T 83966 balkishan chaudhary

Studies on wilt of Pigeonpea caused
by Fusarium udum Butler.
THESIS
Submitted to the
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur
In partial fulfillment of the requirement for
the Degree of
MASTER OF SCIENCE
In
AGRICULTURE
(PLANT PATHOLOGY)
By
BALKISHAN CHAUDHARY
Department of Plant Pathology
College of Agriculture, Jabalpur 482004
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur,
Madhya Pradesh
2016

CERTIFICATE - I
This is to certify that the thesis entitled, “Studies on wilt of Pigeonpea
caused by Fusarium udum Butler.” submitted in partial fulfillment of the
requirement for the degree of MASTER OF SCIENCE in Agriculture (Plant
Pathology) of Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur is a record
of the bonafide research work carried out by Mr. BALKISHAN CHAUDHARY,
I.D. No. AP/JB-469/2014 under my guidance and supervision. The subject of
the thesis has been approved by the Student’s Advisory Committee and the
Director of Instruction.
All the assistance and help received during the course of the
investigation has been acknowledged by him.
Place: Jabalpur
Date:
Dr. Sanjeev Kumar
Chairman of Advisory
Committee
THESIS APPROVED BY THE STUDENT’S ADVISORY COMMITTEE
Committee Name Signature
Chairman Dr. Sanjeev Kumar ……………………………………..
Member Dr. U. K. Khare ……………………………………..
Member Dr. S. K. Singh ……………………………………..
Member Dr. R. B. Singh …………………………………….

CERTIFICATE - II
This is to certify that the thesis entitled “Studies on wilt of Pigeonpea
caused by Fusarium udum Butler.” submitted by Mr. Balkishan Chaudhary
to the Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur in partial fulfillment
of the requirement for the degree of Master of Science in Agriculture in the
Department of Plant Pathology JNKVV, Jabalpur, after evaluation has been
approved by the Examiner and by Student’s Advisory Committee after an oral
examination on the same.
Place: Jabalpur
Date: …………….. Dr. Sanjeev Kumar
Chairman of the Advisory
Committee
MEMBERS OF THE ADVISORY COMMITTEE
Committee Name Signature
Chairman Dr. Sanjeev Kumar …………………………….
Member Dr. U. K. Khare …………………………….
Member Dr. S. K. Singh …………………………….
Member Dr. R.B. Singh …………………………….
Head of the Department Dr. S. N. Singh …………………………….
Director Instructions Dr. Dhirendra Khare …………………………….

Declaration and Undertaking by Candidate
I Balkishan Chaudhary S/o Gopal Lal Certify the work embodies in the
thesis entitled “Studies on wilt of Pigeonpea caused by Fusarium udum
Butler.” is my own first hand bonafide work carried out by under the guidance
of Dr. Sanjeev Kumar at Department of Plant Pathology, JNKVV, Jabalpur
and place during 2014-2016.
The matter embodied in the thesis has not been submitted for the
award of any other degree/diploma. Due credit has been made to all the
assistance and help.
I, undertake the complete responsibility that any act of
misinterpretation, mistakes and errors of fact are entirely of my own.
I, also abide myself with the decision taken by my advisor for the
publication of material extracted from the thesis work and subsequent
improvement, on mutually beneficial basis, provided the due credit is given,
thereof.
Place: Jabalpur
Date: Balkishan Chaudhary

Copyright© Jawaharlal Nehru Krishi Vishwa Vidyalaya,
Jabalpur Madhya Pradesh 2015
Copyright Transfer Certificate
Title of the Thesis : “Studies on wilt of Pigeonpea caused by
Fusarium udum Butler.”
Name of the candidate : Balkishan Chaudhary
Subject : Plant Pathology
Department : Plant Pathology
Year of thesis submission : 2016
Copyright Transfer
The undersigned Balkishan Chaudhary assigns to the Jawaharlal
Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh, all rights under
Copyright Act, that may exists in and for the thesis entitled “Studies on wilt
of Pigeonpea caused by Fusarium udum Butler” submitted for the award
of M.Sc. (Ag.) degree.
Date: / /
Place: Jabalpur
Dr. Sanjeev Kumar Balkishan Chaudhary
(Major Advisor) (Student)

ACKNOWLEDGEMENT
Thanks to God and his blessing by which I was able to complete my
thesis and gave me an opportunity to express my heartful gratitude to all those
who have given me helping hands to make this study success.
It is pleasure for me to express my indebtness to Dr. Sanjeev Kumar,
Chairman of my advisory committee and Asst. Professor, Department of Plant
Pathology in College of Agriculture, J.N.K.V.V., Jabalpur, Madhya Pradesh, India
for initiating flora of research in me, continuing encouragement, insightful
guidance, untiring help, keen attention, constant stimulations and constructive
criticism extended all along during the investigation and for its proper
presentation in the form of thesis.
I wish to remembrance of my venerable of my advisory committee. I am
grateful to Dr. U. K. Khare, Professor, Department of Plant Pathology and Dr. S.
K. Singh, Professor, Department of Plant Breeding and Genetics and Dr. R. B.
Singh, Professor, Department of Agricultural Statistics for their valuable
suggestions, and illuminating guidance, and generous help throughout the course
of this investigation.
I express my sincere thanks to Dr. S. N. Singh, Professor and Head, Department
of Plant Pathology for valuable guidance and generous help. Dr. V.S.Tomar The
Honourable Vice Chancellor; Dr. Ashok Kumar Ingle Register, Dr. Dhirendra
Khare Director of Instruction and Dr. (Mrs.) Om Gupta, Dean, College of
Agriculture, Jabalpur for permitting me to complete the degree programme
successfully.
I sincerely express my appreciation and gratitude to respected teachers
of Plant Pathology, Dr. Jayant Bhatt, Dr. U.K. Khare, Dr. S.P. Tiwari, Dr. M.S.
Bhale, Dr. (Mrs.) Usha Bhale, Dr. A.R. Wasnikar and Dr. (Mrs.) Vibha Pandey
for their time to time suggestions, encouragement and help in various ways.
I am thankful to the office staff and workers of Department of Plant
Pathology for needful co-operation. I also wish to express my feelings towards
my batchmates Prahlad, Shivakant Kushwaha, Prahlad Masumkar, Naresh
Kumar and friends Jitendra Kumar Fogya, Vinod Mehra, for their timely help and
unceasing encouragement throughout the study.
Words are not enough to express my heartiest feelings of humble
gratitude indebtedness and profound sence of appreciation to my beloved father
Shri Gopal lal, mother Chhoti Devi, brothers Mahaveer Singh and Sister Hansha,
Sarita and Sonu for their deep love, blessings, constant inspiration and care
throughout my life which enables me in my ascent to the present
accomplishment.
Place: Jabalpur
Date: …./…./2016 (Balkishan Chaudhary)

LIST OF CONTENTS
Number Title Page
1. Introduction 1-2
2. Review of Literature 3-24
3. Material and Methods 25-41
4. Results 42-55
5. Discussion 56-63
6. Summary, Conclusions and Suggestions
for further work
64-66
6.1 Summary 64-65
6.2 Conclusions 65-66
6.3 Suggestions for further work 66
7. Bibliography 67-76
8. Appendices I - X
Curriculum Vitae

LIST OF TABLES
Number Title Page
3.1 Details of expression of sporulation. 31
3.2 Name of antifungal plant, their doses and formulation. 39
3.3 Name of fungicides, their doses and formulation. 40
3.4 Name of fungicides, their doses and formulation. 41
4.1 Incidence of wilt in different districts of Madhya Pradesh. 43
4.2 Effect of solid media on radial growth and sporulation of
Fusarium udum.
43
4.3 Effect of liquid media on dry mycelial weight of Fusarium
udum.
44
4.4 Effect of various pH on radial growth and sporulation of
Fusarium udum.
46
4.5 Growth of antagonists and pathogen in monoculture. 47
4.6 Growth of antagonists and pathogen in dual culture. 48
4.7
Effect of volatile compounds from Trichoderma on radial
growth of Fusarium udum after five days of incubation.
49
4.8
Effect of non - volatile compounds from Trichoderma on
radial growth of Fusarium udum after five days of
incubation.
50
4.9 Evaluation of antifungal activities of plant extract on radial
growth of Fusarium udum after six days of incubation.
51
4.10 Effect of fungicides on radial growth of Fusarium udum. 52
4.11
Effect of various concentrations of fungicides on the
germination, radicle and plumule length and production of
biomass.
55

LIST OF FIGURES
Number Title
Between
Page no.
1. Incidence of wilt in different districts of Madhya
Pradesh.
42-43
2. Effect of solid media on radial growth and
sporulation of Fusarium udum.
43-44
3. Effect of liquid media on dry mycelial weight of
Fusarium udum.
44-45
4. Effect of various pH on radial growth and sporulation of
Fusarium udum.
46-47
5. Growth of antagonists and pathogen in monoculture. 47-48
6. Growth of antagonists and pathogen in dual culture. 48-49
7. Effect of volatile compounds from Trichoderma on
radial growth of Fusarium udum.
49-50
8. Effect of non-volatile compounds from Trichoderma at
5% concentration on radial growth of Fusarium udum
after five days of incubation.
50-51
50-51
50-51
11. Evaluation of antifungal activities of plant extracts at
after six days of incubation.
51-52
12. Evaluation of antifungal activities of plant extracts at 10
% concentration on radial growth of Fusarium udum
51-52
51-52
14. Effect of fungicides on radial growth of Fusarium udum. 52-53

LIST OF PLATES
Number
Title
Between
Page no.
1. Collection, Isolation and Identification of
Fusarium udum.
42-43
2. Effect of solid media on radial growth and
43-44
3. Effect of liquid media on dry mycelial weight of
Fusarium udum.
44-45
4. Growth of antagonists and pathogen in monoculture. 47-48
5. Growth of antagonists and pathogen in dual culture. 48-49
6. Effect of volatile compounds from Trichoderma on
radial growth of Fusarium udum after five days of
incubation.
49-50
7. Effect of non-volatile compounds from Trichoderma
at 5, 10 & 15% concentrations on radial growth of
Fusarium udum after five days of incubation.
50-51
51-52
10% concentration on radial growth of Fusarium
udum after six days of incubation.
51-52
15% concentration radial growth of Fusarium udum
51-52
11. Effect of different fungicides on radial growth of
Fusarium udum.
52-53
12. Effect of 25 & 50 ppm centrations of fungicides on the
germination, radicle and plumule length and its
biomass.
55-56
13. Effect of 75 & 100 ppm centrations of fungicides on
the germination, radicle and plumule length and its
biomass.
55-56

LIST OF ABBREVIATIONS
cm = Centimetre
mm = Millimetre
g = Gram
lbs = Pound
psi = Pound pressure inch
PDA = Potato dextrose agar medium
PDB = Potato dextrose broth medium
ml = Millilitre
lit = Litre
No. = Number
i.e. = That is
sp. = Species
o
C = Degree Celsius
etc = Extras
et al = Co-worker
eg = As for example
ha = Hectare
% = Percent
@ = At the rate of
viz. = Namely
CD = Critical Difference
df = Degree of freedom
µl = Micro litre
Hrs: min = Hours and Minute
Max. = Maximum
Min. = Minimum
Fig = Figures

