenically, to ensure quality consistency and predictability (Lunau
et al. 1993). A symbiont is extracted from the nematodes and subsequently sterile
nematode eggs are prepared and applied to the medium pre-inoculated with bacterial
symbiont. A comprehensive review of the current situation regarding the in vitro
mass production of EPNs was published by Shapiro-Ilan et al. (2012).
Solid culture was first performed in two dimensional arenas (e.g, Petri dishes),
using various media. A substantial improvement was achieved by adopting a three-
dimensional rearing system with the liquid medium mixed with an inert carrier (e.g.
pieces of polyurethane foam). Media were initially based on animal products (e.g. pig
kidney) but were later improved by including various other ingredients (e.g. peptone,
yeast extract, eggs, soy flour). The culture starts with the inoculation of the sterilized
medium with bacteria followed by the nematodes. Nematodes are then harvested
within 2–5 weeks by placing the foam onto sieves immersed in water. Only a few
companies currently use this approach.
The in vitro liquid culture method is a complex process requiring medium de-
velopment, understanding of the biology of the nematode-bacteria complex, the
development of bioreactors, and understanding and control of the process parame-
ters. The process takes place in large fermentors (up to 80.000 l). It is necessary to
supply enough oxygen, and prevent excessive shearing of the nematodes. Once the
culture is completed, nematodes are removed from the medium through centrifuga-
tion. This method is currently the most cost-effective and thus the majority of EPN
products result from liquid culture. Major producers using this method are E-Nema
GmbH, Koppert B.V., Becker Underwood, Andermatt Biocontrol, BioLogic, etc.
Commercialized EPNs are listed in Table 19.1.
The repeated culturing of nematodes can result in the reduction of benefi-
cial traits such as infectivity, environmental tolerance, or fecundity (Shapiro-Ilan
et al. 2012). Thus precautions against trait deterioration have been proposed consist-
ing of the minimization of serial passages, the introduction of fresh genetic material,
the cryopreservation of stock cultures, and the creation of homozygous inbred lines
which are more resistant to trait deterioration.
Formulation and Application Methods Entomopathogenic nematodes are always
applied as infective juveniles, and are mainly used for controlling the larval or pu-
pal stages of insect pests in the soil or cryptic habitats. Application against foliar
pests may be successful under specific conditions. Application technologies for
entomopathogenic nematodes were thoroughly reviewed by Wright et al. (2005).
According to these authors, EPNs can be applied using the equipment for other con-
trol agents, but EPNs are of the most expensive control agents, and thus application
techniques should be optimized in order to achieve a cost-effective control.
At present, besides a classical aqueous suspension, EPNs are formulated in water-
dispersible granules, nematode wool, gels, vermiculite, clay, peat, sponge, etc. The
shelf life of the EPN based products depends on the formulation and nematode
species. Actively moving nematodes can remain alive and infective for 1–6 months
 ◦
180 V. Puža

Table 19.1 Commercialized EPN species, their bacteria symbionts, target pests and production
status
Nematode Symbiont Target insects On market
H. bacteriophora P. luminescens, P. temperata White grubs, weevils Widely
H. indica P. luminescens White grubs, weevils, In the USA
small hive beetle
H. megidis P. temperata Weevils, white grubs -a
H. zealandica P. zealandica White grubs -a
S. carpocapsae X. nematophila Armyworms, cutworms, Widely
leatherjackets
S. feltiae X. bovienii Fungus gnats, thrips, Widely
weevils, moths
S. glaseri X. poinarii White grubs -a
S. kraussei X. bovienii Black vine weevil Widely
S. kushidai X. japonica Grubs In Asia
S. longicaudum X. bedingii Grubs -a
S. riobrave X. cabanillassii Weevils In the USA
S. scapterisci X. innexi Mole crickets -a
a
Currently unavailable

under refrigeration ranges. EPNs with reduced mobility (formulations in gels) are
still infective after up to 9 months of storage while EPNs formulated in partial
anhydrobiosis (formulations in water soluble powders) remain so for up to 1 year.
After mixing with water, the nematodes are applied using sprayers, mist blowers,
or irrigation systems. The application of infected insect host cadavers can enhance
EPN persistence (Shapiro-Ilan et al. 2001). The recently proposed “lure and kill”
approach based on the application of the nematodes in capsules with insect attractant
may reduce the number of nematodes necessary to control the insect pest (Hiltpold
et al. 2012).
Several recommendations can be made for EPN applications. The application
should target the most vulnerable insect pest stage. Moderate pre-irrigation few hours
before an EPN application will enable the nematodes to move easily through the soil
pores. Adequate moisture should be maintained several days after application. For
any spray application, the nozzle openings should not be smaller than 50 microns, and
operating pressures within a system should not exceed 1000–2000 kPa, depending
on the EPN species.
Genetic Improvement Entomopathogenic nematode strains with improvements of
their pathogenicity, host range, environmental tolerances, and shelf-life are highly
desirable. Such strain enhancement might be achieved through genetic improvement
approaches. These methods were comprehensively reviewed by Burnell (2002).
19 Control of Insect Pests by Entomopathogenic Nematodes 181

Hybridization and selective breeding is the most promising approach to enhance

specific survival attributes, such as desiccation and heat tolerance, cold activity, host-
finding and nematicide resistance. Thanks to recent advances in EPN and bacteria
genomics it will be possible to identify genes from the whole genome that are being
expressed, in order to detect those that are involved in a particular process and target
them via genetic engineering methods. However, at present, it is unlikely that a
transgenic EPN strain would meet regulatory and public acceptance as a control
agent (Perry et al. 2012).
Safety In the past four decades, numerous studies have assessed the effect of en-
tomopathogenic nematodes and their bacterial symbionts on non-target organisms.
A thorough review of this subject was given by Ehlers (2005). The available data
show that EPNs are safe to humans and animals. In laboratory and field tests, non-
target arthropods were less susceptible to EPNs. Also, no adverse effect on the
non-target soil arthropods has been observed after EPN application. According to
Bathon (1996), the mortality of non-target animals in the field may occur, but will
be temporal, spatially restricted, affecting only part of the population, and its impact
can be considered negligible.
A few decades ago, a close relative to symbiotic Photorhabdus species, P. asymbi-
otica was identified as a facultative human pathogen. It was later revealed that strains
of this species are associated with Heterorhabditis indica and H. gerrardi. This fact
raises concerns about the safety of EPNs to humans. However, commercially pro-
duced H. bacteriophora and H. megidis have never been found in association with
this bacterium. According to Grewal (2012), more research is needed on these bac-
teria, but as a precaution it is advisable to avoid contact between these bacteria and
human wounds.
Regulation In general, regulations form a major obstacle in the commercialization
of biopesticides. Luckily, EPNs have been exempted from the registration process
in many countries, e.g. in the USA, while other countries require registration for
the exotic or non-indigenous species/strains. In the EU, EPNs are not covered by
the legislation of plant protection microbial products but are usually covered as
macroorganisms together with beneficial arthropods (Ehlers 2005). Thus in most
EU countries, no registration is required. Those countries that require registration
usually ask for information that is freely available in the scientific literature. Some
countries further require the data of each new product from field trials performed
within their borders. The recent EU directive 2009/128/EC on the sustainable use
of pesticides states that non-chemical methods should be given priority wherever
possible. Demand for EPN-based products is therefore likely to increase in the future.
Control of Target Pests Entomopathogenic nematodes are used for the control of
a variety of insect pests in soil, cryptic, and foliar habitats. EPNs are native to soil
and thus many soil insects have received attention regarding control efforts with
EPNs. The most important beetles targeted by EPNs in soil are weevils (e.g. the
black vine weevil Ottiorhynchus sulcatus, the root weevil Diaprepes abbreviatus,
and the sweet potato weevil Cylas formicarius), and grubs (e.g. the japanese beetle
 ◦
182 V. Puža

Popillia japonica and the garden chafer Phyloperta horticola). EPNs are further used
to control soil-borne dipterans such as fungus gnats (e.g. Bradysia spp., Lycoriella
spp.) and maggots (e.g. Delia radicum). Among lepidopterans, cutworms (Agrotis
spp.) and earworms (e.g. the corn earworm Helicoverpa zea) spend some or all of
their feeding stages in contact with the soil and thus can be controlled by the use of
EPNs.
Cryptic habitats are also considered to be favorable for EPN survival, as UV
radiation and desiccation are minimized. In these habitats, the bark and wood bor-
ing moths (Synanthedon spp., Euzophera semifuneralis), or codling moth (Cydia
pomonella) represent the most important target pests.
Foliar habitats are less suitable for EPN use due to the adverse conditions associ-
ated with them. Thus EPN-based control in these habitats tends to be less efficient.
However, some foliar pests, e.g. dipteran leafminers (e.g. Liriomyza trifolii, Tuta
absoluta), or diamondback moth larvae (Plutella xylostella), can be successfully
controlled with EPNs.

References

Akhurst RJ, Boemare NE (1990) Biology and taxonomy of Xenorhabdus. In: Gaugler R, Kaya HK
(eds) Entomopathogenic nematodes in biological control. CRC, Boca Raton, pp 75–90
Bathon H (1996) Impact of entomopathogenic nematodes on non-target hosts. Biocontr Sci
Techn 6:421–434
Bedding RA, Akhurst RJ (1975) A simple technique for the detection of insect parasitic rhabditid
in soil. Nematologica 21:109
Burnell A (2002) Genetics and genetic improvement. In: Gaugler R (ed) Entomopathogenic
nematology.CABI, New York, pp 333–356
Campbell JF, Gaugler R (1997) Inter-specific variation in entomopathogenic nematode foraging
strategy: dichotomy or variation along continuum? Fundam Appl Nematol 20:393–398
Ciche TA, Darby C, Ehlers RU et al (2006) Dangerous liaisons: the symbiosis of entomopathogenic
nematodes and bacteria. Biol Control 38:22–46
Ehlers RU (2005) Forum on safety and regulation. In: Grewal PS, Ehlers RU, Shapiro-Ilan DI (eds)
Nematodes as biocontrol agents. CABI, Oxford, pp 107–115
Ehlers RU, Stoessel S, Wyss U (1990) The influence of phase variants of Xenorhabdus spp. and
Escherichia coli (Enterobacteriaceae) on the propagation of entomopathogenic nematodes of
the genera Steinernema and Heterorhabditis. Rev Nematol 13:417–424
Grewal PS (2012) Entomopathogenic nematodes as tools in integrated pest management. In: Abrol
DP, Shankar U (eds) Integrated pest management: principles and practice. CABI, Wallingford,
pp 162–236
Hiltpold I, Hibbard BE, French BW et al (2012) Capsules containing entomopathogenic nematodes
as a Trojan horse approach to control the western corn root-worm. Plant Soil 358:11–25
Lewis EE, Gaugler R, Harrison R (1992) Entomopathogenic nematode host finding—response to
host contact cues by cruise and ambush foragers. Parasitology 105:309–315
Lunau S, Stoessel S, Schmidt-PeiskerAJ et al (1993) Establishment of monoxenic inocula for scaling
up in vitro cultures of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis
spp. Nematologica 39:385–399
Perry RN, Ehlers RU, Glazer I (2012) A realistic appraisal of methods to enhance desiccation
tolerance of entomopathogenic nematodes. J Nematol 44:185–190
19 Control of Insect Pests by Entomopathogenic Nematodes 183

Peters A (1996) The natural host range of Steinernema and Heterorhabditis spp. and their impact
on insect populations. Biocontrol Sci Techn 6:389–402
Poinar GO Jr (1979) Nematodes for biological control of insects. CRC, Boca Raton, p 249
Poinar GO Jr (1993) Origins and phylogenetic relationships of the entomophilic rhabditids,
Heterorhabditis and Steinernema. Fudam Appl Nematol 16:333–338
Pu̇ža V, Mráček Z (2010) Mechanisms of coexistence of two sympatric entomopathogenic nema-
todes, Steinernema affine and S. kraussei (Nematoda: Steinernematidae), in a central European
oak woodland soil. Appl Soil Ecol 45:65–70
Shapiro-Ilan DI, Lewis EE, Behle RW et al (2001) Formulation of entomopathogenic nematode-
infected-cadavers. J Invertebr Pathol 78:17–23
Shapiro-Ilan DI, Han R, Dolinksi C (2012) Entomopathogenic nematode production and application
technology. J Nematol 44:206–217
White GF (1927) A method for obtaining infective juvenile nematode larvae from cultures. Science
66:302–303
Wilson MJ, Ehlers RU, Glazer I (2012) Entomopathogenic nematode foraging strategies—is
Steinernema carpocapsae really an ambush forager? Nematology 14:389–394
Wright DJ, Peters A, Schroer S et al (2005) Application technology. In: Grewal PS, Ehlers RU,
Shapiro-Ilan DI (eds) Nematodes as biocontrol agents. CABI, New York, pp 91–106
Chapter 20
Bacillus thuringiensis-Based Products for Insect
Pest Control

Ruud A. de Maagd

Abstract Bacillus thuringiensis (or Bt, as it has become generally known) is one of
the oldest and widely used biological control agents and has a long history of use.
Bt and a number of related bacteria produce a variety of toxins, mostly—but not
exclusively- localized in the parasporal crystals, which are, together with the spores
themselves, the components of the typical spore/crystal mixtures. These are used to
control insect pests in agricultural crops. While Bt products quietly kept holding the
first place in biological pesticide sales, interest in Bt was increased by the production
and commercialization of transgenic crop plants expressing one or more Bt toxins
since 1996. Here I will present a brief overview of the history, biology, and practical
uses of Bt and its toxins.

20.1 History of Bacillus thuringiensis Research

Bt was first isolated in Japan in 1901 by Ishiwata, but first fully described by the
German Berliner and named by him after the province in Germany (Beegle and
Yamamoto 1992). Because of its origin (silkworm), Bt was long considered a risk
for the silk industry, but a first product was launched in France in 1938 under the
name Sporeine. In the 1950s, several studies focussed on the parasporal body or
crystal, which had been noted before but was now characterized in terms of solubility,
content, and insecticidal activity. Several European countries, the USSR, as well as
the USA started commercial production again in the same period. Research and
development received a further boost from formation of large collections of natural
Bt isolates. The common belief that Bt was only active against Lepidoptera (the
larvae of butterflies and moths) was refuted by the discovery of Bt israelensis, with
activity against Diptera (larvae of blackflies and mosquitos) in 1977. Helped by the
growth of large strain collections, held all over the world and thought to run in the
tens of thousands of strains, Bt’s known activity range was further increased with

R. A. de Maagd ()
Plant Research International, Wageningen UR, P.O. Box 619,
6700 AP Wageningen, The Netherlands
Tel.: +31 317 480548
e-mail: ruud.demaagd@wur.nl

© Springer International Publishing Switzerland 2015 185

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_20
186 R. A. de Maagd

Fig. 20.1 a The life cycle of Bacillus thuringiensis. b Primary structure of three-domain protoxin
proteins. The white parts are removed upon activation. Colors of the three domains correspond to
those in the next panel. c Cartoon representation of the three-dimensional structure of the 3-domain
toxins. d Schematic representation of the mode of action of Bt toxins in the insect gut

the discovery of Bt tenebrionis, with activity against Coleoptera (beetles) in 1983.

Strains with further new (claimed) activities followed, such as against Hymenoptera
(bees and wasps), Hemiptera, Mallophaga, Nematoda and Protozoa, although these
have not all been confirmed. With the discovery of methods for genetic modification
of plants in 1982, the idea of using genes encoding Bt toxins in transgenic plants to
render them resistant to insect pests was rapidly put into practice, with the first report
appearing in 1987, and the first transgenic crops in the field by 1996 (de Maagd et al.
1999).

20.2 Phylogeny and Ecology of Bacillus thuringiensis

Bt is a member of the Bacillus cereus group of gram-positive bacteria, and typically

it will form so-called endospores when it senses adverse environmental conditions.
Such spores form a though protective capsule and are dormant, non-reproductive
and very hardy structures, that may “germinate”, i.e. resume vegetative growth and
division, when conditions are favourable again (Fig. 20.1a). Genetically, Bt is very
similar to B. cereus, a causative agent of food poisoning, and to B. anthracis, the
causative agent of anthrax in mammals. The distinction is in the details: only Bt
forms parasporal crystals during sporulation, which make it a (facultative) insect
pathogen, while it lacks the distinctive mammalian-active toxins of B. anthracis.
Toxins are encoded (mostly) by genes on mobile genetic elements, plasmids, so
that Bt strains that have lost those plasmids are indistinguishable from B. cereus.
20 Bacillus thuringiensis-Based Products for Insect Pest Control 187

Although originally associated with insect populations, both Bt as well as B. cereus

are found in a large diversity of habitats, such as the phylloplane, soil, and stored
grain products. Many isolated Bt strains have no demonstrable toxicity for any tested
insect species, and with Bt occurring together with B. cereus, the role of parasporal
crystal production in the bacterium’s ecological niche, at a considerable cost of
resources, remains somewhat a mystery. The consensus is to call Bt a facultative
entomopathogen (Glare and O’Callaghan 2000).
There are several other bacterial species that produce insecticidal, and even paras-
poral crystal proteins related to those of Bt, such as Lysibacillus sphaericus, which
is used against mosquito larvae, several Paenibacillus species, which cause diseases
in honeybee larvae and in the white grubs of Japanese beetles, Brevibacillus lat-
erosporus (toxic to Diptera), and Clostridium bifermentans (toxic to mosquitos) (de
Maagd et al. 2003).

20.3 Bt Toxin Diversity, Mode of Action, and Specificity

Bacillus thuringiensis, as a species, has a vast armoury of different toxins, both

parasporal crystal proteins as well as insecticidal and more general toxins produced
during vegetative growth (de Maagd et al. 2003). The parasporal crystal proteins
(and some vegetative, soluble proteins) make Bt an insect pathogen. In a natural
setting the toxin’s action on the insect gut may help the spore to get access to the
body cavity, where it can germinate and cause septicaemia. In general, toxicity
of a strain can be explained by the activity of the particular toxins that the strain
produces. Each strain can produce one or more toxins, together determining the host
specificity of the strain. By far the most common insecticidal proteins of Bt are the
delta-endotoxins, which together form the crystal. Two major groups, Cry (crystal)
and Cyt (cytolyic) delta-endotoxins, can be distinguished. The largest group are
the Cry proteins, with over 120 different main types known today, and many more
minor variants. While initially Cry proteins were classified according to their general
insect order activity (CryI against Lepidoptera, CryII against Leps and Diptera, CryIII
against Coleoptera, etc.), this classification was found to be untenable in the face of
the growing number of holotypes. A new classification based on amino acid sequence
homology was later accepted (see: http://www.btnomenclature.info/). Although all
Cry1 proteins are still generally active against some Lepidoptera species, and Cry3
proteins against Coleoptera etc., the more recent higher rank number toxins have
more varying activity. In general, each toxin has activity against only one or a few
species within the same order, some toxins have a wider activity spectrum within an
order (particularly so for Lepidoptera) and some have been found to be active against
species from different orders. All this also depends on the number of target species
that have actually been tested (see also: The Bacillus thuringiensis toxin specificity
database at http://www.glfc.cfs.nrcan.gc.ca/bacillus/). Thus, it is important to keep
in mind that to refer to “Bt toxin” is meaningless without knowing the specific type
of toxin and its activity spectrum.
188 R. A. de Maagd

By far the most common type of Cry proteins appears to have a conserved three-
dimensional structure, consisting of three structural domains, and are hence called the
3-domain toxins. These generally occur in crystals as protoxins, with N-terminal and
C-terminal parts that after solubilisation from the crystal are removed by proteolytic
action (Fig. 20.1b). In the remaining activated toxin, the first N-terminal domain
consists of six amphipathic alpha-helices surrounding a central hydrophobic helix,
which are thought to become part of the eventual membrane pore (see below). The
second and third domain contain mostly beta-sheets, and are involved in binding to
specific insect gut receptors (Fig. 20.1c). To add to the complexity of the Bt toxin
arsenal, not all Cry proteins have this conserved structure, and other structures have
been found (de Maagd et al. 2003).
The mode of action and its role in host specificity of the three-domain toxins has
been extensively studied and is schematically shown in Fig. 20.1). Crystal proteins
act on the gut of the insect host, so crystals need to be ingested to become active.
Upon ingestion, the protoxins are solubilized from the crystal. This solubilization is
dependent on pH. Subsequently protoxins are processed by proteolytic enzymes of
the host’s gut, by which the active toxin is produced. The activated toxin subsequently
binds to one or several receptors on the surface of the epithelial cells lining the insect
gut. The specific toxin/receptor-interaction is a major determinant of host-range,
from the toxin side by domains II and III of the activated toxin. In a further not
completely characterized, and somewhat contested sequence of events, possibly
involving further processing, structural conformation changes and oligomerization
of the toxin upon receptor binding, the toxin inserts into the epithelial cell membrane,
forming pores. Both the extensive leakage of electrolytes as well as activation of host
signal transduction pathways may contribute, but do eventually result in the death
of the gut epithelial cells (Vachon et al. 2012). The insect stops feeding almost
immediately, is paralyzed, and dies. From the above description it should be clear
that each step of the mode of action contributes to the unique host specificities of the
different toxins.
The other relatively common components of many parasporal crystals are the Cyt
toxins, so called because in vitro they show general cytolytic activity against many
cells, although in vivo they are generally restricted to Diptera, with some exceptions
for coleopteran species reported. The protein structure is a three-layered α-β-α fold
and it requires proteolytic activation. It acts on target gut epithelial cells by membrane
pore formation, although unlike Cry proteins they have no specific receptors but more
generally interact with membrane phospholipids (Soberón et al. 2013).
Other, less common Bt Cry proteins have very different structures from the 3-
domain toxins, Cyt, and from each other. Some of these show more similarity to
toxins from other pathogens, which may give clues to their mode of action. Except
for Cry34/35 in transgenic plants (see below) these do not appear in products. Non-
crystal toxins include the Vegetative Insecticidal Proteins (VIPs). Of these, VIP3 is
toxic to a range of lepidopterans through pore formation in the insect gut and has
been applied in transgenic plants (see below).
The evolution of the great diversity of strains, toxins and specificities, particularly
for the 3-domain toxins, may be explained by the organization of the toxin-encoding
20 Bacillus thuringiensis-Based Products for Insect Pest Control 189

genes in the bacterium. Most toxin genes are located on large plasmids, which can
be mobilized and transferred between bacteria, thus creating new toxin gene combi-
nations. On the plasmids, cry genes are often clustered in groups, and this may lead
to intergenic recombination between homologous genes, which leads to reshuffling
of gene fragments. This mechanism can be used for creating new domain combina-
tions in the laboratory, and this mechanism probably played an important role in the
generation of structural variation (de Maagd et al. 2001). Furthermore, most toxin
genes are located close to sequences related to DNA transposition. Transposition
may mobilize individual genes between plasmids and assemble new combinations
of genes on plasmids.

20.4 Applications of Bt in Agriculture and Forestry

The first commercial use of microbial Bt-based insecticides occurred as early as

1938 in France, and in the 1950s in the United States. The history of Bt discovery
and use have been extensively reviewed elsewhere (Beegle and Yamamoto 1992;
Glare and O’Callaghan 2000). Commercial viability as an alternative for chemical
insecticides arrived in the 1970s with more potent strains. Bt products are particularly
used in high-margin crops, such as in vegetables and fruits. On the other hand,
Bt has been used against forestry pests such as those caused by gypsy moth and
spruce budworm, by means of wide-scale aerial applications. After its discovery,
Bt israelensis was developed for use in the US and in Germany’s Rhine valley for
control of biting flies and mosquitos, and in Africa for disease vector control. Bt
tenebrionis has been used to some extent for Colorado potato beetle control and for
control of chrysomelid beetles in Eucalyptus plantations (Entwistle et al. 1993). Bt
is the single most successful biopesticide product type on the market, in the 1990s
taking up over 90 % of the global biopesticide market (which by itself is a small part
of the total pesticide market). This number has decreased somewhat to an estimated
55 % in 2012, due both to decreased Bt spray use as well as to an increase in the
number and volume of other biopesticide products.
Microbial Bt products usually consist of dried spore/crystal-mixtures that can be
produced by fermentation on a large variety of media, together with other formu-
lation ingredients. Most information about Bt products comes from the US market,
where 13 different active ingredients in 123 products where registered (Walker et al.
2003). However, many more less well characterized products have been produced,
particularly in the Soviet Union, in China or other countries (Glare and O’Callaghan
2000). Strain improvement for higher insecticidal protein content or wider target
spectrum has been undertaken by conjugation of toxin encoding plasmids and site-
specific recombination, while improvement of individual toxins has been studied
using mutagenesis or domain swapping (Baum et al. 1999).
Despite its prominent position as a biopesticide, and advantages for environmental
impact and non-target effects, Bt products have never taken a large part of the overall
insecticide market. This is partially due to the lack of Bt products with activity
190 R. A. de Maagd

against major insect pest orders such as aphids and white flies. Additionally, Bt is
a not a systemic protectant and in most cases can only be used aboveground and
on the outside of the plant, leaving the plant unprotected for pests attacking roots
(such as Corn rootworm) or burrowing into plant tissues (such as European Corn
borer). Expressing Bt toxins in transgenic crops, where they are expressed in these
otherwise unprotected tissues and are continuously present without the need for
repeated application, has proven to be an effective alternative.
Improvements in transgenic plants focussed on the differences between microbial
and plant gene function, removing cryptic RNA splice sites and other elements that
negatively affect RNA stability, and finally optimization of codon use for the target
plants through the construction of synthetic genes. This results in Bt toxin levels
of 0.2–1 % of total soluble protein and proper resistance to target insect species (de
Maagd et al. 1999). Many different crop/gene-combinations for various pests have
been developed. However, only a handful of these have reached the commercial-
ization stage and are still being cultivated somewhere in the world today. The two
most substantial crops containing Bt genes for insect control today are cotton and
corn. Potato, tomato, eggplant, rice, and soybean varieties with Bt genes have been
approved for cultivation but their application so far has been negligible. Cotton has
been commercialized from 1996 with a varying array of Bt genes, mostly for resis-
tance to cotton bollworm (Lepidoptera) in a large number of countries, including
USA, Australia, India and China, and for Pink bollworm in the USA. This resulted in
a vast majority of cotton in some countries being of a transgenic Bt variety. Bt maize
has also been cultivated since 1996, first for resistance to European corn borer and
Mediterranean corn borer (Lepidoptera), later also for resistance to cutworms (Lep-
idoptera) and for Corn rootworm (Coleoptera) (see also: www.isaaa.org). Recent
developments concentrate on the so-called “stacking” of genes, combining several
Bt genes in one variety by crossing, both for wider target spectrum as well as for resis-
tance management (see below). For an extensive up-to-date overview of approved or
pending Bt crops world-wide, the reader is referred to one of several online databases
(http://www.cera-gmc.org/?action=gm_crop_database).

20.5 Safety and Resistance

Safety issues in relation with the use of Bt sprays and, particularly, with the use of Bt
toxins in transgenic crops are a constant source of study and discussion. With regard
to mammalian and human toxicity or pathogenicity, these issues may be partially
overlapping as both applications involve similar toxins, but other issues are distinct:
sprays may lead to ingestion or inhalation of live bacteria, albeit in usually small
amounts, consumption of transgenic crops mostly leads to ingestion of individual Cry
proteins. These issues are discussed in great detail elsewhere (Glare and O’Callaghan
2000).
Few reports exist of adverse effects of Bt sprays, despite its long history of use. Its
close relation to the recognized food-pathogen B. cereus has raised some concerns.
20 Bacillus thuringiensis-Based Products for Insect Pest Control 191

Some Bt strains produce heat-stable β-exotoxins with demonstrated activity against

many insects and even fish and mammals, and are banned from use as pesticides
by regulatory authorities. The safety of Bt toxins in transgenic crops is part of a
larger discussion of transgenic crop safety. Regulatory authorities such as the US
EPA (Environmental Protection Agency) and EFSA (European Food Safety Agency)
deem the currently approved Bt crops safe for human consumption and see no proof
of adverse effects on the environment. Study of environmental and non-target effects
of Bt toxins has been an integral part of the safety assessment of transgenic crops for
environmental release (EFSA Panel on Genetically Modified Organisms 2010).
As for any insecticide, extensive and uninterrupted use of Bt sprays or toxins as in
transgenic crops exerts strong selective pressure on insect pest populations leading
to the increased presence of resistant individuals that, given time, may replace the
sensitive population. This potential problem was recognized early on and observed in
the field for some Bt sprays, and received considerable attention upon the introduction
of Bt crops. Resistance alleles, for example genetic variants of genes for Bt receptors,
are present in all populations at a (very) low frequency and can be readily selected in
controlled laboratory conditions. Emergence of resistance in transgenic fields seems
rare, although some has been reported. This delay may be partially due to resistance
management strategies aimed at reducing the speed of resistance developing in a
population (Tabashnik et al. 2013).

20.6 Concluding Remarks

Over a hundred years of discovery and use of Bt and its toxins has given us a vast
domain of knowledge on toxin structure and action, as well as a valuable tool for
insect pest control in agriculture. Further commercial development of new strains and
crop varieties has slowed down since their first introduction, due to more stringent
registration demands and high cost of the approval process for crops, in combination
with increased weariness from the consumer side. Still, Bacillus thuringiensis will
continue to fascinate researchers for a long time to come.

References

Baum JA, Johnson TB, Carlton BC (1999) Bacillus thuringiensis—natural and recombinant bioin-
secticide products. In: Hall FR, Mean JJ (eds) Methods in biotechnology, vol 5. Humana,
Totowa, pp 189–209
Beegle CC,Yamamoto T (1992) History of Bacillus thuringiensis berliner research and development.
Can Ent 124:587–616
de Maagd RA, Bosch D, Stiekema WJ (1999) Bacillus thuringiensis toxin mediated insect resistance
in plants. Trends Plant Sci 4:9–13
de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins
to colonize the insect world. Trends Genet 17:193–199
192 R. A. de Maagd

de Maagd RA, Bravo A, Berry C et al (2003) Structure, diversity and evolution of protein toxins
from spore-forming entomopathogenic bacteria. Annu Rev Genet 37:409–433
EFSA Panel on Genetically Modified Organisms (2010) Guidance on the environmental risk
assessment of genetially modified plants. EFSA J 8:1879–1990
Entwistle PF, Cory JS, Bailey MJ et al (1993) Bacillus thuringiensis, an environmental pesticide:
theory and practice. Wiley, Chichester
Glare T, O’Callaghan M (2000) Bacillus thuringiensis: biology, ecology and safety. Wiley,
Chichester
Soberón M, López-Díaz JA, Bravo A (2013) Cyt toxins produced by Bacillus thuringiensis: a protein
fold conserved in several pathogenic microorganisms. Peptides 41:87–93
Tabashnik BE, Brévault T, Carrière Y (2013) Insect resistance to Bt crops: lessons from the first
billion acres. Nat Biotechnol 31:510–521
Vachon V, Laprade R, Schwartz JL (2012) Current models of the mode of action of Bacillus
thuringiensis insecticidal crystal proteins: a critical review. J Invertebr Pathol 111:1–12
Walker K, Mendelsohn M, Matten S et al (2003) The role of microbial Bt products in U.S. crop
protection. J New Seeds 5:31–51
Chapter 21
Post Harvest Control

Emilio Montesinos, Jesús Francés, Esther Badosa and Anna Bonaterra

Abstract Harvested fruits, vegetables, nuts and grains harbour a very reach mi-
crobiota that influence their shelf-life, quality and safety for human consumption.
Spoilage due to fungal or bacterial rot, mycotoxin production and contamination
by food-borne human bacterial pathogens are within the main problems of harvest
products that are consumed fresh. Control of these problems is currently done by
conventional methods of sanitation, disinfection or treatment with chemical fungi-
cides. Biological control of postharvest problems can be achieved with certain strains
of antagonistic viruses, bacteria, yeast and fungi. The mechanisms of action are
very diverse, and several mechanisms may act simultaneously. Mechanisms in-
clude competition for nutrients and niches, antibiosis by means of antimicrobials
and lytic enzymes, inhibitory volatile metabolites, pH decrease, parasitism, and
induction of defence responses in the harvested plant product. Several commercial
products containing strains of biological control agents are available as an alternative
or complement to chemicals for postharvest rot control.

E. Montesinos () · J. Francés · E. Badosa · A. Bonaterra

Laboratory of Plant Pathology, Institute of Food and Agricultural Technology,
University of Girona, Girona, Spain
Tel.: +34639763764
e-mail: emilio.montesinos@udg.edu
J. Francés
Tel.: +34 972419735
e-mail: jesus.frances@udg.edu
E. Badosa
Tel.: +34972418877
e-mail: esther.badosa@udg.edu
A. Bonaterra
Tel.: +34 972419734
e-mail: ana.bonaterra@udg.edu

© Springer International Publishing Switzerland 2015 193

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_21
194 E. Montesinos et al.

21.1 Introduction

Plants provide a physical-chemical environment to complex microbial communities,

comprising of viruses, bacteria and fungi. In addition, the different properties be-
tween aerial and soil plant parts determine that specific microbial communities are
established. Microbiota composition also changes with the soil type, plant species,
cultivar, season of the year, farm practices, and storage conditions (Setati et al. 2012).
The diversity of the plant microbiota in fresh fruits and vegetables is very high as re-
vealed by metagenomics studies (e.g. 100–1000 operational taxonomic units, OTUs)
with only a few dominant groups (20–50 OTUs) (Leff and Fierer 2013) .
Since plants are important sources of fresh products, their in-field microbiota has
a great influence in the harvested produce (see also Chap. 44). Plant products, that
are consumed raw are rich in nutrients and can be contaminated at the field level
or during the processing conditions at postharvest. The harvested produce main-
tains several functional and physiological activities (e.g. respiration, ripening in
climacteric fruits), that can influence its microbiota during postharvest. These mi-
croorganisms can affect the quality and quantity (spoilage) (Snowdon 1990) as well
as safety (food-borne human bacterial pathogens, mycotoxins) (Magan and Aldred
2007). For more details see Table 21.1.
Fresh fruit rot caused by fungi is a typical source of losses that affect all kinds
of pome, stone, and citrus fruits, as well as berries, cucurbits and tropical fruits
(Snowdon 1990; Prusky and Gullino 2010) . Fungal pathogens commonly found are
Penicillium species in apple, pear and citrus fruits, Rhizopus and Monilinia in stone
fruits, and Botrytis and Colletotrichum in berries. Bacterial rot is more common in
vegetables. Dried grains and nuts are affected by fungi and bacteria that due to starch
degradation can cause a decrease in weight, quality and nutritional properties.
Mycotoxigenic moulds, apart from deterioration, can produce toxic secondary
metabolites that are very important in dried cereal grains and nuts stored un-
der improper humidity conditions (Magan and Aldred 2007). The most common
toxinogenic fungi are Penicillium verrucosum (ochratoxin), Aspergillus flavus (afla-
toxins), A. ochraceous (ochratoxin) and several species of Fusarium (fumonisins,
trichotecenes). Mycotoxinogenic fungi are also a source of mycotoxin contamina-
tion in rotten fresh fruits (Barkai-Colan and Paster 2008). Certain strains of Bacillus
cereus produce heat stable toxins in cereals.
Food borne human bacterial pathogens can be found in postharvest produce and
are an increasing problem of concern, especially in ready-to-eat fresh products,
like minimally processed vegetables and sliced fruit, because they provide ideal
conditions for bacterial growth (Badosa et al. 2008). These products have been related
to outbreaks of foodborne diseases caused by the human pathogens Escherichia coli
O157:H7, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus and
Pseudomonas aeruginosa (Lin et al. 1996).
To prevent postharvest losses, the shelf-life of plant produce is increased by means
of cold-storage or controlled atmosphere methodologies (low oxygen and increased
CO2 , inert gases), or combinations of both systems. In spite of these limiting con-
ditions for microbial activity, either the length of storage period or the environment
21 Post Harvest Control 195

Table 21.1 Spoilage and safety problems of postharvest products caused by microorganisms
Product Problem caused Causal microorganism
Citrus fruits Blue and green mold Penicillium italicum, P. digitatum
Black spot Alternaria spp.
Brown rot Phytophthora sp.
Pomefruits Blue mold Penicillium expansum
Neofabraea rot Neofabraea
Bitter rot Colletotrichum gloeosporioides
Mucor rot Mucor piriformis
Stonefruits Brown rot Monilinia spp.
Rhizopus rot Rhizopus stolonifer
Gray mold rot Botrytis cinerea
Berries Antracnose Colletotrichum acutatum
Gray mold Botrytis cinerea
Leather rot Phytophthora sp.
Tropical/subtropical Banana fruit rot Gloeosporium, Colletotrichum, Fusarium
fruits
Mango fruit rot Colletotrichum, Phytophthora, Alternaria
Avocado fruit rot Colletotrichum, Phytophthora
Pineapple rot Fusarium, Phytophthora, Penicillium,
yeasts
Vegetables (leafy, roo- Soft rot Pseudomonas, Pectobacterium, yeasts
ts and tubers, germi- Food-borne human E. coli, Salmonella enterica, Listeria
nated seeds) pathogens monocytogenes
Grains and nuts Spoilage and mycotoxin Aspergillus, Penicillium, Erotium, Fusar-
production ium, Bacillus and other G+ bacteria

may result in produce spoilage, in multiplication of pre-existing human pathogenic

bacteria or in preservation of toxins.
Thus, storage conditions should be preceded by good production practices and
preharvest treatments, either to decrease populations of undesirable microorgan-
isms or to sanitize latent infections. Treatments of disinfection by chemical/physical
means or application of authorized fungicides are a conventional practice. However,
consumer concerns and strong restrictions about pesticide residues in plant products
have stimulated the development of less aggressive and toxic means of preserva-
tion of postharvest products. Biological control, using beneficial microorganisms,
has emerged as an alternative or complement to conventional methods of control of
biotic postharvest losses.
 196

Fig. 21.1 Examples of biological control of postharvest fungal rot. Non-treated control (right panels) and treated with the biocontrol agent (left panels). Pears
and apples were treated with the biocontrol agent Pseudomonas fluorescens EPS288. Peach and strawberries were treated with Pantoea agglomerans EPS125
E. Montesinos et al.
21 Post Harvest Control 197

21.2 Biological Control Agents for Postharvest Control

Biological control agents (BCAs) of postharvest diseases have evolved rapidly since
the first report on control of stone fruit rot with Bacillus subtilis (Pusey and Wil-
son 1984) and pomefruit by a non-pathogenic strain of Pseudomonas syringae
(Janisiewicz and Korsten 2002). The mechanisms of action among BCAs are very di-
verse, and several mechanisms may act simultaneously. These mechanisms include
competition for nutrients and niches (CNN; competitive exclusion), antibiosis by
means of antimicrobials and lytic enzymes, inhibitory volatile metabolites, pH de-
crease, parasitism, and induction of defence responses in the harvested plant product
(Fig. 21.1).
Biological control of fungal rot has been extensively studied using strains obtained
from the microbiota of wild plants or postharvest produce (Janisiewicz and Korsten
2002; Bonaterra et al. 2003; Prusky and Gullino 2010) . A list of relevant BCAs
is described in Table 21.2. Several bacterial strains pertaining to Pseudomonas (P.
syringae, P. fluorescens, P. graminis), Pantoea (P. agglomerans, P. ananatis), Bacil-
lus (B. subtilis, B. amyloliquefaciens), and Rahnella (R. aquatilis), yeast strains,
mostly of Candida (C. famata, C. oleophila, C. saitoiana, C. sake), Kloeckera
apiculata, Metschnickowia pulcherrima, Cryptococcus laurentii, and Rhodotorula
glutinis, have been reported as biological control agents of fungal rot. Fungal strains
like Aureobasidum pullulans and Muscodor albus were also described as effective.
Biocontrol to prevent mycotoxin production in postharvest products has been also
the object of development (Magan and Adler 2007). Generally exclusion of colo-
nization and growth of the toxinogenic fungus and degradation of the mycotoxins are
the main strategies. Most efforts have focused on control of toxinogenic species of
Aspergillus (ochratoxins) in nuts by using atoxigenic strains, lactic acid bacteria and
Flavobacterium aurantiacum. Similarly, Fusarium (fumonisins) in grains is con-
trolled by Bacillus amyloliquefaciens, P. fluorescens and several yeasts. Biological
control of mycotoxigenic fungi in fresh fruit is based in the same BCAs as used for
fruit rot control .
Biological control of food-borne human pathogens in fruit and vegetables has
also been reported (Janisiewicz et al. 1999). Several beneficial bacteria are effective
in preventing or decreasing population levels of E. coli, Salmonella enterica and
Listeria monocytogenes in ready-to-eat vegetables and sliced fruits, like strains of P.
syringae (Leverentz et al. 2006) and of lactic acid bacteria (Trias et al. 2008). Also
lytic bacteriophages have been reported as being effective (Sulackvelidze 2013).
The success of biological control of postharvest losses, diseases and mycotoxins
has stimulated commercial activities to bring products to the market. Thus, several
BCA strains are currently, or have been in the past (some of them are no longer
manufactured), the active ingredients of commercial biofungicide products regis-
tered for postharvest control in various countries. Some examples are Cryptococcus
albidus (Yieldplus), B. subtilis B426 (Avogreen), B. subtilis QST713 (Serenade),
Metschnickowia fructicola 277 (Shemer), P. syringae ESC10 (Biosave), C. oleophila
I-182 (Aspire), C. oleophila O (Nexy) or Aureobasidium pullulans DSM14941
(BoniProtect).
198 E. Montesinos et al.

Table 21.2 Microorganism strains reported to control rotting, toxin production or food-borne human
pathogenic bacteria in harvested products. (For more details the reader is referred to the books from
Barkai-Golan and Paster 2008; Janisiewicz and Korsten 2002; Magan and Aldred 2007; Prusky and
Gullino 2010)
Microbial group Species Strain Pathogen Harvested prod-
controlled uct
Pseudomonas P. syringae ESC10, ESC11 Pi, Pd Citrus
P. fluorescens EPS288 Pe Pome
P.graminis CPA-7 Ec, Se, Lm Sliced fruits
Pantoea P. agglomerans EPS125, CPA-2 Pe, Pd, Bc, Mf, Pome, citrus,
Rs stone fruits
P. ananatis BLBT1-08 Bc Grapes
Bacillus B. subtilis B-3, CPA-8, Mf, Pi, Pd Citrus, stone,
B426 avocado
B. amyloliquefa- QST713 Mu, Bc, Co, Peach, straw-
ciens berry
Rahnella R. aquatilis BNM523 Pe, Bc Pome
Candida C. oleophila O, I-182 P, Bc Pome, citrus,
stone
C. saitoana – Pd, Bc Pome, citrus
C.sake CPA-2 Pi, Pd, Pe Pome, citrus
C. famata – Pd Citrus
Pichia P. guilliermondii M8 Bc Apple
P. anomala K Pe Pomefruits
Cryptococcus C. albidus – Bc, Pe Pomefruits
C. laurentii YY6 Bc Raddish
C. HRA5 Mo, Pe Sweet cherry,
infirmo-miniatus pomefruits
Metsnikowia M. fructicola 277 Pe, Pd, Bc, Rs, Pome, citrus,
Mo, Aa, Fu grapes
Kloeckera K. apiculata 34-9 Bc Citrusfruit
Rhodotorula R. glutinis – Pe Pear, cherry
Muscodor M albus – Co, Bc Pome, stone,
grapes
Aureobasidium A. pullulans DSM14941, L1, Al, Gl, Pn, Bc, Pome, stone,
L8 Mo strawberry
Trichoderma T. harzianum, T. T32 Bc, Co, Pd Strawberry,
viride tomato, apple,
citrus
Lactobacillus L. plantarum CM160 Ec, Se, Lm Lettuce, apple
Leuconostoc L. mesenteroides CM135 Ec, Se, Lm Lettuce, apple
21 Post Harvest Control 199

Table 21.2 (continued)

Microbial group Species Strain Pathogen Harvested prod-
controlled uct
Weissella W. cibaria TM128 Ec, Se, Lm Lettuce, apple
Bacteriophages – Mixtures Ec, Lm Vegetables,
melon
Pi P. italicum, Pd P. digitatum, Pe P. expansum, Ec E. coli, Se S. enterica, Lm L. monocytogenes,
Bc B. cinerea, Mf M. fructicola, Rs R. stolonifer, Mo Monilinia, Aa A. nigricans, Fu Fusarium, Co
Colletotrichum, Al Alternaria, Gl Gloeosporium, Pn Penicillium

21.3 Factors Affecting Efficacy of Postharvest Biocontrol

The efficacy and success of biological control of postharvest diseases is greatly

dependent on intrinsic properties of the BCA, but also on pathogen and host material
(fruit, nut, grain, vegetable), and on environmental factors.
In BCA systems, there is a dose-effect relationship, doses of 106 –107 for yeast
and of 107 –108 for bacteria are efficient for control of postharvest fruit rot (Mon-
tesinos and Bonaterra 1996). The relative dose of pathogen and biocontrol agent
is an important factor determining the efficacy and consistency of biological con-
trol of postharvest pathogens. Infectivity titration of the pathogen in the presence
of varying concentrations of the biocontrol agent provide data that can be fitted to
dose–response models to derive efficacy parameters like the median effective dose
(ED50 ) of pathogen and biocontrol agent. These parameters can be useful to perform
comparisons between strains, pathogens and hosts. Using this approach pathogen
aggressiveness on the host was reported as an influencing factor in the efficiency of
postharvest biocontrol on several fresh fruits (Francés et al. 2006).
The efficacy of control of postharvest problems depends on the time at which the
treatment is applied relative to that of the pathogen arrival, and therefore depends
on the strategy used for treatment, which can be preventive (before the infection of
the pathogen), or curative (after the infection of the pathogen). Major progress in
preventive treatments has been made using microorganisms effective against wound
infecting pathogens, but considerably less advance has been reported in control of
already developed, or in latent, infections (e.g. infections caused by Monilia or
Colletotrichum).
Storage conditions can have a triple effect on the plant produce ecosystem. For
example, reduced oxygen and increased CO2 atmospheres greatly slow down fruit
and seed respiration and pathogen activity, but may have a negative effect on the BCA
in strict aerobes like Bacillus and Pseudomonas or do not affect facultative aerobes
like yeasts. Low storage temperatures, close to 0◦ C, sufficiently slow down the
activity of the BCA, pathogen and plant produce. However, at certain moments both
the pathogen and the BCA may be active due to favourable environmental conditions.
For example, during transition from field harvest to industrial cold-storage or when
cold storage ends for commercial delivery (Fig. 21.2).
200 E. Montesinos et al.

30

25

20
Incidence (%)

15

10

0
NTC
1 2 3 4 ANT
5 6 7 8 FUN
9 10
Orchard 11

Fig. 21.2 Incidence of blue mold rot on Golden apple from eleven commercial orchards upon
wounding, fungicide or biological control treatment with Pseudomonas fluorescens EPS288,
Penicillium expansum inoculation and subsequent storage under Ultra Low Oxygen-cold storage
(0.5–1.0◦ , 0–1.5 % CO2 , 1.25 % O2 ) during 5 months, and a 7-day ripening period at 20◦ . Treat-
ments consisted of either the chemical fungicide imazalil (FUN), the biocontrol agent Pseudomonas
fluorescens EPS288 (ANT ) and non-treated controls (NTC)

21.4 Production, Formulation and Application of Postharvest

Microbial Pesticides

For the commercial development of a postharvest microbial pesticide, cells have

to be grown, preserved, and formulated for storage and delivery (Montesinos and
Bonaterra 2009). Methods used for industrial scale-up are solid or liquid-phase fer-
mentations, but depend on the nature of the microorganism (bacteria, fungi, yeast,
or bacteriophages) (Boytsensko et al. 1998).
Bacteria and yeast are generally grown by liquid fermentation in bioreactors, but
some fungi are fermented by solid-state procedures. Subsequently, cells are har-
vested by centrifugation, either from liquid cultures or solid phase, homogenized
and cleaned to obtain concentrated cell/spore suspensions that often contain some
supernatant metabolites of interest (e.g. antimicrobials, lytic enzymes). Bacterio-
phages are produced in a double stage process in which the first step consists of
preparing the bacterial target (e.g. E. coli), that is later used as the host to multiply
the lytic bacteriophage.
In order to increase the shelf-life and ecological fitness of the BCA, several pro-
cedures have to be carried out for stabilizing the viability of the cells. Generally,
formulations of commercial microbial pesticides consist of liquid-phase suspen-
sions that are maintained under refrigeration, in a frozen state, or as a dried product.
21 Post Harvest Control 201

Dehydration permits optimum storage conditions, handling, and distribution, but the
associated processes are costly, especially lyophilisation, and the prefered method
is spray drying or fluidized bed drying. However, spray drying generally results in a
high loss of viability due to the thermal treatment. The final formulations are com-
posed of an active ingredient (cells or spores and sometimes culture components),
carriers or inert materials used to support cells, and adjuvants. Products can be stable
for several months or even years.
Cell death of the biocontrol agent can occur after dehydration and delivery due to
the sharp change from the optimal laboratory culture conditions to the stressing dehy-
dration process and the growth-limiting fruit surface. However, stress tolerance can
be induced by cultivation under suboptimal conditions by means of osmo-adaptation.
This procedure has been used to improve drought stress tolerance, epiphytic survival
and biocontrol efficacy of the apple blue mold biocontrol agent Pantoea agglomerans
EPS125 (Bonaterra et al. 2005). Another strategy is the amendment of the formula-
tion with specific nutrients that cannot be used by, or are toxic for, the pathogen and
can be used or do not affect the biocontrol agent (Janisiewicz 1994).
The formulated product can be applied during preharvest (field spray) or
before/after storage (spraying or drenching).

21.5 Limitations and Future Trends

The management of postharvest losses tends to use low impact (soft) strategies
that often are less efficient than synthetic antimicrobial products (e.g. fungicides).
Therefore, a multiple barrier strategy is necessary to optimize levels of control. Soft
chemicals acting as barriers or affecting directly the spoilage microorganism (e.g.
bicarbonates, silicates), surface disinfection compounds (e.g. ozone, chlorine, elec-
trolyzed water), physical methods (e.g. hot water, microwaves, UV pulsed light),
and response defence inducers on the host (chitosans, acibenzolar, salicylic acid) are
among the systems used.
Biological control forms part of the list of these technologies. However, many
of these systems are not compatible with the simultaneous use of biocontrol agents
because they can inhibit its colonization, growth and metabolism. Fortunately, others
are compatible, either as previous (e.g. hot water, disinfection), or as simultaneous
treatments (defence inducers).
A limitation of certain BCAs of postharvest diseases is related to the biosafety of
the antagonistic microorganism (see also Chap. 32 and Chap. 33). This aspect has
greatly limited authorizations for commercial use in certain cases (e.g. Burkholderia
cepacia) due to reports on clinical outbreaks associated to this species. Another issue
is the acceptance and safety of improved biocontrol strains using recombinant DNA
technology.
202 E. Montesinos et al.

References

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in Catalonia (Spain) using plate counting normalized methods and QPCR. J Sci Food Agric
88:605–611
Barkai-Golan R, Paster N (2008) Mycotoxins in fruits and vegetables. Elsevier Inc, London
Bonaterra A, Mari M, Casalini L et al (2003) Biological control of Monilinia laxa and Rhizo-
pus stolonifer in postharvest of stone fruit by Pantoea agglomerans EPS125 and putative
mechanisms of antagonism. Int J Food Microbiol 84:93–104
Bonaterra A, Camps J, Montesinos E (2005) Osmotically induced trehalose and glycine betaine ac-
cumulation improves tolerance to desiccation, survival and efficacy of the postharvest biocontrol
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JW (eds) Methods in biotechnology. Humana Press, Totowa, pp 487–508
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efficiency in Pantoea agglomerans. Postharvest Biol Technol 39:299–307
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D-glucose on apples and pears. Appl Environ Microbiol 60:2671–2676
Janisiewicz WJ, Korsten L (2002) Biological control of postharvest diseases of fruits. Annu Rev
Phytopathol 40:411–441
Janisiewicz WJ, Conway WS, Leverentz B (1999) Biological control of postharvest decays of apple
can prevent growth of Escherichia coli O157:H7 in apple wounds. J Food Prot 12:1372–1375
Leff JW, Fierer N (2013) Bacterial communities associated with the surfaces of fresh fruits and
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teria monocytogenes and Salmonella enterica serovar Poona on fresh-cut apples with naturally
occurring bacteria and yeast antagonists. Appl Environ Microbiol 72:1135–1140
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microbiology, Elsevier Inc, Oxford pp 110–120
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Pusey PL, Wilson CL (1984) Postharvest biological control of stone fruit brown rot by Bacillus
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 Part IV
Plant Growth Promotion by Microbes
Chapter 22
The Nitrogen Cycle

Martine A. R. Kox and Mike S. M. Jetten

Abstract This chapter focuses on the nitrogen (N) cycle, a complex network of
mainly microbial transformations in which various nitrogen compounds are inter-
converted. Both microorganisms and plants absorb N from and excrete N into the
environment. First, N assimilation is addressed (22.1), after which N transformations
by microorganisms are described (22.2). In paragraph 22.3 both plant and microbial
N cycling are discussed at the ecosystem level, followed by paragraph 22.4, where
the use of N by humans and the consequences for the N cycle are reviewed. Finally,
in 22.5 the conclusions and outlook are presented.

22.1 N Metabolism of Plants

After the discovery in the early 1900s that N compounds could increase crop pro-
ductivity, this topic was intensively studied. Generally N is a limiting nutrient for
plant production and mineralization, hence N availability is an important controlling
factor for ecosystem processes. The N cycle is also tightly coupled to the carbon
(C) cycle. Access to N dictates both the photosynthetic activity, which is the main
C input in plants, and the production of protein (Larcher 2001). N compounds are
incorporated into plant material when (1) the N compound is available, when (2)
the plant has adequate uptake systems and (3) when all assimilatory complexes are
present and active.
N Sources In terrestrial ecosystems, the soil acts as a nutrient reserve for plants,
where 98 % of the mineral nutrient supply is bound in humus, organic matter and
insoluble compounds and only less than 0.2 % is dissolved in water. The soil’s N

M. A. R. Kox () · M. S. M. Jetten

Department of Microbiology, Institute of Water and Wetland Research,
Faculty of Science, Radboud University Nijmegen, Heijendaalseweg 135,
6525 AJ Nijmegen, The Netherlands
Tel.: +31 24 365 2569
e-mail: m.kox@science.ru.nl
M. S. M. Jetten
Tel.: +31 24 365 2941
e-mail: m.jetten@science.ru.nl
© Springer International Publishing Switzerland 2015 205
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_22
206 M. A. R. Kox and M. S. M. Jetten

content depends mostly on microbial mineralization of organic matter into inorganic

N such as ammonium (NH+ −
4 ) or nitrate (NO3 ). N availability for plants is affected
by the root structure and density, Radial Oxygen Loss (ROL), root exudates and
symbioses with microorganisms (Jackson et al. 2008). In anoxic environments such
as wetlands, ROL is of special interest as it supplies oxygen (O2 ) to microorganisms
(Lamers et al. 2012).
Uptake of N Terrestrial plants control mineral uptake via their roots. Furthermore N
uptake and assimilation are always dependent on the availability of other nutrients,
especially phosphorus. Although both inorganic and organic N (i.e. amino acids)
can be taken up by plants, most studies have focused on inorganic N sources. En-
ergetic costs of N uptake and assimilation are highest for NO− 3 , followed by NH4
+
−
and lowest for amino acids (Lambers et al. 1998). NO3 first has to be reduced to
NH+ 4 before it can be assimilated. Terrestrial plants adapted to low pH and redox
potential preferentially take up NH+ +
4 . Subsequently, the release of H acidifies the
rhizosphere, affecting the rhizosphere microbiome and causing reduced nitrification
rates. Plants adapted to soils with a higher pH use preferentially NO− 3 as it causes
less soil acidification compared to NH+ 4 (Lamers et al. 2012; Larcher 2001; Britto
and Kronzucker 2002).
Terrestrial plants have both high and low affinity transport systems (HATS and
LATS) for the active uptake of NO− +
3 and NH4 . HATS take up inorganic N between 1
μM and 1 mM. LATS are only expressed when inorganic N is above 0.5 mM. Only
for NO− −
3 uptake, inducible transporters are known. Uptake of NO3 induces HATS,
followed by positive feedback on the putative transporters, causing an increased
uptake of NO− −
3 . Subsequently, in the cytoplasm NO3 is first reduced to nitrite (NO2 )
−
+
by nitrate reductase (Nas), followed by nitrite reductase (Nir) into NH4 . Uptake of
NH+ 4 directly from the environment is mediated by ammonium transporters (AmtB)
that are located in the cell membrane. Regulation of NO− 3 absorption via HATS
depends on the N status of the whole plant, whereas NH+ 4 uptake via HATS is locally
regulated in the roots (Jackson et al 2008). Passive uptake of inorganic N is mediated
by LATS, which are energetically costly due to poor regulation.
The uptake and assimilation of N is not very different for (heterotrophic) mi-
croorganisms. AmtB’s are responsible for the uptake of ammonium, NO− 3 uptake is
mediated by ABC transport proteins or exchanged by NarK antiporters. NO2 − uptake
is catalyzed by FocA.
Assimilation and Incorporation of N The different steps in N assimilation in plant
cells are comparable to assimilation in microorganisms. When NO− 3 is transported to
the cytoplasm, it is reduced to NO−2 by an assimilatory Nas. Subsequently Nir at the
expense of ferrodoxin or NAD(P)H reduces NO− + +
2 to NH4 . NH4 is used for the pro-
duction of glutamate for which several systems exist. Glutamine synthetase forms
glutamine from glutamate and NH+ 4 . Next, glutamine 2-oxoglutarate aminotrans-
ferase (GOGAT; glutamate synthase), catalyzes the synthesis of 2 glutamate from
glutamine and 2-oxoglutarate. Furthermore, glutamate dehydrogenase, can convert
glutamate back into 2-oxoglutarate and NH+ 4 during N remobilization. Subsequently,
glutamate and glutamine are used to produce various amino acids (mostly aspartate
22 The Nitrogen Cycle 207

and aspargine) via transamination. Via negative feedback glutamine concentrations

regulate the N uptake by HATS. Internal transport of N occurs primarily as amino
acids via xylem and phloem, and can be stored in young shoots, leaves, buds, seeds
and storage organs of plants, from where it can be remobilized if needed (Jackson
et al. 2008).
N Requirements N requirements differ greatly between plant species. Severe in-
sufficient N supply often results in dwarf growth. Ultimately this leads to sclerosis,
where plants have small cells and thickened cell walls. Excessive amounts of N,
especially of NH+ 4 , can become toxic for plants. Most plausible processes involved
in NH+ 4 toxicity are energetically costly membrane effluxes, reduced photosynthesis,
displacement of cations and interference of hormone metabolism. Long term exces-
sive N uptake will eventually lead to lowered resistance against abiotic and biotic
stressors and delayed reproduction of the plant (Britto and Kronzucker 2002).

22.2 Microbial N Cycle

After reviewing the N processes of plants, the N transformations of microorganisms

will be discussed in the next paragraphs.
N2 Fixation Nitrogen fixation is the only known biological process where dinitro-
gen gas (N2 ) is transformed into NH+ 4 . This energetically costly process (16 ATP
per molecule of N2 fixed) is exclusively performed by N fixing microorganisms
(diazotrophs). The enzyme nitrogenase (nif) catalyzes this reaction and becomes in-
active when exposed to O2 . Diazotrophs occur free living and in symbiosis. The best
studied diazotrophic symbiosis is that between legumes and Rhizobia sp., but it is
also known for other plants, mosses and between microorganisms (Chap. 21). Even
though direct evidence is missing, it seems likely that bryophytes were the first land
plants that started to live in symbiosis with diazotrophs.
Nitrification The subsequent oxidation of ammonium via NO− −
2 to NO3 is being
studied since the nineteenth century, as this process causes significant losses of fixed
N that are no longer available for crops. Early studies focused on the metabolism of
Nitrosomonas spp. that thrive at high NH+ 4 concentrations. The key enzyme ammo-
nium monooxygenase (AmoA) produces hydroxylamine (NH2 OH) and is present
in large amounts in their membrane systems. NH2 OH is converted into NO− 2 by
an hydroxylamine oxidoreductase (Hao). Further studies indicated that Nitrosospira
spp. might be more relevant at lower NH+ +
4 concentrations. NH4 oxidizers mostly
live in close proximity to NO2 oxidizing bacteria (NOB) that convert the toxic NO−
−
2
rapidly into NO− 3 by a nitrite oxidoreductase (NxrAB) system. Nitrospira and Ni-
trospina spp. thrive at low NO− 2 environments like freshwater and marine systems,
respectively. In contrast to the expectation, only low numbers of bacterial nitrifiers
were observed in marine surveys. About 20 % of the cells were of Archaeal origin,
but their metabolism remained unknown until metagenomic inventories showed the
presence of amoA genes on fosmids that also contained 16S rRNA gene copies of
208 M. A. R. Kox and M. S. M. Jetten

Fig. 22.1 Schematic overview of N transformation from ammonium (NH + −

4 ) and/or nitrate (NO3 )
perspective. Corresponding genes are printed in italic (grey), where nif nitrogenase, amo ammonium
monooxygenase, hao hydroxylamine oxidoreductase, nxr nitrite oxidoreductase, nar respiratory
nitrate reductase, nap periplasmic nitrate reductase, nir nitrite reductase, nor nitric oxide reductase,
nos nitrous oxide reductase, nrf multiheme nitrite reductase, nod putative NO dismutase and hzs
hydrazine synthase. Enzymes nar and nap are involved in all conversions of NO− −
3 to NO2 .Glutamine
synthetase (GS) and glutamine 2-oxoglutarate aminotransferase (GOGAT ) genes, both involved in
assimilation of NH+ 4 . Modified and extended with permission after Burgin and Hamilton (2007)

these Archaea. The isolation of the first archaeal NH+

4 oxidizer Nitrosopumilis mar-
itimus from a marine aquarium showed that Archaeal Ammonium-Oxidizers (AOA)
have a very high affinity for NH+ +
4 probably reflecting their low NH4 habitats.

Denitrification Denitrification is the oldest known process of the N cycle. In this

process NO and N2 O are produced from NO− −
3 and NO2 and N2 is the final prod-
uct. Most denitrifiers are facultative anaerobes, but some species may continue to
denitrify in the presence of O2 , a process known as aerobic denitrification. The den-
itrification trait is widespread among Bacteria, Archaea and can even be found in
some Eukaryotes. The reduction of oxidized N species (NOx ) is catalyzed by met-
alloenzymes that contain molybdenum, iron or copper. The electrons needed for the
reduction are derived from oxidation of inorganic or organic sources Fig. 22.1).
Denitrification is probably the most intensively studied process of the N cycle,
because it may be responsible for more than 20 % of fixed N losses in agriculture.
Initial studies focused on the identification of intermediates and later emphasis was
on the regulation and enzymology of the process. Many organisms possess a nitrate
reductase either located at the cytoplasmic membrane (NarGH) or in the periplasm
(NapAB), that produces the important intermediate NO− 2 , which is subsequently
22 The Nitrogen Cycle 209

converted into NO by either a nirK or a nirS nitrite reductase. Various membrane-

bound nitric oxide reductases exist. Finally N2 O is converted by a nitrous oxide
reductase (NosZ).
In laboratory cultures, one single species may convert NO− 3 all the way to N2 , in
nature the reactions are probably divided between different species, and co-cultures
are rather rule than exception. In natural habitats denitrifiers will have to compete
with dissimilatory nitrate reduction to ammonia (DNRA) and anammox for NO− 3
and NO− 2 , and the outcome of the competition may be dependent on the C/N ratio
(Fig. 22.2). For a longtime it was believed that methane (CH4 ) could not be used
by denitrifiers, because its activation would require O2 . However, in 2006 a co-
culture of Bacteria and Archaea was enriched that could perform CH4 dependent
denitrification (Raghoebarsing et al. 2006). By increasing the NO− 2 concentration,
the Archaea disappeared from the community and Bacteria named Methylomirabilis
oxyfera became dominant. M. oxyfera was shown to have a peculiar denitrification
pathway involving the dismutation of NO into O2 and N2 by a putative NO dismutase
(Ettwig et al. 2010). Recently it has been established that Archaea can convert NO− 3
to NO− +
2 and maybe even further to NH4 at the expense of CH4 (Haroon et al. 2013).

DNRA DNRA is one of the least studies aspects of the N cycle. Many microor-
ganisms are able to perform the DNRA reaction especially at low NO− 3 and high C
concentrations. First NO−3 is converted to NO −
2 by a nitrate reductase. In the sec-
ond step a multiheme nitrite reductase (nrfA) converts the NO− 2 directly to NH+ 4.
Electrons needed for reduction are derived by fermentation of organic compounds or
by sulfide oxidation. DNRA is a difficult pathway to detect and needs sophisticated
stable isotope experiments. An elegant example is the study of Lam et al. (2009) that
investigated the N cycle pathways in the Chilean OMZ (Oxygen Minimum Zone).
By applying a complementary array of methods, they were able to show that DNRA
may contribute up to 40 % of the N flux in this OMZ.
Anaerobic Ammonium Oxidation Only in 1995 the first publication on the dis-
appearance of NH+ 4 from an anoxic denitrifying pilot plant was reported. After
complaints by the citizens of Delft that the Gist Brocades pilot plants produced
too much hydrogen sulfide, the waste water engineers added copious amounts of
calcium nitrate to prevent sulfate reduction. Inadvertently they created favourable
conditions for anaerobic ammonium oxidizing (anammox) bacteria to proliferate.
Biomass of the pilot plant was subsequently used to start new more defined en-
richment cultures, first as fluidized bed reactors, later as sequencing batch reactors,
yielding enough anammox biomass to perform the necessary experiments. Inhibitors
studies with antibiotics showed that the process was bacterial, while15 N stable iso-
topes studies indicated the production of the rocket fuel hydrazine (N2 H4 ). As the
enrichments yielded 70–90 % anammox dominance, physical purification methods
based on gradient centrifugation had to be applied. This gave sufficient purified cells
to do crucial15 N and 14 C experiments showing the autotrophic nature of the anam-
mox bacteria. From the purified cells, the 16S rRNA gene could be amplified, and
the anammox bacteria were shown to belong to the phylum of the Planctomycetes.
Electron microscopic analysis showed that anammox bacteria have a unique cell plan
210 M. A. R. Kox and M. S. M. Jetten

Fig. 22.2 Branching diagram with a simplified overview of NO− 3 transformations under different
conditions, indicated by the different colors. Depicted in green is CH4 availability, blue is the carbon
input, red represents iron (Fe) concentrations, in yellow the free sulfide concentrations (H2 S, S 0 ,
FeS), finally in different brown shades are the C/N ratio under the different Fe or free sulfide
concentrations. (Conc. the concentration; DNRA Dissimilatory Nitrate Reduction to Ammonium;
AOM Anaerobic Oxidation of Methane). Adapted and extended with permission from Burgin and
Hamilton (2007)

with a specialized compartment harboring the enzymes responsible for the anammox
reactions (Van Niftrik and Jetten 2012). Analysis of the fatty acids of the cells and
organelle indicated that anammox bacteria have both ether and ester lipids of con-
catenated cyclobutane rings that from a kind of staircase structure, hence their name
ladderane lipids. These are unique and can be used as specific anammox biomarkers.
After the availability of suitable diagnostic tools, several expeditions to OMZs
were organized. Indeed it could be documented that in those OMZs, anammox
bacteria were present and active. Taken together they could account for half of the
loss of fixed N from the systems, making them important players in the global N
cycle. Recently it was also shown that anammox can contribute significantly to the
N-loss in terrestrial ecosystems such as wetlands and river sediments.
After the genome of the first anammox bacterium was resolved, the molecular
mechanisms were elucidated (Kartal et al. 2011). The crucial intermediates were
NO and N2 H4 and a unique enzyme complex hydrazine synthase was identified.
Application of anammox bacteria together with partial nitrification may result in
22 The Nitrogen Cycle 211

more sustainable waste water treatment systems saving on O2 and electricity us-
age, methanol consumption, and ecological footprint (Kartal et al. 2010). Based on
these advantages, already more than 20 full scale anammox plants have been build
worldwide, and many more are commissioned.

22.3 N Cycle in Ecosystems

After the introduction of the N cycle processes, the following section will focus on the
cooperation and competition for N compounds between plants and microorganisms.
The competition is controlled by metabolic limitations and environmental conditions.
Plant vs Plant N Competition Plants have evolved diverse adaptations to cope
with nutrient limitations. Resource depletion has been hypothesized as the strategy
in plant-plant competition for N. By taking up more N compounds than directly
necessary, N can become rapidly depleted in the environment and thus limiting
for competitors. Competition for N resources between plants also occurs indirectly.
Microorganisms flourish in the rhizosphere due to high litter production by roots.
Plants modulate their rhizospheric microbiome (see Chap. 43) by attracting certain
species, which have the potential to enhance N uptake for the plant.
Plant vs Microorganisms The trade-off between plants and microorganisms in
competition for N, is a much debated topic. In terrestrial ecosystems, the classical
paradigm stated that microorganisms are stronger competitors for N than plants,
hence plants would only use the microbial N left-overs. In the late 1990s, a new
hypothesis was developed that put less emphasis on mineralization and underlined
depolymerization of the complex N compounds present in soil by microorganisms,
as the key process and bottleneck in N cycling (Jackson et al. 2008).
Short term experiments with 15 N additions showed that microorganisms take
up organic and inorganic N faster than roots. Microorganisms have high substrate
affinities, low volume to surface ratio’s and fast turnover rates compared to plants and
therefore are stronger competitors. In the long run plants assimilated most of the15 N
due to the gradual release of 15 N that was first mineralized by the microorganisms. It
is this temporal difference that determines the competition for N between plants and
microbes in the end (Jackson et al. 2008; Kuzyakov and Xu 2013). The most direct
competition between plants and microbes occurs at the level of the available inorganic
N. Nitrifiers have to compete for available NH+ +
4 with all NH4 assimilating plants
and microorganisms, whereas the processes of denitrification and DNRA compete
for NO− −
3 with plant and microbial NO3 assimilation.

Competition for NH+ 4 Fertilization experiments with inorganic N have either shown
about equal N uptake rates for both plants and microorganisms, hence both were
simultaneously limited in N. In a NH+ 4 fertilization experiment plant removal resulted
in increased nitrification rates. This indicated that plants and autotrophic nitrifiers
compete for NH+ 4 , with plants being the stronger competitors (Kaye and Hart 1997).
212 M. A. R. Kox and M. S. M. Jetten

Competition for NO− −

3 In rice soils, a study of NO3 assimilation by plant and
microorganisms showed that fertilization with labeled NO− 3 always resulted in more
15
NO−3 ending up in the microbial than in plant N pools. As rice grows in flooded
soils under anaerobic conditions, ROL will release oxygen into the rhizosphere. The
supplied O2 stimulates nitrification, and subsequently NO− 3 can enter the anoxic zone
where denitrification and anammox cause fixed N to be lost to the atmosphere. By
blocking transport of O2 and N2 via the aerenchym through clipping of rice plants
below the water surface, nitrification rates rapidly decreased and in the longer term
also denitrification rates decreased (Arth et al. 1998; Matheson et al. 2002).
O2 is an important regulator of NO− 3 partitioning between denitrification and
DNRA. It was shown that microcosms planted with Glyceria declinata experienced
more O2 intrusion and higher redox potentials compared to unplanted microcosms,
leading to higher denitrification than DNRA rates (Matheson et al. 2002). In a study
with young-barley, short term rewetting increased nitrification rates, whereas long
term rewetting also stimulated denitrification (Højberg et al. 1996). Since the role of
denitrification in N loss might be overestimated (Burgin and Hamilton 2007), future
studies in anoxic systems should also analyze the role of anammox and DNRA.
Microorganism vs Microorganisms Also microorganisms among each other have
to interact and/or compete for NH+ 4 and NOx . Nitrifiers (AOA, AOB, NOB) live in
close proximity to ensure rapid conversion of NH+ −
4 to NO3 without intermediate
−
NO2 accumulation. Availability of O2 is an important factor determining whether
anammox or nitrifiers can convert NH+ 4 first, and whether NOB or anammox can
convert NO− 2 subsequently. In systems with fluctuating O2 and NH+ 4 concentrations
like wastewater systems, sediments or OMZs, consortia of AOA, AOB, NOB and
anammox have been found to be active (Lam et al. 2009). Low ammonium and O2
concentrations seem to favor AOA. Laboratory studies under O2 limitation showed
that AOA can thrive very well in co-cultures with anammox bacteria. Several guilds
may have to compete for NO− 3 and the outcome of the competition is mostly de-
termined by the quality and quantity of the electron donor. Burgin and Hamilton
(2007) made an elegant scheme to predict the occurrence of the various NO− 3 reduc-
ing processes (Fig. 22.2). At low organic carbon, denitrification and anammox will
compete, and above a C/N ratio of 2, denitrification may be favored. Under high en-
ergy or carbon input DNRA may be the most important process, while denitrification
is dominant at low C/N ratios in the absence of NH+ 4 . At high CH4 concentration
AOM Archaea may play a significant role in nitrate reduction and DNRA, and M.
oxyfera in NO− 2 conversion to N2 .

