Compatibility of Temperature and PH Tolerant Trichoderma Strains With Herbicides, and Their Bioefficacy Against Rhizoctonia Bataticola

Compatibility of temperature and pH tolerant Trichoderma
strains with herbicides, and their bioefficacy against

Rhizoctonia bataticola
THESIS
Submitted to the
Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya,

Gwalior
In partial fulfillment of the requirements for the Degree of
MASTER OF SCIENCE
In
AGRICULTURE
(PLANT PATHOLOGY)
By
SWATI PANWAR
Department of Plant Pathology,
Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya,
College of Agriculture, Indore (M.P.)
2017
CERTIFICATE – I
This is to certify that the thesis entitled “Compatibility of temperature and pH tolerant
Trichoderma strains with herbicides, and their bioefficacy against Rhizoctonia bataticola”.
submitted in partial fulfillment of the Degree of MASTER OF SCIENCE IN AGRICULTURE (Plant
Pathology) of Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya, Gwalior is a record of the bonafide
research work carried out by Miss. SWATI PANWAR under my guidance and supervision. The subject
of the thesis has been approved by the Student’s Advisory Committee and the Director of Instruction.
No part of the thesis has been submitted for any other degree or diploma or has been
published. All the assistance and help received during the course of these investigations has been
acknowledged by the scholar.
Place:Indore
Date:---/---/------
Signature
(Dr. A. Krishna)
Chairman of the Advisory Committee
MEMBER OF STUDENT’S ADVISORY COMMITTEE
1. Chairman (Dr. Ashok Krishna ) : -----------------------------
2. Member ( Dr. R. K. Singh) : -----------------------------
3. Membe (Dr. R. K. Choudhary) : -----------------------------

CERTIFICATE – II
This is to certify that the thesis entitled “Compatibility of temperature and pH tolerant
Trichoderma strains with herbicides, and their bioefficacy against Rhizoctonia bataticola”.”.
submitted by Miss. SWATI PANWAR to the Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya,
Gwalior, in partial fulfillment of the requirements for the degree of Master of Science in
AGRICULTURE in the Department of Plant Pathology has been accepted after evaluation by the
external examiner and approved by the Student’s Advisory Committee after on oral examination the
same.
Place:Indore
Date:---/---/------ Signature
(Dr. A. Krishna)
Chairman of Advisory Committee
MEMBERS OF THE ADVISORY COMMITTEE
1. Chairman (Dr. Ashok Krishna) : -------------------------------
2. Member (Dr. R. K. Singh) : ---------------------------------------
3. Member (Dr. R. K. Choudhary) : ---------------------------------------
Head of the Department : ----------------------------------------
Dean of the college : ---------------------------------------
Director Instruction : ----------------------------------------

ACKNOWLEDGEMENT
I praise God for enabling me to accomplish this great task of thesis

work in his grace for his glory. I find no words to express my sincere feelings
of gratitude towards my most esteemed guide and chairperson of my
advisory committee, Dr. Ashok Krishna, Professor of Dept of Plant
Pathology and Dean of College of Agriculture, Indore, who deserves my
most sincere thanks and respect for inspiring and excellent guidance,
contact, encouragement, unceasing interest, constructive criticism and
helping attitude throughout the investigation and preparation of the
manuscript.
I extend my sincere gratitude to Dr. Ashok Krishna Professor, Dept
of Plant Pathology , Dr. R.K. Singh Professor, Department of Plant
Pathology and other members of the Advisory committee, Dr. R.K.
Choudhary Professor, Department of Entomology, Dr. K.S. Kumar,
Professor, Department of Agricultural Statistics, College of Agriculture,
Indore, for their suggestions and helping attitude during the research work.
With profound respect, I wish to express my sincere gratitude to Dr. A.
K. Singh, Vice Chancellor RVSKVV Gwalior, Dr. B.S.Baghel, Director
Instructions (RVSKVV) Gwalior, Dr. H.S. Yadava, Director Research
Services RVSKVV and Dr. Ashok Krishna Dean, College of Agriculture,
Indore, (M.P) for providing necessary facilities during the experiment.
I am also extremely thankful to Shri Shankar lal Choudhary, Lab
technician, Departmen Shri Pradeep Singh Chouhan for extending the
help during my research work.
I extend my thanks to my best Seniors Vibhor Joshi, Tarachand
Waskale Friends’ Neha, Mamta, Arpita, Nitika, Kavi, Ankit, Arvind, Mukesh,
Ravi, Ravi, Anil, heri and all my jhingus members who helped me directly or
indirectly during course of investigation.
I find no rhetorical gems from the ocean of words to express my profound
feeling to my most venerable parents Mr. Kunwarsingh Panwar and Mrs.
Neeta Panwar , Mr. Narsingh Mandloi and Mrs Lalita Mandloi .I also want
to express my profound feelings to my loving brothers Bablu Dada ,Pappu
Bhaia ,Pintu Bhaia Bhayu Dada and family who has piloted me up to this
stage and whose love, devotion, blessing and care throughout my life
enabled me to achieve this seemingly invincible goal.
Date: / /
Place: Indore Swati Panwar
LIST OF CONTENTS
Number Title Page
1. Introduction 1-3
2. Review of Literature 4-13
3. Material and Methods 14-23
4. Result 24-45
5. Discussion 46-50
Summary, Conclusion and Suggestions for further

6. 51-54
work
7. Bibliography i-viii
Appendices ix-xiii
Vita xiv
LIST OF TABLES
Table Page
Title
No. No.
3.1.6 The formulated chemical name of herbicides used along 16
with their sours.
4.3 Recovery of Trichoderma spp. from rhizosphere soils of 25
different districts and crops.
4.4.1 Mycelial dry weight (g) of Trichoderma strains at different 26
temperatures.
4.4.2 Radial growth of Trichoderma strains at different 28
temperatures
4.5 Effect of different pH on the biomass production of 30
Trichoderma strains
4.6(1) Inhibition of radial growth of Trichoderma by glyphosate in 32
vitro. -
4.6.(2) Inhibition of radial growth of Trichoderma by Triflurain 10G 34
in vitro. -
4.6.(3) Inhibition of radial growth of Trichoderma by Atrazine 36
50%wp in vitro. -
4.6.(4) Inhibition of radial growth of Trichoderma by Diuron 80DF 38
in vitro. -
4.6.(5) Inhibition of radial growth of Trichoderma by paraquat 3L in 40
vitro. -
4.6.(6) Inhibition of radial growth of Trichoderma by 2-4-D 38EC 42
in vitro. -
4.7 Effect of different strains of Trichoderma on pre and post 45
mortality of chick pea against dry root rot.
LIST OF PLATES
S.N. Plates
1. Strains of Trichoderma
2. Isolation of Rhizoctonia bataticola.
3. Mycelial dry weight (g) at variable temperatures.

4. Radial growth of Trichoderma strain at different temperatures.
Comparative biomass production ability of Trichoderma strains at
5.
different pH.
Comparative tolerance ability of Trichoderma strains in response of
6.
different herbicides at 500 ppm.
Comparative tolerance ability of Trichoderma strains in response of
7.
different herbicides at 1000 ppm.
Comparative bioefficacy of Trichoderma strains against dry root rot
8.
disease of chickpea.
LIST OF FIGURES
S.N. Title
1. Mycelial dry weight (g) at variable temperatures.
2. Radil growth of Trichoderma strains at different temperatures.
3. Effect of different pH on the biomass production of Trichoderma strains.
4. Comparative tolerance ability of Trichoderma strains in response of

different herbicides at 500 ppm and 1000 ppm.
CHAPTER-1
INTRODUCTION
Chickpea (Cicer arietinum L.) is known in India by different names:
Bengal gram, gram and chana. India is the largest producer of chickpea
accounting for 64% of the global production. It provides a high quality protein
(20-22%) for vegetarians’ diet; is rich in fiber, minerals and β- carotene. It
also fixes the atmospheric nitrogen (~40 kg N/ha) thus reduces the need of
nitrogenous fertilizers. Chickpea can be grown profitably on residual moisture
in heavy soils, in rainfed rice fallow lands (RRFL) without and or with
minimum irrigation. To date, more than 50 pathogens have been reported on
chickpea from different parts of the world (Bayraktar and Dolar, 2009).
However, only a few of them cause serious economic losses, such as
Ascochyta blight caused by Ascochyta rabiei, Fusarium wilt caused by
Fusarium oxysporum f. sp. ciceris, and root rot caused by number of fungi,
including Rhizoctonia solani (Bayraktar and Dolar, 2009). Macrophomina
phaseolina (Tassi), which is pycnidial stage of Rhizoctonia bataticola (Taub)
Butler, seems to be the most important cause of seed rot, root rot of
seedlings, necrosis of the root collar, and suberization of infected tissues
(Dhrub-Sing et al., 1987, Tayars et al., 1988). It is gaining importance in the
present scenario because of climate change when crop is growing in high
temperature and moisture stress. It is mainly a soil-inhabiting pathogen but
many environmental and soil factors influence disease development. The
losses due to M. phaseolina are estimated to be around 10.8 to 24.1 per cent
in India [1,2].
Seed treatment by fungicides is effective to some extent in reducing
losses caused by R. bataticola in crops which are particularly vulnerable at
the seedling stage. Fungicide seed treatment with carbendazim and
thiophanate methyl and vitavax reduced the DRR of chickpea (Taya et al.
1990; Bhardwaj 1995; Singh and Sindhan 1998; Rathore and Rathore 1999;
Sharma and Gupta 2004) but still these fungicides are not enough to control
soil borne diseases sustainably. Bio-control agents offer a sustainable and
ecofriendly way to manage soil born diseases. Bio-control agents exhibit four
forms of antagonism i.e. competition, antibiosis, parasitism and the growth
inhibition of M. phaseolina could be attributed mainly due to antibiosis or
hyper parasitism. Trichoderma spp. are among the most studied fungal
BCAs and commercially marketed as biopesticides and soil amendments
(Harman, 2000; Harman et al. 2004; Lorito et al. 2004). Depending upon the
strain, the use of Trichoderma in agriculture claims and can provide
numerous advantages
(i) Colonization of the rhizosphere by the BCA (‘‘rhizosphere competence’’)
allowing rapid establishment within the stable microbial communities in the
rhizosphere ,
(ii) Control of pathogenic and competitive/deleterious microflora by using a
variety of mechanisms there by improvement of the plant health and
(iii) Stimulation of root growth (Harman et al. 2004).
The success of Trichoderma as biocontrol agents (BCAs) is due to their high
reproductive capacity, ability to survive under quite adverse conditions,
efficiency in the utilization of nutrients, capacity to modify the rhizosphere,
strong aggressiveness against phytopathogenic fungi and efficiency in
promoting plant growth and defence mechanisms. These properties have
made Trichoderma a ubiquitous genus present in any habitat and at high
population density (Misra and Prasad, 2003).
Among different agrochemicals herbicides applied to soil also affects
the soil microflora both infectious and their antagonists. Some of the
herbicides might interfere with growth conditions of Trichoderma. In the
present scenario where focus is on integrated pest management all the plant
protection components should be compatible with each other. In the present
study biocompatibility of Trichoderma with various herbicides is evaluated in
different temperature and pH conditions so as to come up with a well
optimized system to effectively manage the problem of weed and dry root rot
in chickpea. The competition for space, infestation sites and nutrients has
also been shown to be possible mechanisms involved in the biocontrol
activities of Trichoderma spp. (Dennis and W ebster 1971a, b; Chet 1987;
Tronsmo and Hjeljord 1998).Keeping this in view the present investigation
was conducted with the following objectives.
Objectives:
1. Isolation and purification of Trichoderma strains from soil.
2. Evalution of Trichoderma strains for tolerance to temperature and pH

regimes.
3. Assessment of compatibility of different Trichoderma strains with common