1
INTRODUCTION
Pigeonpea (Cajanus cajan), popularly known as tur or arhar is one of
the important pulse crop after chickpea in India. Besides being rich source of
protein (22.3%), essential amino acids particularly lysine, this crop also help in
maintaining the soil fertility through natural biological nitrogen fixation. The
ability of pigeonpea to produce economic yields in soils characterized by
moisture deficient makes it an important crop of dry land agriculture. Farmers
grow it in various production systems as a mixed crop, intercrop and perennial
crop using long established traditional practices (Chauhan, 1990). In India
pigeonpea grown in 36.3 lakh ha and production of pigeonpea is 27.6 lakh
tones. The major pigeonpea growing states are Maharashtra, Madhya
Pradesh, Gujarat, Karnataka, Andhra Pradesh, Uttar Pradesh, Jharkhand,
Orissa, Bihar and Rajasthan contributing 88.2% in total pigeonpea production
(Singh and Singh, 2014). In Madhya Pradesh, area and production of pigeon
pea is 3.50 lakh ha and 2.17 lakh tone (Shrivastava et al. 2016). However, the
average national and state yield of pigeonpea is disappointingly low in
comparison to potential yield. The major factor for low production of
pigeonpea in India are ecological factors, lack of appropriate pulse production
and protection technologies, poor post harvest technologies, less thrust on
basic research, inadequate supply of quality seed to farmers and socio
economic constraints etc. Apart from these, the pest and disease problems
are the major bottlenecks in realizing the higher yields. Pigeonpea crop
suffers from over 210 pathogens (83 fungi, 4 bacteria, 19 viruses and
mycoplasma and 104 nematodes) reported from 58 countries (Reddy et al.
1990; Nene et al. 1996). The major diseases that assume significant
importance include wilt (Fusarium udum Butler), sterility mosaic (Pigeonpea
sterility mosaic virus) and phytophthora blight (Phytophthora drechsleri).
Among these, wilt is the most serious disease causing irreversible losses and
lethal damage to crop. Some of the important diseases are Fusarium wilt,
Phytophthora blight, Cercospora leaf spot, collar rot, dry root rot, Alternaria
leaf spot, powdery mildew, sterility mosaic and phyllody. Incidentally, only a
few of them causes economic losses in India (Kannaiyan et al. 1984). Among

2
the diseases Fusarium wilt caused by Fusarium udum is the most important
soil borne disease and was first reported from Bihar state in India (Butler,
1906). The disease appears on young seedlings but the highest mortality
occurs during flowering and podding stage. Although the disease first appears
in patches in a field and can extend to entire field if pigeonpea is repeatedly
cultivated in the same field. The yield loss of pigeonpea depends on the stage
at which the plants wilt and it can approach 100, 67 and 30 per cent when wilt
occurs at pre-pod, maturity and pre harvest stages, respectively (kannaiyan
and Nene, 1981) and sometimes it causing upto 100% loss in grain yield
(Okiror, 2002). The disease incidence has been increasing year after year and
most of the released cultivars became susceptible to the disease indicating
the development of more virulent races of the pathogen in major pigeonpea
growing areas of the state. In view of significance of pigeonpea and the
enormity of yield loss caused by fusarial wilt, various methods are used to
manage the disease but they fail to give satisfactory control of the wilt.
Chemical control of the disease is difficult, impractical and uneconomical, as
the large scale soil application of chemicals required is expensive, hazardous
and disturbs the biological balance (Songa, 1990). Hence efforts have to be
made to curtail pathogen activity and restricting losses below economic
threshold level by choosing alternative methods like use of botanicals
pesticides, bioagents, manipulation of cultural practices and lastly the need
based use of eco friendly pesticides. So the present investigations were
undertaken with the following objectives:
 Assessment of losses caused by the wilt in major pigeon pea growing
districts of M.P.
 To know the best media and pH for the growth and sporulation of the
target pathogen.
 To find out the management option for disease management.

3
REVIEW OF LITERATURE
Pigeonpea is an important pulse crop grown in the India. Pigeonpea is
attacked by a large number of diseases and therefore, the risk involved in its
cultivation is quite high, Over 63 fungal, 3 bacterial, 19 viral or alike pathogens
and 10 nematodes are reported to infect the crop at various growth stages
(Nene et al. 1996). However, only few of these cause significant yield loss and
are economically important in India. The disease spectrum and severity varies
in different agroclimatic zones and cropping situations. In the main Kharif crop
in northern, eastern and central India, wilt (Fusarium udum), sterility mosaic
(Pigeonpea sterility mosaic virus) and phytophthora blight (Phytophthora
drechsleri f.sp. cajani) are most widely distributed economically important
diseases. The present review pertaining to the wilt of pigeonpea caused by
Fusarium udum Butler.
The disease and the causal organism
Butler (1906) reported that wilt is the most important soilborne disease
of pigeonpea and was first described in 1906 from Bihar state, India.
Butler (1910) first described the pigeonpea wilt pathogen as Fusarium
udum.
Nene et al. (1979) reported that the pathogen can be isolated from all
parts of the host from lateral fine roots to pedicel and pod hull. However, the
pathogen is mostly confined to vascular tissues and is both inter and intra
cellular. The septate hyphae run across the cells, growing rapidly along the
inside of the walls of the large vessels. These vessels often appear pluged
with the hyphae. However, there are instances where none or only few
vessels show presence of hyphae although browning of the tissues is present.
It is a soil borne facultative parasite that enters through roots and then
becomes systemic.
Nene et al. (1980) isolated the fungus from apparently healthy 15 day
old plants from a wilt sick plot. The causal organism is a soil borne facultative

4
parasite that enters through roots and then becomes systemic invading tap
root, lateral roots, main stem, branches, leaflets, petioles, rachis and pedicel.
Kannaiyan and Nene, (1981) revealed that even though plants get
infected at an early stage, they seem able to “keep fighting” with the fungus
until flowering and podding. The yield loss depends on the stage at which the
plants wilt; it can approach 100% when wilt occurs at the pre pod stage, about
67% when wilt occurs at maturity and 30% when it occurs at the pre-harvest
stage.
Rai and Upadhyay, (1982) have reported Gibbrella indica Rai as the
perfect stage F. udum. The perfect stage Gibberella indica is usually found on
exposed roots and collar region of the stem up to the height of 35 cm above
the ground level. The mature perithecia are superficial, commonly
aggregated, subglobose to globose, sessile, smooth walled, dark violet, and
350-550 μm in diameter. Asci are 8 spored, mostly subcylindrical, 60-80 x 6-
10 μm, broader in the middle, with short stalk, a narrow apex, and a central
apical pore. Ascospores are ellipsoidal to ovate, 10-17 x 5-7 μm, hyaline,
commonly 2 celled rarely 3-4 celled and constricted at the septa. In culture
these spores germinate to produce short or long conidiophores bearing micro
and macro conidia which are pathogenic to pigeonpea. The fungus is
heterothallic and single ascospore cultures do not produce perithecia. When
culture from different strains are grown together perithecia are formed after 25
days at 18-22o
C. The ascospores germinate to produce micro and
macroconidia.
Upadhayay and Rai, (1982) reported that the pathogen extends more
rapidly from one place to another along the root than across the soil. It is
dispersed through irrigation, rain water and displacement of host debris by
termites that feed frequently on dead wilted plants.
Kannaiyan et al. (1985) reported that the disease is caused by
Fusarium udum Butler. The pigeonpea wilt fungus is host specific being
pathogenic only on pigeonpea and its wild relative, Atylosia spp.
Upadhayay and Rai, (1989) reported that the pathogen is specific in
parasitism, being pathogenic to pigeonpea only.

5
Upadhyay and Rai, (1989) reported that Fusarium udum shows wide
variation in morphological, cultural and physiological characterstics,
production of enzymes and virulence to different cultivars of pigeonpea and
occurrence of physiologic races. There is no relationship between cultural
characteristics and aggressiveness.
Upadhayay and Rai, (1992) revealed that the pathogen has also been
found to be of a seed borne nature. The mycelium is hyaline and produces 3
types of spores within the host tissue as well as in cultures namely
microconidia, macroconidia and chlamydospores. Microconidia are small,
elliptical or curved, unicellular or 1-2 septate, and measure 5-15 x 2-4 μm.
The macroconidia are long, curved (fusaroid), with prominent epical hook, and
notched at the base, septate (3-4 septa), and measured 15-50 x 3-5 μm. The
presence of the prominent epical hook distinguishes the species from
Fusarium oxysporum. Chlamydospores are also formed in the host as well as
in old cultures. They develop from any cell of the hypha, often from cells of the
macroconidium. The cells round off and become thick walled to form
chlamydospores. These spores are oval to spherical, single or in chains,
terminal or intercalary and persist in the soil for long.
Kumar and Upadhyay, (2014) studied variability among 15 isolate of
Fusarium udum, the pigeonpea wilt pathogen, collected from different
locations of Bihar in respect of cultural and morphological characters, and
pathogenicity. The colony diameter ranged from 42.3 to 70.3 mm 8 days after
incubation at 27 ± 2°C. The colony color varied from white to pink, and back of
the plate showed light to dark yellow to brown pigmentation. The dry
mycelium weight ranged from 98.3 to 201.3 mg, while number of spore
ranged from 0.8 to 3.6 million ml-1
on potato dextrose broth medium after 15
days at 27 ± 2°C. The size of macro conidia and micro conidia ranged from
15.4– 35.0 x 2.0–6.1 µm, respectively. Wilt incidence ranged from 14.3 to
61.9% on the susceptible cultivar Bihar. Isolates which produced abundant
sporulation were highly virulent, while moderately virulent isolates was poor
sporulations.

6
Kumar and Upadhyay, (2014) studied the cultural, morphological and
pathogenic variability in fifteen isolates of Fusarium udum causal agent of
pigeonpea wilt. The isolates were procured from four major Fusarium
udum growing states, Bihar, Jharkhand, Orissa and West Bengal. These
exhibited considerable variations in cultural and morphological characters on
potato dextrose agar medium. The colony diameter ranged from 29.6 to 57.3
mm after eight days of incubation at 27 ± 2°C. The dry mycelium weight
ranged from 98.3 to 201.0 mg, while number of spore ranged from 0.8 to 3.6
million ml-1
from potato dextrose broth medium after 15 days at 27 ± 2°C. The
size of macro conidia and micro conidia ranged from 15.4-45.0 X 2.1-6.2 µm
and 2.5-17.5 X 2.1-6.2 µm, respectively.
Economic Importance of Disease
Kannaiyan et al. (1981) recorded the incidence of the wilt in
Maharashtra (22.6%), Bihar (18.3%), Uttar Pradesh (8.2%), West Bengal
(6.1%), Madhya Pradesh and Gujarat (5.4%), Andhra Pradesh (5.3%), Tamil
Nadu (1.4%), Karnataka (1.1%), Orissa (0.3%) and Rajasthan (0.1%).
Kannaiyan and Nene, (1984) recorded the average incidence of the wilt
disease varies from 0.1% (Rajasthan) to 22.6% (Maharashtra).
Kannaiyan et al. (1984) recorded the annual pigeonpea crop loss due
to wilt in India alone has been estimated at US $ 36 million, while in eastern
Africa the annual losses were estimated at US $ 5 million.
Reddy and Basuchoudhary, (1985) reported upto 22.5 per cent
damage to the pigeonpea crop due to Fusarium wilt.
Khare et al. (1994) reported wilting symptoms at pre-flowering and
podding stage caused 100 per cent, at maturity 67 per cent and at pre
harvesting stage 30 per cent loss.
Nene et al. (1996) reported that the disease occurs in most of the
pigeonpea growing countries of Asia, Africa, Europe and America especially
Bangladesh, Germany, Ghana, Granada, India, Indonesia, Italy, Kenya,
Malawi, Mauritius, Myanmar, Nepal, Tanzania, Thailand, Trinidad and
Tobago, Uganda, Venezuela, Vietnam and Zambia.

7
Singh et al. (2002) observed a yield loss of 10 to 50% and in some
years up to 90% in pigeonpea due to Fusarium wilt in farmer’s fields.
Chauhan and Kumar, (2004) reported that the disease is, however,
more important in India and Eastern Africa. In India the wilt disease occurs in
almost all the pigeonpea growing states but is less prevalent in Southern
states.
Distribution and Survey on Disease Incidence
Booth (1971) reported the wilt disease from Tanzania, Uganda,
Germany, Italy, Vietnam, Kenya, Thailand, Indonesia and Trinidad.
Sharma and Srivastava, (1977) conducted a survey on the wilt
incidence in 27 districts of Madhya Pradesh at the maturity stage of
pigeonpea and reported maximum disease from Shajapur and Baster districts
and minimum disease in rest of the districts.
Kannaiyan and Nene, (1981) reported the pigeonpea wilt from Uttar
Pradesh, Bihar, Madhya Pradesh, Rajasthan, Gujarat, Maharastra, West
Bengal, Orissa, Andhra Pradesh, Karnataka and Tamil Nadu. The average
incidence varied from 0.1% in Rajasthan to 22.6% in Maharastra. The disease
was severe in Maharashtra, Bihar and Uttar Pradesh.
Kannaiyan et al. (1981a) reported the incidence of wilt in Karnataka
varied from 0 to 90 percent. It was severe in major crop growing areas of
Gulbarga, Dharwad, Bidar and Bijapur.
Kannaiyan et al. (1984) reported that the disease is quite serious in
Malawi, Tanzania and Kenya.
Gaur and Sharma, (1989) surveyed major pigeonpea growing districts
of Rajasthan to determine the prevalence of Fusarium wilt at seedling,
flowering and podding stages and indicated that the disease is an important
problem only in Alwar and Dholpur districts.
Saka et al. (1994) conducted a survey on Fusarium wilt of pigeonpea
in 13 districts in northern and southern Malawi in 1993 showed that Fusarium
wilt was the most widely distributed disease, with an average incidence of
5.4%.

