T 83974 shiva kant kushwaha

Studies on Collar rot of Lentil caused
by Sclerotium rolfsii Sacc.
THESIS
Submitted to the
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur
In partial fulfilment of the requirement for
The Degree of
MASTER OF SCIENCE
In
AGRICULTURE
(PLANT PATHOLOGY)
By
SHIVA KANT KUSHWAHA
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 Collar rot of
Lentil caused by Sclerotium rolfsii Sacc.” 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. SHIVA KANT
KUSHWAHA, I.D. No. AP/JB-198/2010 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. (Mrs.) Om Gupta ……………………………………..
Member Dr. Suneeta panday ……………………………………..
Member Dr. R. B. Singh …………………………………….

CERTIFICATE - II
This is to certify that the thesis entitled “Studies on Collar rot of
Lentil caused by Sclerotium rolfsii Sacc.” submitted by Mr. Shiva Kant
Kushwaha 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. (Mrs.) Om Gupta …………………………….
Member Dr. Suneeta Panday …………………………….
Member Dr. R.B. Singh …………………………….
Head of the Department Dr. S. N. Singh …………………………….
Director Instructions Dr. Dhirendra Khare …………………………….

Declaration and Undertaking by Candidate
I Shiva Kant Kushwaha S/o Ram Pal Certify the work embodied in the
thesis entitled “Studies on Collar rot of Lentil caused by Sclerotium rolfsii
Sacc.” is my own first hand bonafide work carried out under the guidance of
Dr. Sanjeev Kumar at Department of Plant Pathology, JNKVV, Jabalpur
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: Shiva Kant Kushwaha

Copyright© Jawaharlal Nehru Krishi Vishwa Vidyalaya,
Jabalpur Madhya Pradesh 2015
Copyright Transfer Certificate
Title of the Thesis : “Studies on Collar rot of Lentil caused by
Sclerotium rolfsii Sacc.”
Name of the candidate : Shiva Kant Kushwaha
Subject : Plant Pathology
Department : Plant Pathology
Year of thesis submission : 2016
Copyright Transfer
The undersigned Shiva Kant Kushwaha 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
Collar rot of Lentil caused by Sclerotium rolfsii Sacc.” submitted for the
award of M.Sc. (Ag.) degree.
Date: / /
Place: Jabalpur
Dr. Sanjeev Kumar Shiva Kant Kushwaha
(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 Assistant 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. (Mrs.) Om Gupta, Professor, Department of Plant Pathology and
Dr. Suneeta Panday, 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 Registrar, 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 Balkishan Chudhary, Prahlad, Omprakash Kapgate, Prahlad
Masumkar, Naresh, Ishwar Patidar, Vipendra Parmar,Riyazuddin khan,Pankaj
Dunge, Mithlesh and friends Brijesh, Sheesh Ram, Jitendra 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 Ram Pal, mother Geeta Bai, brothers Lavkush, Jitendra and Sister Rani 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 (Shiva Kant Kushwaha)

LIST OF CONTENTS
Number Title Page
1. Introduction 1 – 3
2. Review of Literature 4 – 23
3. Material and Methods 24 – 41
4. Results 42 – 59
5. Discussion 60 – 67
6. Summary, Conclusions and Suggestions for
further work
68 – 73
6.1 Summary 68 – 71
6.2 Conclusions 71 – 73
6.3 Suggestions for further work 73
7. Bibliography 74 – 83
8. Appendices I – XI
Curriculum Vitae

LIST OF TABLES
Number Title Page
3.1 Details of expression of formation of sclerotia 29
3.2 Name of antifungal plant, formulation and their doses 38
3.3 Name of fungicides, formulation and their doses 39
3.4
Name of seed treating fungicides, formulation, and their
doses
40
3.5 Disease rating scale 41
4.1
Effect of solid media on radial growth and sclerotia
formation of Sclerotium rolfsii
43
4.2
Effect of liquid media on dry mycelial weight and sclerotia
45
4.3
Effect of various pH on radial growth and sclerotia
46
4.4 Growth of Trichoderma and pathogen in monoculture 47
4.5
Antagonistic activity of Trichoderma against Sclerotium
rolfsii in dual culture
48
4.6
Effect of volatile compounds from Trichoderma on radial
growth and sclerotia farmation of Sclerotium rolfsii
50
4.7.1
Effect of non-volatile compounds from Trichoderma on
radial growth of Sclerotium rolfsii
51
4.7.2
Effect of non-volatile compounds from Trichoderma on
sclerotia formation of Sclerotium rolfsii
52
4.8.1
Effect of plant extracts on radial growth of Sclerotium
rolfsii
53
4.8.2
Effect of plant extracts on sclerotia formation of
Sclerotium rolfsii
54

Number Title Page
4.9
Effect of fungicides on radial growth and sclerotia
formation of Scleroium rolfsii
55
4.10
Effect of seed treatment with fungicides on the
germination, radicle and plumule length and its biomass.
58
4.11
Sceening of available lentil germplasm against
Sclerotium rolfsii
59

LIST OF FIGURES
Number Title Page
1.
Effect of solid media on radial growth of Sclerotium
rolfsii
43-44
2.
Effect of liquid media on dry mycelial weight of
Sclerotium rolfsii
43-44
3.
Effect of various pH on radial growth of
Sclerotium rolfsii
46-47
4.
Growth of Trichoderma and target pathogen in
monoculture.
46-47
5.
Antagonism of Trichoderma against radial growth of
Sclerotium rolfsii in dual culture
48-49
6.
Antagonism of Trichoderma against sclerotia
production of Sclerotium rolfsii in dual culture
48-49
7.
Effect of volatile compounds from Trichoderma on
50-51
8.
Effect of volatile compounds from Trichoderma on
50-51
9.
Effect of non-volatile compounds from Trichoderma at
5% concentration on radial growth of Sclerotium
rolfsii
51-52
10.
rolfsii
51-52
11.
rolfsii
51-52
12.
5% concentration on sclerotia formation of Sclerotium
rolfsii
51-52

Number Title Page
13.
10% concentration on sclerotia formation of
Sclerotium rolfsii
52-53
14.
15% concentration on sclerotia formation of
Sclerotium rolfsii
52-53
15.
Effect of plant extract at 5% concentration on radial
growth of Sclerotium rolfsii
53-54
16.
53-54
17.
53-54
18.
Effect of plant extract at 5% concentration on
53-54
19.
54-55
20.
54-55
21.
Effect of fungicides on radial growth of Sclerotium
rolfsii
55-56
22.
Effect of fungicides on sclerotia formation of
Sclerotium rolfsii
55-56

LIST OF PLATES
Number CONTENT Page
1. Collection, Isolation and Identification of Sclerotium
rolfsii
42-43
2. Effect of solid media on radial growth of Sclerotium
rolfsii
43-44
3. Effect of solid media on sclerotia formation of
Sclerotium rolfsii
43-44
4. Effect of liquid media on dry mycelial weight and
45-46
5. Growth of Tricoderma and target pathogen in
monoculture
47-48
6. Antagonism of Tricoderma against radial growth of
Sclerotium rolfsii in dual culture
48-49
7. Antagonism of Tricoderma against sclerotia
production of Sclerotium rolfsii in dual culture
48-49
8. Effect of volatile compounds from Trichoderma on
50-51
9. Effect of non - volatile compounds from Trichoderma
on radial growth of Sclerotium rolfsii
51-52
10. Effect of non - volatile compounds from Trichoderma
on sclerotia formation of Sclerotium rolfsii
52-53
11.
Effect of plant extracts at 5% concentration on radial
growth of Sclerotium rolfsii after three days of
incubation
53-54
12.
Effect of plant extracts at 10% concentration on
radial growth of Sclerotium rolfsii after three days of
incubation
53-54

Number CONTENT Page
13.
radial growth of Sclerotium rolfsii after three days of
incubation
53-54
14.
sclerotia formation of Sclerotium rolfsii after fifteen
days of incubation
54-55
15.
days of incubation
54-55
16.
days of incubation
54-55
17. Effect of fungicides on radial growth of Sclerotium
rolfsii after three days of incubation
55-56
18. Effect of fungicides on sclerotia formation of
Sclerotium rolfsii after fifteen days of incubation
55-56
19.
germination, radicle and plumule length and its
biomass
58-59
20.
germination, radicle and plumule length and its
biomass
58-59
21. Screening of available lentil germplasm against
Sclerotium rolfsii
59-60

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
DAI = Day after incubation

1
INTRODUCTION
Lentil (Lens culinaris L.) is an edible pulse. It is a bushy annual plant of
the legume family, grown for its lens-shaped seeds. Lentils have been part of
the human diet since the aceramic (before pottery) Neolithic times, being one
of the first crops domesticated in the Near East. Archeological evidence
shows they were eaten 9,500 to 13,000 years ago. In India, lentils soaked in
water and sprouted are offered to gods in many temples. It is also a practice
in South India to give and receive sprouted peas by women who perform
Varalakshmi Vratam. It is considered to be one of the best foods because the
internal chemical structures are not altered by cooking. With about 30% of
their calories from protein, lentils have the third-highest level of protein, by
weight, of any legume or nut, after soybeans and hemp. Proteins include the
essential amino acids isoleucine and lysine, and lentils are an essential
source of inexpensive protein in many parts of the world, especially in West
Asia and the Indian subcontinent, which have large vegetarian populations.
Sprouted lentils contain sufficient levels of all essential amino acids, including
methionine and cysteine. Lentils also contain dietary fiber, folate, vitamin B1,
and minerals. Red (or pink) lentils contain a lower concentration of fiber than
green lentils (11% rather than 31%). Health magazine has selected lentils as
one of the five healthiest foods. The low levels of Readily Digestible Starch
(RDS) 5%, and high levels of Slowly Digested Starch (SDS) 30%, make lentils
of great interest to people with diabetes. The remaining 65% of the starch is a
resistant starch that is classified RS1, being a high quality resistant starch,
which is 32% amylose. Lentils also have some anti-nutritional factors, such as
trypsin inhibitors and relatively high phytate content. Trypsin is an enzyme
involved in digestion, and phytates reduce the bio-availability of dietary
minerals. The phytates can be reduced by soaking the lentil in warm water
overnight. Lentils are a good source of iron, having over half of a person's
daily iron allowance in a one cup serving.

