International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 4, Issue 6, Dec 2014, 127-138
TJPRC Pvt. Ltd.

A CARBENDAZIM TOLERANT MUTANT OF TRICHODERMA HARZIANUM SHOWED

HIGHER BIOCONTROL ACTIVITY
PRANAB DUTTA, HIMADRI KAUSHIK & B. C. DAS
Department of Plant Pathology Assam Agricultural University, Jorhat, Assam, India

ABSTRACT
We hypothesized to develop a new biotype of T. harzianum having higher tolerance to carbendazim, with
enhanced survival ability and increased biocontrol activity against S. sclerotiorum. To test this we induced T. harzianum by
exposing the culture to UV radiation and trained it grow in different concentration of carbendazim. We studied the
biocontrol ability of mutant T. harzianum against S. sclerotiorum, both in vitro and in vivo. We found mutants of
T. harzianum were resistant to higher concentrations of carbendazim than non mutants and continued to grow even up to
0.2%. Mutant of T. harzianum in combination with carbendazim (0.05%) was highly effective for the management of the
disease and increasing the yield. Mutants capable of tolerating wider dosage of the fungicide would ideally have a better
chance of survival than those with low tolerance. Therefore, induction of mutants in T. harzianum paves the way to
develop strains with enhanced survival in the rhizosphere and enhanced capacity to control soil borne diseases under field
condition.

KEYWORDS: Carbendazim, Management, Mutant, Sclerotinia sclerotionum, Trichoderma harzianum, White Mold of
French Bean

INTRODUCTION
White mold of french bean caused by Sclerotinia sclerotiorum (Lib) de Bary is a devastating disease causes yield
loss upto 100 per cent. Chemical methods of disease management particularly carbendazim are available but, due to the
growing concern of environmental hazards people are concentrating on management by biological control. In a preliminary
study Trichoderma harzianum was found to be best in inhibiting the growth of S. sclerotiorum. T. harzianum is a potential
biological control agent of many organism causing plant diseases including S. sclerotiorum. Unfortunately, T. harzianum is
highly sensitive to the agrochemicals more particularly carbendazim fungicides (Papavizas, 1982). Carbendazim is around
in the environment because of its indiscriminate use to curb number plant diseases of other host crops. This problem can be
overcome by developing a biotype with having tolerance to carbendazim. If we had such a tolerant biotype this would open
up the possibility to use as an important component of integrated disease management in which both chemical and
biological agents can be used. Induction of mutants of antagonist is one of the most widely used tools to improve
antagonistic fungi for biological control properties like fungicide tolerance, survival ability, antagonistic and biological
control potential and ability to colonize plant parts.
Other species of Trichoderma have also been used to induce mutants to enhance biocontrol ability. Therefore
T. harzianum is a likely candidate for inducing a mutant strain. We hypothesized to develop a new biotype of T. harzianum
having higher tolerance to carbendazim, with enhanced survival ability and increased biocontrol activity against
S. sclerotiorum. To test this we induced T. harzianum by exposing the culture to UV radiation and trained it grow in
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Pranab Dutta, Himadri Kaushik & B. C. Das

different concentration of carbendazim. We studied the biocontrol ability of mutant T. harzianum against S. sclerotiorum,
both in laboratory and in field condition.

MATERIALS AND METHODS

Culture of Trichoderma harzianum
Culture of T. harzianum was obtained from culture bank of Department of Plant Pathology, AAU, Jorhat and
Potato dextrose Agar (PDA) medium was used for its maintaenance. S. sclerotiorum was isolated from freshly infected
French bean plant (variety contender) from Horticultural Orchard, AAU, Jorhat and maintained on PDA.
INDUCTION