8
Bidari et al. (1995) conducted survey on wilt incidence in 84 fields in
different districts of Karnataka, and the study revealed that wilt incidence
ranging from 0.05 to 67.0 percent with an average of 7.65 percent. The mean
incidence of wilt was lowest in Bijapur (4.25%) when compared to other
pigeonpea growing districts. The maximum incidence of 67% was recorded in
Gulbarga followed by 35.70% in Bidar.
Chauhan and Kumar, (2004) surveyed 15 districts of eastern Uttar
Pradesh, India to record the incidence of wilt in pigeonpea caused by
Fusarium udum. The highest Percent disease incidence was reported from
Ghazipur district (14.7%) and that of the lowest was from Pratapgarh district
(2.4%). Jaunpur, Varanasi, Goarkhpur, Azamgarh were also affected by wilt,
with PDI values ranging from 10.4 to 11.8%.
Srivastava et al. (2008) studied the incidence of wilt disease in
pigeonpea in 14 districts of Uttar Pradesh, India, from July to March 2005-06
and 2006-07. Wilt caused by a Fusarium spp. was present in all the districts
surveyed. Disease incidence ranged from 5 to 18% in 2005-06, and from 7 to
23% in 2006-07. Greatest wilt incidence ranging between 13-18% was
recorded in Mahsi districts in 2005-06 and that of 9-23% was recorded in
2006-07.
Saifulla and Mahesh, (2009) conducted an extensive roving survey and
identified hot spots for Fusarium wilt of pigeonpea, in different districts of
southern Karnataka during three consecutive Kharif seasons from 2004-05 to
2006-07. Among the six districts surveyed during Kharif 2004-05 (first year)
maximum mean wilt incidence of 12.55% was recorded in Kolar district and
disease incidence ranged between 0-90 percent. During the second year
(2005-06), among the five districts surveyed, maximum mean wilt incidence of
13.92% was recorded in Chamarajanagar district and disease incidence
ranged between 0-65 percent. During the third year (2006-07), among the
seven districts of southern Karnataka surveyed, maximum mean wilt
incidence of 8.13% was recorded in Bangalore urban district with a range of
0-96% where as Tumkur district was free from wilt incidence.

9
Upadhyay and Kumar, (2009) collected samples of wilted plants of
pigeonpea during 2004-2007, from 17 districts of Bihar for isolation of the
pathogen. Sixty two cultures of Fusarium udum were isolated, purified by
single spore/hyphal tip method. Final grouping of 62 isolates of F. udum
based on morphological, cultural characteristics and relative pathogenicity
was done and isolates were categorized in 17 groups. A total of 12
representative isolates of F. udum were selected out of these and their
reaction was studied on 9 pigeonpea wilt differentials viz., ICP-7035, ICP-
8858, ICP-8859, ICP-8863, ICP-9174, BDN- 2, C-11, MAL-13 and Bahar.
Based on variation in reaction pattern on wilt differentials especially on C-11,
ICP-9174 and Bahar, 4 variants (variant 1, 2, 3 and 4) in isolates of F. udum
have been identified. These variants of F. udum were widely distributed in
different districts of Bihar.
Pawar et al. (2012) surveyed for the wilt disease in Kharif 2009-10 to
assessed the severity of the problem. The information collected revealed that
incidence of wilt ranged from 1 to 22 percent with mean incidence of 5.09 per
cent. Sole crop of pigeonpea expressed more incidence than the intercrop
with sorghum, soybean or cotton.
Symptoms
The symptoms of disease have been described in detail by several
workers (Butler 1910 and 1918; Subramanian 1963; Chaube 1968; Amin et al.
1976; Upadhyay and Rai, 1989; Nene et al. 1979). Pigeonpea plants may
show wilt symptoms from seedling to maturing stage throughout their life.
Generally wilt symptoms appear 4-6 weeks after sowing but are most
common in reproductive stage. The initial visible symptoms are loss of
turgidity in leaves and slight inter veinal clearing. The foliage shows slight
chlorosis and sometimes becomes bright yellow before wilting. Leaves are
retained on wilted plants. The characteristic internal symptom of wilt is the
browning of the vascular tissues. At later stages, the branches dry up from top
to down wards and finally the whole plant dries up. Lateral root infection
results in partial wilting, whereas, tap root infection results in complete wilting.

10
Different media
Reddy (2002) observed maximum growth of F. udum on Richard’s agar
and potato dextrose agar.
Sataraddi et al. (2003) reported Richard’s mediuum to be best suited
medium for the growth of Fusarium udum the causal agent of Fusarium wilt on
pigeonpea.
Gangadhara et al. (2010) conducted in vitro studies on effect of
different media on mycelia growth of F. oxysporum f. sp. vanillae isolates. The
fungus isolates showed best growth on Richard’s agar and potato dextrose
agar media among five culture media that were tried.
Khan et al. (2011) tested Fusarium oxysporum f.sp. ciceri for variation
in growth and cultural characters on five different solid media, PDA found to
be best for the growth of different isolates.
Khilare and Ahmed, (2012) conducted in vitro study on effect of
different culture, media on mycelia growth of Fusarium oxysporum f.sp. ciceri.
The fungus grew the best on Czapek dox agar and PDA media among six
culture media were tested.
Jalander and Gachande, (2015) conducted experiment to find out the
suitable culture media for the study of cultural variability among seven isolates
of Fusarium oxysporum f. sp. udum isolated from seven different varieties of
pigeonpea [Cajanus cajan (L.) Millsp.] using Czapek’s dox agar (CDA), potato
dextrose agar (PDA) and glucose nitrate agar (GNA). The growth rate and
growth pattern of F. oxysporum f. sp. udum isolates were different on three
types of media. Mycelial colour of the pathogen and substrate colour also
different on different media. The pathogen isolates showed variability in
pigmentation more on CDA as compared with other and the growth rate of the
pathogen isolates per day were high in case of GNA was observed. The
colonies grown on CDA and GNA were showed maximum fluffy growth; on
PDA it was appressed manner.
Singh et al. (2016) studied effect of different solid media, viz., Potato
dextrose agar, Richard's agar, Czapek's dox agar, Asthana and Hawker's
agar and Ashby's agar media on radial growth and sporulation of Fusarium

11
oxysporum f.sp. lentis. Potato dextrose agar and Richard's agar were the best
medium for radial growthand sporutation of Fusarium oxysporum f.sp. lentis
as highest colony diameter (76 and 70 mm) and excellent sporulation were
observed on these media. For biomass production of Fusarium oxysporum
f.sp. lentis, Potato dextrose was found to be best as highest dry mycelial
weight (315.5 mg) was recorded in this.
pH
Chaudhary (1971) and Prasad et al. (1992) reported 6.0 pH levels as
the best for the growth and sporulation of Fusarium moniliforme f.sp.
subglutinanse.
Kishore et al. (2009) found pH 5.5 to 7.0 to be the best for growth and
sporulation of Fusarium oxysporum f. sp. lini (Belley).
Gangadhara et al. (2010) conducted in vitro studies on effect of
different pH levels on mycelia growth of F. oxysporum f. sp. vanillae isolates.
The most suitable pH level for growth of fungus was 5.0 and 6.0.
Jaruhar and Prasad, (2011) studied on the effect of pH on the growth
and sporulation of Fusarium oxysporum f. sp. lentis after incubation of two
weeks in vitro culture in sucrose nitrate medium. pH level 6.0 was found
optimum for the growth as well as sporulation of the fungus. Sporulation of
chlamydospore was however found best in the pH level 4.0. Further increase
in the pH level show retarding effect on growth and sporulation. Size of the
spores also increases with increase in the pH range.
Khilare and Ahmed, (2012) conducted in vitro study on effect of
different pH levels on mycelia growth of Fusarium oxysporum f.sp. ciceri. The
most suitable pH level for growth of fungus was 6.0 and 6.5 with 24.7
conidia/μl.
Siddique et al. (2012) studied the influence of pH levels (4.0, 4.5, 5.0,
5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5) on in-vitro growth of Fusarium oxysporum
f.sp. phaseoli isolated from diseased bush bean (Phaseolus vulgaris L.)
seedlings. The growth was measured in terms of radial colony diameter on
semi solid medium and dry mycelia weight grown in liquid medium. Based on

12
results of the study it was concluded that the optimum pH for mycelial growth
of the fungus was 6.0-6.5.
Tyagi and Paudel, (2014) conducted In-vitro studies to check the effect
of pH on the growth and sporulation of Fusarium oxysporum. They observed
that pH level 6.0 is the optimum pH for the growth as well as sporulation of the
fungus. However chlamydospore sporulation was found best in the pH level
4.5. Further increases in the pH level showed retarding effect on growth and
sporulation.
Yadav et al. (2014) studied the effects of temperature, pH and carbon
source on radial growth rate of Fusarium moniliforme, causal organism of
Bakanae disease on potato dextrose broth medium. Optimum temperature
and pH for growth was 20°C and 5.0 with maximum dry mycelium weight and
sporulation i.e. 2.168 gm 1.806 million spores/100 ml, respectively.
Hossain et al. (2015) studied the effect pH on growth and sporulation of
F. moniliforme. In this study seven (4, 5, 6, 7, 8, 9 and 10) levels of pH were
tested against three period (3, 6 and 9 days) of measurement. F. moniliforme
grew well in all range of pH tested. The widest colony diameter and maximum
sporulation was observed at pH 6 after 9 days of incubation. Number of spore
production sharply decreased after pH 8 and lowest spore/ml was recorded at
pH 10.
Singh et al. (2016) studied effect of different pH levels on radial growth
and sporulation of Fusarium oxysporum f.sp. lentis. Growth of the test fungus
was obtained at all the pH levels tested but it was maximum at pH 6.5 (61.0
mm) after 168 hrs of inoculation. Growth of the test fungus decreased by
increasing or decreasing the pH level from the 6.5 level. The foremost acidic
and alkaline pH is not suitable for the growth of pathogen.
Effect of plant extracts
Singh et al. (1999) studied the effect of Cyperus rotundus rhizome
extract on spore germination, percent germination and germination types of
conidia of F. udum using different solvent. They recorded increased percent
germination with increased in dilution of the extract and was maximum at
extract dilution of 1:10 (extract: water).

