Indian J Microbiol (AprJune 2012) 52(2):137144

DOI 10.1007/s12088-011-0205-3

ORIGINAL ARTICLE

Isolation and Characterization of Trichoderma spp.

for Antagonistic Activity Against Root Rot and Foliar Pathogens
Krishna Kumar N. Amaresan S. Bhagat
K. Madhuri R. C. Srivastava

Received: 22 June 2010 / Accepted: 23 November 2010 / Published online: 13 August 2011
Association of Microbiologists of India 2011

Abstract Trichoderma, soil-borne filamentous fungi, are

capable of parasitising several plant pathogenic fungi.
Twelve isolates of Trichoderma spp. isolated from different locations of South Andaman were characterized for
their cultural, morphological and antagonistic activity
against soil borne and foliar borne pathogens. The
sequencing of these isolates showed seven different species. The isolates revealed differential reaction patterns
against the test pathogens viz., Sclerotium rolfsii, Colletotrichum gloeosporioides and C. capsici. However, the
isolates, TND1, TWN1, TWC1, TGD1 and TSD1 were
most effective in percentage inhibition of mycelial growth
of test pathogens. Significant chitinase and b-1,3-glucanase
activities of all Trichoderma isolates has been recorded in
growth medium. T. viride was found with highest chitinase
whereas T. harzianum was recorded with highest b-1,3glucanase activities.
Keywords Trichoderma  Spice  Biocontrol  Andaman
and Nicobar Islands

K. Kumar (&)  N. Amaresan  S. Bhagat  K. Madhuri 

R. C. Srivastava
Division of Field Crops, Central Agricultural Research Institute,
Port Blair 744 105, Andaman and Nicobar Islands, India
e-mail: [email protected]

Introduction
The anamorphic fungal genus Trichoderma (Hypocreales,
Ascomycota) is a cosmopolitan soil-borne fungi frequently
found on decaying wood [1, 2] of which some are economically important producers of industrial enzymes
(Trichoderma ressei) [3], antibiotics [4] and have been
used as biocontrol (i.e., T. harzianum, T. atroviride and
T. asperellum) agents against plant pathogens [5]. Trichoderma spp. are among the most frequently isolated soil
fungi and present in plant root systems [6]. However, there
is still considerable interest in finding more efficient mycoparasitic fungi especially within Trichoderma spp.,
which differ considerably with respect to their biocontrol
effectiveness. It is important to isolate Trichoderma spp.
having potentially higher antagonistic efficiency by the
selection of isolates with high potential to secret extra
cellular lytic enzymes chitinase and b-1,3-glucanase. The
lytic enzymes break down cell wall polysaccharides into
short oligomers and by this way facilitate the hyperparasite
to penetrate into the cytoplasm of the target fungi [7]. The
aim of this study was screening of Trichoderma spp. for
their antagonistic ability by dual cultures as well as their
capability of producing lytic enzymes against the test
pathogens.

N. Amaresan
e-mail: [email protected]

Materials and Methods

S. Bhagat
e-mail: [email protected]

Soil Samples and Isolation

K. Madhuri
e-mail: [email protected]

Soil samples were collected from different ecological

habitat of spice crops of district, South Andaman, Andaman and Nicobar Islands (India) for the isolation of
Trichoderma spp. (Table 1). Samples were brought to

R. C. Srivastava
e-mail: [email protected]

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Indian J Microbiol (AprJune 2012) 52(2):137144

laboratory and stored at 4C until used. Five-fold serial

dilutions of each soil samples were prepared in sterilized
distilled water and 0.5 ml diluted sample was poured on the
surface of Trichoderma Specific Medium (TSM) [8]. Plates
were incubated at 28 2C for 96 h. Morphologically
different colonies appearing on the plates were purified in
the Potato Dextrose Agar (PDA) (HiMedia, India). The
purified isolates were preserved at 4C and used during the
course of study.

Fungal Growth Conditions and DNA Extraction

Cultures were maintained on PDA at 25C was grown in
potato dextrose broth for 3 days. Mycelial mat was
collected on filter paper, washed with distilled water for
23 times, frozen and were used for DNA extraction.
Genomic DNA was extracted using method of Raeder and Broda [10]. DNA resuspended in 50 ll of TE
buffer and quantified by use of ethidium bromide
fluorescence.

Phenotype Characters of the Trichoderma Isolates

The morphological and cultural characteristics of 12
isolates of Trichoderma were studied in four different
media viz., OMA, CMD, PDA and TSM following the
protocol of Samules et al. [9]. Mycelial discs (6 mm) of
young growing culture of respective isolates of Trichoderma was inoculated in the periphery of the Petri plates
containing above said media and incubated at 28 2C
for one week. Colony radius was measured at 24, 48 and
72 h. each growth rate experiment was repeated three
times in triplicate and the results were averaged for each
isolate. Additional characters include presence of pigments, green conidia, odor and colony appearance were
also noted.
Morphological observations were recorded from cultures grown on PDA plates. The following characters were
measured; Phialide width at the widest pint, phialide
length, conidium length and width and presence of chlamydospores. Each character was measured from water
after initial wetting in 3% KOH for each isolates.

PCR Amplification and Sequencing of Amplification

Products
Primers ITS1 (50 -TCC GTA GGT GAA CCT GCG G-30 )
and ITS4 (50 -TCC TCC GCT TAT TGA TAT GC-30 )
described by White et al. [11] were used to amplify a
fragment of rDNA including ITS1 and ITS2 and the 5.8S
rDNA gene. PCR amplifications were performed in a total
volume of 50 ll by mixing 20 ng of the template DNA
with 2.5 mM concentrations of each deoxynucleotide triphosphate, 1 lm concentrations of each primer and 3 U of
Taq DNA polymerase in 109 Taq buffer A (GeNeI). These
reactions were subjected to initial denaturations of 1 min at
95C, followed by 35 cycles of 1 min at 95C, 30 s at 55C
and 1.5 min at 72C, with a final extension of 10 min at
72C using GeneAmp PCR system 9700 (Applied Biosystems). The PCR products were resolved using a 1%
agarose gel. Sequencing of purified PCR product was
undertaken by SAP services, GeneI, Banglore using ITS1
(forward primer).

Table 1 Trichoderma isolates studied, their identification and host

S. No.

Isolate designationa

Crop nameb

Place of collection

Species identified

Accession number

1
2

TSD1
TWN1

Cinnamon
Nutmeg

Sippighat
Wandoor

Trichoderma inhamatum
T. harzianum

GQ426033
GQ426034

TGN1

Nutmeg

Guptapara

T. harzianum

GQ426035

TWD1

Cinnamon

Wandoor

T. asperellum

GQ426036

TND1

Cinnamon

New Mangulton

T. erinaceum

GQ426037

TJP1

Pepper

Jirkatang

T. harzianum

GQ426038

TWC2

Clove

Wandoor

T. harzianum

GQ426039

TMC1

Clove

Manjery

T. longibrachiatum

GQ426040

TWP1

Pepper

Wandoor

Trichoderma sp.

GQ426041

10

TCC1

Cinnamon

Calicut

T. ovalisporum

GQ426042

11

TGD1

Cinnamon

Guptapara

T. viride

GQ426043

12

TBC1

Clove

Brichgunj

T. brevicompactum

GQ426044

Coding system of isolates T Trichoderma, S place of collection and D crop name

Rhizosphere soil of various spice crops

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Indian J Microbiol (AprJune 2012) 52(2):137144

139

Phylogenetic Analysis

b-1,3-Glucanase Activity

ITS gene sequences of the isolates were compared with

ITS sequences available by the BLAST search in the
NCBI, GenBank database (http://www.ncbi.nlm.nih.gov).
Multiple sequence alignment was performed using
Clustal X (1.1). The method of Jukes and Cantor [12]
was used to calculate evolutionary distances. Phylogenetic dendogram was constructed by the neighbourjoining method and tree topologies were evaluated by
performing bootstrap analysis of 100 data sets using
MEGA 3.1 (Molecular Evolutionary Genetic Analysis).
The sequences were submitted to GenBank under the
following accession numbers GQ426033GQ426044 (12
sequences).

For assay of b-1,3-glucanase enzyme, 0.5 ml laminarin,

1.0 ml of 0.05 M citrate buffer (pH 4.8) and 0.5 ml culture
filtrate was mixed and incubated at 40C for 60 min. An
equal volume of dinitrosalicyclic acid reagent was added to
the reaction mixture and warmed in boiling water for
15 min. The absorbance of reaction mixture was measured
at 575 nm in a spectrophotometer and compared with
standard graph drawn by following the same procedure but
using different concentrations of glucose instead of culture
filtrate. The quantity of reducing sugar was calculated from
the glucose standards used in the assay and activity of b1,3-glucanase was expressed in nkat/ml. One nkatal corresponds to the release of 1 nmol glucose equivalent per
second.