22.4 Anthropogenic N Use and Changes in N Cycle

The global cycling of N has doubled over the last century, starting with the application
of the Haber-Bosch process (N2 + 3 H2 → NH3 ) in 1913. At the expense of fossil
fuel, artificial fertilizer could be produced, and thus increased crop productivity and
22 The Nitrogen Cycle 213

harvest. Though, what was not realized at the time is that the use of (excess) N
fertilizer has severe ecological impacts.
Studies on N fertilizer use have primarily focused on loss of N fertilizers into
other (pristine) ecosystems. Only part of the N that is applied as fertilizer is taken
up by microorganisms and plants and later on removed via harvest of the crops. The
remainder will enter the N cycle of the ecosystem. According to Burgin and Hamilton
(2007) the most desirable way to reduce high N levels in ecosystems is via permanent
removal by denitrification (or anammox), because other N transformations may result
in even more harmful N-compounds.
The main processes studied with respect to N loss from ecosystems are NO− 3
leaching, ammonia volatilization and loss as NOx or N2 gas (Cameron et al. 2013).
NO− 3 leaching depends on nitrate loading of the soil and the levels of drainage that
occur. NO− 3 is a large problem for water quality and can affect human health. Via
groundwater the leached NO− 3 enters rivers and lakes, where it might stimulate algal
blooms and cause biodiversity loss. NO− 3 losses from fertilizer-use can be reduced
by using adequate and efficient fertilizer levels to prevent N excess, by optimizing
plant N uptake to avoid N losses and if all else fails nitrification inhibitors can be
applied. Fertilizer-use is nowadays highly restricted and managed so that fertilizers
are used in an efficient manner, with amounts that are matched to the rate of plant
growth.
Ammonia (NH3 ) volatilization is especially a problem in areas surrounding in-
tensive animal farms. Most important sources are animal urine and feces, but also
N fertilizers contribute to NH3 volatilization. Once volatilized, NH3 deposits cause
acidification and eutrophication. NH3 volatilization can be best reduced by apply-
ing fertilizers beneath the soil surface or just before rain, and by reducing intensive
animal farming.
Ultimately, N can be lost to the atmosphere via nitrification or denitrification
in the form of NO, N2 O or N2 . In particular, NO and N2 O (NOx ) form a serious
problem since they deplete the ozone layer and contribute substantially to climate
change. The global warming potential of N2 O is 298 times that of CO2 . To reduce
NOx formation, nitrification inhibitors combined with optimized fertilizer application
may diminish nitrification. Methods to reduce denitrification include changing the
soil physiochemical parameters (i.e. increasing pH by applying lime, or increase
aeration of the soil, as described in Cameron et al. 2013).

22.5 Conclusions and Outlook

N cycling has been studied intensively for over decades. Although the knowledge on
the N cycle has increased, the role of anammox, the contribution of AOM dependent
conversions of nitrogen and the interactions in the N cycle deserve more attention.
Ultimately, improving our understanding of the N cycle will help to retain and restore
balances in ecosystem N cycles which have been affected by anthropogenic activities.
214 M. A. R. Kox and M. S. M. Jetten

Acknowledgements We would like to thank our co-workers and collaborators and granting agen-
cies for their continuous support (ERC 232937, ERC 339880, Spinozapremie 2012 and OCW-NWO
Gravitation Grant SIAM 024.002.002).

References

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Soil Biol Biochem 30:509–515
Britto DT, Kronzucker HJ (2002) Ammonium toxicity in higher plants: a critical review. J Plant
Physiol 159:567–584
Burgin AJ, Hamilton SK (2007) Have we overemphasized the role of denitrification in aquatic
ecosystems? A review of nitrate removal pathways. Front Ecol Environ 5:89–96
Cameron KC, Di HJ, Moir JL (2013) Nitrogen losses from the soil/plant system: a review. Ann
Appl Biol 162:145–173
Ettwig KF, Butler MK, Le Paslier D et al (2010) Nitrite-driven anaerobic methane oxidation by
oxygenic bacteria. Nature 464:543–548
Haroon MF, Hu S, Shi Y et al (2013) Anaerobic oxidation of methane coupled to nitrate reduction
in a novel archaeal lineage. Nature 500:567–570
Højberg O, Binnerup S, Sørensen J (1996) Potential rates of ammonium oxidation, nitrite oxidation,
nitrate reduction and denitrification in the young barley rhizosphere. Soil Biol Biochem 28:47–54
Jackson LE, Burger M, Cavagnaro TR (2008) Roots, nitrogen transformations, and ecosystem
services. Annu Rev Plant Biol 59:341–363
Kartal B, Kuenen JG, van Loosdrecht MCM (2010) Engineering. Sewage treatment with anammox.
Science 328:702–703
Kartal B, Maalcke WJ, De Almeida NM et al (2011) Molecular mechanism of anaerobic ammonium
oxidation. Nature 479:127–130
Kaye J, Hart S (1997) Competition for nitrogen between plants and soil microorganisms. Trends
Ecol Evol 5347:139–141
KuzyakovY, Xu X (2013) Competition between roots and microorganisms for nitrogen: mechanisms
and ecological relevance. New Phytol 198:656–669
Lam P, Lavik G, Jensen MM et al (2009) Revising the nitrogen cycle in the Peruvian oxygen
minimum zone. Proc Natl Acad Sci U S A 106:4752–4757
Lambers H, Chapin III FS, Pons TL (1998) Plant physiological ecology, 1st edn. Larcher publisher
Springer Verlag, Berlin
Lamers LPM, Van Diggelen JMH, Op den Camp HJM et al (2012) Microbial transformations of
nitrogen, sulfur, and iron dictate vegetation composition in wetlands: a review. Front Microbiol
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Larcher W (2001) Physiological plant ecology, 4th edn. Lambers publisher Springer Science +
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Matheson F, Nguyen M, Cooper A (2002) Fate of 15 N-nitrate in unplanted, planted and harvested
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Van Niftrik L, Jetten MSM (2012) Anaerobic ammonium-oxidizing bacteria: unique microorgan-
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anaerobic methane oxidation to denitrification. Nature 440:918–921
Chapter 23
Biological Nitrogen Fixation

Frans J. de Bruijn

Abstract Biological nitrogen fixation (BNF) is the process of the reduction of dini-
trogen from the air to ammonia carried out by a large number of species of free-living
and symbiotic microbes called diazotrophs. BNF presents an inexpensive and envi-
ronmentally sound, sustainable approach to crop production and constitutes one of
the most important Plant Growth Promotion (PGP) scenarios. Here I will summarize
various aspects of BNF, including the dinitrogen reduction catalysed reaction carried
out by “nitrogenase” and the enzymes/genes involved and their regulation, the in-
herent “oxygen paradox”, the identification of diazotrophs, sustainable agricultural
uses of BNF, symbiotic plant-diazotroph interactions and endophytic diazotrophs,
data from the field, and future prospects in BNF.

23.1 Introduction

Fixed nitrogen is a limiting nutrient in most environments, with the main reserve of
nitrogen in the biosphere being molecular di-nitrogen from the atmosphere, which is
an inert gas with a triple bond, that is energetically unfavourable to break. Nitrogen
availability is limiting for plant growth and has long been overcome through the ap-
plication of synthetic nitrogen-rich fertilizer. Using increasing amounts of fertilizers
the yield of crop plants such as cereals has been greatly augmented, but this has been
at a high economic and environmental cost (Ferguson et al. 2010). The industrial
production of nitrogen fertilizer costs more than US$ 100 billion because the ener-
getically difficult reduction of the triple bond carried out at high temperature and
pressure requires the use of large amounts of fossil fuel, a limited resource. Thus,
fertilizer costs are high and this affects especially resource-poor farmers worldwide.
In addition, the use of fertilizer has a severe environmental impact, due to run-off of
excess non-assimilated nitrate, and concomitant eutrophication of rivers, lakes and
oceans, as well as contamination of the drinking water. Moreover, carbon dioxide is

F. J. de Bruijn ()
INRA/CNRS Laboratory of Plant-Microbe Interactions, 24 Chemin de Borde Rouge,
Auzeville CS 52627, 31326 Castanet-Tolosan Cedex, France
Tel.: +33561285320
e-mail: debruijn@toulouse.inra.fr

© Springer International Publishing Switzerland 2015 215

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_23
216 F. J. de Bruijn

released during fossil fuel combustion which occurs during production of chemical
fertiliser and contributes to the greenhouse effect, as does the decomposition of ni-
trogen fertilizer, which releases nitrous oxide. The latter gas is about 292 times more
active as a greenhouse gas than carbon dioxide (Ferguson et al. 2010).
Biological Nitrogen Fixation (BNF), the reduction of atmospheric dinitrogen to
ammonia, carried out by a large and diverse group of free-living and symbiotic
microorganisms, presents an inexpensive and environmentally sound, sustainable
approach to crop production and constitutes one of the most important Plant Growth
Promotion (PGP) scenarios (see de Bruijn 2015a for a comprehensive coverage of
BNF).
The increased need for fixed nitrogen, be it industrially- or biologically fixed
nitrogen, is exemplified in the case of rice. Rice is the most important staple food
for over 2 billion people in Asia and for hundreds of millions in Africa and Latin
America. To feed the ever-increasing populations of these regions, the world’s annual
rice production must increase from 560 million tons in the year 2000 to 760 million
tons by 2020 (IRRI 1993). If future increases in rice production have to come from
the same or even reduced land area, rice productivity (yield ha-1) must be greatly
increased to meet these goals (Ladha et al.1997). Nitrogen is the major nutrient
limiting rice production. One kg of nitrogen is required to produce 15–20 kg of
grain (Ladha and Reddy 2003). Enhancing rice production from the present 8–12 t
per hectare by 2020 would require an increased application of 400 kg per hectare,
doubling the amount of N fertilizer presently applied (Ladha and Reddy 2003). This
obviates the need for alternative approaches, namely BNF in cereals (see de Bruijn
2015b; Chap. 42).
BNF occurs when atmospheric di-nitrogen is converted to ammonia by an enzyme
called nitrogenase (Postgate 1998). The reaction for BNF is:
N2 + 8 H+ + 8 e− + 16 MgATP → 2 NH3 + H2 + 16 MgADP + 16 Pi
The process is coupled to the hydrolysis of 16 equivalents of ATP and is accom-
panied by the co-formation of one molecule of H2 . In free-living diazotrophs,
the nitrogenase-generated ammonium is assimilated into glutamate through the
glutamine synthetase/glutamate synthase pathway (Postgate 1998). In the case of
associative or symbiotic nitrogen fixing diazotrophs (see below), the ammonia
produced by the nitrogen fixating bacteria is excreted and assimilated by plant
enzymes.

23.2 The Oxygen Paradox

Enzymes responsible for nitrogenase action are very susceptible to destruction by

oxygen leading to the “Oxygen Paradox”: while oxygen and respiration are generally
involved in generating the large amount of ATP required for nitrogenase function,
the enzyme is terminally inactivated by oxygen. How, one can ask, do microbes
fix nitrogen on a planet covered by 20 % oxygen (Postgate 1998)? The problem
23 Biological Nitrogen Fixation 217

is even acerbated in the case of oxygenic photosynthetic diazotrophs, including

cyanobacteria (Flores et al. 2015; see below). The following strategies are used to
deal with the Oxygen Paradox:
1. The simplest strategy is that of avoiding oxygen. For example, some diazotrophs
are obligate anaerobes. Many bacteria are facultative anaerobes: capable of either
aerobic or anaerobic growth. Diazotrophic members of this group usually fix
nitrogen only anaerobically (Postgate 1998).
2. Some strains of rhizobia (see below) show microaerobic diazotrophy. Several
other types of nitrogen-fixing aerobes show oxygen sensitivity amounting to
microaerophily (Postgate 1998).
3. Respiration is another mechanism to allow (micro)aerobic nitrogen-fixation. In
aerobes a high levels of respiration is a highly efficient means of generating ATP
(necessary for nitrogenase action), while protecting nitrogenase from oxygen
damage (respiratory protection). There exist also a “conformational protection”
of nitrogenase (Postgate 1998).
4. The cyanobacteria are oxygenic phototrophic prokaryotes in which the capability
to express the BNF machinery is widespread. Two main strategies have evolved
to allow the two incompatible processes of oxygenic photosynthesis and BNF to
be performed in a given organism: temporal expression in diel cycles and spatial
separation through the formation of specialized cellular structures (heterocysts)
in which BNF takes place (Flores et al. 2015).
5. The root- and stem nodules on leguminous plants are highly specialized structures
in which bacteroids, differentiated forms of the inducing rhizobia fix nitrogen for
the host plant (see below). In these nitrogen fixing nodules leghemoglobin is
produced by the plant cells, which binds and transfers oxygen. Its affinity for
oxygen is so high that it delivers it to the bacteroids at a free concentration
harmless to their nitrogenase (Postgate 1998).

23.3 The Nitrogenase Mediated BNF reaction

Enzymatic conversion of dinitrogen to ammonia is catalysed by nitrogenase, an en-

zyme complex which is highly conserved in free-living and symbiotic diazotrophs.
The convential nitrogenase or Mo-nitrogenase contains a prosthetic group with
molybdenum (Iron-Molybdenum Cofactor; FeMoCo; Newton 2015).
The nitrogenase enzyme is composed of two metalloproteins. Component 1, also
designated MoFe protein, is a tetramer, composed of two non-identical subunits α
and β, while component 2, also designated as the Fe protein, is a dimer consisting of
identical subunits (Franche et al. 2009; Newton 2015). Two FeMoCo’s are bound to
the α subunits of the MoFe protein. Nitrogen fixation is a highly complex process,
which is not yet fully elucidated. For a detailed description of nitrogenases, their
biosynthesis and their mode of action, see Newton (2015).
218 F. J. de Bruijn

The Nitrogen Fixation Genes The genetics of nitrogen fixation was initially elu-
cidated in Klebsiella pneumoniae where the nif genes required for the synthesis of
nitrogenase are clustered in a 24 kb region of the chromosome. This entire region
was sequenced early on by Arnold et al. (1988). The three structural genes encoding
Mo-nitrogenase proteins are nifD and nifK for the Mo protein subunits and nifH for
the Fe protein (Franche et al. 2009). The complete assembly of nitrogenase requires
other nif genes involved in the synthesis of FeMoCo, including nifB, nifQ, nifE, nifN,
nifX, nifU, nifS, nifV, nifY and nifH. In addition nifS and nifU are involved in the as-
sembly of iron-sulfur clusters and nifW and nifZ in the maturation of the nitrogenase
components (Franche et al. 2009). In addition, Klebsiella contains genes required
for electron transport to nitrogenase (nifF and nifJ) as well as the regulatory nifLA
genes involved in the regulation of nif gene expression in response to the oxygen
and nitrogen status of the cell (Franche et al. 2009; Dixon and Kahn 2004; see also
below). The nif gene cluster is not always this complex. Recently, a minimal nitro-
gen fixation gene cluster from Paenibacillus containing only 9 nif genes has been
identified and shown to enable expression of active nitrogenase in Escherichia coli
(Wang et al. 2013). This would greatly facilitate the engineering of nitrogen fixation
in non-nitrogen fixing organisms such as plants (see Sect. 23.11).
Regulation of nif (fix) Gene Expression Nitrogen-fixing bacteria have evolved
several mechanisms to sense multiple environmental signals in order to adapt the
nitrogen fixation process to their physiological constraints. Availability of a nitrogen
source is a key regulatory signal repressing the nitrogen fixation process. In sev-
eral nitrogen-fixing γ-Proteobacteria (e.g.: Azotobacter vinelandii, Pseudomonas
stutzeri, K. pneumoniae) the NifA activator and the anti-activator NifL proteins, en-
coded by the nifLA operon, control the expression of all other nif genes, in concert
with the alternative sigma factor RpoN and the Integration Host Factor (IHF). The
nifLA operon is in turn controlled by the general nitrogen regulatory protein NtrC, in
concert with RpoN, and by the PII protein (GlnB or GlnK) in response to the fixed
nitrogen status (Dixon and Kahn 2004).
In free-living diazotrophs oxygen regulation occurs via the nifL gene product,
which serves as a repressor in the presence of oxygen. In symbiotic nitrogen-fixing
rhizobia, transcription of nitrogen fixation genes (nif and fix genes) is induced primar-
ily by low-oxygen conditions. Low-oxygen sensing and transmission of this signal
to the level of nif and fix gene expression involve at least five regulatory proteins,
FixL, FixJ, FixK, NifA, and RpoN (sigma 54) (Dixon and Kahn 2004).

23.4 Major Diazotropic Players and their Phylogeny

By 1960 the nitrogen-fixation capacities of free-living soil bacteria had been estab-
lished for only a dozen genera. This is a long way from our present knowledge of the
distribution of nitrogen fixation ability in most phyla of the Bacteria and Archaea
domains (Franche et al 2009; see Fig. 23.1).
23 Biological Nitrogen Fixation 219

Fig. 23.1 Simplified phylogenetic 16S tree with prokaryotes carrying nif genes. Reprinted with
permission from Springer from Franche et al. (2009)

The high degree of conservation of certain nif genes and the recent and rapid
increase in the availability of microbial sequences affords novel opportunities to
re-examine the occurrence and distribution of nitrogen fixation genes. The current
practice for computational prediction of nitrogen fixation is to use the presence of
the highly conserved nifH and/or nifD genes (Dos Santos et al. 2012 and references
therein). Dos Santos et al. (2012) searched the fully sequenced genomes of 1002
bacterial and archaeal species for coding sequences for NifD and NifH, and identified
174 species which contain homologous sequences, suggesting that the phylogenetic
distribution of diazotrophs is much broader than previously known. Nitrogen fixation
activity has not been experimentally shown in 92 of these species (Dos Santos et al.
2012). The authors went on to look at the occurrence of nine additional nif genes and
concluded that there existed a minimum gene set for nitrogen fixation, consisting
of nifHDK (catalytic) and nifENB (biosynthetic), which they used to identify 92
species containing coding sequences similar to NifD and NifH, of which 67 met the
minimum set requirement (Dos Santos et al. 2012). Based on gene content, these 67
species were proposed to have the capacity for nitrogen fixation (Dos Santos et al.
2012).

23.5 Sustainable Agricultural Uses of Biological Nitrogen

Fixation; the Legume-Rhizobium Symbiosis

Biological nitrogen fixation is a highly valuable alternative to nitrogen fertilizer. It is

most effective in the interaction between members of the legumes (Leguminosae) and
the well known α-proteobacteria called rhizobia (including the genera Azorhizobium,
Allorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium), as
well as more recently recognized rhizobia belonging to the β-proteobacteria (Moulin
et al. 2015). Once the symbiosis is established, rhizobia fix atmospheric nitrogen
220 F. J. de Bruijn

Fig. 23.2 Lentil field in

Western Manitoba in which
plants on the right received a
commercial rhizobial
inoculants; and in the middle
rows are the control plants.
(Reprinted with permission
from the Crop Science
Society of America from
“Benefits of inoculating
legume crops in the Northern
Great Plains” (Vessey 2004.
Crop Management Vol. 3)

and provide it to their host plant (symbiotic nitrogen fixation), in return for carbon
(energy) provided by the plant. Because nitrogen is a key limiting factor for plant
growth and development, the ability of legumes to enter into a symbiosis with
nitrogen-fixing rhizobia provides them with a distinct advantage over other plant
species (Ferguson et al. 2010) and constitutes highly proficient sustainable agriculture
systems .
Legumes include major food and feed crop species, such as soybean, pea, clover,
alfalfa, lentils and mungbean. They represent the third largest group of angiosperms
and are the second largest group of food and feed crops grown globally (Ferguson
et al. 2010; for the case of soybean, see Chap. 41). They are cultivated on 12–15 %
of all available arable land and are responsible for more than 25 % of the world’s
primary crop production with 247 million tons of grain legumes produced annually
(Ferguson et al. 2010). In addition to food and feed crops, nodulated legumes such
as soybean and Pongamia pinnata have garnered a great deal of attention as future
sustainable biofuel sources because of their high seed oil content (Ferguson et al.
2010; see also Chap. 41). The legume-Rhizobium symbiosis is the most important
symbiotic association in terms of biological nitrogen fixation, producing roughly
200 million tons of fixed nitrogen annually (Ferguson et al. 2010; see Fig. 23.2).

23.6 The Actinorhizal-Frankia Symbiosis

Besides in legumes, symbiotic nitrogen fixation is also found in the Actinorhizal

symbiosis. Actinorhizal plants represent about 200 species distributed among 24
genera in eight angiosperm families (Franche et al. 2009). All actinorhizal plants are
woody trees or shrubs except for Datisca, a genus of flowering plants. Often they are
considered “pioneer plants” in regions with poor soil and examples of well known
genera include Alnus (alder), Eleagnus (autumn olive), Hippophae (sea buckthorn)
and Casuarina (beef wood) (Franche et al. 2009).
23 Biological Nitrogen Fixation 221

The genus Frankia comprises high GC percentage Gram-positive bacteria belong-

ing to the family Frankiaceae in the order Actinomycetales. One striking feature of
Frankia is its ability to differentiate into two unique developmental structures that
are critical to its survival: vesicles and spores. Vesicles are the site for actinorhizal
nitrogen fixation. For an excellent review of this symbiotic system see Franche et al.
(2009).

23.7 Cyanobacteria and Symbiosis

Cyanobacteria are widely distributed in aquatic and terrestrial environments. While

nitrogen fixation occurs both in unicellular and filamentous species, associations
with plants are essentially limited to heterocystous cyanobacteria, primarily of the
genus Nostoc and Anabaena. Besides vascular plants, there exist a wide variety
of non-vascular lower plants belonging to the bryophytes, including liverworts and
hornworts, algae and fungi, that develop associations with cyanobacteria, as well as
many marine eukaryotes (Franche et al. 2009). In terms of symbiotic associations
with vascular plants, Gunnera, a genus of about 40 species, is the only angiosperm
with which the cyanobacterium Nostoc punctiforma is associated and fixes nitrogen.
The mechanism of infection and Nostoc differentiation is described in Franche et al.
(2009) but once intracellular, a high frequency of differentiation of vegetative cells
into heterocysts occurs and nitrogen is fixed at a high rate.

23.8 Nitrogen Fixation in the Ocean

BNF is an integral part of the marine nitrogen cycle and together with N losses
through denitrification and anaerobic ammonia oxidation determines the size of
the oceanic nitrogen pool (Zehr and Bombar 2015). BNF in the oceans is of
a similar magnitude to anthropogenic BNF, and yet many questions remain on
what the major N2 -fixing organisms are, and what controls their distributions and
BNF rates. Using molecular and metagenomic approaches, surprising discoveries
have been made, since many environmental microorganisms have yet to be ob-
tained in pure culture. Cyanobacteria appear to be the main oceanic N2 -fixers, with
several key species that include the filamentous, colonial, nonheterocyst-forming
Trichodesmium, heterocyst-forming strains that are symbiotic with diatoms. The
N2 -fixing microbial taxa generally differ among the different marine habitats, and
include Archaea and diverse (photo)heterotrophic and chemolithotrophic bacteria
and photoautrophic cyanobacteria. For example, archaeal nitrogenase (nifH) genes
have been found in deep water and near hydrothermal vents (Zehr and Bombar
2015). It is accepted that BNF is a key component of the marine nitrogen cycle, but
rather than being an easily quantifiable process carried out by few species in well
constrained areas, the accumulating knowledge shows that we are just beginning to
222 F. J. de Bruijn

understand the impacts of diazotrophs in the pelagic ocean (and other) ecosystems
(Zehr and Bombar 2015).

23.9 Associative and Endophytic Nitrogen Fixation

In addition to strictly associative diazotrophs, there has been an increasing interest

in endogenous BNF systems, particularly nitrogen-fixing endophytic bacteria. En-
dophytic bacteria are defined as bacteria detected inside surface-sterilized plants or
extracted from inside plants and having no visibly harmful effects on the host plant
(see Chap. 5; see de Bruijn 2013).
The extensive Brazilian experience with associative and endophytic diazotrophs
and Plant Growth Promoting Rhizobacteria (PGPR) on sugarcane, rice and other
grasses is highly relevant. This experience is highlighted in a special issue of Plant and
Soil (James and Baldani 2012), based on the BNF with non-Legumes International
Symposium of 2010 in Brazil. In the case of sugarcane and other biofuel crops, the
location of the meeting in Brazil was particularly pertinent since the highly advanced
Brazilian bioethanol program, which produces over 27 billion liters of ethanol per
year, is based on the cultivation of sugarcane, deriving much of its N-requirements
via BNF (James and Baldani 2012). Field-based BNF quantitative studies revealed
very substantial inputs into sugarcane of at least 40 kg N per ha per year (Urquiaga
et al. 2012).

23.10 Field Data

Soybean and BNF have been the focus of many studies worldwide and field data
are presented in Chap. 41. An evaluation of BNF in food grain legumes grown
in experimental plots in Africa revealed high levels of symbiotic dependency on N2
fixation for their N nutrition (Dakora et al. 2015). Cowpea could, for example, derive
30–96 % of its N nutrition from symbiosis, soybean 39–87 %, pigeon pea 27–92 %,
groundnut 24–67 %, mungbean 66–86 %, chickpea (kabuli) 3–92 % and chickpea
(desi) 21–82 % (Dakora et al. 2015).

23.11 Future Prospects in Biological Nitrogen Fixation

Several factors, such as efficient strain selection, inoculum production and quality,
plant breeding for nitrogen fixation etc. can be improved upon, and associative
(endophytic) nitrogen fixation clearly is of importance. However, the “holy grail” of
nitrogen fixation research is the quest for nitrogen fixation in cereals, such as rice
(de Bruijn 2015b). Two ways have been envisioned: (i) the transfer to and expression
of the nif genes in transgenic cereal plants and (ii) the transfer of the ability to fix
23 Biological Nitrogen Fixation 223

nitrogen symbiotically (de Bruijn 2015b). Although tremendous progress has been
made in the characterization of nif genes for transfer into plants (de Bruijn 2015b)
and the elucidation of the Common Symbiotic Signalling Pathway (CSSP or SYM;
de Bruijn 2015b; see Chap. 42) in legumes and cereals, still a considerable amount of
new information will be needed to achieve either goal (de Bruijn 2015b; Chap. 42).
However, experimentation towards elucidating the essential nif genes and custom
tailoring them for expression in plants, as well as studying the SYM pathway genes
and identifying the “missing components” (de Bruijn 2015b; see Chap. 42), are now
supported by large grants, for example from the Bill and Melinda Gates Foundation
the BBSRC (UK) and NSF (USA), raising the hope for a bright future in this field.

Acknowledgements The writing of this review Chapter was supported by the Laboratory of Plant-
Microbe Interactions (LIPM), INRA, CNRS and the Labex Tulip. Springer Verlag is gratefully
acknowledged for their Permission to reprint Fig. 1 and quote and cite excerpts of the text of
Franche et al. (2009). Claude Bruand is thanked for his critical review of the manuscript.

References

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nutrition/health in Africa. In: de Bruijn FJ (ed) Biological nitrogen fixation. Wiley, Hoboken
(in press)
de Bruijn FJ (ed) (2013) Molecular microbial ecology of the rhizosphere. Wiley, Hoboken, pp
1–1269
de Bruijn FJ (ed) (2015a) Biological nitrogen fixation. Wiley, Hoboken (in press)
de Bruijn FJ (2015b) The quest for biological nitrogen fixation in cereals: a perspective and
prospective. In: de Bruijn FJ (ed) Biological nitrogen fixation. Wiley, Hoboken (in press)
Dixon R, Kahn D (2004) Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol
2:621–631
Dos Santos PC, Fang Z, Mason SW et al (2012) Distribution of nitrogen fixation and nitrogenase-like
sequences amongst microbial genomes. BMC Genomics 13:162–174
Ferguson BJ, Indrasumunar A, Hayashi S et al (2010) Molecular analysis of legume nodule
development and autoregulation. J Int Plant Biol 52:61–76
Flores E, Lopez-Lozano A, Herrero A (2015) Nitrogen fixation in the oxygenic phototrophic
prokaryotes (cyanobacteria): the fight against oxygen. In: de Bruijn FJ (ed) Biological nitrogen
fixation. Wiley, Hoboken (in press)
Franche C, Lindstrom K, Elmerich C (2009) Nitrogen-fixing bacteria associated with leguminous
and non-leguminous plants. Plant Soil 321:35–59
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James EK, Baldani JI (2012) The role of biological nitrogen fixation by non-legumes in the
sustainable production of food and biofuels. Plant Soil 356:1–3
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prospects. Plant Soil 252:151–167
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Moulin L, James EK, Klonowska A et al (2015) Phylogeny, diversity, geographical distribution and
host range of legume-nodulating Betaproteobacteria: what is the role of plant taxonomy? In: de
Bruijn FJ (ed) Biological nitrogen fixation. Wiley, Hoboken (in press)
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Biological nitrogen fixation. Wiley, Hoboken (in press)
Postgate J (1998) Nitrogen fixation. Cambridge University Press, Cambridge, pp 1–109
Urquiaga S et al (2012) Evidence from field nitrogen balance and15 N natural abundance data for
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Vessey K (2004) Benefits of inoculating legume crops with rhizobia in the northern great plains,
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Chapter 24
Phosphate Mobilisation by Soil Microorganisms

José-Miguel Barea and Alan E. Richardson

Abstract Microorganisms are fundamental to the cycling of phosphorus (P) in

soil-plant systems as they are involved in a range of processes that govern P transfor-
mations and availability. Soil microorganisms in particular are able to release plant
available P from otherwise sparingly available forms of soil P, through solubilisation
and mineralisation reactions of inorganic and organic P, respectively. The poten-
tial of phosphate solubilising microorganisms (PSM) to improve plant P nutrition is
widely recognised, and the mechanisms involved are being investigated. The feasi-
bility of developing efficient management systems based on PSM as biofertilisers
is of current interest in rhizosphere biotechnology. Mycorrhizosphere interactions
involving PSM and their interaction with AM fungi is of further relevance for the ac-
quisition, transport and supply of P to plant roots, and therefore to soil P cycling and
plant P nutrition. Managing these interactions (mycorrhizosphere tailoring) provides
an environmentally-acceptable agro-technological practice to improve agricultural
sustainability.

24.1 Phosphorus in the Soil-Plant System

Phosphorus (P) is a vital element for life on earth. In particular, P is essential for plant
growth and development, as it is a component of fundamental macromolecules in-
volved in genetic, regulatory, structural, signal transduction and other metabolic
processes. In addition to the orthophosphate anion, other plant P-integrating
molecules include nucleic acids and ADP/ATP, indispensable for photosynthesis,

J.-M. Barea ()

Soil Microbiology and Symbiotic Systems Department, Estación Experimental del Zaidín,
CSIC, Prof. Albareda 1, 18008 Granada, Spain
Tel.: + 34686404966
e-mail: josemiguel.barea@eez.csic.es
A. E. Richardson
CSIRO Plant Industry, PO Box 1600, Canberra 2601, Australia
Tel.: + 61 2 6246 5189
e-mail: alan.richardson@csiro.au

© Springer International Publishing Switzerland 2015 225

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_24
226 J.-M. Barea and A. E. Richardson

respiration and other biochemical processes involved in energy storage and trans-
fer reactions. Plant P also occurs in storage compounds such as phytate and related
compounds, pyrophosphate and as a component of membrane phospholipids and
phosphoproteins (White and Hammond 2008).
Forms of Phosphorus in Soil Soil P occurs as either inorganic phosphates or or-
ganic phosphate derivatives. The primary mineral form of P in soil is apatite. The
weathering of apatite results in the release of orthophosphate anions, primarily as
HPO2− 1−
4 and H2 PO4 to soil solution, but only in small quantities. Soil solution or-
thophosphate content typically ranges from 0.1 to 1 mg P kg−1 which represents
about 1 % of the total soil P. Most orthophosphate in soil undergoes reactions which
makes it only sparingly available to plants. Orthophosphate is rapidly adsorbed on
clay mineral surfaces and other soil particles and colloids or precipitated as inor-
ganic salts (e.g., with calcium in alkaline soils or with aluminum and iron in acidic
soils), which are of low solubility. A significant amount of orthophosphate is also
integrated in complex organic molecules (soil organic P), which can account for
30–60 % of the total soil P. Major identifiable fractions of organic P in soil include
inositol phosphates, such as phytate (salts of myo-inositol hexakisphosphate), nu-
cleic acids, phospholipids and phosphonates. Inositol phosphates are considered to
be the dominant form of organic P in many soils. Phosphorus immobilised within
the living soil microbial biomass is also significant, and typically represents about
5 % of the total soil P (Plante 2007; White and Hammond 2008).
The Soil Phosphorus Cycle From a functional point of view the various forms
of P in soil are interconnected and integrated through the so called soil P cycle.
As outlined by Plante (2007) the soil solution P pool is central to the P cycle and
is the primary source of labile orthophosphate for biological uptake by microor-
ganisms and plants. Soil solution P also provides the interconnection between the
biological subsystem (including plant residues, soil microbial P, labile and stable
organic P) and the geochemical subsystem (i.e., primary minerals, secondary min-
erals and adsorbed P, that includes P occluded with soil constituents). Whilst the
availability of orthophosphate in the geochemical subsystem is mediated largely by
physical-chemical reactions such as dissolution, precipitation, sorption-desorption
and oxidation-reduction, these processes are also influenced strongly by biological
activities. Soil microorganisms are able to interact across both subsystems through
either solubilisation of inorganic P or mineralisation of organic P and thus play a key
role in the cycling of soil P. Furthermore, soil microorganisms interact directly with
soil solution P and may thus directly influence the availability of orthophosphate to
plants through mobilisation or, conversely, in the short term, by competition with
plants for available nutrient through P immobilisation.
Availability of Phosphorus for Plant Nutrition Plant roots acquire orthophosphate
from soil solution via their associated volume of soil through either the rhizosphere
(Chap. 3) or mycorrhizosphere (Chap. 25) (Fig. 24.1) However, because of the high
reactivity of P in soil and the rapid uptake of orthophosphate by roots, the concen-
tration of orthophosphate around roots is often low. This low concentration is further
24 Phosphate Mobilisation by Soil Microorganisms 227

Root Rhizosphere Soil

Root hairs

P uptake Diffusion SOIL SOLUTION P

1 µM
10 mM ~0 µM (Pi and Po)

Root exudates Desorption

Rhizosphere ADSORBED P (Pi)
microorganisms
Sugars Solubilization
Organic anions MINERAL P (Pi)
Organic anions
H+ ions
H+ ions
Siderophores Mineralization
Siderophores ORGANIC P (Po)
Phosphatases Phosphatases Solubilization

Mycorrhizal fungi

Mycorrhizosphere

Fig. 24.1 Root-soil microbiome processes governing transformation and availability of phospho-
rus in soil-plant systems highlighting the importance of the rhizosphere and mycorrhizosphere.
(Reproduced from Richardson et al. 2009 by permission of the publisher)

compounded by slow diffusion of orthophosphate anions in solution which results

in a distinct zone of depletion in soil immediately surrounding the root system. The
low rate of replenishment of orthophosphate in the rhizosphere/mycorrhizosphere
soil solution from the bulk soil is therefore a major factor that regulates P availability
to plants (White and Hammond 2008). Consequently, P-based fertilisers are routinely
used in agricultural production systems to either maintain the P status of fertile soils
or to increase P availability in deficient soils. The efficiency of P fertiliser use in
most systems, however, is low, whereby only 10–50 % of applied P is recovered by
crops in the year of application. The remainder of the P accumulates in soil in either
inorganic or organic P fractions and, subject to efficient mobilisation, can provide a
P benefit in subsequent years. Rock phosphates (RP), which are used for the produc-
tion of water soluble P-fertilisers, are also widely used as a direct source of P albeit
with relatively low agronomic efficiency. Development of strategies to increase the
mobilisation of P from accumulated forms in soil or to enhance the utilisation of RP
are thus promoted as being important to increase the efficiency of P fertiliser use. As
microorganisms are a key component of the soil P cycle they are widely considered
as the basis of some of these alternative strategies to improve the sustainability of P
use in agriculture systems (Zapata and Roy 2004).
228 J.-M. Barea and A. E. Richardson

24.2 Microbial Mobilisation of Phosphorus in Soil

Microorganisms are known to drive plant nutrient cycling and many other funda-
mental processes resulting in plant growth promotion (Barea et al. 2007; Lugtenberg
et al. 2013). In particular, specific soil microorganisms (i.e., plant growth promoting
rhizobacteria; PGPR) change the capacity of plants to acquire P from soil solution
via mechanisms that include; (i) modifying soil sorption equilibria to facilitate P
diffusion, (ii) enhancing mobilisation of poorly available sources of P, (iii) increas-
ing the extension of root surface area, (iv) by stimulating root branching and/or
root hair development and (v) altering root surface properties to enhance P uptake
(Richardson et al. 2009). Here we focus on mechanisms under (ii), whereby micro-
bial activities result in increased release of available P from sparingly available forms
of either inorganic (solubilisation) or organic (mineralisation) P in soil. This has par-
ticular relevance from a sustainability point of view because P mobilisation activities
have broad significance in the maintenance and productivity of both agricultural and
natural ecosystems (Richardson 2007).
Phosphate Solubilisation Bacteria and fungi isolated from plant rhizospheres have
been shown to solubilise in vitro various inorganic phosphates, such as calcium,
aluminum or iron salts. These microorganisms are collectively termed “phosphate
solubilising microorganisms” (PSM). They include Bacillus, Enterobacter, Rhi-
zobium, Bradyrhizobium, Enterobacter, Panthoea, Erwinia, and Pseudomonas as
common bacterial genera, and Aspergillus, Trichoderma and Penicillium as fungal
representatives (Marschner 2008). In the case with sparingly soluble forms of cal-
cium phosphates, the mechanism of solublisation is most commonly associated with
proton release and media acidification. For iron or aluminum phosphates, solubilisa-
tion due to acidification appears to be less effective and production of organic acids is
of greater importance. Organic anions are effective in chelation processes that result
in the sequestration of calcium, iron or aluminum which is associated with a release
of orthophosphate to solution. Commonly reported organic anions include citrate,
oxalate, lactate, succinate, gluconate and 2-ketogluconic acid. Siderophore produc-
tion likewise has been reported to be effective for solubilisation of Fe phosphates
(Marschner 2008). The amount of orthophosphate released from sparingly soluble
forms is dependent on the microorganisms involved, culture conditions and the de-
gree of solubility of the P substrate (Whitelaw 2000). Solubilisation of P is further
dependent on the presence of readily metabolisable carbon sources. As such, isolates
selected as being effective for P solubilistion under laboratory conditions may not
be effective in soil due to either carbon limitation or other unfavorable microhabitat
conditions (Richardson 2007).
Molecular-based approaches have recently been used to investigate the mecha-
nisms involved in P solubilisation by specific microorganisms. For example, one
mechanism is based on the ability of Pseudomonas spp. to produce gluconic
acid from glucose by the oxidation reaction catalysed by glucose dehydroge-
nase which uses pyrroloquinoline quinone (PQQ) as a redox cofactor. Finally,
2-ketogluconate is produced which facilitates both the chelation of calcium and
24 Phosphate Mobilisation by Soil Microorganisms 229

release of protons. A genomic library of Pseudomonas spp. has recently been anal-
ysed for PQQ biosynthetic genes to determine their involvement in P solubilisation
(Browne et al. 2013).
Phosphate Mineralisation The mineralisation of organic P in soil and release of
orthophosphate to soil solution is largely mediated by microbial activities (Richard-
son et al. 2009). Bacteria and fungi isolated from plant rhizospheres have been
shown to have capacity to hydrolyse organic P substrates either in vitro or when
added to soil. Common microorganisms include Bacillus and Pseudomonas as bac-
teria and Aspergillus and Penicillium as fungi (Marschner 2008). Mineralisation of
organic P often first requires solublisation of substrates with subsequent hydrolysis
by phosphatase enzymes, which in many cases is synonymous with the activities of
PSM. Microorganisms produce diverse types of enzymes which include non-specific
acid and alkaline phosphates, and specific enzymes, such as phytases which release
orthophosphate from phytate and other inositol phosphates. The importance of mi-
croorganisms for phytate mineralisation has been demonstrated in various studies,
whereby the availability and plant uptake of orthophosphate can be improved by
inoculation with PSM with P mineralisation capability. Nevertheless, the effective-
ness of phytases in many soil environments remains less clear since enzymes may
also readily be absorbed to soil particles or degraded, and inositol phosphates ad-
sorb strongly or precipitate readily with iron or aluminum oxides and other soils
constituents (Marschner 2008). Nonetheless, microbial utilisation of organic P sub-
strates in soil and its turnover has potential to supply a significant amount of P to
meet plant requirements. This is of particular importance in the rhizosphere and
mycorrhizosphere where metabolisable carbon is more available and there is greater
capacity to capture mobilised P. However, further experimental evidence to quantify
microbial mineralisation of P and the direct value of immobilised P in the microbial
biomass to plant nutrition is required (Richardson et al. 2009).
It is important to note that to date much of the work on P solubilisation and min-
eralisation has involved soil microorganisms that have been isolated and grown in
culture media. More recent culture-independent molecular-based studies have shown
that a high percentage (i.e., greater that 90 %, and possibly as high as 99 %) of soil
microorganisms are unculturable, and that this includes therefore likely microorgan-
isms that are involved in phosphate-solubilisation and P cycling (Barret et al. 2013).
As culture-independent approaches are being used to further dissect plant-microbial
interactions it is evident that plants play a significant role in shaping microbial com-
munities in the rhizosphere and mycorrhizosphere. As such there is new opportunity
for linking the structure and function of the root-soil microbiome to orthophosphate
availability and P-solubilising capacity (Browne et al. 2013).
Significance of PSM in Improving Plant Nutrition While it is clear that soil mi-
croorganisms are integral to the operation of the soil P cycle, the extent to which
P released by soil microorganisms actually benefits plant P acquisition remains to
be more fully elucidated. Indeed because orthophosphate release from sparingly
available soil P sources by microbiological-driven activities may be highly tran-
sient in nature, this has implications for its efficacy in promoting plant growth.
230 J.-M. Barea and A. E. Richardson

In addition, mobilised orthophosphates are also subject to further reaction in soil,

for example through either ‘re-fixation’ reactions or immobilistion into microbial
biomass, and may thus not be considered to be immediately available to plants. Spa-
tial interactions may likewise impede the availability of orthophosphate within the
plant rhizosphere and/or mycorrhizosphere and thereby limit any potential agricul-
tural benefit (Richardson 2007). However, if orthophosphate made available by PSB
can be taken up more efficiently by plant roots or through an effective mycorrhizal
mycelium (Chap. 25), then the resultant microbial interaction could synergistically
act to improve P supply to the host plant (Richardson et al. 2009).
Managing PSM to Improve Plant Phosphorus Nutrition Given the potential of
PSM to contribute to the development of more sustainable agricultural systems, a
number of approaches have been proposed for their management and opportunity
for capture of benefits. These are based on either the manipulation of naturally ex-
isting microbial populations, or by the development of microbial inoculants that
contain specific microorganisms with recognised potential for P mobilisation. Man-
agement of naturally existing populations, whilst attractive, is a relatively untargeted
approach, where it is often difficult to predict the response of microbial populations
as a consequence of different agricultural practices (Richardson 2007). For example,
crop rotation and amendment of soils with organic wastes (e.g., manure crops) is
known to enhance P cycling or to increase the biological activity in soils, although
specific effects on either solubilisation or mineralisation processes remain to be more
fully investigated. Likewise, because of its distinct physical, chemical and biological
properties, biochar (produced by pyrolitic transformations of organic materials) is
also being used widely as a soil amendment that may also facilitate the efficacy of
phosphate solubilising or mineralisation activities in soil (Lehmann et al. 2011).
A range of biofertiliser-based products using selected PSM (both bacteria and
fungi) have been developed as commercial products for use in various agricultural
systems across the world. These products, using specific microorganisms either in-
dividually or as part of a microbial consortium, have in many cases shown positive
effects in various field trials (Antoun 2012). However, inconsistent results are com-
monly observed indicating the complexity of interactions in the soil-plant systems,
where diverse ecological variables need to be considered. In some instances it seems
that benefits of plant growth promotion derived from microbial inoculants occurs by
stimulation of root growth, allowing a greater exploration of soil P, rather than by
direct increase in P-mobilisation (Richardson et al. 2009).
Successful development of PSM requires that appropriate inoculant formulations
and delivery systems be developed for specific microorganisms (Antoun 2012). Fur-
thermore, selected microorganisms must be able to maintain their ability to solubilise
P after repeated sub-culturing under laboratory conditions or following re-isolation
from soil, and exhibit sufficient saphrophytic competence for persistence in soil en-
vironments. To be effective in soil it is critical that selected PSM are able to establish
themselves either on the root surface or within in the root-soil habitat. Rhizosphere
competence is a key trait involved in microbial establishment in the rhizosphere
(Chap. 3) and there is need to develop non-disruptive visualisation techniques
24 Phosphate Mobilisation by Soil Microorganisms 231

for assessing microbial colonisation of the rhizosphere and/or mycorrhizosphere

(Barea et al. 2013).
PSM Interactions to Improve Key Rhizosphere Processes Interaction of PSM
with other PGPR has been shown in several cases to be beneficial for plant growth. For
example, PSM have been used in conjunction with N2 -fixing bacteria and mycorrhizal
fungi to improve N2 -fixation by legumes though a greater P supply (Zaidi et al. 2010).
Inoculation of lucerne (alfalfa) with phosphate-solubilising bacteria (PSB) enhanced
both nodulation and N2 -fixation, as estimated by using 15 N and 32 P isotopic methods.
Using the 32 P dilution approach, Barea et al. (2013) showed that inoculation with
PSB increased the mobilisation of sparingly available P from either endogenous soil
P or from P supplied as rock phosphate.

24.3 Mycorrhizosphere Interactions to Improve Plant

Phosphorus Nutrition

The colonisation of roots by mycorrhiza affects diverse aspects of plant physiology

resulting in quantitative and qualitative changes in root structure and composition of
root exudates. This has a significant effect on the compositional structure and func-
tion of microbial communities in the rhizosphere. In addition, mycorrhizal mycelium
directly modifies the physical characteristics of surrounding soil and interacts further
with soil microorganisms. These mycorrhiza-induced interactions, has led to the de-
velopment of the so-called mycorrhizosphere (Barea et al. 2013). Other rhizobacteria
that favor the formation of mycorrhiza on roots have also been recognised (Frey-Klett
et al. 2007), and are commonly referred to as “mycorrhiza-helper-bacteria” (MHB).
Mycorrhizosphere interactions involving PGPR can be managed (mycorrhizo-
sphere tailoring) to benefit plant growth and health, and soil quality (Barea et al.
2013). Interactions between mycorrhizas and PSB in particular are relevant to P cy-
cling and plant P nutrition (Richardson et al. 2009). PSB interactions with arbuscular
mycorrhizal (AM) associations are considered in more detail below, as association
with AM fungi is common with some 80 % of terrestrial plant species being able to
be colonized, including those of agronomic interest (Chap. 25).
Biological and Ecological Basis of Mycorrhizosphere Interactions The extensive
and highly branched external mycelium of AM fungi characteristically increases the
zone of soil exploration and potential for plant nutrient uptake beyond the rhizosphere
(Chap. 25). External AM mycelium are able to extend several cm (up to 25 cm)
from the root and thus provide for the uptake and transport of P in soil that is
independent of the diffusive rate of orthophosphate in soil solution. In addition, 1 cm
of root length can harbour as much as 1 m of fungal hyphae, with densities of up to
40 m of mycelia per gram of mycorrhizosphere soil (Smith and Smith 2012). These
properties ofAM mycelium are especially relevant for acquisition of P from soil as has
been widely demonstrated using compartmented devices and 32 P isotopic labelling
studies. Importantly, orthophosphate made available by PSM has potential to be
taken up and transported more effectively by roots that are colonised with AM fungi.
232 J.-M. Barea and A. E. Richardson

Shoot dry weight Shoot P content Specific activity

e
1000 4 10 a
e a
800 3 8 b
d

Bq mg of P-1
cbc
mg/plant

mg/plant
600 d 6
c c c c b
2 b
400 b b c 4 e
aa b
1 a f
200 2
0 0 0
-M +M -M +M -M +M -M +M -M +M -M +M

-RP + RP -RP + RP -RP + RP

Amount of P derived from

AM colonization Figure captions
different sources 80 f
3 f
e
= Nonbacterized (PSB)
60 d de
2
mg/pot

e = PSB inoculated
%

e c
40
d c +/-M = +/-AM inoculation
1 c b
20 a a RP = Rock Phosphate
a b
PdfL = Plant P derived from the
0 0 labelled (available) P pool
PdfL PdfRP PdfL PdfRP -M +M -M +M
PdfRP= Plant P derived from RP
-M +M -RP + RP

Fig. 24.2 Interactive effects of PSB and AM fungi in enhancing plant growth and phosphorus (P)
uptake from endogenous soil P or P added as rock phosphate. Plants (onions) were grown in a soil
microcosm system which integrated 32 P isotopic dilution approaches in an agricultural soil with
indigenous microbiota either with or without inoculation. Plants inoculated with both PSB and AM
fungi produced greater biomass, accumulated more shoot P and had a lower 32 P specific activity
than non-inoculated or single-inoculated plants, thus indicating greater access to poorly-available
sources of P. Up to 75 % of the P in dual-inoculated plants was derived from added RP and it was
evident that inoculated PSB behaved as a mycorrhiza helper bacteria by promoting establishment
of both indigenous and inoculated AM fungi. (Reproduced from Toro et al. 1997 by permission of
the publisher)

Agronomical Application of PSB x AM Fungal Interactions The feasibility of

capturing greater benefit for plant P nutrition through synergistic interactions be-
tween PSB and AM fungi has received wide interest since the pioneering work of
Azcón et al. (1976). For example, various co-inoculation studies have shown sig-
nificant increase in the mobilisation and plant uptake of P from sparingly available
sources either directly from soil or when added as RP (Barea et al. 2013). In some
studies the inoculated PSB behaved as MHB by promoting establishment of both
indigenous and inoculated AM fungi (Fig. 24.2). Whilst consistent results have been
reported for glasshouse studies, more variable response occurs under field conditions.
Interestingly, in the study by Barea et al. (2007) the agronomic efficiency of P uptake
by plants from RP was increased with dual inoculation, and was greatest when an
organic matter amendment was applied. Whilst organic matter appeared to enhance
the solubilisation activities of PSM, the underlying mechanisms involved and rela-
tive contribution of the various microbial partners in such interactions remains to be
more fully investigated.
24 Phosphate Mobilisation by Soil Microorganisms 233

The Use of Isotopic Techniques to Assess Mycorrhizosphere Interactions Iso-

topic (32 P and 33 P) dilution approaches have commonly been used to investigate
exchange rates in orthophosphate equilibrium between solution and solid phases of
soil and to measure the availability of P from fertiliser sources (including RP) as in-
fluenced by management practices (Zapata and Roy 2004). Accordingly, 32 P-tracer
methodologies have been used to determine the contribution of AM fungi and PSB
to plant P uptake from different source of P (Toro et al. 1997). These techniques typ-
ically involve labelling of the exchangeable pool of soil P with 32 P-orthophosphate.
Plants subject to different inoculation treatments are then grown in the soil amended
either with or without RP. Difference in isotopic composition, or “specific activity”
(SA = 32 P/31 P quotient) in plant tissues can then be used to assess P uptake, whereby
lower SA of the plant (relative to control plants) is indicative of greater access to
otherwise sparingly available forms of P (Toro et al. 1997).
Results from several isotopic-labeling experiments using different plant species
(mainly legumes) have shown increased biomass and P content of plants co-
inoculated with AM and PSB, along with lower SA of shoot tissues compared with
non-inoculated or singularly-inoculated plants (Azcón and Barea 2010). This sug-
gests that PSB are effective in releasing orthophosphate anions from soil or RP
sources and, that in the presence of AM fungi, the P is more available for plant
uptake (Fig. 24.2). By using isotope dilution concepts (Zapata and Roy 2004) the
relative contribution of the different P sources to plant P content (i.e., from added
RP) can also be determined. As such, it is evident that co-inoculated plants generally
have significantly greater access to P from RP, while plants inoculated with PSB
only showed greater reliance on the exchangeable soil P pool (Barea et al. 2007).
Collectively, plants inoculated with both AM fungi and PSM therefore appear to be
more P efficient compared to those that were non-inoculated or singly-inoculated. In
conclusion this demonstrates that opportunity exists to gain benefit in plant P nutri-
tion from interactive effects of PSB and AM-fungi through tailored management of
the mycorrhizosphere (Azcón and Barea 2010).

References

Antoun H (2012) Beneficial microorganisms for the sustainable use of phosphates in agriculture.
Procedia Eng 46:62–67
Azcón R, Barea JM (2010) Mycorrhizosphere interactions for legume improvement. In: Khan MS,
Zaidi A, Musarrat J (eds) Microbes for legume improvement. Springer, Vienna, pp 237–271
Azcón R, Barea JM, Hayman D (1976) Utilization of rock phosphate in alkaline soils by plant
inoculated with mycorrhizal fungi and phosphate-solubilizing bacteria. Soil Biol Biochem
8:135–138
Barea JM, Toro M, Azcón R (2007) The use of 32 P isotopic dilution techniques to evaluate the
interactive effects of phosphate-solubilizing bacteria and mycorrhizal fungi at increasing plant
P availability. In: Velázquez E, Rodríguez-Barrueco C (eds) First international meeting on
microbial phosphate solubilization. Series: developments in plant and soil sciences. Springer,
Dordrecht, pp 223–227
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Barea JM, Pozo MJ, Azcón R et al (2013) Microbial interactions in the rhizosphere. In: de Bruijn
F (ed) Molecular microbial ecology of the rhizosphere. Wiley, Hoboken, pp 29–44
Barret M, Tan H, Egan F et al (2013) Exploiting new systems-based strategies to elucidate plant-
bacterial interactions in the rhizosphere. In: de Bruijn F (ed) Molecular microbial ecology of
the rhizosphere. Wiley, Hoboken, pp 57–68
Browne P, Barret M, Morrissey JP et al (2013) Molecular-based strategies to exploit the inorganic
phosphate-solubilization ability of Pseudomonas in sustainable agriculture. In: de Bruijn F (ed)
Molecular microbial ecology of the rhizosphere. Wiley, Hoboken, pp 615–628
Frey-Klett P, Garbaye J, Tarkka M (2007) The mycorrhiza helper bacteria revisited. New Phytol
176:22–36
Lehmann J, Rillig MC, Thies J et al (2011) Biochar effects on soil biota—a review. Soil Biol
Biochem 43:1812–1836
Lugtenberg BJJ, Malfanova N, Kamilova F et al (2013) Plant growth promotion by microbes. In:
de Bruijn FJ (ed) Molecular microbial ecology of the rhizosphere. Wiley, Hoboken, pp 561–573
Marschner P (2008) The role of rhizosphere microorganisms in relation to P uptake by plants. In:
White PJ, Hammond J (eds) The ecophysiology of plant-phosphorus interactions series: plant
ecophysiology, vol 7. Springer, Dordrecht, pp 165–176
Plante AF (2007) Soil biogeochemical cycling of inorganic nutrients and metals. In: Paul EA (ed)
Soil microbiology, ecology, and biochemistry. Elsevier, Oxford, pp 389–432
Richardson AE (2007) Making microorganisms mobilize soil phosphorus. In: Velázquez E,
Rodríguez-Barrueco C (eds) First international meeting on microbial phosphate solubilization.
Developments in plant and soil sciences, vol 102. Springer, Netherlands, pp 85–90
Richardson AE, Barea JM, McNeill AM et al (2009) Acquisition of phosphorus and nitrogen in the
rhizosphere and plant growth promotion by microorganisms. Plant Soil 321:305–339
Smith SE, Smith FA (2012) Fresh perspectives on the roles of arbuscular mycorrhizal fungi in plant
nutrition and growth. Mycologia 104:1–13
Toro M, Azcón R, Barea JM (1997) Improvement of arbuscular mycorrhizal development by in-
oculation with phosphate-solubilizing rhizobacteria to improve rock phosphate bioavailability
(32 P) and nutrient cycling. Appl Environ Microbiol 63:4408–4412
White PJ, Hammond JP (2008) Phosphorus nutrition of terrestrial plants. In: White PJ, Hammond JP
(eds) The ecophysiology of plant-phosphorus interactions. Plant ecophysiology, vol 7. Springer,
Netherlands, pp 51–81
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Zaidi A, Ahemad M, Oves M et al (2010) Role of phosphate-solubilizing bacteria in legume im-
provement. In: Khan M, Zaidi A, Musarrat J (eds) Microbes for legume improvement. Springer,
Vienna, pp 273–292
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trition, Bulletin 32. Food and Agriculture Organization of the United Nations and International
Atomic Energy Agency, Rome, pp 148
Chapter 25
Arbuscular Mycorrhizas: The Lives of Beneficial
Fungi and Their Plant Host

Paola Bonfante and Alessandro Desirò

Abstract When plants colonized the land 450 million years ago, they were already
associated to soil fungi, which assisted them in facilitating the uptake of mineral
nutrients. This symbiotic association is known as mycorrhiza, a word that covers
all the symbioses established between plants and beneficial fungi. Their presence in
most environments suggests that evolution has promoted mycorrhizas because of the
benefits gained by both partners. The improved nutrient status has a positive impact
on the overall plant physiology, as it influences growth, water absorption and protec-
tion from root diseases. Mycorrhizal fungi instead acquire organic carbon directly
from their green hosts, and accomplish their life cycle. These features are considered
as landmarks of mutualistic symbioses. Owing to the huge diversity of plant and
fungal taxa involved, and the multiplicity of the resulting interactions, mycorrhizas
are usually classified in two broad categories, known as ecto- and endomycorrhizas,
depending on whether the fungus colonizes the root’s intercellular spaces or develops
inside the plant cells. The aim of this chapter is to focus on arbuscular endomycor-
rhizal symbiosis, which is the most ancient and common plant-fungal association,
and to provide an overview that updates traditional knowledge with recent data.

25.1 Arbuscular Mycorrhiza: At the Root of Endosymbioses

Of all the amazing diversity hidden inside the mycorrhizal world, Arbuscular Myc-
orrhizal (AM) symbiosis is the most widespread type, as it occurs in more than 80 %
of land plants, and involves, as symbiotic fungi, the Glomeromycota, an ancient

P. Bonfante () · A. Desirò

Department of Life Sciences and Systems Biology, University of Torino,
Viale Mattioli 25, 10125 Torino, Italy
Tel.: + 39 011 6705965
e-mail: paola.bonfante@unito.it
A. Desirò
Tel.: + 39 011 6705775
e-mail: alessandro.desiro@unito.it
© Springer International Publishing Switzerland 2015 235
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_25
236 P. Bonfante and A. Desirò

sp

fc

sp
sp
a b

de

b ch
ar

ar

fc ve
rh
cw
c d

pn



ih cc
fn

rc 
hr 


fn
e f 

Fig. 25.1 AM symbiosis main features. a Cluster of asexual spores (sp) of Rhizophagus intraradices
surrounded by extraradical hyphae (arrow head) which represent the extraradical AMF structures. b
A squashed spore (sp) of Gigaspora margarita reveals its multinucleated nature. The nuclei, green
25 Arbuscular Mycorrhizas: The Lives of Beneficial Fungi and Their Plant Host 237

phylum that has coevolved with plants for at least 450 million years. Arbuscular
mycorrhizas (AMs) contribute to the uptake of soil nutrients in plants, thus increasing
their productivity and conferring resistance to stress. At the same time, as obligate
biotrophs, AM fungi (AMF) cannot grow in pure culture, as they are unculturable
in the absence of their host: they depend on the plant for their viability and in
particular for carbohydrates. Their uniqueness is also due to other biological traits:
the concept of species is poorly defined in this fungal group and reflects a high degree
of genetic and functional variability; this situation has also led to difficulties in the
assignment of a phylogenetical position. On the basis of the genome of Rhizophagus
irregularis, a ubiquitous fungus which was the first AMF to be sequenced (Tisserant
et al. 2013; Lin et al. 2014), we can state that Glomeromycota are phylogenetically
closer to Mucoromycotina, a basal fungal group, than to Asco-and Basidiomycota
(Schüβler et al. 2001). A particular feature is their multinucleated status: spores and
syncytial hyphae contain thousands of nuclei (Fig. 25.1a, 25.1b), and this makes
classical genetic approaches unsuitable and opens questions on whether nuclei are
genetically divergent. The genome sequencing of individual nuclei has revealed a
consistent uniformity, thus pointing to the homokaryotic nature of R. irregularis
(Lin et al. 2014). AMF are considered to be asexual, although genetically distinct
strains anastomose with each other exchanging genetic material, and many mating-
type related genes have been described. Finally, many AMF contain endobacteria in
their cytoplasm, and this leads to an unexpected increase in their genetic complexity
(Fig. 25.1c), even if the role of such novel fungal microbiota requires to be better
investigated.
On the other hand, the availability of genetic tools and genomic information for
several host plants, has shed light on the multiple aspects of plant-fungal interactions,
including the process of root colonization, communication between the symbionts
and the contribution of each partner to the functioning of the association (Gutjahr
and Parniske 2013) .

←
Fig. 25.1 (continued) spots (arrow head) within the fungal cytoplasm (fc), were stained using
SYTO 9 fluorescent dye. c AMF spores and mycelia may often contain endobacteria. The
transmission electron micrograph shows a rod-shaped bacterium (b) embedded in the fungal
cytoplasm (fc) which has been named Candidatus Glomeribacter gigasporarum. d AMF colonize
not only roots, but also thalli of basal plants, like the liverwort Conocephalum conicum here
illustrated. Fungal structures are labelled (in green) with an AMF-specific probe. Ventral (e)
and dorsal (de) epidermis, and chlorenchyma (ch) are never colonized. In the inset, a detail of
arbuscules (ar) in the liverwort parenchyma cells. Rhizoids (rh), liverwort cell wall (cw). e A root
of a grapevine colonized by Funneliformis mosseae which produces branched arbuscules (arrow
head) in the cortical cells (cc). Hairy root (hr), rhizodermal cell (rc), intracellular hypha (ih). f
Transmission electron micrograph of an arbusculated cell of Lotus japonicus. The details of the
plant-fungal interface are shown: a membrane of plant origin (arrow head) surrounds the fungal
branches (fb). This area is considered the main site for nutrient exchange between the two partners.
Plant nucleus (pn), fungal nucleus (fn). Scale bars: a 400 μm; b 18 μm; c 0,2 μm; d 90 μm (10
μm in the inset); e 150 μm; f 1,5 μm
238 P. Bonfante and A. Desirò

25.2 AM Fungi are an Important Component

of the Plant Microbiota

Thousands of microbes are associated with plant roots, forming the so-called root
microbiota (Chap. 3; Chap. 43). Among these, AMF assume a prominent position,
as they play an essential role in ecosystem functioning: they influence organism
interactions and provide a range of benefits to the host plant which, in return, sup-
plies carbon to the fungus. AMF are important determinants of plant biodiversity,
ecosystem variability and productivity: it has been demonstrated that an increase in
the number of AMF species corresponds to an increase in aboveground biodiversity
and productivity (van der Heijden et al. 2008). Hence, given their potential bene-
ficial effects, it is essential to understand the factors that control the assembly, the
distribution and the dynamics of AMF in order to identify the main drivers of the
microbial communities in natural and agricultural ecosystems, and to monitor and
maximize their ecosystem functions.
The occurrence of AMF in plants from most parts of the world has been studied
since 1844 when the Tulasne brothers described the first AMF species Glomus macro-
carpum and G. microcarpum. After about 150 years, AMF were grouped within a
monophyletic phylum, the Glomeromycota (Schüβler et al. 2001), which is distinct
from the Zygomycota where they had previously been placed. The phylum Glom-
eromycota is currently represented by about 250 described species. Before the advent
of molecular techniques, AMF identification was based solely on the microscopic
examinations of their spores. However, spores are simple structures that offer a rather
limited number of features to help discriminate among taxa and, in some cases, one
species might easily be mistaken for another. In the nineties, the development of
PCR-based approaches led to novel identification rules for AMF: since then, their
taxonomy has been the subject of several changes and now, more than ever, it is ex-
tensively debated and controversial. New AMF species are frequently described, and
their taxonomy is reorganized at different hierarchical levels (Redecker et al. 2013):
until 2001, AMF were distributed in just 1 class, 1 order, 3 families, and 6 genera;
through the use of molecular phylogeny, they were later assigned to 1–3 classes, 4
to 5 orders, 11–14 families, and to 25–29 genera, depending on the scheme of the
adopted classification. To make matters more complex, the use of DNA sequencing
methods has allowed Glomeromycota to be directly detected from environmental
samples (i.e. soil and plant root), thus the number of retrieved fungal phylotypes
has increased. Many sequences assigned to the “uncultured” or “environmental” cat-
egory have been uploaded in gene banks; however, the AMF phylotypes retrieved
from plant roots exceed the known described AMF species, suggesting that the diver-
sity of these fungi is still underestimated (Öpik et al 2013). Metagenetic approaches
based on new technologies (see Chap. 30), such as high-throughput pyrosequencing
of DNA amplicons (i.e. a fragment of 18S rRNA gene) are excellent tools to in-
crease our knowledge about diversity and distribution the AMF species. Studies on
environmental samples have been carried out in temperate, boreal, tropical African
25 Arbuscular Mycorrhizas: The Lives of Beneficial Fungi and Their Plant Host 239

and American woodlands and in grasslands as well as in natural ecosystems in Eu-

rope and North America. However, Asia, South America, Africa and Oceania are
still poorly studied: a higher molecular diversity of AMF from these climatic and
geographic zones can be expected (Öpik et al. 2013).

25.3 The Colonization Process

AMF establish symbiotic associations with almost 80 % of plants, including basal

plants, such as liverworts and hornworts, ancient trees like Ginkgo biloba, most
herbaceous plants and the cultivated crops that feed the world. Owing to this diver-
sity, several plant tissue colonization patterns have been described that mirror the
root anatomy of the involved host, but in spite of this, many features are shared
(Fig. 25.1d). The propagules of AMF are asexual spores which germinate inde-
pendently of the host plant to develop an asymbiotic mycelium (Fig. 25.1a), which
cannot grow for more than a few days without the host root, due to their obligate
biotrophic nature. When AMF perceive the host exudates, extraradical hyphae start
branching, probably to improve their chances of contacting the root surface. This
hypha-epidermis contact leads to the differentiation of hyphopodia, the swollen fun-
gal structures that adhere to the plant cell wall and produce the first intraradical
structures, i.e. penetration hyphae. Host cell integrity is maintained by the invagina-
tion of its plasma membrane, which proliferates and engulfs the developing hypha,
physically separating the fungus from the plant cytoplasm. Colonization is often
more massive in the cortical tissues, where hyphae spread the infection. Their final
target is the inner cortical cells, where hyphae penetrate and branch repeatedly in
order to differentiate into arbuscules, the small fungal trees that give their name to
this form of mycorrhizas and which represent the main site of nutrient exchange
(Fig. 25.2).
In conclusion, the colonization process involves a host-independent and time-
limited presymbiotic phase, and a symbiotic phase where extra- and intra-root fungal
structures are developed. Root colonization is in fact accompanied by the prolifer-
ation of the extraradical mycelium, which extends beyond the nutrient depletion
zone that surrounds the roots, and provides the structural basis for improving the
ecological fitness of mycorrhizal plants. The description of such a colonization pro-
cess has led to questions concerning the way in which the partners communicate
in the rhizosphere and establish a physical contact at the surface of the roots, and
how the plant accommodates the fungus inside its cells, making the symbiosis func-
tionally active. Replying to these questions has required complementation between
molecular, genetic, biochemical and cell biology approaches.
The Molecular Dialogue The release of soluble signals in the rhizophere is an easy
way of allowing the mycorrhizal partners to be informed of their reciprocal presence,
before physical contact. Plants are known to release a cocktail of molecules that
attract AMF, in combination with chemiotropic responses. Among them, carotenoid-
derived strigolactones (SL) have been well characterized. These molecules which
240 P. Bonfante and A. Desirò

Pi Fungal cell wall

Pi Fungus
Pi Pi Pi
Periarbuscular Plant
Pi space
AMF-mediated
Pi absorption
Spore
Extraradical
Pi Pi
mycelium Periarbuscular
membrane
Nucleus
Cytoplasm

Hyphal
branching Direct Pi
absorption
Strigolactones Fungal Pi
Hyphopodia exudates Pi

PPA Calcium spiking

Arbuscules

Fig. 25.2 The scheme illustrates the different steps of the root colonization process and the main
pathways of the phosphate transfer from the soil to the plant cells. After germination of the resting
spore, the mycelium starts to explore the surrounding soil. The perception of strigolactones, present
in root exudates, induces hyphal branching. In the meantime, plant exudates induce the production
of signalling molecules by the fungus, which activate the symbiosis signalling pathway and trigger
calcium spiking in rhizodermal cells. The contact between the root and the fungus occurs after
the formation of the hyphopodium on the root surface. This induces the aggregation of cytoplasm
and the formation of the prepenetration apparatus (PPA) in the contacted rhizodermal cell and the
underlying outer cortical cell. Thus, the fungus starts the intracellular invasion of the root, following
the route chartered by the PPA from the surface to the inner cortical cells, where hyphae branch to
form arbuscules. Pi is present in the soil at a low concentration. The plant itself directly takes up
the phosphate from the soil, even if this event is limited to the proximity of the root. Differently, the
hyphae of the extraradical mycelium grow over the depletion zone and actively absorb phosphate
from the soil. The phosphate is condensed as polyphosphate granules which move along the hyphae
from the outside to the inside of the root, till the arbuscules. Here (see the arbuscule details) the
phosphate is released from the fungus towards the interface area and actively transferred to the plant
by the AM-inducible plant phosphate transporters which are expressed on the perifungal membrane

were first described as stimulators of seed germination of parasitic plants (Ruyter-

Spira et al. 2013), elicit a branching in AMF and stimulate their metabolism at low
concentrations. In addition, SLs have been shown to be powerful plant hormones,
which are involved in shoot growth regulation by inhibiting the development of
lateral buds. The multiple roles of SLs suggest that they play a basic role in plant
development and that their leakage from the roots into the soil may have become
useful to different root-interacting organisms (symbiotic fungi and parasitic plants) as
they co-evolved with their hosts. Other plant molecules, which have been identified
as cutin monomers, are considered important cues to allow hyphopodium formation
(Oldroyd 2013).
25 Arbuscular Mycorrhizas: The Lives of Beneficial Fungi and Their Plant Host 241

Upon germination, AMF release diffusible signals, which are perceived by host
plants, even in the absence of physical contact. These factors, often referred to as
‘Myc factors’in analogy with the nodulation (NOD) factor of nitrogen fixing rhizobia,
activate a number of plant responses: upregulation of genes involved in signal trans-
duction, stimulation of lateral root development, starch accumulation, and repeated
calcium oscillations in epidermal cells, in analogy with rhizobium-legume symbio-
sis. Bioactive exuded molecules have so far been identified as lipochitoligosaccarides
(Myc-LCOs), which are very similar in structure to the NOD-factor (Maillet et al.
2011), and tetra- and penta-chitooligosaccharides (Genre et al. 2013). These chitin-
related molecules activate a signalling pathway, which depends on the genes that are
required for the establishment of both AM and nodule symbiosis, and for this reason
are known as Common Symbiosis (SYM) genes. Such genes were first discovered
in legumes but were then found to be present in many AM host plants. The proteins
encoded by SYM genes activate a pathway that starts with the perception of micro-
bial signals at the plant plasma membrane, thanks to lysine-motif (LysM) receptor
kinases (Oldroyd 2013). Together with other co-receptors, the LysM proteins trans-
duce the microbial signal to the cytoplasm and then to the nucleus, as suggested by
the nuclear localization of all the downstream elements of the SYM pathway. Here,
the generation of a nuclear calcium spiking is registered, and this offers an easily de-
tectable marker for early symbiotic events. A calcium calmodulin-dependent protein
kinase plays a central role in this cascade, probably decoding calcium oscillations
and regulating a number of downstream genes. The proteins encoded by SYM genes
are also involved in the intracellular fungal accommodation: mutants for SYM genes
are not only defective in the signaling pathway (as testified by the lack of nuclear
calcium spiking), but also in the assembly of the pre-penetration apparatus (PPA)
and the subsequent fungal colonization (Bonfante and Genre 2010) (Fig. 25.2). It has
been demonstrated that when the fungus produces the hyphopodium, the contacted
epidermal cells start to assemble the machinery that is used to build the interface
compartment where the fungus will be hosted. Cytoplasm aggregates at the contact
site, concentrating endoplasmic reticulum membranes, Golgi bodies and secretory
vesicles. Again, intense oscillations of calcium concentrations take place, and require
the presence of SYM genes. Only when the PPA is completed, does the fungus start
growing inside the roots. On this basis we can conclude that the molecular dialogue
between AM partners acts at multiple levels: driving the fungus towards the plant,
regulating its recognition upon physical contact and controlling early colonization
events.
The Genetics and Molecular Basis of Plant-Fungal Interaction The current
knowledge on AMs shows that plant cells actively control the colonization process.
The development of mycorrhiza-defective mutants has resulted to be a powerful tool
in deciphering the genetics basis of such a control. Mutants in SYM genes were
the first to be investigated at the beginning of the twenty-first century: the genetic
dissection of the colonization process became possible through a comparison of the
impact of a specific mutation on nodule and AM formation in legumes (Oldroyd
2013). In recent years, other common genes have been found to be involved in the
242 P. Bonfante and A. Desirò

accommodation of both root microbes. These include Vapyrin, which is essential not
only for arbuscule progression but also for rhizobial infection (Gutjahr and Parniske
2013) and a group of vesicle-associated membrane proteins. The development of the
perifungal membrane which surrounds intracellular hyphae and defines the symbi-
otic interface, is in fact a common feature of all biotrophic interactions (Balestrini
and Bonfante 2014). Other genes are, instead, AM specific and have an impact on
arbuscule morphogenesis and functioning. Among them, the best characterized gene
is the phosphate transporter that is induced by mycorrhization, is expressed at the
periarbuscular membrane and is considered a functional marker for AMs. When si-
lenced there is a block in the branching of the arbuscule which assumes a stunted
morphology (Harrison 2012). Similarly, mutation in two ABC transporters results
in arbuscular defects. Taken together, these findings suggest that the plant proteins
which are essential for arbuscule development are mostly located in the perifungal
membrane, regulate the nutritional exchanges and seem to be independent of the
SYM signalling pathway.
Studies on mutants have been successfully integrated with molecular approaches
based on the description of plant transcriptomic profiles upon AM colonization. The
significant cell reorganization during root colonization is associated with changes
in the transcriptome of AM roots. The pattern of gene expression of different root
cell types during colonization has been investigated through genome-wide transcrip-
tome profiling, combined with quantitative realtime-PCR on several model plants,
including rice and tomato (Salvioli and Bonfante 2013). Thanks to the use of laser
microdissection, the most prominent changes have been identified- as expected- in
the arbusculated cells where hundreds of genes have resulted to be differentially reg-
ulated. Regardless of host and AM fungal identity, a core set of genes are consistently
differentially regulated, leading to the identification of a “mycorrhizal signature”.
This set usually contains some gene classes, such as the nutrient transporters, includ-
ing mycorrhiza-inducible phosphate, nitrogen and sulphate transporters, which are
considered functional markers of an active AM. Another group of plant genes that are
characteristic of mycorrhizal roots is related to membrane and cell wall synthesis:
according to the notion that the fungus is limited by the interface compartment where
cell-wall material is laid down, many cell wall related genes are upregulated, as also
confirmed by immunocitochemical and in situ hybridization observations (Balestrini
and Bonfante 2014) .
Transcriptome analyses have detected the effects of mycorrhization on plant de-
fense mechanisms. Pathogenesis related proteins are reported to be activated in AMs
thus confirming that mycorrhizal plants seem to react more promptly to pathogen
attack, eventually showing enhanced resistance. The underlying mechanisms are not
fully understood, since many events probably overlap: the fungus is perceived as a
foreign microbe, since it releases elicitor molecules. Chitin is one of the best known
fungal elicitors, and many chitin receptors have been identified as regulators of plant
responses during their interaction with pathogens (Chap. 10). AMF are known to re-
lease a cocktail of chitin-related molecules which might be perceived as detrimental
or beneficial signals. In order to respond to the former, the host develops a higher
defense level, which is overcome by the fungal effectors. It is reasonable to presume
25 Arbuscular Mycorrhizas: The Lives of Beneficial Fungi and Their Plant Host 243

that, at the end, positive signals are dominant, since the plant usually allows AMF
colonization. A current hypothesis is therefore that mycorrhizal establishment stim-
ulates the priming of a plant’s innate immune system rather than the induction of
specific defense mechanisms.
Taken as a whole, a combination of genetics, molecular and morhological
approaches has revealed how the process of fungal colonization and arbuscule devel-
opment can be dissected into phases that require a finely tuned activation of specific
plant genes. Although fungal biology is now starting to be unravelled thanks to the
genome sequencing of R. irregularis, a straightforward way of transforming AMF is
still not available, and—as a result—the functional meaning of a fungal gene/protein
cannot be validated easily. All the current knowledge therefore indicates a dominant
role of the plant on fungal development.