herbicides.
4. Bioefficacy of Trichoderma strains against Rhizoctonia bataticola in the

greenhouse.
CHAPTER – II
REVIEW OF LITERATURE
Influence of agrochemicals on soil mycoflora being changed in favour

of the pathogen or their natural antagonist must be the concern of study in
the present day agriculture. Presently indiscriminate use of herbicides forces
the research to draw inference in favour of ecofriendly molecules, to be
advocated to growers in future.
Biological control offers a great promise for disease management
either adopted as a sole measure in integration with other methods, in
general whether in absence of crops, at the level of seed, nursery or in the
main field. Competition and antagonism exhibited by fungi need to be
exploited properly being the need of modern era.
Cook and Baker (1983) biological control is “the reduction of the
amount of inoculum or disease-production activity of a pathogen
accomplished by or through one or more organisms than man”. This
definition includes the use of less virulent pathogen, more resistant cultivars
of the host, and microbial antagonists “that interfere with the survival or
disease production activity of the pathogen”.
Trichoderma spp., are common inhabitant of soil and roots. They are
highly interactive in root, soil and foliar environments. They produce or
release a variety of compounds that induce localized or systemic resistance
response in plants. Trichoderma strains have ability to increase root growth
and development, crop productivity, resistance to abiotic stresses, and
uptake and use of nutrients.
With the advent of bio control as a potential approach to Integrated
Pest Management (IPM) in the area of fungi-mediated plant disease control,
the genus Trichoderma has gained considerable importance in recent times
(Mrinalini and Lalithakumari, 1996; Nagee et al. 2003).
Harman et al. (2004) described that the members of genus
Trichoderma are opportunistic, avirulent plant symbionts, as well as being
hyperparasites of other fungi.
The available literature relating present study is being reviewed and
presented in this chapter.
2.1 Isolation and identification of Trichoderma spp. from soil
Thakur and Norris (1928) isolated the genus Trichoderma in India

from soils of Madras. Mostly identifications were based on the morphological
characters. Various research articles were published in India on the bio-
control efficiency of T. harzianum; T. koningi; T. longibrachiatum; T. virens;
T. hamatum and T. viride.
Rifai (1969) in his monograph on Trichoderma has recognized this
genus into nine aggregates, several biological species under each
aggregate, but was unable to define the limits of individual biological species
(Samuels et al. 1998).
Bisset (1991a, b, c and 1992) elevated Rifai species aggregate to
special level and recognized several species within each of five section of
the genus Trichoderma.
Samuels et al. (1996) identified Trichoderma in culture media, which
produces large number of small green or white conidia from phialides
present on the profusely or meagrely branched conidiophores.
Kader et al. (1999) mixed the soil samples with sterile distilled water
and made a series of dilutions , 0.5 ml was placed on potato dextrose agar
(PDA) and incubated at 30ºC for three days. Fungi were isolated from the
mixed isolates from each plate and subcultured on PDA, until a pure isolate
was obtained.
Sivakumar et al. (2000) isolated Trichoderma from soil samples
collected from three Rambutan fields where leaf litter was allowed to
accumulate. Soil suspensions were prepared by adding 1.0 g soil to 10 ml of
sterile distilled water and shaking for 15 minutes, suspension was serially
diluted to 10 -5 and 0.1 ml of it was spread on a potato dextrose agar (PDA)
plate which was then incubated at 28°C for 5 days. Trichoderma were
isolated in pure cultures.
Cigdem and Merih (2003) collected thirty-one soil samples from
differen agricultural fields and forests in Eskisehir and inoculated on potato
dextrose agar, malt extract agar, rose bengal agar and oat flour agar and
incubated at 28 °C for 5 days. Colonies were purified by subcultures and
Trichoderma spp. were identified according to thekey given by Watts et al.
(1988) and Rifai (1969).
Mustafa et al. (2009) tested mycelial growth, conidal production and
biomass yield of three different Trichoderma species (T. harzianum, T. viride,
and T. longibrachiatum) on five different culture media including Potato
Dextrose Agar, Waksman agar, Agar-agar, Czepak’s agar and Corn Meal
agar. PDA was the best medium in terms of growth spore production and
biomass yield. Trichoderma harzianum outclassed the three in terms of
mycelial growth biomass yield and spore production.
Chakraborty et al. (2010) studied nineteen isolates of Trichoderma
viride and Trichoderma harzianum using RAPD and ITS-PCR. RAPD profiles
showed genetic diversity among the isolates with the formation of eight
clusters. Analysis of dendrogram revealed that similarity coefficient ranged
from 0.67 to 0.95. ITS-PCR of rDNA region with ITS1 and ITS4 primers
produced 600bp products in all isolates. This result indicated the
identification patterns of Trichoderma isolates.
Rahman et al. (2011) worked on strains of T. harzianum (IMI-392432,
392433, 392434); T. pseudokoningii (IMI-392431) and T. virens (IMI-
392430). Among these T. harzianum was the most common in all of the
habitats. Colony forming units (cfu’s) of Trichoderma species varied
significantly in different habitats and were positively correlated with the
physicochemical characteristics of the habitat.
Pingolia et al. (2013) exploited use of different kind of agar medium
like PDA and MEA for the growth of mycelium of Trichoderma viride. The
best mycelium growth observed in PDA with 2.5 g glucose and 1.5g lactose
respectively. Similarly, while using malt extract agar medium the best growth
recorded in MEA with 2.5 g lactose.
2.2 Effect of temperature on Trichoderma spp.

Widden and Abitbol (1980) reported that T. viride was the most
abundant species in early spring and autumn in a spruce forest soil.
Antal et al. (2000) screened 360 Trichoderma strains for cold
tolerance. Fourteen identified as T. aureoviride, T. harzianum and T. viride –
grew well at 50C on both minimal and yeast extract agar media. The
incidence of cold tolerant isolates was the highest in species group T. viride.
Sobieralski et al. (2009) investigated the effect of temperature and
medium on the aggressive Trichoderma aggressivum f.sp. europaeum
isolates mycelium growth. Growth of experimental isolates was examined on
standard agar medium and on manure medium at 15, 20, 25, 30 and 35°C.
The mycelium of the examined isolates grew better on the manure medium
than on standard agar medium and the optimum temperature for all the
examined isolates was 25 and 30°C.
Gupta and Sharma (2013) studied the effect of optimum temperature
on Trichoderma harzianum. Optimum temperature for Trichoderma
harzianum was estimated by monitoring the radial growth rate and colony
morphology in culture plate at 25°C, 30°C, 37°C and 45°C.T .harzianum
hyphal extension grown faster at 25-30°C, slower grown at 37°C and no
growth was observed at 45°C after six day of inoculation. Population
dynamics was maximum in shade followed by 4°C. Hence optimum
temperature of Trichoderma harzianum was found between 25-30°C.
2.3 Effect of pH on Trichoderma spp.
Papavizas (1985) found that pH values higher than 6.5 were needed
for the maximal linear growth of T. harzianum.
Przybylowicz and Donoghue (1988) stated that the optimum pH for
the development of Trichoderma spp is 4.5 to 5.0 in a moist environment.
Jackson et al. (1991) have found that optimum biomass production of
three Trichoderma isolates occurred at pH ranges between 4.6 and 6.8.
Kukuc and Kivanc (2003) studied the ability of T. harzianum strains to
grow at pH 2,10 and 12 was tested in liquid medium containing 0.05 g l -1
bromocresol purple. It was observed that T. harzianum strains showed
different growths at different temperatures but they did not grow on the
media at pH 2, 10 and 12.
Benitez (2004) reported that growth of Trichoderma is more efficient in
acidic than alkaline soils and they modify the rhizosphere soil by acidifying
the soil.
Bonilla (2006) in Trichoderma harzianum, had the highest rate of
development in the control group (18.94mm / day) on PDA medium at pH
5.24, and the lower rate of development was obtained in treatment PDA +
SQ (2.0mm / day) with an initial pH of 11.42.
Miguel et al. (2007) found strain variation among the isolates within
the Trichoderma species. The Trichoderma isolates from rice cultivated soils
showed their preference of pH 5.5 and 6.5 for optimal growth.
Romero (2007) reported that T. harzianum had the highest
development rate (11.5 mm / day) on PDA medium with an initial pH of 5.6.
The lower rate of development was obtained in the NaOH-11 treatment (0.5
mm / day) with a pH of 10.8 in the medium YCM (yeast complete medium).
Arenas et al. (2012) reported that the strain T. viridae (CP-T4) has a
rate of development of 0.41mm/ day at pH 11.2 showing negative effects on
growth and development in alkaline pH.
Kolli et al. (2012) isolated twenty six isolates of Trichoderma from
forest and agricultural soils were tested in vitro for their pH levels, tolerance
and biomass production. Different isolates of same species varied in
biomass production at tested pH levels i.e. 4.5, 5.5, 6.5 and 7.5. Sixteen
isolates showed highest biomass at 7.5 and none at acidic pH i.e. 4.5.
Isolates from agricultural soils have more tolerance towards varied pH levels
than the isolates from forest soils.
2.2.1 Trichoderma species as biocontrol agents:
Ghaffer (1968) observed that, Macrophomina phaseolina was

inhibited as well as over grown by T. viride.
Ahmad and Baker (1987) conducted an experiment that, Trichoderma
species, either added to the soil or applied as seed treatments, grow readily
along with the developing root system of the treated plants .
Chet (1987) reported that fungus in the genus Trichoderma are the
most promising biocontrol agents for plant pathogenic fungi. Specific strains
have the ability to control a range of pathogens, under a variety of
environmental conditions.
Audenaert et al. (1998) reported that application of Trichoderma
harzianum to bean roots resulted in a 25 to 100 per cent reduction in the
severity of foliar gray mold, caused by Botrytis cinerea. Biocontrol fungus T.
harzianum T39 and a chemical BTH (benzothiadiazol) were tested for
induction of resistance in tomato to B. cinerea.
Bailey and Lumsden (1998) identified that specific strains of fungi in
the genus Trichoderma colonize and penetrate plant root tissues and initiate
a series of morphological and biochemical changes in the plant, considered
to be part of the plant defence response, which subsequently leads to
induced systemic resistance.
Howell et al. (2000) demonstrated that seed treatment of cotton with
biocontrol preparations of T. virens or application of T. virens culture filtrate
to cotton seedling radical induced synthesis of much higher concentrations of
the terpenoids desoxyhemigossypol, hemi gossypol and gossypol in
developing roots than those found in untreated controls. They described that
the pre-emergence phase of cotton seedling disease was effectively
controlled by strains of T. virens but they are much less efficient in the
control at post-emergence phase of the disease that is incited by R. solani.
Yedida et al. (2000) investigated that inoculation of cucumber roots
with T. harzianum induced an array of pathogenesis-related proteins,
including a number of hydrolytic enzymes.
Indra et al. (2003) evaluated the fungal antagonistics like T. viride, T.
harzianum, T. hamatum, T. koningii, T. pseudokoningii, T. longibrachiatum
and Gliocladium virens against black gram root rot fungus M. phaseolina. T.
harzianum and T. longibrachiatum were at par in controlling M. phaseolina.
Maheswari and Sankaralingam (2005) isolated six isolates of
Trichoderma spp. and screened preliminarily in vitro for their antagonistic
potential against Alternaria alternata, the causal agent of leaf blight of
watermelon. Though all the Trichoderma spp. were antagonistic to A.
alternata, Trichoderma spp. CIAH-175 recorded maximum reduction in
mycelial growth of pathogen up to 86.3 percent.
Ozbay and Newman (2004) observed that, Rhizoctonia solani,
Pythium ultimum and Chalara elegans were strongly inhibited by
Trichoderma under in vitro conditions. The observed inhibitory action of
Trichoderma was associated with a high rate and extent of CO 2 accumulation
in comparison with the plant pathogenic fungi.
Matroudi et al. (2009) selected three different species T. harzianum-8,
T. atroviride PTCC5220 and T. longibrachiatum PTCC5140, from 30
Trichoderma isolates, on the basis of their high level of chitinase and/or
glucanase activity, along with their rapid growth rate in vitro for the control of
stem rot of canola (Brassica napus ) caused by Sclerotinia sclerotiorum.
These showed high growth inhibition of two phytopathogenic isolates of
Sclerotinia sclerotiorum (S1 and S2), with T. atroviride the greatest effect,
reducing growth by 85-93%.
Mustafa et al. (2009) analysed in-vitro, species of Trichoderma
strongly antagonised six different seed borne pathogenic fungi viz. Fusarium
moniliforme, Fusarium oxysporum, Rhizoctonia solani, Fusarium solani,
Botryodiplodia theobromae and Alternaria alternata in dual culture
assay.Trichoderma harzianum gave maximum inhibition of mycelia growth of
all pathogenic fungi.
Gaigole et al. (2011) showed that Trichoderma viride have the
abilitymto inhibit soil borne pathogen of different crops like Rhizoctonia solani
and most commonly used fungal biocontrol agent and have long been known
as effective antagonist against plant pathogen.
2.4.3 Antagonistic potential of Trichoderma strains against R.