13
Mandhare and Suryawanshi, (2008) studied effect of different plant
extracts against the mycelial growth of F. udum using poisoned food
technique. They found that the extract of Ocimum sanctum, Eucalyptus spp.
and Nerium indicum completely inhibited the growth of F. udum in agar plates.
Singh et al. (2010) reported that aqueous extract of A. indica was most
effective in inhibiting mycelial growth (67.8%) of F. udum followed by Datura
festilosa (61.2%), Tagetes erecta (52.6%), Eucalyptus citridora (52.2%),
Aegle marmelos (47.9%) and Mimusops elengi (45.9%), respectively.
Khandare and Salve, (2011) found that the leaf extracts of Vitex
nigundo, Polyalthia longifolia, Vinca rosea, Adhatoda zylanica and Hyptis
suaveolens inhibited the radial growth of F. udum at 25% concentration of the
extract. They also observed zero conidia per microscopic field per ml of
suspension at 10% concentration of V. negundo leaf extract and leaf extract
of V. negundo, P. longifolia, V. rosea, A. zylanica gave 100% control efficacy
against pathogenic fungi in vivo conditions.
Devi and Chhetry, (2012) screened antifungal effect of plant extracts
against mycelial growth and spore germination of F. udum at different
concentrations of 5%, 10%, 15% and 20% using poisoned food technique and
cavity slide method. Among them A. sativum at 20% alone recorded 100%
inhibition of mycelial growth and spore germination.
Shukla and Dwivedi, (2012) studied In vitro efficacy of different
concentration i.e. 5%, 10% and 15% of plant extracts viz., Bitter guard,
Turmeric, Garlic and Black pepper to control Fusarium udum. All the plant
extracts showed considerable diminution in the growth of pathogens. Growth
of Fusarium udum has been reduced by 15% concentration of turmeric
(89.2%) followed by garlic (88.26%) and black pepper (82.22%).
Gupta et al. (2015) evaluated antifungal activity of crude extracts of 20
plants for their antifungal activity by "food poisoning method", against
Fusarium oxysporum f. sp. udum, a causal agent for wilt disease of pigeon
pea. The crude extract of leaf of Phyllanthus nurai Linn, and Vitex negundo
Linn exhibited maximum toxicity against the test fungus.

14
Suryawanshi et al. (2016) assessed bio-efficacy of 12 aqueous
phytoextracts (each @ 10 and 15%) against C. dematium, applying poisoned
food techniques. All the test aqueous phytoextracts exhibited antifungal
activity against the test pathogen and it was directly proportional to their
concentrations. However, significantly highest mycelial growth inhibition was
recorded with A. indica (60.74%), followed by A. satium (56.48%), O. sanctum
(49.44%) and A. cepa (44.85%) and rest of the test phytoextracts recorded
mycelial growth inhibition in the range of 18.15 to 45.00%.
Biological Control
Garrett (1965) defined biological control as the control of disease by
living microorganisms under their natural or artificial circumstance.
Dennis and Webster, (1971b) revealed that the most frequently studied
fungi in relation to biological control of various plant diseases on several crops
are the species of Trichoderma which are potential biocontrol agents and their
activity in natural soil is firmly established. Trichoderma sp. is the most
common soil inhabitants and gained maximum attention and success for their
highly antagonistic behavior
Sunitha and Gaikwad, (1995) observed a wide zone of inhibition with
the use of Trichoderma harzianum against F. udum. They also found that
pigeonpea seeds coated with the antagonist microorganism germinated better
than untreated seeds and produced longer shoots and roots when shown in
either wilt infested or sterilized soil.
Siddiqui and Mahmood, (1996) found that simultaneous use of
biocontrol agents viz., Glomus mosseae, Trichoderma harzianium and
Verticillium chlamydosporium against wilt disease complex of pigeonpea
caused by Heterodera cajani and Fusarium udum gave better control than
their individual applications in reducing wilting intensity.
Singh et al. 1997 reported that Trichoderma harzianum showed
mycoparasitism against F. oxysporum f. sp. ciceri, causal agent of chickpea
wilt and T. viride exhibited antibiosis.
Singh et al. 2002 reported that the culture filtrates of test fungi
Gliocladium virens, Aspergillus flavus, Trichoderma harzianum, Penicillium

15
citrinum and Bacillus licheniformis (strain-2042) inhibited the radial growth of
F. udum at varying degrees. The maximum inhibition (69%) was recorded in
G. virens, A. flavus and B. licheniformis, P. citrinum and T. harzianum also
showed marked inhibition against the test pathogen (50.6-60.6%).
Jha et al. (2004) studied the antagonistic potential of bioagents viz.,
Trichoderma viride, T. harzianum and T. virens against H. maydis.The result
revealed that none of the individual bioagents was found effective in inhibiting
the spore germination of H. maydis irrespective of their concentrations.
Kumar et al. (2005) studied the effect of three antagonists viz.,
Trichoderma viride, T. harzianum and T. virens on Alternaria leaf blight of
Vicia faba. All the three isolates overgrew the colony of Alternaria alternata
but T. viride parasitized the test fungus earliest. T. viride exhibited highest
growth rate in mono and dual culture.
Kumar et al. (2009) evaluated one isolate each of Trichoderma viride,
Trichoderma harzianum and Trichoderma virens against twelve isolates
of Fusarium udum collected from different districts of Bihar, Jharkhand and
West Bengal by dual culture technique. All the bioagents inhibited the growth
of F. udum in vitro. Dholi isolate of T. harzianum inhibited the growth of F.
udum isolates Fu-5, Fu-12, Fu-14, Fu-21, Fu-24, Fu-27 and Fu-43 by 40.8,
74.5, 49.4, 59.1, 60.5, 40.8 and 57.8 percent, respectively and proved
superior to other antagonists. T. viride was found to have potential in inhibiting
the radial growth of F. udum isolates Fu-29, Fu-37, Fu-49 and Fu-57 while T.
virens was more effective in inhibiting the growth of Fu-61.
Kumar et al. (2009) studied the efficacy of bioagents
against Helminthosporium maydis in vitro condition. Among the three native
antagonist isolates of Trichoderma viride, Trichoderma harzianum and T.
virens screened, T. viride inhibited the radial growth of H. maydis to an extent
of 60.7% followed by T. harzianum (55.1%) and T. virens (52.6%). Studies on
hyphal interaction between antagonists and test fungus revealed
disorganization of protoplasmic content and lysis of host hyphae.
Ajith and Lakshmidevi, (2010) studied the effect of volatile and non-
volatile compounds produced from Trichoderma spp., viz., Trichoderma

16
saturnisporum, Trichoderma harzianum, Trichoderma viride, and Trichoderma
reesei by poisoned food technique against Colletotrichum capsici, fungal
pathogen responsible for anthracnose disease in Bell peppers (Capsicum
frutescence). The results showed that all the selected Trichoderma spp. has
potential to inhibit the mycelial growth of C. capsici. The volatile compounds
produced form all the selected Trichoderma species showed 30 to 67%
inhibition of C. capsici. However, non-volatile compounds or culture filtrate
from Trichoderma viride at 3-4% concentration shows complete mycelial
inhibition of the test fungi. Trichoderma harzianum, T. saturnisporum and T.
reesei also have the ability to control growth of C. capsici by 21 to 68% at a
concentration of 50% culture filtrate. From the results it is clear that all the
isolates taken were effective in controlling the pathogen in-vitro.
Choudhary and Mohanka, (2012) studied the antagonistic potential of
nineteen isolates of Trichoderma ascribed to three species namely:
Trichoderma harzianum (Th), Trichoderma viride (Tv) and Trichoderma
koningii (Tk) against phytopathogen viz., Fusarium oxysporum f.sp. lentis,
causing wilt of lentil, a disease prevalent in Bihar. Among the different isolates
of Trichoderma isolate-5 and 7 of Th, 2 and 18 of Tv and isolate 9 of Tk were
found to be more efficient amongst all, as they showed better antagonism
against the tested phytopathogen. The isolate Th-5 caused maximum
inhibition (82.8%) followed by Th-7 (82.3%), Tv-2(79.2%) Tv-18 (74.4%) and
Tk-9 (71.0%). Rest isolates were moderate in activity. Metabolites extracted
from liquid culture filtrates also depicted almost the same trend of superiority
as mentioned in dual culture i.e. the same isolates further proved its better
potentiality when compared with rest, Th-5 with superior bio-antagonistic
potential.
Kumar et al. (2012) revealed that during the recent past, Trichoderma
has gained maximum popularity as an effective and ideally used biological
control agent for management of soil borne pathogens. They are reported to
have antifungal, anti nematode, plant growth promoting and plant defense
inducing activities. Some important soil borne pathogens against which
species of Trichoderma showed promise are Pythium, Fusarium, Rhizoctonia,

17
Sclerotium and Macrophomina. These pathogens causes diseases like
damping off, wilt, root and collar rot and stem canker.
Ommati and Zaker, (2012) studied about the biocontrol efficacy of
some native Trichoderma isolates against Fusarium oxysporum, an important
causal agent of potato wilt disease under laboratory conditions. Fourteen
isolates were collected among which eight showed promising ability in
inhibiting growth of the pathogen through dual culture and production of
volatile and non-volatile metabolites but T. asperellum and T. atroviride were
almost more efficient than other isolates in inhibiting the mycelial growth of the
pathogen in comparison to control.
Hassan et al. (2013) evaluated Trichoderma hamatum for its
antagonistic potential against Fusarium oxysporum f.sp. lentis, the causal
agent of vascular wilt disease of lentil and found effective.
Kumar (2013) reported that Trichoderma is a genus of asexually
reproducing fungi that is present in all types of soils. Recent discoveries show
that they are opportunistic, avirulent plant symbionts, as well as being
parasites of other fungi. At least some strains establish robust and long-
lasting colonizations of root surfaces and penetrate into the epidermis and a
few cells below this level. They produce or release a variety of compounds
that induce localized or systemic resistance responses.
Kumar et al. (2014) reported that popularization of biopesticides is very
slow as compared to chemicals and only 2% biopesticides are available.
Among the different biopesticides, Trichoderma is most exploited and have
many success stories. Trichoderma viride and Trichoderma harzianum have
curved a niche for themselves in India as important biocontrol agents for
management of various diseases.
Patel and Patel, (2014) tested seven different strains of Trichoderma,
isolated from wilt infected leguminous crops of Madhya Pradesh and tested
for their antagonistic activity against Fusarium (soil borne pathogen), which is
expressed as a zone of inhibition in the culture plates. The seven strains were
identified as Trichoderma viride, T. harzianum, T. asperellum, T. koningii, T.
atroviride, T. longibrachiatums and T. virens. In this study the best strain of

18
Trichoderma species (Trichoderma viride) and then preparing a simple
bioformulation that is cheap, easy to apply and readily accessible to the
farmers.
Raut et al. (2014) screened two strains of Trichoderma aspellerum
isolated from soil for their efficacy against some common soil borne plant
pathogens by dual culture technique. Both antagonists strains produced non-
volatile metabolites and inhibit the mycelia growth of Fusarium graminearum,
Rhizoctonia solani and Pythium umtimum.
Kumar et al. (2015) reported that Trichoderma viride has got
tremendous ability to grow on a wide variety of agricultural by products. The
use of substrates cow dung wheat bran, karanj kake etc in place of
commercial media for mass cultivation may reduce the cost of production.
Thereby, these eco-friendly products can be made available to the farmers at
a lower price.
Ahmad et al. (2016) evaluated two biocontrol agents, Trichoderma
viride and Aspergillus niger for their efficacy against Altemaria altemata dual
culture technique. The results revealed that all the concentrations of
biocontrol agents were significantly effective in inhibiting the mycelial growth
of A. alternate. Maximum inhibition in colony growth of A. altemata was
observed in T. viride (64.14%) and least inhibition was noticed in A. niger
(46.90%) in dual culture technique.
Kumar et al. (2016) extracted volatile organic compounds (VOCs)
emitted by Trichoderma harzainum OTPB3 by head-space solid phase micro
extraction and analyzed by gas chromatography mass spectrometry. About 94
compounds were detected, which includes 16 aliphatic hydrocarbons, 20
aromatic hydrocarbons, one monoterpinoid, 25 sesquiterpenoids, 13 alcohols,
10 each of aldehydes and ketones, one acid, one esters, three nitrogen and
sulphur and four oxides compounds. The effect of VOCs on growth of
Phytophthora infestans ir PIT30 and tomato were assessed. Among them, a-
caryophyllene, 1, 8 cineole, p-cymene and 1-octen-3-ol exhibited 100%
inhibition of pathogen.