2
Of late, lentil production for the major lentil producing nations has been
trending upwards. Among the main producers, production has been trending
upwards in Canada, the US, and Australia, but has been highly variable and
trending down in India, Bangladesh, Syria and Turkey. Lentil production in
India has always been important as it is the one of the most important rabi
crops in the country. India has been producing lentil since 1st
century AD and
has always been an important producer of the crop. In India, lentil is cultivated
in an area of about 1.41 mha, producing 1.08 mt. of grain, with an average
yield of 765 Kg/ha. Lentil crop is grown in India in the winter season in the
states of Uttar Pradesh, Madhya Pradesh, Bihar, West Bengal, Rajasthan,
Haryana, Punjab, Assam and Maharashtra. Around 90% of the production
comes from the first four states pertaining to the eastern and the northern part
of the country. The southern part of the country hardly contributes to India’s
total production. Uttar Pradesh accounts for the maximum production in the
country contributing to around 45% of the country’s production as well as for
the maximum area under lentil cultivation. The crop is both cultivated as a
primary crop and a secondary crop in the country. Sagar, Jabalpur,
Bundelkhand and Bhopal in Madhya Pradesh, Tal lands spread over south
Bihar districts in Bihar, Kanpur in Uttar Pradesh and Kota in Rajasthan are the
districts where Lentil is cultivated primarily. However, in these states, lentil
yield potential is far below than the other cereal crops (Mondal et al., 2013b).
Various causes are associated with its low yield. One of them is the diseases
causing remarkable yield loss. About 17 diseases have been recorded in lentil
of which 12 are caused by fungi, 2 by nematode and 2 by viruses and 1 by
mycoplasma (Baker and Rashid, 2007).
Collar rot disease caused by Sclerotium rolfsii on lentil crop is very
important a polyphagus pathogenic fungus causes substantial losses in
quality and productivity of yield. S. rolfsii Sacco is a nonspecialized soil borne
fungal pathogen of worldwide importance and has a host range of over 500
species (Punja and Grogan, 1988). The fungi can attack the crop during any
time from seedling to flowering stage and are comparatively more destructive
at the seedling stage. The pathogenic fungus is soil-borne in nature and
produces sclerotia, which can survive in the soil for many years. Infected

3
young seedlings show damping-off symptoms. Plants infected at an advanced
stage gradually turn pale, droop and dry (Njambere and Chen, 2011). The
disease causes appreciable loss in yield due to which, area under this crop is
consistently decreasing. For restoring the area, production and productivity of
lentil, it is necessary to reduce the loss caused by this disease. So, the
present study was undertaken to find out the effect of different management
tools viz., chemical, botanicals, bio-control agents and healthy looking seeds
against collar rot of lentil. The major objectives are:
 To ascertain the cultural factors responsible for the growth of the target
pathogen.
 To develop a suitable and feasible management strategy for effective
management of disease.

4
REVIEW OF LITERATURE
The available literature pertaining to research investigation is reviewed
critically under the following major heads:
History and taxonomy of the fungus
Rolfs (1892) for the first time recorded a tomato blight disease caused
by Sclerotium rolfsii Sacc, in Florida and recognised the small, round sclerotia
as the outstanding morphological characteristics of the organism.
Saccardo (1911) has given the name Sclerotium rolfsii and recognised
the fungus as an imperfect form.
Curzi (1932) proposed the generic name Corticium for the fungus,
based on the studies of perfect stage in pure culture.
Taebot (1973) reported that according to the basidial stage of
Sclerotium rolfsii. It is a species of Athelia in corticiaceae.
Pathogen
Oduro and Tetteh (1978) reported Sclerotium rot (S. rolfsii) for the first
time on soybean varieties raised at the University of Science and Technology,
Kumasi, Ghana. Collar and pod rots were observed.
Khare et al. (1979) reported that various diseases such as vascular
wilt, collar rot, root rot, stem rot, rust, powdery mildew, downy mildew, which
are caused by Fusarium oxysporum f.sp. lentis, Sclerotium rolfsii, Rhizoctonia
solani, Uromyces fabae, Erysiphe polygoni and Peronospora lentis,
respectively are known to infect lentil.
Punja (1985) reported that the most common hosts are the legumes,
crucifers and cucurbits. The diseases caused by the fungus are more serious
in tropical and sub-tropical regions. The large number of sclerotia produced by
S. rolfsii and their ability to persist in the soil for several years, as well as the
profuse growth rate of the fungus make it well suited facultative parasite and a
pathogen of major importance through out the world.

5
Ferreira and Boley (1992) reported that Sclerotium rolfsii, a soil-borne
fungal pathogen causes disease in a wide range of agricultural and
horticultural crops. S. rolfsii has at least 500 species hosts in 100 families.
Purification, Isolation and Identification
Sulladmath et al. (1975) reported that oat (Avena sativa) plants grown
in experimental plots, were severely affected by S. rolfsii at the flag leaf stage.
The affected plants indicated root-rot symptoms marked by dark brown
discolouration at the collar region, covered by a white cottony mycelial growth.
On uprooting such plants, the stem breaks easily at ground level. In advanced
conditions, numerous sclerotia were seen on the collar region. Isolation from
the infected portions yielded Sclerotium rolfsii Sacc.
Siddaramaiah et al. (1978) reported that about one per cent of the
Niger plants wilted in 1978 at Botanical Garden of College of Agriculture,
Dharwad, Minute examination of the wilted plants showed that collar portion of
the plants were sunk and covered with white mycelial mat and large number
of sclerotial bodies around the collar portion. Heavily infected plant died within
a week. Repeated isolation of the infected portion of the plant yielded the
fungus Corticium rolfsii.
Padole et al. (2009) studied incidence of collar rot in 15 to 45 days old
chickpea crop which ranged from 5-30% in 51 locations surveyed nearby
Jabalpur. Investigations on variations in 51 isolates of Sclerotium rolfsii
showed considerable variations with regard to cultural and morphological
characters on PDA and grouped them into three pathotypes. The
pathogenicity test showed the isolates to vary in number of days taken to
initiate plant mortality and 100% mortality.
Symptomology8
Wilson (1953) described the symptoms of collar rot as, mycelium
covering the plant stem near the soil surface. The production of abundant
white mycelium, and small brown spherical sclerotia on the infected parts
were characteristic symptoms of the disease. Later, affected plants/branches
turned yellow or drooped while retaining their green colour, followed by drying
and turning straw colored.

6
Kumar and Dubey (2000) described the symptoms of collar rot on pea
as the plants showed dark brown lesions near the collar region which
increased in size and covered half or more of the root and collar region of the
stem. Affected portions rotted after some time, with discoloration of vascular
tissues in the region. In cases where seedling survival was prolonged
because of late infection, plants showed leaf yellowing and dropping, brown
lesions at the nodes, and, ultimately, death of the plant.
Dantas et al. (2002) reported that collar rot of common bean
(Phaseolus vulgaris), caused by Sclerotium rolfsii, can induce high losses.
The isolates were obtained from the stem of infected plants and the
inoculation was done by deposition of ten sclerotia on the collar of previously
wounded plants. Some cultivars and lines showed susceptibility to the isolates
and some cultivars and lines showed resistance to the isolate.
Rao et al. (2002) conducted an experiment on stem rot or collar rot of
flora bean (Dolichos lablab) caused by S. rolfsii. The symptoms of the disease
included appearance of initially small, oval, straw to brown lesions at the collar
region followed by wilting of the lower leaves and gradual drying of the whole
plant. White mycelial growth developed on the rotted portion, small and
brown, round to oval sclerotia were produced. The pathogen was identified
based on morphological observations and pathogenicity test.
Kator et al. (2015) studied the disease causing potential of Sclerotium
rolfsii on some tomato cultivars and bioassay was conducted. The cultivars
showed disease symptoms such as chlorosis, wilting, damping off, blighting
and necrosis.
Pathogenicity test
Kilpatrick and Merkle (1967) reported the effect of different levels of S.
rolfsii inoculum on foot rot of wheat and found that, 0.5 and 1.0% inoculum
was superior to 3, 5 and 10%. However, considerable amount of infection was
recorded in two per cent inoculum and 100% disease in 6% and above
inoculum level.
Sengupta and Das (1970) studied the cross inoculation of isolates of S.
rolfsii from groundnut, wheat, potato, guava and Bengal gram. They

7
concluded that Bengal gram was the most susceptible host against S. rolfsii.
Although isolates were most virulent to their appropriate hosts.
Datar and Bindu (1974) proved the pathogenicity of S. rolfsii on
sunflower by soil inoculation method under glasshouse condition. The
inoculum was prepared by growing the fungus on sterilized maize bran
medium and mixed with the sterilized soil one week before sowing. Typical
symptoms were produced within a week of inoculation in the field.
Sulladmath et al. (1975) reported that for testing pathogenicity,
Sclerotium rolfsii was grown on corn-meal-sand medium for a week and
mixed into the top layer of sterilized soil filled into six inches diameter clay
pots. Typical root rot symptoms were observed 30-35 days after sowing. The
organism was re-isolated from such infected plants.
Roy (1977) tested pathogenicity of Sclerotium rolfsii on pea (Pisum
sativum L), cauliflower (Brassica oleracea var capitata L) and Arum
(Colocassia sp.). Rotting in all the cases was more than 50 per cent in four to
five days except Colocassia sp.
Siddaramaiah et al. (1978) reported that collar rot infection was caused
by Corticium rolfsii Curzi. The pathogenicity was proved by sowing 50 seeds,
artificially inoculated with 20 days old Corticium spp. culture and the same
quantity of the seeds were sown in sterilized soil as control. The fungus
started infection after the third day of seed germination and all the 40
seedlings were infected within a week, causing post emergence death.
Mishra and Bais (1987) used 15 days old fungal culture grown on sand
corn meal medium for studying pathogenicity of root rot of barley caused by S.
rolfsii, by mixing upper 4-5 cm layer of soil with inoculum at the rate of one
flask per pot.
Kulkarni and Kulkarni (1994) studied the most susceptible growth stage
of groundnut to S. rolfsii, maximum mortality was recorded in 15 days old
plants and the least mortality in 105 days old plants.
Singh and Thapliyal (1998) reported that inoculum density levels of 2.5
to 10g/kg soil significantly increased the emergence rot which was ranged
from 36.70 to 90% in seed and seedling rot of soybean caused by S. rolfsii.