AND

ISOLATION

OF

UV-RADIATED

CARBENDAZIM

TOLERANT

MUTANT

OF

T. HARZIANUM
Preparation of Conidial Suspension
Conidial suspension of 6 days old T. harzianum culture was prepared in sterile distilled water (10 ml), centrifuged
twice at 10,000 rpm and pellets of the suspension washed subsequently in phosphate buffer with pH 7.0 (0.067 M). Pellets
of conidia were re-suspended in 4.0 ml of phosphate buffer after second washing, and spore concentration of 4 x 106
conidia/ml were adjusted with sterile water.
UV Irradiation and Induction of Fungicidal Tolerance of the UV Mutant
Mutant of T. harzianum was developed following the method of Papavizas et al, (1990). T. harzianum was
allowed to grow in czapeks dox agar (CDA) medium amended with 0.1% (1000 mg lit-1) with alkylating agent ethyl
methane sulphonate (EMS). Plates were then exposed to UV-radiation chamber at 390 nm / 390000A maintaining the 16.4
cm distance from UV-tube for different periods viz, 0, 1, 2, 4, 6, 8, 12, 16, 20, 24, and 28 h. The cycle of EMS and UV
exposure was repeated once again. The organism were then isolated and grown on CDA to obtain conidia by incubating at
25 1C. Colony developed were picked and transferred to CDA slants, subcultured subsequently for thrice.
Stable colonies developed were picked and grown CDA plates amended with 0.01% carbendazim (100 mg/lit of
CDA) and 500 mg lit-1 of streptomycin. Plates were incubated at 25 1C. To test the stability, colonies developed after 5
days were isolated, purified with serial dilution technique and sub-cultured thrice on CDA without carbendazim. The stable
mutants were grown on CDA with 0.05% carbendazim (500 mg lit-1) and streptomycin (500 mg lit-1). Colonies developed
after 5 days were again isolated, purified with serial dilution and sub-cultured thrice on CDA media without carbendazim
to test their stability after next higher concentration. The same method was repeated for 0.1 per cent (1000 mg lit-1) and 0.2
% carbendazim (2000 mg/lit) and sub-cultured on CDA to test the stability of the mutant. The stable colonies produced
were picked, transferred to CDA and incubated at 25 1C.
Mutants with high degree of carbendazim tolerance were grown for 72 h on fungicide free CDA medium for their
radial growth studies. Twenty five stable colonies of T. harzianum obtained after EMS and UV irradiation were
designated as Th1M1, Th1M2 (after 1 h), Th2M2 , Th2M3 (after 2 h), Th4M1, Th4M2 , Th4M2 , Th4M3, Th4M4, Th4M5
(after 4 h), Th6M1, Th6M2, Th6M3(after 6h), Th8M1, Th8M2(after 8h), Th12M1, Th12M2(after 12 h), Th16M1,
Th16M2 (after 16 h), Th20M1, Th20M2 (after 20 h), Th24M1, Th24M2 (after 24h), Th28M1, Th28M2 (after 28 h). These
were tested for viability with respect to their radial growth (mm) in fungicide free CDA medium for further selection.
From each of the period of exposure (h) one mutant with higher radial growth after 48 h was selected. The selected mutants
were Th1M2, Th2M2, Th4M4, Th6M2, Th8M1, Th12M2, Th16M1, Th20M1, Th24M2, and Th28M1. The phenotypic and
Impact Factor (JCC): 4.3594

Index Copernicus Value (ICV): 3.0

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A Carbendazim Tolerant Mutant of Trichoderma harzianum Showed Higher Biocontrol Activity

