Bamboo inhabiting fungi

and their damage to the substrate

Dissertation

zur Erlangung der Würde des Doktors der Naturwissenschaften

des Fachbereichs Biologie,
der Fakultät für Mathematik, Informatik und Naturwissenschaften
der Universität Hamburg

vorgelegt von
Dongsheng Wei
aus China

Hamburg 2014
USDA Fores t Service
Forest Products
L a b o r a tJanuary
Date:
o r y 21, 2014

Subject: English Assessment by a Native Speaker www.fpl.fs.fed.us

To: University of Hamburg, Ph.D. Dissertation Committee for Dongsheng Wei

As a native speaker of the English language, I have checked and made minor editorial corrections to the
Ph.D. dissertation entitled, Bamboo inhabiting fungi and their damage to the substrate, authored by
Dongsheng Wei. I find that this dissertation is very well written and fulfills the requirements for an English
dissertation.

Respectively,

Daniel Joseph Yelle, Ph.D.

Research Forest Products Technologist

1 GIFFORD PINCHOT DRIVE • MADISON, WISCONSIN • 53726-2398

PHONE 608 231-9200 • FAX 608 231-9592 • TDD 608 231-9544
Acknowledgements

I would like first to express my greatest gratitude to my supervisor, Prof. Dr. Olaf Schmidt,
for giving me the opportunity to work in his laboratory and for guidance in finishing this
research work at the Centre for Wood Science, University of Hamburg. The completion of
this work would not be possible without his assistance and patience.

Furthermore, I would like to express my sincere gratitude to Prof. em. Dr. Dr. h.c. mult.
Walter Liese for his endless supply of information and literature about bamboo. I am
confident that our friendship will be never ending.

Many thanks are to Dr. Uwe Schmitt, Dr Jürgen Plus, PD Dr. Gerald Koch, Dr. Ron Janzon,
Tanja Potsch, Daniela Paul, Karin Brandt, Anne-Marie Wettich, Sergej Kaschuro, Anna
Knöpfle and Nicole Erasmy at the Thünen Institute for their technical help in lab. They have
offered kind and helpful suggestions throughout the duration of the experimental work.

Special thanks are to Dr. Mathias Rehbein and Marie-Therese Lenz not only for helping me
with fungal degradation experiment and data analysis but also for many tips beyond research
that were useful to lead a happy life. I will not forget them for their immense support during
my life in Hamburg.

My sincere thanks are due to former and current PhD candidates, Thi Kim Hong Tang,
Mohsen Bahmani, Stephanie Helmling, Yongshun Feng for providing their constant support
and a stimulating and fun environment in which to learn and grow.

I gratefully acknowledge is extended to Prof. Dr. Robert A. Samson who gave me two times
chances in 2011 to do fungal taxonomy research at the Fungal Biodiversity Centre(CBS),
Institute of the Royal Netherlands Academy of Arts and Science. To Dr. Jos Houbraken, Dr.
Jan Dijksterhuis, Neriman Yilmaz I am very grateful for their help to carry out experiments as
well as for the great working environment. Thanks are also to the Federation of European
Microbiological Societies (FEMS) for the grant during my first stay in the Netherlands.

I gratefully acknowledge the German Academic Exchange Service (DAAD) for the financial
support during my PhD studies.

Last but not least, I would like to thank my parents and my three sisters for their continuous
moral support, encouragement and love which has helped me enormously.

I
Table of contents

Acknowledgements ............................................................................................................... I
Table of contents ................................................................................................................. II
Abstract/Zusammenfassung ............................................................................................... V
List of abbreviations .......................................................................................................... IX
1 Introduction ....................................................................................................................... 1
1.1 Fungi ............................................................................................................................ 1
1.1.1 Characterization ..................................................................................................... 1
1.1.2 Classification ......................................................................................................... 2
1.1.3 Identification.......................................................................................................... 4
1.1.4 Important genera of bamboo inhabiting fungi ......................................................... 6
1.2 Bamboo ........................................................................................................................ 9
1.2.1 Biology ................................................................................................................ 10
1.2.2 Significance ......................................................................................................... 14
1.2.3 Uses ..................................................................................................................... 15
1.2.4 Damage by fungi and insects ................................................................................ 19
1.3 Factors affecting fungal degradation tests.................................................................... 21
1.4 Modern techniques to study the morphology of degraded bamboo .............................. 22
1.4.1 Scanning electron microscopy (SEM) .................................................................. 23
1.4.2 Confocal laser scanning microscopy (CLSM) ...................................................... 23
1.4.3 Transmission electron microscopy (TEM) ............................................................ 24
1.4.4 Ultraviolet microspectrophotometry (UMSP) ....................................................... 24
1.4.5 Fourier transform infrared (FTIR) spectroscopy ................................................... 25
1.4.6 X-ray photoelectron spectroscopy (XPS).............................................................. 26
1.5 Aim of the study ......................................................................................................... 27
2 Material and Methods ..................................................................................................... 28
2.1 Molecular identification of bamboo inhabiting fungi ................................................... 28
2.1.1 Collection of infected bamboo samples ................................................................ 28
2.1.2 Isolation and purification of fungi ........................................................................ 28
2.1.3 DNA extraction and amplification by polymerase chain reaction (PCR) ............... 29
2.1.4 PCR products examination and purification ......................................................... 29
2.1.5 Sequencing and sequence analysis ....................................................................... 29

II
 2.2 Identification of Penicillium and Aspergillus isolates from bamboo ............................ 30
2.2.1 Morphological identification of Penicillium and Aspergillus isolates .................... 31
2.2.2 Molecular identification of Penicillium and Aspergillus isolates ........................... 31
2.3 Blue-stain test ............................................................................................................. 32
2.3.1 Used blue-stain fungi ........................................................................................... 32
2.3.2 Investigated bamboo ............................................................................................ 32
2.3.3 Blue-stain test set-up ............................................................................................ 33
2.3.4 Assessment of hyphal growth and chlamydospores in bamboo ............................. 33
2.4 Degradation tests ........................................................................................................ 34
2.4.1 Preserving jar test................................................................................................. 34
2.4.1 Fungus cellar test ................................................................................................. 36
2.4.3 Kolle flask test ..................................................................................................... 37
2.4.4 Vermiculite test.................................................................................................... 40
2.4.5 Field test .............................................................................................................. 42
2.5 Chemical analysis of bamboo after degradation .......................................................... 43
2.5.1 Degraded bamboo samples ................................................................................... 43
2.5.2 Acetone and ethanol extraction ............................................................................ 43
2.5.3 Hydrolysis ........................................................................................................... 44
2.5.4 HPAEC-borate analysis ....................................................................................... 45
2.6 Transmission electron microscopy (TEM) studies of degraded bamboo ...................... 46
2.7 Ultraviolet microspectrophotometry (UMSP) studies of degraded bamboo.................. 46
2.7.1 Light microscopic analysis ................................................................................... 47
2.7.2 UV-absorbance spectra measurement ................................................................... 47
2.7.3 UV-scanning measurement .................................................................................. 48
3 Results and Discussion .................................................................................................... 49
3.1 Identification of bamboo inhabiting fungi ................................................................... 49
3.1.1 Moulds and Basidiomycetes on bamboo culms .................................................... 49
3.1.2 Identified fungi from bamboo by DNA sequencing .............................................. 51
3.2 Identification of some Penicillium and Aspergillus isolates ......................................... 54
3.2.1 Morphological characterization of Penicillium and Aspergillus isolates................ 54
3.2.2 Molecular identification of Penicillium and Aspergillus isolates ........................... 55

III
 3.3 Blue-stain test ............................................................................................................. 57
3.3.1 Discolouration of samples by blue-stain fungi ...................................................... 57
3.3.2 Microscopic characterises of blue-stain in bamboo ............................................... 58
3.3.3 Development of hyphae and chlamydospores ....................................................... 60
3.4 Degradation tests ........................................................................................................ 62
3.4.1 Preserving jar test................................................................................................. 62
3.4.2 Fungus cellar test ................................................................................................. 65
3.4.3 Kolle flask test ..................................................................................................... 66
3.4.4 Vermiculite test .................................................................................................... 72
3.4.5 Field test ............................................................................................................. 77
3.5 Chemical analysis of degraded bamboo ...................................................................... 78
3.6 Transmission electron microscopical (TEM) studies of degraded bamboo ................... 85
3.6.1 Early stages of decay ........................................................................................... 85
3.6.2 Medium stages of decay ....................................................................................... 86
3.6.3 Late stages of decay ............................................................................................. 87
3.7 Ultraviolet microspectrophotometry (UMSP) studies of degraded bamboo.................. 88
3.7.1 Light microscopy ................................................................................................. 88
3.7.2 UV-absorbance spectra of individual cell wall layers ........................................... 89
3.7.3 Scanning UV-microspectrophotometry ................................................................ 95
4 Conclusions .................................................................................................................... 103
5 References ...................................................................................................................... 106
List of publications ........................................................................................................... 117
Conference contributions ................................................................................................. 118
Appendices........................................................................................................................ 119
Publication I ................................................................................................................... 119
Publication II .................................................................................................................. 126
Publication III ................................................................................................................ 138
Publication IV ............................................................................................................... 145

IV
Abstract
As a fast growing and sustainable grass, bamboo is of increasing interest for construction and
industrial utilization. However, bamboo has a low natural durability and is easily attacked by
fungi during storage, transport, processing and final use. Little is known about the fungi
inhabiting and degrading the bamboo. Therefore, fungal identification and degradation studies
were performed with bamboo species.

Fungi were isolated from culms of bamboo species and identified by rDNA-ITS sequencing.
In total, 150 strains were isolated and 76 isolates were identified representing 16 genera and
37 species. Most isolates were Deuteromycetes/Ascomycetes (moulds, stain fungi). Two
Basidiomycetes, Schizophyllum commune and Cyathus stercoreus, were identified. Genera of
Aspergillus and Penicillium contain closely related species. Therefore, ß-tubulin and
calmodulin sequences were also used for identification.

Staining experiments were performed with pure cultures of blue-stain fungi in Petri dishes.
Colonization of vessels and parenchyma, observed by light microscopy after 4 and 20 weeks
incubation, occurred by thick, brown hyphae, chlamydospores and penetration of lignified cell
walls by transpressoria as it is also found in wood cells.

The degradation of bamboos by white-, brown- and soft-rot fungi was investigated in
preserving jars, Kolle flasks, vermiculite, and in the field.

In preserving jars, the mass loss (ML) was measured with small samples (3 × 1 cm) after 1, 3
and 12 months of incubation with Basidiomycetes for white-rot and brown-rot decay and with
Ascomycetes for soft-rot attack. Whereas the brown-rot fungi Coniophora puteana and
Gloeophyllum trabeum yielded less degradation, the white-rot fungus Trametes versicolor and
the soft-rot species Chaetomium globosum revealed considerable decay. ML was significantly
affected by the water content of bamboos: C. puteana was aggressive in dry samples (approx.
30 % moisture content), the white-rot species Schizophyllum commune produced most ML in
wet substrates (180 %u).

To imitate natural conditions, bigger samples (25 cm long) were used in the ―Fungus cellar-
test‖ where the bamboo is inoculated with pure cultures and incubated in large metal tubes
containing unsterile garden soil. In contrast to results with small samples in the jar test,
samples in the Fungus cellar were considerably more degraded by C. puteana (max. 43 % ML)
and S. commune (max. 20 %). Obviously, the moisture conditions influenced both fungi,
whereby S. commune decayed samples with soil contact and thus with high water content (90-
182 %u) more than samples without soil contact; in contrast, C. puteana produced more ML
in samples placed on wood supports and with lower water content (34-65 %u).

V
The durability of five bamboo species from various origins against brown-, white- and soft-rot
fungi was investigated in Kolle flasks in accordance to the European standards EN 350-1, EN
350-2 and EN 113. Considerable variability exists in the durability of the bamboos: Guadua
angustifolia was rather resistant to T. versicolor and Dendrocalamus asper against Ch.
globosum. Among the brown-rot fungi, four strains of C. puteana and two strains of G.
trabeum produced less ML (max. 2.9 %). Of the white-rot fungi, T. versicolor yielded the
highest ML (max. 15.3 %), whereas S. commune was rather inactive (max. 3.2 %). Of the
soft-rot fungi, Ch. globosum showed medium degradation (max. 9.6 %) and Paecilomyces
variotii low decay (max. 3.1 %).

Proposed is a method to investigate degradation of lignocelluloses by pure cultures of

basidiomycetes using preserving jars with vermiculite as reservoir for water and nutrients.
Samples of Gigantochloa atroviolacea and Phyllostachys pubescens and wood samples of
Fagus sylvatica and Pinus sylvestris were inoculated with C. puteana and S. commune. The
fungi were cultured on vermiculite containing different amounts of tap water or malt extract
solution. ML of the bamboos after 32 weeks was low and did not show a remarkable
influence of moisture content and nutrient addition. However, considerable degradation of P.
sylvestris sapwood occurred by C. puteana whereby moisture and nutrients influenced its
aggressiveness.

Bamboo stake test was conducted in the field. Fungal activity was visually assessed after 3.5
years. The opened stakes showed different stagess of degradation.

Chemical analyses were performed with hydrolysis and HPAEC-borate. Pleurotus ostreatus
and T. versicolor consumed the three cell wall components hemicellulose, cellulose and lignin.
The brown rot fungi C. puteana and G. trabeum were less aggressive. The soft-rot fungus Ch.
globosum consumed the carbohydrates, however with some decrease in lignin content, which
is characteristic of this rot type.

The micromorphology of Bambusa maculata degraded by the white-rot fungi Cyathus

stercoreus, P. ostreatus, T. versicolor and the soft-rot fungus Ch. globosum was studied by
transmission electron microscopy (TEM) and ultraviolet microspectrophotometry (UMSP).
TEM results showed morphological changes in the various cell wall layers. The UMSP area
scans and additional point analyses provided insights into the topochemistry of decay.

In summary, the results show that all bamboo species investigated can be colonized by the
various groups of fungi, moulds, staining and rot fungi. Considerable degradation occurs by
white- and soft-rot fungi.

VI
Zusammenfassung

Bambus ist als schnellwüchsiges, nachwachsendes Gras zunehmend interessant für

Konstruktionen und industrielle Nutzung. Wegen der geringen natürlichen Dauerhaftigkeit
wird Bambus jedoch während Lagerung, Transport, Verarbeitung und Nutzung leicht von
Pilzen angegriffen. Da über Besiedlung und Abbau wenig bekannt ist, erfolgten
Pilzidentifizierungen und Abbauversuche an mehreren Bambusarten.

Von Bambushalmen aus verschiedenen Ländern wurden insgesamt 150 Pilzstämme isoliert
und 76 durch rDNS-ITS-Sequenzierung identifiziert, die zu 16 Gattungen und 37 Arten
gehören. Meist handelte es sich um Deuteromyceten bzw. Ascomyceten (Schimmelpilze und
Bläue-Erreger) sowie um die Basidiomyceten Schizophyllum commune und Cyathus
stercoreus. Da die Gattungen Aspergillus und Penicillium nahe verwandte Arten enthalten,
wurden zusätzlich die ß-Tubulin- und Calmodulin-Sequenzen zur Identifizierung mittels
BLAST verwendet.

In Petrischalen erfolgten Verfärbungsversuche an Bambus mit Bläuepilz-Reinkulturen. Die

Lichtmikroskopie von Gefäßen und Parenchymzellen nach 4 und 20 Wochen Inkubation
zeigte die für Bläuepilze in Holzgewebe typischen dicken, braunen Hyphen und
Chlamydosporen sowie ihr Durchdringen lignifizierter Zellwände mit Transpressorien.

Der Abbau von Bambus wurde mit Weiß-, Braun- und Moderfäulepilzen in Weckgläsern,
Kolleschalen, auf Vermiculit und im Freiland untersucht.

In den Gläsern wurde der Masseverlust (MV) an kleinen Proben (3 x 1 cm) nach 1, 3 und 12
Monaten Kultivierung von Basidiomyceten hinsichtlich Weiß- und Braunfäule sowie
Ascomyceten auf Moderfäule gemessen. Während die Braunfäulepilze Coniophora puteana
und Gloeophyllum trabeum nur wenig Abbau bewirkten, ergaben der Weißfäulepilz Trametes
versicolor und der Moderfäuleerreger Chaetomium globosum beträchtliche Zersetzung. Die
Bambusfeuchte beeinflusste den MV deutlich: C. puteana war in trockenen Proben (etwa
30 % Wassergehalt) aktiver und der Weißfäulepilz Schizophyllum commune in nassem
Substrat (180 %u).

Zur Nachahmung natürlicher Bedingungen wurden größere Proben (25 cm lang) im

„Pilzkeller― auf unsteriler Gartenerde in großen Metallwannen mit Reinkulturen inkubiert. Im
Gegensatz zu den kleinen Abmessungen im Weckglasversuch zeigten hier C. puteana
(maximal 43 % MV) und S. commune (max. 20 %) deutlichen Abbau. Offensichtlich wurden
beide Pilze durch die Feuchtebedingungen beeinflusst: S. commune zersetzte Bambus mit
Erdbodenkontakt und daher höherem Wassergehalt (90-182 %u) stärker als ohne

VII
Bodenberührung, wogegen C. puteana mehr MV bei den auf Unterlagen befindlichen Proben
und somit geringerer Feuchte (34-65 %u) bewirkte.

An fünf Bambusarten verschiedener Herkunft wurde die Dauerhaftigkeit gegenüber Weiß-,

Braun- und Moderfäulepilzen in Kolleschalen gemäß der Europäischen Standards EN 350-1,
EN 350-2 und EN 113 ermittelt. Die Arten unterschieden sich beträchtlich: Guadua
angustifolia war recht dauerhaft gegenüber T. versicolor und Dendrocalamus asper gegen Ch.
globosum. Innerhalb der Braunfäuleerreger ergaben die vier Stämme von C. puteana und die
zwei Isolate von G. trabeum wenig Abbau (max. 2,9 % MV). Von den Weißfäulepilzen
bewirkte T. versicolor den größten MV (max. 15,3 %), während S. commune weniger aktiv
war (max. 3,2 %). Bei den Moderfäuleerregern zeigten Ch. globosum mittleren (max. 9,6 %)
und Paecilomyces variotii kaum Abbau (max. 3,1 %).

Zur Untersuchung des Lignocellulose-Abbaues mit Basidiomyceten-Reinkulturen wurde eine

neue Kultivierungsmethode mit Vermiculit als Reservoir für Wasser und Nährstoffe
vorgeschlagen. Proben von Gigantochloa atroviolacea und Phyllostachys pubescens sowie
Holzproben von Fagus sylvatica und Pinus sylvestris wurden mit C. puteana und S. commune
beimpft und auf Vermiculit mit verschiedenem Gehalt an Leitungswasser oder Malzextrakt-
Lösung inkubiert. Bei Bambus war der Abbau nach 32 Wochen zwar gering und ohne
erkennbaren Einfluss von Wasser- und Nährstoffzugabe, erheblich jedoch bei P. sylvestris-
Splintholz und C. puteana, dies in Abhängigkeit von Bambusfeuchte und Nährstoffen.

Die visuelle Auswertung eines Freilandversuches (stake test) nach 3,5 Jahren ergab
unterschiedliche Zersetzungsstadien sowie meist Moderfäule in der äußeren Bambusschicht
und Weißfäule im Inneren.

Laut chemischer Analysen nach Hydrolyse und HPAEC-Borat-Technik veratmeten Pleurotus

ostreatus und T. versicolor die Zellwandbestandteile Hemicellulose, Cellulose und Lignin,
C. puteana und G. trabeum waren kaum aktiv, Ch. globosum verbrauchte
Fäuletyp-charakteristisch besonders beide Kohlenhydrate und minderte den Ligningehalt.

Die Mikromorphologie wurde an Bambusa maculata nach Abbau durch Cyathus stercoreus,
P. ostreatus, T. versicolor und Ch. globosum mittels Transmissions-Elektronenmikroskopie
(TEM) und Ultraviolett-Mikrospektralphotometrie (UMSP) untersucht. Die TEM zeigte
morphologische Veränderungen der Zellwandschichten, der UMSP-Flächen-Scan und die
Punktmessungen ergaben Einblicke in die Topochemie.

Insgesamt haben die Ergebnisse gezeigt, dass alle untersuchten Bambusarten von den
verschiedenen Pilzgruppen, Schimmelpilze, Bläue-Erreger und Fäulnispilze, besiedelt und
durch Weiß- und Moderfäulepilze stark angegriffen werden.

VIII
List of abbreviations

BLAST Basic local alignment search tool

CLSM Confocal laser scanning microscopy
CML Compound middle lamella region
CREA Creatine agar
CYA Czapek yeast extract agar
FTIR Fourier transform infrared
HPAEC High performance anion exchange chromatography
HPLC High performance liquid chromatography
ITS Internal transcribed spacer
MC Moisture content
MEA Malt extract agar
ML Mass loss
ML-F Middle lamella of fibre
ML-P Middle lamella of parenchyma cell
PCR Polymerase chain reaction
RH Relative humidity
SEM Scanning electron microscopy
TEM Transmission electron microscope
UMSP Ultraviolet microspectrophotometry
UV Ultraviolet
XPS X-ray photoelectron spectroscopy
YES Yeast extract sucrose agar

IX
1 Introduction

1.1 Fungi

Approximately 120,000 fungal species have been described, and new species are added at the
rate of approximately 1,200 per year (Blackwell 2011, Kirk et al. 2008). A conservative
estimate of the total number of fungal species thought to exist is 1.5 million (Hawksworth
1991, 2001). Blackwell (2011) even estimated over 5 million fungal species.

If 1.5 million fungal species is a reasonable estimate, the vast majority of all extant fungi are
yet to be named. Assuming a relatively constant rate at which new species are described, it
will take more than 1,100 years to catalogue and describe all remaining fungi. However, many
of these fungi are likely to become extinct before they are ever discovered given current rates
of habitat and host loss. For example, up to 2% of the tropical forests are destroyed globally
each year (Purvis and Hector 2000). These habitats are exceedingly rich in fungal species
(Hawksworth and Rossman 1997). For example, 15-25 % of the fungi collected in short-term
studies in the tropics are new species (Kirk et al. 2008).

1.1.1 Characterization

The following descriptions are mainly based on Kendrick (1992), Alexopoulos et al. (1996),
Jennings and Lysek (1999) and Schmidt (2006). A fungus is a member of a large group of
eukaryotic organisms with cells devoid of chlorophyll that includes microorganisms such as
yeasts and moulds as well as the more familiar mushrooms. These organisms are classified as
a kingdom: Fungi, which is separate from plants, animals, protists and bacteria. Some
morphological, biochemical, and genetic features are shared with other organisms, while
others are unique to fungi, clearly separating them from the other kingdoms. One major
difference is that fungal cells have walls that contain chitin, unlike the cell walls of plants and
some protists, which contain cellulose, and unlike the cell walls of bacteria. These and other
differences show that fungi form a single group of related organisms, named the Eumycota
(true fungi or Eumycetes), that share a common ancestor. This fungal group is distinct from
the structurally similar Myxomycetes (slime moulds) and Oomycetes (water moulds).

1
Fungi mostly develop from very narrow, tubular, branching filamentous structure called
hyphae, which are the main mode of vegetative growth. A mass of hyphae collectively forms
a spreading network called mycelium. These filaments exude enzymes, and absorb food at
their growing hyphal tips. Moreover, hyphae are collectively very long so that they can
explore and exploit food substrates very efficiently. Reproduction of fungi, usually by means
of spores by which develop directly on substrates, are released by the hyphae or by a range of
unique and complex structures formed on the fungal mycelium that are called fruiting bodies.

Fungi play an essential role in the decomposition of organic matter and nutrient recycling.
They have long been used as a direct source of food, such as mushrooms and truffles, as a
leavening agent for bread, and in fermentation of various food products, such as wine, beer,
and soy sauce. Since the 1940s, fungi have been used for the production of antibiotics, and,
more recently, various enzymes produced by fungi are used industrially and in detergents.
Fungi are also used as biological antagonists to control weeds, plant diseases and insect pests.
Many species produce bioactive compounds called mycotoxins, such as alkaloids and
polyketides, which are toxic to animals including humans. The fruiting structures of a few
species contain psychotropic compounds and are consumed recreationally or in traditional
spiritual ceremonies. Fungi can break down manufactured materials and buildings, and
become significant pathogens of humans and other animals. Losses of crops due to fungal
diseases (e.g. rice blast disease) or food spoilage can have a large impact on human food
supplies and local economies.

1.1.2 Classification

In the classification of fungi, there have benn various attempts that focused on artificial and
natural systems. However, a generally recognized fungal classification system does not exist,
which was ironically argued that there might be as many systems as there are systematists.

The differences between fungi and other organisms regarded as plants had long been
recognized. Whittaker (1969) proposed a five-kingdom system and recognized fungi as an
additional kingdom. It has become a commom standard; some refinement of this standard is
still used in the literature and forms the basis for new multi-kingdom systems. He classified
the polyphyletic origin of the fungi into Protista and Fungi. Protista with about 2,000
described species, are grouped into sex divisions that include slim fungi and ‗lower fungi‘,

2
which are independent from each other. The ‗higher fungi‘, with about 120,000 described
species, group into four division: Zygomycota, Ascomycota, Basidiomycota and
Deuteromycota (Schmidt 2006).

Due to recent research technology based on DNA comparisons, the taxonomy of the fungi is
in a state of constant flux. The molecular revolution in fungal taxonomy commenced in the
early 1990s, with analyses of PCR-amplified ribosomal RNA genes (White et al. 1990). These
current phylogenetic analyses often overturn classifications based on older and sometimes less
discriminative methods based on morphological features and biological species concepts
obtained from experimental matings.

As the broad outlines of fungal phylogeny have come into focus, there have been repeated
attempts to summarize the state of knowledge and to restructure higher-level classifications.
Two important works that have influenced fungal taxonomy in the 21st century are Ainsworth
& Bisby‘s Dictionary of the Fungi (Kirk et al. 2001) and The Mycota VII (McLaughlin et al.
2001a, b). These publications represented major advances toward a phylogenetic
classification of Fungi, but they have become outdated.

There is consequently a pressing need for the fungal systematics community to adopt by
consensus, a higher-level classification for Fungi that is based on well-supported
monophyletic groups, and which can be recommended for general use. Hibbett et al. (2007)
presented a higher-level classification for all groups of Fungi according to recent molecular
phylogenetic studies. Currently, one kingdom, one subkingdom, seven phyla, ten subphyla, 35
classes, 12 subclasses, and 129 orders are accepted. The seven phyla include Microsporidia,
Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Glomeromycota, Ascomycota,
and Basidiomycota. The subkingdom Sikarya contained the Ascomycota and the
Basidiomycota.

As there is no unique accepted system at the higher taxonomic levels, there are frequent name
changes at every level. Researchers are encouraged to use a unified and more consistent
nomenclature. The Amsterdam Declaration on Fungal Nomenclature (Hawksworth et al. 2011)
was agreed at an international symposium convened in Amsterdam on 19-20 April 2011
under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The
declaration recognizes the need for an orderly transitition to a single-name nomenclatural

3
system for all fungi and to provide mechanisms to protect names that otherwise become
endangered. Websites such as Index Fungorum and ITIS list current names of fungal species,
with cross-references to older synonyms.

1.1.3 Identification

1.1.3.1 Traditional methods

Ever since the pioneering 18th and 19th century taxonomical works of Carl Linnaeus,
Christian Hendrik Persoon, and Elias Magnus Fries, fungi have been classified according to
their morphology (e.g., characteristics such as spore colour or microscopic features) or
physiology. The first records of fungi on bamboo are Dothidea goudotii from the leaves of
Chusquea sp. and Sphaeria bambusae from the culms of Bambusa arundinacea (Leveille
1845).

To identify the fungi, fruit bodies are preferentially used (Grosser 1985, Breitenbach and
Kränzlin 1986, Jahn 1990, Ryvarden and Gilbertson 1993, 1994, Bravery et al. 2003, Schmidt
2006, Huckfeldt and Schmidt 2006). However, fruiting bodies are not always present. Fruiting
body formation after isolation in laboratory culture is rare, e.g. in Antrodia vaillantii and
Schizophyllum commune. However, some tree parasites like Armillaria species form
rhizomorphs that can be used for identification. Several indoor species form mycelial strands
(cords) which allow identification by the classical strand diagnosis of Falck (1912) and the
extended version of Huckfeldt and Schmidt (2006). Even for experts, it may be difficult to
recognize closely related species. Morphological characteristics between two related species
are rather difficult to distinguish from each other. Furthermore, morphological characteristics
within a species are quite variable so that two individuals may not necessarily be recognized
as belonging to the same species.

Three Basidiomycetes with their different fruiting bodies and mycelia on malt extract agar
plates are shown in Fig. 1.1.