bataticola:
Shamim et al. (1997) have reported for Rhizoctonia sp., that

Trichoderma spp. could control root rot of cotton, damping-off of tobacco
(Cole and Zvenyika, 1988), peanut (Bertagnolli et al. 1996), Rhizoctonia
black scurf of potato (Papavizas, 1985). In 1989, Chaudhary and
Gangawane concluded that Trichoderma spp. provided high efficacy to
control of charcoal rot of peanut caused by R. bataticola.
Nair et al. (2006) conducted to a pot experiment to determine the
effects of biological control agents (Trichoderma harzianum, Trichoderma
viride and Bacillus subtilis) on Rhizoctonia bataticola infecting sesame VAR-
1. The seeds treated with spore suspension of Trichoderma harzianum were
superior over all the other treatments. The germination percentage was
maximum (92 %) in case of Trichoderma harzianum followed by
Trichoderma viride (89.33 %).
Dubey et al. (2011) reported the secondary metabolites from culture
filtrate and mycelial mass of potential isolates of Trichoderma viride, T. virens
and T. harzianum were extracted by solvent extraction and soxhlet water
both distillation methods and evaluated at different concentrations against
Rhizoctonia bataticola and Fusarium oxysporum f.sp. ciceri. All evaluated
concentrations (500, 250, 125 and 75 ppm) were found to be inhibitory to
these pathogens.
2.3 Compatibility of Trichoderma strains with herbicides:
Chappel and miller (1966) reported herbicides are very effective in
reducing the growth of s. rolfsii and other soil borne pathogens at a very low
concentration.
Bozarth and Tweedy (1971) reported that the radial growth of S.
rolfsii was inhibited by 26-27 percent by atrazin and trifluralin. The
Trichoderma production was also reduced by all the herbicides.
Bagy and Hemida (1993) evaluated primextra and found reduction in
total count of Aspergillus and A. niger and improved counts at A. candidus
and A. corneus. The mycelial growth of A. niger, A. terreus, A. fumigatus
and T. hargianum increased at the high dose.
Macek and Lesnik (1994) found inhibition of mycelial growth of
Trichoderma longibrachatum with triasulfuran herbicide applied at field
concentrations.
Samuels (1996) found herbicide formulation namely glyphosate was
foliar sprayed and oxadiargyl was applied to soil to control herbs in the
research field. T.asperellum isolated in the present study, from herbicide
treated soil has been found to be a new addition to the fungi of india.
Sharma et al. (1999) showed that various herbicides concentrations
exhibited beneficial or harmful effects on growth of Trichoderma harzianum
and moderate compatibility with 2, 4-D.
CHAPTER- III
MATERIAL AND METHODS
The present study on bio efficacy of naive strains of Trichoderma

against Rhizoctonia bataticola was undertaken to isolate Trichoderma spp.
from different soils, characterisation of the isolated strains to assess the
tolerance levels to different herbicides and to test their antagonism against
an important soil borne plant pathogen such as Rhizoctonia bataticola were
carried out in the laboratory in the Department of Plant Pathology,
R.V.S.K.V.V., College of Agriculture, Indore, M.P, India. Details of the
materials used, experimental procedures followed and techniques adopted
are given as under.
3.1 Materials:
The materials used included soil samples, isolates of Trichoderma,
ingredients of culture media, diseased plant samples, glass wares,
equipments, chemicals and a few miscellaneous articles.
3.1.1 Soil samples:
The soil samples were collected from six districts of west M.P. namely
Dhar, Barwani, Indore, Khandwa, Sehore and Khargone districts of Western
Madhya Pradesh, randomly up to 20 cm depth with soil auger for isolation of
the Trichoderma strains and their quantitative and qualitative determinants
using TSM.
3.1.2 Collection of diseased specimens:
Diseased chickpea plants exhibiting typical symptoms of dry root rot
were collected from the research and cultivator’s fields. The diseased plants
were brought to the laboratory. The specimens were critically examined for
the symptoms and the pathogen was isolated from such plants to obtain the
culture of Rhizoctonia bataticola, the dry root rot pathogen.
3.1.3 Different strains of Trichoderma spp:
Six strains of Trichoderma spp. isolated from soils collected from six
districts of M.P. were used for antagonistic potential against Rhizoctonia
bataticola in vitro.
3.1.4 Chemicals:
(a) Cleaning solution:

Cleaning solution containing potassium dichromate 80 g, distilled
water 300 ml and concentrated sulphuric acid 400 ml was used to clean the
glass wares.
(b) Mercuric chloride solution:

A 1:1000 solution of HgCl2 was prepared and used for surface
sterilization of samples during isolation of the pathogen.
(c) Mounting medium:
Lacto phenol and cotton blue with the following composition were
used as the working media for studying the characteristics of the pathogen
and the antagonists.
i. Lacto phenol
Phenol
(pure crystals liquefied by gentle heating on a water 20 g
bath)
Lactic acid 20 ml
Glycerol 40 ml
Distilled water 20 ml
ii. Cotton blue
Anhydrous lacto phenol 67 ml

Distilled water 20 ml
Cotton blues 0.1 g
3.1.6 Herbicides
The Herbicides used in the experiment along with their trade name,
common name and manufacturing time of application were given in the table
3.1.6
Table-3.1.6 The formulated chemical name of herbicides used along

with their sours.
Common Trade name C.C. Time of

name aplication
Glyphosate Roundup 4L Pre -plants
Triflurain Trilin 10 G Pre -planting
Atrazine Many 50 WP Pre-emergence
Diuron Karmex 80 DF Pre-emergence
Paraquat Gramoxone 3L Post-emergence
Max
2-4-D Formula-40 38 EC Post-emergence
3.1.8 Ingredient of culture media:

Two culture media, namely, potato dextrose agar and Trichoderma
selective medium (TSM) with following ingredients were used during the
course of investigation.
(i) Trichoderma selective medium (TSM):

Selective isolations of Trichoderma spp. from soil were made on
modified selective medium (Elad and Chet, 1982) having following
ingredients.
Ingredients Q.
1. MgSO4.7H2O - 0.2 g
2. K 2HPO4 - 0.9 g
3. KCl - 0.15 g
4. NH 4NO3 - 1.0 g
5. Glucose - 3.0 g
6. Chloramphenicol - 0.25 g
7. Rose Bengal - 0.15 g
8. Captan - 0.15 g
9. Apron 35-SD - 0.025 g
10. Agar-agar - 20 g
11. Distilled water - 1l
12. pH - 6.5
(ii) Potato dextrose agar (PDA) medium: (Rangaswami and Mahadeven,

2001)
Ingredients Q.
1. Peeled and sliced potato (extract) - 200 g
2. Dextrose - 20 g
3. Agar-agar - 20 g
4. Distilled water - 1l
5. pH (adjusted to) - 6.5
3.1.9 Glass wares:

Standard "Borosil" make glass wares like Petri dishes, beakers,
funnels, pipettes, Erlenmeyer flask, culture tubes, measuring cylinder etc.
were used during the course of study.
3.1.10 Equipments:
Equipments used during the investigation included research
microscope, refrigerator, autoclave, hot air oven, BOD incubator, laminar air
flow, weighting balance, LPG gas burner, Bunsen burner, hot plate etc. Small
instruments like Inoculation needle, scalpel razor, glass cavity slides,
polythene bags, desiccators, glass marking pencils, cover slips brush
dropper, match box etc. were also used during the study.
3.2 METHODS
3.2.1 Cleaning and sterilization of glass wares:
The glass wares were cleaned by dipping them in cleaning solution for
5 minutes and finally rinsed with running tap water for 30 minutes. The Petri
dishes were sterilized in a hot air oven at 180 ± 2°C for 1 to 2 hours. The
inoculation needle, cork borer and other metallic instruments were sterilized
by dipping them in alcohol and heating red hot over flame of the spirit
lamp/Bunsen burner.
(i) Glassware and their cleaning

Borosil and Corning glassware were kept in the cleaning solution
containing 60 g of potassium dichromate (K 2Cr207) and 60 ml of concentrated
sulphuric acid (H 2SO4) in one litre of water for whole day. Then they were
cleaned by washing with detergent powder followed by rinsing several times
in tap water and finally in distilled water.
(ii) Sterilization
All the glass wares were sterilized in an autoclave at 1.1 kg per sq cm
pressure for 20 minutes. All the media were sterilized for 15 minutes at 1.1
kg per sq cm pressure, except those containing sugars and nitrogen sources
which were sterilized at 0.7 kg per sq. cm pressure for 10 minutes and soil
used for experiment was sterilized at 1.33 kg/sq cm pressure for two hours.
(iii) Surface sterilization of plant parts
Plant materials were surface sterilized using 0.1 per cent mercuric
chloride solution for 20-30 seconds and then washing in sterile water thrice.
3.2.2 Preparation of media:
(i) Potato dextrose agar:

Peeled potatoes cut into triangular pieces were boiled in ½ litre water
in a sauce pan for 8-10 minutes. In another pan, agar-agar was boiled in ½
litre water separately so that it gets completely dissolved. Potato extract was
collected after filtration on muslin cloth over a funnel and added to dissolve
agar-agar along with dextrose. The medium was poured in conical flask of
250 ml, plugged with non absorbent cotton and autoclaved at 121.6ºC (15
lbs) for 25 minutes. Streptomycin sulphate @ 1.0 g/l was added to the
medium before pouring into Petri dishes to avoid bacterial contamination. It
was used during the investigation.
(ii) Trichoderma selective Medium (TSM):

