INTERNSHIP REPORT

Title: “ISOLATION & IDENTIFICATION 0F FUNGAL PATHOGENS

FROM PEA SEEDS

at

National Agricultural Research Centre (NARC)

Islamabad
Submitted By

JAHANGIR KHAN

13-bac-8143

Report submitted in the partial fulfilment of the requirement for the degree of

B.Sc. (Hons.) Agriculture

in

Plant Pathology.

Department of Plant Pathology

Balochistan Agriculture College Balleli, Quetta
2018
 IN THE NAME OF ALLAH,
THE BENEFICIENT, THE MERCIFUL
WHO IS THE OMNIPOTENT AND
OMNIPRESENT
 CERTIFICATE
This is to certify that Mr. JAHANGIR KHAN S/O MEHRAB KHAN

Registered Roll No. 13-bac-8143, Department of Plant Pathology, B.Sc (Hons)

Agriculture, Balochistan Agriculture College, Quetta has successfully completed

internship from _______________ to _________________ at Department of Plant

Pathology, Faculty of Fungal pathology, National Agriculture Research Center

(NARC).

External Supervisor: _________________

Dr. Shahzad Asad
Principal scientific officer
CDRI. NARC. Islamabad

Director: ___________________
Dr. Anjum Munir
Director CDRI.
NARC. Islamabad

Internal Supervisor: ____________________

Mr.Ahmed Khan
Associate Professor/Chairman
Department of Plant Pathology
Balochistan Agriculture
College,Quetta

Dated: __________________
 DEDICATION

I DEDICATED THIS EFFORT

TO

HOLY PROPHET HAZRAT MUHAMMAD (PBUH)

The greatest social reformer

The Prophet of Mercy

AND

My family, specially my parents, sisters and brothers

for their support, for me they all are sprinkling candle in
darkness.
They always pray for my success.

MY RESPECTED TEACHERS

for their accommodative attitude, encouragement, scholastic guidance,

and valuable suggestions during my studies.

 CONTENTS

Topics

ACKNOWLEDGEMENT

INTRODUCTION OF NARC

1 INTRODUCTION

1.1 OBJECTIVES

2 MATERIALS AND METHODS

2.1 SAMPLE COLLECTION

2.2 STERILIZATION

2.3 ISOLATION

2.4 PURIFICATION

2.5 IDENTIFICATION

2.6 MORPHOLOGICAL CHARACTERISTICS

3 RESULTS AND DISCUSSIONS

REFERENCES
 ACKNOWLEDGMENT
First of all I would like to thank Almighty ALLAH, because of his
blessings, I fulfilled my work properly.

I acknowledge with thanks to my Internal Supervisor Mr. Ahmed Khan,

Associate. Prof., and honourable teachers Mr. Muhammad Waris (Assistant
Prof), Syed Zulfiqar Ali (Assistant Prof), Miss. Tahira Nisa (Lecturer), Mr.
Basharat (Lecturer), Mr. Muhammad Ather Shaikh (Lecturer), Department of
Plant Pathology, Balochistan Agriculture College, Balleli Quetta, for their
cooperation, constant support and encouragement.

I would also like to pay attribute to my External Supervisor Dr. Shahzad

Asad principal scientific officer CDRI. NARC. Islamabad, for his full co-operation
in the laboratory in carrying out the training course.

I earnestly and sincerely thank to my affectionate and loving Father and

Mother for their prayers, kind attitude and their inspiration is the sprinkling candle
in darkness.

JAHANGIR KHAN
INTRODUCTION TO (NARC),
Islamabad established in 1984, is the largest research centre of the
Pakistan Agricultural Research Council (PARC). NARC, with a total land area
of approximately 1400 acres, is located near RawalLake, six kilometers south-
east of Islamabad. Physical facilities in term of experimental fields, laboratories,
green houses, gene bank, library/ documentation, auditorium, machinery & lab
equipment repair workshops, stores, hostels, cafeteria, audio visual studios, are
also available at NARC.