19
Kumar et al. (2016) evaluated three biocontrol agent viz., isolates of
Trichoderma viride, Trichoderma harzianum and Trichoderma virens to test
the antagonism against Fusarium oxysporum f.sp. lentis under in vitro
condition. All the three antagonists have showed the potential of parasitizing
the growth of F oxysporum f.sp. lentis when antagonist was grown in dual
culture. The rate of parasitism was faster in T. viride (56.6% over growth in 96
hrs) than T. harzianum and T. virens. The volatile compounds from T. viride
suppress the mycelial growth of F oxysporum f. sp. lentis and found effective
when compare to others. The non-volatile compounds from T. viride
completely inhibited the radial mycelia growth of F. oxysporum f. sp. lentis at a
concentration of 10% as compared to T. harzianum and T. virens.
Meena and Gangopadhyay, (2016) evaluated antagonistic potentiality
of twelve fungal antagonists against M. phaseolina under laboratory condition.
T. viride (Tv-BKN) significantly inhibited the mycelial growth of M. phaseolina.
Mathews and Dhanapar, (2016) evaluated efficient strains of biocontrol
agent (Thichoderma) against rot pathogens of small cardamom viz.,
Phytophthora meadii and Pythium vexans under in vitro. Out of the sixty
isolates, three Trichoderma isolates viz., T17, T34 & T9 proved 73.33, 72 &
70.22 percent inhibition respectively against P. meadii. Among the superior
isolates, percentage inhibition of P. meadii due to volatile and non-volatile
metabolite production varied from 3.3-8.8% and 65.55-67.77% respectively
emphasizing the role on non-volatile metabolite production as the cause of
inhibition. In case of Trichoderma isolates antagonistic to P. vexans, the
inhibition percent due to volatile and non-volatile metabolite production varied
from 1.1-3.3% and 65.55-67.77%.
Mishra and Pandey, (2016) tested T. viride (Dahod isolate) and T.
asperellum (Junagadh isolate). T. asperellum (Sarsa), T. asperellum
(Junagadh) and T. viride (Anand) in in vitro against R. solan and M.
phaseolina causing wet root rot and dry root rot in chickpea, respectively by
dual culture method. Higher inhibition of mycelial growth of these pathogens
was recorded with T. asperellum (Sarsa) i.e. 72 percent and 89.33 percent,
respectively.

20
Nagamani et al. (2016) evaluated antagonistic native Trichoderma spp.
against the wilt through dual culture, volatile and non-volatile. Out of the
twenty isolates, T. asperellum (ATPU 1) was found to be most effective in
inhibiting the growth of F. oxysporum f.sp. ciceri with inhibition of 84.1%.
Volatile metabolites produced by Trichoderma isolates showed significant
effect on the growth of test pathogens. Isolate T. asperellum (ATPU 1) was
found most efficacious against F. oxysporum f.sp. ciceri by 86.7%. The
highest inhibition was recorded with T. viride (KNN 2) against F. oxysporum f.
sp. ciceri with 95.0%. All Trichoderma isolates significantly inhibited test
pathogens by production of non-volatile inhibitors at 10%, 15% and 20%
respectively.
Olufolaji et al. (2016) evaluated Trichoderma harzianum, an
endophyte against cowpea charcoal rot disease in-vitro and in-vivo. In the in-
vitro experiment, the antagonist inhibited the mycelial growth of M. phaseolina
in all the treatment methods applied. The highest percentage inhibition was
recorded on 72 hours prophylactic inoculation of Tharzianum while the lowest
was recorded on 72 hours curative inoculation with 93.3% and 29.4%
respectively. The higher the number of days the antagonist had established
itself before the inoculation of pathogen, the higher the mycelial growth
inhibition on the pathogen.
Singh et al. (2016) evaluated three bioagents namely T. harzianum,
T. viride and P. fluorescence against radial growth of F. oxysporum f. sp. Pisi.
T. harzianum exhibited highest inhibition percentage of radial growth (80.55%)
followed by T. viride (76.00%) and P. fluorescence (70.22%) over control. All
three bioagents exhibited higher degree of antagonism against F. oxysporum
f.sp. pisi.
Sonam et al. (2016) evaluated the production of potential growth-
promoting metabolites (IAA and phosphate) for eight isolates of Trichoderma
against two soils borne plant pathogens (Scelrotium rolfsii and Rhizoctonia
solani) expressed varying degrees of antagonistic responses, in-vitro
antagonism being more effective against R. solani than S. rolfsii.

21
Suryawanshi et al. (2016) assessed bio-efficacy of eight
antagonists against C. dematium, applying dual culture. Highest mycelial
growth inhibition of the test pathogen was recorded with Trichoderma viride
(70.00%), followed by Aspergillus niger (65.93%), T. harzianum (64.07%), T.
hamatum (61.48%), T. longibrachitum (57.78%).
Umbarkarl et al. (2016) evaluated Trichoderma viride against
Altemaria brassicicola and Altemaria raphani isolated from infected leaf
tissue. ln dual culture, inhibition of Altemaria brassicicola was recorded with
Trichoderma viride by 78.78% and growth of Altemaria raphani was inhibited
with Trichoderma viride by 79.46%.
Chemical Control
Vasudeva et al. (1958) reported that griseofulvin and bulbiformin,
two antibiotics, were found very effective against Fusarium udum.
Chakrabarti and Nandi, (1969) reported that synthetic formulations
of antibiotics, griseofulvin and bulbiformin have been reported effective in
controlling the wilt disease.
Agrawal et al. (1974) conducted in vitro studies against Fusarium
oxysporum f.sp. lentis and found Thiram, Benlate, Rhizoctol and Cereson wet
to most effective.
Sen and Kapoor, (1974) reported that soil drench of carbendazim or
benomyl effectively decreased the wilt incidence on tomato and consequently
increased the yield. Carbendazim was found significantly superior to benomyl.
Sinha (1975) observed that a satisfactory control of the disease by
bavistin applied as soil drench at 2,000 ppm, 10 days before inoculation of
pigeonpea with F. udum. In laboratory experiments bavistin was highly
effective in suppressing the mycelial growth.
Agrawal and Khare, (1977) found that the organomercurials and
arsenic fungicides were fungicidal, while Thiram, Captan, Folpet, Difolatan,
Oxycarboxin and Benomyl were fungistatic against Fusarium oxysporum f.sp.
lentis.

22
Haider et al. (1978) reported the disease control over three years by
captan, brassicol (quintozene) and phenyl mercury acetate.
Upadhyay and Rai, (1981a) found a considerable reduction in the
wilt incidence of pigeonpea by Diathane Z-78 and Zincop. Spore germination
of F. udum was completely inhibited by benlate.
Rai and Upadhyay, (1983) also reported a reduction in the
competitive saprophytic colonization of Fusarium udum in the soil-sand
inoculum mixture amended with Bavistin, Dithane Z-78 and Difolatan
fungicides.
Kotasthane et al. (1987) reported that spore germination of
Fusarium udum was completely inhibited by Benlate and Campogram-M at 50
ppm. Bavistin and BAS 38601F were also highly effective in checking the
mycelial growth of Fusarium udum and these fungicides were most effective
as seed treatments.
Sinha and Upadhyay, (1990) conducted experiments with eleven
compounds for control of Fusarium udum. The results revealed an inhibition of
the pathogen growth by Emisan-6 (Methl ethyl mercuric chloride) and sulfex
(80%) at all concentrations, while Dithane M-45 (mancozeb) and Thiram were
relatively less effective.
Haware and Kannaiyan, 1992 reported that effectiveness of
pigeonpea seed treatment with Benlate, Thiram, Benomyl and Thiram
mixtures against wilt.
Sumitha and Gaikwad, (1995) also evaluated few systemic and
non-systemic fungicides against the pathogen in vitro and the results revealed
a complete inhibition in the fungal linear growth by Bavistin (0.1%), Topsin M-
70 (0.1%), Thiram (0.1%), Captan (0.15%) and Dithane 2-78 (0.3%).
Singh (1998) reported that soil application of thiram and benomyl
effectively managed Fusarium wilt.
Pandey and Upadhayay, (1999) also recorded effective control of
F. udum in pigeonpea due to carbendazim (bavistin).

23
Agarwal et al. (2003) reported that wilt incidence F. udum also
decreased by the application of carbendazim as seed treatment resulting to
significant increase in the yield of pigeonpea.
Ingole et al. (2005) also observed that a combination of
Carbendazim + Thiophanate (0.15 + 0.10%) was effective in reducing the
Fusarium wilt.
Shah et al. (2006) conducted experiment to see the effect of some
fungicides viz., Carbendazim, Mancozeb, Sulphur and conjoint Carbendazim
and Mancozeb against F. udum. All the treatments significantly reduced the
growth of the F. udum as compared to control but it was observed that the
growth of the fungus were significantly less in 10,000 ppm concentration as
compared to 10, 100 and 1000 ppm concentration of fungicides. On
comparative analysis of different fungicides tested Mancozeb showed
maximum inhibition of F. udum as compared to other fungicides.
Maheshwari et al. (2008) tested seven fungitoxicants
against Fusarium oxysporum f.sp. lentis in vitro. All these significantly
checked the growth of the pathogen as compared to control. Carbendazim
proved most effective fungitoxicant for checking the fungal growth, followed by
Captan, Hexaconazole and Diniconazole.
Mahesh et al. (2010) recommended integrated management
(systemic fungicide, biocontrol agent and FYM) as the most effective
treatment of Fusarium udum.
Dhanamanjuri et al. (2013) studied the effect of fungicides on the
seed germination, growth and biomass production of Cicer arietinum and Zea
mays under in vitro. The data obtained indicates that germination percentage
of seeds and biomass production were slightly affected with the differences in
the two crops under investigation. The fungicide Bavistin (Carbendazim) at 10
ppm concentration was the best among the treatments of Cicer arietinum
while in case of Zea mays, 1 ppm concentration of Bavistin (Carbendazim)
has shown better stimulating effect on the seed germination and plant growth
(radicle and plumule) as compared to control. Significant differences in the
growth values of seeds between treated and control plants were observed.

24
Patil et al. (2014) conducted storage experiment on influence of
seed treatment chemicals on seed quality in pigeonpea Cv. BRG-1. Thiram @
3 gm/kg of seed + spinosad @ 0.04 ml/kg of seed which were stored in
superbag recorded significantly higher germination (83.50%), seedling length
(30.43 cm), seedling dry weight (28.90 mg), seedling vigour index-I (2555)
and II (2427), seed moisture is lowest recorded 8.45% at the end of sixth
months of storage period compared to control.
Singh et al. (2014) studied the efficacy of different treatment
measures Carbendazim, Benomyl, Vitavax against seed borne fungi viz.,
Fusarium spp. All the three fungicides Benomyl, Carbendazim and Vitavax
showed minimum occurrences against all the seed borne fungi.
Ahmad et al. (2016) evaluated two fungicides namely Mancozeb
and Carbendazim for their efficacy against Altemaria altemata by poison food
technique. The results revealed that all the concentrations of fungicides were
significantly effective in inhibiting the mycelial growth of A. alternata.
Mancozeb caused highest growth inhibition (78.02%) followed by
Carbendazim (75.42%).