All the ingredients except Apron 35-S D, chloromphenicol, rose bengal
and captan were mixed in 1 litre distilled water, which was boiled to dissolve
the agar-agar. The medium was transferred to 250 ml Erlenmeyer flask,
plugged with cotton and autoclaved at 121.6ºC (15 lbs) for 25 minutes. After
cooling (55ºC), the required quantity of Apron 35-S D, chloremphenicol, rose
bengal and captan were added to the medium just before pouring in to the
sterilized Petri dishes.
3.2.3 Collection of soil samples:
Random samples were taken from about 15-20 cm depth by means of
a soil auger. The samples were placed into clean polythene bags. Three
samples from each treatment replication were taken. The sampling of the
rhizosphere soil was done separately. For rhizosphere analysis, only the soil
adhering to the root system was used. The soil samples were taken about 5-
10 cm away from the root. The composite samples were, mixed thoroughly
and screened through 2 mm sieve (Baruah and Dutta, 1978).
3.2.4 Test organism/ Isolation and purification of the pathogen:
The pathogens R.bataticola ware isolated from root region of the
diseased plants by time segment method (Rangaswami and Mahadevan,
2001) and purified by hyphal tip method and maintained on potato dextrose
agar slants. The affected portion (root) of the diseased plants were cut with
the help of sharp razor and rinsed with sterilised water to remove traces of
dirt. These were surface sterilised by dipping in 1:1000 mercuric chloride
solution for one minute and washed twice with sterile water. These pieces
were transferred aseptically to sterilised petri dishes containing solidified
PDA-S in a laminar air flow. The petri dishes were incubated at 26±1ºC. The
appearing fungus was observed after 72 hours and isolations were made
from developing colonies for further study.
3.2.5 Dilution plate count method
Goss (1972) described the dilution plate method for isolation and
enumeration of soil micro-organisms. One gram sieved soil on dry weight
basis was dispensed aseptically in 10 ml sterile water in a culture tube and
shaken for 20 minutes on a mechanical wrist action shaker. The suspension
was serially diluted aseptically till the dilution of 1: 1000 was reached one ml
of the desired end dilution from each replicate was transferred aseptically to
each of the three sterile petridishes. Pre cooled, TSM was poured into each
Petri-dish and the contents immediately mixed by rotating the dishes in a
circular and side to side motion before the medium got solidified. The plates
were incubated at 26±1ºC for seven days. Colonies of Trichoderma were
counted with the help of a magnifier and the average number per gram of soil
was determined. The data were subjected to statistical analysis following the
procedure for analysis of completely randomised design (Panse and
Sukhatme, 1978). Identification of the colonies was made with the aid of a
research microscope, after isolation of individual fungus in pure culture on
PDA by observing the typical characteristics described in the text (Gilman,
1957; Barnett and Hunter, 1987; Webster, 1989).
3.2.7 Single Spore Isolation

Spore suspension was prepared by inoculating small piece of
mycelia with conidia into a Bijoux bottle containing sterile distilled water and
shake vigorously. After that, one loop full of spore suspension was streaked
on Water Agar (WA, Merck) in a zig-zag manner. The inoculated WA plates
were incubated at 28°C for 24 – 48 h. After that, well isolated colonies
germinated from single conidia were sub cultured to new PDA plates. The
inoculated plates were incubated at 28°C for 3 – 5 days and used as
inoculums for further studies and storage of fungal cultures.
3.2.6 Maintenance of cultures

The fungi were sub-cultured on potato dextrose agar slants and
allowed to grow at 27±1 0 C for one week. Such slants were preserved in a
refrigerator at 40C and renewed once in two months to maintain the virulence
of the pathogen and antagonism of the Trichoderma spp.
3.2.7 Physiological studies
(i) Influence of temperature regimes on the growth of Trichoderma spp.

For Trichoderma spp. Potato Dextrose Agar medium and PDA broth
was used in this experiment. The different temperatures tried for the growth
of the fungi were 50C, 250C and 450C. For each treatment level, three
replications were maintained. The flasks were inoculated and incubated as
described earlier. The data on radial growth of colony, and dry weight of the
mycelia mat were recorded and analysed statistically.
(ii) Influence of hydrogen ion concentration on the growth of

Trichoderma spp.
The liquid medium used in this experiment was PDA broth solution.
Adjustment of pH was done by adding 0.1 N alkali (NaOH) or acid (HCl).
Reaction of the medium was adjusted to the desired pH of 4.0, 5.0, 7.0, 9.0
and 10.0. Twenty ml of the medium was pipetted out into each 100 ml
conical flask. Each treatment was replicated thrice. The flasks were sterilized
at 1.1 kg per sq cm pressure for 15 minutes. They were inoculated and
incubated as described earlier. The pH of the culture filtrate was determined
by using Precision pH meter. The data on radial growth of colony, shape and
size of Rhizoctonia were recorded and analyzed statistically.
3.2.10 Evaluation of Trichoderma strains against Rhizoctonia bataticola

in the green house :
Pot culture technique was used for measurement of antifungal activity
and the ability of the Trichoderma strains to reduce the attacks of
Rhizoctonia bataticola in the green house (Pot conditions) to chickpea plants,
in this experiment. The pathogen was multiplied in the PDB medium and
added to the soil at the rate of 10 7 cfu/Kg soil, which was pre sterilized at
1210C and 15 lb pressure for 15 minutes and filled in pots. Chickpea seeds
were treated with spores obtained from the cultures at the rate of 4g/Kg
seed. The seeds were placed in a clean container, sprinkled with water until
they were wet, adequate quantities of the bio-control agent available in a talc
based dry powder form (Devikarani et al., 2009) was added and the
container was agitated thoroughly until the seeds were uniformly coated. The
seeds were sown in the pots which were pre infested with the pathogen. 10
seeds per plot were sown at the 2-3 cm in 3 pots for each of the 16 strains of
Trichoderma and a control in which the seeds are not treated with the strains
of Trichoderma, only treated with water. Watering was done adequately and
regularly observations on disease incidence were recorded as pre
emergence mortality, and post emergence mortality at regular intervals of 7,
15 and 21 days after planting.
Per cent incidence of disease was calculated by the formula –
Disease incidence (%) =
3.2.10 Statistical analysis

Statistical analysis was done for the interpretation of the results
obtained. Completely Randomized Design (CRD) and factorial CRD (for in
vitro experiments) were employed to analysis the data. The critical
differences were worked out at 0.05% probability level. The value in
percentage was transformed by angular transformation. The experiments
were conducted in the PG laboratory, Department of Plant Pathology,
College of Agriculture, Indore (M.P.).
CHAPTER – IV
RESULTS
Studies on “Identification and bio-efficacy of native strains of

Trichoderma tolerant to variable environmental conditions and
effectiveness against Rhizoctonia bataticola” were carried out on six
isolates of Trichoderma on the basis of their wide adaptation involving their
potentiality on different temperatures, pH and tolerant to common herbicides .
Results on these aspects have been reported here under in depicting the
behavior of different strains of the Trichoderma spp.
4.1 Collection of soil samples for isolation and purification of
Trichoderma spp.
Total 55 soil samples collected from six diverse districts of west M. P.
Out of these 10 were collected from Dhar , Barwani (6), Indore (13),
Khandwa (13), Sehore (6) and 7 soil samples from Khargone district of
western Madhya Pradesh.
4.2 Collection of soil samples from rhizosphere of different crops for
isolation of Trichoderma spp.
Soil samples were collected from the rhizospheric soil zone of eight
different crops, out of which eight samples were collected from cotton crop
rhizospheric zone followed by Sorghum (7), Gram, Arhar, Bean, Chilli,
Soybean and Pigeon pea (6).
4.3 Recovery of Trichoderma spp. from soils of different districts of
M.P. and rhizospher of different crops.
Total six strains of Trichoderma were isolated from the collected soils
and rhizospheric samples. The details of the districts and crop rhizospheres
from which strains of Trichoderma isolated were given in detail in the table
4.3. and in plate no 1 .
4.3 Recovery of Trichoderma spp. from rhizosphere soils of different
districts and crops.
Trichoderma strain Place/Crop of isolation
Ts1 Khandwa, Soybean
Ts2 Indore, Gram Crop
Ts3 Bharwani, Soybean
Ts4 Dhar, Arhar
Ts5 Khargone,Chilli
Tc1 Sehore,Gram Crop

4.4 Adaptation potentiality of different Trichoderma strains in variable
temperature regimes.
A detailed study on cultural characteristics of Trichoderma strains
such as mycelial dry weight in grams and radial growth in mm on PDA media
was conducted at different temperatures such as 45 0C, 250C and 50C was
carried out and the procedure was described in detail in the previous chapter
materials and methods.
4.4.1 Mycelial dry weight (g) of Trichoderma strains at different
temperatures.
A separate experiment was carried out to assess the effect of
temperature regimes on the dry weight of strains of Trichoderma. The mean
of three replications have been presented in Table 4.4.1. and in plate-3,
which revealed that among the temperatures maximum growth of
Trichoderma strains (0.32g) at 25 C in comparison to 450C (0.20g) and none
0
of the strains were exhibited growth at 50C. All the strains of Trichoderma
showed significant differences among variable temperature regimes.
Table 4.4.1 Mycelial dry weight (g) of Trichoderma strains at different
temperatures.
Mycelial dry weight (g) at variable temperatures

Strain
450C 250C 50C Mean
Ts1 0.15 0.28 0.00 0.14
Ts2 0.17 0.20 0.00 0.12
Ts3 0.22 0.35 0.00 0.19
Ts4 0.10 0.36 0.00 0.15
Ts5 0.24 0.31 0.00 0.18
Tc1 0.33 0.47 0.00 0.26
Mean 0.20 0.32 0.00
CD(P=0.05) 0.14 0.12 0.00
SEm± 0.04 0.04 0.00
Among the strains of Trichoderma, the maximum dry weight was showed by
Tc1 (0.26g).The next strain which showed the superior effect was the strain
Ts3 (0.19g) while Ts2 recorded the minimum dry weight of (0.12g). All the
strains of Trichoderma showed significant differences in different range of
temperatures.
Strains of Trichoderma at 450C temperature showed different mycelial dry
weights. The maximum and minimum dry weight was showen by Tc1 (0.33g)
and Ts4 (0.10g) respectively. All the strains of Trichoderma showed
significant differences in dry weights with Ts 4 (0.10g).
At 250C temperature the maximum dry weight (0.47g) was recorded with the
strain Tc1 however, minimum (0.20g) in Ts2. All the strains of Trichoderma at
temperature 250C showed significant differences except the strains Ts 2
(0.20g) and Ts1 (0.28g) in which dry weight of the strains ware at par with
each other.
None of the Trichoderma strains, growth at 50C.
Significant difference in biomass production at different temperatures
was observed among the six Trichoderma strains. On PDB medium mycelia
dry weight was recorded maximum at 25 0C. At 450C all the isolates showed
very slow growth rate and no growth in the mycelia dry weight was recorded
at 50C. Results showed that 250C was most suitable for growth of all isolates.
At 450C Tc1 (0.33 g) expressed the maximum dry weight and at 25 0C the
strain Tc1 (0.47g) was recorded with maximium biomass production. The
result here indicates that the strain Tc 1 was favoured by the growth at wide
range of temperatures.
4.4.2 Radial growth of Trichoderma strains at different temperatures.
A detailed study on the radial diameter of different strains of Trichoderma
was carried out by varying temperatures (45 0C, 250C and 50C). The mean of
three replications have been presented in Table 4.4.2, plate-4, which
revealed that the maximum colony diameter of strains of Trichoderma was
recorded (85.72 mm) at 250C followed by the colony diameter 55.38 mm at
450C.Minimum colony diameter 0.00 g was recorded at a temperature of 5 0C.
All the strains of Trichoderma showed significant differences among variable
temperature regimes.
Among the strains of Trichoderma the maximum colony diameter was
showed by Ts2 (57.77 mm) followed by Ts 4 (47.89 mm). The minimum colony
diameter was showed by Ts 1 of (38.55 mm). All the strains of Trichoderma
showed significant differences in different range of temperatures.
Table 4.4.2 Radial growth of Trichoderma strains at different

temperatures.
Colony diameter (mm) isolates at variable

Strain temperatures
450C 250C 50C Mean

Ts1 28.33 87.33 0.00 38.55
Ts2 83.33 90 0.00 57.77
Ts3 51.33 90 0.00 47.11
Ts4 76.67 67 0.00 47.89
Ts5 44.00 90 0.00 44.66
Tc1 48.67 90 0.00 46.22
Mean 55.38 85.72 0.00
CD(P=0.05) 19.48 9.93 0.00
SEm± 6.32 3.22 0.00
Strains of Trichoderma at 450C temperature showed by strain Ts 2