4
Fig. 1.1. Example of Basidiomycetes with characteristic fruiting bodies but similar mycelia.
Left: Pleurotus ostreatus; middle: Schizophyllum commune; right: Trametes versicolor.

1.1.3.2 Modern methods

Classical methods could be rapid if there are fruiting bodies or further characteristics. If only
vegetative mycelia are seen, (e.g., on living trees, on stored and fallen timber, in buildings or
in laboratory culture), keys and books for identification are available (Nobles 1965, Stalpers
1978, Lombard and Chamuris 1990). However, some genera among fungi can barely be
distinguished at the species level in culture, as is true for the indoor genera Antrodia,
Coniophora and Leucogyrophana. Thus, molecular methods, like protein electrophoresis,
isozyme analyses, immunological and DNA-based techniques, were established in the 1980s
(Schmidt 2006) and are used for identification.

Nowadays, the fungal ribosomal DNA (rDNA) most often used for fungal identification.
Particularly, specific DNA-fragments from the cluster of ribosomal RNA genes, the ITS
sequence between the 28S and 18S rDNA within a species are highly conserved but differ
even between closely related species. Sequencing these can allow an unambiguous
identification, irrespective of fungal classification. To amplify the internal transcribed spacer
(ITS) regions, Kim et al. (2011) identified fungi from bamboo isolates in Korea using ITS5

5
and ITS4 as primers for ascomycetes and ITS5 and ITS4B as primers for basidiomycetes.
ITS4 and ITS1 as primers were applied for identifying strain and mould fungi on bamboo in
China (Ma and Jiang 2009).

1.1.4 Important genera of bamboo inhabiting fungi

More than 1,100 species of fungi have been described or recorded from bamboo, which
comprise about 630 Ascomycetes, 150 Basidiomycetes and 330 mitosporic taxa (100
Coelomycetes, and 230 Hyphomycetes) (Hyde et al. 2002). Most species belong to the
Ascomycetes, Basidiomycetes or Deuteromycetes (moulds), but had been only identified by
classical methods, thus may contain false identifications.

In the following notes a description of common and relevant fungi is given which were
identified in this study by DNA techniques. For a more detailed account of the specific
species, see references provided in this section.

Aspergillus

Among the moulds, Aspergillus species are common contaminants on bamboo. They occur
more normally than Penicillium spp in subtropical and tropical areas. It is important to
identify aspergilli that many species are human pathogens (e.g. A. fumigatus, A. terreus) or
notorious mycotoxin producers (e.g. A. flavus, A. versicolor). Some species can produce
useful nature products that can be used into industry (e.g. A. niger).

To all Aspergillus species, aspergillum is still the name of an asexual spore-forming structure;
although one-third of species are known to have a sexual stage. The Colonies of Aspergillus
usually fast growing, coloured white, yellow, yellow-brown, brown to black or shades of
green, mostly consisting of a dense felt of erect conidiophores. Conidiophores are unbranched
with a vesicle. Phialides are grown directly on the vesicle (uniseriate) or on metulae
(biseriate). Conidia are one-celled, smooth or ornamented, and hyaline or pigmented, forming
chains which maybe compact columns (columnar) or diverging (radiate). Species may
produce `Hülle´ cells (large, thick- and smooth-walled cells) or sclerotia (firm, usually
globose, masses of hyphae). Teleomorphs consist of Eurotium, Emericella, Neosartorya and
other genera.

6
Morphological characteristics were mainly used in the identification of Aspergillus. The
genus is divided into 18 groups and 132 species are recongnized (Raper and Fennell 1965).
Up to now, more than 250 species with about 70 named teleomorphs are identified. Modern
molecular technology is also used in the identification (Samson and Varga 2007, Varga and
Samson 2008).

Cladosporium

The genus of Cladosporium is widely distributed in the world. Some species are pathogens or
are saprophytic and more or less host-specific on old or dead bamboo.

The colonies of Cladosporium are usually slow growing, coloured olive-green to blackish-
brown or black. Conidiophores are fragile, erect, straight or flexuous, unbranched or branched
only in the apical region, with geniculate sympodial elongation in some species. Conidia are
one- or 4-celled, ellipsoidal, fusiform, ovoid or (sub) globose, verrucose or echinulate, often
with distinct scars, pale to dark olivaceous-brown, smooth-walled, which are produced in
branched acropetal chains.

In the past decades, only a few indoor species are recognized and identified as C.
cladosporioides, C. sphaerospermum, C. herbarum and C. macrocarpum. The latest
phylogenetic studies demonstrated that many new species may be discovered from these taxa.
(Schubert et al. 2007, Zalar et al. 2007, Dugan et al. 2008).

Penicillium

The genus of Penicillium is composed of over 400 species. Many species are growth on
various substrates. Furthermore, some species are found on bamboo where they spoiled and
coloured the bamboo and bamboo products. Penicillium chrysogenum, P. brevicompactum
and P. citrinum are the most common ones.

The typical mycelium consists of hyphae, which is a highly branched network, septate, and
usually colourless. The conidiospores sprout on the mycelia, which are the main dispersal
organ of the fungi and usually green. Sexual reproduction produces ascospores.The asci
contain eight unicellular ascospores.

7
The morphological characteristics can be used for classification, but the isolates should be
culture under standard conditions. However, the phenotypic characteristics of some species
are very similar so that it is very difficulty to distinguish. The latest identification of
Penicillium is based on a polyphasic approach, using a combination of the morphology of the
conidiophores and conidia, colony characteristics on various media and temperatures and
molecular data (Frisvad and Samson 2004, Samson and Frisvad 2004). An electronic key for
the identification of Penicillium subgenus Penicillium species is provided at
http://www.cbs.knaw.nl/penicillium/, which is based on a database using phenotypical and
molecular (ß-tubulin sequences) characteristics. A database for identifying indoor penicillia is
also obtainable at http://www.cbs.knaw.nl/indoor/ (Adan and Samson 2011).

Schizophyllum

Within the basidiomycetes, Schizophyllum commune is a worldwide distributed fungus,

occurring on every continent except Antarctica. It causes white-rot decay of wood and also
bamboo, acting as a ubiquitous saprophyte and opportunistic pathogen. The basidioma has a
shell-shaped cap, with the tissue concentrated at the point of attachment, resembling a stem. It
is often wavy and lobed, with a rigid margin when old. It is tough, felty and hairy, and
slippery when moist. It is greyish white and up to 4 cm in diameter. The gills producing
basidiospores on their surface are pale reddish or grey, very narrow with a longitudinal split
edge, split when the fruiting body dries out and become inrolled when again wet, which is
unique among the fungi. It is found predominantly from autumn to spring on dead wood or
living plant tissue, in coniferous and deciduous forests. Fig. 1.2 shows fruiting bodies on a
bamboo culm in Nigeria.

Fig. 1.2. Fruiting bodies of Schizophyllum commune on bamboo (photo: W. Liese).

8
1.2 Bamboo

Bamboo is one of the most important non-timber forest products among the world's plant and
forest resources. It is a tribe of flowering perennial evergreen plants and belong to the
Monocotyledons, which are grouped in the kingdom Plantae, order Poales, family Poaceae,
and subfamily Bambusoideae. At present, three tribes are recognized, which are supported by
molecular phylogenetic results, and reflect the three main lineages of Bambusoideae
(Sungkaew et al. 2009): Arundinarieae (temperate woody bamboos, 28 genera, 533 species),
Bambuseae (tropical woody bamboos, 66 genera, 784 species) and Olyreae (herbaceous
bamboos, 21 genera, 122 species). Altogether, 1,439 species in 115 genera of bamboo are
being described and reported worldwide (Clark 2012), which are mainly distributed in the
tropical and sub-tropical zone and a few in the temperate and frigid zone. Bamboo usually
grows with tree species in mixed forests below the main canopy. According to their
geographical distribution, they are divided into three regions: the Asian-Pacific, the American
and the African region (Jiang 2007) (Fig. 1.3). In the 1920s, bamboo was introduced to
Europe, North America and Australia; thus, new cultivating bamboo regions appeared that are
different from their natural distribution regions.

Fig. 1.3. Geographic distribution of bamboo (from W. Liese).

In the past, bamboo did not receive much utilization, but since the start of the 20th century, it
quickly began to appear as a secondary bamboo forest in tropical and sub-tropical forests due
to its fast growth and strong propagation after the upper canopy species were cut. Because of
its diverse usage and the high ecological and economic value, bamboo was planted at a large

9
scale and the artificial bamboo forest was developed (Fig. 1.4). The secondary and artificial
bamboo forest is expanding by the strong underground rhizomes. The total forest area on this
land decreased year by year. According to the statistics in the past 50 years, the cover rate of
the world forest declined from 25 to 17%, the tropical forest cover is disappearing by 240,000
thousand km2 every year and 0.46 km2 every minute (Zhou 1999). On the contrary, the area of
bamboo forests increasingly expands, at a steady annual rate of 3% (Sun and Duan 2004).

Fig. 1.4. Bamboo (Phyllostachys pubescens) forest in China (photo: W. Liese).

1.2.1 Biology

1.2.1.1 Morphology

The main structure of the bamboos consists of three parts: a root system, a subterranean
rhizome system and the aerial culm system.

Bmboo growth originates from a unique rhizome system. There are two different growth
patterns (Fig. 1.5): sympodial species, in which the rhizome remains at the clump, such as
Dendrocalamus gigantea; and monopodial species (`running bamboos´), which spread with
their rhizome widely underground and send up new culms, such as Phyllostachys pubescens.

10
 Fig. 1.5. Sympodial (left) and monopodial (right) bamboo (from W. Liese).

Unlike trees, there is no secondary growth of the culm. The culm emerges from the rhizome at
its full diameter. The typical bamboo culms are composed of nodes and internodes. It is
mostly hollow and separated at the nodes. It grows to its full height in one growing season. Its
maturation takes 3 to 5 years according to different species. Due to lack of a cambium,
bamboo does not possess the ability to enlarge the diameter of the culm. In some species, the
culm can grows to several metres in height; in contrast, some species just grow to a few
centimetres high.

Most bamboo species flower infrequently, at intervals of 30 – 80 years, exhibiting `mass

flowering´with all plants of a particular species flowering worldwide over a period of a few
years. After flowering, they die (Fig. 1.6).

Fig. 1.6. Bamboo flowering.

Left: Fargesia murielae; right: Dendrocalamus strictus (photos: W. Liese).

11
1.2.2.2 Anatomy of culm

The culm is the most important part of bamboo and the main resource for utilization. Due to
rare flowering, classification of bamboo is mainly based on the culm structure. The physical
and mechanical properties of bamboo wood closely correlate to its anatomical structure. From
outer to inner on the cross section, the basic structure of a culm consists of an epidermis,
cortex, ground tissue and lacuna.

The epidermis is the outermost layer of the culm, containing elongated cells, a short cork and
silica cells, and the stomata. The outer cell wall usually is covered by tiny protuberances,
cutinized layer and wax coating. Size, shape, distribution and arranging of protuberances vary
in different bamboo species. Most of them diffuse in aggregates, and the others are reticulate,
grid-shape or undulating. Some species have unicellular hairs on the epidermis. The
characteristics of the culm surface can be considered for taxonomy.

Under the epidermis lies the hypodermis, which consists of 2-3 layers of thick-walled
sclerenchymatous cells. But the hypodermic cell of pachymorph bamboo has thin walls and is
undistinguishable from the cortex. The thickness of cell wall varies depending on its location.
Epidermis and hypodermis are usually called `bamboo green,́ because the cells contain
chlorophyll.

The cortex lies between hypodermis and ground tissue, which consists of several layers of
parenchyma cells.

The ground tissue consists of parenchyma cells embedding the vascular system. The
parenchyma tissue has two types of vertically positioned cells: the long and short parenchyma
cells. The long parenchyma cells, with thickened polylamellated and lignified wall, usually
have starch granules in them. The short parenchyma cells are scattered among the long
parenchyma cells and characterized by dense cytoplasm, thin walls and no lignification. On
average, the culm consists of about 52 % parenchyma, 40 % fibres and 8 % conducting tissue,
but these values vary with species. The cell types vary considerably within a culm, both
transversally across the culm wall and vertically along the culm. The conducting tissue
increases slightly from the outer part in the middle third of the culm wall. The percentage of

12
parenchyma in the inner third is considerably higher than in the outer third. Correspondingly,
the percentage of fibres decreases from the outside to the inside.

Vascular bundles are embedded in the parenchyma tissue. The phloem is on the outside of the
vascular bundle, and the xylem is on the inside. The vascular bundles of the bamboo
internodes are composed of two metaxylem vessels, phloem, protoxylem and attached fibre
sheaths. Some species have additional fibre bundles. The appearance of vascular bundles
across the culm varies continuously from the periphery towards the centre. The bundles in the
middle of the culm wall usually exhibit a maximum size and a characteristic form.

The classical work about vascular bundles types was performed by Grosser and Liese (1971)
analyzing 52 species in 14 genera. They investigated the variability of vascular bundles in
form and size and grouped them into four basic types (Fig. 1.7). In 1984, Wen et al. added the
―semiopen type‖. The five basic types have often been used in further studies.

Fig. 1.7. Basic types of vascular bundles.

Left: type 1 with fibre sheaths; middle: type 2 with enlarged fibre sheath with the phloem;
right: type 3 with two isolated fibre bundles (photos: W. Liese).

1.2.2.3 Chemical composition of the bamboo culm

The main constituents of the culm tissue are cellulose, hemicellulose and lignin; minor
constituents consist of various other soluble polysaccharides, protein materials, resins, tannins,

13
waxes and a small amount of ashes. The main chemical constituents of bamboo are not too
different from those of woody materials (Vena et al. 2013).

Cellulose is the fundamental material of the bamboo cell wall. Cellulose is a macromolecular
polysaccharide. Wood cellulose consists of up to 15,000 ß-1,4 linked glucose anhydride units.
The content of cellulose in bamboo of 40-60% is higher than that in hardwood. Vena et al.
(2013) summarized that for bamboo, glucan content was 40-53 %, whereas the glucan content
of softwoods and hardwoods is typically 40–52 % and 38-56 %, respectively. The content in
14 bamboo species located in the Yunnan Province in China was found to be 48.3 % on
average (Wang et al. 1999).

More than 90 % of bamboo hemicellulose is xylan. Hardwood xylan, like that of Fagus
sylvatica, consists of about 200 ß-1,4 linked xylose units with side chains of arabinose and 4-
O-methyl-D-glucuronic acid (Schmidt 2006). The main hemicelluloses of bamboo are 4-O-
acetyl-4-O-methyl-D-glucuronoxylans, which account for approximately 25 % of the cell wall
material. Bamboo xylan contains 6-7 % acetyl groups, whereas hardwood xylan has 8-17 %
(Vena et al. 2013). The chemical analyses in Chapter 3.5 also revealed that bamboo xylan is
mainly composed of xylose, arabinose, galactose and 4-O-methyl-D-glucuronic acid.

Lignin is a macromolecular three-dimensional polymer. Bamboo lignin belongs to the grasses

(monocotyledons) type of lignin consisting of three types of phenyl-propane units:
p-hydroxylphenyl (H), guaiacyl (G) and syringyl (S) units interconnected through
biosynthetic pathways in a molar ratio of 10:68:22 (Jiang 2007). This shows that bamboo
lignin is qualitatively, but not quantitatively, similar to hardwood lignin.

The distribution of chemical constituents of bamboo cell walls differs and varies in the same
bamboo with age and cell differentiation stages.

1.2.2 Significance

Growing attention of the global markets to bamboo commodities is a relatively new

phenomenon. Developing countries today are not interested in the development at any price.
They are increasingly interested in sustainable economic, social and environmental

14
development. Bamboo is an excellent resource for promoting sustainable growth,
international trade and South-South and South-North cooperation.

The recent development of bamboo is often called a "golden revolution," analogous to that of
the "green revolution," which resolved some world food security problems in the 1970-1980s.
The ongoing golden revolution may help continue to resolve global wood security problems
and reduce environmental pressure on forests. Growing international trade of bamboo
products is an indication of the growing importance of bamboo for the developing and the
developed nations.

1.2.3 Uses

Bamboo has high economic and cultural significance, being used as food source, construction
material, and for versatile products.

Bamboo has traditionally been used to make a wide range of everyday utensils, particularly in
Japan, where archaeological excavations have uncovered bamboo baskets dating to the late
Jomon period (2000-1000 BC). Bamboo has a long history of use in Asian furniture. For
example, Chinese bamboo furniture has a distinct style based on a millennia-long tradition.

Bamboo, like true wood, is a natural composite material with a high strength-to-weight ratio
useful for structures. In its natural form, bamboo as a construction material is traditionally
associated with the cultures of South Asia, East Asia and the South Pacific, to some extent in
Central and South America, and by extension in the aesthetic of the Tiki culture. Bamboo is
used for houses (Fig. 1.8) and structures (Fig. 1.9). In China and India, bamboo is used to
hold up simple suspension bridges, either by making cables of split bamboo or twisting whole
culms of sufficiently pliable bamboo together. Bamboo has also long been used as scaffolding;
the practice has been banned in China for buildings over six storeys, but is still in continuous
use for skyscrapers in Hong Kong. In the Philippines, the nipa hut is a fairly typical example
of the most basic sort of housing where bamboo is used; the walls are split and woven
bamboo, and bamboo slats and poles may be used as its support. In Japanese architecture,
bamboo is used primarily as a supplemental and/or decorative element in buildings such as
fencing, fountains, grates and gutters, largely due to the ready abundance of quality timber.

15
Fig. 1.8. Bamboo for houses in Ethiopia, Bali, China and Germany (from top left to bottom
right) (photos: W. Liese).

Fig. 1.9. Bamboo for structures (photos: W. Liese).

16
Bamboo based panels and floorings (Fig. 1.10) are made through a series of mechanical
and/or chemical processes, including addition of adhesives. They are pressed at a certain
temperature and pressure, and featured in large standard sizes, with good and stable physical
and mechanical properties: There is a big potential for bamboo based panels and floorings to
be used as engineering materials for decorative and/or structural purposes.

Fig. 1.10. Bamboo for furniture and flooring (photos: W. Liese).

Bamboo fibres have been used to make paper in China for centuries. A high-quality,
handmade paper is still produced in small quantities. Coarse bamboo paper is still used to
make spirit money in many Chinese communities.

Bamboo pulps are mainly produced in China, Myanmar, Thailand and India, and are used for
printing and writing papers. The most common bamboo species used for paper are

17
Dendrocalamus asper and Bamboo bluemanea. It is also possible to make dissolving pulp
from bamboo. The average fibre length is similar to hardwoods, but the properties of bamboo
pulp are closer to softwood pulps due to the broad fibre length distribution. With the help of
molecular tools, it is now possible to distinguish the superior fibre-yielding species/varieties
even at juvenile stagess of their growth, which can help in unadulterated merchandise
production.

The rare fruits and young bamboo culms (i.e., the shoots) that sprout from many bamboo
species are edible (Fig. 1.11). They are used in numerous Asian dishes and broths and are sold
in various processed shapes in fresh, dried, and canned versions.

Fig. 1.11. Bamboo fruits and shoots (photos: W. Liese).

Bamboo charcoal is a new environment-friendly biomass material, which is a black porous

solid product made from bamboo. In China and Japan, it is mainly used as fuel for cooking
and drying tea. Most bamboo charcoal for fuel is briquette charcoal, and the rest is raw
charcoal. Bamboo material has an extraordinary micro-structure: it has a high absorptive
capacity after carbonization and becomes even more effective after activation. Moreover,
bamboo charcoal can be used to purify water and eliminate organic impurities and odors.

18
Drinking water sterilized with chlorine can be treated with bamboo charcoal to remove
residual chlorine and chlorides.

Bamboo vinegar is a sort of brown-black liquid made by condensing vapour and gas when
bamboo has been carbonized. It contains more than 100 organic compounds such as saturated
and unsaturated acid, alcohol, methanol, aldehyde, ketone, phenol, etc., and among them
many components have a good capacity for sterilization and restraint. Its composition changes
with collection and storage method. Because of its natural abundance of organic nutrients,
organic bamboo vinegar is a method to pull out toxins from the body. Although acetic and
formic acids are often found in bamboo vinegar, they are relatively mild compounds that
display a variety of health benefits that especially aid in detoxification, sanitation and
improving circulation.

1.2.4 Damage by fungi and insects

Bamboo has low natural durability (Liese and Kumar 2003). As a biological material, bamboo
is in an endangering environment and susceptible to degradation by similar organisms which
attack wood.

1.2.4.1 Damage by fungi

Damage of bamboo by fungi is divided into three main types: mildew, stain and rot. Therefore,
the fungi contain three main types: mould fungi, stain fungi and rot fungi. In general, moulds
belong to Deuteromycetes, stain fungi belong to Deuteromycetes or Ascomycetes, rot fungi
belong to Basidiomycetes (Wang et al. 2000).

Freshly cut bamboo is susceptible to moulds and stain fungi due to the relative high content of
carbohydrates in the tissue and the high moisture content. When bamboo is dried, moulds and
stain fungi are inhibited. The occurrence of moulds and stain fungi differs according to
climate, geographical area, and species. Twenty-two genera, 56 species have been identified
from bamboo and bamboo products from 9 locations of China. The dominating fungal genera
were Trichoderma, Aspergillus, Penicillium, Fusarium, Alternaria and Botryodiplodia (Ma
and Jiang 2009). Fifteen species have been identified from moso bamboo in South China,

19
among which nine common fungi were Alternaria alternata, Aspergillus flavus, Penicillium
citrinum, P. chrysogenum, P. funiculosum, Chaetomium globosum, Fusarium pallidoroseum,
F. camptoceras and Mucor hiemalis (Wu and Wen 2000). 52 species have benn identified
from bamboo in Yunnan Province of China, which belong to 4 subphyla, 6 classes, 10 orders,
18 families and 31 genera (Wang et al. 2000). Twenty five moulds have been isolated and
identified and twenty of them are described from bamboo in the Hunan Province (Wu et al.
1994). Four moulds, Alternaria viticola, Aspergillus restrictus, Cladosporium herbarum,
Penicillium sp., have been reported from Phyllostachys pubescens and P. viridis in the
bamboo garden of Nanjing Forestry University, Jiangsu Province (Zhao et al. 1994).

Decay by rot fungi is a serious problem during storage and in use. The yield may be reduced
by 25% during one year of storage by decay fungi. Various white-rot and some brown-rot
fungi were found to attack bamboo stacks (Guha and Chandra 1979). Trametes versicolor and
the ascomycete Apiospora montagnei were the two most degrading fungi causing mass loss of
21.6 % and 17.9 %, respectively (Kim et al. 2011). The development of fungal colonization
and decay in the culms of Gigantochloa scortechinii during ground contact tests in a tropical
soil were reported by Razak et al. (2002). After 24 months of exposure, untreated and
ineffectively treated culms showed extensive decay. Fungi were observed by scanning
electron microscopy (SEM) in the cell lumina, in the degraded cell walls and in the
intercellular spaces. The morphology of decay was characteristic of white and soft-rot. In
contrast, the tissues of culms with effective preservative treatments showed restricted hyphal
colonization, infrequent hyphal invasion into cells via pits, or no cell wall degradation.

1.2.4.2 Damage by insects

Bamboo is susceptible to pest damage by beetles and termites, depending on the species and
starch content. Because these occur even in dried bamboo and in interior usage, preservative
treatment is necessary to ensure the durability. The bamboo felling season should be during
the period of low carbohydrate content. In India, for example, this content is higher in spring
than in winter, so it is recommended to cut between August and December to reduce the
attack by insects and staining (Kumar et al. 1994). Immersing bamboo in water effectively
prevents powder post beetle (Dinoderus minutus and D. brevis) infestation in cases of lower
starch contents (Sulthoni 1990). The effectiveness of 11 commercial insecticides was tested
by direct application to the beetles and by application to their food. Two environmentally

20
friendly insecticides, Cypermethrine and Permethrine, were the most toxic to the beetles
(Varma et al. 1988).

The durability of species like Bambusa pallida, B. balcooa, Dendrocalamus strictus and D.
stocksii against beetle attack was tested by the adult and larval inoculation technique. The
results showed that preventing attack is highly difficult as the larvae dwell inside the culms
with little exposure to the outside. In his context, fumigants like phosphines can be useful to
penetrate the culms and kill the existing infestations (Remadevi et al. 2012).

Termites are the most aggressive organisms to the bamboo culms or finished bamboo
products. As eusocial insects, termites live in colonies that number from several hundred to
several million individuals. They are capable of utilizing cellulose as a source of food.
Bamboo culms are rich in cellulose so they are easily attacked by termites causing severe
damage. After a rapid deterioration, only a thin outer layer of the culm remains (Liese and
Kumar 2003, Remadevi et al. 2005).

This vulnerability reduces the lifespan of bamboo products, causing a major hindrance to its
applicability. Like with wood, bamboo can be safeguarded against deterioration (Liese 1959a,
Liese and Kumar 2003) by protection and preservation practices during storage and use.
Bamboo for overseas transport can be treated with a borax and boric acid solution by a short-
term dipping or longer soaking period. An effective short-term protection against moulding,
especially in the sensitive first 2 months after culm harvest, is by dipping the culms in
solutions of 10% acetic acid (Tang et al. 2009) or 10% propionic acid (Tang et al. 2012).

1.3 Factors affecting fungal degradation tests

Several factors may influence bamboo degradation, for example site, growth rate, age, portion
of bamboo, extractives content and the microenvironment (Hamaguchi 1953, Liese 1959a,
1985; Abdurachim 1964; Mohanan 1997; Kim et al. 2011; Suprapti 2010; Ma et al. 2010;
Schmidt et al. 2011).

The behaviour of bamboo against decay fungi is an important parameter in bamboo

establishment and use. Most investigations revealed that bamboo degradation is due to white
and soft rot fungi, whereas brown-rot species were less aggressive. Among the investigated

21
fungi, Coniophora puteana and Schizophyllum commune varied with respect to the test
method used: Low maximum mass loss was measured on agar under pure culture condition,
whereas considerable degradation occurred in the `fungus cellar test´where the samples are
placed in large metal containers on unsterile garden soil with different moisture accessibility
(Schmidt et al. 2011). However, C. puteana and S. commune differed in decay activity: C.
puteana produced maximum mass loss at low moisture content of 57 %, whereas decay by S.
commune was highest at 182% moisture content (Schmidt et al. 2011).

Mixed microbial decay in soils with various water holding capacities was studied by Becker
and Kaune (1966). Kaune (1970) and Worrall and Wang (1991) used for wood a vermiculite
system with pure cultures of soft-rot fungi and proved its suitability for wood degradation
tests. Adaskaveg et al. (1991) showed decay of palm wood by basidiomycetes with the
vermiculite-block assay. Curling et al. (2002) exposed basidiomycetes to wood in a
vermiculite-soil system.

Bamboo degradation was also influenced by treatment methods of bamboo. Heat treatment
was performate by Leithoff (2011). Suhirman and Khusniati (1987) studied the durability of
bamboo by mudsubmission treatment.

When comparing data, another difficulty is due to the different tests performed, such as field
and laboratory tests. Also within the same test, the different standards (Asia, European and
American) may differ by sample size, duration of tests and fungal strains. Decay tests for
white-rot fungi on bamboo were performed in 12 weeks according to the American Wood
Preserves Association Standard E10-01, while the soft-rot tests was conducted for 20 weeks
using a modified vermiculite burial test (Kim et al. 2011).

1.4 Modern techniques to study the morphology of degraded bamboo

The knowledge on bamboo degradation by white-rot, brown-rot and soft-rot fungi has been
enlarged considerably in recent years (Cho et al. 2008, Kim et al. 2008, Xu et al. 2013). From
the current research technologies and published literature, six modern methods were used to
study fungal bamboo degradation. They are summarised in the following sections.

22
1.4.1 Scanning electron microscopy (SEM)

A scanning electron microscope (SEM) produces images by scanning the sample with a
focused beam of electrons. The various signals which include information with the sample's
surface topography and anatomy can be produced and detected when the electrons act with
atoms. In general, the electron beam is scanned in a raster pattern, and the image can be
produced when the beam's position is mixed with the detected signal. SEM can gain
resolution better than 1 nanometre. Specimens can be discovered in various conditions, such
as high vacuum, low vacuum, or (with the environmental SEM technique) wet factors.

Using SEM, it was found that attacks to parenchyma cells and places near the inner skin of
bamboo were the most frequent and the vessels were the primary paths for the spread of
mycelium in the bamboo (Xu et al. 2013).

1.4.2 Confocal laser scanning microscopy (CLSM)

Confocal laser scanning microscopy can afford high-resolution optical images with depth
selectivity (Pawley 2006). The ability to obtain in-focus images from selected depths is the
most important character of CLSM, which is known as optical sectioning. The images are
obtained point-by-point and reconstructed with computer so that three-dimensional
reconstructions of topologically complex objects can be observed. Marvin Minsky patednted
the principle of confocal microscopy in 1957, however, the developments of lasers for CLSM
become to standard technique untill the 1980s (Pawley 2006).