morphological characters of the selected mutants were studied after 6 days of incubation.
Antagonistic Effect of Tolerant Mutant of T. harzianum against S. sclerotiorum
T. harzianum mutants (Th-M1, Th-M2, Th-M3, Th-M4, Th-M5, Th-M6, Th-M7, Th-M8, Th-M9, and Th-M10)
were evaluated for the antagonistic activity against S. sclerotiorum and compared with wild T. harzianum (Th-W) by dual
culture method. Seven replications were made and the mycelia growth of S. sclerotiorum was recorded after every 24 h. till
full growth of pathogen occurs in control plates. Per cent inhibition of radial growth of S. sclerotiorum was calculated out.
Preparation of Enzyme Sources
Enzyme source was prepared by following the methods of Dutta and Cheterjee, 2004. Seven days old
T. harzianum (both wild and mutant) cultured on PDA was used for inoculation of Czapeks Dox broth (basal medium).
The later was supplemented with sucrose (3 % w/v), fresh mycelial mat and dry mycelial mat of S. sclerotiorum as the sole
carbon sources.The growth medium in the flasks was passed through filter paper (Whatman No. 1) after 10 days of
incubation. Collected culture filtrates (enzyme source) were preserved in refrigerator at 4 C with 0.02% (W/V) sodium
azide.
- 1, 3-Glucanase
The dinitrosalicylic acid method of Miller, 1959 was used to assay the -1, 3-glucanase (laminarinase).
Chitinase
The method of Ohtakara, 1988 was followed to assay the enzymatsic hydrolysis of colloidal chitin following the
release of free N-acetyl-glucosamine (NAGA) from colloidal chitin. Colloidal chitin was prepared from raw as per the
method of Shimakara and Takiguchi (1988).
Efficacy of Mutant of T. harzianum on Sclerotial Viability of S. sclerotiorum
The experiment was conducted in earthen pot of 25 cm dia. containing 5 kg of soil sterilized with 4% formalin.
Soil was inoculated with 2% (w/w) 15 d old inoculam of S. sclerotiorum grown on maize meal sand medium (MSM).
After 7 days of inoculation of S. sclerotiorum, carrier based formulation of T. harzianum both wild and mutant strain was
inoculated @ 0.2 % (w/w) of soil. The formulation was mixed thoroughly with the soil and covered with polythene sheet.
Pots were watered at regular interval to maintain the moisture. Three sets were maintained for each replication i.e, for 0,
30, 60, 90, 120, 150 and 180 days old formulation. All the treatments were replicated for six times and arranged in
Completely Randomized Block Design. Comparison were made with soil drenching with carbendazim @ 0.3%.
Records on sclerotial survibility percentage on PDA medium was calculated after one month of soil inoculation of
30, 60, and 90, 120, 150 and 180 day old carrier based formulation of T. harzianum for both wild and mutant strain.
For this study sclerotia were collected by sieving the soil then washed with sterile water. Surface sterilized sclerotia
(with 4% NaOCl2) were placed in the centre of PDA plates and incubated at 20 1C. Germination percentage of sclerotia
was recorded after 6 days of incubation.
Field Efficacy of Mutant of T. harzianum against White Mold of French Bean
Field experiments were carried out during rabi seasons continuously for two years. Except the uninoculated
control plots the soils of all the plots were inoculated with the culture S. sclerotiorum (@ 2% w/w) grown on MSM) for 21

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Pranab Dutta, Himadri Kaushik & B. C. Das

days. To get 2% concentration 4.48 kg of pathogen inoculums grown on MSM was added to a plot area of 1 m2
(approximately 224 kg of soil). Talc based formulation of mutant and wild strain of T. harzianum alone and in combination
with methylcellulose (@ 2% w/w) and carbendazim(0.05%) was used for treating the French bean seeds (Var. Contender)
just before sowing. A plot size of 2 x 2 m2 was used for each treatment. FYM @ 20t, N 30kg, P2O5 40 kg and K2O 30
kg/ha were used. Half of N and full dose of FYM, P2O5 and K2O was applied as basal dose and remaining half of N top
dressed at flowering. Irrigation was given regularly. Weeding starting from 25 d after germination was done with
subsequent weeding as per necessity. Three replications were maintained for each treatment and French bean seeds were
sown @ 30 seeds/plot maintaining a spacing of 45 x 35 cm from row to row and plant to plant. Treatments included,
inoculated control (S. sclerotiorum @ 2% alone), un-inoculated control, talc based formulation of mutant T. harzianum
(Th-M) seed treatment with and S. sclerotiorum (@ 2%), seed treated with carbendazim (0.05%) followed by talc based
formulation of mutant T. harzianum (Th- M) and S. sclerotiorum @ 2% (w/w ), talc based formulation of wild
T. harzianum (Th-W) treated seed and S. sclerotiorum (@ 2%), seed treated with carbendazim(0.05%) and then by talc
based Th-W and S. sclerotiorum (@ 2%) (w/w), seed treated with spore suspension of Th-W(4 x 106 spore/ml) and then
methyl cellulose (2% w/v) and S. sclerotiorum @ 2% (w/w), seed treated with carbendazim (@ 0.2% ) and S. sclerotiorum
@ 2% (w/w). Observations were made on seed germination, diseases incidence and yield/ ha.