expressed maximum colony diameter (83.33 mm) followed by the next strain
which showed the superior effect was Ts 4 (76.67mm).The minimum mycelial
diameter was shown by Ts 1 (28.33 mm) .All the strains of Trichoderma
showed significant differences in mycelial diameter in with Ts1 (28.33 mm).
At temperature of 250C all the strains showed almost similar and maximum
mycelial diameter of 90 mm. However, minimum colony diameter was
recorded (67mm) in Ts 4. All the strains of Tricoderma at temperature 25 0C
showed significant difference in mycelial diameter.
None of the Trichoderma strains, grown at 5 0C.
On PDA medium radial diameter of the Trichoderma strains was recorded
maximum at 250C. At 450C all the isolates showed very slow growth rate and
no growth in the mycelia dry weight was recorded at 5 0C. Results showed
that 250C was most suitable for growth of all Trichoderma strains. Growth of
the Trichoderma strains was reduced when the temperature was increased
or decreased from the optimum (25 0C). At 450C the strain Ts2 expressed
maximum colony diameter of 83.33 mm .At 25 0C all the strains exhibited the
maximum colony diameter of 90mm.The result here indicates that the strain
Ts2 were adaptable to wide range of temperatures. Greenish coloured
conidia formed within 4-5 days at 250C and remain white up to 6-10 days at
450C.
4.5 Effect of different pH on the biomass production of Trichoderma
strains
The effect of different pH, 5.0, 7.0, 9.0 on the growth of different
strains of Trichoderma was studied. Results as the mean of three
replications have been presented in table 4.5, plate-5.
The data revealed that the maximum growth of different strains of
Trichoderma was recorded on 7.0 pH (0.39 g) and the minimum colony
diameter was recorded in pH 9 (0.26 g). All the strains of Trichoderma
showed significant differences among variable pH level.
Among the isolates across the pH levels, the maximum dry weight was
recorded by Tc1 (0.37g) and the minimum dry weight was showed by Ts 5
(0.27 g). All the strains of Trichoderma showed significant differences at
different levels of pH.
At pH 5.0 the maximum dry weight was recorded inTs 2 (0.40 g) followed by
TC1 (0.36 g), and the minimum dry weight was recorded by Ts 4 (0.24 g)
which was at par with Ts 5 (0.27 g) followed by Ts 1 (0.31 g), Ts3 (0.32 g). All
the strains of Trichoderma at 5.0 pH level showed significant differences
except the isolate Ts 4 (0.24) which was at par withTs 5 (0.27 g), Ts1 (0.31 g),
Ts3 (0.32 g) .
At pH 7.0 the maximum dry weight was showed by Tc isolate (0.54 g)
followed by Ts1 (0.48 g) and the minimum dry weight was recorded in Ts 2
(0.30 g). All the strains of Trichoderma at 7.0 pH level showed significant
differences except the isolate Ts 2 (0.30 g) which was at par with Ts 5 (0.31 g).
At pH 9.0, the maximum dry weight was showed by Ts 1 strain (0.29
g) followed by Ts 2 and Ts4 (0.27 g) and the minimum was recorded by Tc 1
(0.23 g). All the strains of Trichoderma at pH 9.0 level showed significant
difference.
Table 4.5 Effect of different pH on the biomass production of
Trichoderma strains
Effect of different pH on the biomass of Trichoderma

Strain strain
5 7 9 Mean
Ts1 0.31 0.48 0.29 0.36
Ts2 0.40 0.30 0.27 0.32
Ts3 0.32 0.35 0.26 0.31
Ts4 0.24 0.36 0.27 0.29
Ts5 0.27 0.31 0.24 0.27
Tc1 0.36 0.54 0.23 0.37
Mean 0.31 0.39 0.26
CD(P=0.05) 0.07 0.17 0.01
SEm± 0.02 0.05 0.00
Significant variation in biomass production was observed among all the

strains of Trichoderma at all test pH values of 5, 7, 9 (Table 4.4). Maximum
number of isolates showed high biomass production at pH 7 followed by 5,
minimum at pH 9. The result revealed a variation in mycelia dry weight of
Trichoderma strains at different pH. Mycelial dry weight of a Trichoderma
strains found to be maximum on pH 7 (0.39 g), followed by pH 5 (0.31 g) and
minimum dry weight was recorded with pH 9 (0.26 g). The maximum dry
weight was expressed by the strain Tc 1 (0.54 g) at Ph 7. Maximum dry
weight was recorded by the strain at pH 5.0 were Ts 2, at pH 9.0 were Ts 1
which indicates that the strain Tc 1 was wide adopted .
4.6 Tolerance of Trichoderma spp. to different herbicides.

Six herbicides namely glyphosate 4L, triflurain 10G, atrazine 50WP, diuron
80DF paraquat 3L and 2-4-D 38% EC at two different concentrations of 500
ppm and 1000 ppm were tested against 6 strains of Trichoderma to see their
tolerance levels. The data was given in the table 4. 6 and plate 6 and 7.
(a) Glyphosate 4L
The effect of glyphosate 4L at different concentrations (500 ppm and 1000

ppm) on the per cent inhibition on the colony diameter of different strains of
Trichoderma was studied. The mean of three replications at two different
concentrations have been presented in Table 4.6 (1), which revealed that the
maximum per cent inhibition of strains of Trichoderma was recorded 29.5
(32.68%) at 1000 ppm .Minimum per cent inhibition 1.42 (4.16%) was
recorded at a concentration of 500 ppm. All the strains of Trichoderma
showed significant differences among variable concentration levels.
Among the strains of Trichoderma the maximum per cent inhibition 17.96
(23.49%) was showed by the strains Tc 1 .The minimum per cent inhibition
was showed by Ts 3 11.11 (14.20%) followed by Ts 5 15.00 (16.76%). All the
strains of Trichoderma showed significant differences with each other at
variable concentration levels.
Strains of Trichoderma at 500 ppm concentration showed the maximum and
equal per cent inhibition 5.56 (13.57%) was showed by the strains Tc 1
followed by Ts1 2.96 (9.80%) . Remaining all strains showed the minimum
per cent inhibition of 0.00 (0.41%) .All the strains of Trichoderma showed
significant differences with each other at variable concentration levels except
Tc1 5.56 (13.57%) which was at par with Ts 1 2.96 (9.80%) .
At 1000 ppm the strain that showed maximum per cent inhibition was Ts 4
38.89 (38.46%) followed by Ts 2 33.7 (35.40%) .Minimum per cent inhibition
was recorded as 21.85 (27.75%) by Ts 1 .All the strains of Trichoderma at
1000 ppm showed significant difference with each other.
Table: 4.6 (1) Inhibition of radial growth of Trichoderma by glyphosate
in vitro. -
Inhibition of radial growth in different concentrations of
herbicide (%).
Strain
500 ppm 1000 ppm Mean
Ts1 2.96 (9.80) 21.85 (27.75) 12.40 (18.77)
Ts2 0.00 (0.41) 33.70 (35.40) 16.85 (17.90)
Ts3 0.00 (0.41) 22.22 (28.00) 11.11 (14.20)
Ts4 0.00 (0.41) 38.89 (38.46) 19.44 (19.43)
Ts5 0.00 (0.41) 30.00 (33.11) 15.00 (16.76)
Tc1 5.56 (13.57) 30.37 (33.41) 17.96(23.49)

29.50 (32.68)
Mean 1.42 (4.16)
SEm± CD
Strain 0.47 1.18
Herbicide 0.15 0.39
Strain*Herbicide 0.94 2.37
(* Figures in parentheses are angular transformed values)

(b)Triflurain 10G
The effect of triflurain 10G at different concentrations (500 ppm and 1000
(25.67%) was showed by the strains Ts1 followed by Ts4 23.70 (21.80%).
The minimum per cent inhibition was showed by Ts 3 10.74 (13.8%) followed
by Ts2 9.25 (17.01%). All the strains of Trichoderma showed significant
differences with each other at variable concentration levels except Ts3 10.74
(13.8%) which was at par with Ts 2 9.25 (17.01%).
Strains of Trichoderma at 500 ppm concentration the strain that showed the
maximum per cent inhibition was Ts 1 13.70 (21.55%) followed by Ts 2 4.44
(12.13%), Ts5 2.96 (9.82%).Remaining all strains showed the minimum per
cent inhibition of 0.00 (0.41%) All the strains of Trichoderma showed
significant differences with each other at variable concentration levels except
Ts1 13.70 (21.55%) which was at par with Ts 2 4.44 (12.13%), Ts5 2.96
(9.82%).
47.41 (43.2%) followed by Tc 1 45.56 (42.36%).Minimum per cent inhibition
was recorded as 14.07 (21.90%) by Ts 2 followed by Ts5 19.26 (25.79%). All
the strains of Trichoderma at 1000 ppm showed significant difference with
each other except Ts 2 14.07 (21.90%) which was at par with Ts 5 19.26
(25.79%).
Table: 4.6 (2) Inhibition of radial growth of Trichoderma by Triflurain
10G in vitro. -
Strain Inhibition of radial growth in different concentrations
of herbicide (%).
Ts1 13.7 (21.55) 24.81 (29.79) 19.25 (25.67)
Ts2 4.44 (12.13) 14.07 (21.90) 9.25 (17.01)
Ts3 0.00 (0.41) 21.48 (27.30) 10.74 (13.80)
Ts4 0.00 (0.41) 47.41 (43.20) 23.70 (21.80)
Ts5 2.96 (9.82) 19.26 (25.79) 11.11 (17.80)
Tc1 0.00 (0.41) 45.56 (42.36) 22.78 (21.38)

3.51(7.45) 28.76(31.72)
Mean
SEm± CD
Strain 0.89 2.26
Herbicide 0.29 0.75

(c) Atrazine 50%wp
The effect of atrazine 50%wp at different concentrations (500 ppm and 1000
(18.02%) was showed by the strains Tc 1 followed by Ts 3 7.96 (15.60%) .The
minimum and equal per cent inhibition was 0.00 (0.41%) showed by Ts 2, Ts5.
All the strains of Trichoderma showed significant differences with each other
at variable concentration levels except Ts 2 which was at par with Ts 5.
maximum per cent inhibition was Tc 1 8.52 (16.03%) followed by Ts 1 and Ts4
4.44 (13.29%), Ts3 3.33 (10.49%). Remaining all strains showed the
minimum per cent inhibition of 0.00 (0.41%). All the strains of Trichoderma
were at par with each other except which were 8.52 (16.03%), Ts 1 and Ts4
4.44 (13.29%), Ts3 3.33 (10.49%) showed significant difference.
12.59 (20.72%) followed by Tc 1 11.85 (20.02%) and Ts4 8.89 (16.99%).
Remaining all strains showed the minimum per cent inhibition of 0.00
(0.41%) which were at par with each other.
Table: 4.6(3) Inhibition of radial growth of Trichoderma by Atrazine
50%wp in vitro. -
of herbicide (%).
Ts1 4.44 (13.29) 6.30 (14.55) 5.37 (13.01)
Ts2 0.00 (0.41) 0.00 (0.41) 0.00 (0.41)
Ts3 3.33 (10.49) 12.59 (20.72) 7.96 (15.60)
Ts4 4.44 (13.29) 8.89 (16.99) 6.66 (15.14)
Ts5 0.00 (0.41) 0.00 (0.41) 0.00 (0.41)
Tc1 8.52 (16.03) 11.85 (20.02) 10.18 (18.02)