25.4 The Functioning Mycorrhiza

Although AM host plants can survive without their symbiont, this condition is virtu-
ally unknown in natural ecosystems, where AMF function as helper microorganisms,
and improve the overall plant fitness. AMF have so far proved to be unculturable in the
absence of a host. Being unable to absorb carbohydrates, except from inside a plant
cell, they depend totally on their green hosts for the organic carbon metabolism,
which gives them the status of obligate biotrophs. Although carbon transfer from
plants to AMF was demonstrated already in the 1960s, its molecular mechanisms are
still unclear. The genome sequencing of R. irregularis has provided clear evidence
that this AMF at least does not possess genes coding for polysaccharide and plant-
cell wall degradation (Tisserant et al. 2013), while a high-affinity monosaccharide
transporter MST2 has been shown to play a major role in the uptake of glucose
and xylose during the symbiotic and extraradical phase (Helber et al. 2011). How-
ever, AMF have an excellent capacity to take up all the required minerals from the
soils as well as organic nitrogen products. Their biotrophy therefore seems to be
related to organic carbon, while they may successfully exploit the other soil nutri-
ents, unlike pathogenic biotrophs, that have no access to soil nutrients at all. AMF
were first shown to possess high-affinity inorganic phosphate (Pi ) transporters many
years ago and this provided a breakthrough in the understanding of fungal functions
(Fig. 25.2). Accumulated as polyphosphate, Pi is then rapidly translocated along the
aseptate mycelium to the host plant.
Nitrogen is the other important element taken up by AMF, and the genes that
are involved in the transport of ammonium and amino acids have been identified,
whereas arginine is probably the preferred molecule for long-distance transport to
the host plant. In conclusion, AMF seem to act as an active bridge between the soil
and the plant. However, not all AMF perform equally well in releasing minerals to
the plant: it has been demonstrated using an in vitro system that a reward process
exists thanks to which the fungal partners enforce cooperation by increasing nutrient
transfer to only those roots that provide more carbohydrates. Vice versa, plants can
244 P. Bonfante and A. Desirò

detect, discriminate, and reward the best fungal partners that provide them more
minerals (Kiers et al. 2011). This description which is based on the use of stable
isotope probing to track and quantify both plant and fungal resource allocation to their
reciprocal partners, offers a physiological view of the so-called biological market,
and describes the behavior of mycorrhizal roots at an organismic level. Molecular
approaches have identified the arbusculated cell and, at a sub-cellular level, the
interface area as the compartment in which the nutritional exchanges take place.

25.5 From Root to Food: Conclusions and Perspectives

In addition to the expected effects on roots, mycorrhization also affects gene

expression in shoots, leaves and fruits, and modulates the genes involved in di-
verse metabolic processes, such as photsynthesis, defence, transport and hormonal
metabolism. These systemic effects open new questions on whether mycorrhiza es-
tablishment can improve the nutritional value of specific crops, by enhancing the
content of nutritionally valuable compounds in their edible parts (Salvioli and Bon-
fante 2013). Interestingly, AMF biodiversity itself has been shown to promote plant
productivity, and also to buffer, in some way, the productivity fluctuations that occur
under differing environmental conditions. These latter findings clearly suggest that
the multifunctional ability of AMF to improve plant performance is still far from be-
ing fully understood, and this makes their use in agriculture much more valuable than
as a simple substitute of chemical fertilizers/pesticides (Salvioli and Bonfante 2012) .
Maximizing the beneficial effects of AMF requires a better understanding of
the molecular mechanisms of symbiosis, by on one hand studying the different
responses of plant species and genotypes and on the other hand by developing a deeper
knowledge on the still enigmatic biology of AMF. This could lead to the formulation
of more efficient fungal inoculants that are suitable for specific in field applications:
their availability in the market is thus a urgent need upon scientific validation. As
a prerequisite, a closer link between field research and laboratory studies must be
established, in order to fill the gap that exists between basic knowledge, availability
of novel molecular tools and the systemic effects of AMF in plants of agronomic
relevance.

Acknowledgements Contributions to this review were partly funded by the project MYCOPLANT
(Compagnia di San Paolo and UNITO), MIUR PRIN 2012 and UNITO (60 % 2014).

References

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Genre A, Chabaud M, Balzergue C et al (2013) Short-chain chitin oligomers from arbuscular myc-
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Gutjahr C, Parniske M (2013) Cell and developmental biology of arbuscular mycorrhiza symbiosis.
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in the arbuscular mycorrhizal fungus Glomus sp is crucial for the symbiotic relationship with
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Schüβler A, Schwarzott D, Walker C (2001) A new fungal phylum, the Glomeromycota: phylogeny
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Chapter 26
Plant Hormones Produced by Microbes

Stijn Spaepen

Abstract Plant hormones or phytohormones are historically classified into five ma-
jor classes: auxins, cytokinins, gibberellins, abscisic acid and ethylene. Nowadays,
many other phytohormones have been identified. Diverse microbial species possess
the ability to produce phytohormones, with most data accumulated for the production
and role of auxin. In this chapter the microbial biosynthesis, its regulation and the
role of the different phytohormones in the interaction with the plant are discussed.
Microbial phytohormonal production is a potent mechanism to alter plant physiol-
ogy, leading to diverse outcomes from pathogenesis to promotion of plant growth.
However, genetic evidence for the role of many phytohormones in microbe-plant
interactions is still lacking, thus questioning the importance of the microbial produc-
tion. Targeted approaches focusing on genetic evidence for the role of phytohormones
together with plant experiments in an agronomic setting will allow unraveling the
importance and potential of this fascinating microbial trait.

26.1 Phytohormones in Plants

Hormones are defined as chemical compounds that are produced in small amounts
in a certain tissue controlling and regulating various functions related to growth,
metabolism and reproduction in the receptive tissue. Plants produce different hor-
mones, also called phytohormones, but the structures of these hormones are—in
contrast to those of animals -rather simple and the molecules are not produced and
stored in specific glands.
The five classical phytohormone classes are auxins, cytokinins, gibberellins,
abscisic acid and ethylene. More recently discovered phytohormones include strigo-
lactones, brassinosteroids, jasmonate, salicylic acid, polyamines and nitric oxide.

S. Spaepen ()
Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20,
Box 2460, 3001 Heverlee, Belgium
Tel.: + 32 16321631
e-mail: stijn.spaepen@biw.kuleuven.be; spaepen@mpipz.mpg.de
Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research,
Carl-von-Linné-Weg 10, 50829 Köln, Germany
Tel.: + 32 16321631
© Springer International Publishing Switzerland 2015 247
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_26
248 S. Spaepen

The physiological functions of these hormones have been studied in detail in the past
decades but only recently the molecular mechanisms of how these hormones exert
their effects have been unraveled. (Davies 2010; Santner et al. 2009).
Most phytohormones exert their activity at very low concentrations and changes
in hormone concentrations can drastically alter plant growth and development both
in a positive and negative way as illustrated by the dose-response curve for auxins.
Additionally, most phytohormones do not act alone on a certain growth or devel-
opmental aspect and therefore balances between different hormonal classes can be
important. Another overlooked aspect of phytohormonal action is the conjugation of
the active hormone to other molecules such as sugars and amino acids, (reversibly)
inactivating the hormone.

26.2 Phytohormones in Microbes

Phytohormones have also been detected and identified in the supernatant of culture
medium of many soil and plant-associated bacteria and fungi. In these organisms,
the phytohormones do not induce typical hormonal or major physiological changes.
Microbial phytohormone production has been linked to changes in root architecture
and plant growth promotion. However, the degree of proof for their involvement
can vary a lot depending on the phytohormone and the studied microbial strain.
The presence of a certain phytohormone in the supernatant of a microbial culture
is not enough to prove a functional role of this molecule in its interaction with the
plant. Further evidence can be the correlation of growth responses of plants with
the hormone levels measured in the culture medium or on/in the colonized plant
tissues. The ultimate proof is the inoculation with a bacterial mutant strain, impaired
in the phytohormonal biosynthesis, to directly demonstrate the involvement of the
phytohormone. The five main classes of phytohormones can be identified in the
culture medium of many microbes (see Table 26.1 for an overview of the discussed
phytohormones) but the subsequent analyses necessary for proving their role in plant
growth promotion are lacking for many described cases.

Auxins

This group of phytohormones has the ability to induce cell elongation in the subapical
region of the stem. Besides this ability, auxins are involved in almost all aspects of
plant growth and development such as stem and root elongation, stimulation of cell
division, lateral and adventitious root initiation, apical dominance, vascular tissue
differentiation, gravitropism and phototropism (Davies 2010). The most important
naturally occurring auxin is indole-3-acetic acid (IAA). Other molecules such as
indole-3-butyric acid and phenylacetic acid are also considered active auxins but
their biosynthetic pathways and roles have not been extensively studied.
26 Plant Hormones Produced by Microbes 249

Table 26.1 Most important phytohormones, produced by microbes

Class Structure 1 Effect on plant

Cell elongation and division
Auxin Root initiation
Apical dominance

Inhibition of root elongation

Stimulation of cell division
Cytokinin
Leaf expansion by cell enlargement
Delay of senescence

Seed germination
Gibberellin Stem elongation
Floral induction and fruit growth

Stomatal closure
Inhibition of shoot growth
Abscisic acid
Bud dormancy
Abiotic and biotic stresses
Stress and ripening hormone
Ethylene Senescence and abscission
Abiotic and biotic stresses
1
One representative is shown if multiple compounds belong to one class. Auxin,
indole-3-acetic acid; cytokinin, zeatin; gibberellin, GA3 or gibberellic acid.

IAA Biosynthetic Pathways in Microbes Until now, six biosynthetic pathways

have been described in microbes, with most pathways being suggested based on the
presence of metabolic intermediates in the culture medium (see Spaepen et al. 2007
for details). For many pathways no genetic evidence is available and therefore the
presence and importance of these pathways needs to be uncovered. Most pathways
use the aromatic amino acid tryptophan as precursor. Despite the multitude of
pathways, there are apparently two dominant microbial pathways based on both
the abundance and genetic evidence for these pathways: one via the intermediate
indole-3-acetamide (IAM) and one via the intermediate indole-3-pyruvate (IPyA)
(see Fig. 26.1). In the IAM pathway, tryptophan is first converted by a tryptophan
monooxygenase to IAM, which is then catalyzed to IAA by an IAM hydrolase. The
pathway has been well characterized in many phytopathogenic bacteria and also in
some rhizobia. In the IPyA pathway abundant in beneficial plant-associated bacteria,
tryptophan is in a first step transaminated to IPyA by an aromatic aminotransferase.
In the second, rate-limiting step, IPyA is converted to indole-3-acetaldehyde
(IAAld) by a decarboxylation reaction catalyzed by an IPyA decarboxylase (IPDC,
encoded by the ipdC gene). Finally, IAAld is converted into IAA. In this pathway,
the regulation (see below) and biochemical characterization of the second step
(IpdC protein/ipdC gene) has been intensively studied in multiple bacterial species.
250 S. Spaepen

Trp mono-oxygenase IAM-hydrolase

Indole-3-acetamide

Tryptophan Indole-3-acetic acid

Amino IPDC IAAld

transferase Indole-3-pyruvate Indole-3-acetaldehyde dehydrogenase

Catechol 3-hydroxy-2-oxo-indole-3-acetic acid 2-hydroxy-indole-3-acetic acid

Fig. 26.1 Most important microbial IAA biosynthesis and degradation pathways. The dashed arrow
refers to multiple steps. Intermediates underlined with a dashed line refer to the name of the pathway.
IAAld, indole-3-acetaldehyde; IAM, indole-3-acetamide; IPDC, indole-3-pyruvate decarboxylase;
Trp, tryptophan

Besides the two pathways described above, other microbial pathways for IAA
biosynthesis have been proposed, but for most of these pathways genetic evidence
is lacking. In the tryptamine pathway, tryptophan is first decarboxylated by a tryp-
tophan decarboxylase and subsequently catalyzed to IAAld by an amine oxidase.
Similar to plants, a pathway via indole-3-acetonitrile has been suggested, based
on the conversion of indole-3-acetonitrile by nitrilases or nitrile hydratase to IAA
directly or via IAM respectively.
Important to note is that in one single organism multiple IAA biosynthetic path-
ways may be present and active as demonstrated for Pantoea agglomerans which
genome encodes both the IAM as well as the IPyA pathway (see below for details).
As for plants, some storage products and conjugates also exist in microbes but their
role is still unexplored.
Regulation of IAA Biosynthesis Since IAA is costly to produce, mainly due to the
high cost to synthesize tryptophan, IAA biosynthesis in bacteria is tightly regulated.
One exception is found for pathogenic Agrobacterium strains. The IAA biosynthetic
genes are under control of strong (plant-specific) constitutive promoters. Since this
DNA region is transferred to the plant upon infection, the tryptophan pool of the
plant is used to produce massive amounts of IAA causing gall formation in com-
bination with a high level of cytokinins (Jameson 2000). In other phytopathogens
(not transferring DNA to the plant), expression is mostly linked to other virulence
factors such as the type III secretion system (TTSS). In Ralstonia solanacearum,
auxin biosynthesis is reduced in a TTSS mutant, while in Erwinia chrysanthemi (re-
classified as Dickeya dadantii) the expression of TTSS genes is reduced in an IAA
biosynthesis mutant. In P. agglomerans, IAA is a master regulator not only for TTSS
genes, but also for quorum sensing related genes (Spaepen et al. 2007).
26 Plant Hormones Produced by Microbes 251

The regulation of IAA biosynthesis has also been studied in detail for some benefi-
cial plant-associated strains (see for review Spaepen et al. 2007 and Patten et al. 2013).
However, it is difficult to draw a general consensus and framework based on the
available information. In this section, I will focus on some well-documented cases
to highlight important regulatory factors and mechanisms. In general, the addition
of the precursor tryptophan to the culture medium will increase IAA production.
However, this increase is not always correlated with the induction of expression of
IAA biosynthetic genes by tryptophan as illustrated for the effects of tryptophan
in Pseudomonas putida and Azospirillum brasilense (expression induced and not-
induced by tryptophan respectively). A very particular case of regulation is observed
for A. brasilense: IAA itself is a main regulator. The key gene in IAA biosynthesis
is regulated by a positive feed-back regulation. In this way, the expression increases
with the cell density and reaches its maximum at the stationary phase coinciding
with the highest IAA levels. The autoinduction is specific for auxin molecules and
is not observed for auxin conjugates and tryptophan (Vande Broek et al. 2005).
Many environmental factors are involved in the regulation of IAA biosynthesis
and these factors comprise pH, osmotic and matrix stress and nutrient limitations.
Again, the effect of a certain environmental factor can vastly differ from strain to
strain, but most of the observed effects reflect the environmental cues important for
IAA production in that particular strain. Since IAA is important in the interaction
with the plant, certain plant-derived molecules and cues will induce IAA biosynthesis
and gene expression as demonstrated for flavonoids in Rhizobium, leavy gall extract
for Rhodococcus fascians and the plant surface itself for P. agglomerans. In many
gamma-Proteobacteria, IAA biosynthesis is regulated by the alternative sigma factor
RpoS, a master regulator for the response upon stress conditions and starvation
as demonstrated for different Pseudomonas and Pantoea strains. Other regulators
comprise the GacS/GacA two-component system in Pseudomonas chlororophis and
TyrR (regulatory protein for transport and metabolism or aromatic amino acids) in
Enterobacter cloacae.
Effect of Microbial IAA on Plants and Microbes The effects of auxin production
(mostly in combination with aberrant cytokinin production) by phytopathogens on
plants are very pronounced (gall and tumor formation). Inactivation of auxin biosyn-
thesis leads to a reduced or no gall formation directly demonstrating the link between
bacterial IAA and plant disease. In the gall-inducing bacterium P. agglomerans pv.
gypsophilae both the IAM and IPyA pathway are present, allowing to study the role
of both pathways. Inactivation of the IAM pathway leads to a significant reduction in
gall size without compromising colonization capacity while inactivation of the IPyA
pathway leads to no significant decrease in gall size but a reduced epiphytic fitness
as measured by colonization capacity (Manulis et al. 1998).
In beneficial bacteria, IAA production has always been linked to plant growth
promotion since inoculation experiments with these strains result in increased root
and shoot biomass especially under sub-optimal nitrogen levels. For P. putida and
A. brasilense, mutant strains impaired in IAA production have been used to ana-
lyze the importance of bacterial IAA biosynthesis. For A. brasilense, inoculation of
252 S. Spaepen

wheat roots results in typical auxin-like responses (shortening of primary root and
induced number of lateral roots and root hairs) ultimately leading to an increased
root surface allowing better uptake of nutrients. The changes in root architecture are
also translated into higher biomass of the shoot. From an agronomic point of view,
inoculation of these strains would allow reducing the N fertilizer input without com-
promising plant yield. However, from these inoculation studies it is also apparent
that IAA biosynthesis is not the only mechanism responsible for plant growth promo-
tion since the IAA-impaired mutants still exhibit residual promoting capacity. Also
some fungi such as Trichoderma and Fusarium strains promote plant growth by the
microbial auxin production, although for Piriformospora indica auxin biosynthesis
is only necessary for root colonization.
Auxins not only affect plants, but also induce physiological changes along with
altered gene expression in microbes. For Agrobacterium tumefaciens, it was shown
that IAA shuts down vir gene expression, possibly being a cue for the bacterium for
a successful plant transformation. In yeast, IAA induces adhesion and filamentation
mediated by the surface protein FLO11 in aYAP-1 dependent manner. Here it was hy-
pothesized that IAA can occur at plant wounding sites and serves as attractive signal
for yeast. In Escherichia coli, IAA protects cells against adverse stress conditions. In
addition, genes encoding cell envelope components and proteins overcoming stress
conditions are upregulated upon IAA treatment. In beneficial bacteria, IAA seems
to operate as a signal molecule to alter gene expression for presence of the plant en-
vironment. In Rhizobium etli, IAA-regulated genes are involved in flavonoid signal
processing, attachment to the roots and switching off motility. Also in A. brasilense,
IAA is a signal molecule as assessed by whole genome transcriptional analysis. In
presence of IAA, it adapts itself to the interaction with the plant by altering the expres-
sion of genes coding for transport and cell surface proteins, transcription factors and
the type VI secretion machinery (Spaepen and Vanderleyden 2011; Patten et al. 2013).
Auxin Degradation In P. putida, the iac locus is encoding for the enzymes respon-
sible for IAA degradation. In a three-step reaction, IAA is degraded to catechol
which can be further degraded to β-ketoadipate. In addition, a MarR-type repressor
of the iac gene expression was identified which is probably released in the presence
of IAA. Despite this genetic knowledge on IAA degradation, the ecological function
of auxin degradation in plant interactions is unknown, although it has been suggested
that this activity could alter the plant auxin homeostasis for the benefit of the bac-
terium. Alternatively, bacterial auxin degradation may provide a nutritional benefit
(Scott et al. 2013).

Cytokinins, Gibberellins, Abscisic Acid and Ethylene

Biosynthesis and Role of Cytokinins Naturally occurring cytokinins (CK) are

mostly derived from adenine and modified by substitutions at the N6 , including
the respective ribotides, ribosides and glycosides. CKs promote cell division and
26 Plant Hormones Produced by Microbes 253

differentiation in meristematic tissues both in plant roots and shoots. They are also
involved in processes such as senescence delay, organ formation, root and root hair
development and leaf expansion (Davies 2010).
In plants, CK biosynthesis starts with the transfer of an isoprenoid moiety (mostly
dimethylallyl pyrophosphate) to adenosine phosphate catalyzed by adenosine
phosphate-isopentenyltransferase to generate isopentenyl adenosine-5’-phosphate.
The isoprenoid-derived side-chain can further be modified by hydroxylation. The
cytokinin compound is then hydrolyzed to a free base by a (phospho-)ribohydrolase
(Frébort et al. 2011).
The massive production of auxins and cytokinins by phytopathogenic bacteria or
by the transfer of bacterial oncogenes into the infected plant exemplified by Pseu-
domonas savastanoi and A. tumefaciens are a strategy to induce tumor/gall formation
(Jameson 2000). For the pathogen R. fascians, the fas operon, comprising six genes,
is responsible for the biosynthesis of CKs necessary for leafy gall formation (Pertry
et al. 2010).
For these pathogenic interactions, the role of CKs in their interaction with plants
is clearly demonstrated. Also for many non-pathogenic bacteria, CK production has
been demonstrated. Although plant growth-promoting action is claimed for CKs, firm
evidence is lacking due to the absence of mutant strains defective in CK biosynthesis.
However, it is proven that bacterial CKs are perceived by plant CK receptors, pointing
towards the potential of bacterial CKs to influence CK signaling in plants.
Biosynthesis and Role of Gibberellins The class of gibberellins (GAs) is a broad
group of more than 100 compounds that can be classified as tetracyclic diter-
penoid acids, with ent-gibberellane as backbone. GAs are involved in developmental
processes such as cell division and elongation during almost all stages of plant
growth (from seed germination to fruit growth). In addition, the balance with other
phytohormones is important in determining the role of GAs (Davies 2010).
Both in some fungi as bacteria, GA production has been detected and the biosyn-
thetic pathways have been proposed and/or unraveled. In the fungal rice pathogen
Gibberella fujikuroi, which is used to commercially produce GA3 , GA biosynthe-
sis (starting from the precursor GA12 -aldehyde) has independently evolved from
the plant pathways and differs especially at the stage in which the 3 β- and 13-
hydroxylation occurs. In G. fujikuroi, the biosynthetic genes are clustered together
in the genome (Bömke and Tudzynski 2009). In bacteria, genetic evidence for GA
biosynthesis is minor. Operons containing genes encoding for putative enzymes
involved in GA biosynthesis were identified in some Rhizobium and Bradyrhizo-
bium strains. Only a biochemical analysis of diterpene synthases of Bradyrhizobium
japonicum provides some evidence for the role of this operon in GA biosynthesis.
In Azospirillum lipoferum, a detailed GC-MS analysis could pinpoint the different
synthesized GAs, allowing to suggest a putative pathway for biosynthesis (Cassan
et al. 2014).
Some plant-associated microbes produce GAs as assessed by measuring the GA
content of the culture medium. The best documented case for the role in plant growth
promotion is the reversion of the dwarf phenotype of plants induced by GA inhibitors
254 S. Spaepen

by inoculation with GA-producing Azospirillum strains (Lucangeli and Bottini 1997).

Since most of these strains can also release GAs from conjugated inactive forms, it
is unclear whether bacterial GA production directly or plant endogenously released
GAs are responsible for this reversion. In root nodules of legumes infected with
rhizobial strains, a higher amount of GAs is measured in comparison with non-
infected roots. Since the identified blend of GAs in nodules resembles the GAs found
in the culture medium of the rhizobial strains, it was suggested that nodule-derived
GAs originated from the rhizobial strains.
Abscisic Acid Abscisic Acid (ABA) induces stomatal closure and fruit ripening and
inhibits seed germination. In addition it is involved in bud dormance and protective
responses against abiotic stresses such as drought, salt stress and metal toxicity
(Davies 2010).
In plants, the “indirect pathway” is the route for ABA biosynthesis. In short, the
carotenoid lycopene is modified in several enzymatic steps to violaxanthin, which
is cleaved by a dioxygenase to xanthoxin. The latter compound is further converted
to ABA in two enzymatic steps (dehydrogenase/reductase and aldehyde oxidase)
(Nambara and Marion-Poll 2005).
Bacterial production of ABA has been reported for A. brasilense and B. japon-
icum strains, although the biosynthetic pathways are unknown. Since ABA inhibits
cytokinin biosynthesis, bacterial ABA production can interfere with the cytokinin
levels in plants. In addition under stress conditions, the bacterial ABA production
might sustain the internal ABA pool in plants, alleviating the negative effects of the
imposed stress.
Altered Ethylene Levels by Microbes The gaseous phytohormone ethylene is in-
volved in physiological and developmental processes such as seed germination, cell
expansion, senescence and abscission. It is sometimes called the ripening hormone
since it induces fruit ripening (Davies 2010). Ethylene is also involved in the plant
defense responses against pathogens. It can affect the outcome of the jasmonate-
dependent defense responses by acting synergistically with jasmonate on one branch
of the pathway leading to resistance to necrotrophic pathogens. However, ethylene
antagonizes the MYC branch of the jasmonate pathway, leading to higher suscepti-
bility to insect attacks (Pieterse et al. 2012). Ethylene also has a role in abiotic stress
conditions. Elevated ethylene levels upon stress conditions may lead to inhibitory
effects on plant growth.
Microbial ethylene production has been reported but mainly for bacterial
pathogens such as Pseudomonas, Xanthomonas and Erwinia. The ethylene produc-
tion is contributing to the bacterial virulence by inducing hormonal imbalances in
the plant. The suggested bacterial biosynthetic pathways are distinct from the plant
biosynthetic pathway. In the first bacterial pathway, methionine is the precursor,
while in the second pathway ethylene is produced from 2-oxoglutarate (Tsavkelova
et al. 2006). The full understanding of the putative ethylene biosynthetic pathways
and the role of ethylene production in disease are still unclear.
Since ethylene accumulates in plants under stress conditions (e.g. drought
and wounding), strategies to lower ethylene levels might alleviate stress-induced
26 Plant Hormones Produced by Microbes 255

growth retardation. One such strategy is employed by some beneficial bacteria.

In plants, ethylene is produced from methionine. Methionine is converted to 1-
aminocyclopropane-1-carboxylate (ACC) and 5’-deoxy-5’methylthioadenosine via
S-adenosylmethionine (SAM) by the consecutive action of a SAM synthase and ACC
synthase. Finally ACC oxidase converts ACC to ethylene, CO2 and cyanide. Bacteria
that can lower ethylene levels encode for an enzyme ACC deaminase (AcdS) which
can degrade the direct ethylene precursor ACC to α-ketobutyrate and ammonia, de-
creasing plant ethylene biosynthesis. This strategy has intensively been studied and
is discussed in detail in Chap. 27 by BR Glick.

26.3 Concluding Remarks

The microbial production of phytohormones is a potent mechanism allowing mi-

crobes to alter plant physiology. For some phytohormones, the biosynthesis and role
in interaction with the plant are well studied and sustained by genetic evidence as
discussed above. However, for many examples this is not the case questioning the
importance of this phytohormone in microbe-plant interactions. Further experiments
are necessary to prove whether the microbial production is a real effector in the in-
teraction or whether this is a by-product of the microbial metabolism without any
substantial role. Futhermore, plant experiments in an agronomic setting are neces-
sary. Under these conditions, the introduced organisms will need to compete with
the indigenous microflora, a factor that can explain the low reproducibility and high
variability.

Acknowledgements Stijn Spaepen is a recipient of a postdoctoral fellowship grant from Research

Foundation—Flanders (FWO-Vlaanderen). I wish to thank Jos Vanderleyden for fruitful discussions
on this topic.

References

Bömke C, Tudzynski B (2009) Diversity, regulation and evolution of the gibberellin biosynthetic
pathway in fungi compared to plants and bacteria. Phytochemistry 70:1876–1893
Cassan F, Vanderleyden J, Spaepen S (2014) Physiological and agronomical aspects of phyto-
hormone production by model plant-growth promoting rhizobacteria (PGPR) belonging to the
genus Azospirillum. J Plant Growth Regul 33:440–459
Davies PJ (2010) Plant hormones: Biosynthesis, signal transduction, action! Springer, Dordrecht
Frébort I, Kowalska M, Hluska T et al (2011) Evolution of cytokinin biosynthesis and degradation.
J Exp Bot 62:2431–2452
Jameson P (2000) Cytokinins and auxins in plant-pathogens interactions—an overview. Plant
Growth Regul 32:369–380
Lucangeli C, Bottini R (1997) Effects of Azospirillum spp. on endogenous gibberellin content and
growth of maize (Zea mays L.) treated with uniconazole. Symbiosis 23:63–71
256 S. Spaepen

Manulis S, Haviv-Chesner A, Brandl MT et al (1998) Differential involvement of indole −3-

acetic acid biosynthetic pathways in pathogenicity and epiphytic fitness of Erwinia herbicola
pv. gypsophilae. Mol Plant Microbe Interact 11:634–642
Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol
56:165–185
Patten CJ, Blakney AJC, Coulson TJD (2013) Activity, distribution and function of indole −3-acetic
acid biosynthetic pathways in bacteria. Crit Rev Microbiol 39:395–415
Pertry I, Vaclavikova K, Gemrotova M et al (2010) Rhodococcus fascians impacts plant development
through the dynamic Fas-mediated production of a cytokinin mix. Mol Plant Microbe Interac
23:1164–1174
Pieterse CMJ, Van der Does D, Zamioudis C et al (2012) Hormonal modulation of plant immunity.
Annu Rev Cell Develop Biol 28:489–521
Santner A, Calderon-Villalobos LIA, Estelle M (2009) Plant hormones are versatile chemical
regulators of plant growth. Nat Chem Biol 5:301–307
Scott JC, Greenhut IV, Leveau JHJ (2013) Functional characterization of the bacterial iac genes for
degradation of the plant hormone indole-3-acetic acid. J Chem Ecol 39:942–951
Spaepen S, Vanderleyden J (2011) Auxin and plant-microbe interactions. Cold Spring Harb Perspect
Biol 3 pii: a001438
Spaepen S, Vanderleyden J, Remans R (2007) Indole-3-acetic acid in microbial and microorganism-
plant signaling. FEMS Microbiol Rev 31:425–448
Tsavkelova EA, Klimova SY, Cherdyntseva TA et al (2006) Hormones and hormone-like substances
of microorganisms: a review. Appl Biochem Microbiol 42:229–235
Vande Broek A, Gysegom P, Ona O et al (2005) Transcriptional analysis of the Azospirillum
brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence
involved in auxin responsive expression. Mol Plant Microbe Interact 18:311–323
Chapter 27
Stress Control and ACC Deaminase

Bernard R. Glick

Abstract During its lifetime, a plant is subject to a wide range of both biotic and
abiotic stresses that can limit the growth and development of the plant. The one thing
that all of these stresses have in common is that they induce the plant to synthesize
growth-inhibiting stress ethylene. Plants that are treated with plant growth-promoting
bacteria that synthesize the enzyme 1-aminocyclopropane-1-carboxylate (ACC)
deaminase produce lower levels of stress ethylene as a consequence of the con-
sumption of the ethylene precursor ACC by the enzyme. These treated plants
are damaged/inhibited to a significantly lesser extent following a biotic or abiotic
stress than are plants that are not treated with ACC deaminase-containing plant
growth-promoting bacteria.

27.1 Plant Stress

Plants grown in the field are subject to a large number of both biotic and abiotic
stresses. The biotic stresses include infection by fungal and bacterial pathogens as
well as plant viruses, insect predation and nematode infection. Abiotic stresses in-
clude high and low temperature, drought, flooding, high salt concentrations, high
metal concentrations, organic contaminants, mechanical wounding and excessive
levels of radiation (Abeles et al. 1992). Plants respond to various stresses by synthe-
sizing defensive/stress proteins and by modifying their physiology and biochemistry.
The response of most plants to stress includes the synthesis of increased amounts of
the low molecular weight gas ethylene, a plant hormone that acts to turn on the syn-
thesis of other plant genes. Depending on the conditions, ethylene can both alleviate
and exacerbate many of the effects of plant stress. Within a few hours following a
biotic or abiotic stress, there is typically a small peak of ethylene synthesized by the
affected plant (Glick et al. 2007). This ethylene peak consumes the small amount of
1-aminocyclopropane-1-carboxylate (ACC) that is generally present in non-stressed

B. R. Glick ()
Department of Biology, University of Waterloo, 200 University Avenue West,
Waterloo, ON N2L 3G1, ON, Canada
Tel.: 519-888-4567 x 32058
e-mail: glick@uwaterloo.ca

© Springer International Publishing Switzerland 2015 257

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_27
258 B. R. Glick

Fig. 27.1 Schematic view of how biotic and abiotic stresses cause plants to synthesize ethylene
that increases plant senescence, chlorosis and abscission

plants and is believed to activate the synthesis of plant defensive proteins. The first
ethylene peak is followed (several hours to a few days later) by a much larger peak
of ethylene that results from conversion of newly synthesized ACC, and initiates
processes such as senescence, chlorosis and abscission, all of which are inhibitory
to plant growth and development (Fig. 27.1).
To avoid some of the deleterious effects of various stresses, plants may be treated
with plant growth-promoting bacteria (PGPB) that provide the plant with various
means of altering the plant’s metabolism and thereby decreasing the severity of
the stress. In this regard, biocontrol PGPB may act to inhibit the functioning of
various plant pathogenic organisms by: (i) producing antibiotics or pathogen cell
wall degrading enzymes, (ii) out-competing pathogens, (iii) stimulating induced
systemic resistance, (iv) producing pathogen-lysing volatile organic compounds,
(v) synthesizing iron-sequestering siderophores or (vi) by lowering plant ethylene
levels through the action of the enzyme ACC deaminase (Glick 2010, 2014). On
the other hand, to avoid the deleterious effects of abiotic stresses, plants may be
treated with PGPB that: synthesize (i) siderophores and thereby provide the plant
with iron, (ii) trehalose a non-reducing disaccharide that acts as an osmoprotectant,
(iii) indole-3-acetic acid (IAA), a plant hormone that promotes plant growth, (iv)
cytokinin, another plant hormone [Note that much of what is believed to be the role
of bacterially-produced cytokinin is based on the knowledge of what the addition of
this hormone itself does to plants.] or (v) ACC deaminase which lowers plants ACC
concentrations thereby limiting the extent of plant ethylene synthesis (Fig. 27.2).
27 Stress Control and ACC Deaminase 259

Fig. 27.2 Schematic view

of the interrelatedness
of plant stress, ethylene
and IAA. SAM
(S-adenosyl-methionine)
is converted to ACC
(1-aminocyclopropane-1-
carboxylate) by the plant
enzyme ACC synthase and
ACC is converted to ethylene
by the plant enzyme ACC
oxidase. IAA (some of it
produced by the plant and
some by the bacteria) can
both promote plant growth
and turn on the transcription
of ACC synthase. ACC may
be converted to ammonia and
α-ketobutyrate by the
bacterial enzyme ACC
deaminase. Accumulating
ethylene both inhibits auxin
signal transduction and
increases the effects of the
stress. Exogenous biotic or
abiotic stress can increase the
ethylene and/or IAA

27.2 ACC Deaminase and IAA Working Together

The enzyme ACC deaminase, which catalyzes the cleavage of ACC, into ammonia
and α-ketobutyrate (compounds readily metabolized and completely consumed by
most bacteria and most plants), is commonly found in many soil bacteria as well as
in some soil fungi. In a model that describes the role of ACC deaminase in promoting
plant growth (Glick et al. 2007; Glick 2014), it is envisioned that PGPB bind either
to plant seeds or roots, or colonize the interior surfaces of the plant (i.e. they are
endophytes) and, in response to low levels of the amino acid tryptophan, that are
exuded from the plant, the bacteria produce and secrete low levels of IAA (Fig. 27.2).
The IAA produced by the bacteria and subsequently taken up by plant cells will, to-
gether with the endogenous plant-synthesized IAA, either promote plant growth (cell
260 B. R. Glick

proliferation and cell elongation) or stimulate transcription of plant genes encoding

the enzyme ACC synthase. In the latter instance, bacterial IAA will ultimately act
to stimulate the synthesis of ethylene. In order to facilitate cell elongation, IAA acts
to loosen plant cell walls with the result that root exudation is increased providing
additional nutrients to rhizosphere bacteria. In addition to other small molecules such
as sugars, amino acids and organic acids, plants exude some ACC that is taken up by
rhizosphere bacteria (endophytic bacteria can also do this). If these bacteria contain
ACC deaminase, the exuded ACC will be cleaved and metabolized with the net result
that the bacteria are effectively acting as a sink for ACC. The consequence of the
functioning of bacterial ACC deaminase is that plant ACC levels are decreased so
that the plant cannot produce as much ethylene as it otherwise might (had the ACC
not been cleaved). A plant’s physiology changes as a consequence of a lower level
of ethylene; thus, plants associated with ACC deaminase-containing PGPB typically
have longer and more extensive roots and shoots, a lower amount of leaf abscission,
increased dry weight, and increased chlorophyll and protein content.
Because the plant enzyme ACC oxidase has a much greater affinity (i.e. a lower
Km ) for ACC than does bacterial ACC deaminase, when ACC deaminase-producing
bacteria are present, plant ethylene levels will be dependent upon the ratio of the
amount of ACC oxidase to the amount of ACC deaminase. To effectively reduce
plant ethylene levels, ACC deaminase must function before any significant amount
of ACC oxidase is induced (by either a particular stress or attainment of a specific
developmental phase). Bacterial ACC deaminase is present at a low, basal, level and
is induced by the increasing amounts of ACC that ensue from the induction of ACC
synthase in the plant following a stress (Glick 1995).
Since IAA activates the transcription of ACC synthase and > 85 % of rhizosphere
bacteria synthesize IAA, according to the model that has been described so far,
these bacteria should all eventually produce relatively high concentrations of ACC
within plant cells. The ACC should subsequently be utilized to synthesize plant
inhibitory levels of ethylene. However, the fact that not all IAA-producing bacteria
are inhibitory to plant growth indicates that the model that has been described so
far is incomplete. In this regard, microarray experiments that examined the effects
of wild-type and ACC deaminase minus mutants of PGPB on plants indicated that
with the mutant but not the wild-type bacteria, as plant ethylene levels increased, the
ethylene that is produced feedback inhibits IAA signal transduction thereby limiting
the extent that IAA can activate ACC synthase transcription (Stearns et al. 2012).
However, with plant growth-promoting bacteria that both secrete IAA and synthesize
ACC deaminase, plant ethylene levels do not become elevated to the same extent
as when plants interact with bacteria that secrete IAA but do not synthesize ACC
deaminase. In the presence of ACC deaminase, ethylene levels in the plant are lower
as is the subsequent ethylene feedback inhibition of IAA signal transduction. Thus,
in the presence of ACC deaminase the bacterial IAA can continue to both promote
plant growth and increase ACC synthase transcription with only a small amount
of ethylene feedback inhibition of this pathway. The net result of this synergism
between IAA and ACC deaminase is that by lowering plant ethylene levels, ACC
deaminase permits IAA to (do its job to) stimulate plant growth.
27 Stress Control and ACC Deaminase 261

27.3 Stress Control in Action

A number of different chemical compounds can either lower ethylene levels in plants
or alter a plant’s sensitivity to ethylene. However, most of these chemicals are either
harmful to the environment, expensive or limited in their potential use. However,
if the model shown schematically in Fig. 27.2 has any validity, ACC deaminase-
containing bacteria should effectively decrease the deleterious effects that result
as a consequence of different stresses, and the ability of these bacteria to syn-
thesize IAA should, at the same time, stimulate plant growth. Of course, not all
plants are equally sensitive to ethylene and different plant growth-promoting bacteria
synthesize different amounts of IAA.
When plant roots are stressed because they are exposed to decreased oxygen
concentrations as a result of flooding, ACC synthase is induced and relatively large
amounts of ACC are synthesized. However, the conversion of ACC to ethylene cannot
occur in oxygen-deprived roots so the accumulated ACC is transported through the
plant to the aerobic environment of the shoots where ACC is converted to ethylene
by ACC oxidase. The ethylene synthesis causes wilting, leaf chlorosis and necrosis,
and a significant loss of biomass. However, plants that are first treated with ACC
deaminase-containing PGPB suffer a significantly decreased level of damage from
flooding (Grichko and Glick 2001) .
Various plant diseases may reduce plant biomass yields by ∼ 10 % per year in
more developed countries and by > 20 % per year in less developed countries of
the world. A wide variety of infectious organisms can cause plant diseases including
fungi, oomycetes, bacteria, viruses, phytoplasmas, protozoa and nematodes although
fungi and bacteria cause the majority of common plant diseases. Many PGPB can
act as biocontrol agents, utilizing a wide range of strategies to limit the growth and
pathogenicity of fungal and bacterial phytopathogens. Many disease symptoms of
pathogen-infected plants arise as a consequence of the stress imposed by the infection;
a significant part of the damage to pathogen-infected plants is a result of the response
of the plant to the increased stress ethylene that forms in response to the infection
rather than from the direct action of the pathogen. When PGPB contain the enzyme
ACC deaminase, they can modulate the level of ethylene in pathogen-infected plants
thereby significantly limiting the damage caused by the pathogen. This is the case
for both fungal and bacterial pathogens (Wang et al. 2000; Toklikishvili et al. 2010)
as well as for nematodes (Nascimento et al. 2013).
Some scientists have argued that drought, or soil drying, limits crop yield more
than any other abiotic stress. Moreover, this problem is exacerbated by climate
change that may result in decreased annual rainfall in many agricultural regions that
provide the world’s major staple crops. Thus, as drought is an ethylene-inducing
stress, it is expected that ACC deaminase-containing plant growth-promoting bacte-
ria will facilitate plant growth under drought conditions, and this is precisely what
has been observed (Mayak et al. 2004a; Belimov et al. 2008). In addition, Timmusk
et al. (2011) examined the prevalence and distribution of ACC deaminase-containing
Paenibacillus spp. among bacterial strains isolated from the rhizospheres of wild
262 B. R. Glick

barley (Hordeum spontaneum) growing in Northern Israel. The strains were all iso-
lated from a region termed ‘Evolution Canyon’ in which the South Facing Slope was
sparsely vegetated as a consequence of the harsh conditions on this side of the canyon
including excessive sunlight and frequent drought while the North Facing Slope fea-
tured conditions much more conducive to plant growth including lower levels of
sunlight and the apparent absence of drought. These workers observed that ∼ 50 %
of the bacteria isolated from around the roots of plants from the South Facing Slope
contained ACC deaminase while only approximately 4 % of the bacteria isolated
from the North Facing Slope contained this enzyme. These researchers suggested
that bacterial ACC deaminase was selected for by the plants growing under the more
perennially stressful conditions on the South Facing Slope, thereby protecting plants
and facilitating their survival; without plant growth and root exudation the bacteria
themselves would likely not proliferate. On the other hand, with the much better
growth conditions on the North Facing Slope, there is apparently much less selective
pressure for bacteria to retain genes for ACC deaminase.
Salinity is an enormous worldwide problem for agriculture, especially for crops
grown under irrigation. This is because salt is inhibitory to the growth of a large
number of plants. In fact, it is estimated that around half of the land worldwide
devoted to the growth of irrigated crops is adversely affected by salt. While a number
of strategies have been developed to facilitate the growth of plants in the presence
of salt, arguably one of the most successful approaches has been the use of ACC
deaminase-containing PGPB (Mayak et al. 2004b; Gamalero et al. 2009a). Thus,
researchers have found this strategy to be effective with a wide variety of crops both
in the lab and in the field.
Ethylene is a key signal in the initiation of plant flower senescence although dif-
ferent flowers may be more or less sensitive to ethylene. Chemical ethylene inhibitors
are currently used to prolong the shelf-life of cut flowers, however, many of these
chemicals are either expensive or environmentally hazardous or both. As an alterna-
tive to the use of chemicals, it has been shown that cut flowers may be treated with
endophytic (but not with rhizospheric) ACC deaminase-containing PGPB and these
bacteria can significantly delay the senescence of cut flowers (Ali et al. 2012).
The problem of toxic waste disposal is enormous, with billions of tons of haz-
ardous waste produced worldwide every year. One strategy that has been developed
relatively recently, phytoremediation, includes the use of plants to either remove
or detoxify many environmental contaminants, both metals and organics (Gamalero
et al. 2009b; Glick 2010). Unfortunately, the presence of many of these contami-
nants is inhibitory to plant growth. However, a significant portion of the inhibition
caused by the contaminants may be overcome by the addition of PGPB. Where it
has been examined in some detail, the bacterial traits that are most effective in stim-
ulating plant growth in the presence of environmental contaminants are siderophore
synthesis, IAA production and ACC deaminase activity.
27 Stress Control and ACC Deaminase 263

27.4 Future Prospects

Notwithstanding the fact that there are still only a very limited number of commer-
cialized PGPB, the widespread use of these bacteria is a technology whose time has
come. The current understanding of the mechanisms employed by these bacteria
should hasten the commercial development of this technology, both in agriculture
and in environmental cleanup protocols (phytoremediation). In the short term, it is
likely that ACC deaminase-containing PGPB will be used in conjunction with exist-
ing strains of Rhizobia to inoculate and promote the growth of legumes. Secondly,
the use of ACC deaminase-containing PGPB as an adjunct to the phytoremediation of
organic contaminants such as polycyclic aromatic hydrocarbons already works well
enough for this technology to be commercialized. Third, the use of ACC deaminase-
containing PGPB should be able to effectively complement and augment the job
done by many of the existing commercialized biocontrol strains. Once farmers,
growers and environmental engineers are convinced that this approach works effec-
tively in the field, the use of these organisms, selected for the presence of the enzyme
ACC deaminase and highly effective at lowering plant stress levels, should continue
to grow.

References

Abeles FB, Morgan PW, Saltveit ME Jr (1992) Ethylene in plant biology, 2nd edn. Academic Press,
New York
Ali S, Charles TC, Glick BR (2012) Delay of carnation flower senescence by bacterial endophytes
expressing ACC deaminase. J Appl Microbiol 113:1139–1144
Belimov AA, Dodd IC, Hontzeas N et al (2008) Rhizosphere bacteria containing 1-aminocyclo-
propane-1-carboxylate deaminase increase yield of plants grown in drying soil via both local
and systemic hormone signaling. New Phytol 181:413–423
Gamalero E, Berta G, Glick BR (2009a) The use of microorganisms to facilitate the growth of
plants in saline soils. In: Khan MS, Zaidi A, Musarrat J (eds) Microbial strategies for crop
improvement. Springer-Verlag, Berlin, pp 1–22
Gamalero E, Lingua G, Berta G et al (2009b) Beneficial role of plant growth promoting bacteria
and arbuscular mycorrhizal fungi on plant responses to heavy metal stress. Can J Microbiol
55:501–514
Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41:
109–117
Glick BR (2010) Using soil bacteria to facilitate phytoremediation. Biotechnol Adv 28:367–374
Glick BR (2014) Bacteria with ACC deaminase can promote plant growth and help to feed the
world. Microbiol Res 169:30–39
Glick BR, Cheng Z, Czarny J et al (2007) Promotion of plant growth by ACC deaminase containing
soil bacteria. Eur J Plant Pathol 119:329–339
Grichko VP, Glick BR (2001) Amelioration of flooding stress by ACC deaminase-containing plant
growth-promoting bacteria. Plant Physiol Biochem 39:11–17
Mayak S, Tirosh T, Glick BR (2004a) Plant growth-promoting bacteria that confer resistance to
water stress in tomato and pepper. Plant Sci 166:525–530
Mayak S, Tirosh T, Glick BR (2004b) Plant growth-promoting bacteria that confer resistance in
tomato to salt stress. Plant Physiol Biochem 42:565–572
264 B. R. Glick

Nascimento FX, Vicente CSL, Barbosa P et al (2013) The use of the ACC deaminase producing
bacterium Pseudomonas putida UW4 as a biocontrol agent for pine wilt disease. Biocontrol
58:427–433
Stearns JC, Woody OZ, McConkey BJ et al (2012) Effects of bacterial ACC deaminase on Brassica
napus gene expression. Mol Plant-Microbe Interact 25:668–676
Timmusk S, Paalme V, Pavlicek T et al (2011) Bacterial distribution in the rhizosphere of wild
barley under contrasting microclimates. PLoS One 6. doi:e17968
Toklikishvili N, Dandurishvili N, Tediashvili M et al (2010) Inhibitory effect of ACC deaminase-
producing bacteria on crown gall formation in tomato plants infected by Agrobacterium
tumefaciens or A. vitis. Plant Pathol 59:1023–1030
Wang C, Knill E, Glick BR (2000) Effect of transferring 1-aminocyclopropane-1-carboxylic acid
(ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative
CHA96 on their growth-promoting and disease-suppressive capacities. Can J Microbiol 46:
898–907
Chapter 28
Plant-Microbe Interactions and Water
Management in Arid and Saline Soils

Daniele Daffonchio, Heribert Hirt and Gabriele Berg

Abstract Drought and salinity are major factors limiting agriculture in many regions
in the world, and their importance is predicted to even increase in the near future in
parallel with the ongoing global warming and climate changes. Soil and rhizosphere
microbes are potential resources for counteracting such abiotic stresses in plants.
The knowledge on the roles of root microorganisms in retaining soil humidity and
promoting plant growth under such abiotic stresses is analyzed in this chapter. The
importance of microbial diversity in the rhizosphere for alleviating drought and salin-
ity effects on the plant physiology is discussed in the light of “Desert Farming”, the
general crop management practice that is frequently used in arid regions. The plant
growth promoting functional services exerted by microorganisms within the rhizo-
sphere in arid soils are presented in relation to the plant response under water stress.

28.1 The Effects of Drought and Salinity on Plant Physiology

A major challenge for future agriculture is to cope with the increasing demand for food
production, facing a constantly increasing world population. This growing demand
for agricultural production is paralleled by dramatic losses of arable land due to
enhanced soil destruction and erosion. Drought and soil salinity are the two major

D. Daffonchio ()
DeFENS, Department of Food, Environmental and Nutritional Sciences,
University of Milan, 20133 Milan, Italy
Tel.: + 393339742943
e-mail: daniele.daffonchio@unimi.it
H. Hirt · D. Daffonchio
BESE Division, King Abdullah University of Science and Technology,
Thuwal, 23955-6900, Kingdom of Saudi Arabia
Tel.: + 00966 (012) 808 2959
e-mail: heribert.hirt@kaust.edu.sa
G. Berg
Institute of Environmental Biotechnology, Graz University of Technology,
8010 Graz, Austria
Tel.: + 43664608738310
e-mail: Gabriele.berg@tugraz.at
© Springer International Publishing Switzerland 2015 265
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_28
266 D. Daffonchio et al.

environmental factors that limit plant growth and development, thereby negatively
affecting agricultural yield by more than 60 %. Water shortage is critical in many areas
of the world and is usually countered by extensive irrigation. Although the planet
earth is rich in water, not only the water of the oceans, but also most inland water
resources are highly saline. Moreover, irrigation usually results in soil salinization
making drought and soil salinity increasing agricultural problems (Bartels and Sunkar
2005).
To convert deserts into arable, green landscape is a global vision as well as com-
petent answer to world hunger and climate change (Clery 2011). Desert farming,
which generally relies on irrigation, is part of this vision. Agriculture systems were
already developed in arid landscapes by ancient cultures, yet nowadays, there is a
dramatically increasing need for large-scale desert farming to feed the population.
Desert farming is not only a challenge for irrigation systems with impact on the
global water balance, it can also have an impact on soil microbial diversity. Changes
of soil bacterial diversity, especially reduced beta diversity, was shown to occur in a
semi-arid ecosystem as a consequence of land use for agriculture with potential irre-
versible consequence on the natural soil ecosystem (Ding et al. 2013). To avoid this
risk and to establish sustainable desert farming systems it is important to understand
diversity in arid and saline environments.
Salt and drought stress share some properties and generally result in impaired key
physiological functions. One component of salinity is hyperosmotic stress, resulting
in a water deficit that is comparable to a drought-induced water deficit. The other
component of salt stress is ion toxicity, resulting in metabolic imbalance. Mem-
branes may become disorganized, proteins may become inactive and excess levels
of reactive oxygen species (ROS) can be produced leading to oxidative damage.
As a consequence, inhibition of photosynthesis, metabolic dysfunction, and dam-
age of cellular structures contribute to plant growth inhibition, reduced fertility and
premature senescence.
Plants basically counteract the negative effects of salinity and drought by acti-
vation of genetic and biochemical responses. These responses include the synthesis
and accumulation of osmolytes, such as proline or raffinose, that are able to stabilize
proteins and cellular structures but also to maintain cell turgor pressure by osmotic
adjustment. Moreover, plants also enhance scavenging of ROS, which are generated
as a secondary effect of salt and drought stress. Several plant stress signaling path-
ways have been dissected in detail in the model organisms Arabidopsis thaliana and
rice and a number of central transcription factors have emerged that regulate cohorts
of downstream genes. Despite this tremendous progress, our knowledge on the co-
ordination and integration of these regulatory pathways into the complex matrix of
plant stress physiology is still limited. Moreover, different plant species developed
different strategies to cope with stress. For example, some halophytic Brassicacae,
such as the Arabidopsis-related halophyte Thellungiella halophyla, avoid stress by
salt exclusion, whereas Lobularia maritima accumulates and detoxifies salt by com-
partmentalisation. Moreover, harsh environmental conditions, which are harmful for
one plant species, might not be stressful for another species. These differences cor-
relate with different stress-response mechanisms and two main strategies of stress
28 Plant-Microbe Interactions and Water Management in Arid and Saline Soils 267

avoidance and stress tolerance can be found in the plant kingdom. Stress avoidance
in some species is a genetically inherited trait that delays or prevents the negative
impacts of a stress. For example, cacti show a permanently adapted morphology
and physiology to hot and arid climates. Stress tolerance is an adaptive strategy to
counterbalance stress conditions and most plants can adapt to drought conditions
by closing their stomates to reduce transpirational water loss. Most plants can also
acclimate to stress conditions upon a gradual and repeated increase of a stress fac-
tor. Acclimation induces various physiological changes that are reversed when the
adverse environmental conditions disappear. Overall, whereas the avoidance mech-
anisms are usually constitutive features that are genetically inherited, acclimation
mechanisms are plastic and reversible. At the molecular level, acclimation involves
the modification of gene expression but also epigenetic mechanisms. Stress-inducible
genes comprise genes involved in direct protection from stress, including the syn-
thesis of osmoprotectants, detoxifying enzymes, and transporters, as well as genes
that encode regulatory proteins such as transcription factors, protein kinases, and
phosphatases.
An issue that is worth to be explored for the future of agriculture is the effects
that plant-associated microorganisms may exert on all these metabolic processes for
improving plant resistance and resilience to drought and salinity stresses.

28.2 Rhizosphere Bacterial Diversity in Arid and Saline Soils

and under “Desert Farming”

Arid and Saline Soil Habitats Harbour a Unique Bacterial Diversity Deserts rep-
resent in general extreme environments for microorganisms. Although the conditions
varied strongly in the different regions of the world, all of them are characterized by
a combination of extreme temperatures and desiccation, resulting in arid and saline
soils. These extreme abiotic factors, as well as the absence of plants at many sites,
contribute to the visual appearance of a sterile environment. While early studies
supported this “sterility” by very low levels of viable/cultivable microorganisms,
applications of DNA/RNA-based molecular methods led to interesting new insights
and showed a contrasting picture. For example, in their global-scale study, Fierer and
Jackson (2006) found that the acidic soils of tropical forests harbour fewer bacterial
taxa than the neutral pH soils of deserts. In different sites in the Negev Desert, archaeal
and bacterial diversity was not constrained by precipitation, although the taxonomic
composition differed (Angel et al. 2010). In soil of the Atacama Desert, a high di-
versity of microorganisms known to live in hypersaline environments was found by
analysis of Denaturing Gradient Gel Electrophoresis profiles of amplified 16S rRNA
gene (de Los Ríos et al. 2010). Most of the desert microbial communities seem to
be structured solely by abiotic processes. However, if desert plants are present, they
strongly shape soil microbial diversity. Also desert animals play an important role
as ecosystem engineers and affect the microbial complex of diazotrophs in soils.
All these investigations showed a unique and extraordinary microbial diversity in
belowground desert environments, strongly adapted to their unique conditions.
268 D. Daffonchio et al.

The SEKEM Farms as Example for Sustainable Desert Farming On the SEKEM
farms in Egypt, desert land was converted into arable land, and biodynamic agri-
culture is operated for over 30 years now (www.sekem.com). Today SEKEM is
carrying out organic agriculture on more than 4100 ha and has the largest market for
organic products outside Europe and North America. They produce organic foods,
spices, tea, cotton textiles and natural remedies. However, the cultivation especially
of medical plants is more and more affected by soil-borne phytopathogens, which
lead to significant yield losses. Therefore, this is an excellent study example (i) to
understand the impact of desert farming on microbial diversity and, (ii) to develop a
specific biocontrol strategy for desert agriculture.
To examine the impact of organic agriculture on bacterial diversity and community
compositions in desert soil, soil from a SEKEM farm in comparison to the surround-
ing desert soil from different sites were assessed by pyrosequencing-based analyses
of 16S rRNA and nifH gene sequences (Köberl et al. 2011). In addition, fingerprint-
ing and cultivation-dependent methods were included in this study. Altogether, a
strong impact on the structure and function of the bacterial and fungal communities
was found. The computed Shannon indices of diversity (H’) calculated for bacterial
communities on the basis of amplicon libraries were much higher for agricultural
soil than for desert soil (H’ at a dissimilarity level of 20 %: SEKEM soil: 4.29; desert
soil: 3.54). This indicates a higher bacterial diversity in soil due the agricultural use
of the desert. For both soil types together 18 different phyla were identified; domi-
nant groups present in both soils were especially Proteobacteria (30.2 %), Firmicutes
(27.3 %) and Actinobacteria (10.5 %). In detail, Firmicutes were highly enriched in
agricultural soil (from 11.3 % in desert soil to 36.6 % in SEKEM soil), while Pro-
teobacteria (46.0 % in desert soil and 21.0 % in SEKEM soil) and Actinobacteria
(20.7 % in desert soil and 4.6 % in SEKEM soil) occurred in SEKEM in lower abun-
dances than in the surrounding desert. In addition, in both soils Bacteroidetes (4.6 and
5.3 %) and Gemmatimonadetes (1.4 and 1.9 %) were present. Whereas Acidobac-
teria (7.9 %) and Planctomycetes (1.1 %) were only present in the agricultural soil,
Deinococcus-Thermus (1.1 %) was only detectable in the desert sand. In contrast
to other studies, the most important difference was the high abundance of Firmi-
cutes. Most of the Firmicutes were classified as Bacillus; in the agricultural soil
also the phylogenically related genus Paenibacillus was found (5 % of classified
Firmicutes). These differences between the two soil types found for the bacterial
community could be confirmed for the fungal community analyzed by fingerprinting
and for the functional studies regarding antagonistic microorganisms and diazotrophs
(Köberl et al. 2013a; Köberl et al. 2013b). After long-term farming, a drastic shift in
the microbiomes in desert soil was observed. Bacterial communities in agricultural
soil showed a higher diversity and a better ecosystem function for plant health but
a loss of extremophilic bacteria. Interestingly, we detected that indigenous desert
microbes promoted plant health in desert agro-ecosystems.
In desert soils, plant roots provide important nutrient sources for bacteria and act
as microbial hot spot. The composition of the microbial community in rhizosphere
and endorhiza of three different medical plants (Matricaria chamomilla L., Calen-
dula officinalis L. and Solanum distichum Schumach. & Thonn.) grown under organic
28 Plant-Microbe Interactions and Water Management in Arid and Saline Soils 269

conditions on SEKEM farms confirmed this hypothesis. These results show that dom-
inant bacteria, e.g. Ochrobactrum and Rhodococcus, are taken up by the plants from
the soil and that soil is the main reservoir for plant-associated bacteria. Further, nearly
in all samples Bacillus sp. was found. The fungal community fingerprints included
a quite high diversity in all microenvironments. Verticillium dahliae, a prominent
phytopathogenic fungus, was found nearly in all samples on the SEKEM farms. In
general, mainly potential plant pathogens were found within the fungal communities.
The obligate root-infecting pathogen Olpidium, (fungal phylum Chytridiomycota),
was found especially in the rhizosphere and endorhiza of M. chamomilla. Alternaria
and Acremonium were primarily present in the rhizosphere samples. There were sig-
nificant differences between the rhizosphere and the endorhiza of the medical plants.
In general, samples from the rhizosphere generated more strains than samples from
the endorhiza of the medical plants, which indicate that a sub-set of rhizobacteria
was able to invade the root. Diazotrophs are key organisms for providing nitrogen
in natural or organically managed agricultural ecosystems, especially under desert
conditions. By combining nifH-specific quantitative PCR, fingerprints, amplicon
pyrosequencing and fluorescent in situ hybridization-confocal laser scanning mi-
croscopy, a generally high nifH abundance and diversity in native and agricultural
desert soil and in the rhizosphere of medicinal plants was detected, the highest
reported until now compared with other ecosystems. Statistically significant differ-
ences were found between both soil types (native and agriculturally used), between
the different microhabitats (bulk soil, rhizosphere, endorhiza), and between the three
investigated medicinal plants. Again, a considerable community shift from desert to
agriculturally used soil was observed with higher abundance and diversity in the
agro-ecosystem. In comparison to the rhizosphere, the endorhiza was characterized
by lower abundances and a subset of species. Comparing root-associated communi-
ties, remarkable differences were found. While the microbiomes of M. chamomilla
and C. officinalis were similar and dominated by potential root-nodulating rhizobia
mainly acquired from soil, the perennial S. distichum formed primarily associations
with free-living nitrogen fixers most likely transmitted between plants, possibly by
mean of the seeds, as they are undetectable in soils. The results underline the impor-
tance of diazotrophs in desert ecosystems and moreover identified plants as important
drivers for functional diversity.
The major problems in the cultivation of plants on SEKEM farms are caused by
the soil-borne pathogenic fungi Verticillium dahliae Kleb., Rhizoctonia solani J.G.
Kühn and Fusarium culmorum (Wm.G. Sm.) Sacc. as well as by the soil-borne
pathogenic bacterium Ralstonia solanacearum. Bacterial isolates obtained from the
soil of the SEKEM farm exhibited a higher in vitro antagonistic potential towards
soil-borne phytopathogenic fungi in comparison to the bacteria isolated from the
desert soil (SEKEM 21.2 ± 1.2 %; desert 12.6 ± 0.8 %). From the agricultural soil
17.4 % (27 isolates) demonstrated antagonism towards all three fungal pathogens,
while only 10.6 % (21 isolates) from the desert soil. Already the desert soil harbors
a high proportion of antagonists, which were augmented by organic agriculture in
SEKEM soil. The soil from the farm seems to be supplied with antagonists in such an
optimal way, that there was no detectable enrichment of antagonists in the rhizosphere
270 D. Daffonchio et al.

and endorhiza of the investigated medical plants. In general, M. chamomilla and S.

distichum showed a better antagonistic potential than C. officinalis. Especially the
endorhiza from M. chamomilla harbors a high proportion of antagonists. Whereas
most antagonistic bacteria against Fusarium culmorum were found in the soil and in
the rhizosphere of the medical plants, those against Verticillium dahliae were found
in the endorhiza. A representative selection of promising biological control agents
was identified by partial 16S rRNA gene sequencing as members of the Bacillus
subtilis and Bacillus cereus groups, and of the genera Paenibacillus, Streptomyces
and Lysobacter. Except for one isolate of Lysobacter, only gram-positive antagonists
were found. All microenvironments were dominated by antagonists from Firmicutes
with Bacillus and Paenibacillus isolated from all habitats. Antagonistic Streptomyces
were found exclusively in desert soil. Promising strains of Streptomyces subrutilus,
Bacillus subtilis and Paenibacillus polymyxa were tested for their activity under field
conditions on chamomile seedlings. The Bacillus and Paenibacillus strains enhanced
the production of the bioactive secondary plant metabolite apigenin-7-O-glucoside
(Schmidt et al. 2014). Other Bacillus subtilis isolates were able to control root-knot
nematodes by inducing systemic resistance of tomato plants (Adam et al. 2014).

28.3 Functional Services of Rhizosphere Bacteria under

“Desert Farming”

The wide diversity of bacteria in the rhizosphere is exploited in a series of services

that help plants in counteracting drought and salinity stresses. Such services, ranging
from general ecological services to contributions to the physical protection of the
root from the mechanical stress or in the plant hormone homeostasis, are important
features under Desert Farming.
Microbial Ecological Services The ecology of the arid systems modulates the type
of microorganisms and the extent of the services provided to plants. For instance
the potential plant growth promoting (PGP) services provided by the root associated
bacteria appear to be invariant respect to macroecological factors such as latitude,
soil type and plant cultivar, despite provided by different bacterial communities,
according to observations across an aridity macrotransect from North Italy to North
Tunisia and Egypt (Marasco et al. 2013a). Rhizosphere and endophytic bacteria are
capable of promoting the growth of plants challenged by drought. It has been shown
that such a trait is not a per se capability of the selected microorganisms but it is
associated to the water stress (Rolli et al. 2014). So, root-associated bacteria that
normally do not present in-vivo promotion activities can determine plant resistance
to drought, suggesting that the services to plant are stress-dependent.
Protection from the Soil Mechanical Stress Water evaporation and drought deter-
mine important changes in the soil aggregates with modifications in the soil particle
architecture. Such changes influence the mechanical interactions of the soil with the
28 Plant-Microbe Interactions and Water Management in Arid and Saline Soils 271

root surface and the overall physical condition of the soil including water circula-
tion, air exchanges and temperature in the soil. Rhizodeposition determines favorable
conditions for bacterial growth. Bacterial biofilms produce exopolymers (EPS) that
affect the binding of soil particles to the rhizoplane, improve the water retention in
the rhizosphere and protect the root surface from mechanical damages determined
by the soil hardness. Plants treated with EPS-producing strains and maintained under
drought conditions have increased biomass and cause increased macroporosity in the
root adhering soil compared to the non-treated controls.
Protection from Osmotic and Oxidative Stresses Many bacteria are capable of
mitigating water stress in plants by stimulating the production of osmoprotectants
in the associated plants. The capacity of both Gram negative (Azospirillum and
Pseudomonas) and Gram positive (Bacillus) strains to promote resistance of basil
plants to water stress has been associated to increased concentrations of proline
and soluble carbohydrates in root and leaf tissues. In the treated plants chlorophyll
content increased significantly, confirming the overall beneficial effects of bacteria
against drought (Heidari et al. 2011). Bacteria-mediated protection against water
stress was also associated to the accumulation of anthocyanin in the vacuoles of
mountain laurel (Kalmia latifolia L.), when the stressed plant was colonized by
an endophytic Streptomyces padanus actinomycete (Hasegawa et al. 2004). Bacteria
engineered for overexpressing genes for threhalose synthesis increased their osmotic
stress tolerance as well as resistance to drought of their host plants to which they
were associated to (Rodriguez-Salazar et al. 2009).
Cell turgor is an essential process for maintaining normal physiological activi-
ties in plant tissues. During drought the aqueous vacuoles in the plant cells regulate
the concentrations of osmolytes to decrease the vacuolar osmotic potential and im-
prove water uptake (Park et al. 2005). Different compatible solutes are accumulated
in the vacuoles, including sugars, glycine betaine, amino acids, organic acids and
pigments. These molecules contribute to protection of the plant from stress through
osmotic adjustment, stabilization of the native structure of enzymes and cell mem-
branes and detoxification of ROS induced by the stressful conditions. The increased
concentration of such solutes raises the cell’s osmotic pressure, contributing to
maintain turgor by preventing water loss and promoting water uptake. The over-
expression in Arabidopsis as well as in crop plants (such as tomato) of the vacuolar
H+ -pyrophosphatase, an enzyme implicated in the vacuolar ion turnover, determines
improved resistance to drought and enhanced growth compared to the non-engineered
controls. The engineered plants have enhanced pyrophosphate-driven cation trans-
port into root vacuoles and increased root biomass and are capable of recovering
from exposure to water stress.
The concentration of ROS, such as superoxide, hydrogen peroxide, hydroxyl rad-
ical and singlet oxygen, increases in plant tissues under water stress (Apel and Hirt
2004). ROS react with proteins, lipids and nucleic acids, impairing cell physiological
functions. In parallel to the promotion of the synthesis of osmolytes in plants exposed
to water and salinity stresses, PGP bacteria (PGPB) can enhance tolerance to oxida-
tive stress through the synthesis of stress-related enzymes. For instance, inoculation
272 D. Daffonchio et al.

of drought stressed Lactuca sativa with PGP microbes resulted in increased activity
of catalase enzymes (Kohlera et al. 2009).
Effects on Hormone Homeostasis PGPB are capable of producing plant hormones
including auxins, gibberellins (GAs), cytokinins, abscisic acid (ABA) and ethylene
(see Chap. 26). Despite the difficulty of separating the effects of bacteria vs. plant-
produced hormones, experiments using hormones synthesis inhibitors as well as
Arabidopsis mutants impaired in specific hormone synthetic pathways have shown
some potential effects of different bacteria-produced hormones on stimulating re-
sistance to drought and salinity. By using chemical inhibitors of GAs and ABA
synthesis in planta, Azospirillum lipoferum treated maize seedlings showed drought
stress alleviation, suggesting that bacterial ABA and GAs may play roles in water
stress mitigation. Bacterial production of volatile organic compounds (VOCs) (see
Chap. 8), such as 2R,3R-butanediol, have been proposed to influence the overall plant
hormone balance affecting transpiration and water loss under drought by influencing
stomata closure or contributing to shape root architecture potentiating the uptake of
water, minerals and microelements in water stressed plants (Cho et al. 2008).
Bacteria can also produce enzymes capable of disrupting plant hormone syn-
thetic pathways, such as the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC)
deaminase, which hydrolyzes the plant ethylene precursor ACC to ammonia and α-
ketobutyrate. Such traits may have roles in promoting resistance of plants to drought
and salinity. ACC deaminase activity has been associated with stimulation of resis-
tance to salinity stress. For instance, an ACC deaminase-producing Achromobacter
piechaudii strain decreased the ethylene production by tomato seedlings grown in
the presence of up to 172 mM NaCl, and increased the plant fresh and dry weights
(Mayak et al. 2004).