25
MATERIAL AND METHODS
The investigation has been carried out in the Department of Plant
Pathology College of Agriculture Jabalpur (M.P.) during Kharif 2015-16.
3.1 Disease Survey
An intensive roving survey was conducted in the seven major
pigeonpea growing districts of Madhya Pradesh viz., Jabalpur, Narshingpur,
Satna, Raisen, Betul, Chhindwara and Sidhi during the period from
September to December during the year 2015. The districts were traversed
and observations were recorded in fields after every 15-20 km. Stops were
less frequent in areas where pigeonpea was sparsely grown. The major soil-
borne diseases were Fusarium wilt. Their incidence was assessed by
counting the number of plants showing symptoms in three representative 100
plants randomly chosen in each field. The percentage incidence at each
location in a district was used for calculating the district average. The percent
disease incidence was calculated using the formula,
Percent disease incidence=
No of plants showing wilting symptoms
x100
Total no of plants observed
3.2 Equipments and apparatus
The equipments and apparatus which have been used in the study are
given below:-
Laminar air flow, BOD incubator, Refrigerator, Autoclave, Glassware,
Microscope, Hot air oven, pH meter, Electronic balance, Forceps, Inoculation
Needle, Cork borer, Blade etc.
3.3 Chemicals
The chemicals which have been used in the study are given below:-
Agar–Agar, Dextrose, Sucrose, Mannitol, Di Potassium Phosphate,
Magnesium sulphate, Sodium chloride, Potassium sulphate, Calcium
carbonate, D glucose, Potassium nitrate, Potassium dihydrogen phosphate,
Sodium nitrate, Di potassium hydrogen phosphate, Potassium chloride,

26
Ferrous sulphate, Sucrose, Potassium monobasic phosphate, Ferric chloride,
Hydrogen chloride, Asparagin, Tri basic potassium phosphate, and Sodium
hydroxide.
3.4 Cleaning and sterilization of equipments
Corning make glassware was used during the period of investigation.
All the glassware was cleaned with chronic acid, followed by thorough
washing with detergent powder and then rinsing tap water before use. The
sterilization of media was done at 15 lbs, pressure for 20 min. Petriplates
were sterilized in hot air sterilizer at 180°C for 2 hrs. The petriplates used in
bio control study, were sterilized by alcohol.
The isolation chamber was sterilized by alcohol, followed by ultraviolet
exposure for 20 min. The other equipments used in isolation chamber like
forceps, inoculation needle, cork-borer, blade, etc. were sterilized by dipping
them in alcohol, followed by heating on flame.
3.4.1 Sterilization of glasswares
Glasswares were washed in liquid detergent under running tap water
and rinsed with distilled water 2-3 times. These were air-dried and then kept
in oven for sterilization at 180°C for at least 2 hrs. Plastic wares were
autoclaved at 121.6°C, 15 psi for 20 min.
3.4.2 Sterilization of inoculating needles, forceps, cork–borer and
working table
Clean inoculating needle was sterilized by dipping the loop of needle in
spirit and heating over the flame until red hot. The process was repeated 2–3
times. Forceps and cork-borer were also sterilized in the way of needle. The
working table of laminar air flow was disinfected by sweeping with cotton
soaked in absolute alcohol and exposing it to UV light for 15-30 minutes.
3.4.3 Sterilization of media and distilled water
Sterilized glassware and plastic wares were used for dispensing media
and distilled water. All media were autoclaved at 121.6°C, 15 psi pressure for
20 min.

27
3.4.4 Sterilization of laminar air flow
Prior to the day of inoculation of target pathogen, the laminar air flow
was saturated with alcohol vapors. At the time of inoculation the laminar air
flow chamber was wiped with 70% alcohol or general spirit. Then only
required instruments were kept in the chamber and exposed to UV rays for
15-20 minutes. All the operation viz., transfer, inoculation etc. were done over
a spirit lamp.
3.5 Culture media
All the solid media were sterilized in an autoclave at 121.6°C for 20
minutes. Liquid media sterilized at 10 lbs p.s.i. for 10 minutes and process
was repeated after 24 hrs.
3.6 Isolation of pathogen
3.6.1 Preparation of culture medium
For isolation of target pathogen in vitro condition, potato dextrose agar
(PDA) medium was used. For preparation of PDA, 250 g peeled potatoes
were cut into slices and boiled in 500 ml of distilled water in conical flask. The
extract was strained through a piece of muslin cloth and 20 g dextrose was
added in it. 20 g agar–agar was melted in 500 ml of distilled water separately
and was mixed in potato dextrose solution and the volume was made upto
1000 ml by adding distilled water. PDA was poured in flasks, plugged with
non–absorbent cotton plugs and sterilized in an autoclave.
3.6.2 Preparation of slants
For preparation of PDA slants, 4 to 5 ml medium was poured in each
culture tube and plugged with non–absorbent cotton and sterilized in an
autoclave at 121.6°C for 20 minutes. Later on tubes were kept in slanting
position on wooden support and allowed to solidify. Slants were stored in
refrigerator.
3.6.3 Isolation and purification of the pathogen
Small pieces of infected tissues 1–2 mm dimension from the advancing
margin of the spot, adjacent to healthy portions were cut with blade, washed
well in distilled water to remove dust adhered to the infected pieces. Pieces

28
were dipped in 0.1 percent mercuric chloride solution for 30 seconds and
finally washed well in three changes of sterilized distilled water.
The bits were then transferred to PDA slants with the help of
inoculating needle under aseptic condition and incubated at 28 ± 1ºC. After 72
hrs, fragments of hyphal growth from the growing tips were transferred to
fresh PDA slants. Pure culture was made, following repeated hyphal tip
transfer.
Pure culture was maintained on PDA slants by sub culturing it at 30
days intervals. For preservation of cultures the plugged end of the culture
tubes were dipped in melted wax and stored in a refrigerator at 5 ± 1ºC.
3.7 Morphology
Temporary slides were prepared from pure culture. Calibrated ocular
micrometer was used for measurement of hyphae, conidia and conidiophores.
The length and width of conidia and conidiophores along with width of hyphae
were measured with the help of calibrated ocular micrometer.
3.7.1 Unit of measurement
The unit of measurement was µ (1µ = 1/1000mm = 10‾6
m).
Micrometers
A. Ocular micrometer
The scale contained 100 divisions in grade 10, 20, 30, upto 100. The
value of one division of the scale varied from micrometer to micrometer.
Therefore, calibration of ocular micrometer was made with the help of stage
micrometer to record the value of one division of the ocular.
B. Stage micrometer
It consisted of 1mm scale divided into 100 equal divisions. Therefore, 1
divisions = 0.01mm = 10µm (1mm = 1000µm).
3.7.2 Calibration
For calibration of ocular, it was first placed inside the eye piece of 10X
and stage micrometer was placed on the stage of the microscope. The stage
micrometer was placed under focus and ocular divisions were coincided with

29
divisions of stage micrometer and calculation was made by the following
procedure.
Microscope No. :
Eye piece : 10x
Objective : 10x
Since 100 divisions of stage micrometer = 1mm
Therefore 1 divisions of stage micrometer = 0.01mm
= 0µm (1mm = 1000m)
In the present case 65 divisions of ocular coincided with 100 divisions
of stage micrometer.
1 division of ocular = 100/65
= 1.538 divisions of ocular micrometer
1 divisions of stage micrometer = 10 µm
= 15.38 µm
= 15.4 µm
3.8 Culture media
The various culture media were prepared according to the standard
formulae given by Ricker and Ricker (1936) and Khare et al. (1974). The
constituents and method of preparation of various solid and liquid media used
have been described.
3.8.1 Methods of inoculation
For inoculating different solid media in petriplates, 7 days old culture of
target pathogen grown on potato dextrose agar medium was used. The small
size of the inoculum was cut and placed at the centre of the plate in an
inverted position, so that it came in direct contact with the surface of the
medium. For inoculating different liquid media in 100 ml Erlenmeyer flasks
containing 25 ml broth medium, one disc of 5 mm diameter of target pathogen
mycelium was allowed to float on the medium.
3.8.2 Incubation
The inoculated petriplates and flasks were incubated at 28 ± 1ºC in
B.O.D. incubator for required period.

30
3.8.3 Measurement of radial growth of colony
Radial growth of the regular colonies was measured in two directions at
right angles with help of a linear scale. In case of irregular colonies,
measurements were recorded at the broadest and narrowest diameter and
average of two different directions was taken as growth. In all the cases radial
growth was recorded after 168 hrs of incubation. In case of poisoned food
techniques, it was recorded after 120 and 168 hrs of incubation.
3.8.4 Estimation of dry weight of mycelial growth
The target pathogen was inoculated in liquid media contained in
Erlenmeyer flask. These inoculated flasks were incubated for 21 days at 28 ±
1ºC in order to determine the dry weight of mycelial mat. The mycelial mats
were filtered through previously dried and weighed whatman’s filter paper no.
42 and washed thoroughly with hot distilled water to remove the traces of
suspended sugars. Mycelial mats along with filter papers were dried at 60ºC
for 24 hrs. They were cooled in desiccators. The mycelial mats were weighed
and again dried in oven until the constant weights were obtained. Weight of
mycelial mat was calculated with help of the following formulae:
DW = (W2 – W1)
Where,
DW = Dry weight of mycelial mat
W2 = Weight of test fungus along with filter paper
W1 = Weight of filter paper
3.8.5 Estimation of sporulation
For estimating the sporulation, at the end of the incubation period 5
mm disc was cut and suspended in 10 ml of distilled water and shaken well to
harvest spores. Numbers of spores were counted with the help of
Haemocytometer. The results have been expressed as excellent, good, fair,
poor, and no sporulation on the basis of the following scale.

31
Table - 3.1: Details of expression of sporulation
Sporulation Represented as No. of spores/ microscopic field
Excellent ++++ 61 & above
Good +++ 41 – 60
Fair ++ 21 – 40
Poor + Less than 20
No – –
3.9 Cultural studies
3.9.1 Effect of various solid media on growth and sporulation of
Fusarium udum.
Effect of seven solid media, namely Potato dextrose agar, Asthana and
Hawker’s agar, Czapek’s Dox agar, Richard’s agar, Ashby’s agar, Browns
medium and Coon’s medium on growth and sporulation were studied.
Preparation of media
Potato Dextrose Agar (PDA) medium
Peeled and sliced potato - 200 g
Dextrose - 20 g
Agar-agar - 20 g
Distilled water - 1000 ml
Asbhy’s Agar medium
Mannitol - 20 g
Di potassium phosphate - 0.2 g
Magnesium sulphate - 0.2 g
Sodium chloride - 0.2 g
Potassium sulphate - 0.1 g
Calcium carbonate - 5 g
Agar-agar - 5 g

32
Asthana & Hawker’s medium
D-Glucose - 5 g
Potassium nitrate - 3.50 g
Potassium dihydrogen phosphate - 1.75 g
Agar - agar - 20 g
Czapeks Dox Agar (CDA) medium
Sodium nitrate - 2 g
Di potassium hydrogen phosphate - 1 g
Potassium chloride - 0.5 g
Ferrous sulphate - 0.01g
Sucrose - 30 g
Agar-agar - 20 g
Richards’s Agar (RA) medium
Potassium nitrate - 10 g
Potassium monobasic phosphate - 5 g
Ferric chloride - 0.02 g
Sucrose - 50 g
Agar- agar - 20 g
Browns medium
Dextrose - 2 g
Tri basic potassium phosphate - 1.25 g
Agar- agar - 20 g

33
Coon’s medium
Sucrose - 7.2 g
Dextrose - 3.60 g
Potassium di- phosphate - 2.72 g
Agar- agar - 15 g
Method of preparation
For the preparation of above solid media i.e. Potato dextrose agar,
Asthana and Hawker’s agar, Czapek’s Dox agar, Richard’s agar, Ashby’s agar,
Browns medium and Coon’s medium the constituents were dissolved in 100
ml of distilled water and 2 g agar–agar was added for solidification. The final
volume was made upto 100 ml by adding distilled water.
Sterilization of media
In all cases 100 ml medium was poured in 150 ml Erlenmeyer flask,
separately plugged with non-absorbent cotton and sterilized in an autoclave.
Inoculation, incubation and observations
Medium of each flask was poured into 3 Petri-plates @ 20 ml per plate,
allowed to solidify and inoculated with 5 mm disc of 7 days old culture. Plates
were incubated at 28 + 10
C for 7 days and observations were recorded on
radial growth and sporulation after 96 hrs onwards, respectively.
3.9.2 Effect of various liquid media on growth and sporulation of
Fusarium udum.
Effect of seven liquid media, namely Potato dextrose broth, Asthana
and Hawker’s, Czapek’s, Richard’s, Ashby’s, Browns and Coon’s broth
medium on growth and sporulation were studied.

34
Preparation of media
Potato dextrose broth (PDB) medium
Peeled and sliced potato - 200 g
Dextrose - 20 g
Asbhy’s medium
Mannitol - 20 g
Di potassium phosphate - 0.2 g
Sodium chloride - 0.2 g
Potassium sulphate - 0.1g
Calcium carbonate - 5 g
Asthana & Hawker’s medium
D-Glucose - 5 g
Potassium dihydrogen phosphate - 1.75 g
Czapeks Dox medium
Sodium nitrate - 2 g
Di potassium hydrogen phosphate - 1 g
Potassium chloride - 0.5 g
Ferrous sulphate - 0.01 g
Sucrose - 30 g

35
Richards’s medium
Potassium nitrate - 10 g
Potassium monobasic phosphate - 5 g
Ferric chloride - 0.02 g
Sucrose - 50 g
Browns
Dextrose - 2g
Tri basic potassium phosphate - 1.25 g
Coon’s medium
Sucrose - 7.2 g
Dextrose - 3.60 g
Potassium di- phosphate - 2.72 g
Method of preparation
For the preparation of various liquid media the constituents were
dissolved in 100 ml distilled water. The solutions were heated for sometimes
on a water bath. In each case 25 ml of the medium was pipetted out in 100 ml
Erlenmeyer flask and plugged with non–absorbent cotton. For each medium 4
such flasks were prepared. Media were sterilized as per method described
earlier under section 3.8.