NARC coordinated programmes serve as a common platform for the scientists

working in different federal, provincial agricultural research, and academic
institutions to jointly plan their research activities, avoiding unnecessary duplication
of research efforts. Research which can best be addressed at a national centre rather
than by provincial institutions is undertaken at NARC. The adaptation of technologies
available from the international research system is also managed by NARC, in
collaboration with the provincial research and extension institutions. In particular,
research requiring sophisticated instruments like electron microscopes,
ultracentrifuges, and elaborate analytical and quality testing facilities is undertaken at
NARC, supported by highly qualified and trained manpower.
INTRODUCTION

The pea (Pisum sativum L) is most commonly the small spherical seed or
the seed-pod of the pod fruit Pisum sativum. Each pod contains several peas. Pea
pods are botanically fruit (Rogers and Speed 2007). P. sativum is an annual plant,
with a life cycle of one year. It is a cool-season crop grown in many parts of the
world; planting can take place from winter to early summer depending on location.
The average pea weighs between 0.1 and 0.36 grams. (Anonymous 2017). Peas are
starchy, but high in fiber, protein, vitamin A, vitamin B6, vitamin C, vitamin
K, phosphorus, magnesium, copper, iron, zinc and lutein. (Anonymous 2015). Dry
weight is about one-quarter protein and one-quarter sugar (Jegtvig and Shereen
2007). Pea seed peptide fractions have less ability to scavenge free radicals
than glutathione, but greater ability to chelate metals and inhibit linoleic
acid oxidation. (Pownall et al; 2010). A variety of diseases affect peas through a
number of pathogens, including insects, viruses, bacteria and fungi.(Hagedorn,
1976). In particular, virus disease of peas has worldwide economic importance
(Hagedorn and Donald,1974).
FUSARIUM ROOT ROT

 Caused by Fusarium solani (Mart.) Sacc. f. sp. pisi (Jones) Snyd. and
Hans.
 Yield losses of 26 per cent to 57 per cent have been reported in both
dryland and irrigated areas.
 Fusarium root rot is most prevalent under poor crop rotations, high soil
temperatures (22˚ to 30˚ C), moist soils, acidic soils (pH 5.1 – 6.2) and
low fertility.
 Soil compaction by farm machinery also increases both incidence and
severity.

Symptoms

 Reddish brown streaks initially develop on the primary and secondary

roots.
 These eventually together to form a dark reddish-brown colour on the
primary root up to the soil line.
 Greying, yellowing, death of lower foliage and stunting can occur if
infection is severe.
 Foliar symptoms often appear following warm temperatures and heavy
rainfall.
FUSARIUM WILT

 Caused by Fusarium oxysporum Schl. f. sp. pisi (Van Hall) Snyd. &
Hans.
 Of the disease’s many races, races 5 and 6 are most important in Western
Canada.
 Pathogen can be seed or soil-borne and can infect soils for 10 or more
years.
 Races 5 and 6 thrive under soil temperatures of 20˚ to 21˚ C.

Symptoms

 Race 5 and 6 symptoms are most often found in small, circular patches in
fields.
 In roots cut longitudinally, a yellowish, orange colour is seen in the
vascular tissue, which can extend into the basal area of the stem.
 Downward curling of the leaves and stipules.
 Thickening of the basal internode and brittle leaves and stem.
 Yellowing of the leaves progressing from the base of the plant to the top
of the foliage (plants often die due to loss of the root system).
PYTHIUM ROOT ROT

 Caused by a number of different Pythium species. In Alberta, P. irregulare

and P. ultimum are the most common.
 Infection very dependent on seed quality – cracked seeds leak more
nutrients and are more likely to attract Pythium and be infected than intact
seed.
 More prevalent in cool, wet, poorly drained soils.

Symptoms

 Seeds are rotted – when removed from the soil, they emerge with a layer
of soil around them (this is full of whitish, threadlike fungal hyphae).
 Emerging plant and roots, if produced, may be soft and watery;
cotyledons may or may not be rotted.
 Germinating seeds are only susceptible for 48 to 72 hours – once the root
emerges, the seed is no longer susceptible to infection (new developing
tissue, however, remains susceptible).
 Infection can occur at the tip of feeder roots where young tissue can be
destroyed – this can lead to root pruning and/or reduction in length.
 Depending on the severity of the infection, seedlings may become stunted
and chlorotic and collapse as the root base decays and turns tan to light
brown.
 Infected plants tend to lack vigour and often yield poorly.
RHIZOCTONIA ROOT ROT

 Caused by Rhizoctonia solani Kuehn.

 Seedlings generally less susceptible as they get older.
 High soil temperatures (24˚ to 30˚ C) are known to cause higher rates of
infection.

Symptoms

 Infection occurs (and symptoms appear) close to the soil surface.

 Symptoms on seedlings first appear as water-soaked lesions that
eventually turn reddish brown.
 Plant’s growing point may die as it emerges from the ground – this can
lead to multiple shoots emerging and then dying.
 On older plants, symptoms appear as reddish-brown sunken lesions on the
lower stem, which can cause girdling and often leads to stunted plants.