28.4 The Plant Response to Plant Growth Promoting Bacteria

Under Stress

Plants can generate a large portion of their energy by photosynthesis, but plant growth
requires significant quantities of nitrate (see Chap. 23), phosphate (see Chap. 24),
and other minerals, which are often not freely available in the soil. Root-associated
beneficial microbes are important partners of plants and in exchange to carbohydrates,
provide many of the limiting minerals. The best-known beneficial microbes are
mycorrhizal fungi (see Chap. 25) and rhizobia (see Chap. 23). Mycorrhiza interact
with about 80 % of all terrestrial plant species and provide phosphate and nitrate to
plants. Free-living or endophytic rhizobia can fix atmospheric nitrogen, but only the
family of leguminosae profits from such an interaction through their ability to house
rhizobia in root nodules. Although the interaction of plants with mycorrhizal fungi
and rhizobial bacteria is well understood, other rhizosphere microbes have received
much less attention.
28 Plant-Microbe Interactions and Water Management in Arid and Saline Soils 273

Soil-grown plants are immersed in a sea of microbes and diverse beneficial mi-
croorganisms such as PGPB as well as plant-growth promoting fungi (PGPF) can
stimulate plant growth and/or confer enhanced resistance to biotic and abiotic stresses
(de Zelicourt et al. 2013). The establishment of beneficial plant-microbial interactions
requires the mutual recognition and a considerable orchestration of the responses at
both the plant and the microbial side. Rhizobial and mycorrhizal symbioses share
a common plant signaling pathway that is activated by rhizobial and mycorrhizal
factors (Corradi and Bonfante 2012; Geurts et al. 2012) and this signaling pathway
also seems to be activated by certain beneficial bacteria, suggesting that different
beneficial and pathogenic microbes initiate common plant signaling pathways.
The Role of Plant Growth Promoting Bacteria PGPB belong to a number of differ-
ent bacterial genera, including Rhizobium, Bacillus, Pseudomonas and Burkholderia.
PGPB can improve plant growth under abiotic stress conditions. Enhanced salt toler-
ance of Zea mays upon co-inoculation with Rhizobium and Pseudomonas is correlated
with decreased electrolyte leakage and maintenance of leaf water contents. Some mi-
crobes produce plant hormones, such as indole acetic acid and gibberellic acid, which
induce increased root growth and thereby lead to enhanced uptake of nutrients.
Plants have the ability to acquire a state of induced systemic resistance (see
Chap. 14) to pathogens after inoculation with PGPB. In association with plant roots,
PGPB can prime the plant’s innate immune system and confer resistance to a broad
spectrum of pathogens with a minimal impact on yield and growth. Several PGPB
colonize roots and protect a large variety of plant species, including vegetables, crops
and even trees, against foliar diseases in greenhouse and field trials.
Plant-Associated Fungi Confer Stress Tolerance to Plants Mycorrhizal and/or
endophytic fungi interact with many plant species and significantly contribute to the
adaptation of these plants to a number of environmental stresses including drought,
heat, pathogens, herbivores or limiting nutrients. Some plants are unable to with-
stand stress conditions in the absence of their associated microbes. It appears that
stress tolerance of the host plant can be a habitat-specific feature of the interaction.
For example, Curvularia protuberata confers heat tolerance to its geothermal host
plant Dichanthelium lanuginosum. However, neither the fungus nor the plant can
survive alone at temperatures above 38 ◦ C (Redman et al. 2002). Moreover, only C.
protuberata isolates from geothermal plants can confer heat tolerance (Rodriguez
et al. 2008). A comparison of different fungal endophytes unravels a further layer
of specificity: C. protuberata confers heat but neither disease nor salt tolerance. In
contrast, Fusarium culmorum only confers salt tolerance and Curvularia magna only
disease tolerance. It appears that these specific features contribute to the ability of
some plants to establish and survive in extreme habitats.
Symbiotically conferred disease tolerance appears to involve different mecha-
nisms, depending on the endophyte. For example, a non-pathogenic Colletotrichum
strain that confers disease resistance does not activate host defense in the absence of
pathogen challenge (Redman et al. 1999). Moreover, disease resistance is localized
to tissues that the fungus has colonized, but is not systemic.
274 D. Daffonchio et al.

In contrast, Piriformospora indica confers disease resistance systemically. P. in-

dica colonizes the roots of many plant species and stimulates growth, biomass and
seed production. P. indica promotes nitrate and phosphate uptake and confers resis-
tance against abiotic and biotic stress (Waller et al. 2005). The fungal colonization
stimulates the host to synthesize phosphatidic acid, which triggers the OXI1 pathway
(Camehl et al. 2011). This pathway is usually activated only in response to pathogen
attack to activate host defense. A defect in the OXI1 pathway negatively affects plant
growth by the fungus, resembling a pathogenic interaction. Overall, the differences
between Colletotrichum spp.- and P. indica-conferred disease resistance indicate a
number of different mechanisms yet to be elucidated.
Further evidence indicates that our present concepts of categorizing microbes
as pathogenic or beneficial are inadequate. For example, Fusarium culmorum can
cause disease on a variety of crop plants. However, the F. culmorum isolate FcRed1
is beneficial and confers salt tolerance to its host dunegrass Leymus mollis, but
isolates from non-coastal dunegrass do not. C. protuberata is a plant pathogen for
several monocots, but isolate Cp4666D confers heat and drought tolerance to its
host plant Dichanthelium lanuginosum. While Curvularia species are not known to
have broad disease-host ranges, C. protuberata from the monocot D. lanuginosum
also confers heat tolerance on tomato (Márquez et al. 2007; Rodriguez et al. 2008).
Some microbes can also be present in plants without showing disease symptoms. For
example, Colletotrichum acutatum can colonize pepper, eggplant, bean and tomato
without causing disease, but with other plants, such as strawberry, disease symptoms
become evident (Freeman et al. 2001). So it appears that a number of microbes
have a host-dependent lifestyle as pathogenic or beneficiary partner of plants, but
the molecular basis of the plant-microbe interactions remains to be unraveled.

28.5 Desert Farming Exploits Plant-Microbe Interaction

to Improve Water Management

Beneficial plant-associated microbes can help plants suppress diseases, stimulate

growth, occupy space that would otherwise be taken up by pathogens, promote bi-
otic stress resistance, and increase crop yield and quality by nutrient mobilization
and transport (Berg et al. 2013). Therefore, the plant microbiome is a key determi-
nant of plant health and productivity. While the possibility to control biotic stress by
plant-associated microorganisms is known since more than 100 years, less is known
about controlling abiotic stress. There are many biocontrol products on the market
but our understanding how plant-associated microbes can compensate abiotic stress
is only at the beginning. However, several promising examples are already reported
in the literature. Armada et al. (2014) showed that strains of Bacillus megaterium,
Enterobacter sp., Bacillus thuringiensis, and Bacillus sp. have the potential to alle-
viate drought stress in Lavandula and Salvia by increasing K content, by depressing
stomatal conductance, or by controlled shoot proline accumulation. The production
of osmoprotectans was also identified as a key mechanism of the Stress Protecting
28 Plant-Microbe Interactions and Water Management in Arid and Saline Soils 275

Agent Stenotrophomonas rhizophila DSM14405T (Alavi et al. 2013). In addition,

this strain produced spermidine, which is a general, highly efficient stress protectant.
In another example, pepper plants exposed to bacterial isolates from plants culti-
vated under desert farming exhibited a higher tolerance to water shortage, compared
with untreated control (Marasco et al. 2012; Marasco et al. 2013b). This promo-
tion was mediated by a larger root system (up to 40 %), stimulated by the bacteria
that enhanced the plant’s ability to take up water from dry soil. Altogether, to ex-
ploit plant-microbe interaction to improve water management in desert farming is a
challenging but also promising task for the future.

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Chapter 29
Rhizoremediation

Sofie Thijs and Jaco Vangronsveld

Abstract Over the past centuries, technological revolutions have brought about new
sources of soil and (ground)water pollution. The clean-up costs by conventional
remediation methods are often exorbitantly high, retarding soil remediation if per-
formed at all. This is a severe problem as the health consequences of soil pollution can
be considerable. Against these drawbacks, rhizoremediation which is an inexpensive
and sustainable technology, based on the actions of biodegradative microorganisms
in the rhizosphere and the plant phytoremediation capacity, has gained increased at-
tention. During the symbiotic interactions in the rhizosphere, ectomycorrhizal fungi
extend the belowground surface area of plants where billions of root-associated bac-
teria help to take-up minerals and pollutants, produce vitamins and plant hormones
and degrade organic compounds or sequestrate metals. Genomics technologies and
systems-based approaches have tremendously advanced the way we investigate the
plant “black box” and lead to new insights about how we can exploit the beneficial
plant-microbe association in terms of soil remediation, the topic of this chapter.

29.1 Introduction

The rapid pace of technological advances since the nineteenth century, the Green
Revolution, Second Industrial Revolution, transportation, electronics and medical
developments have brought distinctively benefits in life quality, crop yields and
human health but also have its inextricably drawbacks for the environment: the over-
exploitation of natural resources and the enormous increases in wastes, releases of
pollutants and soil/water quality degradation that occur at high rates. According
to the European Environment Agency (EEA), industrial activities account for 60 %

J. Vangronsveld () · S. Thijs

Centre for Environmental Sciences, Hasselt University, Agoralaan, building D,
3590 Diepenbeek, Belgium
Tel.: +32 11 268 331
e-mail: Jaco.vangronsveld@uhasselt.be
S. Thijs
Tel.: +32 11 268 331
e-mail: sofie.thijs@uhasselt.be

© Springer International Publishing Switzerland 2015 277

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_29
278 S. Thijs and J. Vangronsveld

of Europe’s soil contamination from which the oil sector takes a one fourth part.
According to UNESCO, in the developing world, 70 % of the industrial waste is
dumped untreated in the waterways. In the next decades, it is estimated that each
oil and polycyclic aromatic hydrocarbons (PAHs)-contaminated site will cost hun-
dreds of thousands of euros to treat by conventional techniques and costs can be
upwards to billions when groundwater is also polluted. Besides the high costs, dump
and treat techniques are not always sufficient, they are invasive, time-consuming,
detrimental to soil and biological life and can lead to pollution of the biosphere and
release of greenhouse gasses. With the impetus demand for new agricultural and
industrial areas, the development of cheap, eco-friendly remediation strategies such
as rhizoremediation are important.

29.2 Rhizoremediation

Rhizoremediation is a specific subset of phytoremediation and involves the biodegra-

dation of organic pollutants in the root zone ascribed to the microbes in the
rhizosphere of plants used for phytoremediation or of plants which naturally occur
on contaminated soils (Fig. 29.1).
Plant roots can be considered as a substitute for tilling of soil, to spread the root-
associated microorganisms through the soil and to penetrate layers normally not
accessible to bacteria and to incorporate nutrients, bring oxygen and provide better
redox conditions which helps to stimulate and activate the rhizosphere microorgan-
isms. In addition, the majority of plants, in particular trees, live in symbiosis with
ectomycorrhizal (ECM) and/or arbuscular mycorrhizal (AM) fungi (Chap. 25). ECM
fungi act as a web-like extension of the root system enhancing the absorptive surface
area of plants. ECM play a crucial role in nutrient recycling and they are often more
resistant against (a)biotic stresses. In this mycorrhiza-root continuum, billions of bac-
teria help to take-up minerals and pollutants, produce vitamins and plant hormones
and degrade organic compounds or sequestrate metals. The enhanced degradation of
pollutants in the rhizosphere is beneficial to the plant and results in improved plant
growth on contaminated soils.
Compared to conventional techniques, plants are very attractive clean-up technol-
ogy tools, (1) they provide their own solar energy by photosynthesis and pump–up
gratuity pollutants with the water-stream making it much cheaper (it depends on the
site and pollutant concentration but in general it falls within 10–50 % of the costs for
conventional techniques, (2) remediation is in situ, (3) it generates much less wastes
and instead even creates economic benefits such as wood, feed stock or bioenergy
production. Rhizoremediation is suitable for vast sites with low to moderate pollu-
tant concentrations. Sites are often co-contaminated with organic and heavy metal
pollutants and are characterized by poor soil conditions (pH, texture, low nutrient
availability, salinity) which all have retarded the implementation. However, new
knowledge led to designing novel strategies that can overcome some of the inher-
ent limitations and commercialization of bio-inoculants is taking place, showing the
promising growing interest in rhizoremediation.
29 Rhizoremediation 279

Economic valorisaon
Biofuel producon, bioenergy
Technology plaorm using plants and root-associated
Wood, pulp, feed-stock
microorganisms to clean-up contaminated sites Re-use of agricultural land
B. Strategies to improve rhizoremediaon
Biosmulaon C. Up-scaling
Bio-augmentaon Rhizo-engineering field-tests

Toxic chemical compounds

D. Monitoring
Predictability
Efficiency
organic and heavy
metal chemical
pollutants
A. Rhizosphere interacons
Plant roots act as a natural lling system
Acvaon
Inject biodegradave microorganisms
Detoxificaon
in deeper soil layers
Mineralisaon
Using genomics, transcriptomics, proteomics, stable
isotope probing and culvaon of microorganisms to
understand the pathways of contaminant degradaon

Fig. 29.1 Schematic overview of the processes involved in rhizoremediation. a Summary of the
rhizosphere interactions at the micro-scale. On the bottom, scanning electron microscopy picture
of bacteria (colored in green) on the root surface of the grass Agrostis capillaris, from Thijs, un-
published. Picture of the split-root vertical agar plate system to analyse root-growth responses of
Arabidopsis thaliana, from dr Remans, unpublished. b Engineering techniques to improve rhi-
zoremediation. Picture of the inoculation of Salix sp. cuttings with PGP and heavy-metal resistant
bacteria in cadmium contaminated soil, from Janssen unpublished (Hasselt, Belgium). c The need
for up-scaling laboratory and microcosm tests to the field; picture of the harvest of short-rotation
coppice of willow trees on cadmium polluted soils in Belgium, from Janssen, unpublished.
d The development of effective monitoring methods. (Picture: prof dr Joel Burken (Missouri S&T,
Missouri, US) taking a sample from a tree at the campus of Hasselt University, Belgium, picture
from Vangronsveld unpublished. In the right panel, the economic benefits and value of rhizoreme-
diation are described. (Picture of bio-oil, Hasselt University, Belgium; Picture of the oil tanks is
from Weyens, unpublished; Picture of the roots is from Thijs, unpublished)

In this chapter, we explore the plant-bacteria interactions in rhizoremediation with

a focus on organic contaminants degradation. Next, we comment on strategies how
to successfully improve rhizoremediation and highlight key case studies of bacterial-
ectomycorrhizal interactions in rhizoremediation. We conclude with the perspectives
and needs brought by new analytical techniques.
280 S. Thijs and J. Vangronsveld

29.3 Plant-Bacteria Interactions in Rhizoremediation

Plant-bacteria interactions that lead to the degradation of contaminants in the rhizo-

sphere occur at the soil-root interface. At the microscale, the crucial processes involve
root-colonization by the bacteria, selection and maintenance of the degradation
genes, the role of root exudates in the activation of catalytic genes and inter-kingdom
communication which shape the community.
Colonization The biodegradative bacteria in the rhizosphere can originate from
(bacterized) seeds or they can be recruited from the main soil reservoir during growth
of the plants on the contaminated soil (Fig. 29.1). These bacteria can subsequently
be spread through the soil during root emergence and growth. In addition, bacteria
can also actively colonize the roots by chemotactic movement. Using in vivo ex-
pression technology (IVET), transcriptomics and mutants defective in motility, the
mechanisms of root-colonization and the identification of genes which are activated
during rhizosphere colonization are being unraveled (Ramos-Gonzalez et al. 2005).
Root Exudates Regulate Gene Expression Root exudate compounds such as sug-
ars, organic acids, fatty acids, secondary metabolites, nucleotides and also inorganic
compounds play a crucial role in structuring the rhizosphere microbial population
(Chap. 17, Chap. 23, Chap. 24, Chap. 26, Chap. 27 and references therein). Exu-
dates select those microbial populations which are able to respond to the exudate
buffet with rapid growth responses so that eventually only a small subset of the
whole soil microbial diversity is colonizing the roots and to a much higher density
than the surrounding bulk soil (Bais et al. 2008). A significant role in plant selec-
tion processes is attributed to aromatic compounds that are exuded (i.e. terpenoids,
phenols, flavonoids and other lignin-derived components), the structures are sim-
ilar to those of contaminants and can act as co-substrates for difficult to degrade
compounds such as high-chlorinated biphenyls (PCBs) and PAHs or as inducers of
degradation pathways. Recently, -omics technologies and systems-based approaches
are applied to study the gene-regulation network in contaminant degradation and for
sure they will provide promising new insights about how we can exploit the beneficial
plant-microbe association in terms of soil remediation (Matilla et al. 2007).
Communication and Dynamics Multiple signals are sent and received by plants
and microorganisms. These signals are responsible for recognition of microorgan-
isms, recruitment of catalytic potential, mycorrhization, resistance to stresses and
quorum sensing, i.e. the gene-expression regulation on the population level and in-
cludes bacterial traits such as motility, biofilm formation, symbiosis and conjugation
(Chap. 3). Some of the communication systems, e.g. the interaction between ar-
buscular mycorrhiza (AM) and trees, are symbiosis of several hundreds of millions
years old and probably evolved during co-evolution. Changing a(biotic) factors, e.g.
growth stage of the plant, soil type, contamination type, season, and level and density
of other microorganisms are all thought to cause significant shifts in the rhizosphere
and (root) endophytic community structure. For example, Siciliano et al. (2001)
demonstrated that the catabolic alkane monooxygenase genes were more prevalent
29 Rhizoremediation 281

in root endophytic and rhizospheric microbial communities than in bacteria present

in bulk soil contaminated with hydrocarbons.
Bio-degradation Potential of Bacteria Many bacteria possess the catabolic/
catalytic tools to mineralise/transform recalcitrant pollutants including petroleum
hydrocarbons, PAHS, chlorinated aliphatic compounds, solvents, PCBs, nitro-
aromatics and also for the removal of nitrate and ammonia in waste-streams and metal
immobilization or exclusion. The online Minnesota Biocatalysis/Biodegradation
Database supports almost 1200 compounds, over 800 enzymes, 1300 reactions and
almost 500 microorganisms with bio-degradative characteristics and is increasing to
grow (Gao et al. 2010).

29.4 Strategies to Improve Rhizoremediation

The presence of the biodegradative strains, the expression of the catalytic genes and
maintenance are all crucial factors which determine the success of rhizoremedia-
tion. However under natural conditions, rhizoremediation can be slow, indicating
that some of these factors or others are limiting the removal of pollutants. There-
fore, scientists and biotechnology companies developed strategies to improve the
rhizoremediation efficiency (Fig. 29.1b).
Bio-stimulation Fertilisation of the plants by addition of compost, minerals (ni-
trogen, phosphorous, potassium) or fertilisers on a base of organic carboxylic acids
which can enhance root exudation, has been reported to enhance the degradation
of pollutants in the rhizosphere by stimulating growth and activity of microorgan-
isms. In addition, additives are used to improve the physico-chemical properties of
the soil and to increase the bio-availability of pollutants e.g. biosurfactants for oil
degradation and siderophores for metal-bioavailability.
Bio-augmentation The introduction of a microbial strain or consortium is another
way to enhance degradation of contaminants in the rhizosphere. Delivery methods for
introducing these beneficial degrading microorganisms in soil include seed coating or
inoculation of plant roots with a bacterial suspension by root-dipping or soil drench,
in analogy to probiotics (Chap. 33). Growth of the inoculated root system acts as a
‘bio-injector’ of the degrading strains in the soil.
For ‘newer’pollutants, such as high-level chlorinated PCB’s and chloro-ethylenes,
sometimes no microbial catabolic pathways have evolved. In this situation, construc-
tion of genetically engineered (GE) and genetically modified (GM) strains might be
interesting (Brazil et al. (1995). In addition, in case of co-contamination, construc-
tion of microbial resistance for multiple organics and/or heavy metals is required.
However, genetic modification has also limitations, the costs, the amount of work
involved and the often limited success with the new synthetic vectors make this area
extremely challenging (Hernández-Sánchez and Wittich 2012).
With the introduction of bacteria in the rhizosphere it is important to consider their
rhizosphere competence. Firstly, Kuiper et al. (2002) developed novel enrichment
282 S. Thijs and J. Vangronsveld

techniques whereby the strains are selected based on the combination of effective
root colonization, assessed by sequential bacteria inoculation/re-isolation from the
roots of model plants and the abilities to degrade the pollutant. Secondly, bacteria
which show in vitro PGP-properties and abilities to increase plant biomass in planta
are receiving more attention. We hypothesize that the beneficial PGP-bacteria settle
in the root-zone and increase plant biomass in contaminated soil, alleviate plant
stress by ACC-deaminase and thereby enhance rhizoremediation (Thijs et al. 2014).
Thirdly, it is important to select the appropriate plant-bacterium combinations for
efficient degradation of the contaminants (Kuiper et al. 2004). There exists a plant-
cultivar specificity of microbial communities and furthermore variations in exudates,
contaminant levels and soil conditions influence the microbial community. Fourthly,
inoculating a consortium of bacteria whereby the partners can deliver nutrients and
other growth factors or use the various intermediates in the organic degradation
pathway more efficiently often out-performs the action of single (GM) strains. Fifthly,
because of the complex rhizosphere interactions, also the inoculation with ‘competent
endophytes’ (see Chap. 5) is considered, i.e. bacteria that successfully colonize the
internal plant tissues and which possess the capacity to incite plant physiology can
be selectively favored.
Transgenic Plants Engineering of plants with genes that allow superior degradation
abilities was endeavored, by e.g. overexpression of genes involved in metabolism,
uptake and transport of pollutants. Very often bacterial genes are used whereby
complete degradation pathways are introduced in plants. This was already proven
to be successful for enhanced degradation of highly recalcitrant compounds such
as explosives, PCBs and PAHs (Mackova et al. 2006). Another approach is rhizo-
engineering and is the use of transgenic plants which secrete more root exudates or
other rhizodeposits, which all indirectly have a significant effect on the rhizosphere
microbial community (Van Aken et al. 2010).

29.5 Realisations in the Field

During recent years, numerous studies have been published that describe the com-
bined use of plants and bacteria in rhizo-and phytoremediation through successes
and failures. Studies have focused on phreatophytic trees that are deep-rooting, fast
growing and are ‘pumping’ huge amounts of water such as Populus and Salix or
grasses and small shrubs with an extensive fine root system enhancing the bacterial
transformation of pollutants in the rhizosphere. Good reviews on phyto- and rhizore-
mediation studies exist (Gerhardt et al. 2009; Kanaly and Harayama 2010; Kavamura
and Esposito 2010; Megharaj et al. 2011; Mackova et al. 2006). Bio-augmented rhi-
zoremediation was reported to not always lead to enhanced successes compared with
natural rhizoremediation. Often, this results from the poor root colonization ability
of the introduced strains. Therefore, as mentioned previously, this trait, together with
other rhizosphere competence abilities, is crucial for successes in the field.
29 Rhizoremediation 283

The first studies dealing with ECM-fungi in remediation, mainly focused on the
re-vegetation and stabilization of polluted soils. Most of the results from the involve-
ment of ECM in contaminant degradation originate from laboratory experiments
and microcosm studies but field studies remain scarce. Therefore, up-scaling of
ECM-bacteria rhizoremediation is needed (Bücking 2011). Mycorrhizal fungi can
importantly contribute to rhizoremediation: ECM fungi can directly enable ligni-
nolytic and cell-wall degrading enzymes to degrade various recalcitrant pollutants
such as TPH, PAHs, pesticides, explosives and PCBs, increase the plant tolerance
to withstand toxicity, improve nutrition and protection from pathogens, enhance the
bio-availability of organics and heavy metals or by affecting the composition and
activity of the bacterial contaminant degrading population (Chaudhry et al. 2005).
Under some circumstances, ECM can also inhibit bacterial growth or reduce bacterial
activity, so not only positive and synergistic effects must be considered.
Remediation of total petroleum hydrocarbons (TPH) is often hindered by low plant
growth, particularly when TPH-concentrations cause stress to plants. Application
of PGPR-enhanced phytoremediation (PEP) was demonstrated to be successful in
remediating a highly contaminated petroleum hydrocarbon site in Southern Ontario,
California (Gurska et al. 2009). It was hypothesized that the PGPR-bacteria reduce
the plant stress level and improve plant growth, thereby stimulating rhizoremediation.
When Pinus sylvestris seedlings were inoculated with the ECM Suillus bovinus or
Paxillus involutus and grown in soil contaminated with petroleum hydrocarbons, a
bacterial biofilm of strains was formed on the exterior of the hyphae (Sarand et al.
1998). Moreover, these strains harbored plasmids involved in the degradation of
mono-aromatics.
Because of their hydrophobicity and chemical stability, PCB’s are only slowly
taken up by plants and degraded by associated microbes, resulting in incomplete
degradation or accumulation of toxic intermediates. Narasimhan et al. (2003) demon-
strated that A. thaliana flavonoid-overexpressing mutants were colonized at a much
higher level than non-mutant lines by the PCB-degrading bacterial strain P. putida
PML2, suggesting that rhizo-engineering of plants producing altered exudates is a
valuable tool to enhance PCB-degradation.
Forty percent of the waste sites in the United States are co-contaminated with
organics and trace elements (arsenic, barium, cadmium, chromium, lead, copper,
nickel and zinc). Todd and Reina (2003) described several approaches to increase
organic biodegradation in the presence of metals and include the application of metal
immobilizing additives or clay minerals and the reduction of metal bio-availability
by use of metal-resistant bacteria or mycorrhizae. Dual inoculation with ectomycor-
rhizal fungi and bacteria has been shown to improve growth and metal accumulation
of mycorrhized trees and it is suggested that the bacteria facilitate the ECM colo-
nization and increase the bio-availability of metals to the mycorrhized plant (Zimmer
et al. 2009). In return, the bacteria receive nutrients from the plant.
284 S. Thijs and J. Vangronsveld

29.6 Perspectives and Research Needs

A large number of man-made chemicals still lack good biological catalysts. To date,
for most of the 10 million organic compounds described, biological degradation has
not been investigated. With the intense research that is now occurring in the nan-
otechnology, material sciences and electronics sectors, new challenges are arising
in e.g. the biodegradability of fullerenes and carbon nanotubes, considered as the
ultimate high molecular weight PAHs. More than 6000 ECM fungal species are
likely to exist worldwide and only a fraction of the potential of ECM fungi to de-
grade pollutants has so far been determined. In addition, with the characterization
of cultivable biodegradative isolates we are only scratching the top of the iceberg of
the whole microbial diversity. The detection and capture of novel broad-host range
plasmids from soil bacteria and subsequent sequencing can provide a wealth of new
information and insights into the role of plasmids in bioremediation and ecology
(Heuer and Smalla 2012).
Technological advances and reduced costs of next generation sequencing
technologies, whole-genome approaches, metatranscriptomics, metaproteomics,
metabolomics, advances in micro-arrays (PhyloChip, GeoChip) and ecological tools
such as stable isotope probing (SIP), mark the start of a more wide use of these
NGS-techniques in rhizoremediation, providing unprecedented insights in the com-
plex interactions in the rhizosphere (Ramos et al. 2011). Using gene-expression
technologies, Govantes et al. (2009) elucidated a complex regulator circuit in bac-
teria that involves nitrogen control of the herbicide atrazine utilization. Based on
these findings, they developed valuable strategies to improve atrazine degradation in
fertilized agricultural soil, e.g. they propose the application of inhibitors of nitrogen
assimilation or the use of mutant bacterial strains that are impaired in the nitrogen
control.
In many cases, the most effective remediation solution is a combination of sev-
eral techniques depending on the soil pollution type/degree, its environmental and
health risks, the economic value of the polluted site and juridical and legislative
policies (Segura and Ramos 2013). Because rhizoremediation needs time and is
under influence of environmental factors e.g. temperature, pH, new pollution over
time, it is important to establish reliable monitoring methods to estimate the effi-
cacy of rhizoremediation and to predict the clean-up time. Innovative tools such as
the use of indicator species, phytophorensics i.e. the chemical analyses of xenobi-
otics in the sap-stream of plants to trace contaminants in soil and groundwater and
plant nanobionics, which all take much less time, efforts and costs than traditional
detection methods are being developed (Fig. 29.1d).
A last important consideration is the preservation and maintenance of degrada-
tion genes for rhizoremediation. Noor et al. (2014) pointed out that ongoing selection
is taking place in newly isolated strains from French agricultural soils recurrently
exposed to atrazine and simazine herbicides in 2000, proven by 6 amino-acid dif-
ferences in the atzA dechlorinase gene in the majority of the new strains compared
with the original isolates from 1990. This suggests that the environment remains the
29 Rhizoremediation 285

major reservoir for the discovery of novel strains for rhizoremediation better adapted
to the current conditions. As such, rhizoremediation still is a promising and fertile
research area.

Conclusion

At this point, our understanding of the plant system and its microorganisms in the rhi-
zosphere is fairly good. Studies involving newly isolated organisms have enhanced
our knowledge of degrading activities and regulation in the rhizosphere over an ex-
panding range of contaminants and environmental conditions. At the same time, the
challenge of future investigations is to further unravel how the microorganisms func-
tion together in their natural environment, the mechanisms of ECM in biodegradation
of recalcitrant xenobiotics and fortuitous metabolism, the efficacy of consortia in rhi-
zoremediation and the dynamics in rhizosphere communities. This information will
allow the manipulation of rhizoremediation systems to accomplish soil remediation
to the greatest extent, predictability and over the shortest time periods.

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and promises. Environ Sci Technol 44:2767–2776
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Phytoremediation 11:200–213
 Part V
Important Technologies
Chapter 30
Microbial Communities in the Rhizosphere
Analyzed by Cultivation-Independent
DNA-Based Methods

Susanne Schreiter, Namis Eltlbany and Kornelia Smalla

Abstract The development of methods to extract nucleic acids directly from the
rhizosphere or from microbial cells detached by a mechanical treatment from roots
opened new dimensions to study the rhizosphere microbiome and to overcome lim-
itations of cultivation-dependent methods. This chapter summarizes the potentials
and limitations of cultivation-independent methods used by our group in the last 15
years to investigate microbial communities in the rhizosphere and their response to
changing environmental conditions. We showed that rhizosphere microbial commu-
nities are highly dynamic, and that their composition is mainly shaped by the plant
and the soil type and factors influencing these drivers of microbial diversity in the
rhizosphere.

30.1 Introduction

The importance of the plant microbiome for plant growth and health is increas-
ingly recognized. The fraction of soil influenced by the plant, termed rhizosphere,
is an interface that connects the soil with the plant. Understanding the complex
interactions in the rhizosphere remained a challenge until tools allowing cultivation-
independent analysis of DNA or RNA extracted directly from the rhizosphere
became available. Here we provide a short overview of some of these tools which
were used to study the influence of different factors on the microbial community
compositions in the rhizosphere. The chapter is biased towards our own work
and for more comprehensive compilation the reader is referred to recent reviews

K. Smalla () · S. Schreiter · N. Eltlbany

Julius Kühn-Institut, Federal Research Centre for Cultivated Plants (JKI),
Messeweg 11–12, 38104 Braunschweig, Germany
Tel.: + 49 531 299 3814
e-mail: kornelia.smalla@jki.bund.de
S. Schreiter
e-mail: susanne.schreiter@jki.bund.de
N. Eltlbany
e-mail: namis.eltlbany@jki.bund.de

© Springer International Publishing Switzerland 2015 289

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_30
290 S. Schreiter et al.

(Berg and Smalla 2009; Berendsen et al. 2012; Bulgarelli et al. 2013). Before dis-
cussing the different nucleic acid-based methods and the major findings we would
like to draw the reader’s attention to critical prerequisites for obtaining meaningful
data.

30.2 Experimental Design and Sampling

The adequate experimental design and sampling strategy depends on the hypotheses
to be tested and often pre-experiments might assist in determining the numbers of
samples to be analyzed. Furthermore, the strategy used to sample the rhizosphere
influences the data obtained. Typically composite samples from the root system of
several plants are analyzed for reporting in a representative manner on the structural
and functional diversity in the rhizosphere and on the variation among replicates
within the same treatment and between treatments. We have studied the rhizosphere
microbial communities of different plant species from various sites and geographic
regions. Usually the plants were destructively sampled by uprooting them and vig-
orously shaking the roots. Different protocols have been used also in our laboratory
and the protocols had to be adapted for various reasons. Therefore it is highly recom-
mended for comparison of data from different studies to carefully read the sampling
protocols described, as the fractions of the rhizosphere microbial communities ana-
lyzed differed—depending on the protocols applied—in the proportion of rhizoplane
and bulk soil microorganisms present. Rhizosphere total community (TC-) DNA was
extracted from the soil brushed off from the root (Marques et al. 2014). This technique
is typically used when long-distance transport of samples is needed and microbes
residing on the rhizoplane or in soil particles glued to the roots or from fine roots
were likely missing. However, in most studies performed by our group the complete
root system, after vigorous shaking, was cut into pieces and mixed. Subsamples
placed in plastic bags were treated in the Stomacher® Circulator after adding saline
or water. Via paddle movement cells were detached from the root and soil particles
and the Stomacher treatment step was repeated three times. To obtain the microbial
pellets, the combined supernatants were centrifuged and the TC-DNA was extracted
from the complete pellet with of commercial soil DNA extraction kits (Weinert et al.
2009; Schreiter et al. 2014a). When a combination with cultivation-dependent anal-
ysis was done, e.g. to determine the potentially antagonistic fraction (Berg et al.
2002) or to monitor inoculant strains (Adesina et al. 2009; Xue et al. 2013; Schreiter
et al. 2014a) an aliquot from the combined supernatant was used for plating of serial
dilutions. Recently, we had to modify the protocol in a project aiming to compare the
effect of three soil types on the rhizosphere communities. An additional root washing
was performed in order to remove big clumps of soil adhering to the roots of plants
grown in clay rich soils before the Stomacher® protocol (Schreiter et al. 2014b). The
TC-DNA obtained from the pellet gained with this protocol was assumed to repre-
sent the genetic information of microbes colonizing the rhizoplane and rhizosphere.
Although a complete dislodgment of cells adhering to the roots and soil particles
30 Microbial Communities in the Rhizosphere . . . 291

seems to be impossible, it is important that cells bound to soil particles with different
degrees of strength are released with similar efficiency. Another crucial step for the
recovery of representative DNA that mirrors the genomes of all microbes present in a
rhizosphere sample is the efficient lysis of microbial cell walls. This can be achieved
by mechanical cell disruption and by enzymatic or chemical disintegration of cell
walls, or a combination of these methods. The efficiency of the different methods
used might not only influence the yield but also the presence of genomic DNA in
cells difficult to lyse. However, obviously the strength of lysis needs to be a trade-off
as too rigorous lysing methods might shear DNA released from cells that are easy
to lyse. The DNA yield might vary considerably for different DNA extraction kits
used for the same rhizosphere soils. Commercial kits for extraction from soil after a
harsh lysis with the FastPrep®-24 Instrument were major achievements and allowed
a simplification and miniaturization of the method. Extraction kits are less time-
consuming and efficiently remove co-extracted humic acids which would disturb
PCR-amplification. Finally, it should be stressed that strict precautionary measures
need to be taken to prevent contamination of the DNA during the extraction. In par-
ticular, when PCR is used to amplify a target gene that occurs less frequently, e.g.
antibiotic resistance genes or transgenic DNA, the extraction of DNA, preparation
of PCR reactions and analysis of PCR products need to be done in separate rooms.

30.3 Bacterial and Fungal Community Composition

in the Rhizosphere

PCR-based amplification of 16S and 18S rRNA gene or ITS fragments from rhi-
zosphere DNA and their subsequent analysis by fingerprinting, cloning and/or
sequencing are most frequently used to study the composition of microbes in the
rhizosphere and the effects of treatments. The rapidly growing database of riboso-
mal rRNA gene sequences contains presently more than a million good quality 16S
rRNA gene sequence entries deposited in Ribosomal Database. A disadvantage of
using ribosomal rRNA gene fragments is that bacteria possess different numbers of
ribosomal RNA operons. The numbers of 16S rRNA operons are assumed to reflect
different ecological strategies of bacteria (Klappenbach et al. 2000) and sequence
heterogeneity of the different operons might occur (Nübel et al. 1996). Costa et al.
(2007) proposed the Pseudomonas-specific gacA gene as an alternative marker for
studying their community composition. However, no matter which gene is targeted,
one major limitation that remains is that gene fragments of less common populations
are often not represented in clone libraries or fingerprints, especially when primers
targeting all bacteria, archeae or fungi are used for PCR amplification. Bent and
Forney (2008) termed this problem “the tragedy of the uncommon”. The applica-
tion of group-specific primers targeting the 16S rRNA gene can assist in studying
less common populations (Heuer et al. 1997; Heuer et al. 2002; Gomes et al. 2001;
Costa et al. 2006a; Costa et al. 2006b; Weinert et al. 2009). The sequence diversity
among 16S and 18S rRNA gene or ITS amplicons from TC- DNA can be analyzed
292 S. Schreiter et al.

by various techniques such as the terminal restriction fragment length polymorphism

(T-RFLP), single strand conformation polymorphism (SSCP) or denaturing gradient
gel electrophoresis (DGGE) that were developed in the end of the 1990’s. A com-
parison of these fingerprinting techniques showed that they had similar resolution
levels and provided similar results despite the different 16S rRNA gene regions used
(Smalla et al. 2007). At that time the great advantage of the fingerprinting techniques
was that a sufficient number of replicates could be analyzed in parallel, and when
combined with statistical analysis, testing of different biotic and abiotic factors in-
fluencing the bacterial and fungal community composition became possible (Kropf
et al. 2004). A clear drawback of the molecular fingerprinting techniques was that
bands with treatment-dependent intensity had to be excised from the fingerprints,
re-amplified, cloned and sequenced. Sequencing of dominant bands with identical
electrophoretic mobility detected in the rhizosphere of strawberry and oilseed rape
were shown to represent taxonomically different populations (Costa et al. 2006a).
On the one hand, 16S rRNA gene fragments from taxonomically distinct popula-
tions might have the same electrophoretic mobility due to similar melting behavior
while, on the other hand, one population might generate more than one band due to
operon sequence heterogeneity. The DGGE fingerprints based on 16S and 18S rRNA
gene and ITS fragments were used to study the influence of the following factors on
the composition of the bacterial and fungal communities in the rhizosphere: (i) plant
species (Smalla et al. 2001; Costa et al. 2006a), (ii) plant growth developmental stage
(Smalla et al. 2001; Gomes et al. 2001; Gomes et al. 2003), (iii) the cultivar Weinert
et al. 2009), (iv) the site (Costa et al. 2006a, 2006b) (v), the soil type (Schreiter et al.
2014a) , and (vi) the effects of inoculants or pathogens (Adesina et al. 2009; Xue
et al. 2013).
To obtain information on the taxonomic affiliation of the dominant bacteria in the
rhizosphere cloning and sequencing of 16S rRNA gene amplified from TC- DNA
of three potato genotypes grown at two sites were used. This approach was rather
time and cost intensive and thus typically was not applied for replicates but for
pooled samples. The TC-DNA from the potato rhizosphere of replicates of the same
samples was also analyzed by PhyloChips (DeSantis et al. 2007). The PhyloChip
was hybridized with Biotin-labelled 16S rRNA gene fragments. By means of the
PhyloChip a total of 2432 operational taxonomic units (OTUs) were detected in the
rhizosphere of potatoes and 864 were detected in all replicates. The major limitation
of the PhyloChip approach is that the diversity detected depends on what is on the
Chip, and that the hybridization signal intensity cannot be directly related to relative
abundance. Nevertheless, PhyloChips are great tools for comparing the relative abun-
dance of particular OTUs within and between treatments (Weinert et al. 2011). Thus
OTUs differing in relative abundance in the rhizosphere of the same potato cultivars
between sites and, more importantly, between cultivars could be identified. In recent
years amplicon sequencing technology became an important tool in rhizosphere mi-
crobiology and revolutionized the field. With increasing read length and sequencing
depth this technology now allows analyzing multiple replicates as previously done
by DGGE to determine the effect of various biotic and abiotic factors on the micro-
bial community composition in the rhizosphere (Marques et al. 2014; Schreiter et al.
2014a). The community composition analysis done by pyro- or illumina sequencing
30 Microbial Communities in the Rhizosphere . . . 293

at a much higher resolution level largely confirmed data obtained by DGGE. The
main advantage of amplicon sequencing is that at the same time insights into the
taxonomic composition and identification of genera differing in relative abundance
depending on the treatment becomes feasible. Although the assignment to species
level is only achieved for a fraction of the sequence reads, the situation will improve
with increasing read lengths. However, researchers should keep in mind that there
is a large diversity beyond the 16S rRNA gene level (Eltlbany et al. 2012). Recent
insights come from the determination of the plant microbiome by direct sequencing
of DNA (metagenome) or cDNA (metatranscriptome). Presently, a major limitation
of the direct sequencing approach is that typically no replicates were sequenced.
The enormously large sequence data sets can provide insights into metabolic path-
ways, plant effectors, and mobile genetic elements (MGE) which can be the basis
for generating new hypotheses.
The TC-DNA can be also used to quantify the abundance of beneficial or plant
pathogenic bacteria by PCR-Southern blot hybridization (Eltlbany et al. 2012). The
presence of antibiotic resistance genes and MGE in TC-DNA can be determined
by quantitative real-time PCR (qPCR) and Southern blot hybridization. The latter
approach was shown to be more sensitive and specific than qPCR but remained semi-
quantitative. Quantitative real-time PCR should be done, if possible, with Taqman
probes instead of Evagreen in order to achieve a high specificity.

30.4 Main Findings Obtained by Molecular Analysis

of Rhizosphere Plant Species and Growth
Stage-Dependent Diversity

DGGE fingerprints of bulk soil and rhizosphere samples from strawberry, oilseed
rape and potato plants that were grown in a randomized plot design at the same
field site revealed an enrichment of specific bacterial populations in the rhizosphere
(rhizosphere effect) and plant species-dependent bacterial community composition
(Smalla et al. 2001; see Fig. 30.1). Bulk soil fingerprints were characterized by
many equally intense bands indicating a high evenness while in the rhizosphere fin-
gerprints of the several stronger bands were detected, indicating an enrichment of
some populations in response to root exudates and a reduced evenness. Some bands
showed a plant species-dependent enrichment. Bands that were detected only in the
rhizosphere fingerprints of strawberry plants were identified after cloning of the re-
amplified PCR products and sequencing indicated an enrichment of Actinobacteria in
response to the growing strawberry plants. Furthermore, the early studies by Smalla
et al. (2001) and Gomes et al. (2001) already showed that different plant develop-
mental stages were characterized by different bacterial community compositions.
This finding was also observed for lettuce grown in three soils by means of amplicon
sequencing (Schreiter et al. 2014a) . When strawberry and oilseed rape plants were
grown at different field sites, the rhizosphere fingerprints were influenced by both
the site and the plant species. Interestingly, the actinobacterial DGGE fingerprints of
 294

Fig. 30.1 The effect of Pseudomonas jessenii RU47 (RU47) and Bacillus amyloliquefaciens FZB42 (FB01) on total bacterial community in rhizosphere obtained
by DGGE. The unweighted pair group method with arithmetic mean (UPGMA) analysis of this gel revealed a strong effect of the plant species on the composition
of the rhizosphere bacterial community
S. Schreiter et al.
30 Microbial Communities in the Rhizosphere . . . 295

strawberries grown at three sites displayed highly similar actinobacterial community

compositions indicating that Actinobacteria did strongly respond to the strawberry
exudates (Costa et al. 2006a). Similarly, Pseudomonas populations were enriched in
the rhizosphere of strawberries as revealed by sequencing of bands from the Pseu-
domonas fingerprints from strawberry. The sequences of populations enriched in the
strawberry rhizosphere grown at three sites were identical to those from isolates with
in vitro antagonistic activity towards Verticillium dahliae (Costa et al. 2007).
Plant Genotype-Dependent Diversity In contrast to the effect of the plant species
and the site, the influence of the plant genotype on the microbial community com-
position in the rhizosphere is much more subtle. In order to investigate the effect of
transgenic potato plants, five different potato cultivars and two transgenic lines grown
at two sites were investigated by DGGE fingerprints (Weinert et al. 2009). Transgenic
potatoes were found to be in the normal range of variability among different culti-
vars. PhyloChip analysis revealed that OTUs differing between cultivars belonged to
the Pseudomonadales, Enterobacteriales and Actinomycetales (Weinert et al. 2011).
Moreover, the bacterial community compositions in the tuber rhizosphere of three
sweet potato genotypes were recently compared by DGGE and amplicon sequenc-
ing of 16S rRNA gene fragments. While DGGE fingerprints showed only minor
plant genotype-dependent differences at both sampling times, amplicon sequencing
allowed identifying plant genotype-specific populations which were linked to low
starch content. However, in the tuber rhizosphere of all plant genotypes, Bacillus
and Paenibacillus were significantly enriched compared to bulk soil.
Site- and Soil Type-Dependent Microbial Diversity DGGE and amplicon se-
quencing analysis was used to analyze the effect of soil types on the microbial
community composition in the rhizosphere under field conditions. Earlier studies
from our group had already provided insights into the effects of different sites (Heuer
et al. 2002; Costa et al. 2006a; Weinert et al. 2009) as it was assumed that the mi-
crobial community composition was not only influenced by the soil type but also
by cropping history, agricultural management practices and climate. The study by
Schreiter et al. (2014a) was the first to show under field conditions that three soils
that had been kept in an experimental plot system under identical cropping history
and weather conditions at the same field site for more than 10 years still displayed
a distinct bacterial community composition in the rhizosphere of lettuce, indicating
that the soil properties (mineral and organic composition, pH) are indeed a major
factor shaping the microbial community composition in the rhizosphere.
Taxonomic Composition Cloning and sequencing of 16S rRNA gene fragments
amplified from TC-DNA of the rhizosphere of three potato varieties grown at two
sites showed a similar composition of major phyla and classes in the potato rhi-
zosphere with the phylum Proteobacteria being the most abundant, followed by
Acidobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Verrucomicrobia. In-
terestingly, despite the low coverage (approx. 150 sequences per site) sequences
affiliated to the Acidobacteria, Verrucomicrobia or phylum TM7 were detected in
the rhizosphere of potatoes of all cultivars and from both sites (Weinert et al. 2011).
296 S. Schreiter et al.

Cultivation-independent analysis clearly showed that these organisms which are dif-
ficult to culture are abundant in the rhizosphere but their role, e.g. in the dialogue
with the plant, still needs to be revealed. Amplicon sequencing of 16S rRNA gene
fragments from the rhizosphere of lettuce grown under field conditions in three soils
revealed that Proteobacteria were strongly enriched in the rhizosphere of lettuce in
all three soils compared to bulk soil while the relative abundance of Actinobacteria
decreased. Several genera such as Sphingomonas, Variovorax, Pseudomonas, and
Rhizobium were enriched in the rhizosphere of lettuce, independent of the soil type.
Many dominant OTUs (defined at 97 % sequence identity) in the rhizosphere of let-
tuce were shared among the three soil types although some were soil type-specific
(Schreiter et al. 2014a). Whereas in the tuber rhizosphere of sweet potato in partic-
ular the relative abundance of Firmicutes (Bacillus and Paenibacillus) was enriched
compared to bulk soil (Marques et al. 2014).
Effects of Inoculants DGGE fingerprints and amplicon sequencing of 16S rRNA
gene fragments were also used to investigate the effect of inoculants on the indigenous
microbial communities in the rhizosphere (Götz et al. 2006; Adesina et al. 2009;
Grosch et al. 2012; Xue et al. 2013). Compared to the effect of the plant species, the
soil type or the year-to-year variation, inoculants influenced rhizosphere microbial
communities to a lesser extent. Interestingly, the composition of the indigenous
microbial community was most strikingly influenced by a mixture of Trichoderma
viridae and Serratia plymuthica.
Detection of Antibiotic Resistance Genes and Mobile Genetic Elements The
importance of horizontal gene exchange for short-term bacterial adaptability and
successful colonization of new ecological niches has only recently been fully rec-
ognized (Heuer and Smalla 2012). The rhizosphere provides a natural hot spot of
horizontal gene transfer as nutrient availability, bacterial cell numbers and activity
are increased compared to the bulk soil. We used PCR-Southern blot hybridization
and qPCR to detect resistance genes and MGE-specific sequences. We could show
that the abundance of sulfonamide resistance genes (sul1, sul2) was unexpectedly
lower in the rhizosphere of maize and grass grown in manure-treated soils compared
to control. Another interesting observation was the enrichment of class 1 integrons
and IncP-1 plasmids in the rhizosphere of lettuce grown in three soils (Jechalke et al.
2014). This increased abundance might be caused by aromatic compounds in the
root exudates of lettuce (Neumann et al. 2014).

Conclusion

The analysis of TC-DNA from the rhizosphere became an important component of the
tool set available in rhizosphere microbial ecology and provided important insights
of practical relevance, e.g. for plant breeding or biocontrol. Conclusions from 16S
rRNA gene based analysis should be drawn cautiously as the resolution level is
limited and diversity beyond 16S rRNA gene sequences is high and not captured.
Therefore, methods analyzing TC-DNA should be combined with microscopy and
30 Microbial Communities in the Rhizosphere . . . 297

cultivation approaches. Likely, new image analysis tools and sensitive chemical
detection methods will be more and more integrated to better understand the complex
interactions in the rhizosphere.

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Chapter 31
Visualization of Plant-Microbe Interactions

Massimiliano Cardinale and Gabriele Berg

Abstract Plants form complex mosaics of different microhabitats, each of them

colonized by adapted microorganisms ranging from beneficials to pathogens. Since
only a minor fraction of environmental microbes can be cultivated in the laboratory,
molecular methods are usually employed to characterize the whole microbiome.
However, spatial information at micro-scale, which is fundamental to understand the
dynamics of host-microbe interactions, is usually lost during the sample process-
ing. Therefore, it is useful to complement the indirect molecular techniques with
direct visualization methods. Confocal laser scanning microscopy (CLSM) allows
the exploration of microbial habitats at a spatial resolution level unattainable with
molecular methods. In this chapter, we will show how CLSM played a fundamental
role in understanding plant-microbe interactions and their significance. Moreover,
this chapter is expected to be an inspiration for integrating microscopy with molecular
methods in research on plant microbiology.

31.1 Role of Microscopy in Modern Microbial Ecology

“One look is worth a thousand words”, the famous phrase attributed to Fred R.
Barnard (1921), expresses well the reasons why microscopy is a highly valuable
tool in microbial ecology and plant microbiology. Spatial information, crucial
for understanding microbial interactions, is irretrievably lost after sample process-
ing for molecular analysis (for example PCR- or metagenomic-based approaches),
because the spatial scale of microbes does not match that of the molecular methods.

M. Cardinale ()
Institute of Applied Microbiology, Justus-Liebig-University Giessen,
Heinrich-Buff-Ring 26–32, 35392 Giessen, Germany
Tel.: +49 641 99-37354
e-mail: Massimiliano.Cardinale@umwelt.uni-giessen.de
G. Berg
Institute of Environmental Biotechnology, Graz University of Technology,
Petersgasse 12, 8010 Graz, Austria
Tel.: +43664608738310
e-mail: Gabriele.berg@tugraz.at

© Springer International Publishing Switzerland 2015 299

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_31
300 M. Cardinale and G. Berg

Box 31.1 Basics of Confocal Microscopy and Image Analysis

Confocal laser scanning microscopes are hightech machines based on the

acquisition of fluorescent light and composed of hundreds of mechanical, op-
tical and electronic parts. Confocal microscopy allows the detection of (1)
fluorescently stained molecules, cells and tissues, and (2) organisms able
to synthesize fluorescent molecules, either naturally or after genetic engi-
neering. Usually, CLSM systems give also the possibility to add a further
(non-confocal) transmission light channel to the confocal stacks, such as a
Differential Interference Contrast (DIC) image. The “heart” of a confocal mi-
croscope is the pinhole. Here, the photons of light emitted by the specimen
are filtered, and only those coming from the focal plane can reach the de-
tector (photomultiplier) that converts them into an electron flow, which is
finally digitalized by the software. The result of this process is an extremely
sharp image, representing an “optical slice” of the sample. The thickness of
this optical slice depends on both the magnification and the pinhole diame-
ter. Successive optical slices sequentially acquired along the Z-axis form a
confocal stack, which can then be used to create bi-dimensional projections,
or visualized in the three-dimensional space. Proprietary software, such as
Imaris (Bitplane, Zuerich, Switzerland) and AMIRA (TGS Inc., US), as well as
freeware, such as DAIME (www.microbial-ecology.net/daime/), Image Surfer
(http://imagesurfer.cs.unc.edu/) and ImageJ implemented with appropriate plu-
gins (http://rsbweb.nih.gov/ij/plugins/) allow imaging and quantitative analysis
of confocal stacks. Dedicated software tools can create isosurfaces and spheres,
three-dimensional objects that replace the original fluorescent signals. Such
three-dimensional models facilitate the investigation of host-microbe and
microbe-microbe relationships by dramatically increasing the resolution level
of the image.

Moreover, microorganisms interact with the environment as whole cells and at a

micro-scale. Advanced microscopy methods such as electron microscopy, CLSM,
Raman spectroscopy, super resolution microscopy, atomic force microscope (AFM)
show the world of microbes as observed by microbes or their hosts.
Molecular methods deliver significant results, as it is possible to process numerous
representative (pooled) samples in parallel, which allow robust statistics. In the past
few years, microbial ecology was revolutionized by affordable deep sequencing and
-omics technology (see Chap. 30), which allowed the study of natural microbiomes
at an unprecedented level of depth. Once more, the effect was that the scientists’
attention was focused on the sequence-based information delivered by the new tech-
niques, and microscopy was consequently overshadowed. The direct visualization
at micrometric scale delivers qualitative and quantitative information on bacterial
populations and their variation within the hosts’ tissues. However, a statistical ap-
proach in microscopy analysis of host-associated microorganisms is challenging,
31 Visualization of Plant-Microbe Interactions 301

and extremely time-consuming for two main reasons: (1) it is not possible to pool
samples, and (2) different samples cannot be run in parallel by the same operator, in
the same laboratory. Therefore, only the combination of indirect molecular charac-
terization of microbiomes with direct observation in situ allows drawing conclusions
that are more reliable. CLSM not only remains as a widely applicable methodol-
ogy for studying plant-microbe interactions, but also can be extended and integrated
by other microscopic techniques, and therefore serves as a central technology in
such studies. In the following paragraphs, after a technical box explaining basic
principles of the technique, we will discuss relevant topics and applications of con-
focal microscopy in plant microbiology, including analyses of host-colonization and
interactions with pathogens, critical methodological aspects, and integration with
complementary methods.

31.2 Application of CLSM to Plant-Microbe Interactions

Confocal microscopy is used in plant microbiology with different purposes. The ex-
tremely accurate localization of microorganisms within the host, along with targeted
staining of specific structures or taxa, allow gaining insights into their ecology and
interaction with the host (Cardinale 2014). Plant-associated microbes can exhibit
very diverse behaviors, such as ectophytic vs. endophytic colonization. With molec-
ular approaches, these two groups are often separated by surface sterilization of plant
material, but with CLSM it is possible to discriminate between different colonization
patters. This is relevant, because, in terms of interactions, it makes a huge difference
whether an endophytic bacterium is able to enter the plant cells, or it grows in the
apoplastic spaces only. In the first case, the closer intimacy is expected to confer to
this microorganism the ability to interfere more with the host, at both physiological
and genetic level. Such a case is represented by the colonization of root nodules,
usually considered the unique niche of rhizobial symbiosomes (Chap. 23), by non-
rhizobial symbionts, which were shown by CLSM to actually nodulate Cyclopia
ssp., Macroptilium atropurpureum and Mimosa pigra (Elliott et al 2007), and Paeni-
bacillus polymyxa (Annapurna et al. 2013). Such evidences force us to re-evaluate
the extent of microbial diversity in the context of plant-microbe interactions.
The identification of colonization strategies sheds light on the microbial ecology
of host-associated microbes, and this is important to understand the dynamics of
microbial establishment in plants. This relationship between a microorganism and
its host can indeed exhibit an unexpected level of specificity: Bacillus amylolique-
faciens FZB42 showed three different colonization patterns on three different host
plants (Fan et al. 2012). This suggests that it is imprudent to draw general conclusions
from single studies, and that each plant-microbe system may display unique features.
The interaction with microbes is apparently required for good plant fitness, since it
was recognized that probably all plants in nature host complex microbiomes (Vorholt
2012; Philippot et al. 2013). The nature of these associations and their importance
for the plant just began to be elucidated. Like in animals, the microflora associated
302 M. Cardinale and G. Berg

with the different plant organs seems to contribute to their correct functioning and to
the stability of the host’s immune system. Therefore, to investigate the niche special-
ization vs. ubiquity of a microorganism across the different plant microhabitats, can
help us in understanding its role and possible effects on plant fitness, especially if
coupled with appropriate molecular methods. Such investigations can be performed
under controlled conditions, for evaluating the specific behavior of single strains (or
known mixtures of organisms), or on plants grown in nature, in order to understand
the natural associations with the native microbiota. Bragina et al. (2012) showed
by fluorescence in situ hybridization (FISH)-CLSM that the hyalocytes (dead cells
which contain water) of Sphagnum mosses are the preferred colonization sites of
diverse bacteria. Complementary molecular studies demonstrated their potential in-
volvement in nitrogen fixation and methane degradation. The integration of these
results suggested that Sphagnum hyalocytes are not only water reservoirs, but in-
stead could represent “micro-bioreactors” for nutrient production, which support the
host growth. Comparative analysis of environmental samples can highlight the im-
portance of certain groups within the plant-associated microbiome, as shown in an
Egyptian desert farm for Bacillus and Streptomyces, which appeared to be recruited
by plants as effective biocontrol agents (Köberl et al. 2013; Chap. 28).
Intrusion of transport vessels at root level was shown already by CLSM for several
bacteria, such as Enterobacter gergoviae (An et al. 2006) and Bacillus subtilis (Ji et al
2008). The implication of such observations are particularly relevant in the case of
potential human pathogens, such as Enterobacteriaceae, which were already proven
to persist in the soil, colonize crop roots, and from there move to the edible parts of
the plant (Cooley et al 2007). Thus, shedding light onto the mechanisms underlying
bacterial intrusion and dynamics of their internal translocation can improve food
safety by development of targeted measures for outbreak prevention. Escherichia
coli was recently shown by CLSM to colonize Lactuca sativa (green lettuce) leaves
preferentially beneath the epithelium (Chap. 44); similarly, Salmonella enterica in-
trudes the lettuce leaves via the open stomata (Kroupitski et al. 2009) and resides
in the endosphere. This niche specialization may explain why conventional washing
fails in removing pathogens from raw leaves, which may cause the recurrent bacterial
outbreaks in lettuce.
Pathogens represent a special case of plant-microbe interactions; their virulence
relies on a combination of genetic and ecological traits, since the infections usually
target specific plant tissues, and occur only when the responsible microbial genes
are expressed. Only when the density of the pathogen exceeds a certain critical level,
the plant response will not succeed in containing the disease. CLSM was used as a
tool to investigate such synchronized mechanisms, thus contributing to understand
the dynamics of the infection process. The mechanism responsible for the Xylella
fastidiosa-induced degenerative disease of Vitis vinifera was identified as the vessel-
to-vessel movement of the bacterial cells (Newman et al. 2003), and the modulation
of vir gene expression in Agrobacterium tumefaciens during the infection process
(Chap. 37) was studied after downstream insertion of gfp genes (Li et al. 1999).
CLSM helps elucidating the mechanisms of action of beneficial microbes. Plant
growth promoting bacteria (PGPB; Chap. 22–29) and biocontrol agents (BCAs;
31 Visualization of Plant-Microbe Interactions 303

Chap. 18) caught the attention of the scientific community due to their promising
biotechnological potential for sustainable agriculture. They are regarded as environ-
mental friendly measures to replace chemical fertilizers and pesticides, respectively,
although unfortunately the positive effects observed in laboratory under gnotobiotic
conditions often disappear in the field. Rhizosphere competence (the ability to col-
onize plant roots stably) and endophytism are regarded as indicators of potentially
beneficial bacteria (Zachow et al. 2010). Interestingly, in several case studies, CLSM
showed that neither endophytism nor direct contact with the pathogen was the dis-
criminative feature of efficient biocontrol strains (Maldonado-González et al. 2013;
Gasser et al. 2012). Notably, it was also shown that an in vitro inefficient Collimonas
fungivorans strain, successfully reduced disease incidence in vivo (Kamilova et al.
2007). These results indicate that further research is needed to understand the mecha-
nisms of actions underlying the interactions with beneficial microorganisms, in order
to develop efficient bio-products with consistent results in the field (Chap. 32–34).
Lichens, traditionally considered as the typical example of mutualistic symbiosis
between fungi and algae/cyanobacteria, were intensively studied by CLSM, which
unexpectedly showed them to be microbial hot spots (Grube et al. 2009). A clear
succession of bacterial communities from young to older thallus regions suggests
that the lichens harbor a stable, active and functionally adapted microbiome, which
may contribute to the growth and survival under extreme conditions. Such CLSM
observations lead to the conclusion that the lichen paradigm should be reconsid-
ered: from bipartite symbiosis to autonomous mini-ecosystems supported by high
microbial diversity (Fig. 31.1).

31.3 Methodological Aspects of Confocal Microscopy in Plant

Microbiology

Interpretation of the Images Confocal stacks are usually presented as either maxi-
mum projections or single optical slices. For correct interpretation of the images, the
thickness of the confocal stack, as well as the Z-step dimension, must be given. The
scale bar alone does not inform about the spatial volume included in a bi-dimensional
projection of an image series. This is particularly important when analyzing endo-
phytic microbes, cell-cell interactions and huge micro-colonies. In the case of single
optical slices, the thickness of the section should also be mentioned. However, in
certain cases, a proper interpretation of the confocal images is only possible when
displaying the three-dimensional space, either as volume rendering of the signals or
as three-dimensional model made of isosurfaces and spheres, which can drastically
enhance the digital resolution of the image (Fig. 31.2).
Autofluorescence Biological tissues, including plant roots, are usually autofluores-
cent to some extent. Although in some cases it is useful to eliminate or reduce it
by specific pretreatments, more often it is suitable to exploit the genuine autoflu-
orescence, to achieve a spatial contextualization of the targeted microorganisms.
304 M. Cardinale and G. Berg

a b

Fig. 31.1 Bacterial colonization of the lichen Lecanora polytropa. a and b maximum projec-
tions of confocal stacks showing bacteria stained by FISH with the universal bacterial probe
EUB338MIX (red) and the Alphaproteobacteria-specific probe ALF968 (yellow). Fungal and algal
autofluorescence appear as blue/purple and green, respectively; scale bars: 25 μm. c volume-
rendering/isosurfaces three-dimensional model of panel a; EUB338MIX: cyan; ALF968: yellow;
lichen autofluorescence: red

This applies in most studies of plant-microbe interactions (see Figs. 31.1 and 31.2).
In multichannel confocal systems, one channel can be dedicated to the wavelength
range of autofluorescence. This requires preliminary CLSM observations of un-
stained samples, to define the appropriate autofluorescence range and its intensity
(Cardinale 2014). In case of relatively weak signals, signal accumulation during im-
age acquisition can subsequently help to properly visualize an autofluorescent host
matrix.
Combination of CLSM with Other Microscopy Techniques The optical resolu-
tion of the confocal microscopes is constrained by the physical properties of the
light. Therefore, fluorescent microscopy was used in a correlative approach in com-
bination with electron microscopy, in order to identify target objects first, and then
imaging them at nanometric scale (Jahn et al. 2012). A further correlative approach
already employed in medical sciences but not yet in plant microbiology, is the com-
bination of CLSM with AFM (Haupt et al. 2006), which could potentially deliver
structural information, such as interaction forces between beneficial microorgan-
isms, pathogens and hosts. Correlative FISH-CLSM with nano-SIMS (secondary
31 Visualization of Plant-Microbe Interactions 305

a b c

d e f
Fig. 31.2 Cells of Stenotrophomonas rhizophila P69 colonizing an emerging root hair in tomato
endophytically. Bright field image (a), CLSM stack (b, maximum projection; c, volume rendering)
and corresponding isosurfaces/spots three-dimensional model (d) with respective cutting planes
(e-f), to reveal the endophytic colonization; scale bars: 10 μm

ion mass spectrometry) could be of special interest, as this will provide information
on functional contributions of specific taxonomical groups of bacteria. Until now,
nanoSIMS has been used in plant-microbe interactions only to visualize differential
partitioning of 15 NH4+ between roots and soil microbial communities at nanometric
scale (Clode et al. 2009).

31.4 Conclusions

Investigation of host-microbe systems requires a polyphasic approach to untangle

the relationships and their ecological significance. As a complementary approach
to molecular methods, CLSM is best suited to both integrate and corroborate
results of -omics methodologies. Localization at microscale, colonization pat-
tern and cell-cell interactions are not detectable by cultivation, fingerprinting, or
deep-sequencing analysis, yet they represent the basic information necessary to
understand mechanisms and dynamics of plant-microbe interactions in microbial
ecology studies.
306 M. Cardinale and G. Berg

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introduction of Pantoea ananatis in the grapevine phyllosphere. Int J Wine Res 4:53–63
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diversity of bacterial communities in lichen symbiosis. ISME J 3:1105–1115
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biomedical research. Sci World J 15:1609–1618
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understanding in the biomedical and plant sciences. Micron 43:565–582
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Microbiol 9:1597–1603
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Lett 342:168–178
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 Part VI
Products for Plant Growth-promotion and
Disease Suppression
Chapter 32
Commercialisation of Microbes: Present
Situation and Future Prospects

Willem J. Ravensberg

Abstract Microbes are used in biopesticides and biostimulants. The definition of

biopesticides includes microorganisms, and beneficial arthropods; biopesticides are
developed for the control of biotic stresses which are caused by pests and diseases.
Biostimulants influence the plant’s responses to abiotic stresses. The market for
both categories of products is described. An overview of the biopesticides in Europe
is given as well as their targets and crops. Critical failure and success factors in
terms of commercialisation are discussed. The implementation of a product in an
integrated crop management system needs to be investigated in order to develop
guidance for proper use. Limiting factors and promoting trends determine the growth
of the markets for these products. Continued growth is expected since alternatives to
chemicals are demanded by legislation and consumer demand for residue-free food.
Products based on bacteria are anticipated to increase the most for disease and insect
control as well as for plant growth.

32.1 Definitions of Biopesticides and Biostimulants

The Use of Biopesticides The commercial use of products based on living organ-
isms in agriculture started in 1938 in France with Sporeine. The bacterium Bacillus
thuringiensis was used as a biopesticide for control of caterpillars. Gradually, from
the 1960s on, products based on other bacteria, fungi and nematodes reached the
market, and the range of products for pest and disease control has grown ever since.
Today, the worldwide turnover of biopesticides is approximately 1.8 billion US$
(at grower level). Annual growth for the last decade has been around 15 % (CPL
2013). Precise figures on the global market are hard to find. This is partly due to
the various definitions used for biopesticides. These could include living organisms,
like beneficial arthropods and microorganisms, but also pheromones, natural and

W. J. Ravensberg ()
International Biocontrol Manufacturers Association (IBMA), Rue de Trèves 61,
1040 Brussels, Belgium
Koppert Biological Systems, Veilingweg 14, 2651 BE Berkel en Rodenrijs, The Netherlands
Tel.: + 31651410068
e-mail: willem.ravensberg@ibma-global.org; wravensberg@koppert.nl
© Springer International Publishing Switzerland 2015 309
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_32
310 W. J. Ravensberg

biochemical products, and even plant-incorporated genes. They are applied for bio-
control purposes in crops as well as beyond agriculture. Microbes used for plant
growth promoting purposes are defined as biostimulants.
Biopesticides There is no widely accepted use of the term biopesticide. Various
regulatory authorities have their own definition, some do not even use the word
biopesticide, but refer to active substances based on microorganisms. In EU legis-
lation the word biopesticide is not used. Regulation (EC)1107/2009 distinguishes
chemical substances and microorganisms and defines the latter as any microbio-
logical entity, including lower fungi and viruses, cellular or non-cellular, capable
of replication or of transferring genetic material. The USA EPA definition is as
follows: biopesticides include naturally occurring substances that control pests (bio-
chemical pesticides), microorganisms that control pests (microbial pesticides), and
pesticidal substances produced by plants containing added genetic material (plant-
incorporated protectants or PIPs). A microbial pesticide is a microbial agent . . . . . . .,
that: (1) Is a eucaryotic microorganism including, but not limited to, protozoa, al-
gae, and fungi; (2) Is a procaryotic microorganism, including, but not limited to,
Eubacteria and Archaebacteria; or (3) Is a parasitically replicating microscopic el-
ement, including, but not limited to, viruses. The Canadian authority PMRA uses the
following definition for a microbial pesticide: a naturally occurring or genetically
modified micro organism including but not limited to fungi, bacteria, and viruses.
The OECD has no formal definition for biopesticides despite having a BioPesti-
cide Steering Group (BPSG). Working practice covers microbials, semiochemicals,
botanicals and beneficial arthropods. The FAO describes a biological pesticide as –
A generic term, not specifically definable, but generally applied to a biological con-
trol agent , usually a pathogen, formulated and applied in a manner similar to a
chemical pesticide, and normally used for the rapid reduction of a pest population
for short-term pest control.
In this chapter I will use the term ‘biopesticide’ for products based on microor-
ganisms, arthropods and beneficial nematodes. A ‘microbial pesticide’ or simply a
‘microbial’ is used when a microorganism is the active substance. Products based on
arthropods (predatory mites, parasitoids, predatory bugs, lacewings, etc.) and ento-
mopathogenic nematodes (EPNs) are usually regulated in Europe by national laws.
They are often referred to as Invertebrate BioControlAgents (IBCAs) or ‘macrobials’.
Microorganims used to control pests and diseases are considered plant protection
products (PPPs) and need to be registered as such, following similar procedures as
chemical plant protection products. The EU definition for plant protection products
is, however, wide and includes the use of a substance influencing the life processes
of plants (Article 2.1b of Reg. (EC)1107/2009). That means that any microorganism
used for promoting plant growth, yield enhancement, etc. is considered a PPP. This
is not the case in the USA and Canada. The wider definition of the EU causes consid-
erable confusion and many microorganism-based products are not being registered,
but sold as biostimulants, biofertilizers, soil improvers, etc.
Biostimulants The EU as well as the biostimulant industry have taken the initia-
tive to solve this issue, and a revision of the Fertilizer Regulation (EC)2003/2003
32 Commercialisation of Microbes: Present Situation and Future Prospects 311

offers the opportunity to develop a regulatory framework that defines and covers bio-
stimulants. The proposed definition by the European Biostimulant Industry Council
(EBIC) is the following: plant biostimulants contain substance(s) and/or micro-
organisms whose function, when applied to plants or the rhizosphere, is to stimulate
natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance
to abiotic stress, and crop quality (http://www.biostimulants.eu). Biostimulants dif-
fer from crop protection products because they act only on the plant’s vigour and
do not have any direct actions against pests or diseases. When direct action against
biotic plant stress, like pests and diseases, is claimed the product is considered a PPP.
In order to have clarity on these claim related categories both (EC)1107/2009 and
(EC)2003/2003 need to be amended. This is foreseen to be enforced in 2016–2017.