The parenchyma cells of Phyllostachys pubescens after degradation by white rot fungus
Lentinula edodes was observed by CLSM. The results showed that the preferential
degradation of the culm by L. edodes was mostly confined to the parenchyma cells at the early
stagess (Kim et al. 2008). Cho et al 2008 investigated P. pubescens degraded by the brown-rot
fungus Gloeophyllum trabeum and demonstrated that the narrow layers are resistant against
brown-rot attack and the main degradation occurred in the broad layers.

23
1.4.3 Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) is a method in which a beam of electrons is

transmitted through an ultra-thin specimen and interacted with the specimen as it passes
through. The image is produced by the interaction of the electrons transmitted through the
sample. Furthermore, the image is magnified and focused on an imaging device, such as a
fluorescent screen on a layer of photographic film, or by a sensor such as a CCD camera can
be discovered.

Due to the small de Broglie wavelength of electrons, the resolution of TEM is obviously
higher than light microscopes. Therefore, the user can determine fine details even with a
single column of atoms, which is thousands of times smaller than the smallest resolvable
object in a light microscope.

Phyllostachys pubescens degraded by the brown-rot fungus G. trabeum (Cho et al. 2008) and
by L. edodes (Kim et al. 2008) were examined by TEM, both results showed that the
compound middle lamellae in bamboo fibres were degraded at early stages of decay.

1.4.4 Ultraviolet microspectrophotometry (UMSP)

UMSP is a method to measure the relative lignin content semi quantitatively and to get
information on the lignin structural units. The technique is based on the ultraviolet
illumination of thin transverse sections. Lignin displays a characteristic ultraviolet absorbance
spectrum with absorbance maxima around 280 nm due to the presence of associated
phenylpropane groups with several chromophoric structural elements (Fergus et al. 1969,
Scott et al. 1969, Fergus and Goring 1970a, b, Sarkanen and Hergert 1971, Bauch et al. 1976,
Hesse et al. 1991). No other component of the mature cell wall displays ultraviolet absorbance
properties in the same spectral region, and therefore the intensity of absorbance may be
related to the concentration of lignin across the cell wall.

Koch and Kleist (2001) studied the S2 layer of fibre wall of Phyllostachys edulis. They
showed the lamellar structure with increasing lignin content from the lumen towards the
middle lamella and observed a typical UV spectrum for bamboo fibres with a guaiacyl peak at
280 nm and a shoulder between 310 - 315 nm, which can be linked to the presence of p-

24
coumaric acid esters. Furthermore, discrimination between the types of lignin is possible due
to different ratios of their guaiacyl- and syringylpropane units.

Kim et al. (2008) investigated the changes in lignin content of P. pubescens after degradation
by L. edodes. The lignin concentration in the bamboo fibres gradually increased from the
inner part (lumen side) to the outer part. The lowest absorbance in fibre walls was always
associated with the wall regions adjacent to the lumen, whereas high absorbance was recorded
in the regions close to the middle lamellae.

Phyllostachys pubescens degraded by the brown rot fungus G. trabeum showed that the
absorbance maxima of the compound middle lamella region (CML), including the cell corner
developed at 312 - 315 nm. The result underlines that the CML in bamboo is mainly
composed of p-hydroxyphenyl lignin (Cho et al 2008).

1.4.5 Fourier transform infrared (FTIR) spectroscopy

Fourier transform infrared (FTIR) spectroscopy combines microscopy and imaging techniques
for identification and localization. Generally, FTIR spectroscopy is employed to determine the
chemical composition of organic compounds. By combining FTIR spectroscopy with
microscopy, the chemical FTIR analysis gains local resolution. The development of focal
plane array (FPA) detectors and acceleration of measurement times from days to minutes have
made it possible to image the distribution of chemical compounds in biological materials
(Salzer et al. 2000). FPA detectors consist of an array of several thousand detector elements.
These detector elements simultaneously record spectra, which can be converted into images
displaying the spatial distribution of the chemical properties of a sample.

FTIR spectroscopy has successfully been used for identification of bacteria and also a few
fungi, both at genus and species level (Mariey et al. 2001, Naumann et al. 1991, Ngo-Thi et al.
2003, Naumann et al. 2005). In addition, the lignin distribution in cross sections of beech and
poplar was analysed and the spatial resolution compared using FTIR spectroscopy (Naumann
and Polle 2006).

The changes in lignin content of P. pubescens after degradation by L. edodes were estimated
by FTIR spectra. The absorption bands assigned to GS (1511 cm-1), S (1127 cm-1), SH (834

25
cm-1) and HGS (1166 cm-1) lignin units (Faix 1991) were significantly decreased. The
absorption band at 1650 cm-1, assigned to carbonyl groups (Faix et al. 1991), was also
significantly decreased. His finding can be interpreted that lignin was more degraded by L.
edodes than carbohydrates (Kim et al. 2008). In particular, the band at 1646 cm−1, which was
assigned to conjugated carbonyl groups originating from lignin, significantly decreased,
which suggested that not only H-unit lignin but guaiacyl-type or syringyl-type lignin in P.
pubescens were attacked by G. trabeum (Cho et al 2008). Significant changes in FTIR spectra
could be seen after decay by Phanerochaete chrysosporium (white-rot) and G. trabeum (Xu et
al. 2013). Therefore, FTIR can be applied to investigate changes in the chemical structure of
bamboo before and after decay by white-rot and brown-rot.

1.4.6 X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy has been successfully used for chemical analysis of various
materials. Using XPS, a surface layer is analyzed, and the elemental composition of the
samples is examined. XPS can also disclose information about the chemical bonding
environment of the elements. Furthermore, the percentage of certain chemical components on
a surface can be determined.

In pulp and paper research, XPS demonstrates the energy distribution of photoelectrons
ejected from the inner shells of atoms by a soft X-ray, which is a measurable investigation
technology to study the surface chemistry of complex organic materials (Johansson and
Cambell 2004, Li and Reeve 2004, Fardim et al. 2005). XPS has been used for the study of
the surface of red oak and red pine extracted wood (Nzokou and Kamdem 2005), the surface
of different wood species (Sinn et al. 2001) and wood chemical composition after heat-
treatment (Inari 2006). The changes of rice straw lignin and cellulose after decay by white-rot
and brown-rot fungi were studied by XPS (Dey et al. 1992). The result demonstrated that the
XPS data could be related with the loss of lignin and polysaccharides of straw, which is
degraded by fungi and determined by chemical analyses.

Xu et al. (2013) investigated Phyllostachys edulis decayed by P. chrysosporium and G.

trabeum using XPS spectroscopy and verified that this method can be applied to study the
changes in the chemical structure of bamboo before and after decay by rot fungi.

26
1.5 Aim of the study

Bamboo is a fast growing woody grass of increasing interest for the sustainable production of
materials with many applications for buildings and industrial utilization. In general, bamboo
has generally a low natural durability and is easily attacked by fungi during storage, transport
and final use. However, little is known about the fungi inhabiting and degrading the bamboo.
Furthermore, for applications it is important to know which fungi might cause harm to
potential products.

Like other lignocellulose materials, bamboos are subject to biodegradation by fungi under
particular conditions which affect their quality (Hamid et al. 2003). The resistance of bamboo
against decay fungi serves as an important parameter in bamboo utilization. Several factors
may influence the natural durability of bamboo, such as growth site, growth rate, age, portion
of bamboo, extractives content and the microenvironment.

When comparing data, another difficulty is due to the different tests performed, such as field
and laboratory tests. Also within the same test, the different standards (Asia, European and
American) may differ by sample size, duration of tests and fungal strains. Therefore, it is
important to test the bamboo species using the same standards.

The overall objective of this study is to identify bamboo-inhabiting fungi and evaluate their
damage to the substrates. The study consists of the following specific objectives:
(1) to isolate and identify some bamboo-inhabiting fungi by molecular methods,
(2) to test bamboo degradation by different methods,
(3) to analyze chemical changes of bamboo tissue after degradation,
(4) to study micromorphological cell wall characteristics after degradation.

The results obtained in this work, together with other data reported in literature on the decay
of bamboo (Liese 1959a, 1985, Banerjee and Mukhopathya 1962, Purushotham 1963.
Abdurachim 1964, Wang and Hsieh 1968, Murphy et al. 1991, Leithoff and Peek 2001, Razak
et al. 2002, 2006, Hamid et al. 2003, Zhang et al. 2007, Kim et al. 2008, 2011, Suprapti 2010,
Ma et al. 2010, Schmidt et al. 2011, Wei et al. 2012) may contribute to the characterization
and appreciation for bamboo and bamboo products. Such characterization is important not
only for its correct utilization but also for improved market promotion.

27
2 Material and Methods

2.1 Molecular identification of bamboo inhabiting fungi

Bamboo is easily attacked by fungi during storage, transport, processing and final use. Little
is known about the fungi inhabiting and degrading it. Therefore, those fungi were isolated.
Fungi were identified by molecular techniques because the classical methods may fail to
identify morphologically similar organisms.

2.1.1 Collection of infected bamboo samples

The fungus-infected samples were obtained with the help of Prof. Dr. Walter Liese from his
worldwide colleagues and derived from eight countries, Ethiopia, China, Costa Rica,
Germany, Indonesia, Philippines, Thailand and Vietnam. The samples were mostly gained
from culms for practical usage, so that the bamboo species is only known in a few cases, such
as Bamboo multiplex from Thailand, Dendrocalamus asper from Indonesia and Thailand, D.
brandisii from Costa Rica and Phyllostachys glaucoviridescens from Germany. Fig. 2.1
shows moulded bamboo after sea transport to Germany.

Fig. 2.1. Moulded bamboo after ship transport from Asia (right photo: T.K.H. Tang).

2.1.2 Isolation and purification of fungi

The fungi were isolated from the culm surface by streaking spores or hyphae with an
inoculation needle and were cultivated on the 2 % malt extract agar (MEA, Oxoid) at room
temperature for approx. 5 days and then subcultured on agar plates till pure cultures.

28
2.1.3 DNA extraction and amplification by polymerase chain reaction (PCR)

DNA was extracted from the mycelia with the DNeasy Plant Mini Kit (Qiagen, Hilden,
Germany). The polymerase chain reaction (PCR) was performed with the Qiagen Tag Core
Kit in the PTC-100 thermocycler (MJ Research, Watertown, MA, USA). To amplify the
rDNA-ITS region, the standard primers ITS1 and ITS 4 were used (White et al. 1990).

Sequences of used primers are:

ITS1: 5‘-TCCGTAGGTGAACCTGCGG-3‘ (forward)
ITS4: 5‘-TCCTCCGCTTATTGATATGC-3‘ (reverse)

The PCR protocol was as follows: initial denaturing at 98 °C for 4 min, followed by 35 cycles
of 94 °C denaturing, 55 °C annealing and 72 °C extension, each for 1 min. The final extension
was at 72 °C for 7 min. The PCR products were stored at 8 °C (Schmidt et al. 2002).

2.1.4 PCR products examination and purification

The PCR products were examined prior to sequencing using electrophoresis on 2 % agarose
gels (DNA agarose, Biozym, Hess. Oldendorf, Germany) in 0.5 TBE buffer for 30 min with
the Mupid-ex U system (Advance, Tokyo, Japan). Gels were stained with GelStar Nucleic
Acid Gel Stain (Cambrex, Rockland, ME, USA) and examined using UV transillumination.

PCR products assumed to be suitable for sequencing were purified using the QIAquick PCR
Purification Kit (Qiagen).

2.1.5 Sequencing and sequence analysis

Sequencing of the purified PCR products was performed for both DNA strands by Eurofins
MWG Operon (Ebersberg, Germany). The complete rDNA-ITS sequence was obtained by
overlapping both strands. Once a sequence has been obtained for a sample in question, it can
be identified by comparison with sequences deposited in the international databases. The
NCBI Genbank (http://www.cnbi.nlm.nih.gov/) is one of a public database which is used
worldwide. To find the most identical sequences, the Basic Local Alignment Search Tool
(BLAST) was used identification by comparison.

29
The whole procedure is demonstrated in Fig. 2.2.

Sample DNA
collection extraction DNeasy Plant Kit

PCR

Purification

Sequencing Eurofins MVG Operon

Sequence
analysis BLAST

Fig. 2.2. Procedure for molecular identification of bamboo inhabiting fungi.

2.2 Identification of Penicillium and Aspergillus isolates from bamboo

Aspergillus and Penicillium are the two most important mould genera which inhabit harvested
bamboo culms. However, both genera contain very closely related species of which some also
have various synonyms. This may lead to uncertain results when only the rDNA-ITS
sequences are used for identification. Furthermore, the international DNA databases are man-
made and contain mistakes, such as misidentifications. The following experiments were done
in the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Institute of
the Royal Netherlands Academy of Arts and Sciences.

30
2.2.1 Morphological identification of Penicillium and Aspergillus isolates

Classically, Penicillium and Aspergillus are identified by morphological characters.

Penicillium and Aspergillus isolates obtained from bamboos were inoculated in a three point
position on Czapek yeast extract agar (CYA), yeast extract sucrose agar (YES), creatine agar
(CREA) and malt extract agar (MEA, Oxoid). All media were prepared according to Samson
et al. (2010). Colony characteristics were recorded after 7 d of incubation at 25 or 37 °C.

2.2.2 Molecular identification of Penicillium and Aspergillus isolates

The rDNA-ITS-sequence within a species is highly conserved but differs even between
closely related species. Sequencing these can allow an unambiguous identification,
respectively classification of a fungus. Aspergillus and Penicillium contain very closely
related species of which some also have various synonyms. This may lead to uncertain results
when only the ITS sequence is used for identification. Therefore, ß-tubulin and calmodulin
sequences were obtained for these fungi and also used for BLAST identification. The DNA
regions were amplified with the following PCR primers.

ITS (see 2.1.3),

β-tubulin:
T10: 5‘-ACGATAGGTTCACCTCCAGAC-3‘ (forward),
BT2b: 5‘-ACCCTCAGTGTAGTGACCCTTGGC-3‘ (reverse),

calmodulin:
CMD5: 5‘-CCGAGTACAAGGARGCCTTC-3‘ (forward),
CMD6: 5‘-CCGATRGAGGTCATRACGTGG-3‘ (reverse).

31
2.3 Blue-stain test

2.3.1 Used blue-stain fungi

The 11 strains of blue-stain fungi used are listed in Table 2.1, of which 9 strains are our own
isolates from bamboo and 2 derive from the institute collection.

Table 2.1. Used blue-stain fungi

Species Laboratory coding Origin
Alternaria alternata D84-2 Bamboo
Alternaria tenuissima D82-3-2 "
Aureobasidium pullulans 87 Institute
Botryosphaeria subglobosa D26 Bamboo
Cladosporium cladosporioides D16-2 "
C. cladosporioides D81-2 "
Epicoccum nigrum D20 "
Hormonema dematioides 85 Institute
Pestalotiopsis microspora D24 Bamboo
Phoma macrostoma D64-1 "
Unidentified isolate 1.5 "

2.3.2 Investigated bamboo

Investigations were conducted on culm sections of the bamboo species Bambusa maculata,
Gigantochloa atroviolacea and Phyllostachys pubescens. Bambusa maculata and G.
atroviolacea originating from Indonesia were obtained from CONBAM (Geilenkirchen,
Germany) and P. pubescens originating from Germany were obtained from the Bamboo
Centre (Baden Baden, Germany). The curcuma-test (Peylo 2001) was applied to ensure that
the samples were not contaminated with boron compounds which are commonly used for
preventing mould during storage and transport. The bamboo specimens were cut into samples
of 3×1×thickness cm3.

32
2.3.3 Blue-stain test set-up

The experiment was performed with pure cultures of blue-stain fungi (Table 2.1) in Petri
dishes (9 cm diameter). The culture medium consisted of 2 % malt extract (Merck) and 1.5 %
agar (Bacto) in distilled water. The Petri dishes were inoculated with fresh mycelium-agar
plugs (8 mm2) from pre-cultures and incubated at room temperature for 14 days. When the
agar surfaces were completely covered by mycelium, three bamboo samples sterilized in the
autoclave at 121 °C for 30 min were placed on the mycelium in each dish. Three dishes were
used for each strain. After 4 and 20 weeks of incubation at 20±2 °C and 65±5 % relative
humidity (RH) in a climate room, harvests were made and the mycelium was removed from
the bamboo sample surface. Samples were then cut into small specimens and put into PEG
2000 (polyethylene glycol 2000) (20 ml PEG plus 60 ml distilled water) for infiltration. After
1 week water evaporation period, specimens were cut into 20 µm sections with a microtome.
Since bamboo samples easily break during microtoming, sections were placed on adhesive
plaster. The cut sections were embedded in glycerol, examined with a light microscope
Olympus BX51 and photographed.

2.3.4 Assessment of hyphal growth and chlamydospores in bamboo

The development of hyphal growth and chlamydospores on/in the sample was assessed
according to the scheme in Table 2.2, which values the percentage of the tissue covered with
mycelium and the density of clamydospores in vessels and parenchyma cells.

Table 2.2. Evaluation scheme hyphal growth and chlamydospores on bamboo

Coverage Density of
Description Description
of tissue chlamydospores
0 No 0 No
1 1 - 10 % 1 1 - 10 %
2 11 - 25 % 2 11 - 25 %
3 26 – 50 % 3 26 – 50 %
4 > 50 % 4 > 50 %

33
2.4 Degradation tests

2.4.1 Preserving jar test

2.4.1.1 Used fungi

Eight strains are used, which belong to brown-rot (2 strains), white-rot (4 strains) and soft-rot
(2 strains) fungi. Details are listed in Table 2.3.

Table 2.3. Used fungi

Rot type Species Laboratory Other codings/
coding use for EN
Brown Coniophora puteana 167
Gloeophyllum trabeum 183 Ebw. 109, EN 113
White Pleurotus ostreatus 11 ATCC 44737
Trametes versicolor 63 CTB 863A, EN 113
Schizophyllum commune 87
S. commune 98
Soft Chaetomium globosum 10 ATCC 44753
Paecilomyces variotii 13 ATCC 44741

2.4.1.2 Investigated bamboo and wood samples

Investigations were conducted on culm sections of Bambusa maculata and Gigantochloa

atroviolacea from Indonesia and Phyllostachys pubescens from Germany. The curcuma-test
(Peylo 2001) was applied to ensure that the samples were not contaminated with boron.
Samples (31 thickness cm3) were dried at 103 °C, weighed, and autoclaved. Wood samples
(52.51.5 cm3) from Fagus sylvatica L. and Pinus sylvestris L. were used as controls
according to EN 113.

34
2.4.1.3 Preserving jar test set-up

Household preserving jars with a volume of 500 ml were autoclaved for 20 min and used as
culture vessels (Schmidt 1986). Culture medium for the Basidiomycetes consisted of 110 ml
of 2 % malt extract and 1.5 %. For the Ascomycetes, Abrams agar (Savory 1954) was used,
supplemented with 0.1 % yeast extract as a vitamin source. The jars were inoculated with
fresh mycelial agar plugs (8 mm2) and incubated at 21 °C and 70 % RH for 1, 3, and 12
months, respectively. For each harvest time, two parallels were used to consider possible
contaminations during the long cultivation time (Fig. 2.3).

Fig. 2.3. Preserving jar test set-up.

Left: Preserving jars in the incubation room; right: samples with mycelium of Trametes
versicolor after 1 week incubation.

2.4.1.4 Assessment of degradation in preserving jars

Evaluation of mass loss of bamboo and control wood samples decayed by fungi was done
after 1, 3 and 12 months of incubation. At harvest, the mycelium was removed from the
sample surface; the samples were weighed for final wet weight and oven dried at 103 °C for 3
days. The mass loss (ML) due to fungal decay was determined according to the equation:

M1: dry weight before fungal degradation

M2: dry weight after fungal degradation.

The standard deviation for each 4 specimens per treatment group was calculated according to
ANOVA, SPSS software 2002.

35
2.4.2 Fungus cellar test

2.4.2.1 Used fungi

Two species were used: the brown-rot fungus Coniophora puteana 167 from the laboratory
collection and the white-rot fungus Schizophyllum commune 87 from bamboo (own isolation).

2.4.2.2 Investigated bamboo

The seven bamboo species tested are listed in Table 2.4. Culm sections were obtained from
CONBAM and the Bamboo Centre, Baden-Baden. Sections were proved with the curcuma-
test for boron (Peylo 2001). Sections (25 cm) were longitudinally halved, dried at 103 °C,
weighed, dipped in tap water for two days and autoclaved at 121 °C for 40 min.

Table 2.4. Investigated bamboo

Bamboo species Local name Culm diameter (cm) Origin
Arundinaria amabilis Tonkin 3.5-4.0 Vietnam
Bambusa maculata Tutul 4.5-5.5 Indonesia
Dendrocalamus asper Petung 13.5-14.0 Indonesia
Gigantochloa atroviolacea Wulnung 5.5-7.0 Indonesia
Phyllostachys nigra Nigra 2.0-2.5 Japan
P. nigra ‗Boryana‘ Boryana 3.0-4.0 China
Phyllostachys pubescens 5.5-6.5 Germany

2.4.2.3 Fungus cellar test set-up

Metal tubs (120 cm long, 60 cm wide) were filled with 30 L unsterile compost soil from the
institute garden (Gersonde and Becker 1958). Bamboo samples were placed either on
autoclaved wood supports or directly on the soil (Fig. 2.4). Each sample was infected with
three small inoculation wood pieces (0.5 cm3) containing mycelium. The tubs were covered
with panes of glass and kept at 23 °C and 90 % RH for 1 year. The soil was moistened weekly
with sprayed tap water. The development of fungi on the samples was observed by visual
inspection. After harvest, mass loss and final moisture content of samples were measured and
evaluated.

36
Fig. 2.4. Fungus cellar test set-up.
Left: samples in the Fungus cellar; right: samples with mycelium of Schizophyllum commune
after 1 week of incubation.

2.4.3 Kolle flask test

2.4.3.1 Used fungi

The 10 strains of white-, brown- and soft-rot fungi used derived from the laboratory strain
collection and are listed in Table 2.5.

Table 2.5. Used fungi

Laboratory Other codings/
Rot type Species
Coding use for EN
Brown Coniophora puteana 1 Ebw. 15/ EN 113
C. puteana 159
C. puteana 167
C. puteana 247
Gloeophyllum trabeum 183 Ebw. 109/ EN 113
G. trabeum 259
White Trametes versicolor 63 CTB 863/ EN 113
Schizophyllum commune 87
Soft Chaetomium globosum 10 ATCC 44753
Paecilomyces variotii 13 ATCC 44741

37
2.4.3.2 Investigated bamboo and wood samples

The five bamboo species tested are presented in Table 2.6. Culm sections from CONBAM
and the Bamboo Centre were previously proven for boron. Samples were prepared by cutting
to dimensions of 5 cm (length) × 2.5 cm (width) × 0.5-3.5 cm (wall thickness). Wood samples
from F. sylvatica and P. sylvestris were used as controls according to EN 113. All samples
were put into a climate room at 20±2 °C and 65±5 % RH for 4 weeks, weighed, wrapped in
plastic (Sengewald Flexopeel, Germany) and sent to the BBF sterilization service (Kernen,
Germany) for gamma radiation.

Table 2.6. Investigated bamboo

Bamboo species Local name Origin
Bambusa maculata Tutul Indonesia
Dendrocalamus asper Petung Indonesia
Gigantochloa atroviolacea Wulung Indonesia
Guadua angustifolia Guadua Columbia
Phyllostachys pubescens Moso China
P. pubescens Germany

2.4.3.3 Durability test

The durability experiment was performed with fungal pure cultures in Kolle flasks with
white-, brown-, and soft-rot fungi according to the European standards EN 350-1 (1996), EN
350-2 (1997) and EN 113 (1996). The culture medium consisted of 2 % malt extract (Merck)
and 1.5 % agar (Bacto) in distilled water. Flasks containing 48 ml medium were plugged with
cotton, sterilized in the autoclave at 121 °C for 30 min, inoculated with a mycelial plug of the
test fungus and incubated until mycelial growth covered the surface of the medium (Fig. 2.5).
Each two gamma ray sterilized samples were put aseptically in one flask and each three flasks
were used for one fungal strain, with altogether 480 Kolle flasks. After 16 weeks of
incubation at 20±2 °C and 65±5 % RH for the brown-rot and white-rot fungi and 28±2 °C and
65±5 % RH for the soft-rot fungi, mycelia were removed from the sample surface, the
samples were weighed for final wet weight determination and oven dried at 103 °C for mass
loss (ML) determination. Percentage of ML was calculated by weight comparison before and

38
after incubation. Statistical differences were analyzed using variance analysis (ANOVA,
SPSS software 2002).

Fig. 2.5 shows some cultures with P. pubescens in Kolle flasks.

（a） （b）

（c） （d）

Fig. 2.5. Kolle flask test set-up.

(a): Coniophora puteana; (b): Trametes versicolor; (c): Chaetomium globosum; (d): control.

2.4.3.4 Assessment of fungal growth and classification of bamboo durability

The development of fungal growth on the specimens was valued by the density and the
percentage of the sample surface covered with mycelium. Based on the average mass loss,
bamboo durability against fungi was correlated to durability classes considering EN 350-1
(1996), Abdurachim (1975) and Djarwanto and Suprapti (2004).

39
2.4.4 Vermiculite test

2.4.4.1 Used fungi

The brown-rot fungus Coniophora puteana strain 167 from the laboratory collection and the
white-rot fungus Schizophyllum commune strain 87 from bamboo were held on agar plates of
2 % malt extract (Oxoid) and 1.5 % agar (Oxoid).

2.4.4.2 Investigated bamboo and wood samples

Phyllostachys pubescens (diameter 5.5-6.5 cm) and Gigantochloa atroviolacea (diameter 5.5-
7.0 cm) from CONBAM and the Bamboo Centre proven for boron were used. Culm sections
were cut to 5 cm length, 2.5 cm width and 0.5–3.5 cm wall thickness. Wood samples of F.
sylvatica and P. sylvestris were cut to 5 cm length, 2.5 cm width and 1.5 cm height. Samples
were put into a climate room at 20±2 °C and 65±5 % RH for 4 weeks, weighed, wrapped in
plastic (Sengewald Flexopeel) and send for gamma radiation to the BBF sterilization service.
To calculate the initial weight, moisture content at 20 °C and 65 % was adjusted downward.

2.4.4.3 Vermiculite test set-up

The decay experiment was performed in 500 ml preserving jars (Fig. 2.6). Vermiculite of 2-3
mm particle size was obtained from the Vermiculite-shop (thinex new media, Dortmund,
Germany, www.vermiculit-shop.de) and sieved through a 2 mm sieve. Each preserving jar
was filled with 200 ml vermiculite. Tap water or 2% malt extract solution were added in 80,
100, 120, 140 and 160 ml amounts per jar. The jars covered with glass lids were sterilized in
the autoclave at 121 °C and 2.1 bar for 30 min. Each two bamboo or wood samples were put
aseptically in one jar. Two jars were used for one fungus. Vermiculite and sample were
separated by a metal ring to avoid liquid diffusion in the sample. A plug of the test fungus
from agar plates was then placed on the sample top. After 32 weeks of incubation at 20±2 °C
and 65±5% RH, the mycelium was removed from the sample surface, the samples were
weighed for final wet weight and oven dried at 103 °C for 3 days for ML determination. ML
loss was calculated by weight comparison before and after incubation. Standard deviation for
each 4 specimens per treatment group was calculated according to ANOVA, SPSS software
2002.

40
Fig. 2.6. Vermiculite test set-up.
Left: vermiculite jars in the incubation room; right: preserving jar with vermiculite and
bamboo samples.

2.4.4.4 Assessment of fungal growth and vitality test after incubation

The development of fungal growth on the samples was assessed according to the scheme in
Table 2.7, which values hyphal density and percentage of sample surface covered with
mycelium. After 32 weeks of incubation, small portions of overgrown vermiculite were
transferred from the jars to malt agar plates to prove the vitality of the mycelium after the
vermiculite test.

Table 2.7. Evaluation scheme for hyphal growth on bamboo and wood samples
Hyphal density Description Sample coverage Description
0 no growth 0 no coverage
1 sparse mycelium 1 1 – 33 % coverage
2 normal mycelium 2 34 – 66 % coverage
3 thick mycelium 3 67 – 99 % coverage
4 total coverage

41
2.4.5 Field test

2.4.5.1 Investigated bamboo

A small field test was done with culm sections of 4 bamboo collectives (Table 2.8) obtained
from Rudolf Schachtrupp, Hamburg, Germany. Sections were proven for boron.

Table 2.8. Investigated bamboo

Bamboo Geographical origin Culm diameter Length after sawing
(cm) (cm)
Bambusa vulgaris India 6.0 - 6.5 20
unknown species 3 China 5.5 – 6.0 30
unknown species 5 China 2.0 - 3.5 20
unknown species 10 Thailand 3.0 - 3.5 28

2.4.5.2 Field test set-up

The bamboo-stake specimens were exposed to soil near the greenhouse of the Thünen-
Institute (vTI) in Hamburg-Lohbrügge, in March 2009. The specimens were put by 1/2 of
their length into the soil (Fig. 2.7).

Fig. 2.7. Field test set-up.

42
2.4.5.3 Assessment of stake test

After 3.5 years incubation in field the bamboo samples were harvested, the soil and moulds
removed from the sample and cut into two parts: in ground and above ground. Each part was
cut into three sections: top, middle and bottom. To assess the natural durability in the field,
the decay of the different sections were classified visually into 5 classes according to the
rating scheme of Papadopoulos (2010). The classification is shown in Table 2.9.

Table 2.9. Evaluation scheme for stakes in the field

Rating Description
0 total decay
1 severe decay
2 moderate decay
3 slight decay
4 no decay

2.5 Chemical analysis of bamboo after degradation

2.5.1 Degraded bamboo samples

Degraded bamboo samples from the preserving jar test (Chapter 2.4.1) of the 12 months
incubation were used. The samples were splintered and ground in a mill (MF 20 Basic, IKA
Werke GmbH, Germany), producing particles passing a 10-mesh screen.

2.5.2 Acetone and ethanol extraction

The aim of extraction is to remove extractives, including possibly sugar monomers and
dimers, and phenolic glycosides. After measuring the moisture content of samples, the
extractions were conducted with an accelerated solvent extractor (ASE 200, Dionex) (Fig.
2.8). About 1 g of milled degraded bamboo was extracted by acetone:water (9:1) and
ethanol:water (8:2), respectively. The used extraction parameters are listed in the Table 2.10.

43
Table 2.10. Extraction parameters
Acetone/water Ethanol/water
Preheat phase [min] 5 5
Heating phase [min] 5 5
Static phase [min] 10 10
Purgeing with N [sec] 90 90
Cycles 2 2
Pressure [bar] 100 100
Temperature [°C] 70 70

The total extractive content was determined by adding together the single extraction values.

Fig. 2.8. ASE 200 Accelerated Solvent Extractor.

2.5.3 Hydrolysis

Degraded bamboo samples were ground in a vibration mill (Herzog Maschinenfabrik,

Germany). All samples were climatized. The moisture content was measured and the results
were taken into account. Two step hydrolysis methods were used.

For pre-hydrolysis, 2 ml 72 % H2SO4 were added to 200 mg (+/- 10 mg) (atro) the sample.
The samples were hydrolyzed for 1 h at 30 °C (incubated in a water bath). The reaction vessel
was a short test tube. The sample was stirred during the reaction. After 1 h the reaction was
stopped by addition of 6 ml distilled water. Each sample was transferred into a 100 ml

44
volumetric flask using 50 ml of distilled water. The vessel was closed with a little condenser
(a glass ball).

For post-hydrolysis, samples were hydrolyzed under pressure (0.12 mPa) in an autoclave for
40 min. at 120 °C. After cooling down to room temperature, the flasks were filled up with
distilled water to exactly 100 ml and were shaken. Thereafter, the condensed lignin residue
was removed by filtration over a G4 sinter glass crucible. One ml of the liquid was transferred
into a sample vial for the analysis with the borate system and 1.5 ml was saved in a plastic
tube for second analyses. This sample was frozen. The filtered lignin was washed intensively
with distilled water. After this the crucible was dried at 105 °C and the lignin content was
determined gravimetrically.

2.5.4 HPAEC-borate analysis

The high-performance-anion-exchange chromatography-borate analysis was performed by

using a 5.0 mm bore column of 115 mm length (Omnifit) filled with the strong anion
exchange resin (MCI Gel CA08F (Mitsubishi) at 65 °C) (Fig. 2.9). The mobile phase (0.7 ml
min−1) was made of A: 0.3 M potassium borate buffer pH 9.2 and B: 0.9 M potassium borate
buffer pH 9.2. After sample injection, the separation was started with 90 % A and 10 % B. A
linear gradient was run within 35 min to 10 % A and 90 % B. Data acquisition was stopped
after 43 min. Sugar quantification was obtained by after-column derivatization with
Cu-bicinchoninate (0.35 ml min−1) at 105 °C in a 30 m crocheted Teflon coil of 0.3 mm inner
diameter and the detection at 560 nm, which was according to Willföet al. (2009). Data were
analysed with the Dionex Chromeleon software.

Fig. 2.9. Dionex Ultimate 3000 HPLC.

45
2.6 Transmission electron microscopy (TEM) studies of degraded bamboo

Degraded bamboo samples from the preserving jar experiment (Chapter 2.4.1) were used for
TEM. Sections were taken from the first 0.5 mm of the fungi-infected sample surface. The
samples were air dried and embedded in Spurr‘s epoxy resin under mild vacuum according to
Kleist and Schmitt (1999). Ultrathin (80-100 nm) transverse sections of the embedded
specimens were cut with a diamond knife, placed on uncoated 300-mesh copper grids, and
stained with potassium permanganate according to Donaldson (1992). Examination was
carried out with a Philips CM 12 TEM at accelerating voltages of 40 or 60 kV to enhance
contrast (Fig. 2.10).

Fig. 2.10. Philips CM12 transmission electron microscope.

2.7 Ultraviolet microspectrophotometry (UMSP) studies of degraded bamboo

Sections of degraded bamboo samples from the preserving jar experiment (Chapter 2.4.1) for
UV were taken from the first 0.5 mm of the fungi-infected sample surface. The samples were
fixed in glutaraldehyde/paraformaldehyde solution, dehydrated in a graded series of acetone
and embedded in epoxy resin according to Spurr (1969) to stabilize the degraded cell wall
structure and facilitate the cutting procedure. The embedded samples were cut into 1-μm
semi-thin cross sections with an ultramicrotome Ultracut E (Reichert-Jung) with a diamond

46
knife. Thereafter, the sections were transferred to quartz microscope slides, thermally fixed,
mounted in non-UV-absorbing glycerol, and covered with a quartz cover slip.

2.7.1 Light microscopic analysis

As preparation for the UMSP measurements, 1-μm thin sections from the preserving jar
experiment (Chapter 2.4.1) were stained with toluidine blue (Gerlach 1977) to enhance the
cell wall structure of the tissue. Sections were immersed in glycerol and examined with the
Zeiss Ultraphot II microscope and Olympus BX 51 microscope, adapted with the CELL F
(Olympus) evaluation program.

2.7.2 UV-absorbance spectra measurement

Analyses were carried out using a Zeiss UMSP 80 microspectrophotometer (Fig. 2.11). The
lignin modification process within the S2 layer was additionally investigated by photometric
point-by-point measurements (spot size l μm²) between 240 and 400 nm wavelengths using
the program LAMWIN (Zeiss). This program evaluates the UV absorbance spectra according
to Lambert-Beer‘s law. The point measurements were automatically repeated 6 times at each
spot for individual wall layers: (1) ML-F (middle lamella of fibre), (2) secondary wall of fibre,
(3) ML-P (middle lamella of parenchyma cell), (4) secondary wall of parenchyma cell, (5)
secondary wall of vessel. For quantitative studies, 6 spectra were taken from each cell wall
layer.

Fig. 2.11. Zeiss UMSP 80 microspectrophotometer.

47
2.7.3 UV-scanning measurement

The Zeiss UMSP 80 microspectrophotometer equipped with a scanning stages enables the
determination of image profiles at constant wavelengths using the scan program APAMOS
(automatic photometric analysis of microscopic objects by scanning; Zeiss) as described by
Koch and Kleist (2001). The scan program digitises square fields of the tissue with a local
geometrical resolution of 0.25 x 0.25 μm and a photometrical resolution of 4096 grey scale
levels, which are converted into 56 basic colours to visualise the local absorbance intensities.
The scan can be depicted as a two- or three-dimensional image profile, including a statistical
evaluation (histogram) of the semiquantitative lignin distribution. Ten to 12 measurements of
individual wall layers attacked by fungi were carried out: (1) thick fibres in the vascular
bundle, (2) thin fibres in the ground parenchyma, (3) parenchyma cells, (4) vessel-fibre area,
(5) vessel-parenchyma area.

48
3 Results and Discussion

3.1 Identification of bamboo inhabiting fungi

3.1.1 Moulds and Basidiomycetes on bamboo culms

Many of the obtained culm sections showed spots of different size, mostly on their outer
surface and only rarely inside. The colours varied due to the presence of conidia from whitish,
often gray and green to blackish. Most of these spots consisted of sparse and flat, rarely
woolly mycelia. These features are characteristic of moulds (Deuteromycetes). Fig. 3.1 shows
some examples of stained culm sections.

Fig. 3.1. Examples of stained culm sections from Philippines.

Inoculations were done from the moulded spots on agar to obtain pure cultures for subsequent
molecular identification via the DNA.

Basidiomycete-like fruiting bodies were rarely present. Some could be easily identified by
macroscopic features like Schizophyllum commune (Fig. 3.2). From fruiting bodies, mycelium
was aseptically sampled from the inner to obtain pure cultures for molecular identification and
subsequent bamboo degradation tests. Several fruiting bodies were already dry, and their
subculturing was unsuccessful. Due to the huge amount of samples, it was not tried to extract
DNA directly from those dry fruiting bodies. Examples of culms with fruiting bodies are
shown in Fig. 3.2.

49
Fig. 3.2. Fruiting bodies on bamboo samples.
Left and middle: Schizophyllum commune; right: probably Xylaria hypoxylon (ascomycete).

In total, 150 strains were isolated and brought to pure cultures. Of these, 76 were identified
(Table 3.1).

Table 3.1. Isolated and identified fungi from infected bamboos

Country Number of isolates Number of identified isolates
Ethiopia 5 1
China 25 18
Costa Rica 9 1
Germany 6 5
Indonesia 5 1
Philippines 15 2
Thailand 43 19
Vietnam 42 29
Total 150 76

The most frequent isolates were found on bamboos from Asian countries, China (25),
Thailand (43) and Vietnam (42). Hyde et al. (2002) summarised that the greatest diversity of
fungi on bamboo is known from Asia with about 500 species, followed by South America
(180), India (90) and North America (70). The high number of bamboo inhabiting fungi in
Asia may be attributed to the high diversity of Asian bamboos. Forty-four bamboo genera,
60% of the world's total number, occur throughout tropical, subtropical and temperate Asia.
This great diversity of plant species in an area is likely to support an equally diverse Mycota
occurrence. The lower number of fungi described from non-Asian regions may also be
attributed to limited surveying.

50
However, it should be considered that it is impossible to define the time and locality where
the infection of the culms took place. Mould growth is particularly favoured by high moisture
at the substrate surface (culm epidermis) and high relative air humidity (Schmidt 2006). Thus
in the above cases, infections could have been happened already on the living plant. Several
Deuteromycetes and Ascomycetes have been reported to colonize bamboos in the forests
(Mohanan 1997). However, it is assumed that at least the most infections took place after
harvest and particularly during culm storage in the moist tropical climate. A warm
temperature is favourable but not absolutely necessary for mould development; they can even
grow slightly above the freezing point such as on bread and jam at 4 °C in the refrigerator.
Moulds do not like air circulation. Thus, the oversea transport in containers may have also led
to infections. Altogether, those culms often show severe colonization by moulds (Fig. 2.1).
Under suitable conditions on the storage place after culm arrival, further fungal infection and
development are improbable. With regard to bamboo moisture content, the sensitive phase for
fungal attack lies in the first eight weeks with moisture content above the fibre saturation
point (Tang et al. 2012).

3.1.2 Identified fungi from bamboo by DNA sequencing

A total of 76 fungal isolates were identified via fruiting bodies and mycelial pure cultures
representing 16 genera and 37 species. The most frequent isolates belong to the
Deuteromycetes/Ascomycetes (67 isolates) which accounts to 88 %. The other nine 9 isolates
are basidiomycetes, which account to 12 %. In comparison with previous studies,
Basidiomycetes were only rarely isolated in this study. Kim et al. (2011) obtained 34
basidiomycete isolates from 127 fungal isolations.

Table 3.2 lists the identified fungi.

51
Table 3.2. Identified isolates from bamboo by DNA sequencing
Bamboo origin Deuteromycetes/Ascomycetes Basidiomycetes
(number of strains) (number of strains)
Ethiopia Schizophyllum commune (1)
China Alternaria alternata (1)
Alternaria tenuissima (1)
Arthrinium phaeospermum (1)
Cladosporium cladosporioides (2)
Dothiorella gregaria (1)
Fusarium asiaticum (1)
Fusarium culmorum (1)
Fusarium zeae (1)
Nigrospora oryzae (4)
Penicillium commune (1)
Penicillium chrysogenum (1)
Penicillium tricolor (1)
Penicillium variabile (1)
Phoma macrostoma (1)
Costa Rica Schizophyllum commune (1)
Germany Trichoderma koningiopsis (2)
Trichoderma viride (3)
Indonesia Cyathus stercoreus (1)
Philippines Penicillium citrinum (1)
Penicillium sumatraense (1)
Thailand Aspergillus nomius (1) Schizophyllum commune (6)
Aspergillus repens (1)
Botryosphaeria subglobosa (1)
Cladosporium cladosporioides (2)
Epicoccum nigrum (2)
Penicillium brevicompactum (1)
Penicillium citrinum (2)
Penicillium pinophilum (1)
Trichoderma atroviride (1)
Trichoderma koningiopsis (1)
Vietnam Apiospora montagnei(2)
Arthrinium phaeospermum (1)
Arthrinium sacchari (3)
Aspergillus flavus (5)
Aspergillus niger (2)
Botryosphaeria subglobosa (5)
Epicoccum nigrum (4)
Penicillium bialowiezense (1)
Penicillium biourgeianum (1)
Penicillium brevicompactum (2)
Penicillium expansum (1)
Penicillium islandicum (1)
Pestalotiopsis microspora (1)
Total number of
identified 67 9
isolates

52
Among the 67 Deuteromycetes/Ascomycetes, 35 species representing 14 genera have been
identified. The most frequent genus was Penicillium (24 %, 16 isolates), followed by
Aspergillus (13 %, 9 isolates). Some pure cultures are shown in Fig. 3.3.

Fig. 3.3. Mould isolates from bamboo.

Left: Botryosphaeria subglobosa from Vietnam; middle: Epicoccum nigrum from Thailand;
right: Penicillium commune from China.

Among the nine basidiomycete isolates (Table 3.2), Schizophyllum commune was isolated
eight times and Cyathus stercoreus once. Schizophyllum commune can be easily identified by
its fruiting body. The fungus is common on bamboo (Fig. 1.2) and wood. As a white-rot
basidiomycete, S. commune is capable of utilizing all structural components of the lignified
cell wall. Cyathus stercoreus is a new record for bamboo. Neither soft-rot nor brown-rot fungi
were found in this investigation, as it was already reported by Kim et al. (2001).

The reason of not finding soft-rot fungi can only be assumed. These fungi preferentially attack
lignocelluloses of high moisture content, particularly in substrates with soil or water contact
(Liese 1959b, Schmidt 2006); the investigated culms did not derive from those conditions.
However, soft-rot decay was found in the stakes from the field test (see chapter 3.4.5).

An explanation of not detecting brown-rot fungi in bamboo may be that this fungal group
prefers softwoods and is rarely found on hardwoods, like Daedalea quercina on oak wood,
whereas white-rot species prefer hardwoods. Brown-rot fungi consume the carbohydrates
cellulose and hemicelluloses of the lignified cell wall. To my knowledge, differences of the
cellulose characteristics between soft- and hardwoods are not described. However, the main
hemicellulose of bamboo is of the xylan type (see chapter 3.5) as is also the case for
hardwoods, whereas softwoods contain mainly mannans (Schmidt 2006). Therefore, the

53
hemicellulose xylan of bamboo (Vena et al. 2013: 21.6 %, Table 3.15: 19 to 27 %) may have
favour white-rot species and inhibit brown-rot fungi.

3.2 Identification of some Penicillium and Aspergillus isolates

Some mould genera such as Aspergillus and Penicillium contain very closely related species
of which some also have various synonyms. This may lead to mis-identifications. Therefore,
further morphological and molecular experiments were done.

3.2.1 Morphological characterization of Penicillium and Aspergillus isolates

Some Penicillium and Aspergillus isolates from bamboos were inoculated on different agar
substrates. Fig. 3.4 shows as an example the colony characteristics of the Penicillium
albobiverticillium after one week incubation at 25 °C or 37 °C.

Fig. 3.4. Colony appearance of the Penicillium albobiverticillium on various agars.

From left: creatine agar at 25 °C; Czapek yeast extract agar (CYA) at 25 °C; CYA at 30 °C;
malt extract agar at 25 °C; yeast extract sucrose agar at 25 °C.

Colony characteristics were recorded after 7 d of incubation. Fig. 3.5 shows Aspergillus flavus
growth on different media.

54
Fig. 3.5. Colony appearance of an Aspergillus flavus on various agars.
From left: Czapek yeast extract agar (CYA) at 25 °C, CYA at 37 °C, malt extract agar at
25 °C

3.2.2 Molecular identification of Penicillium and Aspergillus isolates

Particularly both genera Penicillium and Aspergillus contain many closely related and
morphologically similar species, which are hardly to be differentiated by the rDNA-ITS
sequence. Furthermore, the international DNA databases are man-made and contain mistakes,
such as misidentifications, e.g. if a deposited sequence was related to a wrongly named fungus.
Therefore, the sequences of ß-tubulin and calmodulin were additionally used for identification.
DNA extraction, PCR, sequencing and BLAST-identification are described in Chapter 2.1 and
2.2.

Table 3.2 shows the some results of Penicillium and Aspergillus isolates.

55
Table 3.2. Comparison of identifications of Penicillium and Aspergillus isolates obtained by
their rDNA-ITS, ß-tubulin and calmodulin sequences

ITS ß-Tubulin Calmodulin

Fungal
strain Max. Max. Max.
coding Species identity Species identity Species identity
(%) (%) (%)
Aspergillus Aspergillus Aspergillus
100 99 99
nomius nomius nomius
D54
Aspergillus Aspergillus
99 99
zhaoqingensis zhaoqingensis
Penicillium Penicillium Penicillium
100 100 98
D23-1 biourgeianum biourgeianum biourgeianum
Penicillium Penicillium Penicillium
100 98 99
D19-2 brevicompactum brevicompactum brevicompactum
Penicillium sp. Penicillium Penicillium
D36 100 90 82
phialosporum variabile
Penicillium Penicillium Penicillium
100 99
D80-2 commune commune commune
Penicillium Penicillium Penicillium
100 100 100
D46 citrinum citrinum citrinum
Penicillium Penicillium Penicillium
100 99 97
D9-1 expansum expansum expansum
Penicillium Penicillium sp. Penicillium
100 87 87
D57 verruculosum aculeatum
Penicillium Penicillium
98 85
aculeatum pinophilum
Penicillium Penicillium Penicillium
D79-1 100 100 97
expansum expansum expansum
Penicillium Penicillium Penicillium
100 100 92
polonicum polonicum commune
D65-1
Penicillium
92
thymicola

Some isolates like strains D23-1 and D19-2 revealed identical names with all three DNA
sequences, other isolates (like D54) showed quite different naming. However, it has to be
considered that some isolates are obviously not represented in the databases by all three
sequences like Penicillium verruculosum D57.

Altogether, several moulds were isolated from bamboo culms and could be identified by
molecular techniques down to the species level. Some moulds are ubiquitous. Detection of
Fusarium asiaticum and Penicillium sumatraense may be a hint that bamboo infection has
occurred in their home country.

56
The following chapters describe blue-stain and decay experiments with bamboo culm samples.
The fungi used are own identifications from bamboos or derive from the laboratory strain
collection.

3.3 Blue-stain test

3.3.1 Discolouration of samples by blue-stain fungi

Most of the bamboo samples were already overgrown by blue-stain fungi after two weeks of
cultivation and discoloured after four weeks to gray-blue. Bamboo samples inoculated with
Aureobasidium pullulans are shown in Fig. 3.6.

Fig. 3.6. Bamboo samples inoculated with Aureobasidium pullulans 87.

Left: 4 weeks; right: 20 weeks.

Fig. 3.7 shows the light control samples of the three bamboo species tested and the
discoloured samples after 20 weeks incubation. There were differences both in colour
between the bamboos and the fungi, respectively. Because the picture shows the dried
samples, the colours were lighter than directly after incubation.

57
 Fig. 3.7. Dry bamboo samples after 20 weeks of incubation with blue-stain fungi.

3.3.2 Microscopic characterises of blue-stain in bamboo

The colonization of the tissue (vessels and parenchyma) by blue-stain fungi was observed by
light microscopy after 4 and 20 weeks incubation. Fig. 3.8 shows some examples. The
samples were colonized by the typically thick, brown hyphae and chlamydospores of blue-
stain fungi. In some cases, a transpressorium, a thin penetration hypha was found. The
transpressorium is the only fungal `organ´by which blue-stain fungi damage lignified cell
walls (Liese and Schmid 1966). It is still unknown whether the penetration of the cell wall is
by mechanical and/or enzymatic action.

58
(a) (b)

(c) (d)

(e)

Fig. 3.8. Hyphae, chlamydospores and transpressorium of blue-stain fungi in bamboo.

(a): hyphae of the non-identified isolate 1.5 in parenchyma cells of Gigantochloa atroviolacea
(200×); (b): hyphae of Hormonema dematioides in a vessel of G. atroviolacea (100×); (c):
chlamydospores of Phoma macrostoma D64-1 in parenchyma cells of Phyllostachys
pubescens (200×); (d): chlamydospores of P. macrostoma D64-1 in a vessel of P. pubescens
(200×); (e): transpressorium (T) of Alternaria alternata in Bambusa maculata (600×).

59
3.3.3 Development of hyphae and chlamydospores in bamboo

Tables 3.4 and 3.5 summarize the abundance of hyphae in vessels and parenchyma cells.

Table 3.4. Abundance of hyphae in vessels

Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens
Species and isolate
4 20 4 4 20 4
weeks weeks weeks weeks weeks weeks
Alternaria alternata D84-2 1 3 3 4 2 4
Alternaria tenuissima D82-3-2 0 1 2 1 1 1
Aureobasidium pullulans 87 2 3 2 3 2 4
Botryosphaeria subglobosa D26 1 1 1 1 2 4
Cladosporium cladosporioides D16-2 1 1 4 4 2 4
Cladosporium cladosporioides D81-2 1 2 4 4 4 4
Epicoccum nigrum D20 0 0 1 2 1 1
Hormonema dematioides 85 2 2 1 4 1 3
Pestalotiopsis microspora D24 0 0 2 2 0 1
Phoma macrostoma D64-1 1 2 1 3 1 4
Unidentified isolate 1.5 1 1 1 3 1 4

Table 3.5. Abundance of hyphae in parenchyma cells

Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens
Species and isolates
4 20 4 20 4 20
weeks weeks weeks weeks weeks weeks
Alternaria alternata D84-2 2 2 2 4 1 1
Alternaria tenuissima D82-3-2 0 0 1 1 1 1
Aureobasidium pullulans 87 2 2 1 3 1 1
Botryosphaeria subglobosa D26 1 1 2 2 1 3
Cladosporium cladosporioides D16-2 1 1 1 3 2 3
Cladosporium cladosporioides D81-2 1 2 1 1 0 1
Epicoccum nigrum D20 0 0 1 2 0 0
Hormonema dematioides 85 2 2 1 2 1 1
Pestalotiopsis microspora D24 1 1 2 2 0 1
Phoma macrostoma D64-1 1 2 1 1 1 1
Unidentified isolate 1.5 1 1 1 4 1 2

In general, the different bamboo species were colonized to a different degree by the blue-stain
fungi. Most samples were already colonized within 4 weeks. Often, colonization of the tissue
increased with incubation time. The fast colonization of the bamboo tissue by blue-stain fungi
indicates that the culms are highly endangered for blue-stain within the first 2 months after
harvest when the moisture content in the culms is above fibre saturation.

60
Blue-stain fungi form chlamydospores with which can survive unsuitable environments and
germinate again to hyphae under suitable conditions. Chlamydospores were not observed for
all isolates, but were found in all three bamboos. Tables 3.6 and 3.7 summarize their
frequencies in vessels and parenchyma cells.

Table 3.6 Abundance of chlamydospores in vessels

Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens
Species and isolates
4 20 4 4 20 4
weeks weeks weeks weeks weeks weeks
Alternaria alternata D84-2 1 3 3 4 2 4
Alternaria tenuissima D82-3-2 0 0 2 1 0 0
Aureobasidium pullulans 87 2 3 2 3 2 4
Botryosphaeria subglobosa D26 1 1 1 1 0 4
Cladosporium cladosporioides D16-2 0 0 0 0 0 0
Cladosporium cladosporioides D81-2 1 2 4 4 4 4
Epicoccum nigrum D20 0 0 0 0 0 0
Hormonema dematioides 85 2 2 1 4 1 3
Pestalotiopsis microspora D24 0 0 2 2 0 0
Phoma macrostoma D64-1 1 2 1 3 1 4
Unidentified isolate 1.5 0 0 0 0 0 0

Table 3.7 Abundance of chlamydospores in parenchyma cells

Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens
Species and isolates
4 20 4 4 20 4
weeks weeks weeks weeks weeks weeks
Alternaria alternata D84-2 2 2 2 4 1 1
Alternaria tenuissima D82-3-2 0 0 1 1 0 0
Aureobasidium pullulans 87 2 2 1 3 1 1
Botryosphaeria subglobosa D26 1 1 2 2 0 3
Cladosporium cladosporioides D16-2 0 0 0 0 0 0
Cladosporium cladosporioides D81-2 1 2 1 1 0 1
Epicoccum nigrum D20 0 0 0 0 0 0
Hormonema dematioides 85 2 2 1 2 1 1
Pestalotiopsis microspora D24 0 0 2 2 0 0
Phoma macrostoma D64-1 1 2 1 1 1 1
Unidentified isolate 1.5 0 0 0 0 0 0

61
3.4 Degradation tests

For information on the susceptibility of bamboo culms to rot fungi, degradation tests were
performed with various experimental set-ups. Basidiomycetes derived from the laboratory
strain collection or are our own isolations were used.

3.4.1 Preserving jar test

Fig. 3.9 shows the results for the brown-rot fungi Coniophora puteana and Gloeophyllum
trabeum.

Coniophora puteana Gloeophyllum trabeum

10 10
8 8
Mass loss (%)

6
1 month Mass loss (%) 6
1 month
3 month 3 month
4 4
12 month 12 month
2 2
0 0
Bambusa Gigantochloa Phyllostachys Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens maculata atroviolacea pubescens

Fig. 3.9. Mass loss caused by brown-rot fungi in preserving jars.

Both brown-rot fungi were relatively inactive within 12 months (max. 5.7 % ML). This
contrasts to observations by Lee et al. (2006) who reported a 25 % ML by G. trabeum in
Phyllostachys pubescens and by Ma et al. (2010) who found a 35 % ML in P. edulis.
However, strain variation within a species should be considered (Schmidt 2006). Both author
groups used other isolates and also other incubation conditions.

Fig. 3.10 shows the results for the soft-rot fungi Paecilomyces variotii and Chaetomium
globosum.

Paecilomyces variotii Chaetomium globosum

70 70
60 60
Mass loss (%)

Mass loss (%)

50 50
1 month 1 month
40 40
3 month 3 month
30 30
12 month 12 month
20 20
10 10
0 0
Bambusa Gigantochloa Phyllostachys Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens maculata atroviolacea pubescens

Fig. 3.10. Mass loss caused by soft-rot fungi in preserving jars.

62
Of the soft-rot fungi, Paecilomyces variotii was less aggressive, whereas Chaetomium
globosum produced severe degradation with P. pubescens (max. 38.0 %), which also occurred
in other experiments of this study. A Japanese isolate of Ch. globosum caused less decay
(Suprapti 2010).

Fig. 3.11 shows the results for the white-rot fungi Schizophyllum commune, Cyathus
stercoreus, Pleurotus ostreatu and Trametes versicolor.

Schizophyllum commune 87 Schizophyllum commune 98

10 10
8 8

Mass loss (%)

Mass loss (%)

1 month 1 month
6 6
3 month 3 month
4 4
12 month 12 month
2 2
0 0
Bambusa Gigantochloa Phyllostachys Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens maculata atroviolacea pubescens

Cyathus stercoreus Pleurotus ostreatus

70 70
60 60
Mass loss (%)

Mass loss (%)

50 50
1 month 1 month
40 40
3 month 3 month
30 30
12 month 12 month
20 20
10 10
0 0
Bambusa Gigantochloa Phyllostachys Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens maculata atroviolacea pubescens

Trametes vesicolor
70
60
Mass loss (%)

50
1 month
40
3 month
30
12 month
20
10
0
Bambusa Gigantochloa Phyllostachys
maculata atroviolacea pubescens

Fig. 3.11. Mass loss caused by white-rot fungi in preserving jars.

Among the white rot fungi (Fig. 3.11), Schizophyllum commune caused the least decay (max.
6.7 %), Cyathus stercoreus and Pleurotus ostreatus medium degradation (max. 28.2 %) and
Trametes versicolor the highest decay (max. 62.5 %).

63
Cyathus stercoreus (Nidulariaceae) (Fig. 3.12) is found in nature on severely rotten wood
debris and on manured or burned soils (Breitenbach and Kränzlin 1986). It was not yet
reported for bamboo and showed low activity (Fig. 3.11).

Fig. 3.12. Fruiting bodies of Cyathus stercoreus on wood chips.

(photo: www.mushroomexpert.com).

Zhang et al. (2007) investigated 34 white-rot fungi and observed up to 15 % ML in P.

pubescens; Pleurotus ostreatus caused 5.2 % degradation and Trametes versicolor 13.6 %
decay. The white-rot fungus Lentinula edodes produced a 13 % ML in P. pubescens (Kim et
al. 2008). Abdurachim (1964) reported 15 % ML by S. commune. A low decay rate by S.
commune was found by Suprapti (2010) and Kim et al. (2011) and was also reported for wood
samples (Schmidt and Liese 1978). However, this species is commonly found on bamboo
culms during storage and use (Fig. 1.2; Liese 1985, Mohanan 1997, Liese and Kumar 2003).
Kleist et al. (2002) showed that S. commune was the most successful coloniser among some
fungi as it could penetrate bamboo via outer and inner culm cell walls, through cross section
planes as well as through nodal ridges and longitudinally through nodia.

When comparing the results with the literature, the possibility of chemical pretreatment of
bamboo against fungi should be kept in mind. For example, boron is often used before
shipment to Europe and would severely affect subsequent decay experiments. Our samples
were boron-free as proven by the curcuma-test. Furthermore, the results of all experiments on
fungal activity strongly depend on the specific strain of a species. Strain variation is common
among fungi, occurs also in S. commune (Schmidt and Liese 1980). Fig. 3.11, however,
shows similar results for the two S. commune isolates.

64
3.4.2 Fungus cellar test

To imitate natural conditions, the less active fungi S. commune and C. puteana were tested in
the `Fungus cellar.́ With this test set-up (Fig. 2.4), larger samples are incubated on unsterile
soil in large metal tubes (Gersonde and Becker 1958). Samples were either directly placed on
the soil or on wooden supports. Table 3.8 summarizes the obtained results.

Table 3.8. Mass loss (ML, %) and final moisture content (MC, %) of bamboo samples after
one year in the Fungus cellar test
Coniophora Schizophyllum
Soil
Species Tub puteana 167 commune 87
contact ML (%) MC (%) ML (%) MC (%)
Arundinaria 1 + 15.5 187 15.3 148
amabilis 1 - 38.6 41 10.8 31
2 - 43.4 57 8.7 68
2 - 39.0 56 7.1 40

Bambusa 1 + 9.9 159 11.0 174

maculata 1 - 20.4 39 5.1 28
2 - 38.3 48 5.0 33
2 - 34.3 52 3.6 37

Dendrocalamus 1 + 5.4 104 5.1 90

asper 1 - 29.3 42 4.3 28
2 - 26.9 45 3.8 32
2 - 28.5 50 4.3 31

Gigantochloa 1 + 6.1 95 6.1 95

atroviolacea 1 - 18.7 34 4.7 25
2 - 41.6 58 3.0 38
2 - 42.9 57 5.0 43

Phyllostachys 1 + 9.7 112 16.4 126

nigra 1 - 32.6 49 9.1 32
2 - 39.7 65 6.6 40
2 - 40.7 56 6.4 52

Phyllostachys 1 + 35.3 103 19.7 182

nigra `Boryana´ 1 - 38.8 54 6.7 26
2 - 37.9 60 5.7 40
2 - 38.8 46 5.1 35

Phyllostachys 1 + 6.3 61 6.3 63

pubescens 1 - 5.4 32 5.4 24
2 - 38.3 42 5.7 31
2 - 31.2 43 6.3 36

65
Due to the not absolutely identical conditions in the two tubs, Table 3.8 shows the data of all
samples. In contrast to results with small samples in the jar test, bamboo samples in the
Fungus cellar were considerably more degraded by C. puteana (max. 43 %) and S. commune
(max 20 %). Obviously, the moisture conditions in the Fungus cellar test influenced the
activity of both fungi, whereby the white-rot fungus S. commune differed considerably from
the brown-rot species C. puteana. Schizophyllum commune decayed samples with soil contact
and thus with high water content (90-182 % u) more than samples without soil contact. In
contrast, C. puteana produced a higher ML in samples located on wood supports and with
lower water content (34-65 % u). In view of practice, bamboo samples in the moisture range
from 24 to 182 % were attacked by basidiomycetes. However, due to the unsterile soil in the
Fungus cellar, samples became contaminated by moulds, which are capable of excreted
growth promoting substances for S. commune. Components from the soil or vitamins from
soil bacteria may have also affected the fungus (Schmidt et al. 2011).

3.4.3 Kolle flask test

3.4.3.1 Growth of fungi and final moisture content

The development of hyphal growth was measured by recording the density of hyphal mats
and the percentage of covering the sample surface (Table 3.9).

After initial differences between the fungi within the first two weeks, most bamboo species
were covered by mycelium after 16 weeks between two thirds of the surface and total
coverage. An exception with low surface growth occurred on Guadua angustifolia samples. In
general, coverage correlated with hyphal density. The density of mycelium is a specific
feature for many fungi (Stalpers 1978). However, it does not relate to actual fungal activity
within the substrate (Schmidt 2006). Gloeophyllum trabeum 183 did not show any hyphae on
the surface of samples from Bambusa maculata, Dendrocalamus asper and G. angustifolia.
However, this strain produced some degradation (Fig. 3.12), obviously not by surface growth
but by substrate mycelium. The final moisture content of the samples was from 39 to 101%,
which is a suitable range for fungal degradation.

66
Table 3.9. Mycelium growth on bamboos after 16 weeks of incubation

Bambusa Dendrocalamus Gigantochloa Guadua Phyllostachys Phyllostachys

maculata asper atroviolacea angustifolia pubescens pubescens
China Germany
HD HC HD HC HD HC HD HC HD HC HD HC
Coniophora
2 4 1 3 2 4 1 0 2 4 3 4
puteana 15
Coniophora
2 4 1 4 2 4 1 1 2 4 2 4
puteana 167
Coniophora
2 4 2 4 2 4 2 2 2 4 2 4
puteana 159
Coniophora
2 4 2 4 2 4 0 0 2 4 2 4
puteana 247
Gloeophyllum
0 0 0 0 1 4 0 0 1 4 1 4
trabeum 183
Gloeophyllum
1 2 1 4 1 4 0 0 1 3 1 4
trabeum 259
Trametes
1 4 2 4 2 4 1 2 3 4 2 4
versicolor
Schizophyllum
2 4 1 4 2 4 0 0 2 4 2 4
commune
Chaetomium
2 4 2 4 2 4 1 2 2 4 2 4
globosum
Paecilomyces
2 4 2 4 2 4 0 0 2 4 2 4
variotii

HD = Hyphal density: 0 = no growth; 1 = sparse mycelium; 2 = normal mycelium;

3 = thick mycelium.
HC = Hyphal coverage: 0 = no coverage; 1 = 1-33 % coverage; 2 = 34-66 % coverage;
3 = 67 - 99 % coverage; 4 = total coverage.

3.4.3.2 Durability of bamboo against fungi

All five bamboo species were rather resistant to degradation by the various strains of the
brown-rot fungi C. puteana and G. trabeum (Fig. 3.13) without significant variation among
the strains. Both fungi comprised the test strains to be used in EN 113 (1996) for wood
samples.

67
 Coniophora puteana 15 Coniophora puteana 159
Coniophora puteana 167 Coniophora puteana 247
Gloeophyllum trabeum 183 Gloeophyllum trabeum 259
4.0
3.5
3.0

Mass loss (%)

2.5
2.0
1.5
1.0
0.5
0.0
A B C D E F
Brown-rot fungi

Fig. 3.13. Mass loss of bamboos caused by brown-rot fungi after 16 weeks incubation.
Error bars represent means ±standard deviation.
A: Bambusa maculata; B: Dendrocalamus asper; C: Gigantochloa atroviolacea; D: Guadua
angustifolia; E: Phyllostachys pubescens China; F: Phyllostachys pubescens Germany.

Among the four C. puteana strains, maximum ML to bamboo was 2.9 %. According to EN
113, C. puteana strain Ebw. 15 shall produce about 20% mass loss in wood and wood
products. The two G. trabeum strains also did not reveal significant ML in bamboo
degradation, regardless of the species.

Mass losses of the wood controls by C. puteana and G. trabeum isolates ranged from 19.7 to
62.7 %, which shows that the conditions of the test set-up should have been also suitable for
the bamboo samples.

The low activity of brown-rot fungi against bamboo correlates to the results obtained in
preserving jars (Fig. 3.9) where a maximum 5.7 % ML was obtained by both brown-rot fungi
on the same bamboo species. However, samples of Melocanna bambusoides after 6 month
incubation in Kolle flasks had shown up to 13.7 % decay by C. puteana Ebw. 15 (Schmidt et
al. 2011). To prove these earlier results, this degradation test according to EN 350 and EN 113
considered sample sterilization by gamma radiation. However, the possible influence of the
sterilization technique can be only assumed. The highest ML of 3 % by G. trabeum is lower
than the one in preserving jars. However, both results contrast to Lee et al. (2006), who found
25 % degradation by G. trabeum in P. pubescens. The reason for this discrepancy is unknown.

68
 Trametes versicolor Schizophyllum commune
25.0

20.0

Mass loss (%)

15.0

10.0

5.0

0.0
A B C D E F
white-rot fungi

Fig. 3.14. Mass losses of bamboos caused by white-rot fungi after 16 weeks incubation.
Error bars represent means ±standard deviation.
A: Bambusa maculata; B: Dendrocalamus asper; C: Gigantochloa atroviolacea; D: Guadua
angustifolia; E: Phyllostachys pubescens China; F: Phyllostachys pubescens Germany.

Among the two white-rot fungi, Trametes versicolor showed the greatest ML with 15.3 % for
Phyllostachys pubescens from China, followed by P. pubescens from Germany (12.3 %) and
G. atroviolacea (10.4 %). Guadua angustifolia was very resistant (2.3 %). Degradation of the
Fagus sylvatica control was 26.7 %.

Schizophyllum commune behaved rather inactively with maximum ML of only 3.2 %.

Schmidt et al. (2011) obtained with S. commune maximum up to 9.1 % degradation in 6
months in Kolle flasks. A maximum of 5.6 % ML occurred during 1 year in preserving jars
(Fig. 3.11). Abdurachim (1964) reported 15 % ML by S. commune. Low decay rates by S.
commune were found by Suprapti (2010) and Kim et al. (2011). The beech wood control in
this study showed also negligible decay (0.8 %), which corresponds to previous results
(Schmidt and Liese 1978).

69
 Chaetomium globosum Paecilomyces variotii

25.0

20.0

Mass loss (%)

15.0

10.0

5.0

0.0
A B C D E F
Soft rot fungi

Fig. 3.15. Mass losses of bamboos caused by soft-rot fungi after 16 weeks incubation.
Error bars represent means ±standard deviation.
A: Bambusa maculata; B: Dendrocalamus asper; C: Gigantochloa atroviolacea; D: Guadua
angustifolia; E: Phyllostachys pubescens China; F: Phyllostachys pubescens Germany.

The two soft-rot fungi, Ch. globosum and Paecilomyces variotii revealed significant
differences with regard to bamboo decay, with higher ML by Ch. globosum than by P. variotii.
Most ML was caused by Ch. globosum to P. pubescens from China (9.6%), followed by G.
angustifolia (8.1 %) and B. maculata (7.5 %). Schmidt et al. (2011) reported up to 52.7 %
decay by Ch. globosum after 6 months in Kolle flasks. In preserving jars maximum 38 % ML
were measured after 1 year. Surprapti (2010) reported 8.0 % ML. Kim et al. (2011) obtained
up to 17.9 % decay for other soft-rot fungi. Maximum ML by P. variotii was only 3.1 %. The
same strain of the species had shown maximum 3.9 % degradation in preserving jars (Fig.
3.10), but another strain up to 19.7 % decay in Kolle flasks (Schmidt et al. 2011). The wood
controls in our experiment revealed rather low ML around 1 % which explains that soft-rot
degradation of wood samples should be tested by the specific standard EN 807 (2001).

3.4.3.3 Classification of bamboos according durability

The bamboos were grouped into 5 durability classes as it is done for wood species. Table 3.10
shows the durability classification of all bamboo/fungus combinations deriving from Figs.
3.13 to 3.15.

70
Table 3.10. The durability classification of bamboo
Bambusa Dendrocalamus Gigantochloa Guadua Phyllostachys Phyllostachys
maculata asper atroviolacea angustifolia pubescens pubescens
China Germany
Coniophora
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
puteana 15
Coniophora
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
puteana 167
Coniophora
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
puteana 159
Coniophora
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
puteana 247
Gloeophyllum
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
trabeum 183
Gloeophyllum
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
trabeum 259
Trametes
Ⅲ Ⅲ IV Ⅱ IV IV
versicolor
Schizophyllum
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
commune
Chaetomium
Ⅲ Ⅱ Ⅲ Ⅲ Ⅲ Ⅱ
globosum
Paecilomyces
Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ Ⅱ
variotii
II = durable (ML < 5%), III = moderately durable (5 – 10 %), IV = slight durable (10 - 30 %)

There were considerable differences. The resistance of the five bamboo species to the
brown-rot fungi C. puteana and G. trabeum, the white-rot fungus S. commune and the soft-rot
species P. variotii would group them into class II (durable). The soft-rot fungus Ch. globosum
attacked the bamboos according to classes II and III (moderately durable). Considering
white-rot decay by T. versicolor, the five bamboo species varied between classes II and IV
(slightly durable).

Because the investigated samples derived from commercial use, the details on the culm
sections tested are not known. Therefore, the above results should be considered with some
restrictions. The susceptibility of bamboo to fungi is influenced by various factors (Schmidt et
al. 2011). Samples from young culms decayed more than older ones. Specimens from the
culm top were more vulnerable to decay than those from the bottom. The cutting season
influences resistance because starch and protein content vary during the year.

Furthermore, durability classifications are not only influenced by the used fungus and special
fungus strain, but also by the specific test laboratory. For example, a comparative durability
test among different European laboratories revealed discrepant results for several soft- and
hardwoods and their corresponding test strains, even though all institutions involved used the
EN standards (Brischke et al. 2013).

71
3.4.4 Vermiculite test

To measure the possible influence of moisture content and additional nutrients on the activity
of C. puteana and S. commune, vermiculite with different amounts of water or malt extract
solutions was used as support for bamboo samples. Fig. 3.16 shows examples of the
vermiculite incubations and Table 3.11 summarizes the results.

Fig. 3.16. Growth of Schizophyllum commune in preserving jars with vermiculite and
mycelium on Phyllostachys pubescens samples. Left: side view; right: top view.

Table 3.11. Fungal growth on bamboo and wood samples in preserving jars with vermiculite

Liquid (ml) per preserving jar

Bamboo Fungus Liquid 80 100 120 140 160
D H D H D H D H D H
Coniophora tap water 1 4 1 4 1 4 1 4 1 4
Gigantochloa puteana malt extract 2 4 2 4 2 4 2 4 2 4
atroviolacea Schizophyllum tap water 1 4 1 4 1 4 1 4 1 4
commune malt extract 2 4 2 4 2 4 2 4 2 4
Coniophora tap water 1 4 1 4 1 4 1 4 1 4
Phyllostachys puteana malt extract 2 4 2 4 2 4 2 4 2 4
pubescens Schizophyllum tap water 1 4 1 4 1 4 1 4 1 4
commune malt extract 2 4 2 4 2 4 2 4 2 4
Fagus Schizophyllum tap water 1 4 1 4 1 4 1 4 1 4
sylvatica commune malt extract 2 4 2 4 2 4 2 4 2 4
Pinus Coniophora tap water 1 4 1 4 1 4 1 4 1 4
sylvestris puteana malt extract 3 4 3 4 3 4 2 4 2 4
D = density of hyphae; H = hyphal coverage of sample.

72
All bamboo and wood samples were totally covered by mycelium after 32 weeks. Hyphal
density with tap water in vermiculite as a moisture reservoir was less than with malt extract.
However, the density of mycelium is not necessarily related to mass loss in test specimens.

Fig. 3.17 shows the mass loss produced by S. commune.

Schizophyllum commune with tap water

240 60
Sample final moisture content (% u)

200 50

160 40

Mass loss (%)

120 30

80 20

40 10

0 0
80 100 120 140 160
Liquid Added (ml)

Schizophyllum commune with malt extract solution

240 60
Sample final moisture content (% u)

200 50

160 40
Mass loss (%)

120 30

80 20

40 10

0 0
80 100 120 140 160
Liquid Added (ml)

Sample final moisture content M ass loss

Gigantochloa atroviolacea Gigantochloa atroviolacea
Phyllostachys pubescens Phyllostachys pubescens
Fagus sylvatica Fagus sylvatica

Fig. 3.17. Influence of moisture content and nutrients on mass loss by Schizophyllum
commune.

Maximum ML of bamboo was only 4.6 %. Thus, possible influences among the parameters,
bamboo species, moisture content and nutrient addition, were rather indistinct for this fungus.

73
This low activity corresponds to the results on agar in preserving jars (Chapter 3.4.1) and in
Kolle flasks (chapter 3.4.3). Also the beech wood samples revealed negligible decay (0.7 %)
by S. commune, which corresponds to previous results with wood samples (Schmidt and Liese
1978).

Fig. 3.18 shows the results for C. puteana.

Coniophora puteana with tap water

240 60
Sample final moisture content (% u)

200 50

160 40

Mass loss (%)

120 30

80 20

40 10

0 0
80 100 120 140 160
Liquid Added (ml)

Coniophora puteana with malt extract solution

240 60
Sample final moisture content (% u)

200 50

160 40
Mass loss (%)

120 30

80 20

40 10

0 0
80 100 120 140 160
Liquid Added (ml)

Sample final moisture content M ass loss

Gigantochloa atroviolacea Gigantochloa atroviolacea
Phyllostachys pubescens Phyllostachys pubescens
Pinus sylvestris Pinus sylvestris

Fig. 3.18. Influence of moisture content and nutrients on mass loss by Coniophora puteana.

Maximum ML of bamboo was 8.4 %, whereby P. pubescens was more decayed than G.
gigantochloa. Both additions, tap water and malt extract, increased ML from the 80 to 120 ml

74
liquid addition, followed by decrease to 160 ml liquid content. Scots pine wood revealed
significant differences among the liquid additions: With tap water, decay increased stepwise
to 14.0 % maximum at 160 ml water addition. The highest amount of decay displayed a
maximum 52.5 % ML at 120 ml malt extract addition, which produced 104 % u moisture
content of vermiculite. The subsequent decrease of ML may be caused by too much water in
the vermiculite.

The standard deviation is not given in Fig. 3.17 due to the only small mass loss by S.
commune), but for C. puteana (Fig. 3.18) due to its greater decay capacity. Both bamboo
species G. atroviolacea and P. pubescens were rather resistant to degradation by S. commune
and C. puteana over the 32 weeks of incubation, the first bamboo being more resistant than
the latter one. Adding different amounts (80 to 160 ml) of water or nutrient solution to
vermiculite had only a small effect. However, the final bamboo moisture contents were 61 to
84%, which is a suitable range for decay fungi (Schmidt 2006).

Fungi are able to transport water by their mycelia from a moisture source to neighbouring
wood (Schmidt 2006). Figs. 3.17 and 3.18 show that the final moisture content of the bamboo
samples was only slightly influenced by the initial liquid additions. Obviously, both fungi did
not transport water from vermiculite to the samples. The presence of the metal ring between
vermiculite and sample could not be the reason because C. puteana could moisten the P.
sylvestris wood. Fig. 3.18 (bottom) shows that moisture content in Scots pine increased up to
218 % u parallel to water addition. Subsequently, ML decreased because of too much water in
the wood.

To investigate if the fungi were still alive after 32 weeks of incubation, the vitality test
performed proved living mycelium in all culture vessels (Fig. 3.19). Because fruit bodies did
not grow in the jars and because both fungi do not produce asexual spores, survival was not
due to spores. Thus, it can be deduced that mycelia were active over the whole culture period.

75
 Fig. 3.19. Vitality test on malt extract agar after vermiculite incubation.
Left: Coniophora puteana; right: Schizophyllum commune.

Schmidt et al. (2011) assumed that high bamboo ML by C. puteana and S. commune in the
Fungus cellar test was mainly caused by the moisture conditions and much less by
components from the unsterile soil. The vermiculite test as a pure-culture experiment in
closed vessels did not show a significant effect of moisture content and nutrient addition on
bamboo degradation. Thus, bamboo degradation in nature may be also influenced by better air
conditions, soil minerals or vitamins from soil bacteria. Already Leutritz (1946) demonstrated
that soil serves as a water holding substrate and provides substances from the soil. The
influence of minerals and vitamins on soft rot was reported by Worrall and Wang (1991) and
Worrall et al. (1991). However, the small differences of mass loss among the bamboos, fungi
and test parameters make a deeper discussion senseless.

The fungi had been selected due to three reasons: S. commune is very common on bamboo
and the experimental strain was our own isolate from bamboo; C. puteana is an obligatory test
fungus in the European standard EN 113; third, it was hoped to explain the influence of
moisture content and nutrients in the fungus cellar test.

However, with regard to wood, the decay results with C. puteana and P. sylvestris samples
(Fig. 3.18) show the suitability of the proposed technique for degradation studies. Adding 2 %
malt extract solution until a wood moisture content reached approximately 100 % provided
high ML within 32 weeks of incubation. The vermiculite method will most likely produce
significant bamboo decay as well if more aggressive fungi such as P. ostreatus and T.
versicolor are used.

76
3.4.5 Field test

A comparative durability test of Quercus petraea and Q. robur samples revealed for EN
laboratory tests the durability class II (durable), but only class IV (somewhat durable) in the
above-ground test and class V (non-durable) in the in-ground examination, respectively
(Brischke et al. 2009). To compare the bamboo laboratory results (see Table 3.10) to the field,
a small stake test was performed for 3.5 years. Examples of the stakes from Bambusa vulgaris
and three unknown species are shown in Fig. 3.20.

Fig. 3.20. Examples of stakes from the field test.

Fungal activity on the stakes was visually assessed according to the rating of Papadopoulos
(2010). Table 3.12 summarizes the results.

77
Table 3.12. Visual evaluation of susceptibility of bamboo stakes to fungi after 3.5 years field
test
Bambusa
Stake part Species 3 Species 5 Species 10
vulgaris
top 2 4 3 3
Above ground middle 2 4 3 2
bottom 2 3 2 2
top 2 3 2 2
In ground middle 1 2 1 1
bottom 1 1 0 1

The above-ground parts of the stakes revealed no attack (rating 4) to moderate attack (rating
2), the in-ground areas showed slight attack (3) to total destruction (0). Fungal activity varied
between the species and sample regions. Among the bamboos, the unknown species 3 was
most resistant and no. 5 was most susceptible to fungi.

For more information, the below-ground parts were opened to view the inner of the stakes
(Fig. 3.21).

Fig. 3.21 Cross-sectional view of opened stakes from the below-ground part.
Left: Bambusa vulgaris; right: species 10.

The opened stakes showed different stagess of degradation. Some revealed a thin outer layer
of soft-rot decay which is characteristic for lignocelluloses in soil or wet conditions (Liese
1985, Schmidt 2006). In the centre, the presence of white rot was assumed due to the
brightened discolouration of the tissue. Although there is rather limited data of the performed
field tests, the results underline the well-known fungal susceptibility of bamboos in ground
contact (e.g., Liese 1985).

78
3.5 Chemical analysis of degraded bamboo

To obtain knowledge on the specific attack of the various rot fungi, the chemical composition
of the bamboo cell walls was performed. Samples used for the analyses derived from the
preserving jar test (Chapter 2.4.1). Three bamboo species, after 1 year degradation including
control samples, were analyzed. All rot types, different decay fungi and increasing ML of
samples were considered (Table 3.13).

Table 3.13. Characterization of bamboo samples for chemical analyses

Bamboo Fungi Rot type Mass loss (%)

Control
Coniophora puteana brown 4.7
Schizophyllum commune white 5.2
Phyllostachys pubescens Gloeophyllum trabeum brown 5.3
Pleurotus ostreatus white 21.0
Chaetomium globosum soft 38.0
Trametes versicolor white 47.8
Control
Coniophora puteana brown 5.6
Gigantochloa atroviolacea Gloeophyllum trabeum brown 5.7
Schizophyllum commune white 6.7
Trametes versicolor white 51.6
Control
Pleurotus ostreatus white 28.2
Bambusa maculata
Chaetomium globosum soft 31.8
Trametes versicolor white 62.5

For sugar analyses, the borate complex anion exchange chromatography (HPAEC-borate)
analysis using cubicinchoninate for detection was applied for the direct quantification of
underivatized sugars. HPAEC-borate requires a strong anion exchange gel as stationary phase.
Applying a linear gradient between 0.3 M and 0.9 M potassium borate buffer pH 9.2 allows
the separation of the wood sugars as their borate complexes. Due to the high borate
concentration, the column continuously regenerates itself so that acid hydrolysates can be
directly injected without neutralization. In a large range, the colour development, measured at

79
560 nm, is proportional to the sugar concentration (Sinner and Puls, 1978). The wood sugars
including 4-O-Me-glucuronic acid elute within 50 min.

As an example, the results of the chromatogram of the P. pubescens control hydrolysate are
presented in Fig. 3.22.

400 210211b #15 [modified by Erasmy, 1 peak manually assigned] UV_VIS_1

mAU WVL:560 nm

9: Glucose
90,0

8: Xylose
300

200
21: Cellobiose
3: Rhamnose

6: Arabinose
7: Galaktose
4: Mannose

10: 4-O-Me
100
19,0
0,9 mol/l Borat: 10,0 % 5
Flow: 0,700 ml/min
min
-50
0,0 10,0 20,0 30,0 40,0 50,0

Fig. 3.22. HPAEC-borate chromatogram of Phyllostachys pubescens control hydrolysate.

All bamboo samples were measured three times and the average was calculated. The amount
of sugars in the P. pubescens control is listed in Table 3.14.

Table 3.14. HPAEC-borate analysis of the Phyllostachys pubescens control

Peak Retention time Area Height Amount

Component
no. (min) mAU*min mAU (%)
1 Cellobiose 12.39 11.71150 15.22644 0.74
2 n.a.* 13.00 1.57442 4.11063 n.a.
3 Rhamnose 15.33 2.24589 2.85388 0.11
4 Mannose 21.45 2.91764 2.64550 0.20
5 n.a. 22.85 0.88235 0.73170 n.a.
6 Arabinose 24.59 11.06571 6.51314 1.31
7 Galactose 26.72 3.72431 1.42916 0.25
8 Xylose 28.30 323.37909 240.38193 23.85
9 Glucose 31.69 364.64658 246.60522 40.51
10 4-O-Me** 35.11 7.49265 4.06662 0.51
* not analyzed; ** 4-O-methylglucuronic acid.

80
Chemical analysis of Bambusa balcooa (Vena et al. 2013) revealed for the healthy tissue
7.1 % extractives, 54.6 % glucan, 21.6 % xylan, 1.1 % arabinan, 25.2 % Klason lignin and
2.4 % ash.

Table 3.15 summarizes the results of the chemical analyses of samples after 1 year
degradation. Lignin content was determined by hydrolysis. After two step hydrolysis, the
lignin amount was determined gravimetrically. The first three analyses columns contain the
results for dry matter; acetone and ethanol extract, which are included by routine in those
overviews, but are of lower significance for the matter in hand.

The percent loss of chemical constituents after 1 year of degradation is given in Table 3.16.