3.45 (8.68) 6.60(12.18)
Mean
SEm± CD
Strain 0.26 0.67
Herbicide 0.08 0.22

(d) Diuron 80DF
The effect of diuron 80DF at different concentrations (500 ppm and 1000
(71.15%) at 1000 ppm. Minimum per cent inhibition 57.28 (49.43%) was
(64.26%) was showed by the strains Tc 1 followed by Ts5 75.88 (62.23%) and
Ts3 75.40 (61.42%). The minimum per cent inhibition was showed by Ts 4
66.49 (56.32%), followed by Ts 2 70.61 (58.43%). All the strains of
Trichoderma showed significant differences with each other at variable
concentration levels
Strains of Trichoderma at 500 ppm concentration, the strain that showed the
maximum per cent inhibition was Tc 1 67.00 (54.98%) followed by Ts 5 61.11
(52.28%). The strain that showed the minimum per cent inhibition was Ts 4
42.66 (40.76%), followed by Ts 2 52.22 (46.26%). All the strains of
Trichoderma, showed significant difference with each other, except Ts 4
42.66 (40.76%) which was at par with Ts 2 52.22 (46.26%).
At 1000 ppm the strain that showed maximum per cent inhibition was Tc 1
92.00 (73.55%) .Minimum per cent inhibition was recorded as 85.66
(67.72%) by Ts1.All the strains of Tricoderma at 1000 ppm showed
significant difference with each other.
Table: 4.6 (4) Inhibition of radial growth of Trichoderma by Diuron
80DF in vitro. -
Inhibition of radial growth in different concentrations
Strain of herbicide (%)
59.26 (50.46) 85.66 (67.72) 72.46

Ts1 (59.09)
52.22 (46.26) 89.00 (70.61) 70.61
Ts2 (58.43)
Ts3 61.48 (51.88) 89.33 (70.96) 75.40

(61.42)
Ts4 42.66 (40.76) 90.33 (71.89) 66.49

(56.32)
Ts5 61.11 (52.28) 90.66 (72.18) 75.88

(62.23)
Tc1 67.00 (54.98) 92.00 (73.55) 79.50

(64.26)
57.28 (49.43) 89.49 (71.15)
Mean
SEm± CD
Strain 1.60 4.04
Herbicide 0.53 1.34

The herbicides tested in the present study exhibited varying levels of
inhibition on the growth of the Trichoderma strains. The results indicated that
diuron was incompatible with Trichoderma strains. The herbicide diuron
inhibited a growth of 57.28 (49.43%) at 500 ppm followed by 89.49 (71.15%)
at 1000 ppm. On the other hand, lower sensitivity to the other herbicides was
observed. At the 500 ppm the per cent inhibition exhibited by glyphosate,
triflurain, atrazine, paraquat and 2-4-D was 1.42 (4.16%), 3.51 (7.45%), 3.45
(8.68%),3.44 (8.82%) and 4.12 (9.36%) respectively and at 1000 ppm was
29.50 (32.68%), 28.76 (31.72%), 6.60 (12.18%), 29.23 (32.32%) and 40.77
(39.34%) respectively. Although the compound diuron was inhibitory in
action, the strain Ts 4 at 500 ppm concentration showed maximum tolerance
of about 66.49 (56.32%) and Ts 1 showed tolerance of 85.66 (67.72) at 1000
ppm concentration.
(e) Paraquat 3L
The effect of paraquat 3L at different concentrations (500 ppm and 1000
(28.90%) was showed by the strains Tc 1 .The minimum per cent inhibition
was showed by Ts4 11.00 (14.17%) followed by Ts 2 15.00 (16.80%). All the
variable concentration levels.
Strains of Trichoderma at 500 ppm concentration showed the maximum and
equal per cent inhibition 6.66 (14.94%) was showed by the strains Tc 1
followed by Ts 3 5.66 (13.75%). minimum per cent inhibition of 0.00 (0.41%)
by the strains Ts2 and Ts4.All the strains of Trichoderma showed significant
differences with each other at variable concentration levels except Tc 1 6.66
(14.94%) which was at par with Ts3 5.66 (13.75%).
46.33 (42.87%) followed by Ts 5 40.33 (39.41%) .Minimum per cent inhibition
was recorded as 14.75 (22.57%) by Ts 3. All the strains of Tricoderma at
1000 ppm showed significant difference with each other.
Table: 4.6 (5) Inhibition of radial growth of Trichoderma by paraquat 3L
in vitro. -
Strain Inhibition of radial growth in different concentrations of
herbicide (%)
Ts1 4.00 (11.47) 22.00(27.94) 13.00 (19.70)
Ts2 0.00 (0.41) 30.00(33.19) 15.00 (16.80)
Ts3 5.66 (13.75) 14.75 (22.57) 10.20 (18.16)
Ts4 0.00 (0.41) 22.00 (27.94) 11.00 (14.17)
Ts5 4.33 (11.99) 40.33 (39.41) 22.33 (25.70)
Tc1 6.66 (14.94) 46.33 (42.87) 26.49 (28.90)

3.44 (8.82) 29.23 (32.32)
Mean
SEm± CD
Strain 0.15 0.38
Herbicide 0.05 0.12

(f) 2-4-D 38EC
The effect of 2-4-D 38EC at different concentrations (500 ppm and 1000
(33.66%) was showed by the strains Tc 1 followed by Ts2 26.83 (28.13%).
The minimum per cent inhibition was showed by Ts 3 11.87 (18.38%) followed
by Ts1 14.00 (21.04%). All the strains of Trichoderma showed significant
differences with each other at variable concentration levels except Ts 3 11.87
(18.38%) which was at par Ts 1 14.00 (21.04%).
maximum per cent was Tc 1 12.00 (20.24%) followed by Ts 1 6.00 (14.14%).
minimum per cent inhibition of 0.00 (0.41%) by the strains Ts 4 and Ts5 All the
variable concentration levels except Tc 1 12.00 (20.24%) which was at par
with Ts1 6.00 (14.14%).
53.66 (47.08%) followed by Ts 4 50.00 (44.98%).Minimum per cent inhibition
was recorded as 21.00 (27.25) by Ts3 followed by Ts1 22.00 (27.95%). All the
strains of Trichoderma at 1000 ppm showed significant difference with each
other except Ts3 21.00 (27.25) which was at par with Ts 1 22.00 (27.95%).
Table: 4.6 (6) Inhibition of radial growth of Trichoderma by 2-4-D 38EC
in vitro. -
of herbicide(%).
Ts1 6.00 (14.14) 22.00 (27.95) 14.00 (21.04)
Ts2 4.00 (11.47) 49.66 (44.79) 26.83 (28.13)
Ts3 2.75 (9.52) 21.00 (27.25) 11.87 (18.38)
Ts4 0.00 (0.41) 50.00 (44.98) 25.00 (22.69)
Ts5 0.00 (0.41) 48.33 (44.02) 24.16 (22.21)
Tc1 12.00 (20.24) 53.66 (47.08) 32.83 (33.66)

4.12 (9.36) 40.77 (39.34)
Mean
SEm± CD
Strain 0.15 0.38
Herbicide 0.05 0.12

4.7 Evaluation of the bio potentiality of strains of the Trichoderma in
the green house .
Trials to evaluate the ability of the Trichoderma strains to control
Rhizoctonia bataticola under green house (pot) conditions were set up using
different strains of Trichoderma applied as seed treatment and by inoculating
inoculums of the pathogen in the soil, the procedure of which was described
early in the chapter Materials and methods
Against Rhizoctonia bataticola
The antagonistic potential of six strains of Trichoderma were evaluated
under greenhouse (pot) conditions for their effect against dry root rot caused
by Rhizoctonia bataticola on chickpea seedlings. The results of per cent
incidence of disease were presented in plate 8.
The results presented in table 4.7 indicated that all the strains of
Trichoderma effectively reduced the incidence of dry root rot on chickpea
seedlings. 100 per cent pre emergence mortality of chick pea seeds was
observed in control. Whereas, the lowest per cent of pre germination
mortality was recorded in Ts 3 6.45 (14.67%). The highest per cent of disease
incidence was observed with the strain Tc 1 15.76 (23.33%).
The data thus revealed that the strain Ts 3 resulted in effective control of pre
emergence mortality of chickpea seedlings caused by Rhizoctonia bataticola
in the green house.
At 7 days after germination, the results indicated that the strain Ts 3 was
found to be effective and recorded a lowest per cent of disease incidence of
about 14.27(22.17%) followed by Ts 5 18.26 (25.07%) . The strain Ts 3 was
found to be in at par with the strains Ts 5. The highest per cent of disease
incidence was observed with the strain Tc 1 28.33 (32.14%) followed by Ts 2
28.26 (32.10%).
about 22.18 (28.08%) followed by Ts 5 24.27 (29.39%) . The highest per cent
of disease incidence was observed with the strain Ts 2 35.92 (36.79%). There
was significant difference among the strains of Trichoderma in controlling dry
root rot.
about 26.66 (31.07%) followed by Ts 5 30.61 (33.57%) . The strain Ts 3 was
found to be in at par with the strains Ts 5. The highest per cent of disease
incidence was observed with the strain Ts 2 42.85 (40.80%) followed by Ts 1
42.59 (40.63%).
The per cent disease incidence of dry root rot in chick pea seeds caused by
Rhizoctonia bataticola, was evaluated in green house conditions under pot
experiment by using 6 Trichoderma strains along with control were shown in
Table 4.6. The minimum per centage inhibition of dry root rot disease caused
by Rhizoctonia bataticola was exerted by Trichoderma strains Ts3, Ts5 at all
the stages of the crop.
Table 4.7 Effect of different strains of Trichoderma on pre and post

incidence of Rhizoctonia bataticola (%)
Strain Pre Post emergence mortality at

germination
mortality 7 days 15days 21days
Ts1 13.27 (21.26) 27.23 34.91 (36.17) 42.59

(31.26) (40.63)
Ts2 13.94 (21.86) 28.26 35.92 (36.79) 42.85

(32.10) (40.80)
Ts3 6.45 (14.67) 14.27 22.18 (28.08) 26.66

(22.17) (31.07)
Ts4 12.97 (21.01) 25.37 31.90 (34.37) 38.09

(30.06) (38.08)
Ts5 13.26 (21.33) 18.26 24.27 (29.39) 30.61

(25.07) (33.57)
TsC1 15.76 (23.33) 28.33 34.21 (35.78) 41.97

(32.14) (40.36)
SEm+ 1.17 2.12 1.25 2.23
CD (P=0.05) 3.60 6.53 3.85 6.88

Fig.1: Mycelial dry weight (g) of Trichoderma strains at different temperatures
Fig. 2 :Radial growth of Trichoderma strains at different temperatures
Fig.3: Effect of different pH on the biomass production of Trichoderma strains
Fig.4: Comparative tolerance ability of Trichoderma strains in response of different herbicides at 500 ppm and 1000 ppm
CHAPTER- V
DISCUSSION
In recent years, research on biological control has gained momentum

for controlling serious soil borne plant pathogens like Fusarium, Rizoctonia,
Macrophomina, Sclerotium, Pythium and Phytophthora spp. employing
Trichoderma and Gliocladium species and varied success has been
achieved.
In the current study investigations were carried out on the six