32.2 Overview of Commercially Available Biopesticides

Several sources providing overviews of approved biopesticides are available on the

market. The 5th edition of BCPC’s Manual of Biocontrol Agents is an online database
(www.bcpcdata.com) that lists all biocontrol agents, mainly from the Western world.
In the EU Database (www.ec.europa.eu/sanco_pesticides/public/) all registered plant
protection products can be found. A list of active ingredients and products approved
in the USA can be found on the EPA website (www2.epa.gov/pesticide-registration).
Many competent authorities in the EU member states have similar websites. In the EU
there are currently 44 microorganisms approved (on strain level) as active substances,
11 are pending approval. In the USA approximately 110 strains are approved. CPL
(2013) reports the number of registered microorganisms for plant protection world
wide: 77 bacteria, 68 fungi and yeasts, 36 viruses and phages, and 2 protozoa, in
more than 2300 products.
Overview of Microbial Pesticides in Europe The approved and pending microor-
ganisms in the EU (in March 2014) are given below as well as the pest/disease targets
and the main crops in which they are applied. The products based on these active
ingredients are only approved in a given number of EU member states, depending
on the company’s market interests.
Fungi—for Insect and Nematode Control Three entomopathogenic fungi, Beau-
veria bassiana, Lecanicillium muscarium, Isaria (= Paecilomyces) fumosorosea,
have been approved for control of whitefly, thrips and some other soft-bodied insects
in greenhouse crops mainly. Metarhizium anisopliae is approved for control of black
vine weevil in soft fruit and tree nurseries. Paecilomyces lilacinus is approved for
control of root knot nematodes.
Fungi—for Disease Control The majority of approved fungi are Trichoderma (12
strains from 5 species) for use as a fungicide. The target diseases are predominantly
soil diseases caused by pathogens such as Fusarium, Pythium and Rhizoctonia spp.,
in greenhouse vegetables, herbs and ornamentals, turf, field vegetables, and some
are applied for control of wood diseases in grapes. Other fungi used against soil
312 W. J. Ravensberg

diseases are Gliocladium catenulatum and Coniothyrium minitans. The latter is used
to control Sclerotinia sclerotiorum. Fungi for control of foliar diseases are limited to
Ampelomyces quisqualis for powdery mildews in vegetables and grapes. Verticillium
albo-atrum is approved against Dutch elm disease, and Phlebiopsis gigantea against
Heterobasidion root rot of pine.
Bacteria—for Insect and Nematode Control Most bacteria are approved for con-
trol of caterpillars in greenhouse and field vegetables, in orchards, and in forestry.
These are Bacillus thuringiensis strains (11). Bacillus firmus and Pasteuria penetrans
are used to control several nematode species in a variety of plants such as soybean,
cotton, vegetables and turf.
Bacteria—for Disease Control Bacillus amyloliquefaciens and B. subtilis strains
are approved for disease control. They target similar diseases and crops as the above-
mentioned Trichoderma’s. The same holds for Streptomyces K61 and Streptomyces
lydicus. Pseudomonas chloroaphis can be used for the control of diseases in cereals
and Pseudomonas sp. ‘proradix’ for Rhizoctonia control in potatoes. B. pumilus has
been developed for control of powdery mildew in greenhouse crops and grapes.
Viruses—for Insect Control Four species of baculoviruses have been approved for
control of caterpillars like the codling moth (Cydia pomonella) in apples and pears,
the beet army worm (Spodoptera exigua), the cotton leaf worm (S. littoralis) and
the cotton boll worm (Helicoverpa armigera) in greenhouse vegetables. Two plant
viruses have been developed, as weak strains, for control of pepino mosaic virus and
zucchini yellow mosaic virus, in tomato and zucchini respectively.
Yeasts—for Disease Control Four yeast-based products have been developed for
control of diseases. Aureobasidium pullulans is approved for fire blight control
in pome fruit, Candida oleophila for post harvest diseases (Botrytis cinerea (grey
mould), Penicillium expansum (blue mould)) in apple and pear. Pseudomyza floccu-
losa is targeted at powdery mildew in roses and greenhouse vegetables. Registration
for baker’s yeast Saccharomyces cerevisiae is pending as a systemic resistance in-
ducer aimed at bacterial and fungal (downy mildew, botrytis) diseases in field and
greenhouse vegetables.
Microbes used beyond Agriculture Microbial pesticides are also applied in
forestry, in amenity, in home and garden, for vector control (mosquitoes) as well
for exoparasites on livestock. Uses in forestry are considerable for control of cater-
pillars like the gypsy moth and for sawflies. The use in turf for soil diseases is
increasing.
Invertebrate Biocontrol Agents Commercial augmentative biological control is
conducted by releasing invertebrate biocontrol organisms. There are about 230
species of the orders of Nematoda and Arthropoda, one species of Mollusca and
one Chilopoda (Van Lenteren 2012). All these IBCAs are used for control of insect
and mite pests, none for plant parasitic nematodes or diseases. One beneficial nema-
tode (Phasmarhabditis hermaphrodita) is applied for control of slugs. Furthermore,
9 species of beneficial nematodes (5 Steinernema and 4 Heterorhabditis spp.) are
32 Commercialisation of Microbes: Present Situation and Future Prospects 313

used for control of soil-dwelling stages of insects, mainly. The majority of IBCAs are
Hymenopteran parasitoids (120), predatory mites (30), and species of Neuroptera,
Heteroptera, Diptera and Coleoptera.
The first use of augmentative biocontrol ocurred in 1902. The expansion of the
number of used species took place in the 1980 and 1990s. The world market of
macrobials is estimated at 480 million US$ in 2011 (CPL 2013), most IBCAs are
sold for greenhouse crops in Europe.
The Market for Biostimulants There are neither reliable sources on the number
of biostimulants available nor is there a consistent and accepted definition for this
type of products. Products are marketed as soil improvers, biofertilizers, organic soil
amendments, biostimulants, etc. They can be substances, microorganisms or com-
plex mixtures. According to EBIC, the European market for biostimulants reached
500 million € in 2013 of which a part consists of microbes. The area of use in Europe
is estimated at 3 million ha with multiple applications. EBIC foresees a steady annual
growth for biostimulants of 10 % or more. In the USA the BioStimulant Coalition
has been founded with similar goals as EBIC. No data on the use and market of
biostimulants in the USA are available.
Biostimulants based on microorganisms often contain multiple species. A general
purpose is solubilizing nutrients like nitrogen and phosphorus for an improved nu-
trient uptake by the plant, production of hormones for plant growth promotion, yield
enhancement, and making the plant more resilient towards abotic stresses caused
by drought and salt. Here, similar bacteria and fungi are used as in plant protection
products such as Bacillus, Pseudomonas, Rhizobium and Trichoderma spp. as well
as mycorrhizae.

32.3 Critical Failure and Success Factors

The annual growth in the biopesticide market is currently around 15 % and the
prospects are promising. Still it requires a great amount of effort to achieve a suc-
cessful commercialisation. Decisive is customer satisfaction based on consistent
performance of the product in relation to the costs and ease of use. Many factors de-
termine success or failure of a product. The disadvantages of microbial pesticides are
frequently reported as the cause of why biopesticides only reach a small percentage,
today 3 %, of the total crop protection market which was approximately 60 billion
US$ in 2013.
Strengths, Weaknesses, Opportunities and Threats The success or failure of a
microbial is determined by the attributes of a biopesticide. The chance for success,
however, is not only dependent on the product, but also on the macro-environment in
which it is used. A SWOT (strengths, weaknesses, opportunities and threats) analysis
can be made of these internal and external factors in general, and for each product
(Table 32.1). It cannot be ignored that the list of weaknesses of biopesticides is long,
which presently often restricts their use to niche markets. On the other hand, biopes-
ticides have unique strengths, and the opportunities to exploit these will increase.
314 W. J. Ravensberg

Table 32.1 SWOT analysis for biopesticides

Strengths Weaknesses
Unique mode of action Speed of kill slow and insect stage-dependent
Specific host range Narrow target spectrum
No residue (exempt of MRL) Efficacy moderate and variable
No or short pre-harvest interval Short residual activity
No or short worker re-entry interval Sensitive to abiotic factors
Compatible with natural enemies, pollinators Limited storage stability (at low temp.)
Compatible with microbial pest control agents More complicated application technology,
spraying necessary, good coverage essential,
often direct contact needed
Excellent tool in IPM systems Incompatible with chemical pesticides
Excellent tool in resistance management Knowledge-intensive products
programmes
Probability of (cross-) resistance low Often only work as part of IPM programme
Safe for humans and the environment Relatively high end-user price
Safe for plants
Approved for organic production
Opportunities Threats
BCAs are a natural, renewable resource Novel safer chemical pesticides
Environmentally benign, no environmental Growers’ scepticism based on experience with
pollution due to production and use chemicals
Increase or maintain biodiversity Only for niche-markets
Developmental costs low to reasonable Increasing regulatory burden
Demand for residue-free food Transgenic crops
Withdrawal and reduction of chemicals Biodiversity and access benefit sharing regula-
tions
Combination with chemicals extend the product Bioterrorism regulations
life of resistant-risk pesticides
Growing of sustainable agriculture and IPM Fear of microbes by the public
Organic food production Limited public research funding
Increasing availability of high quality Lack of user education and extension services
biopesticides
Increasing users’ confidence in biopesticides Weak economic position of farmers
Increasing number of sector where use of chem- Unregistered ineffective “snake oils”
icals is forbidden such as forestry and amenity
areas
Import of low quality products
32 Commercialisation of Microbes: Present Situation and Future Prospects 315

Still, robust field performance is the key factor. Users may decide to use a biopesti-
cide for other reasons such as a short re-entry period or pre-harvest interval, or for
resistance management. Market demands to deliver residue-free produce becomes a
strong incentive to use microbials. Still, costs and efficacy remain the most important
decision factor for a grower.
The Development of a Microbial Pesticide The development of a biopesticide is an
extremely complex process that includes many phases such as discovery, production,
product development, efficacy testing, registration and finally commercialisation (see
Chap. 33). Developmental time can be between 3–5 years and registration likewise.
It will cost many millions of Euros and adoption in the market and the time to a con-
siderable sale volume usually takes a few years. It is therefore imperative that enough
resources are available as well as a thorough and long term company commitment to
allow for such a long and expensive product development process. The complete pro-
cess from ideation to successful commercialisation has been described by Ravensberg
(2011) (http://link.springer.com/book/10.1007%2F978-94-007-0437-4) for insect-
pathogens as microbial insecticides. Key factors of each phase in the process are
described there in detail, the model can be used for any kind of biopesticide.

32.4 Implementation in an Integrated Crop Management

Programme

Both biopesticides and biostimulants are used in agriculture where a multitude of

practices is applied. Proper imbedding of these products is needed for an optimal
use. Particularly the use of chemical pesticides may interfere with, or even kill, the
beneficial organisms.
Application Strategy In order to achieve the most effective results with a biopesti-
cide or biostimulant an optimal application strategy must be developed. For a good
field performance efficient delivery is essential, both in terms of efficacy and costs.
Furthermore, timing, frequency of the application and intervals between multiple
applications need to be established by field trials and in commercial settings. The
environmental conditions, host plant-mediated effects, crop and cultivation effects
influence the field performance, and for every situation testing is required before
proper advice on the use and expected results can be provided to the farmer. The
most influential factors must be identified and investigated.
Integrated Use Products will be used in an integrated programme which means
many variables and multi-trophic interactions need to be considered and studied.
Compatibility with chemical pesticides need to be investigated as well as with natural
enemies and pollinators. Where necessary a safe interval period has to be established.
For biostimulants, optimal use within a regime of use of artificial fertilizers requires
studies. Combined use of biopesticides, biostimulants, chemicals and IBCAs in an
integrated crop management programme needs to be studied in the developmental
phase of a product.
316 W. J. Ravensberg

Costs of an Integrated Programme Integrated crop management programmes can

become quite complex, and for adopting a new product into such a system, the
monetary aspect is a significant factor for a grower’s decision. This means not just
the product’s end-user price, but also the labour involved in the application and the
potential advantage in the market. Demonstrating the costs and benefits to the grower
is vital for the adoption of the product.
The Role of a Distributor The perception of biopesticides often is a barrier to
the adoption of such products, and the role of distributors is pivotal for a success-
ful biopesticide. Marrone (2007) recommended that biocontrol companies invest
in the education of customers and distribution channel partners to improve adop-
tion of biopesticides. Feedback on the customer’s satisfaction or complaints allows
improvements of the product and its use.

32.5 Conclusions

The driving forces in crop protection and crop health management are many and
varied, they continue to develop and alter. The following macro-environmental fac-
tors play an important role: governmental policy and its influence on crop protection
via legislation, the diminishing funding of research and extension, environmen-
tal programmes minimizing pollution, trading policies, biodiversity topics, and
supermarket demands.
Limiting Factors and Threats Research funding by governments is generally get-
ting reduced and without collaboration between academic institutes and companies
new product development is under threat. The Access and Benefit Sharing of genetic
resources and the “Nagoya Protocol” (Convention of Biological Diversity 1992)
(Cock et al. 2009) makes exploration for new microorganism an administrative
burden and threatens new product development. Biopesticides, integrated crop man-
agement and sustainable agriculture tend to be more expensive for farmers, while
agricultural returns are declining. “Food-scare”, i.e. the risk of a negative associa-
tion with insects and microbes (“germs”) on our food is a risk. The regulatory issue
remains to be critical, there is no or a too slow innovation and – as a consequence –
time to registration is too long and too costly.
Promotional Factors and Trends The demand for reduced levels of residue on food
(legislation, and consumer-retailer demands), the Global-GAP rules for growers, the
resulting supermarket competition on extra-legal residue requirements, resistance to
chemicals and the increase of organic agriculture favour biocontrol. Scientific and
technological trends create a flow of new ideas and results, e.g. insect pathogens used
as antagonists of plant diseases, endophytes against insects (Vega et al. 2009). Other
developments occur in strain improvement (hybridization, genetic modification),
production technology, and formulation (encapsulation, polymers, adjuvants).
The regulatory climate challenges the registration and use of chemicals more and
more. Directive 2009/128/EC on sustainable use of pesticides “encourages the use
32 Commercialisation of Microbes: Present Situation and Future Prospects 317

of alternative approaches. . . ..”, and EU Member States have to make a National

Action Plan showing how they will reach those political goals. As an example,
France launched an ambitious plan -‘Ecophyto’-that should lead to 50 % reduction
of chemicals in 2018. Furthermore, the Convention of Biological Diversity requires
governments to improve biodiversity and to mitigate negative impacts. Chemical
pesticides have a negative influence on biodiversity (Geiger et al. 2009). Directive
2009/128/EC states that the use of pesticides should be minimized due to biodiversity
concerns. National Action Plans should realize this goal.
New invasive pests continue to spread, and the lack of registered chemicals or
resistance creates chances for biocontrol agents. Examples in EU are the red palm
weevil, which can be controlled by nematodes, and the tomato leafminer which is
now controlled by a predatory bug in Spanish greenhouse tomatoes. The biopesticide
industry has made considerable progress in production, formulation, efficacy, quality,
and application strategy. Biopesticides have become more reliable and efficacious,
and markets are expanding, even in agricultural crops. Several large agro-chem
multinational companies acquired biocontrol companies indicating that biocontrol
is a serious trend in crop protection today.
Future Prospects The market for biopesticides is estimated to reach between 3–4
billion US$ in the next five years. The demand for sustainable agriculture and the
requirement of supermarkets and consumers for residue-free food is the incentive
for the use of biopesticides. I anticipate particularly growth in the use of bacteria
for insect and disease control, and of antagonistic fungi for disease control. The
biocontrol and biostimulant industry will continue to grow through new companies,
agro-chem companies’ activities, and expansion of the existing players. New oppor-
tunities, as described in this book, will lead to the development of new biocontrol
and biostimulant products for sustainable agriculture.

References

Cock MJW, van Lenteren JC, Brodeur J et al (2009) Do new access and benefit sharing procedures
under the convention on biological diversity threaten the future of biological control? BioControl
55:199–218
CPL (2013) Biopesticides worldwide market. Wallingford, UK
Geiger F, Bengtsson J, Berendse F et al (2009) Persistent negative effects of pesticides on biodiversity
and biological control potential on European farmland. Basic Appl Ecol 11:95–105
Marrone PG (2007) Barriers to adoption of biological control agents and biological pesticides. CAB
Reviews: perspectives in agriculture, veterinary science, nutrition and natural resources, vol.
2(51). CAB International, Wallingford, pp 1–12
Ravensberg WJ (2011) A roadmap to the successful development and commercialization of
microbial pest control products for control of arthropods. Springer, Dordrecht
Van Lenteren JC (2012) The state of commercial augmentative biological control: plenty of natural
enemies, but a frustrating lack of uptake. BioControl 57:1–20
Vega FE, Goettel MS, Blackwell M et al (2009) Fungal entomopathogens: new insights on their
ecology. Fungal Ecol 2:149–159
Chapter 33
Commercialization of Microbes: Manufacturing,
Inoculation, Best Practice for Objective Field
Testing, and Registration

Faina Kamilova, Yaacov Okon, Sandra de Weert and Katja Hora

Abstract Commercialization of plant growth stimulating and biocontrol microbes

plays a significant role in providing environmentally friendly and efficient alternatives
for chemicals used in agriculture and horticulture. This chapter describes principles
of the manufacturing process, including mass production and formulation, of mi-
crobes and specifies existing inoculation techniques. Moreover, since evaluation of
the efficacy of products is essential for estimation of their commercial potential and
because it is also required for their registration, rules and approaches used for the
official efficacy tests based mostly on guidelines of the European Plant Protection
Organization are included. In order to be placed on the market, microbiological plant
protection products and biostimulants/biofertilizers have to be registered. Therefore,
also requirements for registration of microbial products in different countries are
described and discussed.

F. Kamilova () · S. de Weert · K. Hora

Koppert Biological Systems, Veilingweg 14, 2651 BE Berkel en Rodenrijs, The Netherlands
Tel.: +31 10 5140546
e-mail: fkamilova@koppert.nl
S. de Weert
Tel.: +31 10 5140202
e-mail: sdweert@koppert.nl
K. Hora1
Tel.: +31 10 5140461
e-mail: khora@koppert.nl
Y. Okon
Department of Plant Pathology and Microbiology, Faculty of Agriculture,
Food and Environment, The Hebrew University of Jerusalem, 76100 Rehovot, Israel
Tel.: +972 8 9489216
e-mail: yaacov.okon@mail.huji.ac.il

© Springer International Publishing Switzerland 2015 319

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_33
320 F. Kamilova et al.

33.1 Introduction

Many rhizobacteria (such as strains of Azospirillum, Bacillus, Bradyrhizobium,

Pseudomonas, Rhizobium and Streptomyces) and fungi (such as Clonostachys and
Trichoderma) have the potential to directly promote crop yield, help plants to with-
stand abiotic stress, and/or protect plants from pathogens (Harman 2006; Jensen
et al. 2007; Lugtenberg and Kamilova 2009). Primarily studies on the identification
of the strains and eliminating of putative pathogens, on the evaluation of the mode
of action, and on laboratory and/or small scale efficacy testing can indicate the com-
mercial potential of such strains. Development of the manufacturing process and
efficacy testing of the product and its subsequent registration are critically important
stages for the successful commercialization of beneficial microbes for application in
agriculture and horticulture.

33.2 Manufacturing

The industrial manufacturing process includes several steps: the production of a

starting inoculum, small scale production, big scale (up-scale) production, harvest,
formulation and packaging.
Production After the selection of a microorganism with properties of interest and
proven to be safe for humans, animals and the environment, the screening for a well
defined production medium and for the establishment of the optimal process parame-
ters starts. The goals of industrial or mass production are to minimize the fermentation
costs and to produce the highest quantities of the microbe in the best physiological
and metabolic state. The latter can be illustrated by two examples. Firstly, Bacilli
have a relatively low yield and sporulation level which becomes a serious issue
during mass production. Combination of academic research with modification of
the cultivation process allowed reaching a maximum cell density of approximately
1.0 × 1010 CFU/g, with > 95 % sporulation of the cells. This achievement signif-
icantly stimulated the manufacturing of products containing B. amyloliquefaciens
(Junge et al. 2000). Secondly, for Azospirillum the capability to use intercellular
storage material such as polyhydroxybutyrate (PHB), is an important trait for plant
growth promotion (Dobbelaere and Okon 2007). Thus, industrial cultivation of bac-
teria under conditions providing intracellur accumulation of PHB, is a prerequisite
for a consistantly efficient Azospirillum inoculant.
Production of the starting inoculum begins from a pure culture, usually stored
in glycerol at − 80 ◦ C or as freeze-dried cells, and occurs in laboratory flasks. This
so called pre-culture is used for inoculation of small size (15–50 L) fermenters. The
resulting microbial culture is used for up-scale production. Industrial fermentation
can be done as solid or semisolid state cultivation or as liquid (submerged) cultivation.
During solid state fermentation, with little or no “free” water, the microorganisms
are grown on a solid carrier, either mixed with solid nutrient substrate or impregnated
33 Commercialization of Microbes 321

with liquid nutrient broth. Sometimes solid nutrient can also play the role of a carrier.
The final product includes microbial biomass, exometabolites and solid carrier with
or without remnants of nutrients. Advantages of solid state fermentation are the
use of relatively inexpensive substrates, continuality of the process and low waste
volumes. A disadvantage is the risk of microbiological contamination due to non-
sterile conditions typical for this type of cultivation. The process often includes a lot
of manual operations and thus high labour costs. Solid state fermentation is frequently
used for the production of fungal products, mostly because of the inability of some
fungi to produce robust spores in liquid culture.
Liquid or submerged fermentation allows a wide range of use of water-dissolved
substrates including refined and unrefined carbon and nitrogen sources. This pro-
vides more freedom for optimization of technological process. Other advantages of
liquid fermentation are (i) a high degree of process control due to the use of modern
bioreactors, (ii) a substantially reduced probability of contamination due to sterile
conditions and (iii) the microbial biomass can be separated from culture broth and
concentrated. A disadvantage of submerged cultivation is the high volume of waste.
The choice for either solid state or liquid fermentation is determined by the biology
of the strain and by the economy of up-scaling: low initial investments but higher
labor cost versus high investments and lower labor costs (Friedman 1990).
Formulation The goal of formulation is to preserve the microbial biomass obtained
after fermentation and to deliver the microbes in a good condition to their targets and,
after delivery, to enhance their activity (Burges 1998). The process of production of
dry formulations, such as powders and granules, depends mostly on the biological
properties of the microbial cells and on their ability to withstand the drying proce-
dures (Kamilova and De Bruyne 2013). Formulations may contain various additives
such as carriers, nutrients, stickers, protectants and emulsifiers. Among the most of-
ten used carriers are sterilized peat, conditioned cereal grains, talc, agricultural clays
and diatomaceous earth. Nutrients provide a primary boost for the microorganism
immediately after application of the product. Stickers allow microbes to attach bet-
ter to—and stay on—plant surfaces. Protectants defend microbes from desiccation,
UV light, and temperature changes. Microbial exometabolites that are directly or
indirectly involved in beneficial effects of strains can also be included in the formu-
lations. Microbial products can be formulated as (water or oil) suspensions, powder,
and wettable or insoluble granules. Three important factors determine the choice of
formulation type and composition: biological properties and characteristics of the
microorganism, inoculation techniques and types of irrigation systems involved in
the agricultural practice.
Packaging Only materials that secure intact biological, physical and chemical
properties of the end product during storage and transportation should be used for
packaging. The type of packaging is determined by the physical state of the formu-
lated product. The size of packaging can vary and depends on logistics and on market
demand in different countries.
322 F. Kamilova et al.

33.3 Inoculation

The choice of the inoculation technique depends on the mode of action of the ben-
eficial microbe, the plant growth stage at the time of application, and the type of
formulation. Powder and liquid inoculants can be applied either to the seed or directly
to the soil in the seeding furrow. Seed treatment can be performed by specialized
companies and treated seeds of some crops can be stored for a considerable period of
time. Certain crops can be treated by farmers at site, right before sowing. Granular
formulations are most suitable if slow release of a microbe is needed e.g. in situations
when multiple applications of the product is not feasible. Granular inoculants are
applied in-furrow by using suitable granular applicators. If water dispersible formu-
lations or liquid inoculants are applied directly to soil, they are first suspended in
clean potable water so that they can be evenly distributed over the cropping area by
hand or by mechanical spraying equipment. Suspended products can be delivered
directly to the root zone of individual plants by drip irrigation or by drenching fur-
rows. The latter techniques are particularly suitable for multiple applications during
crop cultivation.

33.4 Best Practice for Objective Field Testing

The term “efficacy” was coined by European legislation authorities to evaluate the
benefit of a plant protection product (PPP) in terms of quality and quantity of the crop
of interest under naturally occurring or artificially introduced disease conditions. It
also includes an estimation of the risk of a phytotoxic effect that a PPP might have
on the treated crop (if the product is applied in excessive amounts) and on adjacent
and successive crops as well as its effect on the quality of the products produced
from the treated crops.
The European Plant Protection Organization (EPPO) has set up a number of guide-
lines or standards (http://pp1.eppo.int/). Products can only come on the market after
their efficacy has been positively evaluated by government bodies. The guidelines
describe in detail what information needs to be presented in order to enable evalu-
ators to weigh the benefits and risks of PPPs. The European legislation uses these
guidelines to prescribe the producers of PPPs how they should test their products.
Two of these standards (PP1/181 and PP1/152) contain a summary of the basic prin-
ciples for the design and performance of agronomically and statistically sound field
trials (OEPP/EPPO 2012a, b). Governmental requirements for the demonstration of
efficacy vary between continents and individual countries. Europe tends to be most
strict, forcing the producer to demonstrate the efficacy level claimed on the label
of a product with a high number of field trials, executed according to the EPPO
guidelines by recognized research organizations (6–8 trials per EPPO climate zone,
for each unique crop/target combination). Canada, Australia, California and Florida
follow the European approach. Other Northern American states do not strictly re-
quire evidence of efficacy, but reserve the right to ask for it. Here, the aspects of
33 Commercialization of Microbes 323

safety of the product for health and environment are more important than efficacy.
Countries in South America, Africa and Asia differ widely in their legislation on this
point, requiring case-by-case studies and consultation with the authorities before
starting field trials. Besides basic guidelines, a number of standards for the individ-
ual combination crop/disease (pathogen) were developed by EPPO. It is clear that
these standards can not cover all possible combinations. Nevertheless, at least some
of them can be adapted or modified for trials with other crops and similar pathogens.
In case no specific crop/disease EPPO standard exists and modification of current
standards is impossible, a scientifically sound experimental protocol for appropriate
testing of the product against the disease of interest in the crop of interest should be
developed in collaboration with a testing institute.
For a good estimation of the efficacy of a product, field trials need to conform
to four basic principles. (i)Trials have to be carried out with the end product, in the
formulation as will be used by the grower.(ii) A dose range around the recommended
label dose should be included in the trial. Establishing the minimal effective dose
provides information on the effectiveness and safety of the product. Moreover, it will
also predict the approximate costs per hectare. This information will be very valuable
for the acceptance of the product by the farmers. Inclusion of higher dosages is equally
important to make sure that, in the event of accidental overdosing by the farmer, there
still will be no detrimental effects on the crop. (iii) Trials should be performed under
a wide range of environmental and agronomic conditions, preferably in the climate
where the product will be used. For example, one cannot compare the effects on the
crop of a large scale corn-grower in the United States with that of a small-holder
with a patch of maize in Africa. EPPO provides a table on comparable climates
on a global level in the standard PP1/269(1) (OEPP/EPPO 2010). To be able to
compare reports or papers on efficacy of microbial products in different geographic
localities, it is vital that the agronomic practice and climate conditions of the trial
are described in detail. (iv) The trial should be carried out as closely as possible to
the local agronomic practice.
The product should be evaluated in relation to farmer’s practice: when the farmer
is looking for a solution of his problem, his scope is wider than the application of
just one product. Effectiveness of the product may be increased or decreased in com-
bination with practices such tilling, the choice of fertilizers, pesticides/herbicides,
soil amendments (compost), irrigation, plant density, hygienic measures, crop rota-
tion, and the use of other products based on microorganisms. To be able to advise
the grower on the correct use of the product, and to be able to predict the effect
that a product will have in his situation, these aspects need to be addressed during
product development. Therefore, a detailed inventory of the local agronomic prac-
tice is important, and a multidisciplinary overview of the agronomic setting needs to
be kept.
What are the hurdles in efficacy trials of microbial products? Why are the full
benefits of the product not always observed in a well designed fully randomized
trial? This could be due to the generally small plot sizes (often practiced because of
economic restrictions), and the tendency to secure the investment in a trial. Testing
institutions generally try to increase this security by increasing the disease pressure
324 F. Kamilova et al.

in case of trials with products aimed at disease control, or growing the crop under
too optimal conditions in case of trials aiming at yield assessments. The level of
disease pressure can play a dual negative role in the evaluation of a product. For
instance, the benefit of root colonizing microorganism for soil borne fungal root
diseases might be hardly visible when the disease level is impractically high. This
will lead to rejection of a product that may have worked very well for growers that
maintain an economic threshold of 5 % disease incidence. On the other hand, a trial
with a disease incidence lower than 10 % in the untreated control may not lead to
statistically significant differences. Another example is that a microorganism meant
to improve phosphate uptake will not demonstrate its beneficial properties under
conditions which do not restrict the availability of phosphate for the plant. However,
a phosphate deficit should not cause severely compromised plants in the untreated
control since this would not reflect a realistic situation in the farms.

33.5 Registration

Plant growth promoting strains can posses properties that help plants (i) to fight bi-
otic stress (biocontrol) (see Chap. 18), (ii) to tolerate abiotic stress (draught, elevated
temperature, salinity) (see Chap. 27), (iii) to directly provide plants with essential
nutrients e.g. nitrogen (see Chap. 23),or (iv) to stimulate natural process which ben-
efit the efficiency of nutrient uptake e.g. of phosphorus (see Chap. 24). The first
category is represented by microbial PPPs, often called biopesticides. The last three
categories are represented mostly by plant strengtheners/ biostimulants and microbial
fertilizers. It seems that only bacterial strains directly involved in nitrogen fixation
e.g. Rhizobium, Bradyrhizobium and Azospirillum are easily fitting in the definition
of microbial fertilizers. For other PGP microorganisms a definition of biostimulants
is more suitable. Many biocontrol strains often combine antifungal activity with
stimulation of plant growth under unfavorable abiotic conditions. Whereas for the
researchers and end-users of the microbial products this combination of beneficial
properties looks very interesting and attractive, it seems to be complicated for the
regulatory authorities worldwide. Regulatory authorities apply to agricultural mi-
crobial products the same approach as to chemical products: PPPs and fertilizers
are regulated differently. So, despite the fact that many beneficial microbes combine
more than one beneficial trait, from the regulatory point of view currently micro-
bial inoculants can be assigned only as either biopesticides or as biostimulants/plant
strengtheners/biofertilizers. This requirement plays a critically important role in the
registration of microbial products and in the way the products can be placed in the
market. Companies should critically evaluate the properties and mode of action of
the potential microorganism from the point of view of the most consistently repro-
ducible effects and the marketability of the future product. More details can be found
in Kamilova and De Bruyne, 2013.
Plant Protection Products Registration of PPPs in the EU is regulated by EU Reg-
ulation 1107/2009 (2009). The procedure includes the authorization of the active
microorganism(s) and formulation(s). The registration dossier consists of two parts:
33 Commercialization of Microbes 325

Active substance (microorganism) dossier and Product dossier. Information on the

identity/biology of microorganism, on the methods used for production of the mi-
crobes as well as of the final product, on analytical methods used for the active
microorganism and formulation are parts of the registration dossier. Safety evalu-
ation of both a microbe and the end product includes data on human toxicology
and ecotoxicology, fate and behavior in the environment. Tests should be performed
by laboratories according to guidelines and standards of international organizations
such as Collaborative International Pesticides Analytical Council (CIPAC), Orga-
nization for Economic Co-operation and Development (OECD) or United States
Environmental Protection Agency Office of Chemical Safety and Pollution Preven-
tion (US EPA OCSPP), and International Organization for Standardization (ISO)
according to Good Laboratory Practice (GLP). Outside Europe these tests can be
asked to be performed according to specific national standards. Recognition of the
microorganism as safe on the EU level involves communications between company
applicant designated Rapporteur Member State, national agencies, and the European
Food Safety Authority (EFSA). This leads to publication of the scientific opinion by
EFSA. Based on this document, the European Commission makes a decision on in-
clusion of a microorganism into the list of safe active substances. Then a submission
of a Product dossier should be done at the national level. In the EU, efficacy trials
must be performed by authorized institutions according to Good Experimental Prac-
tice (GEP) in many respects based on EPPO standards in the intended geographical
Zone as described earlier in this chapter.
In the USA, registration of the active microorganism and the product is
regulated by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA,
http://www.epa.gov/agriculture/lfra.html) and implemented by US EPA. In most as-
pects related to data requirements for the registration dossier there is no difference
between USA and EU. However, a major difference is that in the EU efficacy trials
are an obligatory part of a Product dossier, while in the USA efficacy data are not
required by EPA. However, they are required by the regulatory authorities of some
states e.g. California and Florida.
African and South -American countries, China and Mexico do accepts data present
in the European or the US dossiers. However, all of them require efficacy trials
performed locally.
Biostimulants/Microbiogical Fertilizers Currently in Europe registration of mi-
crobial non-PPP inoculants is regulated at the national level. Details about the
situation in some EU countries has been described in Kamilova and De Bruyne
(2013). To harmonize the legislation basis for these products, the recently established
European Biostimulants Industry Council (EBIC) underlines that plant biostimulants
“contain substance(s) and/or micro-organisms whose function when applied to plants
or the rhizosphere is to stimulate natural processes to enhance/benefit nutrient up-
take, nutrient efficiency, tolerance to abiotic stress, and crop quality. Biostimulants
have no direct action against pests, and therefore do not fall within the regulatory
framework of pesticides” (http://www.biostimulants.eu). It is clear that inoculants
based on Bradyrhizobium, Rhizobium, Azospillum and other PGP microbes fall un-
der this definition. EBIC puts a lot of efforts to include biostimulants into Regulation
326 F. Kamilova et al.

(EC) 2003/2003, which regulates fertilizers in the EU in order to provide among

others safe and efficient microbial products. Nevertheless, it is still unclear how in
practice registration of these products will be regulated in the EU.
The situation in South America with microbial inoculants is somewhat less ob-
scure since all MERCOSUR countries (Argentina, Brazil, Paraguay, Uruguay and
Venezuela) signed “Recommendation on use of inoculants in agriculture” (MERCO-
SUR/GMC/RES # 28/98 1998), which includes the obligation to register inoculants
and to establish a “Reference Institution ” for each country, as well as defining its
responsibilities. Actually, each country has adapted its own regulations according to
this recommendation. The quality of microbial inoculants, particularly of rhizobia, is
a major concern of MERCOSUR countries. For example, the network REDCAI (In-
oculants Quality Control Network) was established as a member of the Division for
Agricultural and Environmental Microbiology of the Argentinian Association of Mi-
crobiology, with the aim of creating and validating a set of methodological tools for
the evaluation of inoculants. The Normative Instructions to the Fertilizer’s National
Laws in the MERCOSUR countries sets the official methods for inoculant analysis.
In these countries a the list of recommended and recognized strains does exist. MER-
COSUR may allow registration of an inoculant with strains still not included in the
said list, but the registrant company shall submit reports of efficacy under laboratory
and greenhouse conditions, together with data from at least 3 years of field trials
in three different agro-ecological zones. For other, non −(Brady)rhizobium strains,
companies must provide evidence on safety and efficacy. Efficacy trials have to be
performed in institutions appointed by the national governments.
The Canadian Food Inspection Agency regulates biofertilizers (to improve plant
growth) via Fertilizers ACT and Regulations C.R.C.666 (http://laws-lois.justice.
gc.ca/eng/acts/F-10/) paying attention to the safety for human and animal health,
and environmental as well as for the quality of the product. Under product quality
the number of viable cells sufficient for providing a significant beneficial effect is
meant. In Canada there are no specific requirements for efficacy data for biofertilizers.
In the USA there is no federal law regulating biofertilizers and microbiologi-
cal soil amendments. The Departments of Food and Agriculture of the individual
states regulate this type of products. Requirements may differ drastically: authorities
in some states can ask to perform local efficacy trials, while in other states only
notification of the fact that a product is on the market is sufficient.
In the countries of the Commonwealth of Independent States (CIS) the registration
of microbiological fertilizers as well as of PPPs is performed by the department of
Phytosanitary and Veterinary Surveillance at the Ministries of Agriculture. Efficacy
tests should be performed in the country of application. In general, all tests are
required to be performed by the national institutions or specialized laboratories.
However toxicological tests performed in the EU or in the USA after evaluation by
the designated national institutions can be accepted.
In the African countries and China, departments responsible for registration of
biostimulants/ biofertilizers may be different from those that regulate PPPs. However,
requirement to perform efficacy tests are common for these countries.
In conclusion, the increasing demand for safe and environmentally friendly mi-
crobial products as alternatives for chemicals stimulates the microbiological industry.
33 Commercialization of Microbes 327

Improvement of the production process, development of the most suitable and stable
formulation, and proof of efficacy of the products, will allow building up the market
and successfully introducing microbials particularly in areas where chemicals are ei-
ther ineffective or not available. Correctly performed registration will provide a legal
basis for the products and will therefore help to fit in—or create—new commercial
niches for beneficial microbes.

References

Burges HD (1998) Formulation of microbial biopesticides. Kluwer Academic, Dordrecht

Dobbelaere S, Okon Y (2007) The plant growth promoting effects and plant responses. In: Elmerich
C, Newton WE (eds) Associative and endophytic nitrogen-fixing bacteria and cyanobacterial
associations (Nitrogen fixation: origins, applications and research progress), vol V. Springer,
Heidelberg, pp 145–170
European and Mediterranean Plant Protection Organization (2010) PP 1/269 (1). Efficacy evaluation
of plant protection products. Comparable climates on a global level. Bulletin OEPP/EPPO
Bulletin 40:266–269
European and Mediterranean Plant Protection Organization (2012a) PP 1/181(4). Efficacy evalua-
tion of plant protection products. Conduct and reporting of efficacy evaluation trials, including
good experimental practice Bulletin OEPP/EPPO Bulletin 42:382–393
European and Mediterranean Plant Protection Organization (2012b) PP 1/152(4). Efficacy evalu-
ation of plant protection products. Design and analysis of efficacy evaluation trials. Bulletin
OEPP/EPPO Bulletin 42:367–381
Federal Insecticide, Fungicide, and Rodenticide Act. (1947) Last amended in 2003, Recent update
27 June 2012. http://www.epa.gov/agriculture/lfra.html. Accessed 15 Sept 2014
Fertilizers ACT and Regulations C.R.C. 666. (1985) Last amended on 28 June 2006. http://laws-
lois.justice.gc.ca/eng/acts/F-10. Accessed 1 Sept 2014
Friedman MJ (1990) Commercial production and development. In: Gaugler R, Kaya HK (eds)
Entomopathogenic nematodes in biological control. CRC Press, Boca Raton, pp 153–172
Harman GE (2006) Overview of mechanisms and uses of Trichoderma spp. Phytopathology 96:190–
194
Jensen DF, Knudsen BIM, Lübeck M et al (2007) Development of a biocontrol agent for plant
disease control with special emphasis on the near commercial fungal antagonist Clonostachys
rosea strain ‘IK726’. Australas Plant Pathol 36:95–101
Junge H, Krebs B, Kilian M (2000) Strain selection, production and formulation of the biological
plant vitality enhancing agent FZB24® Bacillus subtilis. Pflanzenschutz-Nachr. Bayer 1:94–104
Kamilova F, de Bruyne R (2013) Plant growth promoting microorganisms: the road from an aca-
demically promising result to a commercial product. In: de Bruijn FJ (ed) Molecular microbial
ecology of the rhizosphere, vol 1 & 2. Wiley, Hoboken, pp 677–686
Lugtenberg B, Kamilova F (2009) Plant-growth promoting rhizobacteria. Annu Rev Microbiol
63:541–556
MERCOSUR/GMC/RES. N◦ 28/98 (1998) Disposiciones para el comercio de inoculantes
visto: El Tratado de Asunción, el Protocolo de Ouro Preto y la Recomendación N◦ 9/97 del
SGT N◦ 8 “Agricultura”. http://www.mercosur.int/msweb/Normas/normas_web/Resoluciones/
ES/Res_028_098_Disp-Rel_Comercio%20Inoculantes_Acta%202_98.PDF. Accessed 14 Sept
2014
Regulation (EC) No 1107/2009 of the european parliament and of the council of 21 October
2009 concerning the placing of plant protection products on the market and repealing Council
Directives 79/117/EEC and 91/414/EEC Official Journal L 309, 24.11.2009 P. L 309/1-309/50
Chapter 34
Towards a New Generation of Commercial
Microbial Disease Control and Plant Growth
Promotion Products

Rainer Borriss

Abstract Biofertilizer and biocontrol formulations prepared from plant growth-

promoting bacteria are increasingly applied in sustainable agriculture. However,
these bioformulations of the first generation are sometimes hampered in their action
and do not fulfill in each case the expectations of the appliers. Unraveling specific
responses of selected model bacteria on the global level will greatly stimulate the
rational design of a new generation of biological fertilizer and biocontrol agents.

34.1 Plant Growth-Promoting Bacteria Used as Bioinoculants

Selective pressure on the microbial population in the rhizosphere is posed by the plant,
in the form of root exudates containing specific nutrients, secondary metabolites, oxy-
gen radicals (ROS), and other stress signals (Ramos-Gonzalez et al. 2013). Many
of the bacteria, which are able to overcome this selective pressure and to propagate
within the plant rhizosphere, act beneficial on plant growth [this process is designated
as plant-growth-promotion (PGP); Lugtenberg and Kamilova 2009), and, simultane-
ously, suppress growth of plant pathogens (this process is designated as biocontrol,
(BC); Haas and Défago 2005). Microbial inoculants, prepared from such rhizobac-
teria, are presently known under different names, such as bioeffectors, biofertilizers,
biostimulants, or biocontrol agents (biopesticides). In the following, I will use the
general term bioeffector, which includes the biological fertilizer and biocontrol func-
tion, since both functions are difficult to separate in plant-growth-promoting bacteria
(PGPB) (Kloepper et al. 1980).
Since the genomes of Pseudomonas fluorescens Pf5 and Bacillus amyloliquefa-
ciens FZB42 have been sequenced as the first representatives of Gram-negative and
Gram-positive bacteria with BC and PGP activity several years ago (Paulsen et al.
2005; Chen et al. 2007), our knowledge base about plant-bacteria interactions has
been steadily increased. Numerous genes and gene clusters that may contribute to a

R. Borriss ()
ABiTEP GmbH, Glienicker Weg 185, Berlin, Germany
Tel.: + 49 30 67057-14
e-mail: rborriss@abitep.de

© Springer International Publishing Switzerland 2015 329

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_34
330 R. Borriss

plant-associated life-style of beneficial rhizobacteria have been identified, and can

be used in developing improved bioeffectors.

34.2 The ‘Ideal’ Bioinoculant (Bioeffector)

Example Bt Despite increasing acceptance of bioinoculants, many features of this

environmental-friendly agents need to improve before they are widely used as a
promising alternative or substitute of chemical pesticides and fertilizers. Especially
variable application results hinder their further distribution. However, there is one
remarkable exception, which might serve as a guideline for further product de-
velopment: bioinsecticides. These products prepared from endospores of Bacillus
thuringiensis (Bt) and related species are by far the most successful biopesticides
(market share 79 %). Bt insecticides are most commonly used against some leaf-
and needle-feeding caterpillars. In recent years, there has been tremendous renewed
interest in Bt. Several new products have been developed, largely because of the
safety associated with Bt-based insecticides, their stability, and their narrow action
spectrum which is specifically directed to the target organisms, but is not harmful to
other insects. Moreover, the molecular features of their action are well investigated
(Chap. 20).
Bioeffectors of the Next Generation has to be compatible with routine field
practices under different field conditions and types of soil (Fig. 34.1), and should
fulfill the following tasks: (i) the formulation should be easy to handle and to place at
the target region (roots, leaves etc.); (ii) persistence at the plant; (iii) shelf-life should
last for at least one or two seasons; (iv) high efficacy, thereby yielding constant repro-
ducible results in the field based on known action principles; (v) safe: no undesired
side effects and not causing health problems for human beings and animals (e.g. skin
irritation); (vi) no negative impact on the environment and not affecting biodiversity
of concomitant microbial communities; (vii) not allowing development of resistance
of pathogens after application; (viii) economically feasible for the customer.

34.3 Manufacturing of Bioinoculants with Superior Stability

Candidate Microbes At present, representatives of three different groups of mi-

crobes seem to be attractive candidates for developing improved bioinoculants. (i)
Gram-negative Pseudomonas spp., such as P. fluorescens, are one of the most power-
ful colonizers of the plant rhizosphere and are able to enhance crop yield dramatically
and to control many plant diseases (Haas and Defago 2005; Chap. 18; Chap. 38).
(ii) Gram-positive Bacillus spp., such as B. amyloliquefaciens plantarum, are the
main constituents of many biocontrol and biofertilizer agents presently on the mar-
ket (Chap. 40). (iii) Last but not least, beneficial microfungi such as Trichoderma
spp., are also applied in agriculture to support plant growth and to suppress plant
pathogens.
34 Towards a New Generation of Commercial Microbial Disease Control . . . 331

Fig. 34.1 Bioeffectors can

be applied by using a
conventional seed drilling
machinery, AMAZONEN-
WERKE H. Dreyer GmbH &
Co. KG, working width of
6 m, row distance: 75 cm.
Under foot inoculation of the
bioeffector is performed
during maize seed drilling (by
courtesy of Dr. Frank
Eulenstein, ZALF e. V.,
Müncheberg, DE)

Other beneficial microbes, but without biocontrol action, such as rhizobia and
Azospirillum brasiliense (Chap. 23), mycorrhiza fungi (Chap. 25), and Sebacinales,
are not treated in this review.
Carriers Protect Bioinoculants However, in many instances, the numbers of cfu’s
in microbe suspensions without a proper carrier start to decline immediately after
drenching into the soil. Consequently, a major task in formulating inoculants is to
provide a more suitable microenvironment, combined with physical protection for
a prolonged period of time to prevent a rapid decline of the introduced bacteria.
Carriers used for inoculants should be preferentially sterile. They include many
different types, from cheap liquid, organic, inorganic, up to highly sophisticated
encapsulated formulations using several polymers. Very promising for durable and
efficient immobilization of inoculant(s) are alginate beads. Unfortunately, due to
relative high production costs, their use is restricted mainly to medical applications
(Bashan et al. 2014) .
Spore-Forming Microbes There is a possibility to circumvent the relatively
expensive use of carriers, when spore-forming microbes are used. Especially,
endospore-forming Bacillus spp are suitable for preparing simple liquid or dry for-
mulations in which it is not necessary to add stabilizing carriers (Borriss 2011;
Chap. 40). Dormant spores are naturally resistant against extreme temperatures, des-
iccation, ultraviolet radiation, and chemical disinfectants. Formulations consisting
of Bacillus endo-spores can be stored for nearly unlimited time, given that premature
germination of spores is avoided, e.g. by adding alcohols, such as iso-propyl alcohol
in concentrations of less than 1 %, or complete removal of germination stimulating
nutrients. Despite that, Trichoderma conidiospores do not reach the longevity of
Bacillus endo-spores although their shelf-life is much higher than that of vegetative
cells and allows the preparation of formulations with sufficient stability.
332 R. Borriss

34.4 Improved Inoculation Techniques

Appropriate Placement of a sufficient amount of the bioinoculant to the host plant

is crucial for the success of its application. Independent of the technique used, a
threshold concentration of the bioeffector is a precondition for its successful appli-
cation. For bacteria, cell numbers of at least 106 per plant/seed is recommended.
This figure corresponds roughly to 1013 –1014 cfu per ha. May be this figure could be
reduced when more target-specific placement techniques are developed. At present,
several techniques for inoculating bioeffectors are in use, but further development
and refinement is necessary for obtaining reproducible results in their application. It
ruled out that different bioeffectors have specific requirements. For example, experi-
ments performed with Bacillus amyloliquefaciens FZB42 revealed that it is necessary
to use endospores, not vegetative cells, when directly applied to plants. Further-
more, clearly better results in growth promotion of lettuce plants were obtained,
when the bioinoculant was applied twice, before and after transplanting, than the
corresponding amount only once.
Seed and Soil Inoculation Success in ‘coating’of seeds with bioinoculants depends
on proper adhesives and on the surface of the seeds. Therefore, this method works not
always appropriate, especially in small seeds with very smooth surfaces, to which
the bioeffector does not well attach. If the inoculation process damages seed coats
(e.g. in the case of peanuts) this may prevent germination. Some species release
anti-bacterial compounds from their seeds, which can inhibit the inoculant (Bashan
et al. 2014). A common soil inoculation technique is band application in which the
inoculant is placed directly in the seed furrow. Granulated or liquid bioinoculants
can be used. Using an appropriate machine, which allows combining the drilling and
inoculation processes, is desirable (Fig. 34.1). For more details about the inoculation
techniques presently recommended, the reader is referred to Chap. 33.

34.5 How to Improve the Plant Growth-Promoting

Function of Bioinoculants?

It is clear that the PGP effect exerted by every PGPB is due to many different features
within a complex network of plant-bacteria interactions. However, there is common
sense that the first precondition for beneficial actions on plant growth is the ability
to colonize plant roots (rhizosphere competence) and/or other (aerial) parts of the
plant.
Spore Germination and Root Colonization of Fungi, and Bacilli Chemotactic
movement towards roots, and competitive root colonization is essential for root col-
onizing microbes (Chap. 3). In case of plant-associated bacilli it was shown that
spores start to germinate when nutrients present in seed or root exudates become
available. Transposon mutants impaired in chemotactic motility and biofilm forma-
tion were unable to support plant growth, suggesting that rhizosphere competence is
34 Towards a New Generation of Commercial Microbial Disease Control . . . 333

a ‘conditio sine qua non’ for PGP. Therefore, selection of mutant or novel wild type
strains, especially aggressive in root colonization, is a promising tool for developing
enhanced PGPB (Kamilova et al. 2005)
Careful Characterization of the Molecular Principles Underlying PGP Al-
though our present knowledge about the different aspects of PGP is far from complete,
intensive efforts should be made for each PGPB to elucidate the molecular princi-
ples determining its beneficial effect on plant growth. For example, we have to know
whether the PGP effect is due to biofertilizer functions (mobilization of nutrients
for plant nutrition, fixation of nitrogen), general plant strengthening, or biostimulant
function (synthesis of plant growth hormones and/or volatiles as is the case in the B.
subtilis and P. fluorescens groups and so on. Only, when we understand, at least in
part, the mechanisms acting between a single PGPB and the host plant, the customer
will be in a position to use such bioinoculants successfully under the appropriate
conditions.
Mixtures of Different Bioinoculants There are many products on the biofertil-
izer market consisting of several microbes which are claimed to have a beneficial
effect on plant growth. The problem with such products is their variable quality.
It seems impossible to keep a constant product quality when vegetative cells of
beneficial Gram-negative bacteria, such as Pseudomonas, Rhizobium, Azospirillum,
and Agrobacterium, are mixed with dormant spores of Gram-positive bacilli and—
possibly—of some fungi. In addition, PGP effects coming from these beneficial
microbes can only be expected when threshold concentrations, e.g. at least 106 cfu,
become attached at the host plant, which is highly unlikely. There is a better applica-
tion perspective for products consisting of only two plant-beneficial microbes with
similar shelf life, which could act synergistically to each other. In the following a
few examples for promising combinations are given.
PGPB and Phosphate Solubilizing Bacteria Bacteria making available fixed
phosphates for plant nutrition (biofertilizer) are highly desirable (Chap. 24). Their
combination with PGPB should enhance their beneficial effect. An example for com-
bining two spore-forming inoculants is a bioformulation consisting of endospores
of the silicate bacterium Paenibacillus mucilaginosus, which is able to solubilize
phosphate and potassium from minerals, and the PGPB Bacillus amyloliquefaciens
plantarum. The combination was successfully applied in tobacco cultures in Yunnan
province, China.
Bacillus spp. and Trichoderma spp. Many PGP Bacillus spp., including B. amy-
loliquefaciens and B. subtilis, synthesize fungicidal lipopeptides under laboratory
conditions (Chap. 40). It is therefore surprising that mixtures of Bacillus spores and
Trichoderma conidiospores were proven to be extremely efficient bioagents under
field conditions. Possible explanations for ‘coexistence’ of both microbes are that
Bacillus is unable to produce reasonable amounts of fungicides in planta, or that
both bioinoculants occupy different niches within the plant rhizosphere, where they
act synergistically on the host plant.
334 R. Borriss

Mycorrhiza and Mycorrhiza Helper Bacteria (MHB) The same seems to be true
for mixtures of mycorrhiza fungi and Bacilli. It is known that mycorrhizal forma-
tion is enhanced by co-inoculation with MHB, e.g. Bacillus spp., which promote
rapid root colonization by ectomycorrhizal fungi. In addition, some MHB promote
the functioning of the mycorrhizal symbiosis. This was illustrated for three critical
functions of practical significance: nutrient mobilization from soil minerals, fixation
of atmospheric nitrogen, and protection of plants against root pathogens.
Combined Bioformulations Bioeffectors enhancing harvest yield of crops and veg-
etables are based on PGPB, but can also contain plant and seaweed extracts, humic
acids, strigolactones and other organic substances stimulating plant growth. It is
likely that synergistic effects occur when formulations consisting of living microbes
and such organic materials in specific combinations are applied to plants. Exploiting
these possibilities is still in an early stage of research.

34.6 How to Improve the Efficacy of Biopesticides?

B. amyloliquefaciens plantarum and P. fluorescens are prototypes for plant-associated

bacteria with BC activity. It is generally assumed that suppression of plant pathogens
is based on two features of these PGPR: (1) production of antibiotics, cyclic lipopep-
tides, and siderophores—all of which can be efficient against bacterial and fungal
pathogens -, and (2) induced systemic resistance (ISR), which stimulates the plant’s
defense system against harmful microbes and viruses.
Antimicrobial Compounds Secondary metabolites toxic to phytopathogenic fungi,
oomycetes and bacteria synthesized in P. fluorescens are phenazines (phenazine-
1-carboxylic acid, PCA; and phenazine-1-carboxylic acid, PCN), hydrogen
cyanide, the chlorinated tryptophan derivative pyrrolnitrin, and the polyketides 2,4-
diacetylphloroglucinol (DAPG), rhizoxin and pyoluteorin (reviewed in Chap. 18).
In addition, several cyclic lipopeptides (c-LPs) are non-ribosomally synthesized in
some representatives of the P. fluorescens species complex. B. amyloliquefaciens
plantarum devotes around 10 % of its whole genome capacity to the synthesis of
antimicrobial compounds. Non-ribosomal synthesized cLPs, such as iturins and
fengycin, are mainly directed against fungal pathogens, whilst the polyketides dif-
ficidin, macrolactin and bacillaene are mainly efficient against plant pathogenic
bacteria. Bacilysin and some bacteriocins are efficient against bacterial antagonists
(reviewed in Chap. 40).
Expression of Antimicrobial Compounds in the Plant Rhizosphere For a long
time the plant protective activity of PGPR has been correlated with the potential to
secrete a wide array of antibiotic compounds. However, in most cases this ability
was only demonstrated upon growth as planktonic cells under artificial conditions.
Notably, under environmental conditions, fluorescent pseudomonads are able to pro-
duce redox-active phenazines which contribute to the natural soil suppressiveness
34 Towards a New Generation of Commercial Microbial Disease Control . . . 335

of Fusarium wilt disease and may act in synergy with carbon competition by resi-
dent non-pathogenic F. oxysporum. Notably, PCA was detected at up to nanomolar
concentrations in the rhizosphere of wheat plants growing in the suppressive soils
near Lind and Ritzville, WA, U.S.A., suggesting that a natural antibiotic can be tran-
siently accumulated across a terrestrial ecosystem in amounts sufficient for the direct
inhibition of sensitive organisms (Mavrodi et al. 2012). By contrast, except the c-
LP surfactin, secondary metabolites synthesized by PGPR B. amyloliquefaciens are
hardly to detect within the plant rhizosphere, suggesting that the BC effect exerted
by the bacterium is not due to direct antibiosis (Debois et al. 2014). These findings
are important for future strategies for screening of powerful PGPR and BC strains.
It is known for a long time, that high efficiency in suppressing fungal or bacterial
pathogens under laboratory conditions do not necessarily reflect the potential of these
selected strains for their performance under field conditions. Also other mechanisms,
such as siderophore-mediated competition for iron, production of lytic enzymes (e.g.
chitinases), and induced systemic resistance play a role in disease suppression.
Induced Systemic Resistance (ISR) Representatives of PGPR, including Pseu-
domonas spp. and Bacillus spp, are able to trigger plant defense responses against
pathogens. Independent of the location of the bacteria triggering that plant defense
reaction, ISR is effective against pathogens infecting aerial or belowground parts of
the plant. Determinants of Pseudomonas involved in ISR are c-LPs, the siderophore
pseudobactin, salicylic acid, and 2,4 diacetyl phloroglucinol (Bakker et al. 2007). In
case of Bacilli, it seems that ISR stimulation is a multifactorial process dependent
on several compounds produced by the rhizobacteria. Candidate compounds are sur-
factin and volatiles, especially acetoin and 2,3 butanediol (Chap. 8). Taken together,
at least for BC Bacilli it is likely that ISR is more important than direct antibiosis
in suppressing plant pathogens. The recent findings about direct antibiosis and ISR
have to be taken into account when developing improved biopesticides.
Improved Biocontrol Formulations Many of the biocontrol formulations which
are currently in use are based on living microbes, such as Bacilli, Pseudomonas,
Trichoderma, and Paecilomyces. Unfortunately, for many of such formulations the
active principle(s) for their pathogen suppressing action it is not known. c-LPs, for
example, have been claimed to be responsible for the antimicrobial effects exerted
by B. subtilis. However, as shown above, it is very unlikely that the concentra-
tion of antifungal c-LPs (iturins and fengycins) within the plant rhizosphere reach
levels sufficient for antibiosis. A possibility to circumvent this problem are biofor-
mulations consisting of both, Bacillus spores and concentrated culture supernatants
containing antimicrobial metabolites (Serenade, Double Nickel 55, see Chap. 42).
Unfortunately, also in those products, only the number of spores is considered as
active ingredient of the biofungicide by regulators. In contrast to chemical fungi-
cides, there is no indication about metabolites and their concentration, except for a
specific treatment of pathogen-infected plant parts. Improved biopesticides rely on
both principles, direct antibiosis by antimicrobial metabolites, and ISR stimulated by
the plant-associated bacterium. In case that the bacterium does not produce sufficient
amounts of the antibiotic, when growing in planta, external metabolite ‘cocktails’
336 R. Borriss

should be added. But in that case it is necessary, for having a standardized prod-
uct quality, to guarantee a fixed concentration of the active principle for efficiently
suppressing the target pathogen. This would enable comparison of chemical and
biological pesticides.

34.7 Is There Any Perspective for GMO-Bioinoculants

in Agriculture?

We have to acknowledge that at present the use of genetically engineered bioinocu-

lants under field conditions is refused by the public, at least in Europe. It is therefore
not surprising that, by contrast to GM-crops (Chap. 15), until now no GM-PGPB
has been registered by the governmental authorities, neither in Europe nor in the
U.S. However, in the light of a steadily increasing world population, growing from
7 billion now to 8.3 billion in 2025, innovative approaches for getting higher har-
vest yields without using increasing amounts of agrochemicals should not longer be
excluded, given that their use is safe and without harmful consequences for human
beings and nature. Like for other releases, careful environmental studies are a pre-
condition before releasing genetically engineered bacteria into the environment. To
illustrate the possibilities of using of GMOs in agriculture, a short example will be
given here.
Expression of the Harpin Gene Enhances Biocontrol Activity of FZB42 The hrp
(“harp”) genes encode type III secretory proteins enabling many phytopathogenic
bacteria to elicit a hypersensitive response (HR) on non-host or resistant host plants
and induce pathogenesis on susceptible hosts. The HR is a rapid localized death of
the host cells that occurs upon pathogen infection and, together with the expression
of a complex array of defense-related genes, is a component of plant resistance. The
plant genes create a cascade of effects, which promote a Systemic Acquired Re-
sistance (SAR) throughout the plant. Beneficial effects on plant growth and health
have been reported. In the laboratory of Xuewen Gao, Nanjing Agriculture Uni-
versity, China, the PGPR B. amyloliquefaciens FZB42 was engineered to express
the harpin gene product by chromosomal integration of two hpa1 genes cloned from
Xanthomonas oryzae. Greenhouse experiments demonstrated efficacy of FZBHarpin
in bio-controlling of the disease rice bacterial blight in tobacco plants (Qiao et al.
2013).
34 Towards a New Generation of Commercial Microbial Disease Control . . . 337

References

Bakker PAHM, Pieterse CMJ, van Loon LC (2007) Induced systemic resistance by fluorescent
Pseudomonas spp. Phytopathology 97:239–243
Bashan Y, de-Bashan LE, Prabhu SR et al (2014) Advances in plant growth-promoting bacterial
inoculant technology: formulations and practical perspectives (1998–2013). Plant Soil 378:1–33
Borriss R (2011) Use of plant-associated Bacillus strains as biofertilizers and biocontrol agents. In:
Maheshwari DK (ed) Bacteria in agrobiology: plant growth responses. Springer, Germany, pp
41–76
Chen XH, Koumoutsi A, Scholz R et al (2007) Comparative analysis of the complete genome
sequence of the plant growth–promoting bacterium Bacillus amyloliquefaciens FZB42. Nat
Biotechnol 25:1007–1014
Debois D, Jourdan E, Smargiasso N et al (2014) Spatiotemporal monitoring of the antibiome
secreted by Bacillus biofilms on plant roots using MALDI Mass Spectrometry imaging. Anal
Chem doi:10.1021/ac500290s
Haas D, Défago G (2005) Biological control of soil-borne pathogens by fluorescent pseudomonads.
Nat Rev Microbiol 3:307–319
Kamilova F, Validov S, Azarova T et al (2005) Enrichment for enhanced competitive plant root tip
colonizers selects for a new class of biocontrol bacteria. Environ Microbiol 7:1809–1817
Kloepper JW, Leong J, Teintze M et al (1980) Enhancing plant growth by siderophores produces
by plant-growth-promoting rhizobacteria. Nature 286:885–886
Lugtenberg B, Kamilova F (2009) Plant growth-promoting rhizobacteria. Annu Rev Microbiol
63:541–556
Mavrodi DV, Mavrodi O, Parejko JA et al (2012) Accumulation of the antibiotic phenazine-1-
carboxylic acid in the rhizosphere of dryland cereals. Appl Environ Microbiol 78:804–812
Paulsen IT, Press CM, Ravel J et al (2005) Complete genome sequence of the plant commensal
Pseudomonas fluorescens Pf-5. Nat Biotechnol 23:873–878
Qiao J, Wu HJ, Huo R et al (2013) Construction of Harpin expression engineering strain FZBHarpin
and evaluation of its biocontrol activity. J Nanjing Agricult Univ 36:37–44
Ramos-Gonzalez M-I, Matilla MA, Quesada JM et al (2013) Using genomics to unveil bacterial
determinants of rhizosphere life style. In: de Bruijn FJ (ed) Molecular microbial ecology of the
rhizosphere. Wiley-Blackwell, Hoboken, New Jersey pp 7–16
Chapter 35
Important Organizations and Companies

Ben Lugtenberg

Abstract Many people, companies and organizations are professionally involved

in - or are interested in - the field of plant-microbe interactions and in the roles
microbes can play in making agriculture and horticulture more sustainable. These
include academic scientists, industrial professionals working in agriculture, horticul-
ture, biotech and food industry, as well as students, teachers, government officials,
decision makers and consumers who want to make themselves quickly familiar with
particular aspects of this broad field. In this chapter I have listed a rather randomly
chosen number of important organizations and companies.

Novozymes, Monsanto and The BioAg Alliance

www.novozymes.com; www.monsanto.com
As the global population’s rapid growth is set to continue, the need to signifi-
cantly increase agricultural output without increasing pressure on the environment
also grows. Microbial solutions enable farmers to drive yield and productivity in a
sustainable way. Deriving from various naturally-occurring microorganisms such as
bacteria and fungi, these solutions can protect crops from pests and diseases and
enhance plant productivity and fertility.
Microbial solutions make up approximately two thirds of the agricultural bio-
logicals industry. Representing roughly US$ 2.3 billion in annual sales, agricultural
biologicals has posted double-digit sales growth each of the last several years. There
are numerous biological products currently on the market that contain microorgan-
isms as active ingredients, including seed treatment and foliar applied products.
Microbial technologies can help improve nutrient acquisition, promote growth and
yield, control insects and protect against disease. These emerging agricultural bio-
logical technologies complement the integrated systems approach that is necessary
in modern agriculture, bringing together breeding, biotechnology and agronomic
practices to improve and protect crop yields.
In December 2013, Novozymes and Monsanto established The BioAg Alliance,
with a goal to discover, develop and sell microbial solutions that enable farmers

B. Lugtenberg ()
Institute of Biology, Sylvius Laboratory, Leiden University,
Sylviusweg 72, 2333 BE Leiden, The Netherlands
Tel.: + 31629021472
e-mail: Ben.Lugtenberg@gmail.com

© Springer International Publishing Switzerland 2015 339

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_35
340 B. Lugtenberg

Applied Microbiology

Biostimulants for substrates

Biological Control Agents

Contract Fermentation

The agronomic solutions developed at Agrifutur are essential for organic farming and integrated farming,
to reduce or eliminate the use of synthetic pesticides and along with them residues. Agrifutur develops and
MISSION

produces beneficial microorganisms. The agronomic benefits are then reflected in better food safety, environment
sustainability and farmers’ health.
Agrifutur is proud to contribute to sustainable agriculture, with a corporate mission that has not changed since
the company was first established.

Thanks to a brave and innovative vision going back over 30 years, Agrifutur is finally able to make concrete
VISION

what we have been dreaming of for a long time - the implementation of a type of agriculture able to grow healthy
food produce with profitable sustainability. A growing number of achievements increases the commitment and
enthusiasm that enables Agrifutur to cultivate life year after year.
RESEARCH

Applied microbiology is as useful in agriculture as it is complex in all its multiple aspects: identification,
genetic stability, optimisation of microbial growth, final product formulation and application in the field.
From the very beginning we have always favoured the setting up of research networks, in which each
collaborator investigates a key aspect.
EUROPEAN PROJECT

For the past 20 years, Agrifutur has regularly taken part in European and national research projects,
thanks to these it has established enduring cooperation with universities and centres of excellence in Europe
and other parts of the world. Agrifutur coordinated the European project BCA GRAPE (2008-2011) “New
biocontrol agents for powdery mildew on grapevine” (EU-FP7-SME). Agrifutur is currently involved in the new
European project DROPSA “Strategies to develop effective, innovative and practical approaches to protect
major European fruit crops from pests and pathogens” (Seventh Framework Programme KBBE.2013.1.2-04).

AGRIFUTUR srl
Via Campagnole, 8 - 25020 ALFIANELLO (Brescia) - ITALIA
Tel. +39 030 9934776 - Fax +39 030 9934777
www.agrifutur.com

Fig. 35.1 Example of a flyer of a company which sells microbial products for plant growth
promotion

worldwide to increase crop yields with less input. Novozymes brought an established
product portfolio and strengths within microbial discovery, application development
and fermentation to this partnership. Combined with Monsanto’s highly-developed
seeds and traits discovery, field-testing and extensive commercial network, the aim
is to deliver a comprehensive research, development and commercial collaboration
that can benefit of agriculture, consumers, the environment and society at large.
35 Important Organizations and Companies 341

Microbial solutions provide more choice for farmers and help meet the demand
for more sustainable agricultural practices. Such solutions can increase crop yields
and develop a more sustainable industry impact profile, ultimately resulting in more
food to feed the growing world and new opportunities to protect the planet.
BISOLBI-INTER LLC
Bisolbi-inter@rambler.ru www.bisolbi.ru
The innovative company Bisolbi-Inter was established on the basis of the All Rus-
sia Research Institute for Agricultural Microbiology (ARRIAM) in Saint-Petersburg.
The company is developing and producing microbial preparations and fertilizers
for agriculture, horticulture and forestry. Besides in Russia, some products have
been registered in Kazakhstan, Bulgaria, Serbia, and South Africa. Registration in
Australia and Turkey is in progress.
Institute of Biology Leiden
The Institute of Biology is proud and happy with this book on Plant-Microbe
Interactions, a topic of major interest in our past and future. We greatly acknowledge
emeritus professor Ben Lugtenberg for all the work and time that he has put into
this project. We wish Jos Raaijmakers a successful professorship in our Institute and
we look forward to a bright future for Plant-Microbe Interaction research at Leiden
University.
International Society for Molecular Plant-Microbe Interactions
www.ismpmi.org
The International Society for Molecular Plant-Microbe Interactions (IS-MPMI)
is a globally diverse organization of scientists who research molecular aspects of
microorganisms interacting with plants and the consequences of such interactions.
IS-MPMI provides opportunities for building, extending, and nurturing collabora-
tions and scientific community through its congress, society communications and its
journal Molecular Plant-Microbe Interactions (MPMI).
ABiTEP GmbH
www.abitep.de
ABiTEP GmbH is a German biotech company founded in 2005. We produce and
distribute natural microbial products for use in agriculture and gardening as well
as biological cleaning agents. Other important activities are contract production and
research. In addition we are involved in various research projects developing modern
and ecologically beneficial methods of plant production.
INCOTEC Group BV
www.incotec.com
INCOTEC’s Coating and Seed Technology Companies around the world provide
products and services for seed coating, pelleting, seed enhancements like priming,
disinfection, application of actives, additives and beneficial microbes, and analytical
services for genetic analysis. By providing key solutions, INCOTEC contributes
significantly to the development of sustainable agriculture worldwide.
342 B. Lugtenberg

Koppert Biological Systems

info@koppert.nl
Koppert provides biological crop protection and natural pollination for profes-
sional growers worldwide in agriculture and horticulture since 1967. We make use
1f natural enemies and micro-organisms to control and prevent infestations and dis-
eases. In addition we also supply bumblebees for the natural pollination of plants.
Research, production, distribution and advice are all major activities of our company.
The International Biocontrol Manufacturers’ Association (IBMA)
http://www.ibma-global.org/
President: Willem Ravensberg; email: willem.ravensberg@ibma-global.org
International Organisation for Biological Control (IOBC)
http://www.iobc-wprs.org/
IOBC was established in 1955 to promote environmentally safe methods of pest
and disease control in plant protection.
 Part VII
Paradigms in Plant-Microbe Interactions
Chapter 36
Trichoderma: A Multi-Purpose Tool
for Integrated Pest Management

Matteo Lorito and Sheridan L. Woo

Abstract Trichoderma spp. are mainly known as biocontrol and beneficial microbes
useful for a range of applications, from seed coating to post-harvest, from soil to
foliar, and able to provide a variety of benefits by using a plethora of mechanisms.
No other beneficial fungus in the agriculture field has received so much combined
attention from science and the commercial market. However, as indicated from the
many hundreds of related publications normally produced each year, we are far from
fully understanding the potential of these incredibly successful, from an ecological
point of view, bionts. This chapter briefly summarizes the main knowledge of the
interactions established by agriculturally useful Trichodermas, and discusses the next
future scenario of the use of these natural, multi-purpose tools.

36.1 The Multiple Interactor and Integrated Pest

Management Tool

The large body of literature concerning this ubiquitous fungal genus indicates that
Trichodemas are among the most active microbes found in natural environments,
as they manage to modify whatever substrate they colonize and establish func-
tional interactions with a variety of other living entities. Stable root colonization,
endophytism, mycoparasitism, competition for nutrients, symbiosis, pathogenicity,
antibiosis, induced resistance, seed germination and growth promotion, increased
nutritional value of produce; all of these activities or properties have been fully
demonstrated as affecting plants, animals, invertebrates, fungi, bacteria and viruses.

M. Lorito () · S. L. Woo

Department of Agriculture, University of Naples Federico II, and Institute of Plant Protection
IPP—CNR, Via Università, 100, 80055 Portici (NA), Italy
Tel.: + 390812539376
e-mail: lorito@unina.it
S. L. Woo
Tel.: + 390812539010
e-mail: woo@unina.it

© Springer International Publishing Switzerland 2015 345

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_36
346 M. Lorito and S. L. Woo

Further, the capacity of these fungi to substantially modify the biological and chem-
ical characteristics of the colonized substrates, which includes detoxification and
enrichment in organic matter, is a common knowledge.
The many thousands of publications, both scientific and divulgative, made since
Weindling’s first description of the fungus in the mid 1930’s, have produced a variety
of patents filed in dozens of countries, and activated the interest of several hundreds of
small-, middle- and large-size companies. This has created a market for Trichoderma-
based products estimated to be over 1 billion USD worldwide. Virtually, anyone
working in the agriculture sector (including gardening and ornamental crops), is
aware of the usefulness of these fungi, which are by the way making a particularly
notable impact on crop production in developing countries (i.e. see Cumagun 2012;
Ha 2010).
Scientific work on Trichoderma is considered a successful model of “translational
research”, where data obtained by using genomics, proteomics and metabolomics
techniques can be effectively implemented in agricultural practices, i.e. by allowing
fast selection of “elite” strains from the immense natural Trichoderma germoplasm
(Lorito et al. 2010; Mukherjee et al. 2013; Studholme et al. 2013).