8
Effect of different media
Akram et al. (2007) reported that potato dextrose agar was best for the
radial growth and sclerotial production of S. rolfsii.
Bhosale and Verma (2007) showed that at 8 Days after inoculation, mycelial
growth was most pronounced (90%) in Richard's medium. The highest
number of sclerotia (141.66 per plate) was recorded in linseed meal medium.
Chaurasia et al. (2013) studied influence of culture media on mycelial
growth followed by its sclerotia production. Potato-dextrose medium was
found to be more suitable for mycelial growth and sclerotia production.
Zape et al. (2013) reported that the most suitable medium for better
growth of Sclerotium rolfsii was potato dextrose agar (PDA) (90.00 mm). It
was also foundhat potato dextrose agar (PDA) and peptone sucrose agar
(PSA) medium were suitable for the sclerotial production of S. rolfsii.
Sumia and Uzma (2015) reported that malt extract peptone-dextrose
agar was found to be the best culture medium to obtain the maximum radial
growth (59 mm).
Effect of pH
Mishra and Haque (1962) reported that S. rolfsii was able to grow and
produce sclerotia with wide range of pH level that is, pH 4.0 to 8.0 the
optimum pH level for the growth and sclerotia formation by the fungus ranged
from pH 5.5 to 7.5. The fungus showed straight sparse mycelia texture and
with increase or decrease of the pH level increase in compactness of mycelia
texture.
Aycock (1966) reported optimum pH near 6.0 for the growth of various
isolates of Sclerotium rolfsii.
Mathur and Sinha (1968) observed that the infection of S. rolfsii in guar
was maximum (54.2%) at pH 6.6 and in gram (89.6%) at pH 5.7. Alkaline
condition reduced the disease in both the crops.
Mathur and Sarbhoy (1976) reported excellent sclerotia formation at pH
3.5 to pH 6.5 in case of sugar beet isolate of Sclerotium rolfsii.

9
Sharma and Kaushal (1979) reported maximum sclerotial development
between pH 5.2 to 5.8, in Sclerotium rolfsii isolated from sunflower.
Prasad et al. (1986) found best mycelial growth of S. rolfsii at pH 5.0
while sclerotial formation at pH 7.0.
Hari et al. (1991) reported the radial growth of Sclerotium rolfsii causing
collar rot of groundnut at pH range of 2 to 9 but the maximum growth was at
pH 6.
Singh and Gandhi (1991) reported that maximum mortality of guar
seedling was observed at pH 6.1 and an increase in pH to 8.4 significantly
reduced disease incidence of Sclerotium rolfsii.
Kulkarni and Kulkarni (1998) found the maximum saprophytic activity of
Sclerotium rolfsii at pH level of 6.0.
Bhosale et al. (2007) reported that Incubation at pH 4.0 was the most
favourable for the growth of the pathogen.
Chaurasia et al. (2013) studied different levels of pH on radial growth
and sclerotia production of Sclerotium rolfsii, pH 5.0 was optimum for mycelial
growth while pH 4.0 to 7.0 was found to be most favourable for the production
of sclerotia.
Muthukumar and Venkatesh (2013) in vitro studied the effect of pH
levels on the mycelial growth and biomass production of Sclerotium rofsii
Sacc. causing collar rot of mint. pH (5.0) produced maximum mycelial dry
weight.
Zape et al. (2013) revealed that Sclerotium rolfsii had a wide range of
pH. The maximum radial growth and formation of sclerotia of S. rolfsii was
observed at pH 6.5.
Biocontrol
Dennis and Webster (1971b) described isolation technique of resident
Trichoderma isolates from the rhizosphere of healthy plants in the fields
having high incidence of various diseases. Production of volatile metabolites
by six resident Trichoderma isolates was evaluated by Inverted plate
technique.

10
Henis and Chet (1975) reported that antagonists may act against
pathogens in one or more of the following mechanisms, competition,
antibiosis, parasitism, predation or induce resistance in plant; hydrolytic
enzymes excreted by antagonists is a well known feature of mycoparasitism.
Agrawal et al. (1977) reported that Trichoderma harzianum was highly
antagonistic to Sclerotium rolfsii causing collar rot of lentil. Filtrate of this
organism also checked the growth of Sclerotium rolfsii on potato dextrose
agar. Trichoderma harzianum could check the mortality of lentil caused by
Sclerotium rolfsii under pot condition. The culture of Trichoderma spp. was
more effective when used with the seeds as compared to that used in soil.
Mathur and Sarbhoy (1978) tested five fungi viz., Trichoderma viride,
Trichoderma harzianum, Aspergillus flavus, Fusarium oxysporum and R.
bataticola known to have some antagonistic properties. Trichoderma
harzianum and Trichoderma viride were showing antagonism against
Sclerotium rolfsii under laboratory conditions.
Elad et al. (1980) reported that T. harzianum was found to be an
effective biological control agent for protecting a number of crop plants from
damage induced by S. rolfsii under both greenhouse and field conditions.
Mukhopadhyay (1987) reported that Trichoderma spp. has long been
known as effective antagonist against plant pathogenic fungi. Trichoderma
spp. are known to inhibit the growth of Sclerotium rolfsii on lentil.
Claydon et al. (1987) reported antifungal properties of volatile
compounds (Alkyl pyrones) produced by T. harzianum. Species of
Trichoderma have been demonstrated in vitro to act against fungal plant
pathogens by producing diffusible volatile antibiotics.
Gaikwad and Kapgate (1990) reported that the spore suspension of T.
harzianum and P. pinophilum prevented the germination of sclerotia of S.
rolfsii under in-vitro conditions.
Mukhopadhyay et al. (1992) reported that combined application of
biological agents and fungicides as seed treatment, first with T. virens and
then with 0.1% carboxin was effective in controlling Sclerotium rolfsii in
chickpea, lentil and groundnut.

11
Rathore et al. (1992) reported volatile activity of T. viride against F.
solani which vacuolated most hyphae of the pathogen and that the hyphae of
the pathogen were comparatively thin as compared to control.
Sugha et al. (1993) studied the conidial coating of the antagonists
Trichoderma viride, T. harzianum on seeds and found the significant reduction
in seedling mortality (47-65%) of chickpea as compared with the untreated
control under in-vitro conditions.
Michrina et al. (1995) and Pandey and Uapadhyay (1997) reported the
effectiveness of diffusible volatile compounds by T. viride and T. harzianum in
vitro.
Virupaksha (1997) tested the antagonistic organisms against
Sclerotium rolfsii. Among them, Trichoderma harzianum and Trichoderma
viride were found to be effective in inhibiting the mycelium growth and
reducing production of sclerotial bodies irrespective of inoculation periods. He
also observed inhibition zone and reduction in size of sclerotial bodies in
presence of antagonists.
Prasad et al. (1999) reported that isolates of Trichoderma and
Gliocladium spp. inhibited mycelial growth (54.9 to 61.4%) and suppressed
the sclerotial production (31.8 to 97.8%) of Sclerotium rolfsii under in-vitro
conditions.
Arora (1999) found that, T. harzianum significantly inhibited the growth
of S. rolfsii, the causal organism of root disease of lentil (Lens esculenta) on
PDA medium.
Desai and Schlosser (1999) reported that Trichoderma species has
ability to infect, macerate and kill the sclerotia of S. rolfsii.
Mondal (1999) tested 55 isolates of T. harzianum, isolate TF-24
showed 93% inhibition of mycelia growth of S. rolfsii on PDA.
Biswas and Sen (2000) studied the dual culture of 11 isolates of T.
harzianum. Isolate viz., T8, T10 and T12 were effective against S. rolfsii as
they over grew the pathogen up to 92%, 85% and 79%, respectively in vitro
conditions.

12
Das et al. (2000) evaluated Trichoderma harzianum, T. viride and T.
koningii in vitro against Sclerotium rolfsii, causing collar rot of tomato.
Trichoderma harzianum was the most effective in inhibiting the mycelial
growth in dual culture.
Patel and Anahosur (2001) reported that Trichoderma harzianum
showed mycoparasitic property by overgrowing, or ceasing the mycelial
growth and reducing the sclerotial production of S. rolfsii under laboratory
conditions.
Dutta and Das (2002) observed 61.5, 59.1 and 57.2 per cent inhibition
in mycelial growth of S. rolfsii by T. harzianum, T. viride and T. koningii,
respectively. They also found reduction in sclerotia production by all the
antagonists.
Faruk et al. (2002) tested six isolates of Trichoderma harzianum in vitro
and also in vivo against Sclerotium rolfsii causing cabbage seedling disease.
They found the isolates of T. harzianum significantly reduced the radial colony
growth of S. rolfsii.
Pranab et al. (2002) studied the efficacy of Trichoderma harzianum, T.
viride, and T. koningii for the management of collar rot of tomato caused by
Sclerotium rolfsii under in vitro condition. T. harzianum was the most inhibitory
to S. rolfsii, showed 61.5% inhibition in mycelial growth of the pathogen, T.
harzianum inhibited more than 90% sclerotial production.
Yogendra and Singh (2002) studied the effect of Trichoderma based
biocontrol agents, viz., T. viride and T. harzianum on the growth of Sclerotium
rolfsii in vitro. Growth inhibition increased with the increase in culture filtrate
concentration. The maximum (75%) growth inhibition was observed at 50%
concentration of culture filtrate of T. viride, whereas in case of T. harzianum,
maximum growth inhibition was 64.44% after 96 hrs of incubation.
Faruk et al. (2002) tested the isolates of Trichoderma spp. as
biocontrol agent against Sclerotium rolfsii. Four isolates of the antagonist
significantly reduced the radial growth of S. rolfsii in dual culture on PDA. The
Trichoderma reduced the post emergence mortality due to S. rolfsii.