OBJECTIVES

Objectives of the present study were;

1. Isolation of pathogens from seeds.

2. Morphological characterization of isolated pathogens.

MATERIAL AND METHODS

2.1 SEED SAMPLE COLLECTION

The seed samples were collected from horticulture department (NARC) and
brought to the fungal lab and were kept in the cooling incubator at 4 °C for further
studies.

MEDIA PREPARATION

Czepek Dox Agar

Ready Czepek Dox Agar Media to use was prepared by adding 19.5g

(Glucose /10g, KH2PO4 /0.25g, NaNO3 /1.0g, MgSO4.7H2O /0.25g, FeSO4 /0.5g,
Yeast Extact /0.5g, Agar /7g) in 500 mL of distilled water.

Figure 1

Potato Dextrose Agar

Potato dextrose agar ready to use was prepared by adding 19.5g of PDA in 500 mL
of distilled water.
2.2. STERILIZATION

 Washing: The glasswares were washed under tap water and were kept
inverted for drying.
 Wrapping: The cleaned dried petri-plates were wrapped with paper.
 Media preparation:The media was prepared and then kept in the autoclave
for sterilization.
 Autoclave: The materials were placed in the autoclave at 121°C under 15
lbs pressure for 20 minutes.
 Pouring: The autoclaved media was let for cooling till the hands could
handle and then the media was poured in the petri-plates in the laminar flow
cabinet and inverted after solidification.
 Laminar Flow Cabinet: laminar The flow cabinet was cleaned with the
Ethanol before use and the forceps, picking needles and razor was dipped in
the spirit for surface sterilization. The Ultra Violet light was turned on 10
minutes before culturing.

Figure 2 Laminar flow Autoclave

2.3 ISOLATION OF FUNGAL PATHOGEN FROM SEEDS

Inected seeds were placed on Czapek dox agar and Potato Dextrose Agar
(PDA) media in the laminar flow cabinet and then plates were transfered into
incubator for five days.

Procedure

The infected seed were first treated with Chlorox 2% placed in petriplate. Then
these seeds were given two washings of distilled water in petri plates. Then the
infected seeds were dried in another petri-plate containing blotter paper. These
seeds were placed in the culture media plates. These plates were incubated at 25 °C
for 5-10 days and observed visually for the growth of fungi.

Detection of Seed-borne Fungi:

For detection of seed borne fungi from the collected samples, two standard
seed health testing methods i.e., blotter paper and agar plate method were used for
evaluation of their efficiency. Purpose of these methods were to check the fungi
associated with the seeds of Pea, Brinjal, Tomato.

Pre- treatment:

Seed samples were dipped in 2 % chlorox for surface sterilization and kept
for a minute in the laminar flow cabinet. Seeds were washed twice with sterilized
distilled water, so as to remove the excessive disinfectant and dried by placing them
on blotter paper for few minutes.
Blotter paper method:

The blotter papers were first cut according to the size of the petri plates
which were then placed in the petri plates making humid chamber then these plates
were wrapped with newspaper and then were autoclaved at 15 psi pressure and 121
°C for 20 minutes.

6 seeds from each sample were tested by using standard blotter paper
method. 6 seeds from each sample were placed on three Petri plates having blotter
paper. In order to provide sterilized condition all the activity was performed in
laminar flow cabinet. After placing the seeds, the plates were incubated at 25 °C for
a week under alternating cycles of 12 hours day and night under fluorescent tube
light. Confirmation of the disease was accomplished with the help of compound
microscope.

Figure 3: Seed samples on blowtter paper

Agar Plate Method:

In Agar plate method Czepek Media was used. The ingredients were mixed
thoroughly in 400 ml of distilled water and the volume was made up to 500 ml. For
proper dissolution of the components the flask was placed on a hot plate containing
a magnetic stir bar. After dissolution of complete ingredients, media was
autoclaved at 15 psi pressure and 121 °C for 20 minutes. The autoclave was
allowed to cool so that it reached the temperature of 50 °C and then it was poured
in petri plates in sterilized laminar flow cabinet so as to avoid any type of
contamination and left for a hour to solidify properly. After the pouring of media,
ultraviolet light was put on for 10-15 minutes in order to completely eliminate the
chances of contamination.

Pre-treated seeds were directly plated on agar using standard agar plate
method. 5-8 seeds were placed in each petri plate containing solidified PDA and
were incubated at 25 °C for a week. After 7 days, plates were observed for the
growth of fungi. From the composite culture, fungi were identified on the basis of
habit characters and colony color of the individual fungi under stereomicroscope.

Figure 4: Seed samples in culture media

2.4 PURIFICATION

All the isolated fungi were purified by picking up the spore by inoculating
needle of each fungi and inoculating it on the PDA media separately in the petri
plate.