36.2 The Pathogen Killer and Inhibitor

Trichoderma strains seem to have ancestrally evolved with the genome of predators
(Druzhinina et al. 2011). They are able to directly kill other microbes, and notably
other soil and plant-associated fungi, and, in terms of host range, to make interking-
dom jumps as well as major shifts in ecology (Chaverri and Samuels 2013). Different
mycoparasitic strategies may be used (Atanasova et al. 2013), with the role of many
factors still to be clarified (Ramão-Dumaresque et al. 2012) since the first demon-
strations based on gene knock-out (Woo et al. 1999). Aspects involved in the process
include cell wall degrading enzymes acting together with powerful sets of secondary
metabolites (Schirmböck et al. 1994), of which many have been found to affect the
activity of all the “The Top 10 fungal pathogens in molecular plant pathology” (Dean
et al. 2012). Direct killing may occur not only on plant pathogenic microbes, although
some of these appear to be preferred targets, possibly because these microorganisms
are more readily encountered around the plant hosts on which many Trichodermas
act basically as symbionts. Predation is not limited to fungi, since numerous strains
are also proposed on the agricultural market as nematode killers (Spiegel et al. 2007).
Direct inhibition or parasitism of pathogens is not the only process; also the ability
to sequester nutrients and to physically exclude the pathogen from the suitable site
of infection are known to be used.

36.3 The Disease Suppressor and Resistance Stimulator

Mycoparasitism has been reported to be used by over one hundred species of fungi
besides Trichoderma. Obviously, genome studies will reveal that also for these fungi
a major component is related to living as predators. However, only in the case of
36 Trichoderma: A Multi-Purpose Tool for Integrated Pest Management 347

Trichoderma strains such, has a large variety of beneficial effects produced on the
plant been reported. In fact, the latest research reveals more and more genetic char-
acters related to a plant symbiont lifestyle, which, in the case of strains selected for
agricultural applications, may represent a more significant behaviour over mycopar-
asitism (Harman et al. 2004; Lorito et al. 2010; Seidl et al. 2006; Shoresh et al.
2010; Studholme et al. 2013; Vinale et al. 2008). Effects on the plant are typically
divided into those enhancing resistance to disease and those promoting plant growth
(see next section), although they depend on numerous interconnected or interplay-
ing mechanisms (Shoresh and Harman 2008). The Trichoderma-mediated enhanced
resistance is today a paradigm in plant-microbe interaction (see Vos et al. 2014 for a
recent review). Many studies have demonstrated its occurrence even at the level of
open field cultivation, and that this is related to the activation of either the ISR (In-
duced Systemic Resistance) or the SAR (Systemic Acquired Resistance) pathways,
or both, depending on the conditions and the strain used. In fact, the mechanism
of activation is still far from being fully clarified, with the demonstrated involve-
ment of MAMPs (Microbe Associated Molecular Patterns) (proteins, sugars and
secondary metabolites) and effectors recognized by specific plant receptors (in the
case of beneficial interaction effectors; they may be considered equivalent to elici-
tors) as well as defense related hormones (Martinez-Medina et al. 2013). The plant
response to pathogen attack in the presence of Trichoderma may be substantially
affected, with dozens of genes differentially expressed, resulting in enhanced PTI
(PAMP Triggered Immunity) and ETI (Effector Triggered Immunity) (Lorito et al.
2010). Defense priming, possibly divided in a ISR-prime and ISR-boost phase, has
been reported to occur at no metabolic cost (Perazzolli et al. 2011), and is able to
suppress diseases caused also by foliar pathogens (Vos et al. 2014).
Finally, there is mounting evidence that the increased resistance to abiotic stresses
(mainly drought and saline) of a variety of plant species treated with Trichoderma,
that has also been observed in the field, is based on specific mechanisms of interaction
(Brotman et al. 2013; Mastouri et al. 2012).

36.4 The Plant Growth Promoter

Trichoderma spp. may be PGPMs (Plant Growth Promoting Microbes). This property
is widely diffuse among the genus, but not present in every strain. The effect can be
so clear and diffuse, ranging from horticultural crops to trees, that this ability is now
regularly tested in newly selected agricultural strains. The molecular mechanisms
supporting the observed effect are complex and related to the plant genotype (Tucci
et al. 2011). They include increased nutrient availability (Yedidia et al. 2001) and/or
stimulation by fungal metabolites (Vinale et al. 2008), with consequent changes in the
plant hormonal profile (Contreras-Cornejo et al. 2009; Hermosa et al. 2013, Roldán
et al. 2011). The transcriptional changes in the plant resulting in the combined effect
of increased resistance and PGP (Plant Growth Promotion) are extensive and may
be induced by using either the living fungus or some of its secreted metabolites. The
348 M. Lorito and S. L. Woo

recent appearance on the market of new strains specifically selected for biofertilizer
activity has further driven the widespread use of Trichoderma, although this has
raised legal issues about registration for use that need to be resolved.

36.5 The Source of Useful Compounds

The secretome of many Trichoderma strains used in agriculture is a rich source

of bioactive compounds. Over 250 different secondary metabolites and dozens of
different enzymes are known to be produced, although only a limited number of
them are normally associated to a single strain. Cell wall degrading enzymes, peptides
like peptaibols, water soluble metabolites such as harzianic acid and its derivatives,
volatile compounds such as 6-pentyl- α-pyrone and isocyanide derivates, proteins
such as hydrophobins and swollenins etc., have all been demonstrated to strongly
affect plant growth and/or resistance to biotic/abiotic stresses when applied in the
absence of the living fungus (Keswani et al. 2014; Mukherjee et al. 2012; Vinale
et al. 2012). Evidence is accumulating on the role of many of these compounds
in the symbiotic association with plant roots, which further supports the concept
that most Trichodermas selected for agricultural applications have evolved from a
main ancestral mycoparasitic lifestyle to that of a plant beneficial colonizer. The
direct use of Trichoderma metabolites, in alternative or combination with the living
fungus or other beneficials, is a declared future target of several R&D programs in
the public and private sectors worldwide. The usefulness of the rich Trichoderma
genome was first demonstrated by the transgenic use of genes encoding cell wall
degrading enzymes, with several papers published after the first report (Lorito et al.
1998), and this will be further exploited following the now common practice to
sequence the genome of commercially important strains.

36.6 The Soil Cleaner and Enricher

A recently highlighted application of Trichoderma concerns the use of strains as a

soil cleaner and/or soil enricher. Many reports propose selected Trichodermas as
bioremediators of soils polluted with arsenic, cyanide, hydrocarbons—i.e. crude oil,
naphthalene, phenanthrene, benzo[α]pyrene—pesticides, heavy metals—i.e. iron,
lead, copper, manganese, zinc—phenols etc. (Tripathi et al. 2013). On the other
hand, the ability of Trichoderma to simply enrich the quality of soils and composts,
as a saprophyte, decomposer and a major component of a well balanced microflora,
has been known for a long time (Bernard et al. 2012). This has made Trichoderma
one of the key microbial ingredients of commercial composts or products that help
composts to mature, based on the evidence that a high level of Trichoderma is also
a positive indicator of both disease suppressiveness and soil fertility.
36 Trichoderma: A Multi-Purpose Tool for Integrated Pest Management 349

36.7 The Commercially Successful Microbe

Trichoderma spp. and Bacillus spp. could be considered today as the most used IPM
biological tools available for the agriculture industry for infectious disease control,
although other BCAs (Biological ControlAgents) and PGPMs are also widely applied
in the real world for growing food and non-food crops. However, if one considers
the diversity of potential uses, Trichoderma appears as a multi-purpose “Swiss army
knife” compared to other single-use microbial products. The number of commercial
products, microbe combinations, formulations, claims and promises (some realistic,
others not) based on Trichoderma, distributed in over 100 countries worldwide, are
endless and ever increasing. India alone may have over 300 products on the market.
Regardless, a significant positive impact has already been achieved, especially on
food crop production in some developing countries. For instance, there is virtually
no medium-to-large size farm south of California and Texas, all the way to Chile,
that is not using or has not used Trichoderma; often combined with other beneficial
biologicals as well as using low-impact agronomic practices, in an attempt to cut
costs on pesticides and fertilizers. In countries such as Cuba and Venezuela, the
use of Trichoderma and other “insumos biológicos” is a government promoted and
supported agricultural practice (Harman et al. 2010). Actually, it is becoming a
common procedure for larger farms to acquire the necessary technological know-
how and to grow by themselves on-site Trichoderma and other BCAs in fermentors
for direct application of freshly-made cultures to crops (Fig. 36.1). For instance, it
has been calculated that one out of four melons or pineapples consumed in US or
Europe has been treated with Trichoderma alone or in combination with other BCAs.
The applications range includes use in the nursery, seed coating, greenhouse, open
field, soil-less systems, post-harvest, as well as directly on tree trunk and wounds.

36.8 The IPM Tool of the Future

The road taken by many countries toward IPM implementation is accelerating the
development and use of “non-chemical methods of plant protection and pest and
crop management”. For instance, European Directive 128/2009 and the Regulation
1107/2009 impose the use of IPM practices to all 28 EU Countries by 2014, and
determined the loss of registration for an estimated 40 % of the available synthetic
pesticides. This will permit Trichoderma—and other BCAs—based products to fi-
nally hatch from their niche market share, and boost related R&D activities, with the
subsequent expected innovations appearing already in the next few years.
New “multi-action” strains will be selected by using genome era-generated in-
formation; they will be able to kill pathogens, produce large amount of propagules,
act as PGPMs, increase resistance against a variety of pathogens—microbes, insects
(Battaglia et al. 2013), viruses and abiotic stresses, enhance nutrient use efficiency
and soil fertility, degrade pollutants in the soil, and act compatibly with other BCAs.
These strains will have a limited efficacy determined by cultivar/crop-associated
350 M. Lorito and S. L. Woo

Fig. 36.1 Successful performance of a Trichoderma plus Bacillus integrated application made on
a large scale in the field. The picture on the left, shows a 5000 ha production of melons (cv. Gallia
and Cantaloupe) on a farm located in south Honduras for export to the USA and the EU. The plants
treated with a biological product (rows on the left of field), based on an advanced technology (use of
“elite” strains selected on the basis of ‘omics generated information and grown directly on-farm by
a specifically developed method), performed better or as well as the conventional chemically-based
treatment (rows on the right). Note that the vegetation of rows on the left side of field is more
abundant of, or at least equivalent to, that of rows on the right. The regular daily application of
fungicides, antibiotics, insecticides and nematocides was completely interrupted during the last half
of the 60 day crop cycle, and substituted with fresh cultures of the two BCAs plus an occasional spray
of botanicals to control mildew, thus permitting a more than 50 % reduction of the total chemical
input. An average of approximately 15–20 % yield increase and complete lack of chemical residues
on the fruits have now been consistently obtained in the past 5 years. Cultures of the different BCAs
are produced in bioreactors built on site (pictures on the right taken in Honduras and Cost Rica),
then often directly connected to the irrigation system (Pictures taken by M. Lorito)

variability. All of these properties are already considered now in modern screening
programs and could be combined with a few strain mixtures or in new hybrid strains
obtained by fusing isolates with different and complementary beneficial traits. The
presence and activity of the BCAs will be easily tracked by using newly developed
detection kits.
New “exotic” strains will be discovered and selected for their activity against
specific stresses, their particular effect on individual crops, their enhanced compati-
bility with some agrochemicals, their adaptability to global environmental changes,
or their ability to target new invasive pathogens.
New strains will be naturally selected or produced by hybridization for dedicated
applications to staple or particularly important crops. The ability to positively respond
to treatment with Trichoderma and other BCAs will be used by plant breeders for the
production of new cultivars that will fully benefit from this symbiotic interaction.
A plethora of new formulations, mostly in a liquid form, facilitating the application
without the negative impact of drying on propagule viability, will appear. Some of
these will be specifically dedicated to hydroponics, forestry and seed treatment.
36 Trichoderma: A Multi-Purpose Tool for Integrated Pest Management 351

Fig. 36.2 Examples of useful combinations of Trichoderma (central picture) with other beneficials.
Some of these mixtures are already available as commercial formulations (i.e. Trichoderma +
Rhizobia or Trichoderma + Mycorrhizae), whereas other co-applications are de facto obtained by
the concurrent use of different products. The increasing understanding of the interaction mechanisms
and the evaluation of the effects in vivo will provide new opportunities for developing combined
products and novel IPM solutions. Pictures taken by the authors or available from Internet were
used

New biopesticides and biofertilizers based on Trichoderma metabolites, both

secondary and proteins, will be used in alternative or in combination with the liv-
ing fungus, similar to the case of Bt and Bt toxin. The advantages include: both
soil and foliar pathogen inhibition, positive effects on the plant obtained by using
very low doses, more precise dose-response correlation, low susceptibility to envi-
ronmental factors, full compatibilty with agrochemicals, excellent shelf life, easy
integration with current agricultural practices, endless possibilities of discovering
new synergistic mixtures, etc.
Synergistic combinations of Trichoderma and other beneficials (including Rhizo-
bia and Mycorrhizae), plus metabolites, botanicals, inorganics, seaweeds, chitosans,
animal-derived products, etc. with extended activity against diseases caused by mi-
crobes, insects and nematodes, will appear on the market. These new multi-agent
formulations will be constructed, differently from the actual products, on a solid
scientific basis and on a deep knowledge of the interaction mechanisms involved
(Fig. 36.2).
352 M. Lorito and S. L. Woo

The opening of new registration pipelines, that may accommodate the multi-
function properties of BCAs such as Trichoderma, may be expected; for instance
there is not a single track to register a strain acting both as a biofertilizer and biopes-
ticide, which is becoming a constraint in the implementation of new good products.
Many of these possibilities may already become a reality in the near future, while the
use of genetically engineered Trichoderma, although very promising and appealing,
appears more distant ahead. The technology is available and already commonly used
in many laboratories worldwide. The knowledge on the role of specific genetic traits
that could be usefully altered, either in the plant or in the fungus, has never been
so great and is rapidly increasing. To paraphrase a famous quote from N. Bourlag,
beneficial microbes such as Trichoderma will help us to realize the necessary miracle
of feeding a world of 10 billion people in a way that is sustainable for our ecosystem
and survival on the planet Earth.

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Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A et al (2011) Trichoderma: the genomics of
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Harman GE, Obregón MA, Samuels GJ et al (2010) Changing models for commercialization and
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Chapter 37
Agrobacterium, The Genetic Engineer

Paul J. J. Hooykaas

Abstract Agrobacteria are common soil and rhizosphere bacteria. Most strains
are saprophytes, but strains harboring a tumor inducing plasmid (Ti plasmid) are
pathogenic and can induce tumors on plants, called crown galls. The disease may
lead to growth retardation and eventually the death of the host plant and thus can
cause severe damage in horticulture. Crown galls form a favorable niche for Agrobac-
terium as they produce specific chemicals called opines which the bacteria can use
for growth. Nowadays, Agrobacterium tumefaciens is best known as a natural genetic
engineer, which is based on the molecular mechanism which it employs to induce
crown gall. This involves the transfer of an oncogenic segment of the Ti plasmid
(the T-DNA) to plant cells and its stable maintenance as part of one of the plant
chromosomes. Expression of genes on the T-DNA is responsible for the formation
of a tumor.

37.1 Crown Gall Disease

The soil bacterium Agrobacterium tumefaciens is the causative agent of crown gall.
This plant disease is characterized by the formation of tumorous overgrowths at
wound sites on plants (Fig. 37.1). In nature tumors are formed often at the root crown,
hence the name crown gall. However, the bacterium can induce tumors effectively
also on stems, roots or leaves, when these are wounded. The host range is broad:
tumors are formed on many dicotyledonous plant species, but not on monocots. The
disease causes severe damage in horticulture. Effective biological control has been
developed based on non-tumorigenic Agrobacterium strains producing bacteriocins
(such as agrocin 84) that selectively kill tumor inducing strains.
Crown gall cells are tumorous as they are able to grow in in vitro culture in the
absence of the plant growth regulators auxin and cytokinin, in contrast to normal plant
cells. In the tumors specific chemical compounds are present that are characteristic

P. J. J. Hooykaas ()
Institute of Biology, Sylvius Laboratory, Leiden University,
PO Box 9505, 2300 RA Leiden, The Netherlands
Tel.: + 31 71 527 4933
e-mail: p.j.j.hooykaas@biology.leidenuniv.nl

© Springer International Publishing Switzerland 2015 355

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_37
356 P. J. J. Hooykaas

Fig. 37.1 Crown gall tumor formation at wound sites on a potato plant (left) and the molecular
mechanism underlying T-DNA transfer into plant cells (right). Pictures were taken from pages C1
(made by dr HRM Schlaman) and C3 in The Rhizobiaceae. Molecular biology of Model Plant-
Associated Bacteria (1998). Reproduced by permission of the publisher

for the tumors and not found in normal plant cells or tissues. These compounds, with
the generic name of opines, are condensation products of amino acids and keto acids
such as pyruvate and α-keto-glutaric acid or sugars. Tumor inducing agrobacteria,
but almost no other bacteria, are able to catabolize the opines. The tumors therefore
form a favorable niche for Agrobacterium.
The bacterium can only infect plants at wound sites, where it can penetrate the
plant and attach to plant cells. Subsequently, the bacterium introduces part of its own
DNA into these plant cells. The transferred DNA (T-DNA) is derived from a large
plasmid called the Ti (tumor inducing) plasmid, which determines the virulence of
the bacterium. The T-DNA is responsible for the tumorous character of the crown gall
cells and for its ability to synthesize opines. This is because the T-DNA contains a set
of genes that is expressed in plant cells, including genes that mediate the synthesis of
the plant growth regulators auxin and cytokinin and genes that mediate the production
of the opines.
Agrobacteria belong to the α-Proteobacteria and are most related to the nitrogen
fixing bacteria of the genus Rhizobium. Initially classification was based on the
virulence properties: A. tumefaciens for tumor inducing strains, A. rhizogenes for root
37 Agrobacterium, The Genetic Engineer 357

inducing strains and A. radiobacter for avirulent strains. After it became clear that the
virulence properties were determined by a transmissible plasmid this classification
was abandoned.
Below I shall provide more detailed information on Agrobacterium and the molec-
ular mechanism of tumor formation. For further information the reader is referred to
the books edited by Kahl and Schell (1982); Spaink et al. (1998); Nester et al. (2005);
Tzfira and Citovsky (2008). Also the following reviews give a detailed account of
several aspects of the system, briefly discussed in this chapter: Bevan and Chilton
(1982); Binns and Thomashow (1988); Braun (1978); Dessaux et al. (1993); Gelvin
(2000); Hooykaas and Beijersbergen (1994); Morris (1986); Nester et al. (1984).

37.2 The Ti Plasmid

Ti plasmids generally have a size of about 200,000 base pairs. The T-DNA only
represents a minor part of it and has a size of about 20,000 base pairs. In some
Ti plasmids the T-DNA is divided over two segments, which are independently
transferred to plant cells. Only one of such T-DNAs contains the genes provoking
autonomous cell proliferation, while the other harbors genes for opine synthesis.
Genes located elsewhere on the Ti plasmid confer on Agrobacterium the ability to
catabolize the opines which are produced in the tumors. Moreover, the Ti plasmids
are conjugative plasmids which are mobile especially in plant tumors. This is because
not only the opine catabolic genes, but also the transfer genes are activated by the
presence of opines. Transfer is also under the control of quorum sensing (see Chap. 7).
N-acyl-homoserine lactones (AHLs) are produced by the TraI protein located on the
Ti plasmid. These AHLs bind to the transcriptional regulator TraR, which drives
expression of the tra genes. The traR gene itself is under control of a transcriptional
regulator which reacts to the presence of its cognate opine and thus conjugation is
controlled by opines and occurs in vivo specifically in the plant tumors (See for a
recent review: Venturi and Fuqua 2013). Besides the T-DNA the Ti plasmid contains
another set of genes which are involved in virulence, the virulence (vir) genes, which
will be discussed below.

37.3 The Virulence Genes

The translocation of T-DNA into plant cells is brought about by a delivery system
which is encoded by genes located in the virulence region of the Ti plasmid (Fig. 37.1).
The virulence region embraces about 30 genes distributed over a number of operons
(Zhu et al. 2000).
Regulation This vir regulon is controlled by a 2-component regulatory system con-
sisting of a receptor histidine kinase called VirA and an associated transcriptional
358 P. J. J. Hooykaas

regulator called VirG (Winans 1992). The virulence system is only activated in na-
ture when the bacterium encounters plants and more specifically plant wound sites
(Stachel et al. 1985). Phenolic compounds which are released at plant wound sites are
key inducers of the vir genes. They are characterized by the presence of a methoxy
group adjacent to the hydroxy group of the phenolic backbone and include well
known lignin precursors and breakdown products such as coniferyl alcohol, sinap-
inic acid, syringaldehyde and acetosyringone (Melchers et al. 1989). The presence
of particular sugars such as glucose and galactose, glucuronic acid and galactur-
onic acid enhances induction. Such sugars bind to a periplasmically located sugar
binding protein called ChvE, which in turn interacts with the periplasmic loop of
VirA (Cangelosi et al. 1990). Also a low pH (around 5.5) and a temperature below
30 ◦ C are required for vir-induction. All the biochemical and biophysical signals are
perceived by the VirA receptor, which becomes auto-phosphorylated in the presence
of inducing signals. VirA subsequently phosphorylates VirG, which then is able to
bind to the vir-boxes, specific sequences present in the promoters of the vir-genes
(Jin et al. 1990). This leads to transcription of the other vir-genes.
T-DNA Processing After activation of the vir-genes T-DNA processing takes place
in the cells. Single strand (ss) DNA breaks are present at the border repeats, which
are 25 base pairs sequences that surround the T-DNA, and single stranded copies of
the T-DNA (called T-strands) can be isolated from induced bacteria (Stachel et al.
1986). Thus T-DNA transfer resembles the process of bacterial conjugation, by which
ssDNA molecules are transferred into recipient cells. In the presence of vir-inducers
the T-DNA delivery system can indeed mediate the mobilization of incQ plasmids
between bacteria (Beijersbergen et al. 1992).
The protein that is responsible for the border nicking and the formation of the T-
strands is a relaxase called VirD2 (Gelvin 2000). Accessory factors including VirD1,
VirC1 and VirC2 are also needed, but their precise role has not been defined as yet.
The VirC1 protein binds to a DNA sequence called overdrive, which is located close
to the so-called right border repeat from where the production of the T-strands start,
and enhances border nicking (Toro et al. 1989). The mobilization of the CloDF13
and incQ plasmids by the vir-system does not require these virC and virD func-
tions. These plasmids use their own relaxase system for nicking at the cognate oriT
sequence and the formation of T-strands (Escudero et al. 2003).
T4SS For translocation of the T-strand into host cells Agrobacterium uses a Type4
Secretion System (T4SS) which is encoded by the 11 genes of the virB operon
(Alvarez-Martinez und Christie 2009). The T4SS forms a gateway for the T-strand
from the cytoplasm over the inner and outer membrane and through a large T-pilus
structure at the surface of the bacterial cell made up of VirB2 monomers, eventually
into the host cell cytoplasm. How the T-pilus contacts host cells and mediates delivery
of the T-strand is still unknown. For entrance into the T4SS a coupling protein is
required, which is encoded by the virD4 gene. This large hexameric membrane
protein recognizes the substrate and mediates delivery to the first protein of the
T4SS (Cascales et al. 2013).
37 Agrobacterium, The Genetic Engineer 359

Effector Proteins A number of the remaining virulence proteins such as VirE2

turned out to have a function in the plant host cells. The VirE2 protein is a ssDNA
binding protein, which binds to the T-strand in the plant cell to protect it against
nucleases and to assist in nuclear delivery (Duckely und Hohn 2003). In the absence
of VirE2, transformation is 3–4 logs reduced and the T-DNA is truncated.
The VirF protein was the first bacterial F-box protein discovered. F-box pro-
teins are abundant in eukaryotic cells and involved in targeted proteolysis of specific
proteins. Expression of VirF in plant cells could complement virF mutants for tu-
morigenesis. This suggested that VirF was delivered into plant cells by the T4SS and
the fact that VirF is not a ssDNA binding protein suggested that delivery of these
proteins was independent of that of the T-strand (Regensburg-Tuink and Hooykaas
1993). Direct evidence for independent protein delivery by the Agrobacterium T4SS
into plant cells was obtained by the CRAfT (Cre Reporter Assay for Translocation)
assay, which revealed that some virulence proteins (nowadays also called effector
proteins) can be delivered into plant cells by bacterial strains, even by those with a
Ti plasmid from which the T-DNA has been removed (Vergunst et al. 2000). The
CRAfT assay also allowed the identification of so far unknown virulence effector
proteins, including VirE3 and VirD5 (Vergunst et al. 2005). The effector proteins all
have a specific 30 amino acids C-terminal, arginine rich transport sequence, which
is essential for translocation by the T4SS.
T-DNA transfer Interestingly, the VirD2 protein, which upon nicking remains
covalently bound to the T-strand, also has C-terminal sequences outside of its N-
terminal relaxase domain which mediate translocation into plant cells. Deletion of
these sequences from VirD2 prevents translocation and leads to avirulence. Addition
of the C-terminal sequences of one of the effector proteins to such a C-terminally
deleted VirD2 protein leads to the restoration of transport and virulence (van Kregten
et al. 2009). This suggests that the T4SS mediating T-DNA translocation is in fact
evolutionary derived from a protein translocation system, but adapted in such a way
that it allows also the translocation of DNA molecules that are covalently attached
to proteins which are recognized as substrates of the system.
The T-DNA delivery system is very efficient. When using single cells from a plant
(plant protoplasts) in in vitro culture up to 40 % of these cells can become transformed
during co-cultivation with Agrobacterium. This efficiency can be explained by the
protection of the delivered DNA molecules by VirE2, and by their immediate tar-
geting to the nucleus. The latter is due to nuclear localization sequences present in
VirD2, which mediate binding to α-importins of the host cells (Sheng and Citovsky
1996). The VirE2 protein assists in nuclear targeting as its binding to the T-strand
prevents knot formation resulting in a long thin thread that can enter the nuclear pore.
Also VirE2 interacts with a host protein called VIP1 (Tzfira et al. 2000), which is a
transcription factor involved in the defense response. Upon infection VIP1 is phos-
phorylated and subsequently moves into the nucleus to activate defense genes. By
binding to the VirE2 proteins that coat the T-strand, VIP1 may enhance the nuclear
targeting of the long T-complex. Agrobacterium apparently has adopted a Trojan
horse strategy by abusing defense signaling to bring about transformation (Djamei
et al. 2007).
360 P. J. J. Hooykaas

37.4 Applications

The Agrobacterium DNA delivery system has become the prime system for the ge-
netic modification of plants. This is due to the low cost involved as there is no
expensive apparatus required such as with other delivery methods (particle gun,
electroporator), but even more so because of the relative precision of the system. As
compared to other systems in general a lower number of DNA copies are integrated
in the genome (reducing gene silencing problems) and mostly these copies are in-
tact. The host range for which the system can be used is extremely broad. Not only
plants, but also yeasts and fungi turned out to be a host for Agrobacterium-mediated
transformation (Bundock et al. 1995; de Groot et al. 1998). Nowadays, the Agrobac-
terium system is also the prime transformation system for many fungi (Michielse
et al. 2005).

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Chapter 38
Take-All Decline and Beneficial Pseudomonads

David M. Weller

Abstract Crops lack resistance to many soil borne pathogens and rely on an-
tagonistic microbes recruited from the soil microbiome to protect their roots.
Disease-suppressive soils, the best examples of microbial-based defense, are soils
in which a pathogen does not establish or persist, establishes but causes little or no
disease, or establishes and causes disease at first but then the disease declines with
successive cropping of a susceptible host. Take-all decline (TAD) controls take-all
disease of wheat caused by Gaeumannomyces graminis var. tritici. TAD is a sponta-
neous reduction in the incidence and severity of take-all occurring with monoculture
of wheat or barley following a severe disease outbreak. TAD suppressiveness is trans-
ferable, eliminated by soil pasteurization, and reduced by growing non-host crops.
It results from the build-up of populations of 2,4-diacetytlphloglucinol (DAPG)-
producing Pseudomonas spp. to a threshold density of at least105 CFU g−1 root.
TAD protects wheat against take-all on millions of hectares worldwide.

38.1 Defense of Plant Roots Against Pathogens

The rhizosphere is enriched and supported by rhizodeposition, leading to the loss

of as much as 21 % of the plant’s photosynthetically fixed carbon from the roots
(Chap. 3).These nutrients select for a complex rhizosphere microbiome that con-
tributes to important plant functions like growth, vigor and defense (Pieterse et al.
2014).
Microbial-Based Plant Defense Beneficial rhizosphere microorganisms that di-
rectly promote plant growth or defend roots against pathogens are recruited from the
bulk soil. Although the soil type is the most important factor determining the com-
position of the rhizosphere microbial community, the plant also plays a key role in
shaping the microbiome and in recruiting and enriching beneficial microorganisms

D. M. Weller ()
United States Department of Agriculture-Agricultural Research Service,
Root Disease and Biological Control Research Service, Pullman,
Washington 99164-6430, USA
Tel.: +1-509-335-6210
e-mail: David.Weller@ars.usda.gov
© Springer International Publishing Switzerland 2015 363
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_38
364 D. M. Weller

because the quantity and quality of root exudates are genetically regulated by the
plant. Unlike for many foliar diseases, crop plants lack resistance to some of the most
common soil borne fungal pathogens and many species of pathogenic nematodes.
As a result, roots rely on microbial-based defenses consisting of microbial “defend-
ers” or “body guards” that antagonize soil borne pathogens and/or initiate induced
systemic resistance (ISR), as the first and sometimes the only line of defense against
soil borne diseases (Weller et al. 2007).
Disease-Suppressive Soils Suppressive soils provide the best examples of natural
microbial-based defense. They are soils in which, because of their microbial makeup
and activity, a pathogen does not establish or persist, establishes but causes little or
no disease, or establishes and causes disease at first but then the disease declines with
successive cropping of a susceptible host crop (Weller et al. 2002; Weller et al. 2007).
Suppression may be general or specific, with the former owing to the collective com-
petitive and antagonistic activity of the total soil microbiome, and the latter owing to
a specific group(s) of microorganisms acting against a specific pathogen and often on
a specific crop. General suppression is not transferable, is reduced by soil steaming;
and is enhanced by practices that increase soil microbial activity. Highly effective
specific suppression is superimposed over general suppression, is transferable, and
is eliminated by soil pasteurization. Suppressiveness is a continuum from general to
specific, with the former often giving rise to the latter in response to certain cropping
practices. In conducive soils, disease readily occurs. Suppressive soils with activity
against fungi, oomycetes, bacteria or nematodes occur worldwide, and some, like
Fusarium wilt suppressive soils are long standing because the suppressiveness is a
natural characteristic of the soil, its origins are unknown, and it persists in the absence
of the plant. Others like take-all decline (TAD) soils are induced because suppres-
siveness is initiated and sustained by monoculture of susceptible crops. In general,
the microbial basis of most suppressive soils worldwide remains poorly understood
(Weller et al. 2007). However, the application of new ‘omics’ approaches combined
with classical methods of characterizing suppressive soils is providing a platform to
more rapidly identify the microbial basis of suppression (Mendes et al. 2011; Bakker
et al. 2013).

38.2 Take-all

Take-all, caused by the soilborne fungus Gaeumannomyces graminis (Sacc.) von

Arx & Olivier var. tritici Walker (Ggt), is one of the most important root diseases of
wheat worldwide (Cook 2003). It develops at a soil pH of 5.5–8.5 and is most severe
where wheat is grown under high precipitation or irrigation (Cook 2003; Freeman
and Ward 2004). However, it also occurs in wheat grown under dryland conditions
(Cook 2003). Ggt survives saprophytically as mycelium in dead roots and tiller bases,
the inoculum source for the next crop (Paulitz et al. 2002; Cook 2003; Freeman and
Ward 2004). Primary infection of the roots of seedlings occurs with the growth of
dark runner hyphae on the root surface. Hyaline hyphae penetrate into the cortex
38 Take-All Decline and Beneficial Pseudomonads 365

and colonize the vascular tissue, causing characteristic black lesions. Runner hyphae
continue to grow over the root surface, to other roots, and upward to the crown
and stem bases. Early infection of the plant ultimately causes yellowing of lower
leaves, stunting, and premature death of plants in patches. Crop rotation and tillage
are effective approaches to manage take-all, but trends in modern farming systems
are toward less tillage (to control erosion) and two or three crops of wheat before a
break crop (for economic reasons). Both of these practices greatly exacerbate take-
all. Wheat varieties lack resistance to take-all, and methods of chemical control,
although available, have had only moderate success in controlling the disease. The
take-all pathogen also attacks barley, rye, triticale, and other related grasses, but to a
lesser extent than wheat (Cook 2003). G. graminis var. avenae also attacks wheat and
causes typical take-all symptoms but it has a broader host range and more commonly
is a problem on oats and bent grass.

38.3 Take-All Decline

TAD, the best characterized example of induced specific suppression, is defined as

the spontaneous reduction in the incidence and severity of take-all and increase in
yield occurring with continuous monoculture of wheat or barley and following a
severe attack of the disease (Hornby 1998; Weller et al. 2002). TAD is a worldwide
phenomenon (Weller et al. 2002) and it is widely used to manage take-all. For
example, in the Pacific Northwest (PNW) of the USA, about 0.8 million ha of wheat
suffer little damage from take-all, probably due to TAD (Cook 2003).
Although TAD development follows a consistent pattern everywhere, the previ-
ous cropping history, environmental conditions and soil factors in a field impact the
robustness of the take-all suppressiveness and the length of time before its onset,
which can vary significantly but averages 4–6 years (Weller et al. 2002). TAD sup-
pressiveness is transferable to conducive soils (Raaijmakers and Weller 1998; Weller
et al. 2002), eliminated by pasteurizing or fumigating the soil (Raaijmakers and
Weller 1998; Weller et al. 2002), reduced or eliminated by growing a non-host crop
(Weller et al. 2002), and regained when wheat or barley is again grown. Interestingly,
soils in some fields that have transitioned into TAD are imprinted with a memory
of take-all suppressiveness. Thus, TAD soils cropped for long stretches to non-host
crops, resulting in a reduction in suppressiveness, are rapidly reactivated to a state
of suppression by only a couple of cycles of wheat (even in the greenhouse), thus
bypassing the need for years of continuous wheat monoculture to regain suppression
(Weller and Yang, unpublished). In addition, the suppressiveness in TAD soils that
have been stored dry in cans for over 25 years, can be readily reactivated by again
planting one or two cycles of wheat (Allende-Molar and Weller, unpublished).
Microbial Basis of Take-all Suppressiveness Since the discovery of TAD, micro-
bial changes in the bulk soil or rhizosphere resulting in inhibition of G. graminis
var. tritici have most commonly been reported as the mechanism(s) of take-all sup-
pression (Hornby 1998; Weller et al. 2002). The types of antagonists responsible
366 D. M. Weller

for TAD have been thought to differ throughout the world because microbes from
different taxonomic groups have biocontrol activity against take-all and G. graminis
var. tritici is very sensitive to different types of antagonism (destruction of hyphae,
cross protection, antibiosis etc.) (Weller et al. 2002). However it is now known that in
fields in the U.S and The Netherlands, TAD results from the build-up of populations
of 2,4-diacetytlphloglucinol (DAPG)-producing fluorescent Pseudomonas spp. to a
density above 105 CFU g−1 root, the threshold required for take-all control (Raaij-
makers and Weller 1998; Weller et al. 2002, 2007). DAPG producers are commonly
found in the soil microbiome at low densities, but infection of wheat roots by G.
graminis var. tritici leads to a dramatic enrichment in their population size, a process
that is repeated over and over during wheat monoculture. The specific suppression
in TAD soils is lost when DAPG-producing Pseudomonas spp. are eliminated, and
conducive soils gain suppressiveness when DAPG producers are introduced via mix-
ing in small amounts (1–10 % w/w) of TAD soil (Raaijmakers and Weller 1998). G.
graminis var. tritici is highly sensitive to DAPG (Kwak et al. 2009), which is pro-
duced in the rhizosphere in TAD soils (Raaijmakers et al. 1999). Control of take-all
does not occur with DAPG-deficient mutants and is positively related to the sen-
sitivity of the G. graminis var. tritici isolate to the antibiotic (Kwak et al. 2009).
Although DAPG producers are the primary drivers of TAD, a major gap still exists
in our understanding of how other components of the microbiome help to modulate
and promote the development of TAD suppressiveness (Sanguin et al. 2009; Bakker
et al. 2013).
2,4-Diacetylphloroglucinol and Biocontrol DAPG, a very broad spectrum polyke-
tide antibiotic, is a key determinant in the biocontrol of a wide range of root and
seedling diseases: for example, root rots of tobacco and tomato, Pythium damping-
off of cucumber, and take-all of wheat by P. protegens CHA0; damping-off of sugar
beet and cyst nematodes and soft rot of potato by P. fluorescens F113; and take-all
by P. fluorescens Q2-87 and SSB17 and P. brassicacearum Q8r1-96 (Weller et al.
2007). DAPG is also known to be a strong inducer of resistance to foliar pathogens
when produced on the roots (Pieterse et al. 2014). The DAPG biosynthetic locus in-
cludes phlACBDE and phlHGF, which function in synthesis, export and regulation.
These genes are conserved among all DAPG-producing fluorescent Pseudomonas
spp. (Weller et al. 2007).

38.4 A Broad Role for DAPG in the Defense of Roots

Enrichment of DAPG producers by crop monoculture is not limited to cereals.

Evidence is rapidly emerging that globally in agroecosystems, strains of DAPG-
producing pseudomonads form part of the foundation of elite communities of
microbial defenders of roots within the larger rhizosphere microbiome (Weller et al.
2007). For example, DAPG producers contribute to the long-standing suppressive-
ness of soils to root rot of tobacco in the Morens region near Payerne, Switzerland
(Weller et al. 2002). Population densities of DAPG producers at greater than the
38 Take-All Decline and Beneficial Pseudomonads 367

threshold needed for disease suppression occurred on both pea and wheat roots
grown in a pea monoculture soil (> 30 year) from Mt. Vernon, WA, USA and this
soil provides control not only of Fusarium wilt of pea but also take-all (Landa et al.
2002). Threshold densities of DAPG producers also occurred on roots of both flax
and wheat grown in a monoculture flax soil (103 years) from Fargo, ND, but were not
detected on plants grown in soil from an adjacent field that had been in a long crop
rotation for the same amount of time. Cropping systems research needs to focus on
identifying rotations and varieties that can sustain populations of DAPG producers
and thus retain disease suppressiveness after crop monoculture is broken (McSpadden
Gardener et al. 2005). Exciting evidence is also accumulating that DAPG may play
a role in defense against diseases in natural ecosystems. For example, Lysobacter
gummosus appears to protect the red-backed salamander against natural infection by
the fungal pathogen causing chytridiomycosis, a serious skin disease, by producing
DAPG (Brucker et al. 2008).

38.5 Diversity Among DAPG Producers

DAPG-producing pseudomonads that belong to the P. fluorescens complex (Loper

et al. 2012) show a considerable amount of genetic diversity (Weller et al. 2007).
Currently, 22 different genotypes of DAPG producers (designated A-T, PfY and
PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis
or restriction fragment length polymorphism (RFLP) and phylogenetic analysis of
phlD, a key gene in the DAPG biosynthesis locus (De La Fuente et al. 2006; Landa
et al. 2002; Landa et al. 2006). It appears that there are at least six more genotypes
that have not been fully described. More recently, whole genome sequencing has
confirmed these groupings (Loper et al. 2012). DAPG-producing pseudomonads are
now placed in three species: P. fluorescens, P. brassicacearum and P. protegens.
Host Preference Several genotypes of DAPG producers typically occur in a field,
but usually only one or two genotypes dominate on the roots of a crop grown in
that soil. For example, although genotypes D, B, E and L occur in PNW TAD
fields, D-genotype (P. brassicacearum) isolates comprise 60–90 % of the DAPG
producers and are primarily responsible for take-all suppression (Weller et al. 2007).
The ability of P. brassicacearum to be the primary driver of TAD lies in the mutual
affinity or preference that this bacterium and wheat roots have for each other. This
affinity allows P. brassicacearum to aggressively colonize the wheat rhizosphere far
better than other genotypes present in the soil microbiome and to maintain threshold
densities throughout the growing season (Raaijmakers and Weller 1998, Weller et al.
2007). Of the 22 described genotypes of DAPG producers, several besides genotype
D have shown a crop preference. For example, P- and K-genotype isolates have an
affinity for pea and wheat, respectively. The ability of crops to enrich for genotypes
of DAPG producers best adapted to colonize their roots is strikingly demonstrated
in two adjacent fields at Fargo, North Dakota, USA, each with greater than 100
years of continuous monoculture flax or wheat, respectively. Continuous culture of
368 D. M. Weller

flax resulted in a dominance of F- (41 %) and J- (39 %) genotype isolates in the

rhizosphere, whereas growth of wheat led to a dominance of D-genotype isolates
(77 %).

38.6 Robustness of Take-all Suppressiveness

Major gaps in our knowledge of TAD still exist as to the basis for variations in
the amount of time required before TAD onset, fluctuations in the robustness of
suppressiveness among fields and years, and longer term breakdowns of suppression
(Hornby, 1998; Kwak and Weller 2012). Kwak et al. (2009) explored the possibility
that Ggt isolates develop tolerance to DAPG in TAD fields after many years of
monoculture, thus lessening the ability of DAPG producers to suppress the pathogen.
However, although Ggt isolates within a given field differed in antibiotic sensitivity,
isolates from TAD fields and conducive fields did not differ significantly from each
other (Kwak et al. 2009). It is not likely that Ggt will develop tolerance in the field to
DAPG because the antibiotic attacks multiple basic cellular pathways in the pathogen
(Kwak and Weller 2012).
Wheat Cultivars Affect Suppressiveness Variations in TAD onset and robustness
may be due to the differential ability of wheat cultivars to initiate and sustain take-
all suppressiveness. For example, wheat cultivars differ in their supportiveness of
population sizes and genotypes of DAPG-producing pseudomonads, the amount of
DAPG produced in the rhizosphere, and expression of defense genes when colonized
by DAPG producers. Crops and cultivars also affect expression of genes in the phl
operon of DAPG producers in the rhizosphere (Weller et al. 2007; Kwak and Weller
2012; Maketon et al. 2012). At this time, no wheat breeding program is focused
on improving the supportiveness of wheat cultivars to microbes involved in natural
disease suppressiveness.

38.7 Conclusions

By the year 2050, there will be 9–11 billion people on Earth to feed using the same
amount or less land and water as is available today. Farmers are being challenged to
produce more, but to do so using sustainable cropping practices and less fertilizer and
pesticides. Soil-borne pathogens that cause root and crown rots, seed and seedling
damping-off and wilts continue to be major barriers to the expansion of the produc-
tion of food, fiber and ornamental crops. Soil-borne pathogens are challenging to
control because plants often lack resistance to them and chemical seed treatments
are usually only effective during the seedling phase of the disease. The use of broad-
spectrum gaseous soil fumigants (methyl bromide, chloropicrin, metham sodium)
has become increasingly unacceptable in agriculture. It is now thought that the rhizo-
sphere holds the key to the next Green Revolution, whereby innovative new varieties
38 Take-All Decline and Beneficial Pseudomonads 369

and management practices will allow plants to be far more capable of recruiting and
utilizing microbes in the soil microbiome for growth promotion and disease defense.
Disease-suppressive soils like TAD are not only sustainable and practical controls
for soil-borne pathogens, but also are model systems for elucidating how the roots
signals, enrich, and sustain elite microbial defenders from the soil microbiome to
answer the plant’s cry for help when it is attacked.

Acknowledgements Parts of this work were supported by the Orville A. Vogel Wheat Research
Fund, Washington State University, Pullman and the Organisation for Economic Cooperation and
Development (OECD), Paris. Mention of trade names or commercial products in this publication
is solely for the purpose of providing specific information and does not imply recommendation or
endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and
employer. I thank Dr. Linda S. Thomashow for reviewing this manuscript.

References

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diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea
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Chapter 39
The Oomycete Phytophthora infestans, the Irish
Potato Famine Pathogen

Charikleia Schoina and Francine Govers

Abstract The oomycete Phytophthora infestans is a filamentous plant pathogen that

causes the late blight disease in potato worldwide. It has been a favorite subject of
study since the Great Irish Famine in the 1840s and is considered to be a model
species for oomycetes. Its genome of over 240 Mb has a remarkable organization
with gene dense regions interspersed with gene poor regions, the latter harboring
genes involved in host specificity and virulence. These genes are key players in
the arms race with the host. They can easily mutate to avoid recognition by immune
receptors and their abundance shows that P. infestans possesses an impressive arsenal
of weapons for attacking potato and is likely hard to beat.

39.1 Potato and Potato Late Blight

The domestication of potatoes (Solanum spp.) probably started at least 10,000 years
ago around Lake Titicaca in modern-day Peru and Bolivia. Since 1400 BC, when the
earliest farmers settled in the Andes, potato production has been of major importance
for the Andean societies. In the sixteenth century the Spanish conquered South-
America and one of the treasures that they brought to Europe was the potato. By
the late 1700s, potato cultivation was widespread in Europe and today potato is
the third most important food crop worldwide. The first reports of potatoes being
vulnerable to disease appeared in Belgium in June 1845. With an unprecedented
speed a mysterious disease spread over Western Europe and wiped out the entire
potato crop. By mid-October of that same year it had reached Ireland, a country
where the socioeconomic structure forced the poor peasants to solely rely on potato
for their daily food. This led to the Great Irish Famine, a disaster that caused a turning

F. Govers () · C. Schoina

Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1,
6708 PB Wageningen, The Netherlands
Tel.: +31 317 483 138
e-mail: francine.govers@wur.nl
C. Schoina
Tel.: +31 317 483 881
e-mail: charikleia.schoina@wur.nl

© Springer International Publishing Switzerland 2015 371

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_39
372 C. Schoina and F. Govers

point in history and gave birth to Irish America. Apart from the 1 million people that
died, another one and a half million settled as refugees in North America. At that
time the concept that microbes could cause plant diseases was unknown. The sudden
appearance of late blight was blamed on abiotic factors like hidden volcanoes, the
steam machine, electricity or the wet summer and even on the devil. Reverend M.
J. Berkeley however, put forward the hypothesis that the mould flourishing on the
potato foliage was the cause and not the consequence of the disease. He succeeded
in drawing a rather accurate picture of a fungus-like creature growing as mycelium
inside a potato leaf and releasing spore-bearing hyphae through the stomata (Large
1940) (Fig. 39.1). This creature was Phytophthora infestans, widely known as the
Irish potato famine pathogen and nowadays responsible for yearly economic losses
of over 3 billion € worldwide (Fry 2008). It belongs to the oomycetes, a group of
diverse organisms that, similar to fungi, grow as mycelium and produce spores to
propagate. Oomycetes are best known as plant pathogens –there are over a hundred
Phytophthora spp. and many downy mildews and Pythium spp.- but the group also
comprises animal and microbial pathogens as well as saprophytes (Kroon et al.
2012; Kamoun et al. 2014). As such they occupy similar ecological niches as fungi.
However, during evolution oomycetes evolved completely independently from fungi
and this is reflected in differences in e.g. cell wall composition (see Chap. 6), actin
cytoskeleton, biochemical and metabolic pathways, and mating systems (Judelson
and Blanco 2005) .

39.2 Tackling the Pathogen

In order to understand how a pathogen attacks and colonizes its hosts one needs to
be able to investigate the molecular and cellular machinery of the organism and to
gain insight into the type of components that the pathogen produces to cause disease.
Most pathogenicity factors are secreted and often produced specifically or at higher
levels during interaction with the host. Already in the early 1990s the first attempts
were made to identify putative pathogenicity factors using differential gene expres-
sion profiling. At around the same time the first successful DNA transformation of P.
infestans was reported and in the years to follow the molecular toolbox was expanded
with protocols suitable for gene function analyses by a targeted ‘knock-down’ ap-
proach based on RNA interference or by over expression (van West et al. 1999). In
all cases phenotypic analyses of multiple independent transformants is required to
demonstrate that an aberrant phenotype is consistently correlated with reduced or
increased transcript levels of the target gene and not the result of disruption of an-
other unrelated gene. In case the transgene carries a fluorescent tag, overexpression
transformants can be analyzed by microscopy to visualize the subcellular location
of the encoded protein. The transgene can also be modified with targeted deletions
or potential ‘gain- or loss-of-function’ mutations or its anticipated function can be
studied in other organisms enabling even more in depth analyses at the biochemical
or structural level. Although all these approaches have been and are being used
39 The Oomycete Phytophthora infestans, the Irish Potato Famine Pathogen 373

Fig. 39.1 A typical late blight lesion has a necrotic center with heavy sporulation surrounded by
a water soaked zone. Outside these zones the pathogen continues to invade healthy cells and the
lesion expands further. The schematic figure shows the vegetative life cycle of P. infestans. Infection
starts when a spore lands on the leaf and germinates. The germ tube forms an appressorium and an
emerging penetration peg enters the epidermal cell. From there hyphae colonize the inner cell layers
where they grow in between the plant cells and produce finger-like protrusions that penetrate the
plant cell. These so-called haustoria are specialised structures that facilitate the delivery of effectors
into plant cells and the uptake of nutrients from plant cells. P. infestans is a hemi-biotrophic pathogen
that needs living plant tissue for the initial phases of colonization, the biotrophic phase. Gradually
the plant cells die and the leaf necrotizes. In this phase hyphae escape through the stomata and
produce numerous spores named sporangia that easily detach and disperse by wind or water. A
sporangium that finds a new host can either germinate directly and initiate a new cycle or, at lower
temperatures, undergo cleavage resulting in a zoosporangium from which 6–8 flagellated spores are
released. These zoospores can swim for several hours but once they touch a solid surface they encyst
and germinate to initiate new infections. Under favourable conditions the pathogen can complete the
cycle from infection to sporulation in 4 days. In the field this cycle is repeated multiple times during
one growing season resulting in billions of spores and a continuous increase of disease pressure.
Besides leaves also stems and tubers get infected and P. infestans can continue to flourish on the
decaying plant material. If not managed properly, infected seed potatoes or waste on refuse piles
are often the sources of inoculum for new infections in spring. An alternative route for surviving the
winter is via oospores, resting spores that can survive in soil for many years and are produced upon
mating. Most Phytophthora spp. are homothallic which means they produce sexual spores without
the need for a partner. P. infestans is heterothallic; isolates are either A1 or A2 mating type and sex
organs only develop when isolates of opposite mating type sense the sex hormone produced by the
mate. The schematic figure is reproduced from Judelson and Blanco (2005) by permission of the
publisher
374 C. Schoina and F. Govers

Fig. 39.2 The GPCR-PIPK gene PiGK4. A case study of gene discovery and functional gene
analyses in Phytophthora infestans. Heterotrimeric G-proteins and phospholipids are key play-
ers in evolutionary conserved signalling networks in eukaryotes and have been shown to play a
role in pathogenicity. A comprehensive inventory of Phytophthora genes involved in phospholipid
signalling revealed that several indeed encode highly conserved proteins whereas others encode
proteins with conserved catalytic domains but combined with other domains. Examples are the
GPCR-PIPKs (GKs), novel proteins that are composed of a G-protein coupled receptor (GPCR)
domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain (a). Based on this domain
structure GKs are anticipated to link G-protein and phospholipid signalling. It could be that the
GPCR, which normally transmits extracellular signals over the membane to the heterotrimeric G-
proteins, bypasses the G-proteins and activates directly the PIPK domain to phosphorylate either
lipids or other proteins. Phylogenetic analysis showed that each Phytophthora spp. has 12 GKs that
are more highly conserved between species than between GKs within one species (b). GK genes
are differently expressed during the life cycle (c). PiGK4 is most highly expressed in germinated
cysts whereas other GKs are higher expressed in zoospores. Microscopic imaging of P. infestans
transformants expressing PiGK4 with a fluorescent tag showed the subcellular location of PiGK4
(d). In other transformants the PiGK4 expression was silenced (S) or overexpressed (OX). Pheno-
typic characterisation demonstrated a role for PiGK4 in spore development, sporangial cleavage,
hyphal elongation and virulence (e). In these transformants the transgene is expressed in its own
genetic background. Expression in heterologous hosts is another approach to study the gene of
interest. For PiGK4 Escherichia coli was used as host organism to determine the orientation of the
seven transmembrane domains that are characteristic for a canonical GPCR. For more details see
Hua et al. (2013). To test if the PIPK domain in PiGK4 has the anticipated enzyme activity, baker’s
yeast is used as heterologous host. PiGK4 should be able to restore a PIPK deficient yeast mutant
in a similar way as the endogenous yeast PIPK gene. Panels c, d and e are reproduced from Hua
et al. 2013 by permission of the publisher

successful (Fig. 39.2), manipulating genes in P. infestans remains a challenge. Trans-

formation efficiencies are low, primary transformants are often heterokaryons with
only a subset of the nuclei carrying the transgene and transgenes are not always stable
or expression is lost.
39 The Oomycete Phytophthora infestans, the Irish Potato Famine Pathogen 375

Because of these limitations there was an enormous drive to exploit other means
that would lead to insight into the mechanisms underlying pathogenicity. The ge-
nomics era created new opportunities and as early as 1999, when DNA sequencing
was still costly and filamentous plant pathogens were hardly subjected to large scale
sequencing, the first set of a thousand P. infestans Expressed Sequence Tags (ESTs)
was published (Kamoun et al. 1999). This already uncovered a gene repertoire far
more complicated than was envisioned. One example is elicitin, a 10 kD secreted
protein highly abundant in culture medium and an elicitor of necrosis in tobacco that
was a holy grail in plant-pathogen interaction research for many years. It turned out
to be a member of an extensive family with other members also eliciting necrosis
(Jiang et al. 2006). Until today, their function in pathogenicity remains elusive. They
bind sterols and since Phytophthora spp. cannot synthesize sterols themselves, they
may have a role in snatching sterols from the environment. Silencing the elicitin inf1
gene in P. infestans did not change virulence on tomato and potato and no detrimen-
tal effects were observed. Since elicitins are ubiquitous in Phytophthora, they have
the characteristics of a pathogen associated molecular pattern (PAMP). Recently a
receptor for INF1 was identified in a wild potato species (Du et al. 2014). It is a
membrane associated receptor-like protein with extracellular leucine-rich repeats,
typical for a pattern recognition receptor, and enhances resistance to late blight when
expressed in cultivated potato. This receptor is a new holy grail, this time for plant
breeders, to exploit it for boosting late blight resistance in new cultivars.
The great leap forward was the completion of two Phytophthora genome projects
in 2006 (Govers and Gijzen 2006). For the first time the whole repertoire of potential
pathogenicity genes could be mined. The usual suspects were the ones encoding
hydrolases such as e.g. proteases, cutinases, lipases or pectinases, as well as pro-
tease inhibitors, protein toxins, and ABC transporters. Other genes were suspicious
because of their evolutionary trajectory. This could be expansion in number in
comparison to close relatives that are not pathogenic, acquisition specifically in
Phytophthora or oomycetes as is the case for example for the elicitins, or domain
shuffling or fusion resulting in proteins that have uncommon domain compositions.
Oomycetes have a relatively high proportion of such novel proteins and several of
these are truly oomycete-specific (Seidl et al. 2011). They may have evolved along
with the metabolic, biochemical and structural features that are characteristic for
oomycetes and as such they could be ideal targets for disease control.
Comparison of the genomes of two species attacking different hosts revealed
a large family of genes encoding highly divergent secreted proteins that share a
common motif in the N-terminus (Jiang et al. 2008). These proteins were coined
RXLR effectors based on the amino acid composition of the shared motif. A few
years later the P. infestans genome was sequenced and included in the comparison
(Haas et al. 2009). This genome of 240 Mb, at least twice the size of that of the
other two species and much larger than that of most fungal plant pathogens, has
a high repeat content of 74 % and a typical bipartite organization with gene-dense
regions or ‘gene islands’ where highly conserved genes are located, and gene-scarce
regions or ‘gene deserts’. The latter are full of repeats but scattered in these deserts
are the RXLR effector genes that, as we know now, play a role in virulence and host
specificity of these pathogens.
376 C. Schoina and F. Govers

39.3 Controlling the Disease

To prevent late blight infection most farmers use chemical control. When the disease
pressure is high they have to spray their crop once every week to be effective. Be-
cause of the adverse effects of chemicals on the environment and the emergence of
fungicide resistant Phytophthora strains, there is an urgency to find alternatives and
late blight resistant potato cultivars are high in demand. Already in the early twenti-
eth century breeders made their first attempts to cross late blight resistance traits into
cultivated potato. Although they succeeded, the resistance didn’t hold longer than a
few years. New P. infestans races emerged and some potato lines became susceptible
to one race and others to another race (Fry 2008). Genetic analyses confirmed that
potato and P. infestans interact according to the ‘gene-for-gene’ model that was pos-
tulated in the 1940s to explain differential responses to pathogens within the same
plant species. In retrospect, we now understand the reason for the rapid loss of resis-
tance. Nearly all late blight resistance genes encode cytoplasmic ‘nucleotide-binding
leucine-rich repeat’ (NLR) proteins that initiate a resistance response at the moment
they encounter the presence of an RXLR effector. As described above, these effectors
are encoded by genes located in the most dynamic regions of the genome. The RXLR
motif functions as a host cell targeting motif, a kind of ZIP code that tells the protein
where to go (Whisson et al. 2007). By an unknown mechanism the numerous RXLR
effectors are delivered into the host cell. Their function is to suppress host defense by
manipulating the host cell machinery for the wellbeing of the pathogen and as such
it is logical that resistance (R) proteins ring the alarm bell when they sense RXLR
effectors nearby. The presence inside the plant cell of only one matching pair of
R protein and RXLR effector is already sufficient for initiating a hypersensitive re-
sponse culminating in localized cell death that arrests further growth of the pathogen
(Fig. 39.3). The key players in the classical ‘gene-for-gene’ model are the RXLR
effectors and the NLRs. The response of P. infestans to a newly introduced resistant
cultivar is to rapidly evade recognition by the novel NLR and it does so in many
different ways. For example, the matching RXLR gene can be deleted of modified
by point mutations or frame shift mutations or its expression can be suppressed by
gene silencing (Vleeshouwers et al. 2011; Kasuga and Gijzen 2013) . In other cases
the effector is still produced but its activity is suppressed, likely by other RXLR
effectors. Knowing how easily an RXLR effector can adapt has predictive value for
the durability of the matching NLR gene. For example, already a long time ago
breeders experienced that resistance derived from certain wild potato species is less
durable than that from other wild potato species and we can now explain this by the
flexibility of the RXLR effectors matching the NLRs from those species: deletions,
point mutations or frame shift mutations suggesting redundancy in function, versus
suppression of effector activity. In the latter case it seems that the pathogen cannot
simply get rid of the effector without losing viability and, as a result, the matching
NLR confers resistance that is more durable (Vleeshouwers et al. 2011). The current
strategy is to stack multiple NLRs in one cultivar and then preferably NLRs that
recognize RXLR effectors with diverse activities. So far, only a few host targets
39 The Oomycete Phytophthora infestans, the Irish Potato Famine Pathogen 377

infiltration of only NLR or effector

co-infiltration of a matching pair

Fig. 39.3 Functional analysis by in planta expression. For secreted Phytophthora proteins with
an anticipated virulence function heterologous expression in plants by Agrobacterium mediated
transformation is an ideal method for functional analyses. Many of the RXLR effectors promote
virulence because they have the ability to suppress defense in the host. This can be monitored by
transiently expressing the effector gene in leaves. RXLR effector genes can also be co-expressed
with NLR genes. Co-expression of a matching ‘gene-for-gene’ pair results in a hypersensitive
response. Expression in plants is also used for finding host targets of effectors or substrates of
proteases, and for analyzing protein-protein interactions between effector and host target

of RXLR effectors are known but it is already clear that these effectors have the
capacity to manipulate the host cell machinery at all levels and at different sites. It
remains to be seen whether plant resistance conferred by NLRs can fully control late
blight. Although very powerful, RXLR effectors are just part of the weaponry that
Phytophthora utilizes to damage the plant. Besides the cell wall degrading enzymes
and proteases there are also numerous secreted proteins with unknown activity, some
that during infection stay in the apoplast and others, such as Crinklers that, similar
to RXLR effectors, are delivered into the host cell.
For biologists P. infestans and its relatives in the oomycete lineage are intriguing;
there is still a lot to discover that can broaden our view of life on earth. For the
farmers P. infestans remains a nuisance that they want to get rid of. The challenge is
to bridge the gap: deepen our knowledge on the biology and exploit this knowledge
for developing durable and environmental friendly strategies to control late blight.

References

Du J, Verzaux E, Chaparro-Garcia A et al (2014) Elicitin recognition confers resistance to the Irish

potato famine pathogen. (To be submitted)
Fry W (2008) Phytophthora infestans: the plant (and R gene) destroyer. Mol Plant Pathol 9:385–402
Govers F, Gijzen M (2006) Phytophthora genomics: the plant destroyers’ genome decoded. Mol
Plant-Microbe Interact 19:1295–1301
378 C. Schoina and F. Govers

Haas BJ, Kamoun S, Zody MC et al (2009) Genome sequence and analysis of the Irish potato
famine pathogen Phytophthora infestans. Nature 461:393–398
Hua C, Meijer HJG, De Keijzer J et al (2013) GK4, a G-protein-coupled receptor with a phos-
phatidylinositol phosphate kinase domain in Phytophthora infestans, is involved in sporangia
development and virulence. Mol Microbiol 88:352–370
Jiang RHY, Tyler BM, Whisson SC et al (2006) Ancient origin of elicitin gene clusters in
Phytophthora genomes. Mol Biol Evol 23:338–351
Jiang RHY, Tripathy S, Govers F et al (2008) RXLR effector reservoir in two Phytophthora species
is dominated by a single rapidly evolving superfamily with more than 700 members. Proc Natl
Acad Sci 105:4874–4879
Judelson HS, Blanco FA (2005) The spores of Phytophthora: weapons of the plant destroyer. Nat
Rev Microbiol 3:47–58
Kamoun S, Hraber P, Sobral B et al (1999) Initial assessment of gene diversity for the oomycete
pathogen Phytophthora infestans based on expressed sequences. Fungal Genet Biol 28:94–106
Kamoun S, Furzer O, Jones JDG et al (2014) The top 10 oomycete pathogens in molecular plant
pathology. Mol Plant Pathol. doi:10.1111/mpp.12190
Kasuga T, Gijzen M (2013) Epigenetics and the evolution of virulence. Trends Microbiol 21:575–
582
Kroon LPNM, Brouwer H, De Cock AWAM et al (2012) The genus Phytophthora anno 2012.
Phytopathol 102:348–364
Large EC (1940) The advance of the fungi. Alden Press, Oxford
Seidl MF, Van den Ackerveken G, Govers F et al (2011) A domain-centric analysis of oomycete
plant pathogen genomes reveals unique protein organization. Plant Physiol 155:628–644
Van West P, Kamoun S, Van’t Klooster JW et al (1999) Internuclear gene silencing in Phytophthora
infestans. Mol Cell 3:339–348
Vleeshouwers VGAA, Raffaele S, Vossen JH et al (2011) Understanding and exploiting late blight
resistance in the age of effectors. Ann Rev Phytopathol 49:507–531
Whisson SC, Boevink PC, Moleleki L et al (2007) A translocation signal for delivery of oomycete
effector proteins into host plant cells. Nature 450:115–118
Chapter 40
Bacillus, A Plant-Beneficial Bacterium

Rainer Borriss

Abstract Plant growth promotion and biocontrol of plant pathogens are features of
Bacillus inoculants applied for a more sustainable agriculture. Recent results mainly
obtained with Bacillus amyloliquefaciens FZB42 and other representatives of the
B. amyloliquefaciens plantarum subspecies support the hypothesis that stimulation
of plant induced systemic resistance (ISR) by bacterial metabolites produced in the
vicinity of plant roots is the key mechanism in the biocontrol action of Gram-positive
endospore-forming bacteria, whereas a direct effect of the numerous antimicrobial
metabolites in suppressing pathogens in the vicinity of plant roots seems to be of
minor importance.

40.1 Overview About General Properties and Taxonomy

Several representatives of the Gram-positive Bacillus spp. and Paenibacillus spp. are
able to colonize plants and to develop thereby beneficial actions on plant growth and
health. At present, Bacilli are by far the most widely used bacteria on the biopes-
ticide market (Borriss 2011). This is mainly due to their ability to produce durable
endospores, which allows the preparation of stable bioformulations with a long
shelf-life. Especially members of the B. subtilis species complex, such as B. sub-
tilis, B. amyloliquefaciens, and B. pumilus, have been proven to be efficient in plant
growth- promotion and biocontrol against plant pathogens. B. subtilis and B. amy-
loliquefaciens strains are difficult to distinguish, and several bioagents declared as
containing B. subtilis spores are in fact representatives of the plant-associated B.
amyloliquefaciens subsp. plantarum (Borriss et al. 2011).
The Bacillus subtilis Group B. subtilis is the model organism of Gram-positive
bacteria. The strictly aerobe B. amyloliquefaciens plantarum, represented by its type
strain FZB42T , is distinguished from other representatives of the B. subtilis group by
its large capacity to synthesize non-ribosomally a high number of polyketides and

R. Borriss ()
ABiTEP GmbH, Glienicker Weg 185, Berlin, Germany
e-mail: Rainer.Borriss@rz.hu-berlin.de

© Springer International Publishing Switzerland 2015 379

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_40
380 R. Borriss

lipopeptides. Examples of commercial products (biocontrol or biofertilizer) contain-

ing B. amyloliquefaciens plantarum as their main active ingredient are KodiakTM
(Bayer Crop Science), Companion (Growth Products Ltd.), BioYieldTM (Bayer
Crop Science), INTEGRAL® (BASF), VAULT® (BASF), SERENADE Max®
(Bayer Crop Science), CEASE(R) (BioWorks, Inc.), RhizoVital® (ABiTEP GmbH),
RhizoPlus® (ABiTEP GmbH), Double Nickel 55TM (Certis USA), and Amylo-X®
(Certis USA). See also Table 40.1 for commercial Bacillus products for agriculture.
B. licheniformis and B. pumilus are other members of the B. subtilis group. By
contrast to B. subtilis and B. amyloliquefaciens, they are facultative anaerobes. Bio-
control agents based on B. licheniformis SB3086 are Green Releaf and EcoGuard
(Novozyme Biologicals Inc.). B. pumilus strain GB34 (Yield Shield, Bayer Crop
Science) is used as an active ingredient in agricultural fungicides. Other EPA reg-
istered biofungicides are SONATA (Bayer Crop Science), and GHA 180 (Premier
Horticulture).
Other Bacilli, not Belonging to the B. subtilis Species Complex, also stimulate
plant growth and health. B. firmus GB126 isolated from cultivated soil is used to
control root-knot nematodes in glasshouse and field grown vegetable crops (BioNem
AgroGreen, originally from Israel, later acquired by Bayer Crop Science; EPA reg-
istered nematicide). Certis USA is developing a product based on B. firmus named
BmJ WG. The biofungicide BioArc is prepared from B. megaterium, the largest
representative of the genus Bacillus.
Paenibacillus spp. The PGPR Paenibacillus polymyxa, formerly known as Bacil-
lus polymyxa, can promote plant growth by producing plant hormones, such as
IAA, cytokinins, gibberellins, and ethylene, and volatile compounds. The faculta-
tive anaerobe is capable of fixing nitrogen, and of synthesing many antibacterial
and antifungal secondary metabolites. NH is a registered fungicide prepared from
Paenibacillus polymyxa AC-1 by Green Biotech Company Ltd.
The PGPR P. mucilaginosus is able to degrade insoluble soil minerals with the re-
lease of nutritional ions, such as potassium and phosphorous. Similar to P. polymyxa,
P. mucilaginosus is also capable of fixing nitrogen.
In the following, I will shortly highlight the different traits of Bacilli involved
in their beneficial effect on plants, mainly by using results obtained during the
last decade with FZB42T , which has been successfully commercialized by ABiTEP
GmbH (http://www.abitep.de/de/), but is also used as a model strain for scientific
research (Borriss 2011).

40.2 Root Colonization

The ability of FZB42 to colonize the rhizoplane is a precondition for plant growth-
promotion. Using a GFP-tagged derivative (Fan et al. 2011) the fate of bacterial root
colonization was recently studied. The bacterium behaves different in colonizing root
surfaces of different plants. FZB42 colonized preferentially root tips when colonizing
 40

Table 40.1 Examples for commercial use of Bacillus based bioformulations in agriculture
Trade name Bacillus strain Known properties Company
TM
Kodiak Bacillus subtilis EPA-registered (71065–2) biological and seed treatment fungicide Bayer Crop Science,
GB03 former Gustafsson
LLC
Companion Bacillus subtilis EPA-registered (71065–2) biofungicide, prevent and control plant diseases. Growth Products Ltd.,
GB03 It produces a broad-spectrum Iturin antibiotic that disrupts the cell-wall White Plains, NY
formation of pathogens, and it triggers an advantageous Induced Systemic 10603
Resistance (ISR) in plants, whereby a plant’s natural immune system is
activated to fight plant diseases
Yield Shield Bacillus pumilus EPA-registered biofungicide (264–985), Suppression of root diseases Bayer Crop Science,
GB34 (= INR7) caused by Rhizoctonia and Fusarium previously Gustafsson
Bacillus, A Plant-Beneficial Bacterium

BioYieldTM B. amyloliquefaciens Combination of strong ISR activity (GB99) with phytostimulaton (GB122) Bayer Crop Science,
GB99 + Bacillus previously Gustafsson
subtilis GB122
Subtilex®, INTEGRAL® Bacillus subtilis EPA-registered (71840–8.) biofungicide, provides protection against Becker Underwood,
MBI600 soil-borne pathogens such as Rhizoctonia solani, Pythium spp. and Saskatoon, Canada
Fusarium spp. to help prevent damping-off and other root diseases acquired by BASF
VAULT® Bacillus subtilis Produced by “BioStacked®” technology, enhancing growth of soy beans Becker Underwood,
MBI600 and pea nuts Saskatoon, Canada
Bacillus pumilus BU EPA-registered (71840-RG, -RE, 2013) plant growth stimulator, induced Becker Underwood,
F-33 systemic resistance Saskatoon, Canada
SERENADE Max Bacillus subtilis EPA-registered (69592–11) biofungicide, Annex 1 listing of the EU Bayer Crop Science,
QST713 agrochemical registration directive (91/414) previously AgraQuest
SERENADE SOIL(R) Bacillus subtilis EPA-registered (69592-EI, 2012) biofungicide for food crops Bayer Crop Science,
QST713 previously AgraQuest
381
 382

Table 40.1 (continued)

Trade name Bacillus strain Known properties Company
SERENADE Optimum® Bacillus subtilis EPA-registered (2013) biofungicide/bactericide for prevention. It works by Bayer Crop Science,
QST713 stopping spore germination, disrupting cell membrane and inhibiting previously AgraQuest
attachment of the pathogen to leaves. For use in leafy and fruiting
vegetables, strawberries and potatoes. Active against fungal (Botrytis,
Sclerotinia), and bacterial pathogens (Xanthomonas and Erwinia)
CEASE(R) Bacillus subtilis Aqueous suspension biofungicide, recommended for leafy and fruiting BioWorks, Inc.,
QST713 vegetables, herbs and spices, and ornamentals Victor, New York,
USA
SONATA® Bacillus pumilus EPA-registered (69592–13) biofungicide, powdery mildew control Bayer Crop Science,
QST2808 previously AgraQuest
Inc
RhizoVital® Bacillus Biofertilizer, plant growth promoting activity, provides protection against ABiTEP GmbH,
amyloliquefaciens various soil borne diseases, stimulation of ISR Berlin
FZB42
RhizoPlus® Bacillus subtilis Plant growth-promoting rhizobacterium and biocontrol agent. It can be used ABiTEP GmbH,
for potatoes, corn, vegetables, fruits and also turf Berlin
Taegro® Bacillus subtilis EPA-registered biofungicide. FZB24 has been originally isolated by FZB Syngenta, Basel,
FZB24 Berlin, the forerunner of ABiTEP GmbH. Registration as a biofungicide for previously Novozyme,
the US was performed by Taegro Inc. and then sold to Novozymes without Davis, California and
agreement with ABiTEP GmbH where the product is still offered Earth Biosciences
POMEX Bacillus subtilis Microbial fungicide, control and inhibition germination effect on powdery NIN Co. Ltd.,
CMB26 mildew, Cladosporium fulvum and Botrytis cinerea
Bacillus subtilis EPA-registered 71840-RG,-RE (2012) fungicide, bactericide for food crops, Certis Columbia, MD
CX9060 turf and ornamentals USA
R. Borriss
 40

Table 40.1 (continued)

Trade name Bacillus strain Known properties Company
Easy Start® TE-Max Bacillus subtilis Rhizosphere bacterium that competes with harmful pathogens for space COMPO Expert
E4-CDX around the roots of the grass plant. Once established this unique strain GmbH, Münster,
physically protects the roots and inhibits the advance of soil borne fungi Germany
Double Nickel 55TM B. amyloliquefaciens EPA-registered (70051-RNI, 2011) a broad spectrum preventive Certis Columbia, MD
D747 biofungi-cide for control or suppression of fungal and bacterial plant USA
diseases (Powdery mildew, Sclerotinia, Botrytis, Alternaria, bacterial leaf
spot, bacterial spot and speck, Fire blight, Xanthomonas, Monilinia
Bacillus, A Plant-Beneficial Bacterium

Amylo-X® B. amyloliquefaciens Annex 1 listing of the EU agrochemical registration directive. Launched to Certis Columbia, MD
D747 Italy by Intrachem Bio Italia SpA for control of Botrytis and other fungal USA/Intrachem Bio
diseases of grapes, strawberries and vegetables, and bacterial diseases such Italia SpA
as fire blight in pome fruit and PSA in kiwi fruit
BmJ WG Bacillus mycoides It works entirely as a microbial SAR activator with no direct effect on the Certis Columbia, MD
BmJ plant pathogen itself. Under development USA
Bacillus pumilus EPA-registered fungicide (2012), Food crops, seeds, ground cover, and Premier Horticulture
GHA 181 ornamentals
BioNem Bacillus firmus EPA-registered (2008), suppressing plant pathogenic nematodes, Bacillus AgroGreen, Israel
GB-126 firmus creates a living barrier that prevents nematodes from reaching the acquired by Bayer
roots Crop Science
The US governmental EPA registration does not depend on successful field trials; it is only necessary to demonstrate that no negative effects are connected with
the use of the biofungicide
383
384 R. Borriss

FENGYCIN
BACILLOMYCIN D
BACILLIBACTIN
SURFACTIN IAA
sigH sigD sfp tasA
RBAM17410 FZB42 degU
alsS nfrA abrB

DIFFICIDIN ACETOIN 2,3-BUTANEDIOL

MACROLACTIN

BACILLAENE
BACILYSIN

PHYTASE

PLANTAZOLICIN AMYLOCYCLICIN

Pi

Fig. 40.1 Secondary metabolites with biocontrol or PGP activities produced by B. amyloliquefa-
ciens FZB42. Genes involved in plant root colonization (white) and plant growth promotion (yellow)
are listed within the bacterial cell. The cyclic lipopeptides (cLP, blue) surfactin, bacillomycin D,
and fengycin are nonribosomally synthesized by modularly organized, giant peptide synthetases
(NRPSs). Antibacterial polyketides (PK, red) are synthesized by membrane-anchored, polyketide
megasynthases. Synthesis of PKs and cLPs is dependent on functional phosphor-panthetheinyl-
transferase Sfp. NRPSs are also involved in synthesis of the dipeptide bacilysin (blue) and the Fe2+
siderophore bacillibactin (blue). The plant growth-promoting metabolites acetoin, 2,3-butanediol,
and indole-3-acetic acid (IAA) are shown in green. Extracellular phytase (green) makes phosphate
fixed in phytate accessible for plant nutrition. Other extracellular enzymes, which are degrading
macromolecules, and supporting the biofertilizer function of FZB42, are ß-glucan and xylan hy-
drolases, amylases, and proteases, for example. Bacterial metabolites, involved in stimulating plant
induced systemic resistance (ISR), are framed

Arabidopsis thaliana. In lettuce, bacterial colonization occurred mainly on primary

roots and root hairs, as well as on root tips and adjacent border cells. Essential genes
for root colonization are involved in surfactin production, motility, biofilm formation,
and stress response (Fig. 40.1). Mutants containing a transposon insertion in the nfrA
gene, encoding a putative nitro/flavin oxidoreductase, were unable to persist on the
surface of lettuce roots, most likely due to their inability to develop an appropriate
response against the plant’s stress reactions (Budiharjo et al. 2014).
The Rhizosphere Competence of FZB42 was studied by using a combination of
field and greenhouse trials. FZB42 is able to effectively colonize the rhizosphere
(6.61–7.45 Log10 CFU g−1 root dry mass) within the growth period of lettuce in the
field. However, the cell number (CFU) of FZB42 per gram of soil decreased to 14 % of
the initial number of cells after 5 weeks of field cultivation (Chowdhury et al. 2013).
40 Bacillus, A Plant-Beneficial Bacterium 385

The same samples were analyzed more deeply by mapping of the metagenome se-
quences corresponding to FZB42. The method called ‘fragment recruitments’ was
used to track persistence of the inoculant FZB42 within the lettuce rhizosphere.
Five weeks after inoculation, the DNA fragments corresponding to FZB42 were
still traceable, but their number was reduced to around 55 % of the initial number
(Kröber et al. 2014). The results obtained with the two different methods indicate
that the inoculant strain FZB42 was less competitive than the indigenous community
members.