Trichoderma strains, isolated from soils and rhizosphere soil samples from
cultivated lands around west Madhya Pradesh, for the characterisation of the
isolated strains in response of variable temperature and pH, to assess
tolerance levels in response of different agrochemicals and to test
antagonism against Rhizoctonia bataticola for evaluation of antagonistic
potential of Trichoderma strains.
5.1 Recovery of Trichoderma spp. from rhizospheric soil of different

crops
A total of 55 soil samples were collected from rhizosphere of 8
different crops and from 6 districts of west M.P. These six strains of
Trichoderma were isolated through serial dilution technique. The result
indicated that Trichoderma spp. could grow and survive in various kinds of
soil conditions. This evidence agrees with the report of Harman et al. (2004)
who had described the members of genus Trichoderma are free-living fungi
that are common in soil and root ecosystems. Hence, for Trichoderma
isolation, the dilution plating technique remains adequate, agreed with the
findings of Sivakumar et al. (2000).
Thakur and Norris (1928) isolated the genus Trichoderma in India
from soils of Madras. Kader et al. (1999) mixed the soil samples with sterile
distilled water and made a series of dilutions. From the dilutions, 0.5ml was
placed on potato dextrose agar (PDA) and incubated at 30ºC for three days.
Fungi were isolated from the mixed isolates from each plate and sub cultured
on PDA, until a pure isolate was obtained.
Cigdem and Merih (2003) collected thirty-one soil samples from
different agricultural fields and forests in Eskisehir and inoculated on potato
dextrose agar (PDA, MERCK), malt extract agar (MEA; MERCK), rose
bengal agar and oat flour agar and incubated at 28 °C for 5 days. Colonies
were purified by subcultures and Trichoderma spp. were identified according
to the key given by Watts et al. (1988) and Rifai (1969).
5.2 Effect of temperature of growth of Trichoderma strain.
Significant difference in biomass production at different temperatures
was observed among the six Trichoderma strains. On PDB medium mycelia
dry weight was recorded maximum at 25ºC. At 45ºC all the isolates showed
very slow growth rate and no growth in the mycelia dry weight was recorded
at 5ºC. Results showed that 25ºC was most suitable for growth of all isolates.
At 45ºC Tc1 (0.33 g) expressed the maximum dry weight and at 25ºC the
strain Tc1 (0.47 g) was recorded with maximum biomass production. The
result here indicates that the strain Tc 1 was favoured by the growth at wide
range of temperatures. The result indicated that Trichoderma spp. could
grow and survive in various kinds of temperature regimes.
On PDA medium radial diameter of the Trichoderma strains was
recorded maximum at 25ºC. At 45ºC all the isolates showed very slow
growth rate and no growth in the mycelia dry weight was recorded at 5ºC.
Results showed that 25ºC was most suitable for growth of all Trichoderma
strains. Growth of the Trichoderma strains was reduced when the
temperature was increased or decreased from the optimum (25ºC). At 45ºC
the strain Ts2 expressed maximum colony diameter of 83.33 mm. At 25ºC,
Ts2, Ts3, Ts5 and Tc1 strains exhibited the maximum colony diameter of 90
mm.The result here indicates that the strains were adaptable to wide range
of temperatures. Greenish coloured conidia form within 4-5 days at 25°C and
remain white up to 6 days at 45°C.
This evidence agrees with the report of Sobieralski et al. (2009) who
had reported that the mycelium of the examined Trichoderma isolates grew
better on the manure medium than on standard agar medium and the
optimum temperature for all the examined isolates was 25 and 30°C.
Gupta and Sharma (2013) reported that the optimum temperature of
Trichoderma harzianum was found between 25-30°C.Trichoderma
harzianum hyphal extension grown faster at 25-30°C, slower grown at 37°C
and no growth was observed at 45°C after six day of inoculation.
The result of the current experiment differs with the report of Antal et
al. (2000) which states that T. aureoviride, T. harzianum and T. viride – grew
well at 5ºC on both minimal and yeast extract agar media. The incidence of
cold tolerant isolates was the highest in species group T. viride. This may be
because of the strain isolation from the soil of the forest of Ásotthalom
(southern Hungary) where cold exists or may be because of the use of
different media such as minimal and yeast extract agar media.
5.3 Effect of pH on the biomass production of Trichoderma strains.
Significant variation in biomass production was observed among all
the strains of Trichoderma at all test pH values of 5, 7 and 9 (Table 4.5).
Maximum number of isolates showed high biomass production at pH 7
followed by 5 and minimum at pH 9. The result revealed a variation in
mycelia dry weight of Trichoderma strains at different pH. Mycelial dry
weight of Trichoderma strains found to be maximum on pH 7 (0.39 g),
followed by pH 5 (0.31 g) and minimum dry weight was recorded with pH 9
(0.26 g). The maximum dry weight was expressed by the strain Ts 1 (0.36 g).
Maximum dry weight was recorded by the strain at pH 5.0 were Ts 2 at pH 7.0
were Ts1and Tc1 at pH 9.0 were Ts 2 ,Ts4 which indicates that the strains Tc 1
and Ts1 were widely adopted to different ranges of pH.
The result was supported by the Benitez (2004) who had reported the
growth of Trichoderma is more efficient in acidic than alkaline soils and they
modify the rhizosphere soil by acidifying the soil. This explains the reason for
isolates which prefer acidic pH. Even though the change in pH in the culture
medium was not tested at the end of our experiment, it is attributed that the
change in preference of Trichoderma strains from higher to lower pH is due
to the change in pH levels.
Papavizas (1985) found that pH values higher than 6.5 were optimum
for the maximal linear growth of T. harzianum.
Kolli et al. (2012) isolated twenty six isolates of Trichoderma from
forest and agricultural soils were tested in vitro for their pH levels, tolerance
and biomass production.
5.4 Tolerance level of Trichoderma strains in response to different
herbicides.
Diuron 80DF was incompatible with Trichoderma strains. The herbicide
Diuron 80DF inhibited a growth of 57.28 (49.43%) at 500 ppm followed by
89.49 (71.15%) at 1000ppm. On the other hand, lower sensitivity of
Trichoderma strains to the other herbicides was observed. At the 500ppm
the percent inhibition exhibited by glyphosate 4L, triflurain 10G, atrazine
50%WP, paraquat 3L and 2-4-D 38% EC was 1.42 (4.16%), 3.51 (7.45%),
3.45 (8.68%), 3.44 (8.82%), 4.12 (9.36%) respectively and at 1000ppm was
29.50 (32.68%), 28.76 (31.72%), 6.60 (12.18%), 29.23 (32.32%) and 40.77
(39.34%) respectively. Although the compound Diuron 80 DF was inhibitory
in action, the strain Ts 4 at 500 ppm concentration showed maximum
tolerance i.e minimum inhibition of radial growth of about 42.66 (40.76%) and
Ts1 showed minimum inhibition of radial growth of about 85.66 (67.72%) at
1000 ppm concentration. The herbicides glyphosate 4 L, triflurain 10 G,
atrazine 50% WP, paraquat 3L and 2-4-D 38% EC showed lesser degree of
toxicity towards Trichoderma strains, which indicated their compatibility with
the test fungus.
Sunil and Kulkarni (2004) reported that following agrochemicals were
highly inhibitory to T. harzianum alachlor, carbendazim, chlorpyriphos,
organomercurial, thiram and trifluralin. Inhibitory effects increased with
increase in concentrations from 500 to 2000 ppm.
5.5 Evaluation of bio potentiality of Trichoderma strains against R.

bataticola in green house condition.
Green house studies were taken up to evaluate the effect of six
strains of Trichoderma against root rot and dry root rot diseases in chickpea.
Under greenhouse, all tested strains of Trichoderma have significantly
reduced dry root rot disease compared to control plants. Data also indicated
that the strains of Trichoderma varied in their reduction of Disease severity
incidence in Chikpea plants. The highest reduction was achieved by the seed
treatment with Ts3 and Ts4.
The strains that showed minimum per cent of disease incidence at
7days after emergence were Ts 3 14.27 (22.17%), and Ts 5 18.26 (25.07%), at
15 days after emergence were Ts 3 22.18 (28.08%), and Ts 5 24.27 (29.39%)
and at 21 days after emergence were Ts 3 26.66 (31.07%), and Ts 5 30.61
(33.57%). This indicated that the results obtained in the dual culture
experiment were same as that of green house (pot) experiment.
This evidence agrees with the report of Poddar et al. (2004) and
Rojo et al. (2007) who reported the potentiality of Trichoderma spp. as
biocontrol agents of phytopathogenic fungi in several crops is well known
especially to Fusarium spp. and Rhizoctonia spp.
Nair et al. (2006) who conducted to a pot experiment to determine
the effects of biological control agents (Trichoderma harzianum, Trichoderma
viride) on Rhizoctonia bataticola infecting sesame VAR-1.The seeds treated
with spore suspension of Trichoderma harzianum were superior over all the
other treatments. The germination percentage and percent of disease control
was maximum (92%) in case of Trichoderma harzianum followed by
Trichoderma viride (89.33%).
CHAPTER -VI
SUMMARY, CONCLUSIONS AND SUGGESTIONS
FOR FURTHER WORK
6.1 Summary
Several species of Trichoderma have been extensively studied for

their ability to control several fungal plant pathogens. In fact, the antifungal
abilities of these beneficial microbes have been known since the 1930s, and
there have been extensive efforts to use them for plant disease control since
then. In the present study an attempt was made to isolate Trichoderma spp.
from different soil samples, to screen them for their tolerance levels to
different temperatures, pH and herbicides and to test the antagonism against
soil borne plant pathogen Rhizoctonia bataticola. The results obtained are
summarized below.
6.1.1 Isolation of Trichoderma spp.
Total 6 strains of Trichodema were isolated from 55 soil samples
collected from 8 different districts and 8 different crops rhizospheres of west
M.P, namely Ts1, Ts2, Ts3, Ts4, Ts5, Ts6 and Tc1.
6.1.2 Temperature sensitivity of Trichoderma strains
The temperature studies revealed that, 250C was suitable for the
growth of the Trichoderma strains, at 450C all the isolates showed very slow
growth rate and no growth in the mycelia dry weight was recorded at 50C. At
450C the strain Tc1 (0.33g) expressed the maximum dry weight and at 250C
the strain Tc1 (0.47 g) was recorded with maximum biomass on PDB
medium. On PDA medium at 450C the strains Ts2, Ts4 expressed maximum
colony diameter of 83.33mm and 76.67mm respectively, at 250C strains Ts2,
Ts3, Ts5 and Tc1 exhibited the maximum colony diameter of 90mm and at
50C there was no growth in the strains. The result here indicates that the
strains Ts2,Ts3, Tc1 were adaptable towide range of temperatures. Greenish
coloured conidia form within 4-5 days at 25°C and remain white up to 6 days
at 45°C.
6.1.3 Tolerance of Trichoderma strains to pH
The result revealed a variation in mycelia dry weight of Trichoderma
strains at different pH. Mycelial dry weight of Trichoderma strains found to be
maximum on pH 7 (0.39 g), followed by pH 5 (0.31 g) and minimum dry
weight was recorded with pH 9 (0.26 g). The pH of 7.0 was found suitable for
the growth of Trichoderma strains and they mostly prefer acidic pH for their
growth. The strains Tc 1 and Ts1 were wide adapted to pH range 7 where as
the strains Ts2 expressed maximum biomass production at pH5.
6.1.4 Effect of herbicides
The results indicated that diuron was incompatible with Trichoderma