36
Inoculation, incubation and measurement of growth
Each flask were inoculated with 5 mm mycelial disc and incubated at
28 ± 1ºC for 21 days and dry mycelial weights were determined as per
method described under section 3.4.4.
3.10 Effect of various pH on growth and sporulation of Fusarium udum.
The set of different pH viz., 5.0, 5.5, 6, 6.5, 7, 7.5, 8 and 8.5 were
prepared and pH was adjusted by adding appropriate amount of HCl and
NaOH in the PDA medium. For each pH value, there were three replications.
PDA was taken as basal medium. The medium as pipetted in 100 ml
Erlenmeyer flask and the pH of medium was adjusted to desired level by
using N/10HCl or N/10NaOH. The flasks containing sterilized medium was
inoculated with 5 mm mycelium disc and incubated at 28 + 10
C. At the interval
of 24 hrs, the linear growth was measured till 7 days. At the interval of 24 hrs,
the linear growth was measured till 7 days. The range of sporulation test
ranges on various pH was recorded after 7 days. Sporulation was calculated
with the help of haemocytometer.
3.11 Biological studies
Three biocontrol agent viz., isolates of Trichoderma viride, Trichoderma
harzianum and Trichoderma virens were evaluated to test the antagonism
against Fusarium udum.
3.11.1 Growth of antagonist and the pathogen in monoculture
To study the growth of antagonists and the test fungus in monoculture,
5 mm mycelial discs of Trichoderma viride, Trichoderma harzianum,
Trichoderma virens and Fusarium udum were inoculated centrally on sterilized
PDA in Petri-dishes. Then plates were incubated in BOD incubator at 28 +
10
C. Observations on colony diameter of individual antagonist and the
pathogen were recorded after 72 hrs of incubation.
3.11.2 Growth of antagonist and the pathogen in dual culture
For screening of the antagonists against Fusarium udum, dual culture
technique developed by Morton and Straube (1955) was adopted. Twenty ml
sterilized melted PDA medium was poured into sterilized Petriplates @ 20

37
ml/plate aseptically, allowed to solidify, then 5 mm discs of the fungus and the
antagonistic cut with the help of sterilized cork borer were placed on PDA
approximately 4 cm apart each other and incubated in BOD incubator at 28 ±
1ºC for 72 hrs. Three replications were maintained for each treatment.
Observation on colony diameter of bioagents and test fungus was recorded.
Inhibition of mycelial growth of test pathogen over check was calculated by
following formula (Vincent 1947).
Inhibition of growth
of pathogen =
Colony diameter of
pathogen in check -
Colony diameter of
pathogen in dual culture
x100
Colony diameter of pathogen in check
In order to study the viability of test fungus, reisolation was done by
transferring 5 mm mycelial disc cut by cork borer from the zone where the test
fungus was already overgrown by the antagonist on PDA medium.
3.11.3 Effect of volatile compounds from antagonist(s) on the radial
growth of Fusarium udum.
The effect of volatile compounds from Trichoderma viride,Trichoderma
harzianum, Trichoderma virens on radial growth of Fusarium udum were
studied as per the method given by Dennis and Webster (1971). The two
bottom portion of Petriplates containing PDA were inoculated with a 5
mm disc of pathogen and antagonist, respectively and both inoculated
bottom plates were placed facing each other and sealed with cellophane
adhesive tape and incubated in BOD incubater at 28 ± 1ºC. The petriplate
containing PDA without antagonist serves as control. The observations on the
radial growth of the test fungus were recorded after 5 days of incubation at 28
± 1ºC. The colony diameter of the test fungus in the treatment in
comparison with that of check gave percent growth inhibition.
3.11.4 Effect of non-volatile (culture filtrate) compounds from
antagonist(s) on the radial growth of Fusarium udum.
The biocontrol agents were grown in Potato dextrose broth at 27ºC with
intermittent shaking at 150 rpm. The metabolites were collected after 15 days
and filtered. The sterilized filtrate were amended in PDA to make 5,10

38
and 15% concentration in petriplates. The solidified agar plates in triplicates
were inoculated at the centre with 5 mm diameter mycelial disc of pathogen
and incubated at 28 ± 1ºC for 7 days. The Plates without filtrate served as
control. The colony diameter was measured and percent inhibition of radial
growth was calculated using the formula given by Vincent, 1947.
3.12 Evaluation of antifungal activities of plant extracts against
Fusarium udum.
Seven locally available plants viz., Citrus limon, Azadirachta indica,
Allium cepa, Allium sativum, Polyalthia longifolia, Ricinus communis and
Parthenium hysterophorus were tested for their antifungal activity against F.
udum. Extracts of plant parts such as leaf, bulb and clove etc were prepared
by the standard method used by Gerard et al. (1994). Fresh plant parts were
washed with tap water followed by sterile distilled water, processed with
sterile distilled water @1mlg-1 of plant tissue (1:1v/w) with pestle and mortar
and filtered through a double layered cheese cloth. The filtrate so obtained
formed the standard plant extract solution. The plant extract so prepared were
screened in vitro against F. udum using poisoned food technique (Mortan and
Straube, 1955). Stock solution 5, 10 and 15 ml were mixed respectively with
95, 90 and 85 ml of sterilized molten Potato Dextrose Agar (PDA) media to
obtained 5, 10 and 15 percent concentration of plant extract. The mixed
medium was thoroughly shaken to ensure uniform mixing of extract. 20 ml of
poisoned PDA was poured into sterile petriplates. Three replications were
maintained for each concentration. After solidification of poisoned media, the
plates were inoculated with mycelium disc (5 mm diameter) of vigorously
growing pure culture colony of F. udum. The control petriplates in three
replications were maintained using only sterile water without any plant extract
but with mycelium disc (5 mm) for comparison. Plates were incubated at 28 ±
of pathogen =
Colony diameter of
pathogen in check
-
Colony diameter of
pathogen in dual
culture
x100

39
1ºC and observation on radial growth of test fungus will be recorded after 168
hours. Recorded data on radial growth was converted into percent growth
inhibition by using following formula given by Vincent, 1947.
of pathogen =
Colony diameter of
pathogen in check
- Colony diameter of
pathogen in dual culture
x100
Table 3.2: Name of antifungal plant, their doses and formulation
S.No. Name of plant Formulation Doses (%)
1. Citrus limon Powder 5, 10, 15
2. Azadirachta indica Powder 5, 10, 15
3. Allium cepa Powder 5, 10, 15
4. Allium sativum Powder 5, 10, 15
5. Polyalthia longifolia Powder 5, 10, 15
6. Ricinus communis Powder 5, 10, 15
7. Parthenium hysterophorus Powder 5, 10, 15
8. PDA as control
-
-
3.13 Fungicidal studies
3.13.1 Effect of fungicide on radial growth and sporulation of F. udum
In order to find out a suitable fungicides for management of wilt of
Pigeonpea, Eight fungicides, namely Captan, Blue copper, Carbendazim,
Carbendazim + Mancozeb, Mancozeb, Fipronil, Thiophanate methyl and
Pyraclostrobin along control was evaluated against Fusarium udum in by
following the poisoned food technique under in vitro condition. PDA poisoned
with each fungicide will be poured into three sterilized Petriplates @ 20
ml/plate and allowed to solidify. Plates containing PDA without fungicide
served as check. After solidification each Petriplate was inoculated with 5 mm
mycelial disc aseptically. Plates were incubated at 28 + 10
C and observation
on radial growth of test fungus will be recorded after 168 hours. Recorded

40
data on radial growth was converted into percent growth inhibition by using
following formula:
Percent growth inhibition (I) =
C - T
x 100
C
Where,
C = Colony diameter in check plate (mm)
T = Colony diameter in the treated plate (mm)
The details about fungicides are given below:
Table 3.3: Name of fungicides, their doses and formulation
S.No. Name of fungicides Formulation Doses (gm/ liter)
1. Captan Powder 2.5g
2. Blue copper Powder 3.0g
3. Carbendazim Powder 1.0g
4. Carbendazim + Mancozeb Powder 2.5g
5. Mancozeb Powder 2.5g
6. Fipronil Liquid 1.0ml
7. Thiophanate methyl Powder 1.0g
8. Pyraclostrobin Granules 0.2g
9. PDA as control - -
3.13.2 Effect of some fungicides on the germination and other growth
parameters of pigeonpea seeds.
Seeds of pigeonpea (TJT-501) were collected from the vendor. Seeds
were carefully selected with no apparent infection/damage and sterilized with
2% sodium hypochloride for 15 minutes. The solution of five fungicides
namely Thiophanate methyl, Pyraclostrobin, Blue copper, Carbendazim and
Carbendazim + Mancozeb was prepared at different concentrations (25, 50,
75 and 100 ppm). Then the selected seeds were soaked overnight (24 hours)
in different flasks containing the test solution of various concentrations. For

41
germination, the treated seeds were placed uniformly in sterilized Petri-dishes
lined with double layer of blotting paper and wetted with 10 ml of different
concentration of the fungicide test solution. For each replicate 10 nos. of
treated seed were used, so total no. of seeds used for each treatment has
been 30 (10×30). One treatment was run as control and treated with distilled
water only. Three replicates for each of the treatment including control was
maintained. All the Petri-dishes were maintained under room temperature.
The seeds were kept under moist condition with the test solutions and equal
volume (i.e. 10 ml) of distilled water. Water was added when the moisture
content of the blotting paper declined. The number of seeds germinated in
each treatment was counted and the germination percentage was calculated
by using the following formula.
Germination (%) =
No. of seeds geminated
x100
Total no. of seeds planted
The radicle and plumule growth of the seedlings exposed to various
concentration of fungicide solution was measured for each germinating seed.
At the end of the experiment, all the radicle and plumule was harvested
separately and oven dried at 700
C for 48 hours to get the biomass of the
same.
Table – 3.4: Name of fungicides, their doses and formulation
S.No. Name of fungicides Formulation Doses (ppm)
1. Thiophanate methyl Powder 25, 50, 75, 100
2. Pyraclostrobin Granules 25, 50, 75, 100
3. Blue copper Powder 25, 50, 75, 100
4. Carbendazim Powder 25, 50, 75, 100
5. Carbendazim + Mancozeb Powder 25, 50, 75, 100
6. Control - -

42
RESULTS
4.1 Collection, Isolation and Identification of Fusarium udum
The wilt fungus was isolated from the diseased pigeonpea plants
collected from research farm of Jawaharlal Nehru Krishi Vishwa Vidyalaya,
Jabalpur and was identified as Fusarium udum on the basis of cultural and
morphological characters. The fungus grew upto 55 mm in 5 days on potato
dextrose agar (PDA) medium. It produced extensive and cottony mycelium in
culture, often with a purple tinge in the mycelium or medium. The mycelium
was septate, hyaline and produced three types of spores. Microconidia were
small elliptical or with 1-2 septa, whereas the macroconidia were long or
curved (fusaroid) (Plates-1). Chlamydospores were oval or spherical and
formed in older cultures from any cell of the hyphae. Pathogenicity of F. udum
isolate was established by verifying the Koch’s postulates. The inoculum of F.
udum prepared on sorghum grains was inoculated in pots containing
autoclaved soil. Healthy and surface sterilized seeds were sown and the
symptoms of disease were observed 30 days after sowing. The wilt symptoms
were identical to those recorded in naturally infested plants. In addition,
blackening was sometimes visible through the bark as streaks or bands.
4.2 Incidence of wilt in different districts of Madhya Pradesh
Survey was conducted in seven districts viz., Jabalpur, Narshingpur,
Satna, Raisen, Betul, Chhindwara and Sidhi of Madhya Pradesh to study the
incidence of disease during 2015-16 to find out the distribution of disease and
the observed data are presented in Table-4.1 and illustrated in Fig-1. The
disease was prevalent in all the districts surveyed however, incidence of
disease varied in different districts. The highest incidence of wilt was recorded
in Betul district with an incidence of 28.8% followed by Raisen district with an
incidence of 26.8%. Least incidence was noted in Narsinghpur (8.03%)
district. The disease incidence recorded in other districts was ranged between
9.3 to 15.0 percent.

43
Table 4.1: Incidence of wilt in different districts of Madhya Pradesh
Sl. No. Name of District Percent wilt incidence
1 Betul 28.8
2 Raisen 26.8
3 Satna 15.0
4 Chhindwara 14.3
5 Sidhi 10.6
6 Jabalpur 9.3
7 Narshingpur 8.03
4.3 Effect of solid media on radial growth and sporulation of Fusarium
udum
Effect of seven solid media, viz., Potato dextrose agar, Asthana and
Hawker’s agar, Czapek’s Dox agar, Richard’s agar, Ashby’s agar, Browns and
Coon’s ager medium on radial growth and sporulation of Fusarium udum were
studied and observations have been presented in Table-4.2 and illustrated in
Fig. 2 & Plate - 2.
Table 4.2: Effect of solid media on radial growth and sporulation of
Fusarium udum
S.No Name of the medium
Radial growth (mm)
Sporulation
After 120hrs* After 168hrs*
1 Potato dextrose agar 61.21 82.00 ++++
2 Richard’s agar 59.96 79.33 ++++
3 Czapek’s Dox agar 37.12 56.83 +++
4 Asthana and Hawker’s
agar
29.86 49.66 ++
5 Browns medium 27.33 47.00 ++
6 Ashby’s agar 47.00 46.33 +
7 Coon’s medium 26.11 45.00 +
CD (0.05) 2.331 2.596
*Average of 3 replications.

44
4.3.1 Effect on radial growth
Maximum colony diameter (82.0 mm) was recorded on PDA medium
followed by Richard’s agar and Czapek’s Dox agar medium which yielded
79.33 mm and 56.83 mm colony diameter, respectively. Least colony
diameter (45.00 mm) of the test fungus was recorded on Coon’s medium. The
colony diameter recorded on Asthana and Hawker’s agar, Ashby’s agar and
Browns medium were respectively, 49.66, 47.0 and 46.33 mm. This indicates
that maximum growth of Fusarium udum was supported by PDA medium.
4.3.2 Effect on sporulation
The test fungus sporulated in all medium tried but excellent and sporulation
were observed in PDA and Richard’s agar medium while good sporulation
was recorded in Czapek’s Dox agar medium. Fair sporulation was observed in
Asthana and Hawker’s agar and Browns medium. Asbhy’s agar medium and
Coon’s medium supported poor sporulation.
Data presented in Table–4.2 clearly indicate that potato dextrose agar
medium is best for radial growth and sporulation of Fusarium udum, followed
by Richard’s agar medium.
4.4 Effect of liquid media on dry mycelial weight of Fusarium udum
Effect of different liquid media namely Potato dextrose broth, Richard’s,
Czapek’s, Asbhy’s, Asthana and Hawker’s, Browns and Coon’s broth medium
on biomass production and sporulation of Fusarium udum were studied and
data have been presented in Table-4.3 and illustrated in Fig. 3 & Plate – 3.
Table 4.3: Effect of liquid media on dry mycelial weight of Fusarium
udum
S.No. Name of the medium Dry weight (mg) after 21 days * Sporulation
1 Potato dextrose broth 348.30 ++
2 Richard’s 375.00 +++
3 Czapek’s Dox 302.16 ++
4 Asthana and Hawker’s 202.23 ++
5 Browns 166.16 ++
6 Ashby’s 125.66 +
7 Coon’s 75.00 +
CD (0.05) 1.951
*Average of 3 replications

45
4.4.1 Effect on mycelial weight
Maximum dry weight (375 mg) of Fusarium udum was recorded in
Richard’s broth medium which was significantly superior to the dry mycelial
weight recorded in rest of the medium. Next best medium supporting the
growth of Fusarium udum was Potato dextrose broth medium, which yielded
348.30 mg dry mycelial weight. The dry mycelial weight recorded on Czapek’s
broth medium (302.16 mg) was significantly lesser to the dry mycelial weight
recorded in Richard’s broth medium in supporting biomass production. Dry
mycelial weight of 202.23, 166.16, 125.66 and 75.00 mg were recorded in
Asthana and Hawker’s, Browns, Ashby’s and Coon’s broth medium,
respectively.
The test fungus sporulates in all tested medium but excellent
sporulation was not observed in any medium. Good sporulation was recorded
in Richard’s broth medium, while Potato dextrose broth and Czapek’s broth
medium supported fair sporulation. Poor sporulation was recorded on Asbhy’s
and Coon’s broth medium, respectively.
Data presented in Table–4.3 clearly indicate that Richard’s broth
medium is best for dry mycelial weight and sporulation of Fusarium udum.
4.5 Effect of various pH on radial growth and sporulation of Fusarium
udum
4.5.1 Effect on radial growth
Effect of different pH viz., 5, 5.5, 6, 6.5, 7, 7.5, 8 and 8.5 on radial
growth and sporulation of Fusarium udum were studied and observations
have been presented in Table–4.4 and illustrated in Fig. 4. Growth of the test
fungus was obtained at all the pH levels tested but it was maximum at pH 6.0
(84.33 mm) after 168 hrs of inoculation followed by pH 6.5 (78.33 mm) and pH
7 (75.16 mm) respectively. Growth of the test fungus decreased by increasing
or decreasing the pH level from the 6.0 level. The foremost acidic and alkaline
pH is not suitable for the growth of pathogen.

46
Table 4.4: Effect of various pH on radial growth and sporulation of
Fusarium udum
S.No. pH
Radial growth (mm)
Sporulation
After 120 hrs* After 168 hrs*
1 5.0 39.30 63.00 ++
2 5.5 58.00 70.33 +++
3 6.0 65.50 84.33 ++++
4 6.5 61.00 78.33 ++++
5 7.0 59.00 75.16 +++
6 7.5 35.00 51.00 ++
7 8.0 33.00 45.00 ++
8 8.5 15.6 20.00 -
CD (0.05) 1.483 1.773
Excellent sporulation was observed at pH 6.0 and 6.5. Good
sporulation was recorded at pH 5.5 and 7.0, respectively. pH 5.0, 7.5 and 8
supported fair sporulation while, no sporulation was observed at pH 8.5.
Data presented in Table–4.4 clearly indicate pH 6 is best for growth and
4.6 Biocontrol study
4.6.1 Growth of antagonists and target pathogen in monoculture
Trichoderma harzianum, Trichoderma viride and Trichoderma virens
were inoculated centrally on PDA to compare their growth rate. Observation
on the radial growth was recorded after 48 and 72 hrs of incubation and
presented in Table–4.5, Fig. 5 & Plate-4. Maximum radial growth of 90.00 mm
was recorded in Trichoderma viride after 72 hrs, followed by 86.83 mm in
Trichoderma virens and 84.66 mm in Trichoderma harzianum. Minimum radial
growth of 84.66 mm was recorded in Trichoderma harzianum after 72 hrs.

47
The test fungus, Fusarium udum showed 36.66 mm growth with white cottony
colony on PDA medium.
Table 4.5: Growth of antagonistic and pathogen in monoculture
Treatment
Radial growth (mm)*
48 hrs 72 hrs
Trichoderma viride 65.16 90.00
Trichoderma virens 62.66 86.83
Trichoderma harzianum 62.00 84.66
Fusarium udum 25.16 36.66
CD (0.05) 1.434 1.723
The study revealed that among the antagonists Trichoderma viride was
fastest in growth. Growth of other antagonists like Trichoderma virens and
Trichoderma harzianum was also faster than the growth of Fusarium udum.
4.6.2 Antagonism studies
4.6.2.1 Trichoderma viride vs. Fusarium udum
When Trichoderma viride and Fusarium udum were grown in dual
culture, the two colonies come in contact, the growth of the test fungus
ceased and the antagonist continued its growth. The mycelial growth of
Trichoderma viride and Fusarium udum in dual culture were 75.67 mm and
14.33 mm, respectively after 72 hrs of incubation (Table–4.6 and illustrated in
Fig. 6 & Plate-5). A clear advancing yellowish green growth of antagonist was
observed over the colony of Fusarium udum.
4.6.2.2 Trichoderma virens vs. Fusarium udum
In dual culture of Trichoderma virens and Fusarium udum also the
fungi grew after inoculation, colonies came in contact, the growth of fungus
ceased and the antagonist continued it growth. The mycelia growth of
Trichoderma virens and Fusarium udum in dual culture were 70.0 mm and
20.0 mm, respectively after 72 hrs of incubation (Table–4.6, Fig. 6 & Plate-5).

48
Table 4.6: Growth of antagonists and pathogen in dual culture
Treatment
Colony
diameter of
antagonist
(mm)*
Colony
diameter
Fusarium
udum (mm)*
Percent
Growth
inhibition
Trichoderma viride 75.67 14.33 61.12
Trichoderma virens 70.00 20.00 45.74
Trichoderma harzianum 67.17 22.83 38.06
Fusarium udum -- 36.86 --
CD (0.05) 1.773
4.5.2.3 Trichoderma harzianum vs. Fusarium udum
In Trichoderma harzianum and Fusarium udum the similar pattern of
mycoparasitism was noted. The colony diameter of Trichoderma harzianum
and Fusarium udum in dual culture were 67.17 mm and 22.83 mm
respectively after 72 hrs of incubation (Table–4.6, Fig. 6 & Plate–5).
It is obvious from this experiment that all the three antagonists viz.,
Trichoderma viride, Trichoderma virens and Trichoderma harzianum have the
potential of parasitizing the growth of Fusarium udum in vitro. The rate of
mycoparasitism was fastest in Trichoderma viride (61.12% over growth in 72
hrs) than Trichoderma virens (45.74%) and Trichoderma harzianum (38.06
%).
4.6.3 Effect of volatile and non - volatile compounds on radial growth of
Fusarium udum
4.6.3.1 Effect of volatile compounds
The volatile compounds from three biocontrol agents viz., Trichoderma
viride Tricoderma virens and Trichoderma harzianum were evaluated against
the Fusarium udum by recording their radial growth. After 5 days of
incubation, it was observed that volatile compounds from Trichoderma viride
exhibited maximum growth inhibition (43.13%) of Fusarium udum when
compare to others. Trichoderma virens exhibited 31.79% growth inhibition.

49
whereas, Trichoderma harzianum shows least growth inhibition of test fungus
which is about 17.52%. (Table–4.7 and Fig. 7 Plate–6)
Data presented in Table – 4.7 clearly indicate that volatile compounds
from Trichoderma viride exhibited maximum growth inhibition (43.13%) of
Fusarium udum.
Table 4.7: Effect of volatile compounds from Trichoderma on radial
growth of Fusarium udum after five days of incubation
S.No. Treatment
Radial growth of
target pathogen
(mm)*
Percent Growth
inhibition
T1 Trichoderma viride 35.16 43.13
T2 Trichoderma virens 42.17 31.79
T3 Trichoderma harzianum 51.0 17.52
T4 Fusarium udum 61.83 --
CD (0.05) 1.851
4.6.3.2 Effect of Non volatile compounds
The non-volatile compounds from three biocontrol agents Trichoderma
viride, Trichoderma virens and Trichoderma harzianum at 5, 10 and 15
percent concentration were evaluated against the Fusarium udum by
recording their radial growth. The culture filtrate (Non–volatile compound)
from all the Trichoderma species exhibited growth inhibition. The culture
filtrate of T. virens was highly effective in inhibiting the radial growth of F.
udum as it produced 57.67, 66.84 and 100 percent growth inhibiton at 5, 10
and 15 percent , respectively (Table–4.8, Fig – 8, 9 & 10 and Plate–7).
The culture filtrate of T. viride and T. harzianum were found moderately
effective as they produced growth inhibiton ranging respectively, between
54.98 to 82.50 and 48.24 to 59.31 percent depending upon the concentration
of culture filtrate.

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