Antagonistic Activity of Trichoderma Isolates

The dual culture technique described by Morton and
Stroube [13] was used to test the antagonistic ability of
Trichoderma spp. against soil borne pathogen S. rolfsii
and foliar borne pathogens viz., C. capsici and C. gloeosporioides. The pathogens and Trichoderma were
grown on PDA for a week at room temperature
(28 2C). Small blocks of the target fungi (C. capsici
and C. gloeosporioides) cut from the periphery were
transferred to the Petri dish previously poured with PDA.
After 2 days of Colletotrichum spp. growth, the Trichoderma spp. was transferred aseptically in the same plate
of opposite end and were incubated at room temperature
with alternate light and darkness for 7 days and observed
periodically. For S. rolfsii both pathogen and Trichoderma
were inoculated at the same time. The experiment was
replicated thrice and percent growth inhibition was calculated by the formula of I = (C-T)/C 9 100, where C
is mycelial growth in control plate, T is mycelial growth
in test organisms inoculated plate and I is inhibition of
mycelial growth.
Assay for Extra Cellular Enzyme Activity
Twelve isolates of Trichoderma were separately inoculated
into 100 ml broth media with four combinations viz.,
Czapek Dox Broth alone, CDB ? mycelial powder of S.
rolfsii (10 g), CDB ? mycelial powder of C. gloeosporioides (10 g) and CDB ? mycelial powder of C. capsici
(10 g) in Erlenmeyer flasks and incubated at 28 1C for
7 days with intermittent shaking at 125 rpm twice a day.
The culture filtrate each isolate was harvested, filtered
through the Whatman Filter Paper 42, centrifuged and
assayed for chitinase and b-1,3-glucanase enzyme activities immediately.

Chitinase Activity
A mixture of 0.5 ml culture filtrate, 0.5 ml suspension of
colloidal chitin and 1.0 ml of Mcllvaines buffer (pH 4.0)
was mixed and incubated at 37C for 2 h in a water bath
with constant shaking. The reaction was stopped by boiling
3 min in heated water bath. 3 ml potassium ferricyanide
reagent was added and warmed in boiling water for 15 min.
The amount of N-acetyl glucosamine (NAG) released was
estimated following the methods of Reissing et al. [14].
The absorbance of reaction mixture was measured in a
spectrophotometer at 420 nm. The amount of reducing
sugar released was calculated from standard curves for
NAG and chitinase enzyme activity was expressed in pkat
(pmol/s) per millitre.
Statistical Analysis
Statistical analysis were performed with the Agres and
Agdata using completely randomized analyses of variances
(ANOVA) was used to compare the biocontrol efficacy of
Trichoderma isolates and means separated by Fishers
protected least significant difference (LSD). The significance of effects of Trichoderma on growth characteristics
was determined by the magnitude of the F value
(P = 0.05).
Results and Discussion
Morphological Characterization
Based on the observation of the conidia, phialides, colony
texture, chlamydospore, conidiophore morphology the
isolates were grouped into section Pachybasium B (clade
Lixii/catoptron), Trichoderma (Rufa), Trichoderma

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(Pachybasium A), Longibrachiatum and Pachybasium B

(Lutea). Section Pachybasium consists of the isolates
TSD1, TWN1, TGN1, TJP1 and TWC1 where the condiophores were much branched, which arise in distinct and
continues ring like zones. The main branches mostly in
groups of 23 and stand at right angle to the bearer and
their length increased with the distance from the tip of the
main branch which gave a conical or pyramidal appearance. In Trichoderma section (Rufa) (TWD1, TGD1 and
TCC1) the hyphae, branched, smooth walled and colourless. The condiophores were observed as less complicated
and formed aerial hyphae. They produced smaller branches
and ultimately a conifer-like branching system is formed.
In Pachybasium B (Lutea) (TBC1) the conidiophores are
smooth walled, verticiliately branched in the Pachybasium
type pattern. Trichoderma (section of Pachybasium A)
consists of T. asperellum seen the conidiophores have
terminating 2 or more philides and primary branching
arising at nearly 90 degrees to the main axis. In section
Longibrachiatum (H. orientalis), the conidiophores were
complicated and progressively longer, often paired, secondary branches. Phialides arising directly from secondary
branches, typically not in whorls. Using the key of Rifai
[15] in the earlier most of the strains isolated in these
regions were identified as T. harzianum. T. hamatum and T.
viride [16]. In fact, apart from T. harzianum, which, nevertheless accounted for the majority of isolates, a very
interesting finding of this study was the coexistence of
several species of Trichoderma (e.g. T. harzianum, T. erinaceum, T. asperellum, T. ovalisporum, T. viride and
T. brevicompactum) in the Bay Island ecosystem.
Molecular Identification of Trichoderma Species
Sequence analysis of twelve isolates was done to confirm
species identity, which initially has been done based solely
on morphological parameters. Comparison of oligonucleotide fragments of rDNA sequences, which included the
5.8S gene and the flanking ITS1 and ITS2 regions, with
reference sequences from public databases, showed that
they were very similar.
Phylogenetic Analysis
The phylogenetic tree obtained by sequence analysis of ITS1
and ITS4 of 12 of our isolate sequences isolates and the
sequence of 22 Trichoderma spp. obtained from NCBI,
GenBank is represented in Fig. 1. The ITS sequence was
chosen for this analysis because it has been showed to be
more informative with various sections of the genus Trichoderma [1719]. Bootstrap analysis with 100 bootstrap
replications demonstrated three major branches. On the
basis of the bootstrap values, 34 Trichoderma spp. could be

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Indian J Microbiol (AprJune 2012) 52(2):137144

divided into 5 groups. Group 1, the T. harzianum complex

includes 6 of our isolates. This group is supported by bootstrap value of 92% contains 3 subgroups supported by
bootstrap values more than 90%. Group II (Lutea) includes 3
strains representing T. brevicompactum. This cluster is
supported by a bootstrap value of 83%. Group IV (Rufa)
includes 3 different isolates includes T. viride, T. ovalisporum and T. erinaceum, this cluster is supported by more than
60% of bootstrap values. In the case of group III cluster
which, consists of section Longibrachiatum (H. orientalis)
and Pachybasium A (T. asperellum). In this analysis no
grouping among the isolates was observed, and did not show
any bootstrap supports suggests instability of the clade.
Antagonistic Activity and Enzyme Production
of Trichoderma Isolates
Results from the dual culture test showed that all isolates of
Trichoderma inhibited mycelial growth of S. rolfsii more
than 50%. Two isolates TGD1 and TWN1 (T. viride and T.
harzianum) showed statistically significant inhibition of
mycelial growth (76.3%) over control against S. rolfsii
(Table 2). The isolates TND1, TBC1, TCC1, TGN1 and
TWC2 (T. erinaceum, T. brevicompactum, T. ovalisopum,
H. lixii and T. harzianum) were next best isolates in
inhibiting the growth of pathogen i.e., 73.3, 70.8, 72.5, 70.8
and 72.1 per cent over the control, respectively. Isolates
TSD1 (T. inhamatum), TJP1 (H. lixii) and TND1 (T. erinaceum) showed statistically significant inhibition of
mycelial growth of C. gloeosporioides in dual culture
(Table 2). The growth inhibitions of these isolates were
45.6, 43.3 and 42.2% respectively over the control. Similarly in the case of C. capsici, isolates TWC2 (T. harzianum) and TSD1 (T. inhamatum) showed significant
inhibition of mycelial growth.
The production of chitinase and b-1,3-glucanase are
summarized in Tables 3 and 4. All isolates showed significantly higher chitinase and b-1,3-glucanase activities
with supplement of different carbon sources as substrates in
the basal media. The highest chitinase enzyme activity was
recorded with T. viride (42.0 U) in CDB ? mycelial
powder of S. rolfsii, followed by T. harzianum (40.3 U), T.
erinaceum (37.1 U), H. lixii (36.4 U), T. brevicompactum
(35.1 U), T. ovalisporum (34.2 U) and Trichoderma sp.
(33.2 U). Chitinase activity of test isolates of Trichoderma
was relatively less when the basal medium was added with
mycelial powder of C. gloeosporioides and C. capsici as
compare to S. rolfsii whereas highest chitinase activity was
recorded with T. harzianum. The partial substitution of
sucrose by same carbon sources in the basal medium has
profound effect as the b-1,3-glucanase activity as compared to basal media (CDB). Highest b-1,3-glucanase
enzyme activity was recorded with T. harzianum (22.6,