40.3 Plant-Growth Promotion

Although the ability of FZB42 to support growth of potato, maize, cotton, tobacco,
leafy and fruiting vegetables, and ornamentals is well documented (Borriss 2011),
our knowledge about the molecular basis of the ‘biofertilizer’ effect of beneficial
plant-associated Bacilli are far from complete. Several traits (Fig. 40.1) are involved
in the complex interplay between root-colonizing bacteria and plant.
1. Tryptophan-Dependent Synthesis of Indole-3-Acetic Acid. Inactivation of
genes involved in tryptophan biosynthesis and in a putative tryptophan-dependent
IAA biosynthesis pathway led to reduction of both IAA levels and plant growth-
promoting activity in the respective mutant strains (Idris et al. 2007). Notably, seed
treatment with FZB42 increased root production, an indicator of auxin production,
but significantly repressed root Pi uptake at low environmental Pi concentrations
(Talboys et al. 2014).
2. Volatiles, such as 2,3-Butanediol and Acetoin, released by B. subtilis and B.
amyloliquefaciens, enhance plant growth. To synthesize 2,3-butanediol, pyruvate
is converted to acetolactate by acetolactate synthase (AlsS), which is subsequently
converted to acetoin by acetolactate decarboxylase (AlsD). FZB42 mutant strains,
deficient in the synthesis of volatiles due to mutations in the alsD and alsS genes,
were impaired in plant growth-promotion (Borriss 2011).
3. Phytase-Producing Bacteria Enhance Phosphorous Availability. Soil phos-
phorous is an important macronutrient for plants. Improved phosphorous nutrition
is achievable by ‘mobilization’ of phosphorous fixed as insoluble organic phos-
phate in phytate (myo-inositol-hexakisphosphate); see also Chap. 24. The
extracellular 3-phytase of the PGP B. amyloliquefaciens FZB45 hydrolyzed phy-
tate to InsP5 and phosphate in vitro (Fig. 40.1). A phytase-negative mutant strain,
whose phyA gene was disrupted, did not stimulate plant growth under phosphate
limitation (Idris et al. 2002). Further experiments under field conditions revealed
that FZB45 only stimulates plant growth when phytate is present in soils which
are poor in soluble phosphate.
Other mechanisms that are involved in biofertilizer function of Bacilli include ni-
trogen fixation, mineral solubilization, and secretion of macromolecule degrading
enzymes (Borriss 2011).
386 R. Borriss

40.4 Biocontrol by Antimicrobial Compounds

B. amyloliquefaciens FZB42 was successfully applied to suppress the plant pathogen

Rhizoctonia solani on lettuce (Chowdhury et al. 2013). Genome analysis revealed that
nearly 10 % of the FZB42 genome is devoted to synthesizing antimicrobial metabo-
lites and their corresponding immunity genes (Borriss 2013). This antibiotic arsenal
(Table 40.2) makes B. amyloliquefaciens FZB42 and related B. amyloliquefaciens
plantarum strains promising microbial biopesticides.
Cyclic Lipopeptides Five gene clusters involved in non-ribosomal synthesis of c-
LPs and of the iron-siderophore bacillibactin were identified in the genome of FZB42
(Table 40.2). Three of the respective gene clusters were assigned to the syntheses of
surfactin, fengycin, and bacillomycin D. The iturin bacillomycin D was identified
as the most powerful fungicide produced by FZB42. An early surfactin secretion
could be of biological relevance since this c-LP, although less fungitoxic than iturins
and fengycins, is essential for moving of the bacteria on plant tissues and for matrix
formation in biofilms (Chen et al. 2009).
Polyketides The three gene clusters encoding the modularly organized polyketide
synthases (PKS) for syntheses of bacillaene, macrolactin, and difficidin cover nearly
200 kb. Difficidin is the most effective antibacterial compound produced by FZB42T ,
but also macrolactin and bacillaene possess antibacterial activity. Difficidin is effi-
cient in suppressing the plant pathogenic bacterium Erwinia amylovora, which causes
fire blight disease in orchard trees. Macrolactin A (MA) and 7-O-succinyl macro-
lactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show
antibiotic effects superior to those of teicoplanin against vancomycin-resistant ente-
rococci and methicillin-resistant Staphylococcus aureus. MA and SMA are currently
being evaluated in preclinical studies in Korea as anti-tumor agents.
Bacilysin Another product of non-ribosomal synthesis, the dipeptide bacilysin was
found as also being involved in the suppression of Erwinia amylovora. Recent ex-
periments demonstrated that bacilysin, besides difficidin, is efficient in suppressing
Microcystis aeruginosa, the main causative agent of cyanobacterial bloom in lakes
and rivers (Liming Wu et al. unpublished).
Ribosomally Synthesized Antimicrobial Peptides remained unknown in B.
amylolique-faciens plantarum for a long time with one remarkable exception:
mersacidin, a B-type lantibiotic, was detected in strain HIL Y85, later clas-
sified as being B. amyloliquefaciens plantarum (Herzner et al. 2011). Ri-
bosomally synthesized antibacterial peptides (bacteriocins) were detected in
FZB42 by using a mutant strain devoid in non-ribosomal synthesis of polyke-
tides, lipopeptides and bacilysin, which still possessed some remaining an-
tibiotic activity. Plantazolicin (PZN) displayed antibacterial activity towards
closely related gram-positive bacteria, especially against B. anthracis. In ad-
dition, PZN displayed a moderate nematicidal activity (Liu et al. 2013).
Due to its extensive degree of modification, PZN is well protected from premature
40 Bacillus, A Plant-Beneficial Bacterium 387

Table 40.2 Genes and gene cluster encoding for secondary metabolites in selected Bacillus spp.
Metabolite Occurrence Gene cluster Size Effect against Reference
Sfp-dependent non-ribosomal synthesis of lipopeptides
Surfactin BAP, BAA, srfABCD 32.0 kb Virus Stein 2005
BSU
Iturin BAP, BAA, bmyCBAD 39.7 kb Fungi Chen et al.
BSU 2007
Fengycin BAP, BSU fenABCDE 38.2 kb Fungi Chen et al.
2007
Polymyxin PPO pmxABCDE 40.7 kb Bacteria Niu et al.
2013
Fusaricidin PPO fus GFEDCBA 32.4 kb Fungi Li and
Jensen 2008
Bacillibactin BAP, BAA, dhbABCDEF 12.8 kb Bacterial Chen et al.
BSU competitors 2007
Sfp-dependent non-ribosomal synthesis of polyketides
Macrolactin BAP mlnABCDEFGHI 53.9 kb Bacteria Chen et al.
2007
Bacillaene BAP, BAA, baeBCDE, acpK, 74.3 kb Bacteria Chen et al.
BSU baeGHIJLMNRS 2007
Difficidin BAP dfnAYXBCDEFGHIJKLM 71.1 kb Bacteria Chen et al.
2007
Sfp-independent non-ribosomal synthesis
Bacilysin BAP, BSU bacABCDE, ywfG 6.9 kb Bacteria, Chen et al.
cyanobacteria 2007
Ribosomal synthesis of processed and modified peptides (bacteriocins)
Plantazolicin BAP FZB42 pznFKGHIAJC DBEL 9.96 kb B. anthrax, Scholz et al.
nematodes 2011
Amylocyclicin BAP FZB42 acnBACDEF 4.49 kb Closely related Scholz et al.
bacteria 2014
Mersacidin BAP Y2 mrsK2R2FGEAR1DMT 12 kb Gram-positive Stein 2005
bacteria
Amylolysin BAP GA1 amlAMTKRIFE 9.36 kb Gram-positive Arguelles
bacteria Arias et al.
2014
Subtilin BSU ATCC spaBTCAIFGRK 12 kb Closely related Stein 2005
6633 bacteria
Ericin BAP A1/3 eriBTCASIFEGRK 12.5 kb Closely related Stein 2005
bacteria
Sublancin BSU sunAT bdbA yolJ bdbB 4.5 kb Closely related Stein 2005
bacteria
Subtilosin A BSU sboA albABCDEFG 7.0 kb Closely related Stein 2005
bacteria
BAP B. amyloliquefaciens plantarum, BAA B. amyloliquefaciens amyloliquefaciens, BSU B.
subtilis subtilis, PPO Paenibacillus polymyxa
388 R. Borriss

degradation by peptidases within the plant rhizosphere (Scholz et al. 2011). A cir-
cular bacteriocin, named amylocyclicin, was recently identified (Scholz et al. 2014).
The peptide suppressed growth of the plant pathogenic actinobacterium Clavibacter
michiganensis and of several Gram-positive bacteria.
Importance of Secondary Metabolites for Biocontrol For a long time one has
thought that the plant protective activity of FZB42 and other PGPR is due to the
antibiotic activity of a wide array of antibiotic compounds upon growth under labo-
ratory conditions. However, in recent years, this became doubtful due to pioneering
work of Ongena et al. They investigated antibiotic production by MALDI MSI in
a gnotobiotic system in which the plantlet and the associated B. amyloliquefaciens
S499, a close relative of FZB42, were growing on a gelified medium covering the
MALDI target plate. Surfactins were detected during early biofilm formation in the
rhizosphere in relatively high amounts, representing more than 90 % of the whole
c-LP production. In contrast, the synthesis of iturin and fengycin was delayed until
the end of the aggressive phase of colonization (Debois et al. 2014).

40.5 Induced Systemic Resistance

Due to the low concentration of antimicrobial compounds detectable in the rhizo-

sphere, it is tempting to speculate that ISR is the main factor for suppressing plant
pathogens by PGPR Bacilli. ISR occurs when the plant’s defense mechanisms are
stimulated and primed to resist infection by pathogens (Doornbos et al. 2012). It has
been demonstrated that Bacillus derived volatiles and cLPs trigger ISR.
Volatiles Several Bacillus PGPR strains emit VOCs that can elicit plant defenses.
Exposure to VOCs consisting of 2,3-butanediol and acetoin (3-hydroxy-2-butanone)
from PGPR Bacillus amyloliquefaciens activates ISR in plants (see Chap. 8). In this
context it is worth to mention that expression of AlsS of FZB42, involved in the
synthesis of acetoin (Fig. 40.1), was triggered in the presence of maize root exudate
(Kierul et al. unpublished), suggesting that root exudates play a role in the elicitation
of acetoin biosynthesis in FZB42.
Circular lipopeptides surfactin and fengycin act as elicitors of host plant im-
munity and contribute to increased resistance toward further pathogenesis ingress in
bean and tomato plants (Raaijmakers et al. 2010).

40.6 Effect of Bacillus Inoculants on the Environment

The impact of beneficial Bacillus inoculants on the root microbiome is important

for their plant health effect. Terminal-restriction fragment length polymorphism, T-
RFLP, and metagenome analyses of lettuce rhizosphere samples inoculated with B.
amyloliquefaciens FZB42 vs. non-treated samples revealed that the inoculant strain
40 Bacillus, A Plant-Beneficial Bacterium 389

only had a minor impact on the community structure within this habitat, while inocu-
lation with the pathogen R. solani did significantly change the rhizosphere microbial
community structure (Chowdhury et al. 2013; Kröber et al. 2014). A significant in-
crease in gamma-proteobacterial diversity was detected in samples inoculated with
the pathogen. However, in the presence of FZB42 this increase was less distinct,
suggesting a selective compensation of the impact of a pathogen on the indigenous
plant-associated microbiome by FZB42 (Erlacher et al. 2014). The results of these
metagenome studies suggest that the application of the commercially available in-
oculant strain FZB42 can be considered as a safe method to promote the health
of the economically important lettuce plant and reduce severity of infections by
phytopathogens like R. solani.

40.7 Conclusions

The beneficial effect of Bacillus PGPR on plant health relies on at least three main
factors:
1. In previously published studies the set of secondary metabolites described here
was suspected to mediate mainly the antibiosis function of Bacillus bioinoculants.
However, the amounts of the relevant antibiotics found in the vicinity of plant
roots were relatively low, making a significant antibiosis function doubtful.
2. These metabolites were also suspected to induce changes within the micro-
bial rhizosphere community, which might affect the health of environment and
plant. However, sequence analysis of rhizosphere samples revealed only marginal
changes in the root microbiome, suggesting that secondary metabolites are not
the key factor in protecting plants from pathogenic microorganisms. On the other
hand, adding FZB42 to lettuce plants compensate, at least in part, global changes
in the community structure caused by the pathogen, indicating an interesting
mechanism of plant protection by beneficial Bacilli.
3. Recent results support hypothesis, that stimulation of plant ISR by bacterial
metabolites, such as VOCs and c-LPs, produced in the vicinity of plant roots,
is the key mechanism in the biocontrol action of Bacilli.

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Chapter 41
Soybean Production in the Americas

Woo-Suk Chang, Hae-In Lee and Mariangela Hungria

Abstract Soybean (Glycine max (L.) Merr.) is one of the most important legume
crops in the world. Approximately 80 % of the world’s soybean is produced by
countries in North and South America. Biological nitrogen fixation (BNF) in soy-
beans, due to the symbiosis with Bradyrhizobium, is economically and ecologically
beneficial because it reduces the need for synthetic N-fertilizers. This chapter de-
scribes history and trends of soybean production, influence of soybean BNF, and
development of inoculants to increase the crop yield in North and South America.

41.1 History of Soybean Cultivation in North

and South America

Farmers grow soybeans for various purposes such as human food, animal feed, and
industrial applications. Since the soybean contains an average of 40 % protein and
20 % oil, it can be a great nutrient source for both humans and animals. A number of
soy foods such as tofu, soy sauce, and soymilk are popular in many Asian countries,
while in the United States most soybeans for human consumption are used to produce
edible oil products such as cooking oils, margarine, mayonnaise, and vegetable
shortening. In South America, in addition to the use of oil, soybean-based milk and
soups are part of the daily meals of children in public schools. Soybeans are largely
added to a variety of processed foods, from meat-derived to crackers. They are

W.-S. Chang () · H.-I. Lee

Department of Biology, University of Texas-Arlington, Arlington, Texas 76019, USA
Tel.: + 1 8172723280
e-mail: wschang@uta.edu
H.-I. Lee
Tel.: + 1 8172723264
e-mail: hilee@uta.edu
M. Hungria
Embrapa Soja, Cx. Postal 231, Londrina, Paraná 86001–970, Brazil
Tel.: + 55 4333716206
e-mail: mariangela.hungria@embrapa.br

© Springer International Publishing Switzerland 2015 393

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_41
394 W.-S. Chang et al.

also considered as an excellent protein source for livestock and are used to produce
industrial products such as biodiesel, soy-based lubricants, and soy inks.
Soybean originated in East Asia. It was first cultivated in China around 1100
BC and had spread to Japan and many other countries by the first century AD. In
the 1980s, the genus Glycine was split into the subgenera Glycine (wild perennial
species) and Soja (including both wild-Glycine soja Sieb. and cultivated-Glycine
max genotypes). G. max was introduced into Europe (Paris) only in 1740. It was first
introduced to North America from China by Samuel Bowen in 1765 (Hymowitz and
Harlan 1983). Soybean became a popular crop in the U.S. between the mid-19th to
early 20th centuries. The American Soybean Association was founded in 1920 by
soybean farmers and extension workers. World War II kindled the prosperity of the
soybean farming in the U.S. as the drastic increase in demand for lubricants and oils
by the war increased the soybean demand. The U.S. has been the leading country
for soybean production in the world. In Canada, soybeans were first cultivated at
the Ontario Agricultural College in 1881, and at present Canada is the world’s 7th
largest soybean producing country.
Glycine spp. were introduced to South America by the end of the 19th century
(Argentina, 1880; Brazil, 1882). Seeds from the U.S., Brazil, Argentina, and Japan
were taken to Paraguay in the 1920s, and later to Bolivia, Colombia, Uruguay,
and Venezuela. Commercial scale production started in the 1940s in Brazil and
Argentina, which led to increased production by the 1960s. One major event for
soybean expansion in South America was breeding of soybean cultivars with a long
juvenile period, which allowed for the production at very low latitudes.

41.2 Soybean Production in North and South America

World soybean production has been rapidly increasing since 1990, mainly due to
increased production in North and South America. The top five countries for soybean
production include the U.S., Brazil, Argentina, China, and India, which represent
more than 90 % of world production (Fig. 41.1).
The soybean cultivation area in the U.S. has rapidly expanded since the mid-20th
century and reached about 31 million ha in 2013. Soybeans are produced in more than
one third of the states but mostly in the Midwestern states Iowa, Illinois, Minnesota,
and Indiana (order of top producing states). The soybean cultivation area declined
in 2007, when many farmers turned to the cultivation of corn to supply the growing
bioethanol industry (Salvagiotti et al. 2008). Nevertheless, the total production of
soybean and the crop yield has been increasing steadily, mostly due to improved
varieties and advances in biotechnology. The national average yield in the U.S. has
increased from 1581 kg ha−1 in 1960 to 2919 kg ha−1 in 2013. In Canada, the average
crop yield has reached 3300 kg ha−1 in 2012. Bioengineered soybeans are one of
the most successful crops commercially in North and South America. They account
for 93 % of the soybean produced in the U.S., and for about 90 and 100 % in Brazil
and Argentina, respectively. Most of these bioengineered soybeans are improved
41 Soybean Production in the Americas 395

USA
Brazil
80 Argentina
China
Production (million metric tons)

India

60

40

20

0
1970 1980 1990 2000 2010
Year

Fig. 41.1 Soybean production of the top 5 countries in the world. The data for the U.S. were retrieved
from the USDA statistics for the annual production from 1970 to 2013 (http://www.nass.usda.gov),
while the data for the other countries represent occasional years (1970, 1980, 1990, 1995, 2000,
2005, and 2009–2013)

varieties with high resistance against herbicides, and now double resistance to both
herbicides and insecticides.
Soybean production continues to grow at impressive rates in the South Ameri-
can countries that account for more than half of the global production (Fig. 41.1);
estimates are that soon Brazil may replace the U.S. as the leading producer in the
world. National average yield in Brazil increased from 1166 kg ha−1 in 1968/1969
to 3115 kg ha−1 in 2010/2011. The potential soybean production has been estimated
at 8000 kg ha−1 , and field trials in North and South America have reported yields of
4000 to 6000 kg ha−1 . In addition, while not scientifically proven, there are reports
of U.S. farm yields exceeding 10,000 kg ha−1 (Hungria and Campo 2004; Hungria
et al. 2005, 2006; Hungria and Mendes 2014).

41.3 Nitrogen Fixation Associated with Soybean in North

and South America

In 1981, LaRue and Patterson estimated that the average amount of BNF associated
with soybean in the U.S. might not exceed 75 kg N ha−1 (Larue and Patterson 1981).
However, Salvagiotti and colleagues (2008) recently reviewed soybean nitrogen fix-
ation more comprehensively by analyzing 637 data sets from field studies published
396 W.-S. Chang et al.

in international journals between 1966 and 2006. They calculated the average BNF
in soybeans to be as high as 111–125 kg N ha−1 . Although their analysis was per-
formed with international data sets from many different countries, almost half of the
data sets were from studies in the U.S. and Canada.
Interestingly, Herridge et al. (2008) reported that the percentage of N derived from
air (%Ndfa) by soybean in the U.S. is on average 60 % of the total Ndfa, which indeed
gives around 140 kg N ha−1 . Estimates of soybean-associated BNF contribution to
the total Ndfa in South America are greater, around 80 % in both Brazil and Argentina
(Herridge et al. 2008). This would correspond with 190 kg N ha−1 ; however, there
are reports of contributions higher than 300 kg N ha−1 (Hungria et al. 2006). The
lower contribution of BNF in the U.S. might be attributed to heavy applications of
N-fertilizers in agriculture, with leftovers for the soybean, as well as to low adoption
of inoculation by the farmers.

41.4 Inoculants and N-fertilizers

Bradyrhizobium In the Americas all inoculant strains belong to the genus Bradyrhi-
zobium, including the species B. japonicum, B. diazoefficiens, and B. elkanii
(Delamuta et al. 2013). The Bradyrhizobium-soybean symbiosis requires specific
signal exchange between the two partners. Soybean secretes isoflavonoids, such as
genistein and daidzein, into the rhizosphere and these substances trigger nodulation
(nod) genes in Bradyrhizobium. These nod genes encode Nod factors, which initiate
root hair curling and formation of infection threads. Bradyrhizobium cells invade
the host plant root cells through the infection threads and subsequently form a new
organelle (i.e., the nodule), in which bacteria develop into bacteroids. Within the
nodule, the bacteroids can express oxygen-sensitive nitrogenase (nif ) genes due to
the microoxic condition (see Chap. 23).
The Agricultural Research Service (ARS) of the U.S. Department of Agriculture
(USDA) has collected Rhizobium isolates, including Bradyrhizobium, since the early
1900’s, and the collection is well known internationally. As of March 2014, the
Germplasm Resources Information Network (GRIN) database of the USDA/ARS
reports 534 strains isolated from soybeans. Many of the strains were isolated by
research programs in the 1930s and 1940s, and the USDA began to produce inoculants
for the small research field in the 1950s and 1960s. The need for a Rhizobium culture
collection was emphasized as the importance of BNF was recognized as a means to
supplement hydrocarbon-based fertilizers.
Inoculants The first U.S. patent for pure cultures of rhizobia to be used in conjunc-
tion with artificial inoculation was issued in 1896. Two years later the first inoculant
company, Nitragin, was established in the U.S. Inoculants for soybean were commer-
cialized by the company in the early 1900s. The search for an adequate medium to
carry rhizobia focused on peat which ultimately became globally established as the
“gold standard”. By the 1950s, inoculant industries expanded into South America.
41 Soybean Production in the Americas 397

Particularly, in the last two decades there has been a shift away from peat towards
liquid formulations, as they are easily applied to the seeds and preferred by the farm-
ers. However, concerns about liquid inoculants were raised long ago; Burton and
Curley (1965) reported inferior performance of liquid-based inoculants even though
2.5 times as many rhizobia as in peat-based inocula were applied to the seeds. Several
decades later, although protective molecules, adhesives and several polymers have
been introduced into inoculants, innovation in liquid, gel or other non-solid formu-
lation has been modest. Therefore, a need for a second generation of inoculants has
arisen in the soybean industry (more information in section 41.5).
Most farmers in South America are convinced of the benefits of soybean reinoc-
ulation with elite strains selected through research programs and registered in
governmental agencies. This results in a market size estimated at over 50 million
doses of inoculants applied by about 60 % of the farmers (some farmers use multiple
doses, especially in first-year planting areas). In contrast, the use of inoculants in
the U.S. is a common practice for only about 15 % of the soybean production area,
and is perhaps due to low prices of N-fertilizers or a perception of their insignificant
performance. An evaluation of the effect of inoculants used in Midwestern states,
including Indiana, Iowa, Minnesota, Nebraska, and Wisconsin, between 2000 and
2008 revealed that the use of inoculants was not successful in enhancing crop yield or
economic return when used in soils that already had a history of soybean cultivation
(De Bruin et al. 2010). Rising prices of N-fertilizers and concerns about the resultant
nitrate contamination of the environment may shift this inoculation panorama.
Competitiveness and effectiveness of inoculants have been considered key prop-
erties to guarantee the success of nitrogen fixation with the soybean crop. There
are long time concerns, however, about highly competitive but low effective indige-
nous/naturalized Bradyrhizobium population in the soybean rhizosphere (Baldwin
and Fred 1929). One main example is of the USDA 123 serocluster in the Midwestern
U.S., given rise to 60 to 80 % of the nodules, while more effective inoculant strains
result in only 10 to 20 % of the nodules formed. This serogroup is also a concern in
Canada, Korea, and Brazil (Hungria and Mendes 2014). Several laboratories have
tried to identify bacterial properties implicated in competitiveness, such as mobil-
ity, chemotaxis, exopolysaccharides, bacteriocins, capacity to respond to several
substrates. Interestingly, molecular approaches have also been considered (Hungria
et al. 2006); however, application of new strategies or genetically engineered strains
to agriculture has been limited.
Reports of the impossibility of displacing competitive strains established in the
soils (Thies et al. 1991) have probably discouraged research to select elite strains, and
farmers to adopt inoculation in the U.S. Currently, in South America probably more
than 90 % of the areas cropped to soybean have been previously inoculated, showing
naturalized bradyrhizobial populations ranging from 103 to up to 106 cells g−1 of
soil. Dozens of field experiments performed in the last 20 years have consistently
shown that reinoculation of soybean results in yield increases. The analysis of sets of
experiments indicates that the average increase in yield due to annual reinoculation is
8 % and 14 % in Brazil and Argentina, respectively, compared to the non-inoculated
control (Hungria et al. 2006; Hungria and Mendes, 2014). In addition, it is worth
398 W.-S. Chang et al.

noting that massive reinoculation reported in South America may cause the replace-
ment of persistent strains with more efficient strains (Hungria et al. 2006; Hungria
and Mendes 2014).
N-fertilizers Less than 40 % of the soybean cultivation area in the U.S. is supple-
mented with chemically synthesized fertilizers. Nevertheless, as soybean represents
a high profit crop, there is also increasing pressure for farmers to use N-fertilizers.
Soybeans apparently do not require additional N-fertilizers in normal soil conditions
due to BNF with its symbiotic partner Bradyrhizobium. Additionally, one of the
factors confounding our understanding of both the efficiency of inoculants and the
use of N-fertilizers is the effect of crop rotation. High residual soil N as a result of
corn cultivation, usually in the Midwest states (i.e., Corn Belt area), may influence
interpretation of soybean yield when soybean is planted behind corn (Stewart Smith,
personal communication). In South America, studies performed over the last two
decades demonstrated that soil N availability as low as 20–40 kg of N ha−1 may
decrease nodulation and N2 fixation in soybeans with no benefits to crop yield. In
addition, no benefits have been reported with the addition of 30 to 50 kg of N ha−1 at
flowering, early or late pod filling stages. Indeed, applications of up to 400 kg N
ha−1 , split across ten applications did not result in higher yields in comparison to
inoculation with elite strains (Hungria et al. 2006; Hungria and Mendes 2014).

41.5 A Second Generation of Inoculants

Promising results have been reported with the use of other microorganisms and
molecules as inoculants for soybean. Co-inoculation with plant-growth-promoting
rhizobacteria (PGPR) such as Azospirillum brasilense may increase yield (Hungria
et al. 2013). One of the most exciting new concepts is to take advantage of the
molecular dialogue between plant and bacterium. Spaink and colleagues (Spaink
et al. 1992) separated Nod metabolites from several Rhizobium and Bradyrhizo-
bium strains using thin-layer chromatography (TLC) and found that common nod
genes, such as nodABC, play a key role in Nod metabolite production. The Nod
metabolite produced by Bradyrhizobium strain USDA 110 was subsequently identi-
fied as a lipochitooligosaccharide (LCO) signaling molecule, similar to those from
Rhizobium species (Sanjuan et al. 1992). The LCO from USDA 110 was able to
promote the growth of soybean at a low concentration (100 nM) in hydroponic con-
ditions (Souleimanov et al. 2002). The Novozymes’ patented LCO molecule product,
Optimize®, has been commercially available both in North and South America. Ap-
parently, field responses with LCO have shown slight but consistent increases in
soybean yields by an average of ca. 2–3 %, although the increases may depend on
specific conditions (Leibovitch et al. 2002). Additionally, it can bring other benefits
such as promoting root growth (Souleimanov et al. 2002). The positive effects of
Nod factors or bacterial metabolites can be also observed in non-legumes such as
corn (Liang et al. 2013; Marks et al. 2013).
41 Soybean Production in the Americas 399

41.6 Perspectives on Economical and Ecological Benefits

of BNF with Soybeans

Enhancement of soybean BNF has profound economical and ecological benefits by

reducing the use of chemical N-fertilizers that are a significant source of greenhouse
gas emissions and cause degradation of water sources ranging from groundwater,
where derivatives of nitrogen cause the “blue-baby” syndrome (Knobeloch et al.
2000), to oceanic systems where increased nitrogen leads to hypoxia (lack of oxygen)
and large scale “dead zones” in the Northern Gulf of Mexico (Burkart and James
1999).
The enormous demand for N by the soybean crop results from the need of about
80 kg of N per 1000 kg of grains; considering that the efficiency of N-fertilizers is
rarely more than 50 %, it is calculated that there is a need of 480 kg N-fertilizer ha−1 ,
or about 700 kg of urea, the most broadly used N-source (Hungria et al. 2006). In an
exercise to quantify the global contribution of BNF with the soybean crop, Herridge
et al. (2008) concluded that 16.4 Tg of N is fixed annually, representing 77 % of the N
fixed by all crop legumes. Considering the high price of N-fertilizers in Brazil—70 %
of which is imported—in combination with the area cropped, the average BNF rate,
and the national average yield, BNF is estimated to save about US$ 15 billion yearly.
Not least important, by using a conservative rate of 4.5 kg of e-CO2 kg−1 (CO2 equiv-
alent) of N-fertilizer, the replacement of BNF by N-fertilizers in Brazil would result
in the emission of about 45 million tons of e-CO2 (Hungria and Mendes, 2014). The
importance of this approach, specifically in South America, with a strong partnership
between plant breeders and microbiologists towards improving the contribution of
BNF should be highlighted. Outstanding symbiotic performances as those reported
in South America can be lost in a few years if plant breeders and microbiologists
do not continue the long-term and successful partnership towards increasing BNF
contribution. A continuous pressure from companies to supply N-fertilizers to the
soybean crop claiming higher yields can also have profound impacts in BNF con-
tribution. Another important consideration is the increasing use of pesticides and
other chemicals in the seed treatment, which can drastically affect Bradyrhizobium
survival and impair BNF (Mendes and Hungria 2014). On the contrary, disclosure
and dissemination of the successful results such as those achieved in South America
can stimulate more farmers to use inoculants.

Acknowledgement Critical review and editing was provided by Dr. Thomas Chrzanowski from
the Department of Biology at UT-Arlington

References

Baldwin IL, Fred EB (1929) Strain variation in the root nodule bacteria of clover, Rhizobium trifolii.
J Bacteriol 17:17–18
Burkart MR, James, DE (1999)Agricultural-nitrogen contributions to hypoxia in the Gulf of Mexico.
J Environ Qual 28: 850–859.
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Burton JC, Curley RL (1965) Comparative efficiency of liquid and peat-base inoculants on field-
grown soybean (Glycine max). Agron J 57:379–381
De Bruin JL, Pedersen P, Conley SP et al (2010) Probability of yield response to inoculants in fields
with a history of soybean. Crop Sci 50:265–272
Delamuta JR, Ribeiro RA, Ormeno-Orrillo E et al (2013) Polyphasic evidence supporting the
reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens
sp. nov. Int J Syst Evol Microbiol 63:3342–3351
Herridge DF, Peoples MB, Boddey RM (2008) Global inputs of biological nitrogen fixation in
agricultural systems. Plant Soil 311:1–18
Hungria M, Campo RJ (2004) Economical and environmental benefits of inoculation and biological
nitrogen fixation with soybean: situation in South America. In: World Soybean Research Con-
ference, 7., International Soybean Processing and Utilization Conference, 4., Brazilian Soybean
Congress, 3. Proceedings, Embrapa Soja, Londrina, Brazil, pp 488–498
Hungria M, Mendes IC (2014) Nitrogen fixation with soybean: the perfect symbiosis?. In: de Bruijn
F (ed) Biological nitrogen fixation. Wiley-Blackwell, New Jersey. (Wiley Publisher, Hoboken)
Hungria M, Franchini JC, Campo RJ et al (2005) The importance of nitrogen fixation to soybean
cropping in South American. In: Werner D, Newton WE (eds) Nitrogen fixation in agriculture,
forestry, ecology, and the environment. Springer, Dordrecht, pp 25–42
Hungria M, Campo RJ, Mendes IC et al (2006) Contribution of biological nitrogen fixation to the
N nutrition of grain crops in the tropics: the success of soybean (Glycine max L. Merr.) in South
America. In: Singh RP, Shankar N, Jaiwal PK (eds) Nitrogen nutrition and sustainable plant
productivity. Studium Press/LCC, Houston, pp 43–93
Hungria M, Nogueira MA, Araujo RS (2013) Co-inoculation of soybeans and common beans with
rhizobia and azospirilla: strategies to improve sustainability. Biol Fertil Soils 49:791–801
Hymowitz T and Harlan JR (1983) Introduction of soybean to North America by Bowen, Samuel
in 1765. Econ Bot 37:371–379
Knobeloch L, Salna B, Hogan A et al (2000) Blue babies and nitrate-contaminated well water.
Environ Health Perspect. 108:675–678.
Larue TA, Patterson TG (1981) How much nitrogen do legumes fix. Adv Agron 34:15–38
Leibovitch S, Migner P, Zhang F et al (2002) Evaluation of the effect of SoyaSignal technology on
soybean yield [Glycine max (L.) Merr.] under field conditions over 6 years in eastern Canada
and northern United States. J Agron Crop Sci 187(4):281–292
Liang Y, Cao Y, Tanaka K et al (2013) Nonlegumes respond to rhizobial Nod factors by suppressing
the innate immune response. Science 341:1384–1387
Marks BB, Megías M, Nogueira MA et al (2013) Biotechnological potential of rhizobial metabo-
lites to enhance the performance of Bradyrhizobium japonicum and Azospirillum brasilense
inoculants with the soybean and maize crops. Appl Microbiol Biotechnol Express 3:21
Salvagiotti F, Cassman KG, Specht JE et al (2008) Nitrogen uptake, fixation and response to fertilizer
N in soybeans. A review. Field Crops Res 108:1–13
Sanjuan J, Carlson RW, Spaink HP et al (1992) A 2-O-methylfucose moiety is present in the lipo-
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89:8789–8793
Souleimanov A, Prithiviraj B, Smith DL (2002) The major Nod factor of Bradyrhizobium japonicum
promotes early growth of soybean and corn. J Exp Bot 53:1929–1934
Spaink HP, Aarts A, Stacey G et al (1992) Detection and separation of Rhizobium and Bradyrhizo-
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Appl Environ Microbiol 57:19–28
 Part VIII
Future Prospects and Dreams
Chapter 42
Exploring the Feasibility of Transferring
Nitrogen Fixation to Cereal Crops

Muthusubramanian Venkateshwaran

Abstract Among the land plants, legumes are unique as they establish symbiotic
relationship with soil-borne, nitrogen-fixing bacteria known as rhizobia to meet their
nitrogen demand. This symbiotic interaction is considered a promising component
of sustainable agriculture due to its economic and ecological benefits. However,
the scope of this symbiosis, which is currently limited to legumes, needs to be
extended to non-legumes, in particular to economically important cereal crops, to
achieve sustainable production of staple crops. This chapter explores the feasibility
of transferring the symbiotic nitrogen fixing machinery to non-legume crops.

42.1 Definitions

Pre-penetration Apparatus During infection by arbuscular mycorrhizal fungi, af-

ter appressorium formation, a precise succession of processes that are coordinated
by the nucleus leads to the formation of the prepenetration apparatus (PPA). This
apparatus appears as a cytoplasmic column containing microtubule and microfila-
ment bundles, very dense endoplasmic reticulum cisternae and a central membrane
thread. Only after the PPA column has been formed does the fungus enter and grow
across the cell (see also Chap. 25).
Pre-infection Thread During rhizobial invasion in legume roots, the cytoplasms in
these activated outer cortical cells align with each other, giving rise to columns of
cytoplasmic bridges called preinfection threads (PITs) through which the inwardly
growing infection thread propagates.
Spontaneous Nodules In certain legumes, such as alfalfa, nodules are formed in the
roots in the absence of rhizobia or an infection thread. These white single-to-multi-
lobed nodules contain a nodule meristem, cortex, endodermis, central zone and
vascular strands. The deregulation of calcium- and calmodulin-dependent protein

M. Venkateshwaran ()
School of Agriculture, University of Wisconsin-Platteville,
Platteville, WI 53818, USA
Tel.: + 001-608-3421898
e-mail: venkateshwam@uwplatt.edu

© Springer International Publishing Switzerland 2015 403

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_42
404 M. Venkateshwaran

kinase leads to the formation of spontaneous nodules in the model legume Lotus
japonicus.

42.2 Commonality in the Molecular Mechanism

of the Legume-Rhizobia and Plant-Arbuscular
Mycorrhizal Fungi Symbioses

More than 80 % of land plants establish a symbiotic association with arbuscular my-
corrhizal (AM) fungi to meet the demand of the plant for phosphorous and other
micronutrients. In addition, legumes establish a symbiotic association with rhizo-
bia to meet the demand of the plant for nitrogen. These mutualistic associations
are established by signal exchanges between plants and microbes. Legumes secrete
flavonoids and isoflavonoids, which induce the expression of nodulation (nod) genes
and the production and secretion of symbiotic signalling molecules, known as Nod
factors. Similarly, strigolactones, which are secreted by the majority of land plants,
trigger spore germination in AM fungi and the subsequent release of Myc factors.
Both Nod and Myc factors are lipochitooligosachcharide (LCO) molecules that are
perceived by LysM-type receptor kinases that are expressed in plant root hairs. Nod
factor receptors have been characterized in model legumes, such as Lotus japonicus
(NFR1 and NFR5) and Medicago truncatula (NFP and LYK3), along with an LRR
receptor-like kinase (LjSYMRK/MtDMI2) that acts as co-receptor. Although Myc
factor receptors have not yet been identified, in M. truncatula, a LysM-type receptor
kinase (LYR1) is being investigated as a Myc factor receptor candidate because its
expression is upregulated during AM symbiosis. These LysM-type receptor kinases
are conserved across all land plants. Cross-talk in Nod and Myc factor perception
has been reported in M. truncatula (Rose et al. 2012). The perception of rhizobial
Nod factors and AM fungal Myc factors triggers symbiotic signalling events, such as
nuclear calcium spiking, i.e., oscillations in the calcium concentration in and around
the nucleus. Nuclear calcium spiking is regulated by nuclear cation channels (LjPOL-
LUX/MtDMI1, LjCASTOR), a calcium pump (MtMCA8) and yet-to-be-identified
calcium channels. Nod and Myc factor-induced calcium spiking is decoded by a
calcium- and calmodulin-dependent protein kinase (LjCCaMK/MtDMI3), which is
a central regulator of nodulation- and mycorrhization-associated gene expression.
This signalling pathway that is shared between legume nodulation and AM is termed
the common symbiotic pathway (CSP). This early symbiotic signalling pathway has
been thoroughly discussed in many recent reviews (Venkateshwaran et al. 2013;
Delaux et al. 2013; Oldroyd et al. 2013). The commonality in the nodulation- and
mycorrhization-associated symbiosis pathway (SYM pathway) suggests the possi-
bility for expanding the already existing mycorrhization-specific SYM pathway to
accommodate and regulate nodulation-specific events in non-legumes (Charpentier
and Oldroyd 2010; Venkateshwaran and Ané 2011; Oldroyd and Dixon 2014; Rogers
and Oldroyd 2014; Fig. 42.1).
42 Exploring the Feasibility of Transferring Nitrogen Fixation to Cereal Crops 405

42.3 Specificity Associated with the Legume-Rhizobia

Symbiotic Signalling Pathway

Although commonality exists between the legume-rhizobia and AM symbiotic sig-

nalling pathway, specificity exists in the signalling pathway that is crucial for the
establishment of root nodule symbiosis (RNS; Venkateshwaran et al. 2013 and De-
laux et al. 2013; Fig. 42.1). There is a logical correlation between the existence of
genes that belong to the symbiosis tool kit and the prevalence of either or both nodu-
lation and mycorrhization (Venkateshwaran et al. 2013; Delaux et al. 2013; Delaux
et al. 2014). Plants belonging to the Brassicaceae, such as Arabidopsis thaliana,
cabbage and cauliflower, lack many key genes of this symbiosis tool kit and are,
thus, unable to establish even AM association, one of the most prevalent symbioses
among land plants (Fig. 42.1) Comparative phylogenomic studies performed among
legumes, non-legume AM hosts (corn and tomato), and non-legume non-AM hosts
(Brassicas) have uncovered the impact of symbiotic associations on the host genome
evolution and have helped identify key genes that are required for nodulation (Delaux
et al. 2014). Therefore, engineering nitrogen fixation in cereals largely relies on the
molecular dissection of the symbiotic signalling pathway, which in turn relies on
genetic, genomic and comparative phylogenomic studies.

42.4 Transferring Nitrogen Fixation to Non-Legumes

Often considered as a search for the Holy Grail, engineering nitrogen fixation in
cereals has been the dream of scientists for many decades, with the ultimate goal of
dissecting the molecular mechanism of RNS to reconstitute this symbiotic signalling
machinery in non-legume plants. Although this goal is long-term and extremely
challenging, such a transfer of the legume-rhizobia symbiotic machinery to non-
legume crops could significantly minimize the exogenous application of energy-
expensive nitrogen fertilizers. In addition to reducing the cost of cultivation for food,
feed and bioenergy, this strategy would also provide protection for the environment
against pollution from chemical nitrogen fertilizers. This task will be extremely
challenging but the availability of advanced molecular tools in rhizobia and host
plants make this goal more realistic now than ever before. Several strategies are
currently underway by international research groups and can be broadly grouped
into the following two major approaches.
Engineering Cereal Crops to Mimic Legumes to Accommodate Endosymbiotic
Nitrogen-Fixing Rhizobia The high specificity of the legume-rhizobia interaction
makes it difficult to nodulate one group of legumes with the symbiotic partner of
another group. Most rhizobia have a very narrow host range, making studies across
species difficult. However, the rhizobial strain NGR234, with a wide host range of
353 legume species representing 122 genera, seems an interesting candidate for the
transfer of root nodule symbiosis (RNS) to non-legume crops. To reconstitute the
406 M. Venkateshwaran

Fig. 42.1 The elaborate symbiotic signalling pathway (SYM pathway) that is conserved in legumes
allows these plants to associate with both rhizobia and AM fungi, whereas the SYM pathway that
is present in non-legume cereal crops enables these plants to associate with AM fungi alone but not
with nitrogen-fixing rhizobia. Plant species belonging to Brassicaceae lack a sophisticated SYM
pathway and are, therefore, unable to establish associations with either rhizobia or AM fungi

RNS machinery in non-legume plants, the genetic make-up of the host needs to be
fine-tuned to allow major nodulation events, such as infection thread formation, in-
tracellular uptake and the formation of root nodules (Markmann and Parniske 2009).
42 Exploring the Feasibility of Transferring Nitrogen Fixation to Cereal Crops 407

The prevalence of CSP in the majority of land plants suggests that this pathway
could be adjusted to accommodate RNS. The homologue of MtDMI2/LjSYMRK
is necessary for another nitrogen-fixing root endosymbiosis, actinorhizal symbio-
sis (ARS), in Casuarina glauca and in the cucurbit Datisca glomerata (Gherbiet al.
2008; Markmann et al. 2008). The events leading to the intracellular accommodation
of the symbiont share remarkable similarities in these three mutualistic interactions
(RNS, AM and ARS). Prior to invasion by the respective endosymbiont, the host
cell prepares to accommodate the invading partner by cytoskeletal and organellar
rearrangements and nuclear movements. Similarities exist in the formation of the
‘pre-penetration apparatus’ (PPA) in AM symbiosis and the ‘pre-infection threads’
(PIT) in RNS and ARS. Because AM symbiosis is prevalent in the majority of land
plants, the components that are necessary for the intracellular accommodation of
symbionts may already exist in non-legume crops. Therefore, in my opinion, engi-
neering a receptor-mediated perception of nitrogen-fixing symbionts in non-legume
plants is not just a dream and will be possible in the near future (Venkateshwaran
and Ané 2011).
The recognition of symbiotic partners by the host is a key factor for the estab-
lishment of mutualistic interactions. Such microsymbiont recognition is mediated by
receptor kinases in plants. The LysM receptor kinases NFR1 and NFR5 in L. japon-
icus and NFP and LYK3 in M. truncatula are Nod factor receptors. The introduction
of L. japonicus Nod factor receptors into M. truncatula enables the latter to enter
into a symbiotic association with the L. japonicus symbiont, Mesorhizobium loti.
Such an extreme specificity of receptors towards Nod factors needs to be relaxed
by genetic manipulations, which would enable the host plant to recognize a wide
array of rhizobial species. The possibilities for the presence of additional Nod factor
receptor complexes and crosstalk between Nod and Myc factor perception have been
reported in M. truncatula (Rose et al. 2012). Homologues of LysM receptor kinases
are present in non-legume plants, such as rice and A. thaliana, where they trigger
the defence reaction upon the perception of chitin oligomers, common elicitors of
fungal pathogens. Even a non-AM host, such as Arabidopsis, can perceive rhizobial
Nod factors that suppress microbe-associated molecular pattern (MAMP)-triggered
immunity in the plant (Liang et al. 2013). Utilizing such pre-existing recognition ma-
chinery without compromising the defence-related role of this machinery presents
additional challenges. Alternatively, Nod factor-independent infection strategies,
such as those observed in certain species of the photosynthetic Bradyrhizobium,
have also been considered as avenues for non-legume plants. Although it is not clear
whether LysM receptor kinases play a role in the recognition of the yet unknown
actinorhizal factors, the dual infection in Parasponia (Ulmaceae) by both Frankia-
and Nod factor-dependent rhizobia suggests this possibility (Venkateshwaran and
Ané, 2011).
Rhizobial infection and nodule organogenesis are two separable events in RNS.
The genes that are involved in the spatio-temporal synchronization of these two
distinct events are mandatory for successful RNS. The further characterization of mu-
tants, such as IPD3/CYCLOPS or NIN, might identify the coordinators of epidermal
(infection) and cortical (infection and nodule organogenesis) events. As previously
408 M. Venkateshwaran

discussed, the commonalities between legume root nodule symbiosis and actinorhizal
symbiosis should be considered to identify the key components and missing links in
non-nodulating plants (Markmann and Parniske 2009). Likewise, the molecular dis-
section of the dissimilarities between nodulating and non-nodulating legumes seems
a promising approach to identify the master regulators of RNS that are missing in
non-legume plants (Sprent and James 2007). The molecular dissection of the RNS
machinery in the non-legume plant Parasponia could also provide clues as to the
master players in symbiotic signalling that are lacking in non-nodulating plants.
The chemical reaction of biological nitrogen fixation (BNF; see also Chap. 23)
is catalyzed by the molybdenum-dependent nitrogenase enzyme, which comprises
two component proteins, the ‘Fe protein’ and the ‘MoFe’ protein (Burgess and Lowe
1996). Several genes are involved in the synthesis and assembly of functional ni-
trogenase in rhizobia, albeit with a high level of variations among different species.
Photosynthetic Bradyrhizobium and Azorhizobium caulinodans carry approximatly
15 nif genes that are required for nitrogenase synthesis, while Rhizobium and Sinorhi-
zobium possess 8 and 9 genes, respectively. Some of the core nif genes that are
conserved in the majority of the rhizobia are nifH (dinitrogenase reductase), nifDK
(α and β subunits of dinitrogenase), nifEN (scaffold for iron-molybdenum cofac-
tor), nifB (P-cluster) and the regulatory gene nifA. The high level of intricacy and
sophistication in the functionality of nitrogenase in BNF enables legumes to occupy
a unique niche among land plants. The possibility of introducing these genes en-
coding functional nitrogenase assembly in non-legume host plants, such as cereals,
and expressing the functional protein complexes in a tissue-specific manner (roots
or spontaneous nodules) for de novo BNF would be another promising strategy.
In addition to having the genes that are necessary for rhizobial infection,
nodule organogenesis and their coordination, legumes possess symbiosis-specific
leghaemoglobin genes for the protection and function of nitrogenase activity. Homo-
logues of leghaemoglobin genes are present in the majority of land plants, including
the evolutionarily older Bryophytes and Pteridophytes. In non-legume plants, includ-
ing rice and maize, homologues of leghaemoglobins are present as non-symbiotic
haemoglobins (nsHbs). Such nsHbs from Parasponia are expressed in the nodules
that are formed during rhizobial interaction and facilitate the diffusion of oxygen to
bacteroids. The role of nsHbs in symbiosis in actinorhizal plants, such as Causarina
glauca, Alnus glutinosa and Myrica gale, is still unknown. Similarly, leghaemoglobin
genes are induced during AM, but their role in this widespread ancestral symbiosis
is unknown. These observations provide encouraging evidence that nsHbs can be
utilized in the assembly of the nodulation pathway in non-legume plants.
Engineering Natural Endophytes of Cereal Crops to Mimic the Nitrogen Fix-
ing Ability of Rhizobia Another approach in enabling endosymbiotic BNF in
non-legume crops is to manipulate the microsymbiont/natural endophytes. The en-
dophytic colonization of plant roots by rhizobia has been demonstrated several times
in the past decade (Gutierrez-Zamora and Martinez-Romero 2001; Lundberg et al.
2012), suggesting that rhizobia can utilize the pre-existing infection machinery in
non-legume plants to colonize these plants as endophytes. Many plants, including
42 Exploring the Feasibility of Transferring Nitrogen Fixation to Cereal Crops 409

the non-AM host Arabidopsis, naturally accommodate endophytes in their roots

(see also Chap. 5). These endophytes belong to a wide range of genera, including
Rhizobiaceae.
Several endophytic nitrogen-fixing bacteria have been isolated from sugarcane
(Saccharum spp.) tissues, including Gluconacetobacter diazotrophicus, Herbaspir-
illum seropedica, and H. rubrisubalbicans. These intracellular associations seem
to be beneficial for the plant, as bacteria promote plant growth when inoculated in
sugarcane plantlets by nitrogen fixation and/or the production of hormones, such as
auxin and gibberellin (Sevilla et al. 2001; see also Chap. 26). The plant mechanisms
that permit bacterial colonization in an endophytic (intracellular spaces and vascular
tissues) and non-pathogenic manner, establishing a beneficial association, are not
yet clear. Different sugarcane genotypes have different rates of BNF, suggesting that
plant genetic factors might control the process of bacterial recognition, colonization,
and/or nitrogen fixation (Urquiaga et al. 1992).
Rhizobium sp. strain IRBG74 is a nitrogen-fixing symbiont in the Agrobac-
terium/Rhizobium clade that nodulates the aquatic legume Sesbania sp. and can
infect rice endophytically, improving plant growth, health, and yield, making it
a good model system for determining the mechanisms of Rhizobium-cereal inter-
actions. Recently, the sequence of the IRBG74 genome, which is composed of a
circular chromosome, a linear chromosome, and a symbiotic plasmid (pIRBG74a),
has been released (Crook et al. 2013). This strain carries all of the necessary nod
genes for the production and secretion of Nod factors that mimic Myc factor LCOs
(unpublished). Preliminary results suggest that the Nod factors of Rhizobium sp.
IRBG74 can trigger nuclear calcium spiking in M. truncatula and that rice is able
to perceive the signalling molecules of this strain (unpublished), further confirming
that the search for natural endophytes of cereals, particularly those that belong to
the Rhizobium clade with an ability to fix atmospheric nitrogen, is the best alternate
avenue to achieving the goal of transferring nitrogen fixation to cereal crops. The
release of the IRBG74 genome sequence enables the manipulation of this strain at
the molecular level to enhance its nitrogen-fixing ability in rice.

42.5 Conclusion

Enabling endosymbiotic nitrogen fixation in cereal crops is a promising component

of sustainable agriculture. Although this goal is challenging and long-term, it could
be achieved by manipulating host and microbial partners. The advent of molecular
tools in both host plants and microsymbionts and our ever-expanding knowledge of
the symbiotic signalling pathways provide hope that the re-constitution of the root
nodule symbiotic machinery in non-legume plants is a feasible yet distant goal.

Acknowledgements I thank Dr. Jean-Michel Ané, University of Wisconsin-Madison, USA for

his guidance in the preparation of this chapter and the figure. I thank Kari Parthasarathy for the
critical reading of the chapter. I thank Katherine Baldwin for technical help in the preparation of
the Fig. 42.1
410 M. Venkateshwaran

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Chapter 43
The Minimal Rhizosphere Microbiome

Jos M. Raaijmakers

Abstract The rhizosphere provides a home to numerous (micro) organisms that

in turn may affect plant growth, development, and tolerance to abiotic and biotic
stresses. How plants shape the rhizosphere microbiome has been subject of many
past and present studies with the ultimate goal to identify plant genetic traits that
select and support beneficial microorganisms. Novel ‘omics technologies have pro-
vided more in-depth knowledge of the diversity and functioning of the rhizosphere
microbiome and significant advances are being made to uncover mechanisms, genes
and metabolites involved in the multitrophic interactions in the rhizosphere. To better
understand this intriguing complexity, both reductionists’ and systems ecology ap-
proaches are needed to identify the biotic and abiotic factors involved in microbiome
assembly. Here, different strategies are discussed to re-shape the rhizosphere mi-
crobiome in favour of microbial consortia that promote root development and plant
growth, and that prevent the proliferation of pests and diseases.

43.1 Introduction

Currently more than one third of the crop yields worldwide are lost due to abiotic and
biotic stress factors, such as drought, salinity, pests and diseases. Future increases
in crop yields will have to be achieved on sub-optimal soils with reduced input
of fertilizers and pesticides (‘more with less’). These challenges have increased the
awareness of the importance of the plant microbiome (i.e. the collective communities
of microorganisms on and in plants, their genomes and interactions) for improved
and sustainable agricultural practices. Plants are colonized by an astounding number
of microorganisms that can have profound effects on seed germination, plant growth
and development, nutrition, diseases and productivity. In this context, plants can be
viewed as superorganisms that rely in part on their microbiome for specific functions

J. M. Raaijmakers ()
Department of Microbial Ecology, Netherlands Institute of Ecology,
Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands
Tel.: + 31317473497
e-mail: j.raaijmakers@nioo.knaw.nl

© Springer International Publishing Switzerland 2015 411

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_43
412 J. M. Raaijmakers

and traits. In return, plants deposit a substantial part of their photosynthetically fixed
carbon into their direct surroundings (spermosphere, rhizosphere, phyllosphere),
thereby feeding the microbial community and influencing their activities and com-
position (Mendes et al. 2013). For many plant-associated microorganisms, however,
there is still little knowledge of their impact on plant growth and health. Hence,
deciphering the plant microbiome is critical to identify beneficial microorganisms
that can be used as an integral component of future agriculture and horticulture.

43.2 The Rhizosphere

The rhizosphere is the narrow zone surrounding and influenced by plant roots via
the release of so-called rhizodeposits (i.e. exudates, border cells, mucilage) (Lynch
1990). The nutrients, trace elements, volatile organic compounds and other metabo-
lites deposited by plant roots attract many (micro)organisms such as bacteria, archaea,
fungi, nematodes and protozoa, making the rhizosphere a hot spot of microbial activ-
ity and interactions (Raaijmakers et al. 2009; Buée et al. 2009; Mendes et al. 2013).
Following the terminology used for microorganisms colonizing the human body, the
collective communities of microorganisms on and also inside plant root tissue, their
genomes and interactions are now referred to as the rhizosphere microbiome. Over
the past five decades, numerous studies have shown that specific members of the
rhizosphere microbiome can affect plant growth and development, plant nutrition
and stress tolerance (Berendsen et al. 2012; Mendes et al. 2013; Philippot et al.
2013). In this context, Cook et al. (1995) postulated that plants may ‘cry for help’ by
selectively stimulating microorganisms that protect them from invading pathogens.
Rhizosphere microorganisms that have been well studied for their beneficial effects
on plant growth and health are the nitrogen-fixing bacteria, mycorrhizal fungi, plant
growth-promoting rhizobacteria (PGPR), and saprophytic and mycoparasitic fungi
(Mendes et al. 2013). For the vast majority of rhizosphere (micro)organisms, how-
ever, there is still little to no understanding of their metabolic potential and functions.
This lack of knowledge has led to numerous studies to catalogue microbial commu-
nities in the rhizosphere of different plant species, to elucidate which microbes are
active during plant development and to unravel which functions and biosynthetic
pathways are displayed in time and space (Mendes et al. 2013; Philippot et al. 2013).
To go beyond ‘collecting stamps’, several meta-‘omics’ approaches (transcrip-
tomics, proteomics, metabolomics) have been and are still being developed to identify
gene transcripts, proteins and metabolites in the rhizosphere. For example, Wang
et al. (2011) adopted a metaproteomics approach to unravel interactions between
plants and rhizosphere microorganisms in different cropping systems. They found,
among others, that approximately half of the bacterial groups classified by proteomic
analysis were not found in the DNA-based metagenomic analyses of the rhizosphere
bacterial community and vice versa (Wang et al. 2011), emphasizing the need to im-
prove the resolution and sensitivity of these approaches. Also, stable isotope probing
(Prosser et al. 2006) has provided new opportunities to identify microorganisms that
43 The Minimal Rhizosphere Microbiome 413

are metabolically active in the rhizosphere. These and other technologies revealed
that also fungi make up a significant part of the rhizosphere microbial biomass, es-
pecially during flowering and senescence (Hannula et al. 2010). Hence, top-down
approaches such as metagenomics and bottom-up approaches targeting individual
microbial species or strains should be integrated to provide a comprehensive cover-
age and understanding of the microbial community and their activities as a whole
(see also Zengler and Palsson 2012; Mendes et al. 2013).

43.3 Shaping the Rhizosphere Microbiome

Several species and strains of rhizobacterial and fungal genera, including Bacillus,
Pseudomonas, Collimonas, Trichoderma, Piriformospora and nonpathogenic Fusar-
ium oxysporum, have been shown to promote plant growth and to protect plants from
stress by different mechanisms (Lugtenberg and Kamilova 2009; Raaijmakers et al.
2009; Raaijmakers and Mazzola 2012; Chap. 3) . These include biofertilization
(Chaps 23, 24 and 25), stimulation of root growth (Chap. 26), antibiosis (Chap. 18),
induced systemic resistance (Chap. 14), parasitism and rhizoremediation (Chap. 29).
These mechanisms are well documented for rhizobacteria belonging to the Proteobac-
teria and Firmicutes, i.e. Pseudomonas (Chap. 18) and Bacillus (Chap. 40), as well as
for the mycoparasitic fungi Trichoderma (Chap. 36) and Gliocladium. Hence, there
is a major interest to develop strategies that re-shape the rhizosphere microbiome in
favour of microorganisms that promote root development and plant growth, and that
prevent the proliferation of pests and diseases.
The first and most obvious strategy to re-direct the microbial composition and
activities in the rhizosphere is changing the quality and/or quantity of root exudates
via plant breeding or via genetic modification. This form of ‘rhizosphere engineer-
ing’ requires detailed knowledge of the exudate composition (spatial, temporal) and
their effects on microbial growth and activity (Bakker et al. 2012). Although our
understanding of exudate chemistry and microbial interactions in the rhizosphere
has improved considerably, there are, to my knowledge, no specific breeding pro-
grams yet that evaluate plant lines for their broad interaction with the rhizosphere
microbiome. More than a decade ago, Smith et al. (1999) investigated the genetic
basis in plants for interactions with beneficial rhizobacteria. They discovered sub-
stantial variation among recombinant inbred lines of tomato and identified loci that
were associated with growth of and disease suppression by a beneficial Bacillus
cereus. Rudrappa et al. (2008) further showed that plants can stimulate, via malic
acid, the protective effects of a beneficial Bacillus subtilus strain in the rhizosphere.
Similarly, Neal et al. (2012) showed that a beneficial Pseudomonas putida strain
was attracted to 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one (DIMBOA), the
allelopathic compound that is exuded in relatively high quantities from roots of young
maize seedlings. These and other studies exemplify that specific phenotypic traits and
genetic variation in host plant species can be exploited to enhance beneficial associ-
ations of plants with rhizosphere microorganisms. To date, however, our knowledge
414 J. M. Raaijmakers

of root exudation in situ is still too limited to provide specific targets that can be used
in plant breeding programs.
The second strategy to re-direct the rhizosphere microbiome is to introduce se-
lected beneficial microorganisms at high densities in soil, onto seeds or other planting
materials (Mendes et al. 2013). Over the past decades, many bacterial and fungal
strains with different beneficial traits have been studied for their ability to boost plant
performance and to control pests and diseases. Although there are several successful
cases (e.g. Agrobacterium radiobacter, Bacillus subtilis), many of the promising
microbes tested to date were less effective in disease control than their chemical
counterpart and therefore not commercially attractive enough for product develop-
ment and implementation in practice. The observed inconsistency in performance of
various promising microbial agents has been attributed to various reasons, including
poor establishment on/in seed or plant tissue, poor survival or lack of expression of
the desired microbial trait/activity at the right time and place.

43.4 Reconstructing a ‘Minimal Rhizosphere Microbiome’

To date, there has been a strong emphasis on ‘one-microbe-at-a-time’ applications,

whereas many ecosystem functions, including nutrient cycling and disease sup-
pression, are generally driven by the (sequential) activity of microbial consortia.
Furthermore, several microorganisms only exhibit a specific activity when they are
part of a consortium (Garbeva and de Boer 2009; Garbeva et al. 2011). Hence, the
use of assemblages of different rhizosphere microorganisms with complementary
or synergistic traits may provide a much more effective and consistent effect. This
concept of so-called ‘reconstructed microbiomes’ or ‘synthetic communities’ (De
Roy et al. 2013; Grosskopf and Soyer 2014) is gaining momentum not only in plant-
microbe interactions but also in the fields of probiotics and natural product discovery.
However, to find and select the right players and microbial composition of a rhizo-
sphere consortium for a specific function (e.g. disease suppression) is still a puzzle
and requires more fundamental understanding of the temporal and spatial dynamics
of the rhizosphere microbiome, the chemistry, the underlying communication and
beneficial activities.
Natural disease suppressive soils (Chap. 38) provide a very good ‘model system’to
unravel and design the optimal microbial consortium to protect plants from infection
by soil-borne pathogens. Studies by Kyselkova et al. (2009); Mendes et al. (2011)
and Rosenzweig et al. (2012) on soils suppressive to different fungal and bacterial
plant pathogens pinpointed multiple bacterial genera that were more abundant in the
suppressive than in the corresponding disease conducive soils. Although the potential
role of the identified bacterial communities in disease suppressiveness was addressed
for only a few genera, these studies do provide a framework to reconstruct microbial
consortia for disease control. Clearly, there is a need for a community systems
approach to resolve the interplay between individual community members, the host
plant and the soil environment (Zengler and Palsson 2012). In this context, Kinkel
43 The Minimal Rhizosphere Microbiome 415

et al. (2011) proposed a co-evolutionary framework for inducing or managing natural

disease suppressiveness of soils. They also argued that control of different plant
pathogens on different crops most likely requires a different subset of microorganisms
(Kinkel et al. 2011). Ideally, the ultimate goal is to design a so-called ‘minimal
rhizosphere microbiome’ that is effective against multiple soil-borne pathogens in
different agro-ecosystems. Based on the concept of the minimal genome (Moya
et al. 2009; Juhas et al. 2011), the minimal rhizosphere microbiome is defined
here as the minimal set of microorganisms, microbial traits and genomes that are
needed to effectively and consistently execute a specific function in the rhizosphere,
e.g. protection of plant roots against fungal infections or rhizoremediation of toxic
compounds (Mendes et al. 2013).
For controlling plant diseases, designing a separate minimal rhizosphere micro-
biome for each of the major pathogen groups (bacteria, fungi, nematodes, oomycetes)
may be feasible. This assumption is based on the fact that various studies have pointed
to common players and mechanisms in different soils that are naturally suppressive
to specific fungal pathogens. For example, Pseudomonas species have been shown
to contribute to suppressiveness of soils to either Fusarium wilt disease or to take-all
disease of wheat (Weller et al. 2002). Furthermore, the onset of natural disease sup-
pressiveness of soils follows a similar pattern for different fungal pathogens (Weller
et al. 2002), suggesting that similar cues, mechanisms and microbes play a role in
the transition of a soil from a conducive to a suppressive state. An in-depth under-
standing of the shifts in community composition and microbial activities during this
transition will be required to select the right microbiome members. Selection and
assembly of minimal rhizosphere microbiomes should be based on functional traits
and genes rather than on taxonomic classification only (Burke et al. 2011; Boon
et al. 2013). Using a modelling approach, Scheuring and Yu (2012) suggested three
easy steps to assemble a beneficial microbiome. In their models, the first step is that
the new host’s microbiome starts with a higher proportion of beneficials either by
vertical transmission or by a higher immigration rate. The second step involves a
high resource supply from the host to the beneficials, which in turn (third step) fu-
els intense interference competition via antibiotic production leading to competitive
dominance of the beneficial microbes. Although Scheuring and Yu (2012) focused
primarily on antibiosis as a key function of a beneficial microbiome, their models
are highly instrumental to identify major processes that drive assembly of a benefi-
cial microbiome. Whether these models could also be used for other important traits
of beneficial rhizosphere microbiomes, such as parasitism, induced resistance and
resource competition, remains to be determined.
In conclusion, the rhizosphere is a diverse and dynamic habitat with multiple
microorganisms that affect plant growth, development and tolerance to abiotic and
biotic stresses. To better understand the multitrophic interactions in the rhizosphere,
both reductionists’ and systems biology/ecology approaches are needed to resolve
the underlying mechanisms involved in microbiome assembly and activity.
416 J. M. Raaijmakers

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beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol Rev
37:634–663
Moya A, Gil R, Latorre A et al (2009) Toward minimal bacterial cells: evolution vs. design. FEMS
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Chapter 44
The Edible Plant Microbiome: Importance
and Health Issues

Gabriele Berg, Armin Erlacher and Martin Grube

Abstract Plants live together with microbial communities to form tight interactions
that are essential for the performance and survival of the host. In recent decades,
many studies have discovered a vast plant-associated microbial diversity. However,
even though plants are a substantial part of a balanced diet including raw-eaten
vegetables, fruits and herbs, the plant-associated microbial diversity has been largely
ignored in this context. We hypothesize that the edible plant microbiome and its
diversity can be important for humans as (i) an additional contributor to the diversity
of our gut microbiome, and (ii) as a stimulus for the human immune system. Two
specific examples for plant microbiomes, of lettuce and banana, are discussed in
comparison with other relevant studies to explore these hypotheses. Moreover, the
biotechnological potential of the edible plant microbiome is evaluated.

44.1 Plant-Associated Microbial Diversity

All Food Plants are Associated with a High Diversity of Microorganisms This
diversity is still currently only partly characterized and is, to a certain degree, spe-
cific for the host species or even cultivars of food plants (Berg and Smalla 2009).
This diversity is also specific for each microhabitat of plants which are usually dis-
tinguished as: the rhizosphere (roots), the phyllosphere (leaves), the caulosphere
(stem), the anthosphere (flowers), the carposphere (fruits), and the endosphere

G. Berg () · A. Erlacher

Institute of Environmental Biotechnology, Graz University of Technology, 8010 Graz, Austria
Tel.: + 43664608738310
e-mail: Gabriele.berg@tugraz.at
A. Erlacher · M. Grube
Institute of Plant Sciences, University of Graz, 8010 Graz, Austria
Tel.: + 43 (316) 873-4312-8423
e-mail: armin.erlacher@tugraz.at
M. Grube
Tel.: + 43 (0)316 380-5655
e-mail: martin.grube@uni-graz.at

© Springer International Publishing Switzerland 2015 419

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_44
420 G. Berg et al.