strains. On the other hand, lower sensitivity of Trichoderma strains to the
other herbicides glyphosate, triflurain, atrazine, paraquat, 2-4-D was
observed. Although the compound diuron was inhibitory in action. The strain
Ts4 at 500 ppm concentration showed maximum tolerance of about 42.66
(40.76%) and Ts1 showed of about 85.66 (67.72%) at 1000 ppm
concentration.
6.2 Bio-efficacy of Trichoderma strains against R. bataticola in green
house.
The results indicated that all the strains of Trichoderma effectively
reduced the incidence of dry root rot on chickpea seedlings. 100 per cent pre
emergence mortality of chick pea seeds was observed in control. Whereas,
the lowest per cent of pre germination mortality was recorded in Ts 3 6.45
(14.67%). The highest per cent of disease incidence was observed with the
strain Tc1 15.76 (23.33%).
The data thus revealed that the strain Ts 3 resulted in effective control
of pre emergence mortality of chickpea seedlings caused by Rhizoctonia
bataticola in the green house.
At 7 days after germination, the results indicated that the strain Ts 3
was found to be effective and recorded a lowest per cent of disease
incidence of about 14.27(22.17%) followed by Ts 5 18.26 (25.07%) . The
strain Ts3 was found to be in at par with the strains Ts 5. The highest per cent
of disease incidence was observed with the strain Tc1 28.33 (32.14%)
followed by Ts2 28.26 (32.10%).
incidence of about 22.18 (28.08%) followed by Ts 5 24.27 (29.39%) . The
highest per cent of disease incidence was observed with the strain Ts 2 35.92
(36.79%). There was significant difference among the strains of Trichoderma
in controlling dry root rot.
incidence of about 26.66 (31.07%) followed by Ts5 30.61 (33.57%) . The
strain Ts3 was found to be in at par with the strains Ts5. The highest per cent
of disease incidence was observed with the strain Ts 2 42.85 (40.80%)
followed by Ts1 42.59 (40.63%).
6.2 Conclusions
A total of six Trichoderma strains were isolated from 55 soil samples
collecte from rhizosphers of eight crops and six districts of west M.P.
revealed the presence of Trichoderma spp. in wide range of soils. From the
above findings, it can be concluded that a significant number of Trichoderma
species occupy in different habitats. The 6 strains of Trichoderma performed
better at 250C temperatures and 7.0 pH. The strain Ts 2 performed well at
different ranges of temperature and pH. Some species of Trichoderma were
found to be adapted to acidic habitats.
diuron was incompatible with Trichoderma strains. The herbicide diuron
inhibited a growth of 57.28 (49.43%) at 500 ppm followed by 89.49 (71.15%)
at 1000 ppm. On the other hand, lower sensitivity to the other herbicides was
observed. At the 500 ppm the per cent inhibition exhibited by glyphosate,
triflurain, atrazine, paraquat and 2-4-D was 1.42 (4.16%), 3.51 (7.45%), 3.45
(8.68%),3.44 (8.82%) and 4.12 (9.36%) respectively and at 1000 ppm was
29.50 (32.68%), 28.76 (31.72%), 6.60 (12.18%), 29.23 (32.32%) and 40.77
(39.34%) respectively. Although the compound diuron was inhibitory in
action, the strain Ts 4 at 500 ppm concentration showed maximum tolerance
of about 66.49 (56.32%) and Ts1 showed tolerance of 85.66 (67.72) at
1000 ppm concentration.
Under greenhouse, all tested strains of Trichoderma have
significantly reduced dry root rot disease compared to control plants. Data
also indicated that the strains of Trichoderma varied in their reduction of
Disease severity incidence in chick pea plants. The highest reduction was
achieved by the seed treatment with Ts 3, and Ts5. This indicated that the
results obtained in the dual culture experiment were same as that of green
house (pot) experiment.
6.3 Suggestions for further work
1. More soil samples from different cropped soils and geographical areas
need to be collected and tested for their bio control potential against the
major soil borne diseases.
2. The present findings need to be supported by molecular characterization
so as to differentiate them in to segregates with genetic diversity.
3. Need the analysis of the biochemical behaviour of the strains to
characterize them as different variants
4. Need to execute more number and diverse range of field experiments for
evaluation of real effectiveness of selected strains of Trichoderma.
5. More studies such as in vivo tests have to be conducted using these
Trichoderma isolates before they can be used as a biological control agent in
agricultural field.
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APPENDICES
ANOVA table for effect of temperature on growth of Trichoderma strains
Analysis of Variance Table 4.4.1-Mycelial dry weight(g) of Trichoderma

strains at different temperatures.
(a)At 450C
SV DF SS MSS Fcal Ftab
Treatment 5 0.09 0.01 2.93 2.80
Error 12 0.07 0.00
Total 17 0.16 0.01
(a)At 250C
Treatment 5 0.12 0.02 4.59 2.80
Error 12 0.06 0.00
Total 17 0.18 0.02
Analysis of Variance Table 4.4.2 Radial growth of Trichoderma strains

at different temperatures.
(a)At 450C
Treatment 5 6471.29 1294.26 10.78 2.80
Error 12 1439.82 119.98
Total 17 7911.18 1414.24
(a)At 250C
Treatment 5 1278.91 255.78 8.19 2.80
Error 12 374.50 31.20
Total 17 1653.41 286.98
ANOVA table for Hydrogen ion concentration (pH)

Analysis of Variance Table 4.5- effect of different pH on the biomass
production of Trichoderma strains
(a)At PH 5
Treatment 5 0.05 0.01 6.41 2.80
Error 12 0.01 0.00
Total 17 0.06 0.01
(a)At PH 7
Treatment 5 0.14 0.02 3.01 2.80
Error 12 0.11 0.00
Total 17 0.25 0.02
(a)At pH 9
Treatment 5 0.01 0.00 7.41 2.80
Error 12 0.07 0.00
Total 17 0.08 0.00
ANOVA table for tolerance potentiality of Trichoderma strains against

variable herbicides.
Analysis of Variance Table – 4.6(1) Glyphosate 4L (Inhibition per cent)

Factor A 1 17097.19 17097.19 2147.54 3.84
Factor B 5 286.03 57.20 7.18 11.07
Interaction A X B 5 904.15 180.83 22.71 11.07
Error 24 191.07 7.961
Total 35 18478.44 17343.18
Analysis of Variance Table – 4.6(2) Triflurain 10G (Inhibition per cent)

Factor A 1 14865.26 14865.26 510.27 3.84
Factor B 5 526.96 105.39 3.61 11.07
Interaction A X B 5 2962.07 592.41 20.33 11.07
Error 24 699.16 29.13
Total 35 19053.45 15592.19
Analysis of Variance Table – 4.6(3) Atrazine 50%wp (Inhibition per cent)

Factor A 1 2125.17 2125.17 820.93 3.84
Factor B 5 1885.04 377.00 145.63 11.07
Interaction A X B 5 1100.55 220.11 85.02 11.07
Error 24 62.12 2.588
Total 35 5172.88 2724.86
Analysis of Variance Table – 4.6(4) Diuron 80DF (Inhibition per cent)
Factor A 1 71814.15 71814.15 776.16 3.84
Factor B 5 248.67 49.73 0.53 11.07
Interaction A XB 5 429.04 85.80 0.92 11.07
Error 24 2220.59 92.52
Total 35 74712.45 72042.2
Analysis of Variance Table – 4.6(5) paraquat 3L (Inhibition per cent

Factor A 1 15073.43 15073.43 18379.54 3.84
Factor B 5 945.18 189.03 230.49 11.07
Interaction A X B 5 1377.98 275.59 336.04 11.07
Error 24 19.68 0.82
Total 35 17416.27 15538.87
Analysis of Variance Table – 4.6(6) 2-4-D 38EC ( Inhibition per cent)
Factor A 1 22816.46 22816.46 27545.89 3.84

Factor B 5 928.80 185.76 224.26 11.07
Interaction A X B 5 2334.29 466.85 563.62 11.07
Error 24 19.87 0.82
Total 35 26099.42 23469.89
ANOVA table for Effect of different strains of Trichoderma on pre and post
Analysis of Variance Table – 4.7 (Disease incidence (%) at pre

germination)
Treatment 5 135.85 27.17 6.60 2.80
Error 12 49.36 4.11
Total 17 185.21 31.28
Analysis of Variance Table – 4.7(Disease incidence (%)at 7 DAS)

Treatment 5 262.42 52.48 3.88 2.80
Error 12 162.10 13.50
Total 17 424.52 65.98

Treatment 5 210.18 42.03 8.95 2.80
Error 12 56.35 4.69
Total 17 266.53 46.72

Treatment 5 257.90 51.58 3.44 2.80
Error 12 179.49 14.95
Total 17 437.39 66.53
VITA
Swati Panwar, the author of thesis was born on 14th June 1993-Khargone
(M.P.). She completed her High School Examination from J. N. V. Junapani
(Sanavad) DT. Khargone (M.P.) with (55.2%) in Central Board of Secondary
Education and Higher Secondary School Examination from Sun Shine H S School,
Indore with (70.80%) first division in M.P. Board of Secondary Education.
She was selected through entrance examination (P.A.T.) and joined the
College of Agriculture, Indore (M.P.) in 2011 and obtained B.Sc. (Ag.) degree in
2015 with 7.31 OGPA out of 10.00 point scale.
The author continued her post graduation from College of Agriculture,

Indore (M.P.), to specialize in “Department of Plant Pathology” and for partial
fulfillment of the requirements for the award of the same, she was allotted with
interesting problem as “Compatibility of temperature and pH tolerant Tricoderma
strains with herbicides, and their bioefficacy against Rhizoctonia bataticola. " for
thesis work which has been duly completed by her and presented in this thesis.
She actively participated in all the cultural activities of the college. Now,
she is submitting the thesis after completing the course with 7.79 OGPA out of
10.00 scale.
Swati Panwar

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Compatibility of temperature and pH tolerant Trichoderma

strains with herbicides, and their bioefficacy against

Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya,

In partial fulfillment of the requirements for the Degree of

Department of Plant Pathology,

Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya,

College of Agriculture, Indore (M.P.)

MEMBER OF STUDENT’S ADVISORY COMMITTEE

1. Chairman (Dr. Ashok Krishna ) : -----------------------------

2. Member ( Dr. R. K. Singh) : -----------------------------

3. Membe (Dr. R. K. Choudhary) : -----------------------------

MEMBERS OF THE ADVISORY COMMITTEE

1. Chairman (Dr. Ashok Krishna) : -------------------------------

2. Member (Dr. R. K. Singh) : ---------------------------------------

3. Member (Dr. R. K. Choudhary) : ---------------------------------------

Head of the Department : ----------------------------------------

Dean of the college : ---------------------------------------

Director Instruction : ----------------------------------------

I praise God for enabling me to accomplish this great task of thesis

Number Title Page

2. Review of Literature 4-13

3. Material and Methods 14-23

Summary, Conclusion and Suggestions for further

2. Isolation of Rhizoctonia bataticola.

3. Mycelial dry weight (g) at variable temperatures.

1. Mycelial dry weight (g) at variable temperatures.

2. Radil growth of Trichoderma strains at different temperatures.

3. Effect of different pH on the biomass production of Trichoderma strains.

4. Comparative tolerance ability of Trichoderma strains in response of

1. Isolation and purification of Trichoderma strains from soil.

2. Evalution of Trichoderma strains for tolerance to temperature and pH

3. Assessment of compatibility of different Trichoderma strains with common

4. Bioefficacy of Trichoderma strains against Rhizoctonia bataticola in the

Influence of agrochemicals on soil mycoflora being changed in favour

2.1 Isolation and identification of Trichoderma spp. from soil

Thakur and Norris (1928) isolated the genus Trichoderma in India

2.2 Effect of temperature on Trichoderma spp.

2.2.1 Trichoderma species as biocontrol agents:

Ghaffer (1968) observed that, Macrophomina phaseolina was

2.4.3 Antagonistic potential of Trichoderma strains against R.

Shamim et al. (1997) have reported for Rhizoctonia sp., that

MATERIAL AND METHODS

The present study on bio efficacy of naive strains of Trichoderma

(a) Cleaning solution:

(b) Mercuric chloride solution:

ii. Cotton blue

Anhydrous lacto phenol 67 ml

Table-3.1.6 The formulated chemical name of herbicides used along

Common Trade name C.C. Time of

3.1.8 Ingredient of culture media:

(i) Trichoderma selective medium (TSM):

7. Rose Bengal - 0.15 g

9. Apron 35-SD - 0.025 g

11. Distilled water - 1l

(ii) Potato dextrose agar (PDA) medium: (Rangaswami and Mahadeven,

1. Peeled and sliced potato (extract) - 200 g

5. pH (adjusted to) - 6.5

3.1.9 Glass wares: