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Advances in Environmental Biology, 5(9): 2631-2638, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Antagonistic Ability Against Rhizoctonia Solani And Pesticide Tolerance Of
Trichoderma Strains
Sarojini Chakravarthy K. , Nagamani A., Rahel Ratnakumari Y. and Bramarambha S.
Department of Botany, Post Graduate College of Science, Saifabad, Osmania University, Hyderabad-500 004,
India.
Sarojini Chakravarthy K. , Nagamani A., Rahel Ratnakumari Y. and Bramarambha S.: Antagonistic
Ability Against Rhizoctonia Solani And Pesticide Tolerance Of Trichoderma Strains.
ABSTRACT
Twenty six isolates of Trichoderma spp. were tested in vitro for their tolerance against pesticides and on the
antagonistic activity against sheath blight pathogen of rice, Rhizoctonia solani. The growth of all Trichoderma
isolates was inhibited by three fungicides tested. Organophosphorus insecticide (monocrotophos) had less
inhibitory effect on Trichoderma spp. than chlorinated insecticide (Endosulfan). The botanical pesticide
(Azadirachtin) showed least inhibitory effect on the Trichoderma isolates growth. The pyrethroid insecticide (λcyhalothrin) significantly enhanced the growth of all Trichoderma isolates. Among all the isolates, T. reesei
(TK) and T. longibrachiatum (M1Ps) were more effective in inhibiting the growth and sclerotial formation of R.
solani.
Key words: Trichoderma, Rhizoctonia solani, fungicides, insecticides.
Introduction
The use of fungicides is the best known and
popular strategy method in the Integrated Disease
Management (IDM) for the management of plant
diseases. However, inspite of their efficient means of
controlling the plant pathogens and the cost effective
nature, the environment and sociological concerns
led to the idea of minimizing fungicide usage which
coupled with the appearance of new resistant strains
of pathogens [12]. Other pesticides also affect the
microbial community in natural ecosystem as they
build up in the soil [26]. Thus, a reduction or
elimination of chemical pesticide applications in
agriculture is highly desirable and is only possible
with the adoption of integrated disease management.
One of the components of integrated management of
plant pathogens is biocontrol strategy. One of the
most promising biological control means is the
application of Trichoderma as a biocontrol agent
alone or integrated with minimal fungicide dosage to
control plant pathogens [9,19,31]. In order to use
both reduced dosage of fungicide and Trichoderma
spp. as biocontrol agents (BCA’s) as components of
IDM, it is essential to use Trichoderma isolates
tolerant to pesticides [34,17,23].
Trichoderma strains with greater biocontrol
efficiency should also have better tolerance against
pesticides for be used as component in Integrated
Pest management (IPM) [18]. Insecticides and
fungicides are commonly used by farmers to control
insects, pests and diseases respectively in rice [24].
Hence, the knowledge on tolerance of effective
antagonistic isolates of Trichoderma against
pesticides commonly used is essential for their
practical utilization.
Hence, the present studies were conducted to study in
vitro, the efficiency of collected strains of
Trichoderma in inhibiting the sheath blight pathogen
of rice, the Rhizoctonia solani Kühn and to assess the
effect of pesticides on the mycelial growth of
Trichoderma isolates.
Material and Methods
Isolation of Trichoderma:
Trichoderma species were isolated from soil
samples collected from rice fields in two districts
(Nalgonda and Ranga Reddy districts) of Andhra
Pradesh, India and forest soil samples of Uttaranchal
(Chamoli district), India. All the isolates were
identified up to species level based on phenotypic
characters like colony colour and growth; size and
shape of conidiophore, phialides and conidia. The
cultures were identified using the available literature
[5,6,7,25,28,29].
Screening Of Trichoderma Isolates Against R. Solani
By Dual Culture Technique:
Corresponding Author
Sarojini Chakravarthy K, Department of Botany, Post Graduate College of Science, Saifabad,
Osmania University, Hyderabad-500 004, India.
E-mail: [email protected]; Tel: +91 9849 370624
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Adv. Environ. Biol., 5(9): 2631-2638, 2011
The biocontrol ability of 26 isolates of
Trichoderma spp., isolated was tested against the
pathogen Rhizoctonia solani, using dual culture
technique [10]. The pathogen Rhizoctonia solani
(AG1) was procured from Directorate of Rice
Research, Hyderabad, India. The mycelial discs (5
mm diameter) of Trichoderma isolates and R. solani
from a four-day-old culture on 2% water agar were
placed on PDA plates on one side, 1 cm away from
the edge of the plate. The disc of the R. solani was
placed on the opposite side 6 cm apart. The plates
were incubated at 24 ± 2 C for one week. The radial
growth of the pathogen as well as antagonist was
observed at regular intervals. The inhibition of
sclerotial formation of the pathogen was also noted.
The percentage inhibition of growth of pathogen in
the presence of biocontrol agent was calculated over
control. The average of three replications for each
treatment was calculated and presented in the data in
all experiments of the study.
Evaluation Of Pesticides On The Mycelial Growth Of
Trichoderma Isolates:
In this study, fungicides and insecticides,
commonly used by farmers were selected to assess
their effect on the growth of the Trichoderma
isolates.
The
seven
pesticides
Contaf
(Hexaconazole), Bavistin (Carbendazim), Sivic
(Tricyclazole), Endosol (Endosulfan), Achook
(Azadirachtin), Monodhan (Monocrotophos) and
Reeva (λ-cyhalothrin) were tested against the
mycelial growth of Trichoderma strains using
poisoned food technique [30]. All the fungicides and
insecticides were added to the Potato Dextrose Agar
(PDA) according to the recommended doses that
were used by the farmers. The concentrations of
pesticides added to the culture medium were: Contaf
– 2 ml/l; Bavistin – 1 g/l; Sivic – 0.6 g/l; Endosol – 2
ml/l; Achook – 5 ml/l; Monodhan – 1 ml/l and Reeva
- 0.2 ml/l.
Petri plates with PDA along with pesticides were
inoculated with Trichoderma isolates and incubated
at 27±2 C. Radial growth of the colony was
measured in millimeters on 3rd, 5th, and 7th days of
incubation. Petri plates without pesticides served as
control. Three replications were maintained and the
experiment was repeated twice. Average of the data
was presented.
Statistical Analysis:
All the data was subjected to analysis of
variance (ANOVA) and significance of variance was
presented at 5% levels using IRRISTAT windows
ver. 4.1.
Results and Discussions
Isolation of Trichoderma:
A total of twenty six isolates of Trichoderma
were isolated in this study. Eighteen isolates were
from rice cultivated soils and eight from forest soil
samples. The isolates from rice cultivated soils
belong to T. asperellum (TS, M3M & M4Ps), T.
harzianum (TH1, TH2, M10P), T. reesei (TK), T.
longibrachiatum (TL2, TL3, M1Ps, M3P, M3Ps,
M5Ms, M6Ms, M8Ps, M9Ms), and T. viride (TD2,
M1M). Isolates from forest soils were identified as T.
aureoviride (TA1, TA2, TA3, TA4), T.
longibrachiatum (TL1), T. virens (TV1, TV2) and T.
viride (TD1).
Screening Of Trichoderma Isolates Against R. Solani
By Dual Culture Technique:
All the tested isolates of Trichoderma in dual
culture significantly inhibited the mycelial growth of
Rhizoctonia solani and the inhibition ranged from
62-78% (Table 1). Variation was observed among the
species of Trichoderma, in their ability to inhibit
radial growth and sclerotia formation by the
pathogen. T. harzianum (M10P), T. reesei (TK) and
T. aureoviride (TA1) were more aggressive in
inhibiting the growth of the pathogen compared to
other isolates and they caused a maximum
percentage inhibition of 77.7%. The isolates T.
harzianum (M10P) and T. reesei (TK) were obtained
from rice cultivated soils of Nalgonda district
whereas T. aureoviride (TA1) was from forest soil
samples. T. aureoviride (TA4), T. longibrachiatum
(TL1, M1Ps), and T. virens (TV2) were found to be
less effective in inhibiting the pathogen growth. The
complete inhibition of the sclerotial formation by the
pathogen in the test plates was found with T. reesei
(TK)
and
T.
longibrachiatum
(M1Ps).
Effect Of Fungicides On Trichoderma Growth:
Effect Of Insecticides On Trichoderma Growth:
The three tested fungicides (hexaconazole,
carbendazim, tricyclazole) completely inhibited the
growth of all Trichoderma isolates in vitro. However,
a few isolates belonging to T. aureoviride (TA1, TA2,
TA3), T. longibrachiatum (M1Ps), T. virens (TV1)
and T. viride (TD1, M1M) showed slight growth
initiation after 10 days, but the growth was very
slow.
A significant variation was observed on the
effect of insecticides on the growth of Trichoderma
isolates. The variation was significant between the
species and even between the different isolates of
same species. However, Trichoderma isolates
collected from two sources did not differ in their
growth response against pesticides.
Azadirachtin,
monocrotophos,
endosulfan
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Adv. Environ. Biol., 5(9): 2631-2638, 2011
significantly reduced the growth of all the isolates of
Trichoderma (Table 2, 3). Among insecticides tested
endosulfan had greater inhibitory effect on
Trichoderma isolates growth. T. harzianum (TH2)
and two isolates of T. longibrachiatum (M3Ps,
M8Ps) growth was relatively less affected by
endosulfan. The inhibition on Trichoderma isolates
growth due to endosulfan ranged from 11-80%.
Highly significant variation in growth inhibition was
observed among different isolates of T.
longibrachiatum and T. harzianum. There was a
visible morphological distortion and uneven growth
pattern in the agar plates treated with insecticides
(Fig 1). Greater inhibitory effect of monocrotophos
was observed with the isolates of T. harzianum
(TH2), T. longibrachiatum (M3Ps) and T. asperellum
(M4Ps).
Table 1. Effect Of Trichoderma On The Growth Of Rhizoctonia Solani Through Dual Plate Culture Technique
Trichoderma species
, 2,
Isolate
% inhibition of R. solani growth
Overgrowth of Trichoderma
Sclerotial formation
T. asperellum1
TS
70
0
10
T. asperellum1
M3M
71.1
14
8
T. asperellum1
M4Ps
72.2
15
6
2
T. aureoviride
TA1
77.7
0
5
T. aureoviride2
TA2
71.1
0
7
T. aureoviride2
TA3
72.2
0
6
T. aureoviride2
TA4
62.2
0
11
T. harzianum1
TH1
71.1
0
6
T. harzianum1
TH2
72.2
0
10
T. harzianum1
M10P
77.7
20
15
T. reesei1
TK
77.7
25
0
T. longibrachiatum2
TL1
63.3
0
6
T. longibrachiatum1
TL2
68.8
0
8
T. longibrachiatum1
TL3
65.5
0
4
T. longibrachiatum1
M1Ps
63.3
33
0
T. longibrachiatum1
M3P
75.5
25
6
T. longibrachiatum1
M3Ps
73.3
20
15
T. longibrachiatum1
M5Ms
75.5
27
12
T. longibrachiatum1
M6Ms
76.6
24
9
T. longibrachiatum1
M8Ps
68.8
20
4
T. longibrachiatum1
M9Ms
74.4
20
8
T. virens2
TV1
67.7
0
8
T. virens2
TV2
62.2
0
13
T. viride2
TD1
73.3
0
12
T. viride1
TD2
68.8
0
9
T. viride1
M1M
76.6
22
4
Control
-
0
0
40
CD (P ≤ 0.05)
-
-
-
-
CV (%)
-
-
-
-
denotes soil used for Trichoderma spp. isolation as 1=Rice cultivated soils and 2=Forest soils respectively.
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Table 2. Effect Of Insecticides At 3rd Day On The Growth (mm) Of Trichoderma Isolates
Insecticides
Trichoderma
Trichoderma species
Isolate
Azadirachtin
Monocrotophos
Endosulfan
Control
T. asperellum
TS
54
45
27
78
T. asperellum
M3M
51
46
25
82
T. asperellum
M4Ps
50
15
19
47
T. aureoviride
TA1
55
35
30
89
T. aureoviride
TA2
52
35
32
89
T. aureoviride
TA3
59
39
35
83
T. aureoviride
TA4
57
35
15
74
T. harzianum
TH1
55
42
35
89
T. harzianum
TH2
44
30
31
89
T. harzianum
M10P
59
32
23
89
T. reesei
TK
88
81
39
89
T. longibrachiatum
TL1
53
33
17
74
T. longibrachiatum
TL2
52
38
26
84
T. longibrachiatum
TL3
64
45
41
89
T. longibrachiatum
M1Ps
41
23
21
49
T. longibrachiatum
M3P
55
37
37
71
T. longibrachiatum
M3Ps
54
21
25
89
T. longibrachiatum
M5Ms
42
20
16
60
T. longibrachiatum
M6Ms
41
34
17
77
T. longibrachiatum
M8Ps
66
34
70
89
T. longibrachiatum
M9Ms
41
26
14
74
T. virens
TV1
63
42
23
79
T. virens
TV2
63
42
29
75
T. viride
TD1
56
45
21
85
T. viride
TD2
54
35
26
85
T. viride
M1M
59
56
35
70
Table 3. Effect of insecticides at 5th and 7th days on the growth (mm) of Trichoderma isolates
Trichoderma
Insecticides
Trichoderma species
Isolate*
Azadirachtin
5th day
7th day
Monocrotophos
5th day
7th day
Endosulfan
5th day
7th day
T. asperellum
TS
66
83
70
88
58
75
T. asperellum
M3M
62
80
72
84
56
78
T. asperellum
M4Ps
44
59
21
29
69
90
T. aureoviride
TA1
66
77
57
71
69
87
T. aureoviride
TA2
57
71
57
73
71
85
T. aureoviride
TA3
73
90
53
72
68
90
T. aureoviride
TA4
69
78
36
74
74
90
T. harzianum
TH1
65
79
61
74
54
75
T. harzianum
TH2
55
73
47
70
61
88
T. harzianum
M10P
70
88
44
60
65
87
T. reesei
TK
90
90
90
90
62
85
T. longibrachiatum
TL1
62
74
49
63
57
86
T. longibrachiatum
TL2
63
80
57
75
48
74
T. longibrachiatum
TL3
75
90
60
81
90
90
T. longibrachiatum
M1Ps**
51
67
34
39
37
57
T. longibrachiatum
M3P
69
78
56
74
90
90
T. longibrachiatum
M3Ps
67
90
37
51
53
90
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Adv. Environ. Biol., 5(9): 2631-2638, 2011
T. longibrachiatum
M5Ms
50
65
25
45
28
75
T. longibrachiatum
M6Ms
51
64
53
66
36
57
T. longibrachiatum
M8Ps
78
84
49
70
90
90
T. longibrachiatum
M9Ms
46
57
35
46
25
36
T. virens
TV1
45
90
63
84
62
90
T. virens
TV2
74
82
63
77
60
83
T. viride
TD1
72
83
53
72
53
90
T. viride
TD2
65
77
52
70
44
65
T. viride
CD (P≤ 0.05)
CV (%)
M1M
72
1.92
1.79
84
2.50
1.94
78
2.00
2.27
85
2.52
2.22
89
2.17
2.21
90
2.22
1.68
* All isolates in control plates reached maximum growth of 90 mm in 4 or 5 days except M1Ps.
** In control M1Ps isolate growth 5th day = 58 mm and 7th day = 74 mm.
Fig 1. Effect of endosulfan on the growth of T. reesei (TK) and T. longibrachiatum (M1Ps)
Right plate: Test plate, Left Plate: Control plate
Azadirachtin had least inhibitory growth effect
on all the isolates. Least growth reduction (1.1%) of
T. reesei (TK) was recorded with Azadirachtin. The
growth inhibition of all other isolates was more than
25% and maximum inhibition was noted in T.
longibrachiatum (M9Ms) as well as T. harzianum
(TH1, TH2). In contrast to inhibitory effect of other
insecticides, λ-cyhalothrin treatment resulted in the
enhanced growth in all the isolates (Fig 2). The
percentage increase in growth of Trichoderma
isolates ranged from 1.5-63%. The percentage
increase of growth was highest in T. asperellum
(M4Ps)
and
T.
longibrachiatum
(M1Ps).
Fig. 2. Effect of λ-Cyhalothrin on the growth of Trichoderma isolates
Discussion:
All twenty six isolates of Trichoderma inhibited
the growth of the pathogen, Rhizoctonia solani
confirming the observations of [20] who stated that
several isolates of Trichoderma were highly
antagonistic to this pathogen. The pathogen growth
inhibitory ability of different Trichoderma strains of
same species and among different species varied
significantly. [13] proved that different strains have
very different biocontrol capabilities. In dual culture
greater numbers of sclerotia were observed in
association with isolates of T. viride from forest soils
than the isolates from rice cultivated soils. [14]
emphasized that Trichoderma species isolated from a
given habitat are expected to cause better disease
control in that habitats. In this study also, the
Trichoderma isolates from rice cultivated soils
showed greater antagonistic effect on Rhizoctonia
solani than the isolates from the virgin soils i.e.,
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Adv. Environ. Biol., 5(9): 2631-2638, 2011
forest soils, where the rice was never cultivated.
The
commercially
available
fungicides
Carbendazim (benzimidazole carbamate), Contaf
(hexaconazole), Sivic (tricyclazole) are known to
control various plant fungal pathogens [2]. The
fungitoxic effects of fungicide resulted in inhibition
of Trichoderma isolates tested in this investigation.
[27] observed that carbendazim, propiconazole,
chlorothalonil and hexaconazole were retarding the
fungal growth totally. [4] opined that soil fungi and
actinomycetes are not as susceptible to herbicides
and insecticides as they are to fungicides. In this
study also, Trichoderma spp. showed greater
inhibitory effect was observed with fungicides than
with herbicides and insecticides. [11] reported the
inhibitory growth of the T. asperellum with
propamocarb, carbendazim, and diethofencarb in
vitro while in vivo it was opposite.
Among insecticides tested endosulfan (chlorine
compound) had greater growth inhibitory effect on
all Trichoderma isolates than monocrotophos
(organophosphorus compound). [1,22,36] reported
that
chlorinated
hydrocarbon
insecticides
(endosulfan) are more deleterious than other
insecticide groups like organophosphates to the
mycopathogen and moreover they were highly
detrimental to the fungus. Significant variation
showed in response was observed among different
Trichoderma species and strains. The pyrethroid
insecticide, cyhalothrin caused an increased soil
fungal population probably due to stimulation of
mineralization
processes,
respiration
and
oxidoreductase processes in the soil. This might be
due to different metabolic processes that enable
utilization of the insecticides as nutrients by different
isolates of Trichoderma[36]. In this study, certain
isolates of T. harzianum and T. longibrachiatum
tolerant against monocrotophos and endosulfan
(Table 1) and they may be used as components in
integrated pest management.
Azadirachtin had a least effect on the growth of
Trichoderma isolates when compared with the other
insecticides. [15,16] reported that reduced sheath
blight incidence with Azadirachtin. Similarly,
[35]have found that neem leaf extract reduced the
mycelial growth of yam rotting fungus, Alternaria
solani. Azadirachtin resistant isolates of Trichoderma
identified in this study may be used in Integrated Pest
Management (IPM). Stimulatory effect of
azadirachtin on the growth of Trichoderma isolates
can be attributed to stimulate the mineralization and
the availability of C, N and P in soil [32]. Thus,
Trichoderma isolates not affected by azadirachtin
might have a synergistic effect in controlling plant
disease when both are used together at the same time
and may serve as low monitory input in integrated
sheath blight management of rice. Hence, the isolates
of Trichoderma viz., T. reesei (TK), and T.
longibrachiatum (M1Ps) which were least affected
by azadirachtin may be applied along with
azadirachtin to manage both sheath blight and other
pests of rice, as these two isolates proved to be
highly antagonistic to R. solani in in vitro studies
with maximum inhibitory effect on growth and
sclerotial formation.
λ-cyhalothrin was enhancing the growth of
Trichoderma isolates in this study. However,
Schuster and Schroder, 1990 reported that it has
inhibitory effect. Cyhalothrin is having negative
effect on the pathogen growth and positive effect on
bioagents in some cases [8]. Thus, cyhalothrin is a
better insecticide to use in IPM than any other tested
insecticides and it can be recommended to use along
with Trichoderma bioagents in IDM.
Conclusions:
Isolates of T. longibrachiatum (M1Ps) and T.
reesei (TK) isolated from rice cultivated soils showed
maximum overgrowth on the sheath blight pathogen
R. solani than Trichoderma isolates from forest soils.
The complete inhibition of sclerotial formation was
observed by these isolates due to maximum
overgrowth on the pathogen in dual culture.
Trichoderma strains identified in this study may be
used for biocontrol of sheath blight after their further
screening under field condition. Among pesticides,
fungicides were highly inhibitory and toxic to all the
isolates of Trichoderma followed by insecticides and
botanical insecticide. Trichoderma aureoviride, T.
longibrachiatum, T. virens, and T. viride strains
growth initiation 10 days, after fungicide application
indicates that they may be applied 10 days after
fungicide application in order to use them in soils for
biocontrol. The pyrethroid insecticide, cyhalothrin,
significantly enhanced the growth of all Trichoderma
isolates suggesting the possibility of integrated use of
pyrethroids with Trichoderma in the disease
management.
Acknowledgements
Authors are grateful to University Grants
Commission (UGC), New Delhi, India for providing
financial assistance and the Principal, Post Graduate
college of Science, Osmania University, Saifabad,
Hyderabad, India for providing laboratory facilities.
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