Indian J Microbiol (AprJune 2012) 52(2):137144

141

Fig. 1 Phylogram based on the

ITS region of the genomic
rRNA gene of 12 isolates and 22
representative strains of
Trichoderma with isolate
number indicated in
parenthesis. The phylogram was
generated after DNA distance
based and neighbor-joining
analysis of the data. Bootstrap
50% majority-rule consensus
tree (1,000 replications),
indicated above the nodes, was
generated using MEGA 3.1

H. lixii (T18-1) EU744189

Hypocreaceae sp. (LM230) EF060566
H. lixii (ATCC 20847) FJ545255
H. lixii (FJA6) FJ998176
Hypocrea sp. (CCN11) DQ993643
H. lixii (DAOM 167671) AY605740
T. harzianum (GJS 99-225) AY027781
H. lixii (PPRC-ET39) FJ461572
Lixii

Hypocrea sp. (HKB12) EF029811

H. lixii (GJS 05-98) FJ442274
H. lixii (SWFC8952) GQ203555
H. lixii (TGN1)
Hypocrea sp (TWP1)

92

H. lixii (TJP1)
T. harzianum (TWC2)
91 T. harzianum (TWN1)

99

T. inhamatum (TSD1)
H. lixii (UNISS 13b-13) EF392739
T. brevicompactum (TBC1)
Lutea

T. brevicompactum (D6) FJ610290

83

91 T. brevicompactum (C8) FJ610288

H. orientalis (TMC1)
T. asperellum (TWD1)
T. asperellum (T4) GQ265954

Longibrachiatum & Pachybasium A

H. orientalis (ICMP 1694) EU401549

T. asperellum (B51) GQ131393
T. asperellum (CPK2722) FJ412053
T. viride DQ888741
T. erinaceum (DAOM 231241) EU280130
84

T. erinaceum (DAOM 237546) EU280134

T. ovalisporum (GJS 06-31) FJ442652

Rufa

T. ovalisporum (TCC1)
60

T. erinaceum (TND1)
T. viride (TGD1)

0.02

31.5, 28.6 and 30.2 U) in CDB, CDB ? S. rolsii,

CDB ? C. gloeosporioides and CDB ? C. capsici followed by T. viride, T. erinaceum and T. inhamatum.
Among all carbon sources as amendment of media,
mycelial powder of S. rolsii induced higher b-1,3-glucanase and chitinase activity than C. gloeosporioides and C.
capsici.
The application of Trichoderma as biocontrol agent
has been focused mainly in controlling root and soilborne diseases while there are few reports on their
application in controlling foliar diseases [20]. This stems
from the fact that Trichoderma sp. are native (residents)
in the soil but transients in the phylloplane [21]. The

antagonism by Trichoderma spp. against many soils

borne plant pathogen has been established [6, 22, 23].
Strong antagonism by Trichoderma spp. against a range
of soil borne plant pathogens has been reported [6, 23].
It is well known that Trichoderma spp. have the potential
to produce cell wall degrading enzymes by using the
materials that are present in the growth medium [6].
Production of hydrolytic enzymes such as b-1,3-glucanase, chitinase, cellulase and proteinase increased significantly when Trichoderma spp. were grown in media
supplemented with either autoclaved mycelium or purified host fungal cell walls [7, 24]. Ulhoa and Peberdy
[25] found that products of chitin degradation also

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Table 2 Antagonistic potential
of Trichoderma isolates against
S. rolfsii, C. gloeosporioides
and C. capsici

Indian J Microbiol (AprJune 2012) 52(2):137144

S. No.

1
2
3

Means of four replications;

CDB Czapek Dox Broth, S
substrate, SR S. rolfsii, CG C.
gloeosporiodes, CC C. capsici

T. longibrachiatum
T. brevicompactum

C. capsici

69.6bc

45.6a

48.9ab

cd

67.1
70.8

ab
abc

T. viride

76.3a

T. erinaceum
T. harzianum
T. asperellum

35.6

36.7e
43.3

27.8
30.0

70.8

ab

69.6

bc
d

28.9
34.4

62.1

10

T. harzianum

76.3a

cd

ab

42.2
35.6

cd

cd

46.7bc
42.2cd
46.7bc

bc

38.9de

42.2de

40.0

cd

27.8
43.3ab

50.0a
46.7bc

Control (mm)