81
Table 3.15. Chemical composition (%) of bamboos after 1 year of fungal degradation
Hemicellulose Cellulose Lignin
4-O-
Bamboo Dry Acetone Ethanol
Fungi methyl- Arabi- Galac- Man- Rham- Cello-
weight extract extract Xylose Total Glucose Total
glucuronic nose tose nose nose biose
acid
Control
95.05 4.61 1.05 0.52 1.37 0.26 0.19 0.11 24.38 26.82 0.69 41.50 42.19 26.25
Coniophora
94.87 3.45 0.80 0.40 1.19 0.24 0.20 0.10 23.86 25.99 0.21 41.11 41.32 25.64
puteana
Schizophyllum
94.52 2.70 0.65 0.39 1.05 0.21 0.19 0.09 23.09 25.03 0.21 39.68 39.89 25.81
commune
Phyllostachys
Gloeophyllum
94.38 2.83 0.60 0.39 1.14 0.33 0.20 0.09 23.65 25.80 0.21 41.02 41.23 25.88
pubescens trabeum
Pleurotus
94.44 2.42 0.79 0.32 0.80 0.16 0.19 0.07 18.68 20.22 0.19 35.34 35.53 20.91
ostreatus
Chaetomium
94.35 4.24 1.39 0.24 0.65 0.19 0.17 0.06 14.05 15.36 0.11 22.62 22.73 20.63
globosum
Trametes
94.45 3.34 0.96 0.19 0.55 0.10 0.15 0.05 12.20 13.24 0.11 24.24 24.35 14.09
versicolor
Control
94.42 3.52 1.34 0.46 1.13 0.61 0.10 0.09 16.14 18.53 0.73 47.86 48.59 29.46
Coniophora
94.44 1.98 0.94 0.36 0.88 0.50 0.11 0.08 15.69 17.62 0.21 47.74 47.95 27.69
puteana
Gigantochloa
Gloeophyllum
94.55 2.06 0.74 0.38 1.05 0.59 0.12 0.08 16.04 18.27 0.21 45.95 46.16 27.91
atroviolacea trabeum
Schizophyllum
94.24 2.05 0.74 0.38 0.99 0.49 0.11 0.08 15.62 17.67 0.21 46.54 46.75 28.34
commune
Trametes
94.68 1.70 1.03 0.16 0.43 0.18 0.11 0.03 7.09 8.00 0.12 25.01 25.12 14.37
versicolor
Control
94.65 3.05 1.20 0.41 0.95 0.50 0.10 0.08 18.13 20.17 0.54 52.15 52.69 26.43
Pleurotus
Bambusa 94.95 2.06 1.36 0.21 0.45 0.19 0.07 0.05 11.36 12.33 0.20 40.53 40.73 16.54
ostreatus
maculata Chaetomium
93.74 3.53 1.23 0.22 0.66 0.28 0.15 0.06 12.12 13.49 0.14 28.90 29.04 23.32
globosum
Trametes
94.70 2.46 1.33 0.12 0.24 0.11 0.10 0.03 5.69 6.29 0.09 20.75 20.84 9.41
versicolor

82
Table 3.16. Percent loss of chemical constituents of bamboos after 1 year of fungal degradation

Component loss (%)

Bamboo Fungi Rot type
Mass Hemicellulose Cellulose Lignin
Control
Coniophora puteana brown 4.7 3.09 2.06 2.32
Schizophyllum commune white 5.2 6.67 5.45 1.68
Phyllostachys pubescens Gloeophyllum trabeum brown 5.3 3.80 2.28 1.41
Pleurotus ostreatus white 21.0 24.61 15.79 20.34
Chaetomium globosum soft 38.0 42.73 46.12 21.41
Trametes versicolor white 47.8 50.63 42.28 46.32
Control
Coniophora puteana brown 5.6 4.91 1.32 6.01
Gigantochloa brown
Gloeophyllum trabeum 5.7 1.40 5.00 5.26
atroviolacea
Schizophyllum commune white 6.7 4.64 3.79 3.80
Trametes versicolor white 51.6 56.83 48.30 51.22
Control
Pleurotus ostreatus white 28.2 38.87 22.70 37.42
Bambusa maculata
Chaetomium globosum soft 31.8 33.12 44.89 11.77
Trametes versicolor white 62.5 68.82 60.45 64.40

83
The sugar composition of hemicellulose (Table 3.15) mainly comprising xylose, arabinose
and 4-O-methylglucuronic acid agrees to the results by Vena et al. (2013) that bamboo
hemicellulose is of the xylan-type, similar as in hardwoods.

Pleurotus ostreatus and T. versicolor (Table 3.16) consumed the three cell wall components
hemicellulose, cellulose and lignin to nearly the same extent which is characteristic of white-
rot fungi of the simultaneous type (Schmidt 2006). Maximum lignin degradation of 64%
occurred by T. versicolor in B. maculata. Due to the low mass loss of 5.2 to 6.7% by the less
aggressive fungus S. commune (Table 3.11) there were only minor changes to the
hemicelluloses, cellulose and lignin content, respectively.

The less destructive brown-rot fungi C. puteana and G. trabeum (Table 3.11) revealed slight
changes in the cell wall components. G. atroviolacea displayed remarkably high lignin
degradation values relative to the other brown-rot fungi.

The soft-rot fungus Ch. globosum preferentially consumed the carbohydrates, which is
characteristic of this rot type. However, there was also some decrease in lignin content, which
may be due to the cleavage of methoxy groups from the aromatic rings by these fungi
(Schmidt 2006).

84
3.6 Transmission electron microscopy (TEM) studies of degraded bamboo

For detailed knowledge on the micromorphology of bamboo cell wall degradation by fungi,
samples from the degrading test in preserving jars (like as for chemical analyses, chapter 3.5)
were investigated with the transmission electron microscope (TEM). Different degrees of
degradation were used to group the results into early, medium and late decay stagess.

3.6.1 Early stages of decay

The TEM observations on early stages of decay (one month of incubation) of Bambusa
maculata (Fig. 3.23) show that degradation was mostly confined to the parenchyma cells.
Parenchyma cells with laminated wall layers (Liese 1998) contained hyphae in the lumen as
well as in the cell wall. Fig. 3.23a shows hyphae eroding from one lumen through the wall in
the neighbouring cell. The vessel (Fig. 3.23b) contained hyphae and holes in the secondary
wall layer, this would occur as cavities in longitudinal sections, a unique characteristic of soft
rot. The vessel seemed to be degraded more easily than the fibres. Most fibres in healthy
bamboo were composed of 3-5 concentric layers. The secondary wall of some fibres near
parenchyma cells consisted of more than 7 layers (Fig. 3.23c).

(a) 5µm (b) 2µm (c) 2µm

Fig. 3.23. Early soft-rot symptoms by Chaetomium globosum.
(a): parenchyma cells; (b): vessels with neighbouring parenchyma; (c): fibres.

85
3.6.2 Medium stages of decay

Fig. 3.24a, b shows medium stages of soft rot after 3 month of incubation. Chaetomium
globosum degraded the cell corner between fibres, and the S2 layer was decayed into many
holes (cavities; Fig. 3.24a). The remaining compound middle lamella region (CML) and S 3
layer are shown in Fig. 3.24b.

(a) 5µm (b) 5µm

(c) 5µm (d) 5µm

Fig. 3.24. Medium stages of decay by soft rot (Chaetomium globosum) and white rot
(Trametes versicolor).
(a): many hyphae of C. globosum in the S2 layer of a fibre and cell wall residues; (b): only
some remaining S2 layer residues in a fibre degraded by C. globosum; (c): secondary wall of
fibres with erosion and wall decay after attack by T. versicolor with hyphae in the lumina; (d):
parenchyma cells degraded by T. versicolor.

86
Generally, cell wall degradation by Basidiomycetes begins in the lumen (Liese 1970, Schmidt
2006). Fig. 3.24c shows the medium stage of white-rot attack by erosion and decay of the cell
wall by T. versicolor. Hyphae migrated through pits, and the diameter of pits in parenchyma
cells was enlarged (Fig. 3.24d).

3.6.3 Late stages of decay

In Fig. 3.25, late stages of soft- and white-rot decay after 12 months of incubation are
presented. Fibres and parenchyma cells were severely decayed by soft-rot (Fig. 3.25 a, b). The
S2 layer was completely decayed. Only the CML and S3 resisted attack, which is typically true
for soft-rot attack. The white rot fungus P. ostreatus degraded the fibres completely so that
only granular residues of the cells remained with some included hyphae.

(a) 5µm (b) 5µm (c) 5µm

Fig. 3.25. Late stages of soft rot (Chaetomium globosum) and white rot (Pleurotus ostreatus).
(a): fibres with totally degraded S2 by C. globosum; (b): parenchyma cells with severe
degradation by C. globosum; (c): fibres totally degraded by P. ostreatus.

TEM studies on P. pubescens decayed by L. edodes (Kim et al. 2008) indicated that the CML
in bamboo fibres was degraded at early stages of decay. This would indicate the selectivityof
this type of white-rot (Liese 1998): L. edodes belongs to the simultaneous white-rot.

Brown-rot fungi were not included in the TEM investigations in this thesis study due to the
low ML measured (chapter 3.5). To my knowledge, the only TEM investigation on brown-rot
decay was performed by Cho et al. (2008) using P. pubescens and G. trabeum.

Altogether, the TEM studies on degraded bamboo revealed a rather similar micromorphology
of cell wall decay as it occurs in wood (e.g., Liese 1998).

87
3.7 Ultraviolet microspectrophotometry (UMSP) studies of degraded bamboo

To investigate in situ the lignification of bamboo cell walls and fungal lignin degradation
within them by white-rot fungi, UMSP studies were performed. Samples used derived from
Bambusa maculata of the preserving jar test. The soft-rot fungus Ch. globosum was included
for comparison. Table 3.17 lists characteristic ML of the investigated samples.

Table 3.17. Characteristics of Bambusa maculata samples used for ultraviolet

microspectrophotometry
Mass loss (%)
Fungus
1 month 3 months 12 months
Cyathus stercoreus 0.2 1.6 17.4
Pleurotus ostreatus 0.8 5.8 28.2
Trametes versicolor 0.7 25.1 62.5
Chaetomium globosum 4.7 23.6 31.8

3.7.1 Light microscopy

Previously to the UMSP studies, sample sections were mapped out by light microscopy to
determine the areas for subsequent UMSP (Fig. 3.26).

88
 V
FV V
FV FV

FP P
P
FP
200 µm 200 µm 200 µm

Fig. 3.26. Light microscopy of transverse section of healthy Bambusa maculata (left) and
after degradation by Trametes versicolor for 3 months (middle) and for 12 months (right).
V: vessel, P: parenchyma, FV: fibres in the vascular bundle, F: fibres in ground parenchyma.

Additionally, Fig. 3.26 clearly demonstrates the progressive tissue degradation from healthy
tissue via 3 months to 12 months incubations.

3.7.2 UV-absorbance spectra of individual cell wall layers

Generally, the lignin molecule exhibits an absorption maximum at 280 nm wavelengths and a
broad shoulder between 310 and 320 nm (Fig. 3.27). This shoulder is typical for
monocotyledons and can be linked to the presence of p-coumaroylation as demonstrated by
Higuchi (1987).

The UV absorbance of a particular anatomical region depends both on the concentration of

the various structural units of lignin, and the extinction coefficient of each structural unit.
There is an absorbance maximum at 280–282 nm, which indicates the presence of the strong
absorbing guaiacyl (G) lignin (Fergus and Goring 1970b, Musha and Goring 1975). The
extinction coefficient of the G unit at 280 nm is 3.5 times that of the S (syringyl) unit (Fergus
and Goring 1970a), and the extinction coefficient of the H (p-hydroxyphenylpropan) unit is
lower than that of the G unit, but higher than that of the S unit (Faix and Schweers 1974).

89
The following figures show the absorbance curves between 240 and 400 nm wavelength.

0.8
0.7
Log absorption

0.6
0.5
0.4
0.3
0.2
0.1
0
240 260 280 300 320 340 360 380 400
Wavelengh (nm)

Control Pleurotus ostreatus 3 months

Pleurotus ostreatus 12 months Trametes versicolor 12 months

Fig. 3.27. UV-absorbance spectra of the secondary walls of fibres of Bambusa maculata
samples.

Fig. 3.27 shows that the absorption at 280 nm decreases by the influence of the white-rot
fungus P. ostreatus. The high absorption by T. versicolor may be due to the fact that the tissue
after 12 month of incubation was strongly decayed (62.5 % mass loss) so that it had not been
possible to select the correct cell wall layer. The UV spectra of the secondary wall of fibres
have only a slight shoulder at 310-320 nm.

90
 0.8
0.7
Log absorption

0.6
0.5
0.4
0.3
0.2
0.1
0
240 260 280 300 320 340 360 380 400
Wavelength (nm)

Control Pleurotus ostreatus 3 months Pleurotus ostreatus 12 months

Fig. 3.28 UV-absorbance spectra of the compound middle lamella of thin-walled fibres within
the ground parenchyma of Bambusa maculata samples.

The spectra of the compound middle lamella region (CML) of fibres within the ground
parenchyma (Fig. 3.28) have the typical shoulders at 280-282 nm and 310-320 nm. These
fibres have relatively thin secondary walls compared to the fibres in the vascular bundles. The
spectra are rather similar. Obviously, there was no effect of the fungus on the lignin in the
CML of these thin-walled fibres. The reason may be that P. ostreatus belongs to the
simultaneous white-rot fungi whose enzyme activity occurs preferentially in the S 2 layer. No
spectrum for T. versicolor was measured because there were no measurable remnants after
culturing the fungus for 12 months.

91
 0.8
0.7
0.6
Log absorption

0.5
0.4
0.3
0.2
0.1
0
240 260 280 300 320 340 360 380 400
Wavelength (nm)

Control Pleurotus ostreatus 3 months

Pleurotus ostreatus 12 months Trametes versicolor 12 months

Fig. 3.29 UV-absorbance spectra of the compound middle lamella of thick-walled fibres in
the vascular bundle of Bambusa maculata samples.

The spectra of the CML of thick-walled fibres in the vascular bundles (Fig. 3.29) with the two
shoulders at 280-282 nm and 310-320 nm reveal a higher lignin content after fungal culture
than in the control. An explanation may be that the content of carbohydrates in the CML of
thick-walled fibres is higher than in thin-walled fibres. Fungi may have degraded these
carbohydrates so that their relative lignin content increased.

92
 0.8
0.7
0.6
Log absorption

0.5
0.4
0.3
0.2
0.1
0
240 260 280 300 320 340 360 380 400
Wavelength (nm)

Control Pleurotus ostreatus 3 months

Pleurotus ostreatus 12 months Trametes versicolor 12 months

Fig. 3.30 UV-absorbance spectra of the secondary walls of vessels of Bambusa maculata
samples.

The spectra of S2 of vessel cell walls (Fig. 3.30) show a lignin decrease by both white-rot
fungi. The absorbance decreased with incubation time of P. ostreatus.

93
 0.8
0.7
Log absorption

0.6
0.5
0.4
0.3
0.2
0.1
0
240 260 280 300 320 340 360 380 400
Wavelength (nm)

Control Pleurotus ostreatus 3 months Pleurotus ostreatus 12 months

Fig. 3.31 UV-absorbance spectra of the compound middle lamella of parenchyma cells of
Bambusa maculata samples.

The spectra of the CML of parenchyma cells (Fig. 3.31) have only the shoulder of 310-320
nm. The typical peak at 280 nm is not present. The lignin content of parenchyma cells
decreased with increasing mass loss. There were no parenchyma cells remaining after 12
months of incubation with T. versicolor.

94
3.7.3 Scanning UV-microspectrophotometry

Using the scanning mode of the ultraviolet microspectrophotometer, tissues can be scanned at
the absorbance of 280 nm in situ for the degree of lignifications in different cell walls and
wall layers.

In the following figures, typical two- and three-dimensional UV microspectrophotometric

scanning profiles of lignin distributions in the various bamboo tissues of Bambusa maculata
are visualized. The colour pixels indicate different intensities of UV absorbance at 280 nm.
The high resolution (0.25 μm x 0.25 μm per pixel) enables the differentiation of UV
absorbance within different cell wall layers (Koch et al. 2003a). The two-dimensional UV
image profiles reveal the average lignin concentration in a respective area. The three-
dimensional presentations allow an improved evaluation of the topochemical distribution of
lignin within the individual cell wall layers, including more than 47,000 measuring points.

95
 (a)

(b)

(c)

(d)

(e)

(f)
Fig. 3.32. UV micrographies and 3D profiles of thick-walled fibres in the vascular bundle of
Bambusa maculata attacked by fungi measured at 280 nm.
(a): control; (b): Cyathus stercoreus 12 month; (c): Pleurotus ostreatus 3 months; (d): P.
ostreatus 12 months; (e): Trametes versicolor 3 months; (f): T. versicolor 12 months.

Fig. 3.32 shows that the lignin content of the thick-walled fibres in the vascular bundle
decreased with the decrease of ML produced by C. stercoreus and the two white-rot species.
The delignification was higher in the S3-S2 wall layers. The cell corners were more lignified

96
than the S2 layer. The image profiles of P. ostreatus 3 months and 12 months incubation
differed only slightly, which does not correspond to different ML of 5.8 and 28.2 %,
respectively (Table 3.17).

(a)

(b)

(c)

(d)

(e)
Fig. 3.33. 280 nm-UV micrographies and 3D profiles of thin-walled fibres within the ground
parenchyma of Bambusa maculata attacked by fungi.
(a): control; (b): Cyathus stercoreus 12 month; (c): Pleurotus ostreatus 3 months; (d): P.
ostreatus 12 months; (e): Trametes versicolor 3 months.

Like with the thick-walled fibres (Fig. 3.32), the lignin content of thin-walled fibres within
the ground parenchyma decreased with degradation time/ML (Fig. 3.33). Obviously,
delignification was parallel to ML. Fungal activity became very obvious already by the great

97
reduction of the wall thickness by the fungi, particularly visible at the P. ostreatus 3 months
(34c) and 12 months incubations (34d), respectively. The twelve-months cultivation of T.
versicolor was not measured because there were no measurable remnants.

(a)

(b)

(c)

(d)

(e)
Fig. 3.34. 280 nm-UV micrographies and 3D profiles of parenchyma cell of Bambusa
maculata.
(a): control; (b): Cyathus stercoreus 12 month; (c): Pleurotus ostreatus 3 months; (d): P.
ostreatus 12 months; (e): Trametes versicolor 3 months.

The parenchyma cell walls revealed considerable decay and delignification by the fungi,
particularly after incubation with both white-rot fungi (34c-e). Cell wall areas of adjacent
cells were completely degraded (d, e) and the cell corners much less degraded.

98
 (a)

(b)

(c)

(d)

(e)

(f)
Fig. 3.35. 280 nm-UV micrographies and 3D profiles of the vessel-fibre area of Bambusa
maculata.
(a): control; (b): Cyathus stercoreus 12 month; (c): Pleurotus ostreatus 3 months; (d): P.
ostreatus 12 months; (e): Trametes versicolor 3 months; (f): T. versicolor 12 months.

The cell walls measured in the vessel-fibre area revealed a similar delignification as observed
in Figs. 3.32 and 3.33 for the fibres. The vessel walls were also attacked (e, f).

99
 (a)

(b)

(c)

(d)

(e)
Fig. 3.36. 280 nm-UV micrographies and 3D profiles of the vessel-parenchyma area of
Bambusa maculata.
(a): control; (b): Cyathus stercoreus 12 month; (c): Pleurotus ostreatus 3 months; (d): P.
ostreatus 12 months; (e): Trametes versicolor 3 months.

The cell wall of the vessel and the surrounding parenchyma cells became delignified even in
the early stages of degradation (3.36c).

100
Fig. 3.37 summarizes the results of the absorbance values at 280 nm of the different Bambusa
maculata cell wall areas.

0.47 0.5
0.47 0.33
0.42 0.45 0.44 0.4
0.34 0.40 0.38
0.37 0.33 0.3
0.34
0.25 0.32 0.32
0.31 0.26
0.26 0.24 0.2
0.24 0.25
0.24 0.25
0.21 0.1
0.21 0.18
0.20
0
A fibres in the
B vessel-fibres vascular
C fibres in the
D area bundles
E parenchyma ground
F vessel-fibres cells parenchyma
area

Fig. 3.37. Absorbance values at 280 nm wavelength of cell wall areas of Bambusa maculata
attacked by fungi after different months of incubation.
A: control; B: Cyathus stercoreus 12 months; C: Pleurotus ostreatus 3 month; D: P. ostreatus
12 months; E: Trametes versicolor 3 months; F: T. versicolor 12 months.

Altogether, the UMSP results correspond to many former investigations, which revealed the
suitability of the technique for studies on the distribution pattern of
lignification/delignification within the cell walls of wood (Scott et al. 1969, Fergus et al.
1969b, Bauch et al. 1976, Koch and Kleist 2001, Takabe, 2002, Koch and Grünwald 2004,
Singh et al. 2006) and bamboo (Lybeer and Koch 2005a, 2005b, Lybeer et al. 2006, Kim et al.
2008). The white-rot fungi investigated, P. ostreatus and T. versicolor, produce a
simultaneous-type of white-rot. Their progressive degradation/delignification starting from
the cell lumen towards the compound middle lamella region became visible in some UMSP

101
pictures. However, interpretation may become problematic when the measured wall area
comprised different cell types like in the vessel/fibre and vessel/parenchyma region.

The topochemical analyses showed that lignin attack on lignin by simultaneous white-rot
fungi occurs in parallel to ML. The beginning of delignification was evidenced at the
outermost part of the secondary wall layer. UMSP proved that even at advanced decay the
degradation was not homogeneous throughout the tissue, as there were still apparently less
attacked cells among significantly damaged ones.

102
4 Conclusions

The bamboo culm is susceptible to infection and colonization by fungi during the first two
months after harvest due to the fungus-suitable moisture content above the fibre saturation
point. Consequently, culms after sea transport from Asia often show infection by moulds and
other staining fungi. Moulds and Basidiomycetes were isolated from culms of several bamboo
species from various countries after arrival in Germany. In total, 150 strains were isolated and
76 isolates were identified by rDNA-ITS sequencing. Most isolates were Deuteromycetes
(moulds)/Ascomycetes. To aid in the identifications of closely related species from the genera
Aspergillus and Penicillium, the ß-tubulin and calmodulin sequences were used for BLAST
identification.

With regard to the discolouration of bamboo by blue-stain fungi, laboratory experiments

showed that the samples were colonized by blue-stain fungi within four weeks of incubation.
The bamboo tissue contained the typical thick, brown hyphae and chlamydospores of
blue-stain fungi, as well the thin perforation hyphae, transpressoria, with which staining fungi
penetrate lignified cell walls, similar to that observed in as it wood cells.

As is true for all lignocellulosic materials, bamboo (during use and storage) is susceptible to
wood-rot fungi. Literature observations describe fungal attack on bamboo to be mainly white-
rot Basidiomycetes and soft-rot Ascomycetes. These results identified the white-rot species
Schizophyllum commune and the secondary saprobiont Cyathus stercoreus from imported
culms. S. commune is often found on bamboos; however, the fungus revealed only low
activity in decay tests.

To investigate the deterioration of bamboo culms by rot fungi, degradation studies were
performed with samples of different bamboo species. Laboratory experiments included the
EN 113 standard test in Kolle flasks and an agar test in preserving jars. The novel technique
with vermiculite in preserving jars as a moisture and nutrient reservoir proved to be
particularly suitable for wood samples as shown by the considerable mass loss of Pinus
sylvestris wood by Coniophora puteana. The reason that the technique failed for bamboo may
be due to the used fungi C. puteana and S. commune. Future studies should include other
Basidiomycetes.

103
To imitate more natural conditions for fungi, larger-sized bamboo samples were tested in the
―Fungus cellar‖, where the bamboo is inoculated with fungal pure cultures and placed on
unsterile soil. In addition to the EN standard fungi, several white-, brown- and soft-rot fungi
were used, along with different strains to consider strain variation.

The investigated bamboo species differed in their susceptibility to fungal deterioration.

However, because the samples were derived from imported culms, important factors
influencing the durability, like culm age, harvest season and position in the culm, were
unknown. Future studies should consider these parameters.

With regard to the different groups of rot fungi, bamboo behaved rather resistant against
brown rot, whereas soft-rot and white-rot fungi produced considerable deterioration.

As reason for the absence of brown-rot fungi among the isolations and for their low activity in
the decay tests, it is assumed that the major bamboo hemicellulose is of the xylan-type,
similarly as in hardwoods, which are the preferred wood substrates for white-rot fungi,
whereas brown-rot fungi prefer softwoods with mannan as the hemicellulose. Soft-rot fungi
have not been isolated because these fungi mainly occur in culms with soil and water contact.
Correspondingly, a field test revealed soft-rot in the outer layer of the in-ground stakes. The
stake centres showed white-rot.

For information on the enzymatic events during bamboo rotting, samples of known mass loss
were subjected to chemical analyses. The investigated white-rot fungi Pleurotus ostreatus and
Trametes versicolor consumed the three cell wall components (cellulose, hemicelluloses and
lignin) almost uniformly at the same time and at a similar rate during the decay stages, which
is characteristic of the simultaneous type of white-rot. Remarkable is the attack of the soft-rot
fungus Chaetomium globosum on the lignin component, probably mainly due to
demethylation of aromatic rings.

For microscopic information on the micromorphology of fungal attack, subcellular analyses

of the cell tissue of the Bambusa maculata culm degraded by C. stercoreus, the white-rot
fungi P. ostreatus and T. versicolor as well the soft-rot fungus Ch. globosum were carried out
using light microscopy, TEM and UV microspectrophotometry. The TEM results provided

104
insight into morphological changes of the cell wall structure during decay. The UMSP area
scans and additional point analysis measurements revealed insights into the topochemistry in
situ. In particular, the latter technique gave information on the lignin modification process
during cell-wall decomposition. The combination of TEM investigation and UV
microspectrophotometry constituted a useful approach to determine topochemical variations
of lignin distribution within individual cell wall layers.

Altogether, the results improved the basic view on the degradation of bamboo by fungi, which
are needed for a better understanding of bamboo degradation by fungi.

105
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116
List of publications

Publication I
Schmidt O, Wei D, Liese W. 2011. Fungal degradation of bamboo samples. Holzforschung,
65(6): 883-888.

Publication II
Wei D, Schmidt O, Liese W. 2012. Susceptibility of bamboo to fungi. 9th World Bamboo
Congress, 235-245.

Publication III
Wei D, Schmidt O, Liese W. 2013. Durability test of bamboo against fungi according to EN
standards. European Journal of Wood and Wood Products, 71(5): 551-556.

Publication IV
Wei D, Schmidt O, Liese W. 2013. Method to test fungal degradation of bamboo and wood
using vermiculite as reservoir for moisture and nutrients. Maderas. Ciencia y
tecnologí
a, 15(3): 349-356.

117
Conference contributions

Oral presentations

Wei D, Schmidt O, Liese W. Susceptibility of bamboo to fungi. 9th World Bamboo Congress,
Antwerp, Belgium, 10 April, 2012.
Wei D. Pilzempfindlichkeit von Bambus. Wood Biology Colloquium, University of Hamburg,
Germany, 03 May, 2012.
Schmidt O, Wei D, Tang TKH, Liese W. Bamboo and fungi. 2nd International Conference of
Biodeterioration of Wood and Wood Products BWWP, Tartu, Estonia, 24-27 April,
2013.

Poster

Wei D, Schmidt O, Liese W. Molecular identification of bamboo-inhabiting and degrading

fungi. Annual Conference of the Association for General and Applied Microbiology,
Karlsruhe, Germany, 3-6 April, 2011.
Wei D, Schmidt O, Schmitt U, Liese W. Fungal decay studies on bamboo species.
4th Congress of European Microbiologists, Geneva, Switzerland, 26-30 June, 2011.
Wei D, Schmidt O, Liese W. Fungal degradation studies on bamboo. 15th International
Biodeterioration and Biodegradation Symposium, Vienna, Austria, 19-24 September,
2011.

118
 Appendices

Publication I

119
 Article in press - uncorrected proof
Holzforschung, Vol. 65, pp. 883–888, 2011 • Copyright
by Walter de Gruyter • Berlin • Boston. DOI 10.1515/HF.2011.084

Fungal degradation of bamboo samples

Olaf Schmidt*, Dong Sheng Wei, Walter Liese and by sampling (the height of the culm, where the sample was
Elisabeth Wollenberg taken), the cutting season, moisture content, and the age of
the plants. The intention of the present paper is a contribution
Division of Wood Biology, Department of Wood Science,
to this complex subject. The results obtained from a large
University of Hamburg, Hamburg, Germany
number of bamboo species under well-defined sampling and
*Corresponding author. test conditions will be described. Though data from field
Division of Wood Biology, Department of Wood Science, tests are very useful, data obtained from laboratory experi-
University of Hamburg, Leuschnerstr. 91d, D-21031 Hamburg,
Germany ments under controlled conditions are more precise and have
E-mail: o.schmidt@holz.uni-hamburg.de a better reproducibility. This approach was chosen for the
presented results.

Abstract
Materials and methods
The degradation of several Asian bamboo species by white-,
brown-, and soft-rot fungi was investigated under laboratory Investigated fungi
conditions by means of different test methods. Severe dete-
The fungi in focus are listed in Table 1. The isolates are stored in
rioration was caused by all three fungi types. The bamboo our strain collection.
species differed in durability. Samples from 6 months young
culms decayed more than older ones. There were no signif-
Degradation tests in preserving jars
icant differences between 1- and 3-year-old culms. Samples
taken from the culm top were more vulnerable to decay than Investigations were conducted on culm sections of Bambusa macu-
those from the bottom. Wet bamboo samples with soil con- lata and Gigantochloa atroviolacea from Indonesia and Phyllosta-
tact were especially degraded by the white-rot fungus Schi- chys pubescens from Germany. Bamboo specimens were obtained
zophyllum commune, whereas the brown-rot fungus from CONBAM (Geilenkirchen, Germany) and the Bamboo Centre
Coniophora puteana produced the greatest mass loss in drier (Baden-Baden, Germany). The curcuma-test (Peylo 2001) was
samples. The sealing of bamboo crosscut ends reduced the applied to ensure that the samples are not contaminated with boron
compounds which are common for moulding prevention during stor-
rate of decay.
age. Samples (3=1 cm2) were dried at 1038C, weighed, and auto-
claved. Household preserving jars with a volume of 500 ml were
Keywords: bamboo; bamboo moisture content; brown-rot autoclaved for 20 min and used as culture vessels (Schmidt 1986).
fungi; culm age; fungal degradation; soft-rot fungi; white-rot Culture medium for the basidiomycetes and ascomycetes were
fungi. 110 ml malt (2%)-agar (1.5%) and Abrams agar (Savory 1954),
supplemented with 0.1% yeast extract as vitamin source, respec-
tively. The jars were inoculated with fresh mycelial agar plugs
(8 mm2) and incubated at 218C and 70% RH for 12 months.
Introduction
Evaluation of fungal mass loss in all experiments was done
according to EN 113 (1996). Minimum and maximum mass loss
Bamboo is an abundant natural product with excellent tech- values, species, the special fungal strains tested, and the number of
nological properties. However, the use of bamboo is restrict- sample replicates are presented in the Tables.
ed due to its susceptibility to deteriorating organisms and its
generally low durability (Liese and Kumar 2003). Several Degradation tests in Kolle flasks
publications are available on the decay of bamboo by fungi
and on its natural durability (Banerjee and Mukhopadhyay To test the influence of culm age and sample location, the species
1962; Purushotham 1963; Abdurachim 1964; Liese 1985; Bambusa polymorpha, Dendrocalamus strictus, Melocanna bam-
Murphy et al. 1991; Leithoff and Peek 2001; Hamid et al. busoides, Oxytenanthera nigro-ciliata, and Thyrsostachys oliveri
2003; Zhang et al. 2007; Kim et al. 2008, 2011; Suprapti were collected at the Experimental Forests of the Forest Research
2010; Ma et al. 2010). Such information can improve pro- Institute (Dehra Dun, India), and of the College of Forestry (Laguna),
during consultancy trips by one of the authors (W. Liese) between
tection measures and lead to better utilisation of bamboo.
1960 and 1970. Specimens with a wall thickness of 5=2 cm were
Many data are available from field tests (i.e., ‘‘graveyard’’
autoclaved for 15 min at 1218C. The experiments with five parallels
tests: Liese 1959; Abdurachim 1964; Wang and Hsieh 1968; were performed according to the Kolle flask-method with malt-agar
Razak et al. 2002). for the basidiomycetes (Liese et al. 1935, EN 113 1996) and
The large body of literature data is not always consistent Abrams-agar for the soft-rot fungi. A number of experiments were
because of the variability of the bamboo species and fungi conducted on samples with sealed crosscut ends (KAHAPON, Kurt
tested. Moreover, it is probable that the results are influenced Herberts Coating Company, Wuppertal, Germany). Kolle flasks

2011/005
 Article in press - uncorrected proof
884 O. Schmidt et al.

Table 1 Fungi investigated.

Codings for
Type of Lab.
Species rot isolate EN Origin and isolation by
Pleurotus ostreatus White 11 ATCC 44737 Tree, Steinach, Germany, W. Luthardt f1949
Schizophyllum commune White 3 Wood, Spain, 1966
Schizophyllum commune White 4 Philippines, 1966
Schizophyllum commune White 87 Dendrocalamus brandisii, Costa Rica, D.S. Wei 2009
Schizophyllum commune White 98 Dendrocalamus asper, Thailand, D.S. Wei 2009
Trametes versicolor White 63 CTB 863A, EN 113 France
Coniophora puteana Brown 1 Ebw. 15, EN 113 Wood, Berlin, J. Liese 1930
Coniophora puteana Brown 167 Wood, Hamburg, O. Schmidt 1997
Gloeophyllum trabeum Brown 183 Ebw. 109, EN 113 Wood, Eberswalde, Germany
Oligoporus placenta Brown 120 FPRL 280, EN113 Wood, Berlin, J. Liese 1938
Chaetomium globosum Soft 10 ATCC 44753 Wood, München 1963
Chaetomium globosum Soft 76 ATCC 6205, EN 807 Wood, Berlin, BAM 1989
Paecilomyces variotii Soft 13 ATCC 44741 Sugar cane bagasse, Trinidad, K. Walter 1977
Paecilomyces variotii Soft 92 DSM 1961 Wood, Berlin, BAM 1990

were kept at 208C for the basidiomycetes and at 288C for the soft- infected with three small inoculation wood pieces (0.5 cm3) con-
rot fungi in a period of 4–6 months. taining mycelium. The tubs were covered with panes of glass and
kept at 238C and 90% RH for 1 year. The soil was moistened weekly
Degradation tests on samples with larger dimension with sprayed tap water.
(Fungus cellar-test)

Culm parts of Arundinaria amabilis (Vietnam), Bambusa maculata Results and discussion
(Indonesia), Dendrocalamus asper (Indonesia), Gigantochloa atro-
violacea (Indonesia), Phyllostachys nigra (Japan), Phyllostachys The fungi investigated (Table 1) are not endemic in the nat-
nigra ‘Boryana’ (China) and Phyllostachys pubescens (Germany)
ural distribution area of bamboo. However, several strains
were obtained from the CONBAM company and the Bamboo Cen-
served for decades in wood degradation experiments (Liese
tre (see above). Sections (25 cm) were longitudinally halved, dried
at 1038C and weighed, dipped in tap water for two days and auto- et al. 1935), which led to the German standard DIN 52 176
claved at 1218C for 40 min. Metal tubs (120 cm long, 60 cm wide) and to the current European standard EN 113 (1996). Some
were filled with 30 l of unsterile compost soil from the institute fungi occur on bamboo (e.g., Mohanan 1997) or have been
garden (Gersonde and Becker 1958). Samples were placed either on isolated from bamboo culms in their natural habitat by the
autoclaved wood supports or directly on the soil. Each sample was authors of the present study.

Table 2 Decay of bamboo species (min–max and average) expressed as % mass

loss of three replicates after 1 year of incubation in preserving jars.

Mass loss (%) caused on the species

Bambusa Gigantochloa Phyllostachys
Fungus, code maculata atroviolacea pubescens
Pleurotus ostreatus 11 22.9–35.7 8.5–13.6 19.7–22.3
28.2 10.6 21.0
Schizophyllum commune 87 2.7–2.9 6.7–6.8 4.7–5.6
2.8 6.7 5.2
Schizophyllum commune 98 1.6–2.0 4.5–7.3 3.6–5.1
1.8 5.6 4.4
Trametes versicolor 63 60.2–64.0 45.7–55.4 44.5–54.0
62.5 51.6 47.8
Coniophora puteana 167 2.8–4.2 5.2–5.9 4.6–4.8
3.6 5.6 4.7
Gloeophyllum trabeum 183 1.8–2.0 4.8–6.7 4.3–7.2
1.9 5.7 5.3
Chaetomium globosum 10 31.2–32.7 7.6–12.7 36.9–39.7
31.8 9.4 38.0
Paecilomyces variotii 13 0.8–1.4 3.3–4.0 3.4–4.2
1.2 3.6 3.9
 Article in press - uncorrected proof
Fungal degradation of bamboo samples 885

Table 2 shows the results of bamboo degradation after rot fungi in samples of three Phyllostachys species and a
1 year of incubation in preserving jars. Among the white-rot maximum of 18% by 12 soft-rot fungi.
fungi, Schizophyllum commune caused the least decay (max The tested bamboo species differed in susceptibility to
6.7% mass loss, ML), Pleurotus ostreatus medium decay fungi (see also Tables 4 and 5). However, more comparative
(max 28.2% ML), and Trametes versicolor the highest (max studies are necessary. Suprapti (2010) showed Bambusa vul-
62.5% ML) decay. Abdurachim (1964) reported a 15% ML garis, Gigantochloa apus and G. atroviolacea to be moder-
by S. commune. A low decay rate by S. commune was found ately resistant, whereas G. pseudoarundinacea and
by Suprapti (2010) and Kim et al. (2011) and was also Dendrocalamus asper were not resistant. Hamid et al. (2003)
reported for wood samples (Schmidt and Liese 1978). How- found G. scortechinii less susceptible to brown-rot (Conio-
ever, this species is commonly found on bamboo culms dur- phora puteana) compared to white-rot (Trametes versicolor).
ing storage and use (Liese 1985; Mohanan 1997; Liese and Differences obtained by field tests have been also reported.
Kumar 2003). Kleist et al. (2002) showed that S. commune Phyllostachys makinoi and Ph. edulis were more resistant to
was the most successful coloniser among some fungi as it sapstain and decay fungi than Leba dolicochoclada and espe-
could penetrate bamboo via outer and inner culm walls, cially Sinocalamus latiflorus (Wang and Hsieh 1968).
through cross section planes as well as through nodal ridges It is likely that the cutting season has an influence on
and longitudinally through nodiums. Both brown-rot fungi, decay resistance because starch and protein content vary dur-
Coniophora puteana and Gloeophyllum trabeum, were rela- ing the year and change with culm age (Magel et al. 2006).
tively inactive (causing max 5.7% ML). This finding is in Hamaguchi (1953) investigated the decay resistance of three
contrast to observations made by Lee et al. (2006), who Phyllostachys species at monthly intervals and obtained the
reported a 25% ML by G. trabeum in Phyllostachys pubes- lowest mass loss in samples cut between September and Feb-
cens, and by Ma et al. (2010), who found a 35% ML in P. ruary, the time after sprouting.
edulis. Among the soft-rot fungi, Paecilomyces variotii was When comparing the results of mass loss experiments in
less aggressive, whereas Chaetomium globosum produced the literature, the possibility of chemical pretreatment of
severe degradation, which also occurred in the other tests. A bamboo species against fungi should be kept in mind. For
Japanese isolate of Ch. globosum caused less decay (Suprapti example, boron treatment is often used before shipment to
2010). Experiments by Leithoff and Peek (2001) on P. pubes- Europe and would severely affect subsequent decay experi-
cens and P. virideglaucescens in Petri dishes also gave com- ments. Our samples were boron-free as proven by the cur-
parable results with moderate decay by S. commune and C. cuma-test. Furthermore, the results of all experiments on
puteana and stronger decay by soft-rot according to the stan- fungal activity strongly depend on the type of isolate
dard ENV 807 (2001). Zhang et al. (2007) investigated 34 involved; strain variation is common among fungi (Schmidt
white-rot fungi and observed up to 15% ML in P. pubescens; and Liese 1980). In our tests, Tables 2 and 3 show similar
Pleurotus ostreatus caused 5.2% and Trametes versicolor results for the two S. commune isolates.
13.6% ML. The white-rot fungus Lentinula edodes produced The influence of culm age on decay resistance was inves-
a 13% ML in P. pubescens (Kim et al. 2008). Suprapti (2010) tigated for Melocanna bambusoides (Table 3). Samples from
observed intense decay by the white-rot fungus Pycnoporus the youngest culm (6 months) were more susceptible to fun-
sanguineus and the brown-rot species Tyromyces palustris. gi, particularly to Trametes versicolor and the two soft-rot
Kim et al. (2011) observed a ML of up to 22% by six white- fungi. The decay of samples from 1-, 2-, and 3-year-old

Table 3 Influence of culm age on decay of Melocanna bambusoides samples

(min–max and average) expressed as % mass loss of five replicates after 6 months of
incubation in Kolle flasks; unpublished data from 1970.

Mass loss (%) as a function of the culm age in years

Fungus, code 0.5 years 1 year 2 years 3 years
Schizophyllum commune 3 7.1–7.8 6.1–7.6 4.8–5.3 4.1–5.1
7.4 6.9 5.0 4.6
Schizophyllum commune 4 8.1–10.2 5.8–7.0 5.1–5.8 3.1–5.0
9.1 6.4 5.6 4.8
Trametes versicolor 63 21.6–42.6 15.8–17.8 11.5–13.4 12.7–16.9
28.3 16.9 12.5 14.7
Coniophora puteana 1 8.5–15.3 10.3–17.0 6.8–9.5 6.3–12.8
11.2 13.7 8.4 9.9
Oligoporus placenta 120 4.3–7.0 4.9–11.2 4.8–6.1 4.6–9.9
5.6 6.8 5.4 6.0
Chaetomium globosum 76 51.2–54.5 30.2–32.8 31.1–34.7 25.4–30.4
52.7 31.4 32.3 27.9
Paecilomyces variotii 92 18.1–20.2 8.4–10.7 6.2–10.3 6.6–10.4
19.7 9.6 8.2 7.5
 Article in press - uncorrected proof
886 O. Schmidt et al.

Table 4 Influence of culm section (top/base) and sample accessibility (all sample sides open/crosscut areas sealed) on decay
(min–max and average) expressed as % mass loss of 5 replicates after 6 months of incubation in Kolle flasks; unpublished data
from 1970.

Mass loss (%) caused by the fungi

Culm Sample Schizophyllum Coniophora Oligoporus Chaetomium Paecilomyces
Bamboo species section sealing commune 4 puteana 1 placenta 120 globosum 76 variotii 92
Bambusa Top No 4.6–6.1 11.5–14.4 7.2–13.4 24.3–41.0 12.0–16.3
polymorpha 5.9 12.1 12.8 33.7 14.1
Yes 4.0–4.6 9.4–13.8 4.0–4.6 11.9–14.5 8.6–10.5
4.4 12.4 4.4 13.3 9.7
Base No 3.6–4.3 6.2–16.3 3.1–5.7 20.7–26.6 7.9–10.2
4.1 15.5 5.7 23.3 9.4
Yes 2.8–3.7 4.4–6.2 1.4–2.3 11.7–14.2 5.8–9.9
3.3 5.5 1.9 12.6 8.1
Dendrocalamus Top No 2.5–3.0 22.9–45.9 5.7–13.6 21.6–33.5 6.8–11.7
strictus 2.7 38.2 11.3 28.2 9.3
Yes 1.8–3.0 6.9–11.3 3.6–12.6 13.5–19.3 3.2–5.2
2.4 8.8 7.7 15.9 4.1
Base No 0.1–10.9 8.3–18.7 3.5–11.7 6.2–7.3 1.8–2.3
4.7 12.7 9.8 6.7 2.1
Yes 1.4–3.5 1.5–3.6 1.1–7.9 6.0–8.0 1.6–2.0
2.0 2.7 3.0 7.2 1.8
Oxytenanthera Top No 0.7–3.0 26.8–51.0 0.7–17.5 38.5–43.6 1.7–17.5
nigro-ciliata 2.1 38.5 7.4 41.1 7.4
Yes 1.8–2.7 8.9–15.1 0.2–4.1 8.2–13.8 1.6–3.6
2.2 11.8 1.4 11.1 2.2
Base No 2.6–3.1 3.2–5.4 1.3–1.9 19.2–22.8 4.5–7.1
2.8 4.0 1.6 21.1 6.0
Yes 1.8–2.7 2.3–2.8 0.5–1.3 5.8–7.5 3.1–3.8
2.2 2.6 0.8 6.5 3.5
Thyrsostachys Top No 5.2–8.5 11.9–26.9 3.4–8.9 38.7–54.2 6.5–8.9
oliveri 6.8 18.5 6.4 47.2 7.7
Yes 6.0–7.7 9.5–11.5 4.8–6.4 13.0–13.7 7.8–9.5
7.0 10.4 5.7 13.3 8.5
Base No 2.7–3.6 5.2–10.0 1.8–2.8 23.0–29.3 4.7–6.4
3.2 8.2 2.3 27.2 5.2
Yes 0.4–2.6 4.7–5.7 1.5–1.8 8.5–9.1 2.4–3.1
1.9 5.3 1.6 8.8 2.7

culms differed only minimally. In Gigantochloa scortechinii, culm, whereas the bottom and top parts were not resistant.
younger culms (6 months) were more susceptible to Conio- Sealing the ends may force the hyphae to penetrate through
phora puteana and T. versicolor than 6.5-year-old culms the epidermis of the samples and not through the vessels at
(Hamid et al. 2003). The higher decay rate in young culms the crosscut plane. In most cases, samples accessible from
can be attributed to a higher content of carbohydrates and all sides were more decayed than sealed ones (Table 4).
proteins in parenchyma cells. On the other hand, Schwarze Kleist et al. (2002) also found the transverse faces to be the
(2007) observed negligible degradation of balsa wood con- easiest route for fungi penetration into the culm.
sisting of 92% parenchyma cells and explained this finding To investigate less active fungi, e.g., S. commune and C.
by inhibiting effect of parenchyma cells against brown-rot puteana, in more detail and to imitate natural conditions,
decay. decay tests with bigger sized samples were performed in the
To investigate the influence of sample location within a ‘Fungus cellar’ by incubating bamboo samples in unsterile
culm, samples were taken from the top and bottom of a culm compost soil in large metal tubs (Table 5). Due to the not
(Table 4). Half of the samples had sealed ends because bam- absolutely identical conditions in the two tubs, the Table
boo cross ends have large vessels and are easily accessible shows the data of all samples. In contrast to results with
for penetrating hyphae and therefore this fact may have an small samples in the jar test, bamboo samples in the Fungus
influence on decay. In general, samples from the top were cellar were considerably more degraded by C. puteana (max
more susceptible to decay than from the bottom. Top culms 43%) and S. commune (max 20%). Obviously, the moisture
have the highest starch content (Abd Latif et al. 1992; Liese conditions in the Fungus cellar-test influenced the activity of
and Abd Latif 2000; Okahisa et al. 2007). Suprapti (2010) both fungi, whereby the white-rot fungus S. commune dif-
reported moderate resistance for the middle portion of a fered considerably from the brown-rot species C. puteana.
 Article in press - uncorrected proof
Fungal degradation of bamboo samples 887

Table 5 Decay (% mass loss, ML) and final moisture content (MC, %) of bamboo samples in
metal tubs after 1 year in fungus cellar-test.

Coniophora Schizophyllum
puteana 167 commune 87
Soil
Species Tub contact ML (%) MC (%) ML (%) MC (%)
Arundinaria amabilis 1 Yes 15.5 187 15.3 148
1 No 38.6 41 10.8 31
2 No 43.4 57 8.7 68
2 No 39.0 56 7.1 40
Bambusa maculata 1 Yes 9.9 159 11.0 174
1 No 20.4 39 5.1 28
2 No 38.3 48 5.0 33
2 No 34.3 52 3.6 37
Dendrocalamus asper 1 Yes 5.4 104 5.1 90
1 No 29.3 42 4.3 28
2 No 26.9 45 3.8 32
2 No 28.5 50 4.3 31
Gigantochloa 1 Yes 6.1 95 6.1 95
atroviolacea 1 No 18.7 34 4.7 25
2 No 41.6 58 3.0 38
2 No 42.9 57 5.0 43
Phyllostachys nigra 1 Yes 9.7 112 16.4 126
1 No 32.6 49 9.1 32
2 No 39.7 65 6.6 40
2 No 40.7 56 6.4 52
Phyllostachys 1 Yes 35.3 103 19.7 182
nigra ‘Boryana’ 1 No 38.8 54 6.7 26
2 No 37.9 60 5.7 40
2 No 38.8 46 5.1 35
Phyllostachys 1 Yes 6.3 61 6.3 63
pubescens 1 No 5.4 32 5.4 24
2 No 38.3 42 5.7 31
2 No 31.2 43 6.3 36

Schizophyllum commune decayed samples with soil contact durability of this bamboo and the wood of Hevea brasilien-
and thus with high water content (90–182% MC) more than sis. Hamid et al. (2003) classified the resistance of Gigan-
samples without soil contact. In contrast, C. puteana pro- tochloa scortechinii against brown-rot and white-rot among
duced a higher mass loss in samples located on wood sup- 24 Malaysian heavy and medium hardwoods. When com-
ports and with a lower water content (34–65% MC). For paring Dendrocalamus asper, D. gigantochloa and Albizia
practical purposes, bamboo samples in the range of falcata, Suhirman and Khusniati (1987) attributed the low
24–182% MC were attacked by basidiomycetes. However, mass loss of bamboo species (18–21%) to their relatively
due to the unsterile soil in the Fungus cellar, samples became high lignin content (24–27%). After six weeks, our decay
contaminated by moulds which may also have excreted a tests comparing bamboo and wood revealed a 25% ML by
growth promoting substance for S. commune. Substances Chaetomium globosum in Oxythenanthera abyssinica and
from the soil or vitamins from soil bacteria may have also 45% ML in the non-resistant hardwood beech.
affected the fungus. In summary, the results show that all bamboo species
When comparing the decay resistance of bamboo in com- investigated in this study can be considerably degraded by
parison to that of wood, the different anatomical and chem- white-, brown- and soft-rot fungi with distinct differences
ical composition of both tissues must be taken into account. between species, culm location and sample moisture content.
Whereas wood contains about 80–95% sclerenchyma cells
in form of fibres and tracheids, the amount of these types of
cells is in bamboo much lower. The culm consists of roughly Acknowledgements
50% parenchyma cells, 40% fibres and 10% conducting cells
(vessels and phloem; Grosser and Liese 1971; Liese 1998).
We thank the Forest Research Institute, Dehra Dun and the FOR-
These proportions tend to vary greatly within the culm; at PRIDECOM, Laguna for providing local specimens, Wolfgang
the culm base, the quantity of parenchyma cells is highest Eberts (Bamboo Centre Germany) and Christoph Tönges
and decreases with increasing height with a corresponding (CONBAM, Germany) for the other samples as well as the Gesell-
increase in the proportion of fibres. Field tests with Gigan- schaft der Freunde und Förderer des Zentrums Holzwirtschaft der
tochloa apus by Abdurachim (1964) showed a similar low Universität Hamburg (GFF) for financial support.
 Article in press - uncorrected proof
888 O. Schmidt et al.

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AD
BNMRHCDQDC
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HRNK@SDR
@QD
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MNS
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C@S@
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AX
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RDPTDMBDR
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4DRRHNM
  #HNKNFX
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XWZW]US[\ %
@MC
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SG@S
LNRS
NE
SGD
HRNK@SDC
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ETMFH
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TMCDQ
SGD
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TRD
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NE
SGD
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#KTDRS@HM
%HRBNKNTQ@SHNM
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4@LOKDR
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R@LOKDR
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56>2E:@:56D '6DE2=@E:@AD:D
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SN
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NE
HMBTA@SHNM

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DWODQHLDMS@K
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HR
CDLNMRSQ@SDC
HM
'HFTQD

'HFTQD

RGNVR
DW@LOKDR
NE
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KHFGS
LHBQNRBNOHB
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» ½

<:HE8
H?GHE8F
B9
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4
4A7

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5
B9
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B?BA<M4G<BA
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5GD
SHRRTD
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AX
SGD
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SGHBJ
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@MC
BGK@LXCNRONQDR
NE
AKTD
RS@HM
ETMFH
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RNLD
B@RDR
@
SQ@MROQDRRNQHTL
@
SGHM
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GXOG@
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QHFGS
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5GD
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HR
SGD
NMKX
ETMF@K
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AX
VGHBG
AKTD
RS@HM
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C@L@FD
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BDKK
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%DFQ@C@SHNM
NE
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AX
'TMFH
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RGNVR
SGD
DWODQHLDMS@K
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CDB@X
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5@AKD

RTLL@QHYDR
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L@RR
KNRR
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4DRRHNM
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»
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;p./6N=/ ./0 z/4.0 <6. 3Q09403 *49:K@A9J==F>
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@
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 926E@>:F>
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4
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4
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NE
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SGD
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T
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4DRRHNM
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UDRRDKR
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A
NE
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HM
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V@KKR
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SGD
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SGD
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65@56D .@
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» ½ ¼

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4
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5EBJAEBG
FL@CGB@F
<A *("-3.$)+."
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1QNEDRRNQ
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4DRRHNM
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HIåI(I&,IJ

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#@LANN
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 Publication III

138
Eur. J. Wood Prod. (2013) 71:551–556
DOI 10.1007/s00107-013-0707-2

ORIGINALS ORIGINALARBEITEN

Durability test of bamboo against fungi according to EN standards

Dongsheng Wei • Olaf Schmidt • Walter Liese

Received: 25 January 2013 / Published online: 19 June 2013

Ó Springer-Verlag Berlin Heidelberg 2013

Abstract The durability of five bamboo species from stärksten Abbau (max. 15,3 % ), während Schizophyllum
various origin against brown-, white- and soft-rot fungi was commune kaum aktiv war (max. 3,2 %). Bei den Moder-
investigated in Kolle flasks in accordance with the Euro- fäulepilzen ergab Ch. globosum mittleren (max. 9,6 %) und
pean standards EN 350-1, EN 350-2 and EN 113. Con- Paecilomyces variotii geringen Abbau (max. 3,1 %).
siderable variability exists in the durability of the bamboo
species. Guadua angustifolia was rather resistant to Tra-
metes versicolor and Dendrocalamus asper against Chae- 1 Introduction
tomium globosum. Among the brown-rot fungi, the four
strains of Coniophora puteana and two strains of Gloeo- Bamboo is a fast growing woody grass of increasing interest
phyllum trabeum produced low mass loss (maximum for the sustainable production of materials with many
2.9 %). Of the white-rot fungi, T. versicolor yielded the applications for buildings and industrial utilization. How-
highest decay (max. 15.3 %), whereas Schizophyllum ever, bamboo has generally a low natural durability and is
commune was rather inactive (max. 3.2 %). Of the soft-rot easily attacked by fungi during storage, transport and final
fungi, Ch. globosum showed medium degradation (max. use. For applications it is important to know its susceptibility.
9.6 %) and Paecilomyces variotii low decay (max. 3.1 %). Like other lignocellulose materials, bamboos are subject
to biodegradation by fungi under particular conditions
Dauerhaftigkeitsprüfung von Bambus gegenüber Pilzen which affects their quality (Hamid et al. 2003). The resis-
gemäß EN-Standards tance of bamboo against decay fungi serves as an important
parameter in bamboo uses. Several factors may influence
Zusammenfassung Fünf Bambus-Arten verschiedener bamboo natural durability, for example site, growth rate,
Herkunft wurden in Kolleschalen auf ihre Anfälligkeit für age, portion of bamboo, extractives content and the
Braun-, Weiß- und Moderfäulepilze gemäß EN 350-1, EN microenvironment. The results obtained in this work,
350-2 und EN 113 untersucht. Die Bambus-Arten unter- together with other data reported in literature on the decay
schieden sich in ihrer Pilzanfälligkeit. Guadua angustifolia of bamboo (Liese 1959, 1985; Banerjee and Mukhopad-
war besonders widerstandsfähig gegen Trametes versicolor hyay 1962; Purushotham 1963; Abdurachim 1964; Wang
und Dendrocalamus asper gegen Chaetomium globosum. and Hsieh 1968; Murphy et al. 1991; Razak et al. 2002,
Bei den Braunfäule-Erregern ergaben die vier Stämme von 2006; Hamid et al. 2003; Zhang et al. 2007; Kim et al.
Coniophora puteana und beide Isolate von Gloeophyllum 2008, 2011; Suprapti 2010; Ma et al. 2010; Schmidt et al.
trabeum nur geringen Masseverlust (maximal 2,9 %). Von 2011; Wei et al. 2012) may contribute to the character-
den beiden Weißfäulepilzen zeigte T. versicolor den ization and appreciation of bamboo species. Such charac-
terization is important not only for its correct utilization but
also for the market promotion.
D. Wei (&)
O. Schmidt
W. Liese
When comparing data, another difficulty is due to the
Section Wood Biology, Centre for Wood Science, University of
Hamburg, Leuschnerstr. 91d, 21031 Hamburg, Germany different tests performed, such as field and laboratory tests.
e-mail: dongsheng.wei@uni-hamburg.de Also within the same test, the different standards (Asia,

123
552 Eur. J. Wood Prod. (2013) 71:551–556

Table 1 Fungi investigated

Tab. 1 Untersuchte Pilze
Rot type Species Laboratory isolate coding Other codings/ Origin/isolation by
use for EN

Brown Coniophora puteana 1 Ebw. 15/EN 113 Building, Berlin, Germany/J. Liese 1930
Coniophora puteana 159 Wood, Soerfosa, Sweden/T. Nilsson 1995
Coniophora puteana 167 Building, Hamburg, Germany/O. Schmidt 1997
Coniophora puteana 247 Building, Karlsruhe, Germany/K. Grimm 1990
Gloeophyllum trabeum 183 Ebw. 109/EN 113 Building, Eberswalde, Germany/J. Liese
Gloeophyllum trabeum 259 Cellar stairs, Lörrach, Germany/K. Grimm 1999
White Trametes versicolor 63 CTB 863/EN 113 France
Schizophyllum commune 87 Dendrocalamus brandisii, Costa Rica/D. Wei 2009
Soft Chaetomium globosum 10 ATCC 44753 Wood, Munich, Germany/1963
Paecilomyces variotii 13 ATCC 44741 Sugar cane bagasse, Trinidad/K. Walter 1977

European and American) may differ by sample size, were used as controls according to EN 113. All samples
duration of tests and fungal strains. Therefore, it is were put into a climate room at 20 ± 2 °C and 65 ± 5 %
important to test the bamboo species using the same stan- relative humidity (RH) for 4 weeks, weighed, wrapped in
dards. The aim of the study is to test bamboo durability plastic (Sengewald Flexopeel, Germany) and send to BBF
using European standards as used for wood species. Sterilisationsservice GmbH, Kernen, Germany for gamma
radiation.