For this purpose the needle was red hot on flame and allowed to cool. Then the
needle was touched vigilantly and slightly to the specific fungal colony. Needle was
then inoculated on solidified PDA media in the petri plate. Plate was sealed with
the paraflim tape so as to avoid any contamination. All this activity was performed
in the laminar flow cabinet. Prior to the activity, laminar was completely sterilized
with the spirit and by keeping ultraviolet light on for 5-10 minutes. After the plates
were sealed by paraflim tape, these plates were incubated at 25 °C for 7 days. Sub
culturing was performed for further purification of the cultures.

IDENTIFICATION:

Morphological and cultural characteristics of isolated fungal pathogens on

different media will be recorded by using taxonomic keys (Ellis, 1971).

Slide preparation:

The slides were prepared by placing a drop of lactophenol at the centre of

slide and the fungal growth on the culture media was placed on the lactophenol
drop by the help of lactophenol and observed under microscope by different
magnifications. The species were identified on account with the techniques used by
(Cheesbrough, 2000).

Figure 5 Lactophenol Slide

 IDENTIFICATION AND SLIDE PREPARATION

Figure 6 : Microscopic study for identifying of seed borne pathogens

2.5 MORPHOLOGICAL CHARACTERISTICS OF ALTERNARIA

Conidiophore

 Pale brown to olive brown

 25–60 x 3–3.5 µm
 Straight or flexuous
 Individual conidiophores arise directly from substrate forming bushy heads
consisting of 4–8 large catenate conidia chains
 Secondary conidiophores are generally short and 1-celled
Conidia

 Pale brown to light brown

 Obclavate to obpyriform orellipsoid, short conical beak at the tip, or beakless
 Surface smooth to verruculose
 Size
 20–63 x 9–18 µm in size
 (on PCA) mature conidia typically 10–30 x 5–12 µm
 Septa
 Several vertical and −8 transverse septa
 (on PCA) 3–7 transepta, 1–5 longisepta
 Chains
 Produced in an often branched, long chain more than 5 conidia.
 (on PCA) individual chains of 5–15 conidia, complex of branching chains
may contain up to 50–60 conidia

Figure 7 : Alternaria alternata

 RESULTS AND DISCUSSION
The seed samples were collected from horticulture department (NARC) and
brought to the fungal lab and were kept in the cooling incubator at 4 °C for further
studies.

Ready Czepek Dox Agar Media to use was prepared by adding 19.5g in 500 mL of
distilled water.

For detection of seed borne fungi from the collected samples, two standard seed
health testing methods were used i.e., blotter paper and agar plate.

Infected seeds were placed on Czapek dox agar media and blotter paper in the

laminar flow cabinet and then plates were transfered into incubator for incubation at

25 °C for 5-10 days.

All the isolated fungi were purified by picking up the spore by inoculating needle
of each fungi and inoculating it on the Czepek Dox Agar media separately in the
petri plate.

The slides were prepared and observed under microscope by different

magnification.

Alternaria alternata was isolated and characterized on the basis of cultural and

morphological studies.

Seed Pathogen Media Colony size No. of days in

incubator

Pea Alternaria PDA Media 5 cm 7

alternata

Pea Alternaria Czepek Media 7 cm 10

alternata
The pea seeds infected with Alternaria alternata were grown in two media .The

media were kept for 7 days in incubuter. The colony size in agar media was

measured as 5cm while the best result was observed in Czepek Media with colony

of fungus with 7cm diameter. the fungal cultures grown in Czepek Media were

subcultured for further clarification.

 REFERENCES

Hagedorn, D. J. (1976). Handbook of pea diseases (PDF). University of Wisconsin

- Extension.

Hagedorn, Donald J. (1974). Virus Diseases of Pea, Pisum sativum. St. Paul,

Minnesota: American Phytopathological Society. p. 7.

Jegtvig, Shereen (2007). "Peas". Nutrition. About.com. Retrieved 2011-01-28.

Nutrition Facts: Peas. Nutrition. vegonline.org. Retrieved, 2015.

Pownall TL, Udenigwe CC, Aluko RE (2010). "Amino acid composition and

antioxidant properties of pea seed ( Pisum sativum L.) enzymatic protein

hydrolysate fractions". Journal of Agricultural and Food

Chemistry. 58 (8): 4712–4718. doi:10.1021/jf904456r. PMID 20359226

Rogers, Speed (2007). Man and the Biological World Read Books. pp. 169–

170. ISBN 978-1-4067-3304-4 retrieved on 2009-04-15.

Solanum lycopersicum- Tomato". Encyclopedia of Life. Retrieved 1 January 2014.