80

30

30

CD (0.05)

0.4387

0.3025

0.1946

SEd

0.2126

0.1466

0.0943

T. harzianum
T. harzianum

45.6bc

72.1
56.3e

11
12

ab

42.2de

38.9

73.3

Trichoderma sp.

cd

bc

ab

Isolate

CDB

CDB ? SR

CDB ? CG

CDB ? CC

T. inhamatum

22.3

32.5

28.4

30.1

T. longibrachiatum

19.2

30.2

25.3

28.1

T. brevicompactum

23.9

35.1

26.4

23.5

T. ovalisporum

22.7

34.2

22.3

19.6

T. viride

28.3

42.0

24.5

27.8

T. erinaceum

23.5

37.1

28.0

28.6

T. asperellum

21.0

22.0

28.0

26.5

T. harzianum

25.1

36.4

26.4

24.8

Trichoderma sp.

20.6

33.2

19.6

21.3

T. harzianum

32.2

40.3

30.2

32.6

T. harzianum

30.5

38.6

29.4

31.2

T. harzianum

20.0

29.6

22.7

20.5

SEd

0.403

0.321

0.369

0.426

CD (0.05)

0.833

0.878

0.763

0.878

regulate the chitinase synthesis in T. harzianum. Kumar

and Gupta [2] reported that cell wall of M. phaseolina
and S. rolfsii is known to have glucan and chitin that
should have resulted in the induction of glucanase and
chitinase in mycelial mat amended media. High b-1,3glucanase and chitinase activities were detected in dual
culture when T. harzianum parasitized R. solani and
S. rolfsii compared with low levels of substrates or
in absence of pathogen [26]. In present investigation, it
was found that chitinase and b-1,3-glucanase enzyme
activities was increased with the substitution of specific
carbon source at 1% concentration. These findings is in

123

C. gloeosporioides

72.5

Table 3 Chitinase enzyme

activity of Trichoderma isolates
from Andaman and Nicobar
Islands (pkat/ml)

T. inhamatum

S. rolfsii

T. ovalisporum

* Values are mean of three

replications

Mycelial growth inhibition* (%)

4
6

Data were analyzed using oneway analysis of variance (values

with different lower case letters
are significantly different; P B
0.05%)

Isolate name

accordance with Ulhoa and Peberdy [25] where they

suggested that chitinase activity was substrates concentration dependent above 0.5% (w/v) chitin there was no
further synthesis of chitinase in the growth medium by
T. harzianum was increased up to 1% concentration,
whereas b-1,3-glucanase enzyme production was increase
up to 1% concentration of laminarin but decreased at
higher concentrations. This may be due to the fact that at
higher concentration of sugar activity of this enzyme was
inhibited. The degree of mycelial inhibition of target
pathogen by T. erinaceum is notable example as biocontrol against pathogens tested.

Indian J Microbiol (AprJune 2012) 52(2):137144

Table 4 b-1,3-Glucanase
enzyme activity of Trichoderma
isolates from Andaman and
Nicobar Islands (nkat/ml)

Means of four replications;

CDB Czapek Dox Broth, SR S.
rolfsii, CG C. gloeosporiodes,
CC C. capsici

143

Isolate

CDB

CDB ? SR

CDB ? CG

CDB ? CC

T. inhamatum

15.2

22.3

24.9

22.5

T. longibrachiatum

17.4

23.5

19.4

21.0

T. brevicompactum

14.8

15.0

17.5

18.6

T. ovalisporum

9.5

18.1

21.2

19.6

T. viride

20.5

24.2

25.5

28.3

T. erinaceum

11.3

29.8

22.3

24.0

T. asperellum

11.5

12.8

16.5

20.0

T. harzianum

13.4

19.3

24.3

25.0

Trichoderma sp.

11.2

16.4

17.6

15.3

T. harzianum

22.6

31.5

28.6

30.2

T. harzianum

19.4

28.1

26.8

27.7

T. harzianum

9.3

17.5

18.7

21.0

SEd

0.372

0.320

0.408

0.406

CD (0.05)

0.768

0.654

0.843

0.837

Acknowledgments This research was supported by grants from the

project Application of Microorganisms in Agriculture and Allied
Sectors of the NBAIM-ICAR, UP, Mau, India. We thank the Head
Division of Field Crops, CARI for constant support and encouragements during the course of study.

12.

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