13
Revathy and Muthusamy (2003) studied the antagonistic effect of
Trichoderma harzianum, T. hamatum and T. viride on Sclerotium rolfsii. T.
viride was the most effective in inhibiting the growth of S. rolfsii (55.8%
inhibition over the control).
Prasad et al. (2003) tested the efficacy of isolates of Trichoderma spp
in suppressing the growth of Sclerotium rolfsii, the cauliflower collar rot
pathogen by dual culture method. They found that T. harzianum (44.1%)
isolate was superior to T. viride (39.1%) isolate in reducing the colony
diameter of S. rolfsii.
Kashem (2005) conducted experiments to determine the efficacy of
Trichoderma in controlling foot and root rot and collar rot of lentil. He found
that Trichoderma harzianum and Trichoderma viride as seed treatment, soil
treatment, seed + soil treatment were effective in controlling collar rot of lentil.
Hannan (2005) studied integrated management of foot rot of lentil,
chickpea and grasspea. He found that post-emergence death of lentil plants,
chickpea and grasspea due to foot rot (Fusarium oxysporum and Sclerotium
rolfsii) was reduced by treating seeds with BAU Biofungicide and BINA-
fertilizer either alone or in combination.
Rudresh et al. (2005) conducted experiment with inhibitory effect of
Trichoderma culture filtrate and non-volatiles on the growth of S. rolfsii.
Amin et al. (2010) tested six isolates of Trichoderma viride. for their
ability to produce volatile metabolites against seven fungal plant pathogens
viz., Fusarium oxysporum (causing chilli wilt), Rhizoctonia solani (causing
sheath blight of rice), Sclerotium rolfsii (causing collar rot of tomato),
Sclerotinia sclerotiorum (causing web blight of beans), Colletotrichum capsici
(causing anthracnose of chilli fruit), Helminthosporium oryzae (causing brown
spot of rice) and Alternaria brassicicola (causing Alternaria blight of cabbage).
Studies indicated that T. viride (Tv-1) was most effective in reducing the
mycelial growth and sclerotia production.
Kashem et al. (2011) conducted experiment with 14 isolates of
Trichoderma spp. (Trichoderma harzianum and T. viride) for control of foot

14
and root rot of lentil (Lens culinaris Medik). The isolate TG-2 of T. harzianum
showed the highest inhibition of the pathogen in field condition.
Rawat et al. (2012) sceened ten Trichoderma isolates for their
antagonistic potential against two major soil borne plant pathogens viz.,
Sclerotium rolfsii and Fusarium oxysporum causing root rot and wilt in lentil
and chickpea, respectively. Under laboratory conditions, high antagonistic
activity against both the test pathogens by all the Trichoderma isolates was
observed in lentil and chickpea.
Bhuiyan et al. (2012) reported that total of 20 T. harzianum isolates
collected from rhizosphere and rhizoplane of different crops were screened
against S. rolfsii following dual plate culture technique. The screened isolates
of Trichoderma significantly reduced the radial growth of S. rolfsii. The isolate
TH-18 of T. harzianum showed the highest inhibition of radial growth of S.
rolfsii.
Darvin et al. (2013) evaluated the effect of Trichoderma spp. on radial
growth of Sclerotium rolfsii. The results from this experiment revealed that T.
viride (TvL), T. harzianum 4 (Th4) and T. harzianum 14 (Th14) isolates were
found effective and showed lowest radial growth of 3.50 cm and highest per
cent inhibition (56.25%) of S. rolfsii.
Darvin et al. (2013) evaluated the effect of Trichoderma spp. on radial
growth of Sclerotium rolfsii Results from non-volatile assay indicated that
irrespective of concentration, culture filtrate of T. viride (TvL) was found to be
most effective.
Pan et al. (2013) isolated five isolates of Trichoderma (TvO, TvG, ThC,
ThR and ThM) from rhizosphere soils of okra, cauliflower, rice, maize and
groundnut using TSM modified medium. In case of S. rolfsii, the maximum
inhibition was recorded with TvG (42.4%).
Pandey (2013) developed clay soil (CS) based bioformulation of
Trichoderma harzianum (Indian Type Culture Collection No. 6797), T. viride
(ITCC No. 2109), and T. virens (ITCC No. 4177) and tested for their ability to
infect, macerate and kill the sclerotia of Sclerotium rolfsii alone and in
integration with Bavistin @2.5g/kg, Metalaxyl + Mancozeb @1.5g/kg, Thiram

15
@2.5g/kg on lentil crop under sick plot. Out of all tested combination of
Trichoderma spp. along with above fungicides, clay soil based T. virens +
Metalaxyl + Mancozeb was found most significantly effective to manage the
collar rot disease caused by S. rolfsii on lentil crop.
Nawar (2013) reported that the bioagent Bio-arc (T. albium) was the
most effective against S. rolfsii growth responsible for 44.66% inhibition.
Maximum inhibition was observed in the culture filtrate of T. harzianum drawn
from potato dextrose broth with mean reduction of 52.33%. Among the tested
saprophytic fungal isolates, Aspergillus ochraceus and Rhizopus nigricans
showed high mean reduction of 47.18 and 46.71% respectively.The
treatments with culture filtrates of all tested fungal isolates were effective in
reducing mycelia growth of S. rolfsii.
Padmaja et al. (2013) studied the antagonistic potential of native
Trichoderma isolates, in vitro against S. rolfsii the causal agent of stem rot
disease in groundnut. Ten native Trichoderma isolates and a commercial
formulation of Trichoderma were screened. Volatile & non-volatile compounds
produced from these isolates were evaluated along with the commercial
formulation through poisoned food technique and culture filtrate studies.
Among them two isolates proved to produce volatile and non-volatile
compounds that inhibited the pathogen effectively.
Sab et al. (2014) studied the bio efficacy of eight antagonists through
poison food technique and dual culture technique against S. rolfsii causing
collar rot of chickpea. Trichoderma harzianum-55 IIHR recorded maximum
inhibition of 70%.
Yasmin et al. (2014) studied the antagonistic potentiality of
Trichoderma harzianum against Sclerotium rolfsii. Dual culture technique was
followed to evaluate the effect of antagonist. The highest percent inhibition
(76.76%) by Trichoderma harzianum was found against Sclerotium rolfsii.
Kashem et al. (2014) reported that macerated extract of Fusarium
solani + Trichoderma harzianum showed the best result in controlling root rot
of lentil.

16
Hoque et al. (2015) tested six selected isolates of three bio-control
agents against foot and root rot pathogens of lentil. S. rolfsii, 80% and 37.85%
inhibition zones were measured against P. fluorescens and T. harzianum,
respectively.
Swathi et al. (2015) reported that volatile metabolites of Tv5 isolate of
Trichoderma viride were more effective against S. rolfsii growth (54.6%
inhibition).
Swathi et al. (2015) also reported that non-volatile metabolites of Th4
isolate of Trichoderma harzianum was more effective against S. rolfsii with
100% growth inhibition at 60 and 80% concentration.
Bhatt et al. (2015) evaluated bioagents against Sclerotium rolfsii
causing southern blight on bell pepper (Capsicum annuum L.). Trichoderma
harzianum isolates 1 and 5 and T. viride isolates 1 and 3 inhibited mycelia
growth by more than 60% and completely inhibited sclerotial production in
vitro.
Effect of plant extracts
Dubey and Kishore (1990) reported that volatile fraction of two
medicinal plants; Azadirachta indica and Eucalyptus globules were more
effective in suppressing the sclerotial germination of Macrophomina
phaseolina than non-volatile fractions.
Jalal and Ghaffar (1992) studied antifungal characteristics of Ocimum
sanctuml L. and found that its leaf extract completely inhibited the growth of S.
rolfsii and other fungi. Ethanol extracts of Aframomum melegueta and
Ocimum gratissimum at 3 to 5% concentration showed total inhibition (100%)
of the mycelia growth of S. rolfsii.
Mahfuzul (1997) tested plant extract like garlic (Allium sativum), ginger
(Zingiber officinale), nisinda (Vitex negundo), dolkalmi (Ipomoea fistulosa) and
marigold (Tagetes erecta) against major seed borne fungal pathogens of chilli.
Among these plant extracts, garlic was found to be most effective.

17
Kurucheve and Padmavathi (1997) reported that, Allium sativum
(garlic) clove recorded the minimum mycelial growth of (176 mg). Under the in
vitro condition.
Prithiviraj et al. (1998) reported that antifungal properties of Allium
sativum are well known against plant pathogens. It contains different
antimicrobial components like allicin, E-and Z-ajoene, iso-E-10-devinylajoene,
and so forth, which are effective against bacteria, yeasts and phytopathogenic
fungi.
Yoshida et al. (1999) studied antifungal properties of Allium sativum
against plant pathogens. It contains different antimicrobial components like
allicin, E-and Z-ajoene, iso-E-10-devinylajoene, and so forth, which are
effective against bacteria, yeasts and phytopathogenic fungi.
Hanthegowda and Adiver (2001) tested different plant extracts against
S. rolfsii. Among different plant extracts, 1:20 dilution of Parthenium
hysterophorus, Polyalthia longifollia and Azadirachta indica significantly
inhibited the mycelial growth of S. rolfsii.
Okereke and Wokocha (2006) reported that the inhibition of damping-
off disease of tomato incited by S. rolfsii was highest with soil drenching of
neem seed (62.4%).
Suleiman and Emua (2009) reported that 55% growth inhibition of Pythium
aphanidermatum with neem leaf extract and followed by ginger rhizome
extract (70%).
Aslam et al. (2010) observed 44.73% mycelial growth inhibition of
important damping-off pathogen, Rhizoctonia solani in vitro when neem leaf
extract was supplemented in potato dextrose agar medium.
Farooq et al. (2010) reported the maximum inhibition of mycelial growth
of Sclerotium (Athelia) rolfsii causing southern Sclerotium rot in sugar beet, by
Azadirachta indica (73.8%).
Yeni (2011) reported that 80% concentration aqueous extract of Z.
officinale inhibited Fusarium oxysporum to 66.70%, 80% aqueous extract of
O. gratissimum inhibited Botrydioploidia theobromae to 60.00% also 73.33%

18
inhibition of Aspergillus flavus was recorded using 30% ethanol extract of Z.
officinale, the same concentration of Ocimum gratissimum inhibited A. niger to
70.00%.
Islam and Faruq (2012) studied effect of seed treatment with neem
leaf, garlic clove, allamonda leaf, ginger rhizome, kalijira seed, bel leaf,
turmeric rhizome, katamehedi leaf and onion bulb against damping-off
disease of some winter vegetable in the net house. All the treatments were
significantly reduced percent damping-off of cabbage, tomato and egg plant
over untreated control. The most effective treatment was neem leaf extract
followed by garlic clove and allamonda leaf extracts in terms of suppressing
damping-off disease incidence with increasing plant growth characters.
Darvin (2013) evaluated extracts of eight plant species viz., Ashoka
(Polyalthia longifolia), Garlic (Allium sativum), Ginger (Zingiber officinalis),
Neem (Azadirachta indica), Seetha Phal (Annonas squamosa), Tulasi
(Ocimum sanctum), Milk weed (Calotropis gigantean) and Peri Winkle (Vinca
rosea) at 10% concentration on development of Sclerotium rolfsii causing
stem rot in groundnut. Among these plant extracts, clove extract of garlic was
most effective and recorded lowest mycelial growth (0.0 cm) and highest per
cent inhibition (PI) (100%) under in vitro condition.
Mahato et al. (2014) studied the effect of different plant oils and plant
extracts on radial growth of Sclerotium rolfsii Sacc. Among the plant oils and
plant extracts, Karanja oil (88.49%) and Murraya exotica leaf extract (86.15%)
were found effective in reducing the growth of S. rolfsii.
Sab et al. (2014) studied the bio efficacy of ten botanicals through
poison food technique and dual culture technique against S. rolfsii causing
collar rot of chickpea. Among the ten botanicals tested, cent per cent mycelial
inhibition was observed in aqueous extract of Agave at different
concentrations.
Gupta et al. (2015) studied the antifungal activity of crude extracts of
some plants against Fusarium udum in pigeonpea. On screening, the crude
extracts of 20 plants for their antifungal activity, the crude extract of leaf of

19
Phyllanthus nursi Linn, and Vitex negundo exhibited maximum toxicity against
the test fungus.
Effect of fungicides
Harlapur (1988) reported the efficacy of thiram in inhibiting the growth
of S. rolfsii, the casual agent of foot rot of wheat.
Mishra and Bais (1987) found that soil treatment with thiram (2000
ppm) minimized pre and post-emergence mortality of barley caused by S.
rolfsii and reported the efficacy of different fungicides hexaconazole (0.1%
and 0.2%), carbendazim (0.2%), and thiophanate-methyl (0.2%) under in vivo
conditions against S. rolfsii of gram and sunflower. Hexaconazole was found
highly effective.
Vanitha and Suresh (2002) reported that, seed treatment with
carbendazim recorded significantly lowest incidence (10.83%) of collar rot of
brinjal caused by S. rolfsii compared to control (39.30%).
Tiwari and Singh (2004) reported that, fungicides like carboxin,
epoxiconazole, hexaconazole, propiconazole and triadimefon which were
found highly effective against Rhizoctonia solani and S. rolfsii, and can be
formulated as seed dresser either with thiram or mancozeb to control both
collar rot and root rot as well as seed mycoflora effectively.
Sheoraj et al. (2005) studied the efficacy of mancozeb, thiram,
carboxin, Dithane M-45, sulfur dust, carbendazim, ziram, streptocycline,
thiophanate methyl and blue copper at 2500 ppm in controlling S. rolfsii
causing collar rot of lentil in vitro. Mancozeb, thiram and carboxin performed
100% control against the pathogen.
Yaqub and Saleem (2006) tested six fungicides viz., Benomyl,
Sancozeb, Thiovit, Dithane M-45, Carbendazim and Topsin-M against
Sclerotium rolfsii by food poison method. At low concentration, no fungicide
inhibited the growth of S. rolfsii, however, at high concentration Dithane M-45
and Sencozeb significantly reduced the growth of sclerotium rolfsii.
Toorray et al. (2007) evaluated seven fungicides (each at 1000, 1500
and 2000 ppm) against Sclerotium rolfsii under in-vitro condition. Complete

20
inhibition of growth of S. rolfsii was recorded by Captan, Thiram, Mancozeb,
Hinosan (edifenphos) and antracol where as Chlorothalonil showed partial
inhibition at low concentration. Bavistin (carbendazim) did not show much
inhibition at all concentrations.
Bhuiyan et al. (2012) screened six fungicides namely Provax-200,
Bavistin, Ridomil, Dithane M-45, Rovral 50 WP and Tilt at 100, 200 and 400
ppm concentration for their efficacy against the radial colony growth of S.
rolfsii. The complete inhibition was obtained with Provax-200 at all the
selected concentrations.
Singh et al. (2012) evaluated different fungicides against Sclerotium
rolfsii Sacc. causing collar rot under in vitro conditions. Out of all ten tested
fungicides at 2500 ppm concentration, four showed 100 per cent suppression
of the pathoegen over the control while in rest others significant reduction in
radial growth and size of sclerotia were observed.
Dhanamanjuri et al. (2013) studied the effect of fungicides on the seed
germination, growth and biomass production of Cicer arietinum and Zea mays
in vitro. The fungicide Bavistin (Carbendazim) at 10 ppm concentration was
the best among the treatments of Cicer arietinum while in case of Zea mays,
1ppm concentration of Bavistin (Carbendazim) has shown better stimulating
effect on the seed germination and plant growth (radicle and plumule) as
compared to Control.
Elizabeth et al. (2013) reported seed treatment with Indofin resulted in
reduction of total seed mycoflora and enhanced the germination percentage
of JG11 (64 to 69%), ICCV 95311(68 to 87%), KAK2 (58 to77%), ICCV92944
(70 to78%). The seedling vigor index was maximum (976.8) in the seeds
treated with Carbendazim of ICCV92944 variety followed by JG11 variety
treated with Captan.
Nawar (2013) studied the inhibitory effect of fungicides against the
growth of Sclerotium rolfsii under in vitro condition. Rhizolex fungicide was
found highly effective and gave 100% reduction in growth when used at lower
concentration 12.5ppm

21
Begum et al. (2014) tested eight fungicides in vitro against S. rolfsii.
The result revealed that maximum (100%) inhibition was observed in
Carboxin, Propiconazole, Hexaconazole, Difenconazole and Carbendazim at
all three concentrations viz., 500, 1000 and 1500 ppm followed by captan
(79.30, 82.76 and 85.23%) and Triadimenfon (49.13, 60.23 and 65.33%) over
control.
Chaurasia et al. (2014) tested nine fungicides, viz., Bavistin, Brassicol,
Captan, Dithane M-45, DM-145, Fytolan, Manzate, Parasan and Sulfex
against Sclerotium rolfsii in vitro by food poison method. All the fungicides
have showed adverse effect on the growth of Sclerotium rolfsii. Brassicol was
found to be significantly effective against inhibition of growth, even in higher
doses i.e., up to 3.0% concentration. Next to Brassicol, Manzate has been
found to be the best as it gave 100% inhibition of growth at 0.1%
concentration. After Manzate, the Parasan and DM-145 have been found to
be the next effective fungicides against Sclerotium rolfsii. The 0.25%
concentration of these two fungicides resulted in 100% inhibition of growth.
Dithane M-45 and Captan were also found to be toxic resulting in 100%
inhibition in growth at 0.5% and 2.0% concentration, respectively.
Hoque et al. (2014) studied the efficacy of four fungicides in controlling
foot and root rot of lentil under field condition. The test fungicides were Rovral
(0.2%), Secure (0.2%), Bavistin (0.2%), Captan (0.2%). Tested fungicides
significantly decreased incidence of foot and root rot of lentil and increased
yield. Best performance was found with Secure (0.2%) in controlling the
incidence of foot and root rot.
Das et al. (2014) evaluated the potential of six systemic fungicides (i.e.
Propiconazole, Hexaconazole, Mycobutanil, Thiophanate Methyl,
Tebuconazole & Carbendazim); three non-systemic fungicides (i.e. Captaf,
Mancozeb & Copper oxychloride) and three combo fungicides(i.e. Metalaxyl
8% +Mancozeb 64% , Carbendazim 12% + Mancozeb 63% & Carboxin
37.5% + Thiram 37.5%) against Sclerotium rolfsii using poisoned food
technique, in vitro. The result showed that the effect of Hexaconazole
(systemic) has been highly effective in suppressing radial expansion of
hyphae.

22
Mahato et al. (2014) studied the effect of different fungicides, on radial
colony growth of Sclerotium rolfsii Sacc. One systemic (Carbendazim 50%),
three contact (Mancozeb, Copper oxychloride, Chlorothalonil) and three
combinations of systemic and contact fungicides (Carboxin 37.5% + Thiram
37.5% WP, Metalaxyl 8% +Mancozeb 64%, Cymoxanil 8% + Mancozeb 64%
WP) were evaluated against S. rolfsii in laboratory. Carboxin+Thiram was the
best combination of fungicides to restrict the fungal growth effectively.
Khan and Javaid (2015) carried out in vitro bioassays using four
fungicides namely Tegula (Tebuconazole), Thiophanate Methyl, Ridomil Gold
(metalaxyl + mancozeb) and Mancozeb at 50, 100, 250 ppm concentrations.
All the concentrations of these fungicides significantly decreased radial growth
of S. rolfsii over control.
Bhatt et al. (2015) reported that chemicals, Strobulirin, Triazole and
Carboxamides had ED 50< 0.5 μg/ml against S. rolfsii in vitro.
Shahiduzzaman (2015) tested fungicides Provax 200 (Carboxin +
Thiram) and Bavistin 50 WP (Carbendazim), against Sclerotium rolfsii the
maximum and significant growth reduction was achieved with only Provax 200
compared to control.
Khalequzzaman (2016) studied the effect of chemical, against foot and
root rot of lentil. The lowest foot and root rot (21.67%) was obtained when
seed treatment was done with Provax 200 (2.5 g/kg seed).
Screening
Akram et al. (2008) evaluated ninety-eight chickpea germplasm
accessions under greenhouse conditions to identify sources of genetic
resistance against collar rot disease incited by the fungus Sclerotium rolfsii.
Out of 98 germplasm accessions only 5 genotypes viz., FLIP 97- 132C, FLIP
97-85C, FLIP 98-53C, ILC -5263 and NCS 9905 exhibited highly resistant
response to disease.
Amule et al. (2014) conducted the experiments during 2009 and 2010
to find out the most effective screening techniques for identifying host plant
resistance against chickpea collar rot caused by Sclerotim rolfsii in pot house.
Out of four techniques employed, chickpea ‘grain inoculation technique’ was

23
found best. The minimum post emergence mortality (6.7%) occurred at 4.0
percent concentration of Pyraclostrobin which is significantly relatively less in
comparison to control (26.8%) during the two consecutive year of testing.
Among 88 chickpea desi genotype GNG 1958 was found resistant to disease,
in Kabuli types, two entries i.e GNG 1969, BG 2086 were resistant.

24
MATERIAL AND METHODS
The investigation has been carried out in the department of Plant
Pathology College of Agriculture Jabalpur (M.P.) during 2015-16.
3.1 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.
3.2 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,
Ferrous sulphate, Sucrose, Potassium monobasic phosphate, Ferric chloride,
Hydrogen chloride, Asparagin, Tri basic potassium phosphate, and Sodium
hydroxide.
3.3 Cleaning and sterilization of equipments
Corning make glassware was used during the period of investigation.
All the glasswares were cleaned with chromic acid, followed by thorough
washing with detergent powder and then rinsing in tap water before use. The
sterilization of the 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.

25
3.3.1 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.
3.3.2 Sterilization of inoculating hard wares
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 disinfested by sweeping with cotton
soaked in absolute alcohol and exposing it to UV light for 15-30 minutes.
3.3.3 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.
3.3.4 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.
3.4 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.
3.5 Isolation of pathogen
3.5.1 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

26
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.
3.5.2 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.
3.5.3 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
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 48
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.
3.6 Morphology
Temporary slides were prepared from pure culture. Calibrated ocular
micrometer was used for measurement of hyphae and sclerotia. The length
and width of sclerotia and width of hyphae were measured with the help of
calibrated ocular micrometer.

27
3.6.1 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).
3.6.2 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
divisions of stage micrometer and calculation was made by the following
procedure.
Microscope No. :
Eye piece : 10 x
Objective : 10 x
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

28
= 15.38 µm
= 15.4 µm
3.7 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.
3.7.1 Methods of inoculation
For inoculating different solid media in petriplates, three 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
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.
3.7.2 Incubation
The inoculated petriplates and flasks were incubated at 28 ± 1ºC in
B.O.D. incubator for required period.
3.7.3 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 72 hrs of incubation. In case of poisoned food
technique, it was recorded after 48 and 72 hrs of incubation.
3.7.4 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.

29
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
3.7.5 Estimation of sclerotia formation of Sclerotium rolfsii
For study the formation of sclerotia of Sclerotium rolfsii, all petridishes,
after measuring the radial growth were kept in the incubator for 15 days. After
15 days of incubation, number of sclerotia per petridish was counted and
categorized as Nil (-), Poor (1+), Fair (2+), Good (3+) and Excellent (4+)
according by the table is given below (Churasia et al., 2013).
Table - 3.1: Details of expression of formation of sclerotia
No. of sclerotia per petridish
Degree of sclerotia
formation
Symbol
0 Nil -
1-100 Poor 1+
101-200 Fair 2+
201-300 Good 3+
More than 300 Excellent 4+

30
3.8 Cultural studies
3.8.1 Effect of various solid media on growth and formation of sclerotia
of Sclerotium rolfsii.
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 agar
and Coon’s agar on growth and sclerotia formation 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
Asthana & Hawker’s medium
D-Glucose - 5 g
Potassium nitrate - 3.50 g
Potassium dihydrogen phosphate - 1.75 g
Agar - agar - 20 g

31
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 agar
Dextrose - 2 g
Tri basic potassium phosphate - 1.25 g
Agar- agar - 20 g
Coon’s agar
Sucrose - 7.2 g
Dextrose - 3.60 g

32
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 agar and Coon’s agar the constituents were dissolved in 100 ml of
distilled water and 2g agar–agar was added for solidification. The final volume
was made upto 100 ml by adding distilled water.
Sterilization of media
In all the 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 three Petri-plates @ 20 ml per
plate, allowed to solidify and inoculated with 5 mm disc of seven days old
culture. Plates were incubated at 28 + 10
C for seven days and observations
were recorded on radial growth after 72 hrs and formation of sclerotia after 15
days onwards.
3.8.2 Effect of various liquid media on growth and sclerotia production
of Sclerotium rolfsii.
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 sclerotia development were studied.
Preparation of media
Potato dextrose broth (PDB) medium
Peeled and sliced potato - 200 g
Dextrose - 20 g

33
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
Richards’s medium
Potassium nitrate - 10 g
Potassium monobasic phosphate - 5 g
Ferric chloride - 0.02 g
Sucrose - 50 g

34
Browns medium
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 sometime 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
four such flasks were prepared. Media were sterilized as per method
described earlier under section 3.8.
Inoculation, incubation and measurement of growth
Each flask was 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.
3.9 Effect of various pH on growth and sclerotia formation of
Sclerotium rolfsii.
The set of different pH viz., 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

35
and the pH of medium was adjusted to desired level by using N/10HCl or
N/10NaOH. The Petriplates containing sterilized medium were inoculated with
5 mm mycelium disc and incubated at 28 +10
C. At the interval of 24 hrs, the
linear growth was measured till 3 days. The number of sclerotia formation per
plate was recorded after 15 days.
3.10 Biological studies
Three biocontrol agents Trichoderma viride, Trichoderma harzianum
and Trichoderma virens were evaluated to test the antagonism against
Sclerotium rolfsii.
3.10.1 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 Sclerotium rolfsii 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.
3.10.2 Growth of antagonist and the pathogen in dual culture
For screening of the antagonists against Sclerotium rolfsii, dual
culture technique developed by Morton and Straube (1955) was adopted.
Twenty ml sterilized melted PDA medium was poured into sterilized
Petriplates @ 20 ml/plate aseptically, allowed to solidify, then 5 mm discs of
the fungus and the antagonist were cut with the help of sterilized cork borer
and placed on PDA approximately 4 cm apart each other and incubated in
BOD incubator at 28 ± 1ºC . Three replications were maintained for each
treatment. Observation on colony diameter of bioagents and test fungus was
recorded after 72 hours and sclerotia production after 15 days. Inhibition of
mycelial growth and production of sclerotia of test pathogen over check was
calculated by following formula (Vincent 1947).
Percent growth inhibition (I) =
C - T
x 100
C

36
Where,
C = Colony diameter in check plate (mm) / no. of sclerotia formed
T = Colony diameter in the treated plate (mm) / no. of sclerotia formed
In order to study the viability of the 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.
3.10.3 Effect of volatile compounds from antagonist(s) on the radial
growth and sclerotia formation of Sclerotium rolfsii.
The effect of volatile compounds from Trichoderma viride,Trichoderma
harzianum, Trichoderma virens on radial growth of Sclerotium rolfsii were
studied as per the method given by Dennis and Webster (1971a). The two
bottom portion of Petriplates containing PDA were inoculated with a 5
mm disc of the pathogen and the antagonist, and both inoculated bottom
plates were placed facing each other and sealed with cellophane adhesive
tape and incubated in BOD incubator at 28 ± 1ºC. The petriplate containing
PDA without antagonist served as control. The observations on the radial
growth of the test fungus were recorded after three days and formation of
sclerotia after 15 days of incubation at 28 ± 1ºC. The colony diameter and
sclerotia farmation of the test fungus in the treatment in comparison with
that of check gave percent growth and sclerotia inhibition.
3.10.4 Effect of non-volatile (culture filtrate) compounds from
antagonist(s) on the radial growth and sclerotia formation of
Sclerotium rolfsii.
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 filtrates were amended in PDA to make 5,10 and
15% concentration in petriplates. The solidified agar plates in triplicate were
inoculated at the centre with 5 mm diameter mycelial disc of the pathogen and
incubated at 28 ± 1ºC for 72 hours. The Plates without filtrate served as
control. The colony diameter and sclerotia formation was recorded and
percent inhibition of radial growth and sclerotia was calculated using the
formula given by Vincent, 1947.
C - T
x 100
C

37
Where,
3.11 Evaluation of antifungal activities of plant extracts against
Sclerotium rolfsii.
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 S.
rolfsii. Extracts of plant parts such as leaf, bulb and clove 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 S. rolfsii 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 S rolfsii. 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 ±
1ºC and observation on radial growth after 72 hours and sclerotia formation
after 15 days of the test fungus was recorded. Recorded data on radial growth
and sclerotia formation was converted into percent inhibition by using
following formula given by Vincent, (1947).
C - T
x 100
C

38
Where,
Table 3.2: Name of antifungal plant, formulation and their doses
S.No. Name of plant Local name Formulation Doses (%)
1. Citrus limon Citrus Powder 5, 10, 15
2. Azadirachta indica Neem Powder 5, 10, 15
3. Allium cepa Onion Powder 5, 10, 15
4. Allium sativum Garlic Powder 5, 10, 15
5. Polyalthia longifolia Ashok Powder 5, 10, 15
6. Ricinus communis Caster Powder 5, 10, 15
7. Parthenium
hysterophorus
Pathenium Powder 5, 10, 15
8. PDA as control
- -
-
3.12 Fungicidal studies
3.12.1 Effect of fungicide on radial growth and Sclerotia formation of S.
rolfsii
In order to find out a suitable fungicide for management of collar rot of
Lentil, eight fungicides, namely Captan, Blue copper, Carbendazim,
Carbendazim + Mancozeb, Mancozeb, Fipronil, Thiophanate methyl and
Pyraclostrobin along with control was evaluated against S. rolfsii by following
the poisoned food technique under in vitro condition. PDA poisoned with each
fungicide was 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 the test fungus was recorded after 72 hours and sclerotia formation

39
after 15 days. Recorded data on radial growth and sclerotia formation was
converted into percent growth inhibition by using following formula:
C - T
x 100
C
Where,
The details about fungicides are given below:
Table 3.3: Name of fungicides, formulation and their doses
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 - -
3.12.2 Effect of some fungicides on the germination and other growth
parameters of lentil seeds.
Seeds of lentil (local variety) were collected from the vendor. Seeds
were carefully selected with no apparent infection/damage and treated with
2% sodium hypochlorite for 15 minutes. The solution of five fungicides namely
Carbendazim + Mancozeb, Pyraclostrobin, captan, and mancozeb was
prepared at different concentrations (25, 50, 75 and 100 ppm). Then the
selected seeds were soaked overnight (24 hours) in flasks containing the test
solution of various concentrations. For germination, the treated seeds were

40
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 ten treated seeds were used, so total no. of seeds
used for each treatment was 30 (10×30). One treatment was run as control
and treated with distilled water only. Three replicates for each of the treatment
including control were 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 exposed to various concentrations of
fungicide solution was measured for each germinating seed. At the end of the
experiment, all the radicles and plumules were harvested separately and oven
dried at 700
C for 48 hours to get the biomass of the same.
Table–3.4: Name of seed treating fungicides, formulation, and their
doses
S.No. Name of fungicides Formulation Doses (ppm)
1. Carbendazim + Mancozeb Powder 25, 50, 75, 100
2. Pyraclostrobin Granules 25, 50, 75, 100
3. Captan Powder 25, 50, 75, 100
4. Mancozeb Powder 25, 50, 75, 100
5. Control - -
3.13 Screening
To find out resistant sources against collar rot disease, 34
genotypes/lines of lentil were screened under field condition at the Breeding
Farm JNKVV Jabalpur (M.P.) during rabi 2015-16.The row to row distance

41
was maintained 30 cm and plant to plant was 10 cm. Data on seedling
mortality were recorded 15 days after sowing. The percentage of mortality for
each germplasm line was calculated and the level of resistance/susceptibility
was grouped according to disease rating scale of Akram et al. (2008) where: 0
= No mortality, 1= less than 1% mortality, 3 = 1-10% mortality, 5 = 11-20%
mortality, 7 = 21-50% mortality and 9 = 51% or more mortality. (Table- 3.5)
Table – 3.5: Disease rating scale
Disease Disease reaction Level of resistance/susceptibility
rating
1 Highly Resistant (HR) Less than 1% mortality
3 Resistant (R) 1-10 % mortality
5 Tolerant (T) 11-20 % mortality
7 Moderately Susceptible (MS) 21-50 % mortality
9 Highly susceptible (HS) 50 % or more mortality

42
RESULTS
4.1 Collection, Isolation and Identification of Sclerotium rolfsii
The collar rot pathogen was isolated from the diseased lentil plants
collected from breeding research farm of Jawaharlal Nehru Krishi Vishwa
Vidyalaya, Jabalpur. The crop season was rabi- 2015-16. The infected tissues
with healthy tissues were cut into small pieces of 0.5 to 1.0 cm long bits. The
bits were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl)
solution for 1 min. The excess water on the surface of the pieces was
removed by blotting on sterile blotting paper. The sterilized pieces were
placed on potato dextrose agar medium supplemented with 200 ppm
streptomycin. The plates were incubated at 28 ± 1°C and examined daily for
the growth of mycelium of Sclerotium rolfsii. Hyphal tip transfer was made
aseptically to potato dextrose agar (PDA) plates amended with 200 ppm
streptomycin. After subsequent growth, to ensure it is not contaminated with
bacteria, the isolates were transferred to PDA slants in test tubes and were
periodically transferred to new slants and stored at 10°C. The pathogen was
identified following a standard key (Bernett, 1980). The fungus grew upto 90
mm in 3 days on potato dextrose agar (PDA) medium. It produced extensive
and white mycelium in culture medium. The mycelium was hyaline, much
branched and hyphae thin walled, septate, connection and sclerotia were
small, mustard shape, white round bodies with clamp in the beginning, later
becoming light to dark brown with shine and measuring 1.0 to 1.15 mm in size
(Plate-1).
4.2 Pathogenicity test
Isolate of S. rolfsii was tested for their ability to cause collar rot disease
of lentil by soil infestation method in pot culture experiment under shade
condition in front of the plant pathology laboratory at College of Agriculture
Jabalpur. Each earthen pot was filled with 1.0 kg sterilized soil. S. rolfsii was
thoroughly mixed with sterilized soil at the rate of 20 g/kg soil. Controls were
prepared using sterilized soil only. Seven seeds (Lentil) were sown in each
pot. Disease development was observed regularly and recorded at seven to

43
30 days after sowing to estimate the effect of pathogens in causing disease.
The causal agent of collar rot was confirmed after re-isolation of the pathogen
from infected root and stems.
4.3 Effect of solid media on radial growth and sclerotia formation of
Sclerotium rolfsii
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 sclerotia formation of Sclerotium
rolfsii were studied and observations have been presented in Table-4.1 and
illustrated in Fig. 1 & Plate – 2 & 3.
Table 4.1: Effect of solid media on radial growth and sclerotia
S.No
Name of the
medium
Radial growth (mm)
Type of
colony
Pigmentation
Degree of
Sclerotia
formation
(After 15
days)
After 48
hrs*
After 72
hrs*
1 Potato dextrose
agar
65.00 90.00 Appressed White Fair
2 Asthana and
Hawker’s agar
49.00 58.16 Appressed Dull white Poor
3 Czapek’s Dox
agar
43.50 51.00 Fluffy Dull white Poor
4 Richard’s agar 37.16 47.50 Fluffy Dull white Poor
5 Browns agar 37.00 39.83 Appressed White Fair
6 Coon’s agar 15.16 19.66 Fluffy White Fair
7 Ashby’s agar 10.83 12.50 Fluffy White Fair
CD (0.05) 2.433 2.81
*Average of 3 replications.
4.3.1 Effect on radial growth
Maximum radial growth (90.0 mm) was recorded on PDA medium
followed by Asthana and Hawker’s agar and Czapek’s Dox agar medium
which yielded 58.16 mm and 51.00 mm radial growth, respectively. Least
radial growth (12.50 mm) of the test fungus was recorded on Ashby’s agar.
The radial growth recorded on Richard’s agar, Browns agar medium and
Coon’s agar medium were 47.50, 39.83 and 19.66 mm respectively. The

44
fungus produced appressed to fluffy type of growth and dull white to white
pigmentation on all the media tested. This indicates that maximum growth of
Sclerotium rolfsii was supported by PDA medium.
4.3.2 Effect on sclerotia formation
The test fungus produced sclerotia on all the media tried but excellent
sclerotia production was not observed in any medium. Fair sclerotia
production was observed on PDA, Brown’s, Coon’s medium and Asbhy’s agar
medium whereas Asthana and Hawker’s agar, Czapek’s Dox agar, Richard’s
agar supported poor sclerotia formation.
Data presented in Table–4.1 clearly indicate that potato dextrose agar
medium was best for radial growth and sclerotia production of Sclerotium
rolfsii.
4.4 Effect of liquid media on dry mycelial weight and sclerotia
Effect of seven 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 sclerotia formation of Sclerotium rolfsii were
studied and data are presented in Table-4.2 illustrated in Fig. 2 & Plate– 4.
4.4.1 Effect on dry mycelial weight
Maximum dry mycelial weight (453.33 mg) of Sclerotium rolfsii was
recorded in Potato dextrose broth medium which was significantly superior to
the dry mycelial weight recorded in rest of the media. Next best medium
supporting the growth of Sclerotium rolfsii was Richard’s broth medium, which
yielded 231.00 mg dry mycelial weight. The dry mycelial weight recorded on
Browns broth medium (170.00 mg) was significantly lesser to the dry mycelial
weight recorded in Potato dextrose broth medium in supporting biomass
production. Dry mycelial weight of 23.66 mg was recorded in Czapek’s broth.
Asthana and Hawker’s, Ashby’s and Coon’s broth media did not support the
growth, as in all these three media Sclerotium rolfsii was unable to grow.

45
Table 4.2: Effect of liquid media on dry mycelial weight and sclerotia
S.No. Name of the medium
Dry mycelial weight
(mg) after 21 days *
Degree of sclerotia
formed after 21
days *
1 Potato dextrose broth 453.33 Excellent
2 Richard’s broth 231.00 Poor
3 Browns broth 170.00 Poor
4 Czapek’s Dox broth 23.66 Poor
5 Asthana and Hawker’s broth 0.00 Nil
6 Ashby’s broth 0.00 Nil
7 Coon’s broth 0.00 Nil
CD (0.05) 2.645
*Average of 3 replications
4.4.2 Effect on sclerotia formation
Excellent sclerotia formation was observed only in PDA medium. Poor
sclerotia formation was recorded in Richard’s, Browns and Czapek’s Dox
broth medium while no sclerotia formation was observed in Asthana and
Hawker’s, Ashby’s and Coon’s broth medium.
Data presented in Table–4.2 clearly indicate that Potato dextrose broth
medium was best for dry mycelial weight and sclerotia formation of Sclerotium
rolfsii.
4.5 Effect of various pH on radial growth and sclerotia formation of
Sclerotium rolfsii
4.5.1 Effect on radial growth
Effect of seven pH levels viz., 5.5, 6, 6.5, 7, 7.5, 8 and 8.5 on radial
growth and sclerotia formation of Sclerotium rolfsii was studied and
observations are presented in Table–4.3 and illustrated in Fig. 3. Growth of
the test fungus was observed at all the pH levels tested but it was maximum
at pH 6.5 (90.00 mm) after 72 hrs of inoculation. pH 6.0 (86.33 mm) and pH 7
(78.33 mm) were also found favorable. Growth of the test fungus decreased

46
by increasing or decreasing the pH level from 6.5 level. Highly acidic and
alkaline pH is not suitable for the growth of pathogen. The fungus produced
fluffy, appressed and dense compact type of growth pattern at different levels
of pH.
Table 4.3: Effect of various pH on radial growth and sclerotia formation
of Sclerotium rolfsii
S.No. pH
Radial growth (mm)
Type of colony
Degree of
sclerotia
formed after 15
days*
After 48 hrs* After 72 hrs*
1 5.5 41.66 65.00 Fluffy Fair
2 6.0 58.66 86.33 Fluffy Excellent
3 6.5 65.33 90.00 Fluffy Excellent
4 7.0 53.00 78.33 Appressed Excellent
5 7.5 44.00 66.66 Appressed Fair
6 8.0 17.33 26.00 Dense compact Poor
7 8.5 15.00 24.66 Dense compact Poor
CD (0.05) 2.617 2.939
4.5.2 Effect of pH on sclerotia formation
Excellent sclerotia formation was observed at pH 6.0, 6.5 and 7.0 while
fair sclerotia production was recorded at pH 5.5 and 7.5. pH 8.0 and 8.5
supported poor sclerotia formation.
Data presented in Table–4.3 clearly indicate that pH 6.5 was best for
growth and sclerotia formation of Sclerotium rolfsii.

47
4.6 Biocontrol study
4.6.1 Growth of Trichoderma and pathogen in monoculture
Trichoderma harzianum, T. viride and T. 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–4.4, Fig. 4 & Plate-5. Maximum radial growth of 90.00 mm was
recorded in Trichoderma viride after 72 hrs followed by 87.33 mm in
Trichoderma virens. Minimum radial growth of 86.50 mm was recorded in
Trichoderma harzianum after 72 hrs. The test fungus, Sclerotium rolfsii
showed 89.33 mm growth on PDA medium.
Table 4.4: Growth of Trichoderma and pathogen in monoculture
S. No Treatment
Radial growth (mm)*
48 hrs 72 hrs
1 Trichoderma viride 65.16 90.00
2 Trichoderma virens 62.66 87.33
3 Trichoderma harzianum 60.33 86.50
4 Sclerotium rolfsii 64.66 89.33
CD (0.05) 2.358 2.039
The study revealed that among the antagonist Trichoderma viride was
fastest in growth. Growth of other antagonists like Trichoderma virens and
Trichoderma harzianum were significantly at par with the growth of Sclerotium
rolfsii.
4.6.2 Antagonism of Trichoderma against radial growth and sclerotia
4.6.2.1 Trichoderma viride vs. Sclerotium rolfsii
When Trichoderma viride and Sclerotium rolfsii 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 Sclerotium rolfsii in dual culture were 45.76 mm and
44.23 mm, respectively after 72 hrs of incubation (Table–4.5, Fig. 5 and
Plate–6).

48
4.6.2.2 Trichoderma virens vs. Sclerotium rolfsii
In dual culture of Trichoderma virens and Sclerotium rolfsii grew after
inoculation, colonies came in contact, the growth of the fungus ceased and
the antagonist continued its growth. The mycelia growth of Trichoderma
virens and Sclerotium rolfsii in dual culture were 46.83 mm and 43.16 mm,
respectively after 72 hrs of incubation (Table–4.5, Fig. 5 and Plate–6).
Table 4.5: Antagonistic activity of Trichoderma against Sclerotium
rolfsii in dual culture
S.
No.
Treatment
Radial growth
of antagonist
(mm)*
Radial
growth
Sclerotium
rolfsii (mm)*
Percent
inhibition
No. of
sclerotia
formed
Percent
Inhibiton
over
contol
1 Trichoderma
harzianum
56.46 33.53 63.60 63.66 84.92
2 Trichoderma
virens
46.83 43.16 51.55 66.33 84.29
3 Trichoderma
viride
45.76 44.23 50.85 36.66 91.31
4 Sclerotium
rolfsii
-- 90 -- 422.33
CD (0.05) 2.556 3.619
4.6.2.3 Trichoderma harzianum vs. Sclerotium rolfsii
In case of Trichoderma harzianum and Sclerotium rolfsii also similar
pattern of growth was noted. The radial growth of Trichoderma harzianum and
Sclerotium rolfsii in dual culture were 56.46 mm and 33.53 mm respectively
after 72 hrs of incubation (Table–4.5, Fig. 5 and Plate–6).
All the three antagonists viz., Trichoderma viride, Trichoderma virens
and Trichoderma harzianum showed more than 50% inhibition of the radial
growth and sclerotia formation of the test pathogen S. rolfsii over control.
Among the tested isolates, Trichoderma harzianum showed the highest (63.6
%) inhibition of the radial growth followed by Trichoderma virens (51.55 %).
The lowest radial growth inhibition of S. rolfsii was observed in Trichoderma
viride (50.85%) which is significantly at par with Trichoderma virens.

49
Results of the experiment also showed that all the three antagonists
were effective in reducing sclerotia formation of S. rolfsii on culture media
Among the tested species, Trichoderma viride showed the highest (91.31 %)
reduction of the sclerotia formation followed by Trichoderma harzianum (84.92
%).The lowest inhibition of sclerotia formation in S. rolfsii was observed in
Trichoderma virens (84.92%) which is significantly at par with Trichoderma
harzianum (Fig-6 and Plate 7).
Data presented in Table – 4.5 clearly indicate that all the three
Trichoderma species showed variable antagonism ranging from 50.85 to
63.60% and 84.29 to 91.31% inhibition in radial growth and sclerotia
formation, respectively. Among the screened antagonists, the Trichoderma
harzianum showed the highest (63.60%) inhibiton of the radial growth of S.
rolfsii while Trichoderma viride showed the highest (91.31%) inhibition of the
sclerotia formation.
4.6.3 Effect of volatile and non - volatile compounds from Trichoderma
on radial growth and sclerotia formation of Sclerotium rolfsii
4.6.3.1 Effect of volatile compounds
The volatile compounds from Trichoderma virens, Trichoderma viride
and Trichoderma harzianum were evaluated against Sclerotium rolfsii by
recording their radial growth and no of sclerotia formed. All the Trichoderma
species produced toxic volatile metabolites having significant effect in
reducing the radial growth and sclerotia formation of the test pathogen.
Trichoderma viride was most effective antagonist producing volatile
metabolites, thereby inhibiting the mycelial growth and sclerotia production by
51.11 and 95.90 per cent respectively. T. virens and T. harzianum caused
inhibition of mycelial growth to the tune of 50.00 and 40.00 per cent and
sclerotia production to the tune of 64.86 and 49.96 percent, respectively.
(Table–4.6 and Fig. 7&8 and Plate–8)
Data presented in Table – 4.6 clearly indicate that volatile metabolites
from Trichoderma viride were most effective in inhibiting the mycelial growth
(51.11%) and sclerotia production (95.90%).

50
Table 4.6: Effect of volatile compounds from Trichoderma on radial
growth and sclerotia formation of Sclerotium rolfsii
S.No. Treatment
Radial
growth(mm) of
target pathogen
(3 DAI)*
Percent
growth
inhibition
No. of
sclerotia
formed (15
DAI)*
Percent
Inhibiton
1 Trichoderma
viride
44.00 51.11 17.66 95.90
2 Trichoderma
virens
45.00 50.00 151.66 64.86
3 Trichoderma
harzianum
54.00 40.00 218.00 49.49
4 Sclerotium rolfsii 90.0 -- 431.66 -
CD (0.05) 2.325 4.167
4.6.3.2 Effect of non volatile compounds
The non-volatile compounds from Trichoderma virens, Trichoderma
viride and Trichoderma harzianum at 5, 10 and 15 percent concentration were
evaluated against Sclerotium rolfsii by recording their radial growth and no of
sclerotia formed. The culture filtrate (non–volatile compound) from all the
Trichoderma species were not found effective in inhibiting the radial growth at
5% and 10% concentrations, respectively. However, the culture filtrates of
Trichoderma species at 15% concentration were found highly effective in
inhibiting the radial growth of Sclerotium rolfsii. Maximum inhibition of radial
growth of the test fungus was recorded in T. harzianum (57.46%) followed by
T. viride (49.62%) and T. virens (26.49%). (Table– 4.7.1, Fig– 9, 10, 11, and
Plate– 9).
Further, the culture filtrate (non–volatile compound) from all the
Trichoderma species were found effective in inhibiting the sclerotia formation
of S. rolfsii at 10% and 15% concentrations. However, the culture filtrates of
Trichoderma species at 15% concentration were found highly effective in
inhibiting the sclerotia production of Sclerotium rolfsii. Maximum inhibition of
sclerotia production of the test fungus was recorded in T. viride (99.83%)

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