2 Materials and methods 2.3 Durability test

2.1 Investigated fungi The durability experiment was performed with fungal pure
cultures in Kolle flasks with white-, brown-, and soft-rot
The ten strains of white-, brown- and soft-rot fungi used fungi according to the European standards EN 350-1
derive from the laboratory strain collection and are listed in (1996), EN 350-2 (1997) and EN 113 (1996). The culture
Table 1. medium consisted of 2 % malt extract (Merck) and 1.5 %
agar (Bacto) in distilled water. Flasks containing 48 ml
2.2 Bamboo and wood samples medium were plugged with cotton, sterilized in the auto-
clave at 121 °C for 30 min, inoculated with a mycelial plug
The five bamboo species tested are presented in Table 2. of the test fungus and incubated until mycelial growth
Culm sections were obtained from CONBAM, Geilenkir- covered the surface of the medium. Each two gamma ray
chen, Germany and the Bamboo Centre, Baden–Baden, sterilized samples were put aseptically in one flask and
Germany. Sections were proved with the curcuma-test each three flasks were used for one fungal strain, with
(Peylo 2001) to ensure that no boron was present which is altogether 480 Kolle flasks. After 16 weeks of incubation
commonly used against moulding during storage and at 20 ± 2 °C and 65 ± 5 % RH for the brown-rot and
transport. Samples were prepared by cutting to dimensions white-rot fungi and 28 ± 2 °C and 65 ± 5 % RH for the
of 5 (length) 9 2.5 (width) 9 0.5–3.5 cm (wall thickness). soft-rot fungi, the mycelium was removed from the sample
Wood samples from Fagus sylvatica and Pinus sylvestris surface, the samples were weighed for final wet weight
determination and oven dried at 103 °C. Percentage of
Table 2 Bamboo species tested mass loss was calculated by weight comparison before and
Tab. 2 Untersuchte Bambus-Arten after incubation. Statistical differences were analyzed using
Bamboo species Local name Origin variance analysis (ANOVA, SPSS software 2002).
Bambusa maculata Tutul Indonesia (Java)
2.4 Assessment of fungal growth and classification
Dendrocalamus asper Petung Indonesia (Java)
of bamboo durability
Gigantochloa atroviolacea Wulung Indonesia (Java)
Guadua angustifolia Guadua Columbia
The development of fungal growth on the specimens was
Phyllostachys pubescens Moso China
evaluated by the density and the percentage of the sample
Phyllostachys pubescens Germany
surface covered with mycelium (Table 3). Based on the

123
Eur. J. Wood Prod. (2013) 71:551–556 553

Table 3 Mycelium growth and durability class after 16 weeks of incubation

Tab. 3 Mycelwachstum und Dauerhaftigkeitsklasse nach 16 Wochen Kultivierung
Bambusa Dendrocalamus Gigantochloa Guadua Phyllostachys Phyllostachys
maculata asper atroviolacea angustifolia pubescens pubescens
China Germany
HD HC DC HD HC DC HD HC DC HD HC DC HD HC DC HD HC DC

Coniophora puteana 1 2 4 II 1 3 II 2 4 II 1 0 II 2 4 II 3 4 II
Coniophora puteana 159 2 4 II 2 4 II 2 4 II 2 2 II 2 4 II 2 4 II
Coniophora puteana 167 2 4 II 1 4 II 2 4 II 1 1 II 2 4 II 2 4 II
Coniophora puteana 247 2 4 II 2 4 II 2 4 II 0 0 II 2 4 II 2 4 II
Gloeophyllum trabeum 183 0 0 II 0 0 II 1 4 II 0 0 II 1 4 II 1 4 II
Gloeophyllum trabeum 259 1 2 II 1 4 II 1 4 II 0 0 II 1 3 II 1 4 II
Trametes versicolor 1 4 III 2 4 III 2 4 IV 1 2 II 3 4 IV 2 4 IV
Schizophyllum commune 2 4 II 1 4 II 2 4 II 0 0 II 2 4 II 2 4 II
Chaetomium globosum 2 4 III 2 4 II 2 4 III 1 2 III 2 4 III 2 4 II
Paecilomyces variotii 2 4 II 2 4 II 2 4 II 0 0 II 2 4 II 2 4 II
HD Hyphal density: 0, no growth; 1, sparse mycelium; 2, normal mycelium; 3, thick mycelium
HC Hyphal coverage: 0, no coverage; 1, 1–33 % coverage; 2, 34–66 % coverage; 3, 67–99 % coverage; 4, total coverage
DC Durability class: II, durable (mass loss \5 %); III, moderately durable (5–10 %); IV, little durable (10–30 %)

average mass loss, bamboo durability against fungi was 3.2 Durability of bamboo against brown-rot fungi
correlated to durability classes (Table 3) considering EN
350-1 (1996), Abdurachim (1975) and Djarwanto and All five bamboo species were rather resistant to degrada-
Suprapti (2004). tion by the various strains of the brown-rot fungi Conio-
phora puteana and Gloeophyllum trabeum (Fig. 1) without
significant variation among the strains. Strain variation
3 Results and discussion with regard to wood mass loss was shown for both species
(Schmidt et al. 2002a, 2002b). Both fungi comprised the
3.1 Growth of fungi and final moisture content test strains to be used in EN 113 (1996) for wood samples.
of bamboo samples In this standard, C. puteana strain Ebw. 15 produces about
20 % mass loss in wood and wood products. Among the
The development of hyphal growth was measured by four C. puteana strains, the maximum mass loss of bamboo
scoring the density of hyphal mats and the percentage was 2.9 %. The two G. trabeum strains also revealed no
coverage of the sample surface. After initial differences significant difference in bamboo degradation, regardless of
between the fungi within the first 2 weeks, most bamboo the species. Mass losses of the wood controls for the four
species were covered by mycelium after 16 weeks C. puteana strains and two G. trabeum isolates ranged from
between two-thirds of the surface and total coverage 19.7 to 62.7 % of decay. This shows suitable incubation
(Table 3). An exception with low surface growth occurred conditions for bamboo. The low activity against bamboo
on Guadua angustifolia. In general, coverage correlated correlates with previous work by the authors (Schmidt et al.
with hyphal density. The density of mycelium is a specific 2011; Wei et al. 2012) reporting maximum 5.7 % mass loss
feature for many fungi (Stalpers 1978). However, it must by both fungi for the same bamboo species of the same
not relate to fungal activity (Schmidt 2006). Among the origin after 1 year of incubation in preserving jars. How-
ten strains, Gloeophyllum trabeum 183 did not show any ever, samples of Melocanna bambusoides after 6 month
hyphae on the surface of samples from Bambusa macu- incubation in Kolle flasks had shown up to 13.7 % decay
lata, Dendrocalamus asper and Guadua angustifolia. by C. puteana Ebw. 15 (Schmidt et al. 2011). To prove
However, this strain produced some degradation (Fig. 1), these earlier results, the degradation test according to EN
obviously not by surface growth but by substrate myce- 350 and EN 113 considered sample sterilization by gamma
lium. The final moisture content of the samples ranged radiation. The highest mass loss of 3 % by G. trabeum is
from 39 to 101 %, which is generally a suitable range for lower than the one reported by Schmidt et al. (2011) with
fungal degradation. Former experiments showed fungal maximum 5.7 % of decay after 1 year in preserving jars.
bamboo degradation in the moisture range from 24 to However, both results contrast with Lee et al. (2006) who
187 % (Schmidt et al. 2011). used potato dextrose agar in petri dishes and found 25 %

123
554 Eur. J. Wood Prod. (2013) 71:551–556

Coniophora puteana 1 Coniophora puteana 159

Coniophora puteana 167 Coniophora puteana 247
Gloeophyllum trabeum 183 Gloeophyllum trabeum 259
4.0

3.5

3.0

Mass loss (%) 2.5

2.0

1.5

1.0

0.5

0.0
A B C D E F
Brown-rot fungi

Fig. 1 Mass loss caused by brown-rot fungi in 5 bamboo species after 16 weeks of incubation. Error bars represent mean ± standard deviation.
A Bambusa maculata; B Dendrocalamus asper; C Gigantochloa atroviolacea; D Guadua angustifolia; E Phyllostachys pubescens China;
F Phyllostachys pubescens Germany
Abb. 1 Masseverluste durch Braunfäulepilze bei 5 Bambus-Arten nach 16 Wochen Inkubation. Fehlerbalken zeigen ± Standardabweichung.
A Bambusa maculata; B Dendrocalamus asper; C Gigantochloa atroviolacea; D Guadua angustifolia; E Phyllostachys pubescens China;
F Phyllostachys pubescens Deutschland

degradation by G. trabeum in Phyllostachys pubescens. Trametes versicolor Schizophyllum commune

The reason for this discrepancy may be the use of different 25.0
isolates and cultivation methods.
Mass loss (%)

20.0
15.0
3.3 Durability of bamboo against white-rot fungi 10.0
5.0
Among the two white-rot fungi, Trametes versicolor
0.0
showed the greatest mass loss with 15.3 % for P. pubes- A B C D E F
cens from China, followed by P. pubescens from Germany White-rot fungi
(12.3 %) and Gigantochloa atroviolacea (10.4 %) (Fig. 2).
Guadua angustifolia was very resistant (2.3 %). Degrada- Fig. 2 Mass loss caused by white-rot fungi in 5 bamboo species after
16 weeks of incubation. Error bars represent mean ± standard
tion of the Fagus sylvatica control was 26.7 %. Schizo- deviation. A Bambusa maculata; B Dendrocalamus asper; C Gigan-
phyllum commune behaved rather inactive with maximum tochloa atroviolacea; D Guadua angustifolia; E Phyllostachys pu-
mass loss of only 3.2 %. Schmidt et al. (2011) obtained bescens China; F Phyllostachys pubescens Germany
with S. commune a maximum of up to 9.1 % degradation in Abb. 2 Masseverluste durch Weißfäulepilze bei 5 Bambus-Arten
nach 16 Wochen Inkubation. Fehlerbalken zeigen ± Standardabwei-
6 months in Kolle flasks and a maximum of 5.6 % mass chung. A Bambusa maculata; B Dendrocalamus asper; C Giganto-
loss during 1 year in preserving jars. Abdurachim (1964) chloa atroviolacea; D Guadua angustifolia; E Phyllostachys
reported 15 % mass loss by S. commune. Low decay rates pubescens China; F Phyllostachys pubescens Deutschland
by S. commune were found by Suprapti (2010) and Kim
et al. (2011). The beech wood control in this experiment 3.4 Durability of bamboo against soft-rot fungi
showed also negligible decay (0.8 %), which corresponds
to previous results (Schmidt and Liese 1978). However, The two soft-rot fungi, Chaetomium globosum and Paeci-
this species is common on bamboo during storage and use lomyces variotii revealed significant differences with
(Liese 1985; Mohanan 1997; Liese and Kumar 2003). regard to bamboo decay, with higher mass loss by Ch.
Kleist et al. (2002) showed that S. commune was the most globosum than by P. variotii (Fig. 3). Highest mass loss
successful coloniser among some fungi as it could pene- was caused by Ch. globosum to P. pubescens from China
trate bamboo via outer and inner culm walls, through cross (9.6 %), followed by Guadua angustifolia (8.1 %) and
section planes as well as through nodal ridges and longi- Bambusa maculata (7.5 %). Schmidt et al. (2011) reported
tudinally through nodes. up to 52.7 % decay by Ch. globosum after 6 months in

123
Eur. J. Wood Prod. (2013) 71:551–556 555

Chaetomium globosum Paecilomyces variotii bamboo seems to be rather resistant against brown-rot
25.0 fungi, whereas some soft-rot and white-rot fungi produce
considerable deterioration. Results correspond to findings
Mass loss (%)

20.0
15.0 by other authors in laboratory studies and to observations
10.0 in the practice, where bamboo is often colonized and
deteriorated by fungi.
5.0
0.0 Acknowledgments We thank Wolfgang Eberts (Bamboo Centre
A B C D E F Germany) and Christoph Tönges (CONBAM, Germany) for provid-
Soft-rot fungi ing bamboo sections and Marie-Therese Lenz for technical help.

Fig. 3 Mass loss caused by soft-rot fungi in 5 bamboo species after

16 weeks of incubation. Error bars represent mean ± standard
deviation. A Bambusa maculata; B Dendrocalamus asper; C Gigan- References
tochloa atroviolacea; D Guadua angustifolia; E Phyllostachys pu-
bescens China; F Phyllostachys pubescens Germany
Abdurachim MRA (1964) Bamboo preservation in Indonesia. Rimba
Abb. 3 Masseverluste durch Moderfäulepilze bei 5 Bambus-Arten
Indonesia 9:66–76
nach 16 Wochen Inkubation. Fehlerbalken zeigen ± Standardabwei-
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wood against fungi. Ministry of Agriculture, Jakarta
chloa atroviolacea; D Guadua angustifolia; E Phyllostachys
Banerjee S, Mukhopadhyay S (1962) A study on Merulius similis
pubescens China; F Phyllostachys pubescens Deutschland
B. & BR. and the associated bamboo-rot. Öster Bot Zeitschr
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Kolle flasks and maximum 38 % after 1 year in preserving Djarwanto, Suprapti S (2004) Laboratory testing on the resistance of
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19.7 % decay in Kolle flasks (Schmidt et al. 2011). The durability of solid wood. Part 1: Guide to the principles of testing
wood controls in this experiment revealed only rather low and classification of natural durability of wood. Beuth, Berlin
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EN 807 (2001). tance in Europe. Beuth, Berlin
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ness against soft rotting micro-fungi and other soil inhabiting
3.5 Classification of bamboo durability micro-organisms. Beuth, Berlin
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bamboo (Gigantochloa scortechinii) compared to 24 Malaysian
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resistances of the five bamboo species to the brown-rot Kim JJ, Lee SS, Ra JB, Lee H, Huh N, Kim GH (2011) Fungi
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S. commune and the soft-rot species P. variotii would schung 65:271–275
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group them into class II (durable). The soft-rot fungus Ch. bamboo culm material by stain and decay fungi. The Interna-
globosum attacked the bamboos according to classes II and tional Research Group on Wood Preservation, IRG Document
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T. versicolor, the five bamboo species varied between Lee KH, Cho CH, Kim YS (2006) Micromorphology of bamboo
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4 Conclusion ture Organization Report to the Government of India, FAO
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Bamboo species differ in their susceptibility to fungal Schriftenreihe Gesellschaft für Technische Zusammenarbeit,
decay. With regard to the different groups of rot fungi, GTZ Document No. 180

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Preservation, IRG Document No. IRG/WG 10-10712 schung 56:563–571
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Development Research Centre, New Delhi Charakterisierung von Gloeophyllum-Arten in Gebäuden.
Murphy RJ, Alvin KL, Tan YF (1991) Development of soft rot decay Z Mykol 68:141–152
in the bamboo Sinobambusa tootsik. IAWA Bulletin n s Schmidt O, Wei D, Liese W, Wollenberg E (2011) Fungal degrada-
12:85–94 tion of bamboo samples. Holzforschung 65:883–888
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Erhalten:25–27 in pure culture. Studies Mycol 16:1–248
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Assoc India 9:2–19 against fungi. J Trop For Sci 22:287–294
Razak W, Hashim WS, Murphy RJ (2002) SEM observation on the Wang SF, Hsieh RC (1968) Durability records of treated and
decay of bamboo Gigantochloa scortechinii exposed in tropical untreated bamboo. Coop Bull Taiwan For Res Inst 15:26
soil. J Trop For Prod 8:168–178 Wei D, Schmidt O, Liese W (2012) Susceptibility of bamboo to fungi.
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and use. Springer, Berlin

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 Publication IV

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ISSN impresa 0717-3644 Maderas. Ciencia y tecnología 15(3): 349-356, 2013
ISSN online 0718-221X
DOI 10.4067/S0718-221X2013005000027

METHOD TO TEST FUNGAL DEGRADATION OF BAMBOO AND WOOD

USING VERMICULITE AS RESERVOIR FOR MOISTURE AND NUTRIENTS

Dongsheng Wei 1,♠, Olaf Schmidt1, Walter Liese1

ABSTRACT

Proposed is a method to investigate degradation of lignocelluloses by pure cultures of basidiomycetes using

preserving jars with vermiculite as reservoir for water and nutrients. Bamboo samples of Gigantochloa atroviolacea
and Phyllostachys pubescens and wood samples of Fagus sylvatica and Pinus sylvestris were inoculated with the
brown-rot fungus Coniophora puteana and the white-rot fungus Schizophyllum commune. The fungi were cultured
on vermiculite containing different amounts of tap water or malt extract solution. Mass loss of the bamboos after
32 weeks was low and did not show a remarkable inluence of moisture content and nutrient addition. However,
considerable degradation of Pinus sylvestris sapwood occurred by C. puteana whereby moisture and nutrients
inluenced aggressiveness.

Keywords: Bamboo degradation, vermiculite, moisture content, nutrient addition.

INTRODUCTION

Bamboo is a fast growing woody grass with increasing importance for sustainable production of materials with
many applications for structures and industrial utilization. However, bamboo has a low natural durability and is
attacked by fungi during storage, transport, processing and inal use (Liese and Kumar 2003). There are several
reports on its degradation by fungi (Liese 1959, 1985; Abdurachim 1964, Mohanan 1997, Kim et al. 2011, Suprapti
2010, Ma et al. 2010, Schmidt et al. 2011). The behaviour of bamboo against decay fungi is an important parameter
in bamboo establishment and use. Most investigations revealed that bamboo degradation is due to white and soft
rot fungi, whereas brown-rot species were less aggressive. Among the investigated fungi, Coniophora puteana and
Schizophyllum commune varied with respect to the test method used: Low maximum mass loss was measured on
agar under pure culture condition, whereas considerable degradation occurred in the `fungus cellar test´ where the
samples are placed in large metal containers on unsterile garden soil with different moisture accessibility (Schmidt
et al. 2011). However, C. puteana and S. commune differed in decay activity: C. puteana produced maximum
mass loss at low moisture content of 57%, whereas decay by S. commune was highest at 182% moisture content
(Schmidt et al. 2011).

Mixed microbial decay in soils with various water holding capacities was studied by Becker and Kaune (1966).
Kaune (1970) and Worrall and Wang (1991) used for wood a vermiculite system with pure cultures of soft-rot
fungi and proved its suitability for wood degradation tests. Adaskaveg et al. (1991) showed decay of palm wood
by basidiomycetes with the vermiculite-block assay. Curling et al. (2002) exposed basidiomycetes to wood in a
vermiculite-soil system. The American standard soil-block test ASTM D1413 was not used for our study because
it is principally similar to our former fungus cellar test (Schmidt et al. 2011).

The aim of this study was to test bamboo and wood degradation by pure cultures of basidiomycetes with
vermiculite as reservoir of moisture and nutrients.

1
Section Wood Biology, Centre for Wood Science, University of Hamburg, Leuschnerstrasse 91d, 21031 Hamburg, Germany
♠
Corresponding author: dongsheng.wei@uni-hamburg.de
Received: 05.11. 2012. Accepted: 17.01. 2013

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 Maderas. Ciencia y tecnología 15(3): 349-356, 2013 Universidad del Bío - Bío

MATERIALS AND METHODS

Fungi
The brown-rot fungus Coniophora puteana strain 167 from the laboratory collection and the white-rot fungus
Schizophyllum commune strain 87 isolated from bamboo (Schmidt et al. 2011) were held on agar plates of 2% malt
extract (Oxoid) and 1.5% agar (Oxoid).

Bamboo and wood samples

Bamboo species Gigantochloa atroviolacea and Phyllostachys pubescens were used. Culm sections of G.
atroviolacea (diameter 5.5-7.0 cm) and P. pubescens (diameter 5.5-6.5 cm) were obtained from CONBAM,
Geilenkirchen, Germany and the Bamboo Centre, Baden-Baden, Germany, respectively. Culms were proven with
the curcuma-test (Peylo 2001) to ensure that no boron was present which is commonly used against moulding. The
samples were cut to 5 cm length, 2.5 cm width and 0.5–3.5 cm wall thickness. Wood samples of Fagus sylvatica
and Pinus sylvestris were cut to 5 cm length, 2.5 cm width and 1.5 cm height. Samples were put into a climate room
at 20±2ºC and 65±5% relative humidity (RH) for 4 weeks, weighed, wrapped in plastic (Sengewald Flexopeel,
Germany) and send for gamma radiation to BBF sterilization service GmbH (Kernen, Germany). To calculate the
initial weight, moisture content at 20ºC and 65 % was adjusted downward to the oven-dry value.

Vermiculite test
The decay experiment was performed in 500 ml preserving jars (Fig. 1). Vermiculite of 2-3 mm particle size
was obtained from vermiculite-shop (thinex new media, Dortmund, Germany, www.vermiculit-shop.de) and sieved
through a 2 mm sieve. Each preserving jar was illed with 200 ml vermiculite. Tap water or 2% malt extract solution
were added in 80, 100, 120, 140 and 160 ml amounts per jar. The jars covered with glass lids were sterilized in the
autoclave at 121°C and 210 kPa for 30 min. Each two bamboo or wood samples were put aseptically in one jar.
Two jars were used for one fungus. Vermiculite and sample were separated by a metal ring to avoid liquid diffusion
in the sample. A plug of the test fungus from agar plates was then placed on the samples top. After 32 weeks of
incubation at 20±2ºC and 65±5% RH, the mycelium was removed from the sample surface, the samples were
weighed for inal wet weight and oven dried at 103ºC for 3 days. Mass loss was calculated by weight comparison
before and after incubation. Standard deviation for each 4 specimens per treatment group was calculated according
to ANOVA, SPSS software 2002.

Figure 1. Vermiculite test setup.

(a) bottle with vermiculite and bamboo samples. (b) incubation room.

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Assessment of fungal growth and vitality test after incubation

The development of fungal growth on the samples was assessed according to the scheme in table 1, which values
hyphal density and hyphal coverage of sample. After 32 weeks of incubation, portions of overgrown vermiculite
were transferred from the jars to malt agar plates to prove the vitality of the mycelium after the vermiculite test.

Table 1. Evaluation scheme for hyphal growth on bamboo and wood samples.

RESULTS AND DISCUSSION

Figure 2 shows details of fungal growth on bamboo samples and on vermiculite particles.

Figure 2. Details of a preserving jar with mycelium of Schizophyllum commune

covering Phyllostachys pubescens samples.
(a) side view. (b) top view.

All bamboo and wood samples were totally covered by mycelium after 32 weeks (Table 2). Hyphal density
with tap water was less than with malt extract. However, the density of mycelium is not necessarily related to mass
loss in test specimens (Schmidt 2006).

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Table 2. Fungal growth on bamboo and wood samples after 32 weeks incubation.

D = hyphal density, C = hyphal coverage of sample

Results of the degradation tests are summarized in igures 3 and 4. Standard deviation is not shown for the
less mass loss by S. commune (Fig. 3), but is shown for C. puteana (Fig. 4) due to its greater decay capacity. Both
bamboo species Gigantochloa atroviolacea and Phyllostachys pubescens were rather resistant to degradation by the
white-rot fungus S. commune and the brown-rot fungus C. puteana over 32 weeks of incubation, the irst bamboo
being more resistant than the latter one. Adding different amounts (80 to 160 ml) of water or nutrient solution to
vermiculite had only small effect. The inal bamboo moisture contents were 61 to 84 %, which is a suitable range
for decay fungi (Schmidt 2006).

Figure 3 shows the results for S. commune. Maximum mass loss of bamboo was only 4.6%. Thus, possible
inluences among the parameters, bamboo species, moisture content and nutrient addition, were rather indistinct
for this fungus. Low decay was also measured on agar in preserving jars and in Kolle lasks (Schmidt et al. 2011)
which was already reported by Suprapti (2010) and Kim et al. (2011). Abdurachim (1964) obtained 15% mass
loss in Gigantochloa apus. Also beech wood showed neglectable decay (0.7-2 %) by S. commune (Fig. 3), which
corresponds to previous results on low degradation of wood samples (Schmidt and Liese 1978). However, this
fungus is common on bamboo culms during storage and use (Liese 1985, Mohanan 1997, Liese and Kumar 2003).

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Figure 3. Inluence of moisture content and nutrients on mass loss by Schizophyllum commune.
(a) incubation with tap water. (b) incubation with malt extract solution.

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Figure 4. Inluence of moisture content and nutrients on mass loss by Coniophora puteana.
(a) incubation with tap water. (b) incubation with malt extract solution.

Figure 4 shows the results for C. puteana. Maximum mass loss of bamboo was 8.4%, whereby P. pubescens
was more decayed than G. gigantochloa. Both liquid additions, tap water and malt extract, increased mass loss from
80- to 120 ml-addition followed by decrease to 160 ml. Scots pine wood revealed signiicant differences among the
liquid additions: With tap water, decay increased stepwise to 14.0 % maximum at 160 ml water addition. Most decay
with 52.5% maximum at 120 ml (104 % moisture content) occurred with the malt extract culture. The subsequent
decrease of mass loss may be caused by too high moisture content.

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Fungi are able to transport water by their mycelia from a moisture source to neighbouring wood (Schmidt 2006).
Figures 3 and 4 show that the inal moisture content of the bamboo samples was not inluenced by the different
initial liquid additions. Obviously, both fungi did not transport water from vermiculite to the samples. Presence
of the metal ring between vermiculite and sample could not be the reason because C. puteana could moisten the
Scots pine wood. Figure 4 (bottom) shows that moisture content increased up to 218 %u parallel to water addition.
Subsequently, mass loss decreased because of too much water in the wood.

The vitality test after 32 weeks of incubation proved living mycelium in all culture vessels (Fig. 5). Because
fruit bodies were not grown in the jars, survival was not due to resistant spores. Thus, it can be deduced that mycelia
were active over the whole culture period.

Figure 5. Vitality test on agar after vermiculite incubation.

(a) Coniophora puteana. (b) Schizophyllum commune.

Schmidt et al. (2011) assumed that high bamboo mass loss by C. puteana and S. commune in the fungus cellar
test was mainly caused by the moisture conditions and lesser by components from the unsterile soil. The vermiculite
test as pure-culture experiment in closed vessels did not show a signiicant effect of moisture content and nutrient
addition on bamboo degradation. Thus, bamboo degradation in nature may be also inluenced by better air conditions,
soil minerals or vitamins from soil bacteria. Already Leutritz (1946) demonstrated that soil serves as water holding
substrate and provides substances from the soil. The inluence of minerals and vitamins on soft rot was reported
by Worrall and Wang (1991) and Worrall et al. (1991). However, the small differences of mass loss among the
bamboos, fungi and test parameters make further discussion problematic. The fungi had been selected due to three
reasons: S. commune is very common on bamboo and the experimental strain was isolated by us from bamboo; C.
puteana is an obligatory test fungus in the European standard EN 113; third, it was hoped to explain the inluence
of moisture content and nutrients in the fungus cellar test. However, with regard to wood, the decay results with C.
puteana and P. sylvestris samples (Fig. 4) show the suitability of the proposed technique for degradation studies.
Assumably, our vermiculite method will also show greater fungal activity against bamboo if more aggressive fungi
such as Pleurotus ostreatus and Trametes versicolor (Schmidt et al. 2011) are used.

CONCLUSION
The preserving jar/vermiculite technique is suitable for fungal degradation studies of lignocelluloses as shown
by the mass loss of Pinus sylvestris wood by Coniophora puteana. Adding 2% malt extract solution till wood
moisture content around 100% provided high mass loss within 32 weeks of incubation. However, with regard to
bamboo, interpretations are problematic due to the low activity of both fungi on bamboo.

ACKNOWLEDGEMENTS
We thank Wolfgang Eberts (Bamboo Centre Germany) and Christoph Tönges (CONBAM, Germany) for
providing the bamboo and Marie-Therese Lenz for technical help.

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