Fig. 44.1 Interactive microbiomes. Examples of the edible plant microbiome of fruits, herbs,
nuts and drugs. We eat all parts of the plants including the phyllosphere (lettuce, cabbage), the
rhizosphere (carrots, turnip), the carposphere (tomato, banana) as well as seeds (beans, peas)
including all endospheres

(all inner parts). Although the discovery of specific microbiomes is primarily as-
sociated with the rhizosphere, there are currently only a few other compartments
where species-specific diversity was detected, e.g. the carposphere (Leff and Fierer
2013). Given the estimated number of 370 000 species of higher plants, a great deal
of work is still required before the details of global plant microbiome diversity will
be fully understood.
Plants are a basic and substantial part of our daily diet. Vegetables, fruits, herbs,
nuts, and medicinal herbs belong to the raw-eaten plants; several examples of each
group are shown in Fig. 44.1. Our food thus comprises all parts of plants that include
their microbial habitats and microhabitats that can be colonized by up to 104 –1010 mi-
croorganisms per gram of plant. These microbial habitats include the phyllosphere
(lettuce, cabbage), the rhizosphere (carrots, turnip), the carposphere (tomato, ba-
nana), as well as the seeds (beans, peas) and corresponding endospheres. An initial
study published by Leff and Fierer (2013) demonstrated that fruits and vegetables
harbored distinct and diverse bacterial communities, and interestingly showed that
vegetables or fruits grown primarily close to the soil surface (i.e., sprouts, spinach,
44 The Edible Plant Microbiome: Importance and Health Issues 421

lettuce, tomatoes, peppers, and strawberries) appear to share communities charac-

terized by high relative abundance of Enterobacteriaceae. The authors concluded
that humans are exposed to substantially different bacteria depending on the types
of fresh produce they consume.
What could the effect of this exposure be on humans? Plant-associated microor-
ganisms could have both a direct and indirect influence on human health. Indirect
positive effects are linked to organisms that enhance the quality factors (including
the content of active principles). Only a few examples are known for such effects,
and most are related to medicinal plants and their bioactive substances (Köberl et al.
2013). For example, microorganisms are involved in the production of antimicrobial
substances, e.g. taxol in endophytic fungi of Taxus baccata (Garyali et al. 2013),
apigenin in Chamomille matricaria (Schmidt et al. 2014), or maytansine in Putter-
lickia verrucosa (Wings et al. 2013). Moreover, fruit-associated bacteria seem to
influence the aroma expression in strawberries, where Methylobacterium treatment
has been shown to enhance the production of aromatic furaneol substances (Verginer
et al. 2010a). Evidence was also provided by Verginer et al. (2010b) for an influ-
ence of grape-associated microorganisms on the aroma of wine, indicating that the
“terroir” effect can to some extent be attributed to bacteria. The indirect negative
effects caused by plant-associated microorganisms are well-studied. The outbreak
of plant pathogens is often associated with a microbiome shift and accompanied
with minor pathogens. They do not only contribute to bad odor and taste, but also
to the expression of mycotoxins which are among the world’s most toxic and car-
cinogenic compounds (Wu et al. 2014). They have been responsible for numerous
foodborne diseases and epidemics throughout history including Claviceps purpurea,
the causative agent for the infamous Saint Anthony’s Fire in Medieval times that oc-
curred after eating contaminated bread (Belser-Ehrlich et al. 2013). Although such
problems could be primarily solved by food hygiene, Fusarium mycotoxins still play
an important role for our health (Wu et al. 2014). There is still very little knowledge
concerning the long term effects of bioactive compounds at low concentration, and
only recently has evidence been introduced for endophytes that produce novel and
still poorly understood compounds. New technologies will contribute to increase the
detection rate of specific beneficial plant-microbe interactions that are also relevant
for human health.
What do we know about the direct effects of plant microbiomes that we consume
along with our food? Most of our existing knowledge concerns fermented food,
such as yoghurt as the foremost example for sources of probiotic strains. However, a
substantial part of our plant diet is consumed fresh and may possibly include trillions
of microorganisms during each meal. Even after washing or rinsing food surfaces,
a substantial number of bacteria is expected to enter the body with our food. Our
primary hypothesis is that the edible plant microbiome and its microbial diversity is
important for humans as: (i) a contributor to the diversity within our gut microbiome,
and (ii) as a stimulus for our immune response. We will present two examples for
crop-associated microbiomes which are eaten raw by humans: of lettuce and banana.
Furthermore, we will discuss our hypotheses as well as the impact of microbial
diversity in general.
422 G. Berg et al.

The Specific Structure of the Lettuce Microbiome Lettuce species such as Lac-
tuca sativa L., Eruca sativa Mill., and their varieties belong to the most important
raw-eaten vegetables world-wide and are a substantial part of a balanced, healthy
diet. Several beneficial effects on health and lifestyle are attributed to the consump-
tion of lettuce as it contains several vitamins, and is also a source of manganese and
high amounts of dietary fibers. The relatively low amount of carbohydrates and fats
correlates with its low calorie value. Lettuce provides habitats for a diverse range
of microbes (Rastogi et al. 2012; Rastogi et al. 2013). Lettuce-associated microor-
ganisms have currently only made it into the headlines in the context of scattered
pathogen outbreaks. There are two crucial features that may be responsible for let-
tuce’s vulnerability to pathogens: the variability and specificity of the associations
within the microbial communities. Overall, a proportion of 12.5 % cultivar-specific
bacteria were identified for the rhizosphere of eight different Lactuca sativa culti-
vars as well as the wild relative L. serriola. In addition, a large core microbiome
was identified that includes 68 operational taxonomic units from nine major phyla
(Proteobacteria the most abundant), and represents 48.8 % of the microbiome. A cor-
relation analysis showed that within the lettuce microbiome co-occurrence prevailed
over co-exclusion. Although predominant taxa (e.g. Pseudomonas, Flavobacterium,
and Sphingomonadaceae) showed positive interactions, they were not necessarily
involved in highly correlated modules of species. This loose bacterial network ob-
served for lettuce allowed allochthonous organisms to colonize lettuce to interactive
niching in microbial communities.
Little is known about the impact of biotic factors on the lettuce microbiota. Our
hypothesis was that any disturbance of the native microbiomes (i) can induce drastic
shifts in the community and that each pathogen outbreak (ii) could be accompanied by
“minor”, less virulent pathogens. In mesocosm and field experiments by using a com-
bined approach including network analyses of 16S rRNA gene amplicon libraries and
FISH microscopy (see Chap. 31), we found substantial impacts detectable as micro-
biome shifts by a plant pathogenic fungus, herbivorous gastropoda, or visiting pets.
Although the genera Enterobacter, Stenotrophomonas, Pseudomonas, and Acineto-
bacter form a core microbiome, all three disturbing factors induced significant shifts
in the community and increased species richness. In Lactuca, this was strongly cor-
related with an increase of Enterobacter and in Eruca with Escherichia/Shigella and
Pantoea—all genera contain potential pathogens. A bacterial diversity associated
with leaves is detectable by cultivation and bacterial DNA analysis, but very few
bacteria are detected on the surface as only a few colonies occupy cavities along the
external surface and in the vicinity of stomata (Fig. 44.2a). Through colonization ex-
periments, we revealed unexpected colonization patterns of enteric species in lettuce
leaves and found that bacterial populations do not colonize the surface, but rather
intrude into the endosphere (Fig. 44.2b).
The Specific Structure of the Banana Microbiome Bananas and plantains are
among the most important crops in the tropics and sub-tropical regions world-wide.
Microhabitat-specific microbial communities for the rhizosphere, phyllosphere, and
endosphere of bananas grown in three different traditional farms in Uganda were
44 The Edible Plant Microbiome: Importance and Health Issues 423

Fig. 44.2 Visualization of the lettuce microbiome. a Naturally occurring Gammaproteobacterial

micro-colonies on the lettuce surface and in the vicinity of stomata visualized by FISH and CLSM.
b Colonization patterns on lettuce leaves treated with E. coli cells. Both experiments are explained
in detail in Erlacher et al. (2014)

detected (Rossmann et al. 2010). Interestingly, the banana stem endosphere showed
the highest bacterial counts (up to 109 gene copy numbers g−1 ), and Enterobacteri-
acaea provided 1/3 of the total bacteria. They comprise 14 genera including potential
human pathogens, (Escherichia, Klebsiella, Salmonella, Yersinia) plant pathogens
(Pectobacterium), but also disease-suppressive bacteria (Serratia). This dominant
role of enterics can be explained by their permanent nature and the vegetative prop-
agation of banana plants, as well as the addition of human and animal manure in
traditional cultivations.

44.2 The Edible Plant Microbiome: Diversity and Human

Health

Concerning our first hypothesis of a link between the plant and human gut micro-
biome, there is an interesting overlap between the plant and human gut microbiome
with respect to species composition and function (Ramírez-Puebla et al. 2013). Re-
cent studies showed that the stomach is colonized by a higher diversity of microbial
species than has long been expected, and explained by the hostile conditions of
low pH values. The stomach milieu thus does not pose a strict barrier for microbial
passage as was previously thought (von Rosenvinge et al. 2013). Even though the
effects of probiotics are often controversially discussed, it has now been shown that
strains, including probiotics, survived the stomach passage to establish successfully
in the gut (Iqbal et al. 2014). David et al. (2014) also recently provided additional
evidence for the survival of foodborne microbes (both animal- and plant-based diet)
424 G. Berg et al.

after transit through the digestive system, and that foodborne strains may have been
metabolically active in the gut.
Our second hypothesis is that bacteria, associated with our diet, such as Enter-
obacteriaceae, act as stimuli for our immune system. Recently, Hanski et al. (2012)
showed a correlation between bacterial diversity and atopy as shown through sig-
nificant interactions with Enterobacteriaceae. Furthermore, they showed a positive
association between the abundance of Acinetobacter and Interleukin-10 expression
in peripheral blood mononuclear cells in healthy human individuals. Interleukin-10
is an anti-inflammatory cytokine and plays a central role in maintaining immuno-
logic tolerance to harmless substances (Lloyd and Hawrylowicz 2009). Endotoxin
derived from Gram-negative bacteria, such as Enterobacteriaceae, is known to have
allergy-protective and immunomodulatory potential (Doreswamy and Peden 2011).
Microhabitats of plants are a reservoir for Enterobacteriaceae (Leff and Fierer
2013, Rastogi et al. 2012), which also include potentially human pathogenic bacte-
ria such as human enteric pathogens (Brandl 2006). Particularly after intermediate
disturbances, these human enteric pathogens are enhanced (Erlacher et al. 2014).
Although outbreaks of enteric pathogens associated with fresh produce in the form
of raw or minimally processed vegetables and fruits have recently increased (Holden
2010), the ecology of enteric pathogens outside of their human and animal hosts is
less understood (Teplitski et al. 2011). If plants are a natural reservoir of Enterobac-
teriaceae, then these bacteria must have been a “natural” part of our diet for a long
time. Taking into account how many vegetables and fruits are eaten by people world-
wide, these outbreaks seem to be more of an accident than the norm, particularly
considering that traditionally, food was not processed and sterilized before eating.
A function of the plant- associated microbiome as an immunostimulant or “natural
vaccination” is more likely than their pathogenic role.

44.3 Conclusions

Members of the prokaryotic and eukaryotic domains of life are often tied together
by intricate interactions. While past research has paid much more attention to the
pathogenic interactions, the results obtained over the last decade have taught us much
more about a beneficial balance between microorganisms and their hosts (Blaser et al.
2013). It seems that in developing these interactions, diversity plays an incredibly
important role. Diversity is intrinsically correlated with a low incidence of pathogen
outbreaks in both plants and humans. Where does microbial diversity come from? The
plants themselves as well as their secondary metabolites and microbiomes co-evolved
together; microbes contribute to the diversification of plants and vice versa and
continue to add to the high plant-associated microbial diversity. Interesting examples
are medical as well as endemic plants which harbor a unique microbiome (Zachow
et al. 2009; Köberl et al. 2013). Conversely, crops cultivated in intensive agriculture
are often characterized by a reduced diversity in comparison with organic agriculture
or natural ecosystems. In the past, breeding strategies induced a specific microbiome
44 The Edible Plant Microbiome: Importance and Health Issues 425

as cultivar-specificity was very often reported (Berg and Smalla 2009). Our lettuce
example revealed a higher diversity in comparison to its wild ancestor as well as a
loose bacterial co-occurrence network in the modern cultivars. This could explain
its susceptibility for pathogens as well as for biocontrol agents. Efficient biocontrol
approaches were already shown for lettuce (Scherwinski et al. 2008; Erlacher et al.
2014). The enhancement of plant-associated microbial diversity is important for the
sustainability of future agriculture. In addition, for human food and health, microbial
diversity is an important issue, and we should take care of plant-associated diversity
and produce our food in a way that is optimal for this purpose. Biotechnological
strategies can be developed to contribute to this purpose. For example, “microbiome
therapies” are a promising method to maintain or enhance plant-associated microbial
diversity in combination with quality control (Gopal et al. 2013). Another interesting
example is the biocontrol agent Bacillus amyloliquefaciens FZB42, which was able to
enhance the overall plant-associated diversity (Erlacher et al. 2014). Next generation
microbial inoculants should take both the diversity as well as human health issues into
consideration (Berg et al. 2013), and someday in the future should have the potential
to control plant diseases, generally enhance microbial diversity, and stimulate our
immune system.

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of microbial communities in the rhizosphere. FEMS Microbiol Ecol 68:1–13
Berg G, Zachow C, Müller H, Philipps J et al (2013) Next-generation bio-products sowing the seeds
of success for sustainable agriculture. Agronomy 3:648–656
Blaser M, Bork P, Fraser C et al (2013) The microbiome explored: recent insights and future
challenges. Nat Rev Microbiol 11:213–217
Brandl MT (2006) Fitness of human enteric pathogens on plants and implications for food safety.
Annu Rev Phytopathol 44:367–392
David LA, Maurice CF, Carmody RN et al (2014) Diet rapidly and reproducibly alters the human
gut microbiome. Nature 505:559–563
Doreswamy V, Peden DB (2011) Modulation of asthma by endotoxin. Clin Exp Allergy 41:9–19
Erlacher A, Cardinale M, Grosch R et al (2014) The impact of the pathogen Rhizoctonia solani
and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome.
Front Microbiol 5:175. doi:10.3389/fmicb.2014.00175
Garyali S, Kumar A, Reddy MS (2013) Taxol production by an endophytic fungus, Fusarium
redolens, isolated from Himalayan yew. J Microbiol Biotechnol 23:1372–1380
Gopal M, Gupta A, Thomas GV (2013) Bespoke microbiome therapy to manage plant diseases.
Front Microbiol 4:355
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and allergy are interrelated. Proc Natl Acad Sci U S A 109:8334–8339
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Ramírez-Puebla ST, Servín-Garcidueas LE, Jiménez-Marín B et al (2013) Gut and root microbiota
commonalities. Appl Environ Microbiol 79:2–9
Rastogi G, Sbodio A, Tech JJ et al (2012) Leaf microbiota in an agroecosystem: spatiotemporal
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Rastogi G, Coaker GL, Leveau JH (2013) New insights into the structure and function of phyl-
losphere microbiota through high-throughput molecular approaches. FEMS Microbiol Lett
348:1–10
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are highly diverse but dominated by Enterobacteriaceae. Appl Environ Microbiol 78:4933–4941
Scherwinski K, Grosch R, Berg G (2008) Effect of bacterial antagonists on lettuce: active biocontrol
of Rhizoctonia solani and negligible, short-term effects on nontarget microorganisms. FEMS
Microbiol Ecol 64:106–116
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microbiome and secondary metabolites of chamomile plants. Front Microbiol 5:64
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Chapter 45
From Nodulation to Antibiotics

Eva Kondorosi

Abstract The importance and future potential of biological nitrogen-fixation is

widely recognized. A further, yet unexplored and less known value of nitrogen-fixing
root nodules is the presence of hundreds of plant peptides with antimicrobial activities
and novel modes of action. These nodule-specific plant peptides are only produced
in the Rhizobium-infected symbiotic cells where they abolish the endosymbiont’s
cell division ability, transforming them to non-cultivable polyploid nitrogen-fixing
cells. The symbiotic cationic peptides are able to kill a wide range of microbes,
including important human pathogens. The peptides are highly stable and their inter-
actions with multiple bacterial targets reduce the probability to develop resistance.
Worldwide spreading of antibiotic resistant microbes became a major cause of mor-
tality. Therefore there is an urgent need for novel antibiotics. The characteristics and
multifaceted action of symbiotic peptides make them excellent antibiotic candidates.

45.1 Introduction

This chapter focuses on host-governed terminal differentiation of the endosym-

bionts and the roles of host-symbiotic peptides. The morphology and physiology
of nitrogen-fixing Rhizobium bacteria, called bacteroids are not uniform (Kondorosi
et al. 2013). In certain legumes the bacteroids are similar to the cultured bacteria
which maintain their cell proliferation potential and can return to the free-living
life-style. In other legumes, the endosymbionts undergo drastic irreversible mor-
phological and physiological changes, transforming them to non-cultivable enlarged
polyploid nitrogen-fixing cells. Terminal bacteroid differentiation is host-controlled
and has multiple origins in the Leguminosae family indicating that it might be advan-
tageous for the host plants, for example because of more efficient nitrogen-fixation
or consumption of the bacteroid’s cell content during senescence (Oono et al. 2010).
These bacteroids are significantly larger than the free-living bacteria and their shape

E. Kondorosi ()
Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences,
Temesvári krt. 62, Szeged 6726, Hungary
Tel.: +36 62 599 673
e-mail: kondorosi.eva@brc.mta.hu

© Springer International Publishing Switzerland 2015 427

B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3_45
428 E. Kondorosi

Fig. 45.1 Different forms of nitrogen-fixing bacteroids. a similar to free-living cells, b swollen,
and c elongated morphology. Bacteroids were stained with PI (red color) in a and b, and with Syto9
(green color) in c (Kondorosi et al. unpublished)

can be elongated and even branched or spherical (Fig. 45.1). Cell growth is associated
with the amplification of the bacterial genome (Mergaert et al. 2006). The degree of
genome amplification varies in the different legume hosts and its significance has not
been elucidated yet. The features of terminal bacteroid differentiation and the plant
effectors have been discovered in the Sinorhizobium meliloti-Medicago truncatula
symbiosis. The M. truncatula nodules are of the indeterminate type in which cells
below the nodule meristem become infected with S. meliloti which first multiplies
in the young symbiotic cells but, once in the older cells of the infection zone, the
endosymbiont’s cell division becomes arrested and definitively lost while in parallel
their elongation begins. The fully differentiated nitrogen fixing bacteroids are 5- to
10-fold longer than the cultured bacteria and their tripartite genome (the chromo-
some and two megaplasmids) is uniformly amplified at least 20-fold (Mergaert et al.
2006). Comparison of nodule transcriptomes with reversible and terminal bacteroid
differentiation revealed the presence of several hundreds of small genes encoding
nodule-specific secreted peptides in the genome of those legumes in which terminal
differentiation of bacteroids occurred (Alunni et al. 2007). During their translation,
these peptides enter the endoplasmic reticulum where the signal peptidase cleaves off
the signal peptide and the processed mature peptide is delivered via the secretory path-
way to the endosymbionts. The essential role of nodule-specific peptides has been
demonstrated in a signal peptidase mutant of M. truncatula in which the unprocessed
peptides remains in the endoplasmic reticulum and the lack of their interaction with
the endosymbionts abolished bacteroid differentiation (Van de Velde et al. 2010).

45.2 Medicago truncatula Symbiotic Peptides: The NCR

and the GRP Families

In the M. truncatula genome, 600 small genes code for secreted nodule specific
peptides. The NCR family, encoding nodule-specific cysteine rich (NCR) peptides,
consists of more than 500 members (Mergaert et al. 2003; Nallu et al. 2014). The
45 From Nodulation to Antibiotics 429

peptides are composed of a relatively conserved signal peptide and a 30–50 amino
acid long mature peptide with four or six cysteine residues at conserved positions
(Mergaert et al.2003). The highly diverse amino acid composition and sequences,
and thereby charge and hydrophobicity of the NCR peptides, result in a range of
anionic, neutral and cationic peptides with pI values from 3 to more than 11. The
NCR peptides are unique but show structural resemblance to defensins, the largest
class of antimicrobial peptides (AMPs) in plants that protect the plants from bacterial
and fungal pathogens (Silverstein et al. 2005).
The GRP family is less numerous and comprises nodule-specific glycine rich
peptides (GRPs) (Kevei et al. 2002; Alunni et al. 2007). GRPs also contain a signal
peptide while the mature peptides have more than 100 amino acid residues and, simi-
larly to NCRs, could be cationic, neutral or anionic. GRPs represent a unique class of
glycine-rich proteins which share certain similarity with glycine-rich antimicrobial
peptides but evolved for symbiosis in galegoid legumes.
Both the NCR and the GRP genes are exclusively expressed in the S. meliloti-
infected symbiotic nodule cells. However, different sets of genes are activated during
the early and late stages of symbiotic cell development. A single symbiotic cell
produces all the 600 peptides during its maturation but at a given stage probably only
several dozens. Combined and consecutive actions of these peptides drive bacteroid
differentiation.

45.3 NCR Peptides Operate With Numerous Bacterial Targets

Affecting Multiple Pathways

Due to the extremely high numbers and recent discovery of the nodule-specific pep-
tides, the knowledge on the peptide actions is rudimentary. It is unclear how the
peptides enter the bacteria. Cationic peptides can interact with the bacterial mem-
branes and penetrate the cells without pore formation but neutral and anionic peptides,
which did not show interaction with the membranes, are also present in the bacteroids
(Van de Velde et al. 2010; Farkas et al. 2014). The presence of early NCR peptides in
the fully differentiated nitrogen⣳fixing bacteroids indicates their high stability and
resistance against bacterial proteases. Therefore, it is possible that the functions of
the peptides may be required beyond the site of their production. The present studies
are focused on those whose absence provokes symbiotic phenotypes or which have
well-defined in vitro activities. While the peptides are produced effectively in the
polyploid symbiotic cells, experiments with heterologous expression systems were
so far unsuccessful. Therefore chemically synthetized peptides (mostly NCRs), la-
beled with various tags allowing their detection and affinity purification, have been
used for functional analysis. The NCR-bound protein partners have been identified
with LC-MS/MS and Western analysis. In the following the bacterial targets of the
cationic NCR247 peptide will be presented as an example for NCR actions (Farkas
et al. 2014). NCR247 is one of the smallest members of the family which exhibits
antimicrobial activities in vitro and its bioinformatics analysis predicted exceptional
430 E. Kondorosi

protein-binding properties. Identifying its bacterial targets revealed unexpectedly

complex interactions and various ways to modulate the bacterium’s physiology at
multiple sites.
NCR247-FtsZ Interaction: Inhibition of Bacterial Cell Division NCR247 is ex-
pressed in those symbiotic cells where bacterial cell division becomes arrested and
the endosymbionts undergo remarkable elongation and genome amplification (Farkas
et al. 2014). One of the NCR247’s interactors was the FtsZ, the primary protein re-
quired for septum formation and cell division in bacteria. FtsZ is present in the cytosol
but preceding cell division it polymerizes and forms a Z-ring at the future site of bac-
terial cell division which serves as a platform for septum assembly. When the Z-ring
is absent, there is no cell division while filamentous growth of microbes can occur.
Treatment of S. meliloti with NCR247 abolished Z-ring formation demonstrating
that one of the NCR247 peptide’s functions is to inhibit bacterial cell division prob-
ably by preventing FtsZ polymerization. In agreement with this finding, Penterman
et al. (2014) showed that treatment of S. meliloti with sublethal levels of NCR247
provoked cell division block, attenuated expression of critical cell cycle regulators
and antagonized Z-ring function.
NCR247-Ribosomal Protein Interactions: Negative Effect on Bacterial Pro-
tein Synthesis The most numerous interactors of NCR247 are ribosomal proteins.
From the NCR247-treated bacteria, 14 small subunit and 12 large subunit proteins
were identified in the NCR247 complex. As NCR247 has exceptional protein-
binding capacity, it might interact directly with all these ribosomal proteins but
more likely a few—yet unidentified—ribosomal proteins are the primary targets that
bind additional ribosomal proteins. Ribosomal proteins are known targets of many
antibiotics (like streptomycin, neomycin, spectinomycin, tetracyclin, hygromycin
B, chloramphenicol, erythromycin, etc. . .) and these interactions interfere with dif-
ferent phases of translation and inhibit protein synthesis (Wilson 2014). Similarly,
NCR247 slows down or fully inhibits protein synthesis in a concentration-dependent
manner. The protein synthesis is ongoing in the nitrogen-fixing bacteroids, but the
complexity of the proteome is reduced and might be influenced by the NCR pep-
tides. Transcriptome analysis of bacteria and bacteroids revealed in the bacteroids
10 to 20-fold lower expression of ribosomal protein genes as well as altered rel-
ative abundance of transcripts that could provoke ribosome diversification in the
bacteroids. Accordingly, NCR247 attracted fewer ribosomal proteins from the bac-
teroid extracts. Ribosomal heterogeneity represents another level of translational
regulation in bacteria which in the polyploid bacteroid genome could be a major
determinant in selective translation of symbiosis-specific proteins (Byrgazov et al.
2013). Treatment of bacteria with NCR247 resulted also in down-regulation of ribo-
somal protein genes contributing to ribosome diversification in the bacteroids. Thus
NCR247, by controlling the expression of ribosomal protein genes and interfering
with translation, plays multiple roles in the reduced and specific proteome of the
bacteroids.
45 From Nodulation to Antibiotics 431

NCR247-GroEL Interaction: Multiplication of Bacterial Targets Another bind-

ing partner of NCR247 is the bacterial chaperone GroEL. GroEL interacts with
hundreds of proteins and is required for their proper folding (Kerner et al. 2005).
GroEL has multiple roles in various symbioses; it is required for maintenance of
endosymbionts in the host cells and in the rhizobia. It is required for full induction
of nodulation genes and for the assembly of the nitrogenase complex. Moreover, its
interaction with NCR247 shed light on its role in efficient bacterial infection and
terminal bacteroid differentiation (Farkas et al. 2014). While the significance of the
GroEL-NCR247 interaction needs to be discovered, NCR247—and perhaps other
NCRs as well—may modify the affinity of GroEL toward various substrate proteins
and thereby may influence the repertoire of active proteins in the endosymbiont in a
host-governed manner.

45.4 Antimicrobial Properties of NCR Peptides

The definitive loss of the endosymbiont’s cell division ability and structural resem-
blance of the nodule-specific symbiotic peptides to antimicrobial peptides raised the
possibility that at least some of them may have antimicrobial activities outside their
natural nodule environment. Using a set of chemically synthesized mature NCR
peptides it has been proven that cationic NCRs have indeed antimicrobial activi-
ties, killing not only the symbiotic bacterium partner but also other microbes. The
strength and spectrum of cell killing activities are dependent on both the charge and
the amino acid sequence of the peptides. This is illustrated on the example of two
cationic peptides NCR335 (pI: 11.22) and NCR247 (pI: 10.15), both of which have
antimicrobial activities at 50 μg/ml concentration but differing in their antibacterial
spectrum (Tiricz et al. 2013). These peptides eliminated the bacteria more rapidly
than the classical antibiotics kanamycin and tetracycline. Rapid killing of bacteria
was associated with increased permeability and disintegration of the bacterial mem-
branes, allowing penetration of the membrane impermeable dye, propidium iodide
(PI) into the cells. Membrane damage is a characteristic killing action of the posi-
tively charged cationic AMPs whose interaction with the negatively charged bacterial
membranes leads to pore formation and subsequently to cell lysis. However, in the
symbiotic cells the bacteroids remain alive and the bacteroid membrane is intact,
indicating that the peptide concentrations during symbiosis might be significantly
lower than those used in the in vitro assays where the actions of peptides are different
from the membrane damaging AMPs.
Besides the bactericidal effect, several NCR peptides proved to be efficient killers
of various fungi, including important human and plant pathogens (unpublished data
from the laboratory). The bactericidal and fungicidal activities were usually as-
sociated in the tested NCRs but some peptides exhibited primarily anti-fungal or
anti-bacterial activities.
432 E. Kondorosi

45.5 Urgent Need for New Antibiotics

Since their introduction ∼70 years ago, antibiotics have been the most powerful
weapons against microbial invaders. The use of antibiotics such as penicillin, strep-
tomycin, tetracycline and chloramphenicol correlates with a sudden increase of the
human population. These classical antibiotics cured and even led to the eradication
of many infectious diseases, saving the lives of millions. However the uncontrolled
use of antibiotics in humans and in livestock led to the appearance of a large number
of antibiotic-resistant bacteria which became a major cause of mortality and morbid-
ity worldwide. The original antibiotics and their successors are largely ineffective
against the antibiotic resistant bacteria. Therefore new antimicrobial compounds are
needed with novel modes of action, good efficacy and low resistance profile. “The
10 × 20 initiative” aims to develop ten new systemic antibacterial drugs by 2020,
with particular focus on the ESKAPE pathogens (Enterococcus faecalis, Staphy-
lococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa and Enterobacteriaceae) (Bassetti et al. 2013).

45.6 Antimicrobial Peptides as Potential Antibiotics Candidates

AMPs are produced by all living organisms and are effectors of the innate immune
system. AMPs are composed of 10 to 50 amino acids and can be cationic or anionic.
The cationic AMPs show broad-spectrum antimicrobial activities against bacteria
and fungi but anionic AMPs can have also antimicrobial activities or can improve
the activities of cationic AMPs (Guilhelmelli et al. 2013). The classical mode of
their antimicrobial activities is membrane damage but several AMPs have intracel-
lular targets and attack microbes simultaneously at multiple sites which provokes
the microbe’s death. These parallel alternative mechanisms for killing and reduce
the tendency for resistance development. However, microbes co-evolved with AMPs
developed strategies to resist AMPs by surface modifications, biofilm formation, or
the AMPs’ increased efflux, proteolytic degradation, trapping or neutralization. Nev-
ertheless, resistance to AMPs is less common than to conventional antibiotics. These
characteristics of AMPs draw increasing attention on them as promising therapeutic
drugs for the treatment of infectious diseases.

45.7 Benefits of NCRs: Novel Therapeutic Drugs and Versatile

Applications

The symbiotic plant peptides in the nitrogen-fixing nodules represent a gold mine of
bioactive molecules. At least one third of them are cationic and potentially antimi-
crobial peptides. Microbes—except the endosymbionts—have never been exposed
to these symbiotic peptides, thus microbial resistance could not have evolved against
45 From Nodulation to Antibiotics 433

them. Broad spectrum activities of NCRs include killing of antibiotic resistant and
ESKAPE pathogens and their long persistence in the endosymbionts indicates re-
markable stability of these plant peptides against proteolytic degradation. Moreover,
treatment of human cell cultures or injection of mice with NCRs so far did not provoke
symptoms of cytotoxicity (unpublished data from the laboratory) while many antibi-
otics used in therapy—severe side effects and toxicity. These beneficial properties
of the symbiotic plant peptides open multiple possibilities for their application.
Therapeutic Potential Antimicrobial NCRs are new antibiotic drug candidates. The
most active and preferably the smallest peptides that are non-toxic for human cells
can function as lead molecules for drug design. Antibacterial and antifungal NCRs
could be used without modifications for topical treatments of skin, nail and epithelial
infections. The oral or intravenous administration of NCRs requires, however, further
exploratory studies on whether the peptides remain stable and active in the body. It
is known that the activity of cationic AMPs is attenuated in the serum and inhibited
by Mg2 + and Ca2 + ions and by high salt concentrations. Similarly, NCRs are also
sensitive to high salt, Mg2 + and Ca2 + levels. Thus their internal use may require
their stabilization and/or specific delivery to the site of infection.
Non-Medical Applications Pests and pathogens reduce the crop yields with 30–
80 % (Becker-Ritt and Carlini 2012). Therefore, hundreds of synthetic organic
compounds are used in agriculture for protection of plants against microbes and
pests. The NCRs, based on their broad range activity against Gram-positive and
Gram-negative bacteria and fungi, could be alternatives for the organic chemical
products. Constitutive or organ/tissue-specific production of selected NCR peptides
could greatly increase the plant’s resistance against pathogens; but this would require
production of transgenic plants at a large scale which is not feasible for several rea-
sons. A realistic alternative solution is the external treatments of plants with the most
effective broad-range antimicrobial NCRs. Spraying the plants with peptides, ob-
tained by chemical synthesis or heterologous expression, could serve for prevention
and elimination of pathogens.
Among many others, a further possible application could be in the food industry
where microbial contamination, unavoidably present on the raw materials, should
be decreased and optimally eliminated. To date there are no standardized, widely
accepted methods for perfect elimination of microbial contamination. The fast act-
ing, non-toxic antimicrobial NCR peptides introduced at various phases of food
processing could contribute to solving this problem.

Acknowledgements I am grateful to Pal Venetianer for critical reading of the manuscript. Our
work is supported by the “SYM-BIOTICS” Advanced Grant of the European Research Council to
EK (grant number 269067).
434 E. Kondorosi

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Index

2,4-diacetylphloroglucinol (DAPG), 127, 171, Antimicrobial peptide (AMPs), 70, 386, 429,
366 431, 432
2-component regulatory system, 357 Antoun, H., 230
Antunez-Lamas, M., 72
Apel, K., 271
A
Appressorium, 80, 373, 403
Abad, P., 156
Arbuscular mycorrhiza, 235, 237
Abeles, F.B., 257
fungi symbioses, 404
Abscisic acid (ABA), 253, 254, 272
Ardanov, P., 29
Adam, M., 270
Arena, J.P., 162
Adesina, M.F., 290
Arguelles Arias, A., 387
Agrios, G.N., 80, 83, 84
Arimura, G.I., 59
Agrobacterium spp., 46, 137–139, 141, 252,
Armada, E., 274
333, 356, 357, 359
Arnold, W., 218
Agrobacterium radiobacter, 414
Aronson, J.M., 42
Agrobacterium tumefaciens, 13, 36, 49, 69,
Arroyo, J., 41
70, 74, 75, 355
Arth, I., 212
Aguilar, C., 46
Arthrobacter spp., 20, 157
Aguinaldo, A.M.A., 92, 94
Arthrobacter chlorophenolicus, 20
Akhurst, R.J., 177, 178
Ascomycetes, 40, 80, 156
Alavi, P., 275
Atanasova, L., 346
Aldred, D., 194, 197
Atibalentja, N., 159
Ali, S., 28, 29, 31, 262
Auxin, 71, 248, 409
Allen, C., 72
biosynthesis, 251, 252
Allmann, S., 112
degradation, 252
Alström, S., 127
manipulation, 100
Alunni, B., 428, 429
Azcón, R., 232, 233
Alvarez-Martinez, C.E., 358
An, Q., 302
Ané, J.M., 404, 407 B
Angel, R., 267 Bömke, C., 253
Annapurna, K., 301 Büttner, C., 117
Antibiotic, 7, 13, 42, 45, 70, 80, 127, 167, 169, Bacillus spp., 8, 127, 162, 295
430 Bacillus amyloliquefaciens, 301, 388
classical, 432 Bacillus cereus, 186
conventional, 432 Bacillus firmus, 312
resistant bacteria, 57, 433 Bacillus Pseudomonas, 413
© Springer International Publishing Switzerland 2015 435
B. Lugtenberg (ed.), Principles of Plant-Microbe Interactions,
DOI 10.1007/978-3-319-08575-3
436 Index

Bacillus subtilis, 35, 55, 57, 105, 139, 159, Bioeffector, 329, 332, 334
302 of next generation, 330
Bacillus subtilus, 413 Biofertilization, 30, 413
Bacillus thuringiensis, 274, 309, 312 Biofertilizer, 8, 310, 313, 324, 329, 333, 351,
Bacillus thuringiensis (Bt)-genes, 105 385
Bacon, C.W., 26, 27 Biofilm, 9, 14, 19, 35, 166, 167
Bacterial ice nucleation, 19 agents, 51
Bacteriophage, 35, 68, 200 formation, 36, 45–48, 50, 71, 73, 169, 280,
Bacteroid, 13, 49, 217, 396, 408, 427, 429, 430 432
Badosa, E., 194 Bioinoculant, 330, 333, 336
Bailly, A., 55 carriers protect, 331
Bakker, P.A.H.M., 127, 335, 364, 366 mixtures of different, 333
Baldani, J.I., 222
Bioinsecticide, 330
Baldwin, I.L., 397
Biological control, 104, 156, 169, 171, 176,
Baldwin, I.T., 112
201
Balestrini, R., 242
mechanism of, 167
Balmer, D., 84
of mycotoxigenic fungi, 197
Barea, J.M., 228, 231–233
Barkai-Golan, R., 194 of plant diseases, 1
Barnard, A., 299 Biological control agent (BCA), 168, 169, 185,
Barret, M., 229 270, 310, 349
Bartels, D., 266 for postharvest control, 197
Bashan, Y., 331, 332 Biological nitrogen fixation (BNF), 216, 219,
Basidiomycete, 40, 80, 156 220, 393, 408
diseases caused by, 83 future prospects in, 222, 223
Bassetti, M., 432 Biopesticide, 8, 171, 181, 189, 310, 313, 335
Bateson, P., 139 Bioreporters, 8
Bathon, H., 181 Biosafety, 201
Battaglia, D., 349 Biostimulant, 310, 311, 315, 329
Baum, J.A., 189 market for, 313
Bazaka, K., 35 Blanco, F.A., 372
Beattie, G.A., 72 Blaser, M., 424
Beauvais, A., 42 Blaxter, M.L., 93, 94
Becker-Ritt, A.B., 433 Bleves, S., 38
Beckers, G.J.M., 130 Bloemberg, G.V., 10
Bedding, R.A., 178 Blok, V.C., 97
Beegle, C.C., 185, 189 Boemare, N.E., 177
Beijersbergen, A., 358 Bojsen, R.K., 41
Beijersbergen, A.G.M., 357
Boller, T., 123, 127
Belimov, A.A., 261
Bolwerk, A., 10
Belser-Ehrlich, S., 421
Bombar, D., 221, 222
Bent, S.J., 291
Bonaterra, A., 197, 199–201
Berendsen, R.L., 127, 290, 412
Berg, G., 8, 274, 290, 419, 425 Bonfante, P., 241, 242, 244, 273
Bernard, E., 348 Bonfim, K., 146
Bernier, S.P., 57 Bongers, T., 92
Bevan, M.W., 357 Boon, E., 415
Binns, A.N., 357 Borriss, R., 331, 379, 380, 385, 386
Bio-augmentation, 281, 282 Bottini, R., 254
Bio-stimulation, 281 Boyetchko, S., 200
Biocontrol, 9, 13, 14, 156, 162, 167, 169, 178, Brader, G., 28, 30
201, 261, 310, 313, 316 Bradyrhizobium spp., 48, 219, 228, 253, 320,
Biodiversity, 21, 238, 317 393, 398
Index 437

Bradyrhizobium diazoefficiens, 396 degrading enzyme, 50, 68, 69, 258, 348
Bradyrhizobium elkanii, 396 eukaryotic, 39
Bradyrhizobium japonicum, 396 fungal, 40
Bragard, C., 117 of oomycetes, 42
Branching factor, 13, 228 Cha, C., 46
Brandl, M.T., 20, 424 Champigny, M., 125
Braun, A.C., 357 Chang, J.H., 37
Britto, D.T., 206, 207 Charkowski, A., 71
Brotman, Y., 347 Charpentier, M., 404
Browne, P., 229 Chaverri, P., 346
Bruce, T., 142 Chemoattraction, 11, 12
Brucker, R.M., 367 Chemotaxis, 7, 36, 166, 397
Bruijn, F.J., 216, 222, 223 Chen, X.H., 329, 386
Brunner, S., 88 Chilton, M.D., 138, 357
Budiharjo, A., 384 Chin-A-Woeng, T.F.C., 10
Bulgarelli, D., 290 Chitin, 40, 42, 80, 85, 161, 168, 242
Bull, C.T., 66 oligomers, 407
Bundock, P., 360 Chitwood, B.G., 93
Burdsall, H.H., 80 Chitwood, M.B., 93
Burges, H.D., 321 Cho, S.M., 57, 272
Burgess, B.K., 408 Chowdhury, S.P., 384, 386, 389
Burgess, J.G., 57 Christiaensen, L.J., 140
Burgin, A.J., 208, 210, 212, 213 Christie, P.J., 358
Burgyan, J., 120 Christina, A., 30
Burkart, M.R., 399 Chung, S.H., 113
Burke, C., 415 Ciche, T.A., 177
Burnell, A., 180 Citovsky, V., 357, 359
Burton, J.C., 397 Clark, W.M., 53
Byrgazov, K., 430 Clery, D., 266
Climate change, 142, 213, 261, 266
C Clode, P.L., 305
Cabib, E., 41 Cock, M.J.W., 316
Cai, D.G., 155 Cold storage, 199
Calcium spiking, 241, 404, 409 Collinge, D.B., 76, 145, 148
Camehl, I., 274 Colonization, 10, 11, 19, 22, 48, 73, 165, 301
Cameron, K.C., 213 endophytic, 27, 28, 408
Campbell, C., 105 process, 239, 241
Campbell, J.F., 178 root, 380
Campo, R.J., 395 Commercialisation, 309
Cangelosi, G.A., 358 Commercialization, 121, 139, 162, 190, 278,
Canker, 66 313, 320
tree, 82 Common symbiotic pathway (CSP), 404
Capsule, 35, 180, 186 Compant, S., 27
Cardinale, M., 301, 304 Comparative genomics, 28, 29
Carlini, C.R., 433 Competition for nitrogen, 209, 211, 212
Carrier, 73, 179, 201, 320, 331 Conesa, A., 42
Cascales, E., 358 Confocal laser scanning microscopy (CLSM),
Cassan, F., 253 269, 299
Cawoy, H., 170 Confocal stack, 303
Cell envelope, 35, 36, 42 Conjugation, 13, 14, 36, 45, 248, 280
fungal, 39 bacterial, 358
of bacteria, 33 Conn, V.M., 29
Cell wall, 33–35, 39, 40, 43, 84 Conrath, U., 129
438 Index

Constitutive defense, 110, 112 DeAngelis, K.M., 47

Contreras-Cornejo, H.A., 347 Debois, D., 335, 388
Convergent evolution nematode, 100 Dechesne, A., 167
Cook, R.J., 364, 365, 412 Defensin, 429
Cooley, M., 302 Delaux, P.M., 404, 405
Cooley, M.B., 21 Dellagi, A., 71
Coomans, A., 96 Delmotte, N., 20
Corradi, N., 273 Delta-endotoxin, 187
Costa, R., 291, 292, 295 Dempsey, D.A., 125
Cotton, J.A., 100 Denitrification, 208, 209, 212, 221
Crickmore, N., 161 Dent, D., 104
Crook, M.B., 409 Depicker, A., 138
Crown gall, 49, 69, 75, 355 DeSantis, T.Z., 292
Crystal protein, 187, 188 Desert farming, 266, 268, 270
Cumagun, C.J.R., 346 Dessaux, Y., 357
Curley, R.L., 397 Dethier, J.J., 140–142
Cuticle, 18, 82, 109 Di-nitrogen, 215, 216
nematode, 157, 160 Diamond, J., 136
waxiness, 18 Diazotroph, 207, 216, 268, 269
Cyclic dimeric guanosine phosphate microaerobic, 217
(c-di-GMP), 166 symbiotic, 217
Cyclic lipopeptide (cLP), 46, 166, 170, 386 Ding, G.C., 266
Cytokinin, 71, 250–252, 380 Disease resistance, 51, 76, 124, 130, 274
Disease-conducive soil, 8
Disease-suppressive soil, 8, 363, 364
D
Dixon, R., 218
D’aes, J., 170
Djamei, A., 359
D’Alessandro, M., 56
Dobbelaere, S., 320
Défago, G., 7, 329, 330
Dodds, P.N., 124
da Silva, J.G., 140
Dong, X., 124, 125, 128
Dakora, F., 222
Doreswamy, V., 424
Dangl, J.L., 85, 87, 123, 146
Dose-effect relationships, 199
Danhorn, T., 49
Dover, G.A., 94
David, L.A., 423
Dow, J.M., 50
Davies, P.J., 248, 253, 254
Downie, J.A., 48
Davis, T.S., 53
Downy mildew, 80, 84, 312
De Block, M., 139
Drought, 2, 8, 18, 31, 106, 257, 265, 271, 272,
De Boer, M., 168 411
de Boer, W., 414 Druzhinina, I.S., 346
De Bruin, J.L., 397 Du, J., 375
de Bruyne, R., 320, 321, 324, 325 Duckely, M., 359
De Groot, M.J.A., 360 Dulla, G., 48
De Groot, P.W.J., 40, 41
De Jonge, R., 87 E
De Ley, P., 93 Effector-triggered immunity (ETI), 70, 79, 87,
De Los Ríos, A., 267 124
de Maagd, R.A., 186–190 Effenberger, A., 140–142
De Olivera, C.F., 118 EFSA Panel on Genetically Modified
De Roy, K., 414 Organisms, 191
De Vleesschauwer, D., 127, 128 Egamberdieva, D., 8
De Weert, S., 11, 12 Ehlers, R.U., 177, 181
De Wit, P.J.G.M., 84, 85, 87 Elasri, M., 46
de Zelicourt, A., 273 Elliott, G.N., 301
Dean, R., 346 Ellis, R.E., 93
Index 439

Elsevier, 150 Frost, C.J., 129

Eltlbany, N., 293 Fruit and vegetable rot, 66, 197, 199
Endophyte, 18, 26, 28, 30, 408, 421 Fry, W., 372, 376
non-deleterious, 25 FtsZ, 430
obligate, 29 Fu, Z.Q., 99, 124, 125, 128
Endophytic colonization, 301, 408 Fuentes, I., 140
Endosymbiont, 37, 407, 428, 431, 433 Fuqua, C., 49, 70, 357
Enterobacteriaceae, 70, 71, 302, 421, 424, 432 Fuqua, W.C., 45
Entwistle, P.F., 189
Epiphyte, 48, 73, 80, 84 G
Erlacher, A., 389, 424, 425 Götz, M., 296
Erwinia spp., 20, 21, 46, 228 Gaiero, J.R., 137
Erwinia amylovora, 66, 69, 71, 73, 75 Gamalero, E., 262
Erwinia chrysanthemi, 250 Gan, Z.W., 161
Escudero, J., 358 Gantner, S., 47
Ethylene, 13, 56, 69, 71, 247, 254, 257, 260, Garbeva, P., 414
380 Garyali, S., 421
Ettwig, K.F., 209 Gasser, M., 303
Exchangeable soil P pool, 233 Gassmann, A.J., 105
Gaugler, R., 178
Geiger, F., 317
F Gelvin, S.B., 357, 358
F-pilus-mediated conjugation, 13 General suppression, 364
Fagan, R.P., 35 Generalist herbivores, 107, 110
Fairweather, N.F., 35 Genetic modification, 105, 186, 281, 360, 413
Fan, B., 301, 380 Genome amplification, 428, 430
Farag, M.A., 53, 55, 59 Genomics, 162, 346, 375
Farkas, A., 429–431 Genre, A., 241
Felix, G., 123, 127 Geurts, R., 273
Fengycin, 170, 334, 335, 386, 388 Gherbi, H., 407
Ferguson, B.J., 215, 216, 220 Giamoustaris, A., 108
Ferris, H., 92 Gibberellin, 253, 409
Fierer, N., 194, 267, 420, 424 Gijzen, M., 375, 376
Finkel, O.M., 23 Girard, G., 10
Flagellum, 36, 39 Glare, T., 187, 189, 190
Flor, H.H., 83 Glick, B.R., 257–259, 261, 262
Flores, E., 217 Global agriculture, 155
Floyd, R., 93 Glomeromycota, 235, 237, 238
Fluorescence in situ hybridization (FISH), 302 Glucosinolates, 107, 109
Food-borne human pathogen, 197 GM-crops, 141, 336
Ford Doolittle, W., 98 Gomes, N.C.M., 291–293
Formulation, 179, 201, 316, 317, 321, 324, Gonsalves, D., 121
349, 350 Gonthier, P., 118
biocontrol, 335 Gopal, M., 425
granular, 322 Gourion, B., 20
Forney, L.J., 291 Govers, F., 375
Frébort, I., 253 Goverse, A., 81–83, 88, 100
Francés, J., 199 Grewal, P.S., 181
Franche, C., 217–221, 223 Grichko, V.P., 261
Fred, E.B., 397 GroEL, 431
Freeman, J., 364 Grosch, R., 296
Freeman, S., 274 Grosskopf, T., 414
Frey-Klett, P., 231 Grube, M., 303
Friedman, M.J., 321 Guerrero, R., 137
440 Index

Guilhelmelli, F., 432 Hornby, D., 365, 368

Gullino, M.L., 194, 197 Hoth, S., 97
Gutierrez-Zamora, M.L., 408 Howe, G.A., 110, 124
Gutjahr, C., 237, 242 Hua, C., 374
Huang, X.W., 159
H Hudson, L.C., 146
Höfte, M., 127, 128, 168 Human health, 30, 213, 277, 421
Højberg, O., 212 Hungria, M., 395–399
Ha, T.N., 346 Hurek, T., 26, 27, 29, 30
Haas, B.J., 375 Hydrophobin, 42, 348
Haas, D., 7, 329, 330 Hymowitz, T., 394
Hallmann, J., 26 Hypersensitive response (HR), 70, 79, 84, 124,
Hamelin, R.C., 149 336, 376
Hamilton, S.K., 208, 210, 212, 213
I
Hammond, J.P., 226, 227
Idriss, E.E.S., 385
Handelsman, J., 65
Image analysis, 297
Hannula, S.E., 413
Immune system, 34, 40, 107, 109, 123, 432
Hanski, I., 424
Induced systemic resistance (ISR), 123, 124,
Hao, Y.E., 156
167, 168, 273, 335, 364, 388, 413
Hardoim, P.R., 26, 27, 30, 31
by beneficial microbes, 127
Harlan, J.R., 394
Inducible defense, 110
Harman, G.E., 320, 347, 349
Ingwell, L.L., 118
Haroon, M.F., 209
Innerebner, G., 21
Harpin, 336
Inoculant, 397
Harrison, M.J., 242
effects of, 296
Hart, S., 211 fungal, 244
Hartmann, A., 51 granular, 322
Hartney, S.L., 168 microbial, 230, 326
Hasegawa, S., 271 Insect, 5, 18, 72, 73, 109
Haupt, B.J., 304 herbivores, 112
Haustoria, 80, 81, 84 larvae, 8
Havelda, Z., 120 parasitoids, 105
Hawksworth, D.L., 80 pathogens, 104
Hawrylowicz, C.M., 424 resisitance, 148
Heger, P., 139 Insecticidal toxin, 161, 162
Heidari, M., 271 Integrated pest management, 345
Helber, N., 243 Interface, 42, 242
Henkels, M.D., 168 root-microbe, 168
Hermosa, R., 347 soil-root, 280
Herridge, D.F., 396, 399 Invertebrate biocontrol agent (IBCA), 310, 312
Herzner, A.M., 386 Iqbal, M.Z., 423
Heterocyst, 217, 221 Irrigation, 22, 73, 121, 140, 266, 323
Heterorhabditis spp., 175, 177, 312 Isotopic (32P and 33P) dilution approaches,
Heuer, H., 291, 295, 296 231, 233
Hinton, D.M., 26, 27 Iturin, 170, 334, 335, 388
Hirt, H., 271
Hoffland, E., 127 J
Hogenhout, S.A., 67, 71, 117 Jackson, L.E., 206, 207, 211
Hohn, B., 359 Jackson, R.B., 267
Holden, N.J., 424 Jahn, K.A., 304
Holterman, M., 92, 94, 96 James, D.E., 399
Hong, C., 121 James, E.K., 222, 408
Hooykaas, P.J.J., 357, 359 Jameson, P., 250, 253
Index 441

Jander, G., 110, 124 Kotze, A.C., 159, 161

Janisiewicz, W.J., 197, 201 Kouser, S., 141
Jaskiewicz, M., 130 Kröber, M., 385, 389
Jasmonic acid (JA), 56, 69, 71, 123, 168 Kronzucker, H.J., 206, 207
Jaubert, S., 98 Kroon, L.P.N.M., 372
Jechalke, S., 296 Kropf, S., 292
Jensen, D.F., 320 Kroupitski, Y., 302
Jensen, S.E., 387 Kudva, R., 36
Jetten, M.S.M., 210 Kurkcuoglu, S., 21
Ji, X., 302 Kuzyakov, Y., 211
Jiang, R.H.Y., 375 Kwak, Y.-S., 366, 368
Jin, S., 358 Kwon, Y.S., 56
Jones, J.D.G., 85, 87, 123 Kyndt, T., 99
Joosten, M.H.A.J., 84
Judelson, H.S., 372 L
Juhas, M., 415 Lòpez-Llorca, L.V., 157
Junge, H., 320 Ladha, J.K., 216
Lagopodi, A.L., 10
Lam, P., 209, 212
K Lambers, H., 206
Köberl, M., 268, 302, 421, 424 Lamers, L.P.M., 206
Kachroo, A., 125 LamovŠek, J., 162
Kado, C.I., 65, 66, 68, 72 Landa, B.B., 367
Kahl, G., 357 Larcher, W., 205, 206
Kahn, D., 218 Large, E.C., 372
Kai, M., 57 Larue, T.A., 395
Kalia, V.C., 51 Lee, B., 127
Kamilova, F., 7, 9, 11, 127, 166, 167, 303, 320, Lee, B.Y., 55, 56
321, 324, 325, 329, 333, 413 Lee, C., 100
Kamoun, S., 372, 375 Leff, J.W., 194, 420, 424
Karanastasi, E., 96, 100 Leghaemoglobin, 408
Kartal, B., 210, 211 Legume, 48
Kasuga, T., 376 nodulation, 49, 404
Kaye, J., 211 Lehmann, J., 230
Keen, N.T., 98 Leibovitch, S., 398
Kerner, M.J., 431 Leveau, J.H.J., 17, 18
Keswani, C., 348 Leverentz, B., 197
Kevei, Z., 429 Lewis, E.E., 178
Kiers, E.T., 244 Li, G.H., 161
Kikuchi, T., 99 Li, J., 387
Kim, K.S., 57, 59 Li, L., 302
King, A.M.Q., 115, 117 Li, Y., 69
Kinkel, L.L., 415 Liang, Y., 398, 407
Klappenbach, J.A., 291 Lichen
Klessig, D.F., 125 paradigm, 303
Kliebenstein, D.J., 142 Liebrand, T.W.H., 85, 88
Klis, F.M., 40 Lin, C., 194
Kloepper, J.W., 30, 329 Lin, K., 237
Knobeloch, L., 399 Lindow, S.E., 18
Koenig, R., 117 Lipke, P.N., 40, 42
Kohlera, J., 272 Lipochitooligosaccharide (LCO), 398, 404
Kombrink, A., 87 Lipopolysaccharide, 10
Kondorosi, E., 427 Liu, Z., 157, 386
Korsten, L., 197 Lloyd, C.M., 424
442 Index

Loof, P.A.A., 96 McSpadden Gardener, B.B., 367

Loper, J.E., 168, 367 Meadows, D.H., 136
Lorenzen, S., 93 Mechanisms of action, 197, 302, 303
Loria, R., 67, 71 Medicago truncatula, 51, 70, 404
Lorito, M., 346–348 Melchers, L.S., 358
Lowe, D.J., 408 Mendes, I.C., 395, 397–399
Lozano-Torres, J.L., 85 Mendes, R., 127, 364, 412, 414
Lucangeli, C., 254 Mentlak, T.A., 87
Lugtenberg, B., 7, 9, 10, 26–28, 30, 31, 127, Mercado-Blanco, J., 26–28, 30, 31
166, 167, 228, 320, 329, 413 Mergaert, P., 428, 429
Luna, E., 129, 130 Mescher, M.C., 129
Lunau, S., 179 Methylobacterium, 20, 421
Lundberg, D.S., 408 Methylobacterium extorquens, 20
Luo, H., 162 Meyer, K.M., 17
Lynch, J.M., 412 Meziane, H., 168
Mi, Q.L., 161
M Michielse, C.B., 360
Márquez, L.M., 274 Microbial, 2, 20
Mélida, H., 42 control, 39
Macel, M., 107 nitrogen metabolism, 282
Macho, A.P., 88 pesticide, 171, 200, 310, 312
Macrobial, 310, 313 development of, 315
Magan, N., 194, 197 phytopathogens, 26
Microbiome, 8, 25, 289, 299, 423
Mahmood, I., 157
endophytic, 25, 29, 31
Maignien, L., 19
phyllosphere, 18
Maillet, F., 241
Microbiota, 17, 22
Maize, 56, 104, 106, 146, 413
endophytic, 31
Maketon, C., 368
phyllosphere, 18, 22, 23
Maldonado, A.M., 125
Mishina, T.E., 125
Maldonado-González, M.M., 303
Mithen, R., 108
Malfanova, N., 28
Mitreva-Dautova, M., 98
Manulis, S., 251
Mitter, B., 28–31
Marasco, R., 270, 275
Molinari, S., 156
Mariani, C., 139
Montesinos, E., 199, 200
Marine nitrogen cycle, 221
Morris, R.O., 357
Marion-Poll, A., 254
Moulin, L., 219
Markmann, K., 406–408 Moya, A., 415
Marks, B.B., 398 Mráček, Z., 178
Marques, J.M., 290, 292, 296 Mukherjee, P.K., 346, 348
Marrone, P.G., 316 Mutualism, 127, 130, 407
Marroquin, L.D., 161 Myc factors, 13, 241, 404
Marschner, P., 228, 229 Mycorrhiza, 231, 239
Marshall, R., 87 helper bacteria, 231, 334
Martínez-Gil, M., 166 Mycorrhizosphere, 7, 226, 229–231, 233
Martínez-Romero, E., 26, 408 Mycotoxin, 194, 197
Martinez-Medina, A., 347
Mass production, 157, 175, 178, 179 N
Mastouri, F., 347 N-acyl homoserine lactone (AHL), 13, 127
Matheson, F., 212 N-fertilizer, 393, 396–399
Mavrodi, D.V., 169, 335 Nübel, U., 291
Maxmen, A., 103, 106 Nadarasah, G., 73
Mayak, S., 261, 262, 272 Nallu, S., 428
Mazzola, M., 169, 171 Nambara, E., 254
Index 443

Nascimento, F.X., 261 P

Neal, A.L., 413 Pu̇ža, V., 178
Nematode, 27 Palsson, B.O., 413, 414
Nematophagous microorganism, 155, 159, 161 Palukaitis, P., 120
Nester, E., 357 Pantoea spp., 20, 21, 46
Neumann, G., 296 Pantoea agglomerans, 201
Newman, K.L., 302 Pantoea herbicola, 75
Newman, M.A., 70 Pantoea stewartii, 50
Newton, W.E., 217 Park, S., 271
Nicolotti, G., 118 Parniske, M., 237, 242, 406, 408
Niederweis, M., 35 Paster, N., 194
Niftrik, L., 210 Pastor, V., 124, 129, 130
Nijland, R., 57
Patel, H.K., 51
Nitrate leaching, 213
Pathogen-associated molecular patterns
Nitrification, 206, 207
(PAMPS), 69, 79, 85
Nitrogen fixation, 49, 127, 207, 217–219, 324,
Pathogen-triggered immunity (PTI), 68, 79
397, 427
Pathogens, 17, 20, 21, 29, 30, 39, 45, 69, 433
Nitrogenase, 216–218, 408, 431
animal, 42
Nitrous oxide emission, 216
Niu, B., 387 bacterial, 66, 69
Nod factor, 396, 398, 404, 407, 409 of plant surface, 49
Nodulation, 7, 13, 49, 398, 405 vascular, 50
Nodule-specific cysteine rich (NCR), 428 Patten, C.J., 251, 252
peptides, 429–431, 433 Pattern-triggered immunity (PTI), 124
Nodule-specific glycine rich peptide (GRP), Patterson, T.G., 395
429 Paulitz, T.C., 364
Nodules, 13, 48, 272 Paulsen, I.T., 329
nitrogen-fixing root, 45 Peden, D.B., 424
spontaneous, 403, 408 Pel, M.J.C., 124
Nuringtyas, T.R., 110 Penterman, J., 430
Nutritional enhancement, 139 Perazzolli, M., 347
Perez-Velazquez, J., 18
Perry, R.N., 181
O
Pertry, I., 253
Öpik, M., 238, 239
Pest control, 105, 191
O’Callaghan, M., 187, 189, 190
Peters, A., 177
Oerke, E.C., 146
Phase variation, 177
Okmen, B., 85
Okon, Y., 320 Phenazine −1-carboxylic acid (PCA), 171, 334
Oldroyd, G.E.D., 240, 241, 404 Philippot, L., 301, 412
Olive, 27 Phosphate
Oliver, J.D., 74 immobilisation, 226
Oliver, R.P., 88 metabolism, 226
Omura, S., 162 mineralisation, 229
Oomycete, 8, 18, 33, 79, 80, 170, 364, 375, 415 solubilisation, 228, 229
Oono, R., 427 solubilising microorganism (PSM), 225,
Oort, A.J.P., 83 228
Opine, 357 transporter, 242
Orthophosphate availability, 229 Phosphorus transformations, 225
Osmo-adaptation, 201 Photorhabdus, 175, 177, 181
Outer membrane (OM), 11, 33–35, 70, 358 Photosynthate, 18
Oxygen paradox, 215–217 Phylloplane, 17, 187
444 Index

Phyllosphere, 17, 18 Pozo, M.J., 130

functional genomics, 20 Pre-infection thread (PIT), 403, 407
Phylloxera, 105, 107 Pre-penetration apparatus (PPA), 241, 403, 407
Phylogenomics, 405 Prieto, P., 27
Phylogeny, 218, 238 Priming, 123, 129, 130, 168
Physiological strain improvement, 316 Prosser, J.I., 412
Phytohormone, 30, 68, 72, 253 Protein secretion, 11, 37
in microbes, 248 Prusky, D., 194
in plants, 247, 248 Prusky, P.L., 197
Phytopathogenic nematode, 92, 156, 159, 161 Pseudomonas spp., 8, 9, 11, 14, 27, 28, 170
Phytostimulation, 30 Pseudomonas aeruginosa, 36, 432
Phytotoxin, 71 Pseudomonas fluorescens, 127
Pieterse, C., 166, 167 Pseudomonas putida, 166, 168
Pieterse, C.M.J., 28, 124, 127–129, 131, 254, Pseudomonas syringae, 20, 48
363, 366 Pumplin, N., 120
Pigliucci, M., 139 Pusey, P.L., 197
Pili, 35, 36, 38, 73
Pinton, R., 7, 8 Q
Pitzschke, A., 141 Qaim, M., 141
Qiao, J., 336
Plant
Qin, L., 98
cell re-differentiation, 100
Quinones, B., 48
cell wall degrading enzyme (PCWDE), 50,
Quorum sensing (QS), 45, 70, 357
71
defense response, 29, 67, 254, 335 R
genetic engineering, 409 Raaijmakers, J., 388
growth and health, 130, 231, 289, 336, 379, Raaijmakers, J.M., 166, 167, 169–171,
412 365–367, 412, 413
growth promotion (PGP), 30, 165, 215, 216, Raetz, C.R.H., 34
228, 230, 248, 313 Raghoebarsing, A.A., 209
growth-promoting bacteria (PGPB), 257, Ramírez-Puebla, S.T., 423
258, 260, 261 Ramão-Dumaresque, A.S., 346
used as bioinoculants, 329, 330 Ramos-Gonzalez, M.-I., 329
growth-promoting rhizobacteria (PGPR), Rasmann, S., 130
127, 412 Rastogi, G., 21–23, 422, 424
hormones, 13, 67, 72, 130, 273, 278, 380 Rathjen, J.P., 124
immune system, 123, 124, 127 Ravensberg, W.J., 315
nitrogen metabolism, 205 Records, A.R., 69
protection product (PPP), 1, 8, 310, 311, Reddy, P.M., 216
322, 324, 325 Redecker, D., 238
Plant-associated bacteria, 30, 46, 51, 248, 249, Redman, R.S., 273
269 Regensburg-Tuı̈nk, A.J.G., 359
Plant-microbe interaction, 2, 274, 275, 301, Registration, 181, 312, 324
305, 421 Rehman, S., 99, 100
Plant-parasitic nematode, 91, 96, 100 Reinhold-Hurek, B., 26, 27, 29, 30
Plante, A.F., 226 Remus-Emsermann, M.N.P., 19
Pliego, C., 12, 166, 167 Rhizo-engineering, 282, 283
Poinar, G.O.Jr., 175, 177 Rhizobacteria, 51, 53, 159, 166
Pollution Rhizoremediation, 31, 278, 284, 415
chemical, 2, 143, 277, 405 strategies to improve, 281
Polyploid, 429, 430 Rhizosphere, 7, 8, 26, 56
Popeijus, H., 98 biotechnology, 225
Postgate, J., 216, 217 effect, 7, 293
Postma, W.J., 100 gene transfer in, 14
Index 445

microbiome, 8 Schell, J.S., 357

QS in, 46, 47 Scherwinski, K., 425
war in the, 13, 14 Scheublin, T.R., 20
Ribosome diversification, 430 Scheuring, I., 415
Rice, 70, 75, 105, 139, 242 Schikora, A., 51
Richardson, A.E., 227–231 Schilirò, E., 29
RNA silencing, 118, 120 Schirmböck, M., 346
Robert-Seilaniantz, A., 124 Schloss, P.D., 65
Roberts, P.A., 98 Schmidt, R., 270, 421
Robin, G.P., 125 Scholthof, K.B., 116
Rodelo-Urrego, M., 120 Scholz, R., 388
Rodriguez, R.J., 273 Schoonhoven, L.M., 109
Rogers, C., 404 Schreiter, S., 290, 292, 293, 295, 296
Roldán, A., 347 Schulz, B., 26
Rolli, E., 270 Scott, J.C., 252
Romeis, J., 151 Secondary metabolite, 84, 109, 177, 194, 280,
Rooney, H.C.E., 85 334, 347
Root exudate, 9, 10, 13, 171, 280, 329, 413 antifungal, 380
maize, 388 Secretion system, 11, 29, 68, 69
Root hair, 27, 252, 384, 404 Segarra, G., 129
Root nodule symbiosis, 405, 408 Seidl, M.F., 375
Root rot, 11, 83 Seidl, V., 347
Rose, C.M., 404, 407 Seligman, S.J., 34
Rosenberg, E., 69, 71, 137 Seo, J.K., 137
Rosenblueth, M., 26 Setati, M.E., 194
Rosenzweig, N., 414 Sevilla, M., 409
Ross, A.F., 125 Shah, J., 125
Rossmann, B., 423 Shapiro-Ilan, D.I., 179, 180
Roy, R., 227, 233 Sheng, J., 359
Rudrappa, T., 56, 413 Shinya, R., 92
Russel, B., 138 Shiomi, K., 162
Rust, 83 Shoresh, M., 347
Ruyter-Spira, C., 240 Siddiqui, Z.A., 157
Ryan, R.P., 26, 30 Siderophore, 71, 168, 169, 258
Rybarczyk-Mydłowska, K., 99 biosynthesis, 50
Rybicki, E.P., 117 Sietsma, J.H., 43
Ryu, C-M., 127 Silverstein, K.A., 429
Ryu, C.M., 30, 53, 55, 56, 58, 59 Simons, M., 10
Sinorhizobium meliloti, 428
S Slaughter, A., 130
Séralini, G.E., 150 Smalla, K., 290, 292, 293, 419, 425
Salvagiotti, F., 394, 395 Smant, G., 98
Salvioli, A., 242, 244 Smets, B.F., 167
Samuels, G.J., 346 Smith, F.A., 231
Sanchez-Contreras, 49 Smith, K.P., 413
Sanglestsawai, S., 141 Smith, S.E., 231
Sanguin, H., 366 Smut, 83
Sanjuan, J., 398 fungi, 83
Sankaran, S., 75 Snowdon, A.L., 194
Santner, A., 248 Soberón, M., 188
Santos, P.C., 219 Soil phosphorus cycle, 226
Sastry, K.S., 117 Song, G.C., 58
Scab, 71, 82 Souleimanov, A., 398
446 Index

Soybean production, 394, 395, 397 Tholl, D., 55

Soyer, O.S., 414 Thomashow, L.S., 171
Spaepen, S., 249, 250, 252 Thomashow, M.F., 357
Spaink, H.P., 357, 398 Thomma, B., 87, 88
Spanu, P.D., 85 Thomma, B.P.H.J., 87
Specialist herbivore, 109, 110, 112 Three-dimensional model, 303
Specific suppression, 364, 366 Tian, B.Y., 159
Spiegel, Y., 346 Timmusk, S., 261
Spoel, S.H., 126, 128 Tiricz, H., 431
Spontaneous nodule, 403, 408 Toklikishvili, N., 261
Sprent, J.I., 408 Tommassen, J., 37
Stachel, S.E., 358 Toro, M., 232, 233
Stavrinides, J., 73 Toro, N., 358
Stearns, J.C., 260 Transferred DNA (T-DNA), 69, 356, 357, 359
Stefani, F.O.P., 149 processing, 358, 359
Steidle, A., 46 Transgenic plant, 139
Stein, E., 128 Transmission, 54, 106, 115, 117, 118
Stein, T., 387 Transport vessel, 302
Steinernema, 175, 176, 178 Trias, R., 197
Stergiopoulos, I., 85, 87 Tripathi, P., 348
Stress controllers, 8 Tritrophic interaction, 112
Strigolactone, 13, 239, 247, 334, 404 Trudgill, D.L., 97
Studholme, D., 346, 347 Tsavkelova, E.A., 254
Sulakvelidze, A., 197 Tucci, M., 347
Sunkar, R., 266 Tudzynski, B., 253
Suppiger, A., 46 Type 4 secretion system (T4SS), 69
Suppressive soil, 335, 364, 369 Tzfira, T., 357, 359
Surfactin, 170, 335, 386, 388
U
Sustainable agriculture, 135, 150, 220, 303,
Urquiaga, S., 222, 409
329, 409
Symbiont, 113, 177
V
rizobial, 29
Vachon, V., 188
Symbiosis, 45, 49, 113, 142, 207, 345
Vaeck, M., 139
Rizobium, 219, 220 Van Dam, N.M., 110
Symbiotic nitrogen fixation, 220 Van de Mortel, J.E., 128
Symbiotic signalling, 409 Van de Velde, W., 428, 429
Synthetic community, 414 Van den Burg, H.A., 85
Synthetic fertilizer, 136 Van der Ent, S., 129, 130
Systemic acquired resistance (SAR), 123–125, van der Heijden, M.G.A., 238
336, 347 Van der Wal, A., 18
Systemic infection, 118, 120 Van Elsas, J.D., 14, 29
Szopinska, A., 39 Van Esse, H.P., 85
Van Hulten, M., 129
T van Kregten, M., 359
Tabashnik, B.E., 191 Van Lenteren, J.C., 312
Take-all decline (TAD), 363–366, 368, 369 Van Loon, L.C., 125, 128
Takken, F.L.W., 81–83, 88 Van Meer, G., 39
Talboys, P.J., 385 Van Megen, H., 94
Tanaka, S., 104 Van Moorhem, M., 142
Teplitski, M., 424 Van Oosten, V.R., 129
Terminal bacteroid differentiation, 427, 428, Van Overbeek, L., 29
431 Van Peer, R., 127
Thies, J.E., 397 Van Wees, S.C.M., 128, 129
Index 447

Van West, P., 372 Whisson, S.C., 376

Van, Gerven N., 36 White, G.F., 178
Vande Broek, A., 251 White, P.J., 226, 227
Vanderleyden, J., 252 White, T.C.R., 106
Vector, 73, 106, 115, 117, 118 Whitelaw, M., 228
population, 121 Whitfield, C., 34
Vega, F.E., 316 Wiehe, T., 139
Venkateshwaran, M., 404, 405, 407 Wilson, C.L., 197
Venturi, V., 70, 357 Wilson, D.N., 430
Verginer, M., 421 Wilson, M.J., 178
Vergunst, A.C., 359 Winans, S.C., 358
Verhagen, B.W.M., 128–130 Wings, S., 421
Vermeulen, S., 140, 142 Woo, S.L., 346
Vernooij, B., 125 Wright, D.J., 179
Viable but nonculturable (VBNC), 74 Wu, F., 421
Vinale, F., 347, 348 Wu, Y., 126
Virulence, 13, 14, 41, 45, 50, 67, 71, 87, 359 Wyss, U., 100
factors, 13, 14, 45, 66, 67, 70, 71, 155, 171
genes, 357 X
Virus-host interaction, 116, 118–120 Xenorhabdus, 175–177
Vleeshouwers, V.G.A.A., 88, 376 Xiao, Z.J., 55
Vlot, A.C., 125 Xie, X., 40
Voinnet, O., 120 Xu, P., 55
Volatile, 13, 50, 53, 57 Xu, X., 211
bacterial, 60 Xu, Y., 171
leaf, 109 Xue, Q., 290, 292, 296
Volk, T.J., 80
Y
Vollmer, W., 34
Yamamoto, T., 185, 189
Von Bodman, S.B., 50
Yang, J., 56
Von Rosenvinge, E.C., 423
Yang, J.K., 162
Vorholt, J.A., 17, 301
Yang, M-M, 170
Vos, C., 347
Yao, J., 72
Vos, I.A., 129
Yeast, 18, 39, 42, 43, 200, 252
Vrieling, K., 107
Yeates, G.W., 92
VV.AA., 26, 30
Yedidia, I., 347
Yu, D.W., 415
W Yu, X.L., 20
Wösten, H.A.B., 42
Walker, K., 189 Z
Waller, F., 274 Zachow, C., 303, 424
Walters, D.R., 124, 129 Zaidi, A., 231
Wang, C., 261 Zambryski, P., 138
Wang, H.B., 412 Zamioudis, C., 28, 127–129
Wang, L., 218 Zapata, F., 227, 233
Wang, L.F., 156 Zehr, J., 221, 222
Wang, M.B., 120 Zeier, J., 125
Ward, E., 364 Zeng, W., 69, 72
Wei, G., 127 Zengler, K., 413, 414
Wei, J.Z., 159 Zhang, H., 56
Weinert, N., 290–292, 295 Zhang, Q., 76
Weisskopf, L., 55 Zhang, Y., 157
Weller, D.M., 171, 364–368, 415 Zhao, Z.T., 84
Wessels, J.G.H., 43 Zhou, X.S., 157
448 Index

Zhu, D.H., 104 Zipfel, C., 88

Zhu, J., 357 Zolla, G., 166
Zhu, S.X., 88 Zoller, H.F., 53
Zilber-Rosenberg, I., 137 Zunke, U., 97