RESULTS
Induction of Carbendazim Tolerance in Mutant
Twenty five numbers of stable carbendazim tolerant colonies of T. harzianum were developed when conidial
suspension were exposed to EMS and UV radiation for different period and different concentrations of carbendazim.
Colony growth of UV-irradiated T. harzianum at 0.2% concentration of carbendazim was only developed by the isolates of
different h of UV irradiation (Table 1). Wild T. harzianum was unable to produce any colony growth. However, maximum
stable colony growth (5 X 10 6 cfu ml-1) was found after 4 h UV irradiation. This was followed by both 2h and 6 h with 3 X
10 6 cfu ml-1.
The growth of stable colonies was inversely proportionate to the concentrations of carbendazim. This means
higher was the concentration, lower was the stable colony growth. The stable colonies of T. harzianum obtained after EMS
and UV irradiation were designated and are presented in Table 2. These were tested for viability with respect to their
radial growth (mm) in fungicide free CDA medium for selection as mutant. Among all stable mutant of T. harzianum
irradiation at 4 h showed significantly higher radial growth as compared to 1, 2, 6 , 8, 12, 16, 20, 24 and 28h UV irradiation
at all period of incubation (Table 3). However, mutant of T. harzianum at 48 h of incubation showed higher radial growth
as compared to 24h of incubation while the radial growth became almost equal and completely covered whole petriplate
(90 mm) in both wild and mutants of T. harzianum at 72 h of incubation. Hence, the mutants induced at 48 h of incubation
were selected for the present study (Table 2).
Ten stable mutants were selected on the basis of their radial growth after 48 h. of incubation from 25 numbers of
stable colonies after 3rd generation. Maximum radial growth at 48 h was recorded in Th4M4 (88.00 mm). This was
designated as ThM1. This was significantly followed by Th2M2 (72.50 mm) and was designated as ThM2. Similarly,
Th5M2 (67.75 mm), Th1M2 (48.00 mm), Th8M1(33.50mm), Th20M1 (32.50 mm), Th24M2 (30.25 mm), Th28M2 (23.25
mm) were designated respectively as ThM3, ThM4, ThM5, ThM5, ThM6, ThM7, ThM8, ThM9 and ThM10 and the wild
T. harzianum as Th-wild (Table 2).

Impact Factor (JCC): 4.3594

Index Copernicus Value (ICV): 3.0

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A Carbendazim Tolerant Mutant of Trichoderma harzianum Showed Higher Biocontrol Activity

Radial Growth of Stable Mutant of T. harzianum at 0.2% Carbendazim Amended Mediums

The rate of radial growth of mutant T. harzianum was slower in carbendazim (0.2 % ) amended media (Table 3)
than in media without carbendazim. Significantly, more radial growth of mutant ThM1 (46.50 mm) was recorded in the
CDA media amended with 0.2% carbendazim. This was significantly followed by ThM2 (44.50 mm), ThM3 (43.00 mm)
and ThM4 (42.80 mm) without any significant difference among them. Radial growth recorded was inversely proportionate
with the increasing h of exposure to UV radiation means higher the exposure period, lower the radial growth.
Wild T. harzianum (0.00 mm) was unable to produce any radial growth in 0.2% carbendazim amended media even after 6
d on incubation. But in CDA media without carbendazim the same was able to fully cover the whole petriplate (90.00 mm)
in 72 h. Phenotypic and morphological characters were also recorded after 72 h are tabulated in Table 4.
Antagonistic Effect of Tolerant Mutant of T. harzianum against S. sclerotiorum
Antagonistic activity in dual culture showed considerable differences between the ten stable mutants and the wild
T. harzianum in their ability to inhibit the radial growth of S. sclerotiorum. Growth inhibition of Th-M1 and Th-M2 were
higher, 53.53% and 48.19% respectively then those of ThM3, ThM4, ThM5, ThM5, ThM6, ThM7, ThM8, ThM9 and
ThM10 and Th-W (wild) (Table 5).
Considering -1, 3-glucanase, mutant strains exhibited no more enzyme activity than the wild types (Table 6).
Maximum chitinase production by wild and mutant strains may be obtained by supplementing dry mycelia mat.
Efficacy of Mutant of T. harzianum on Sclerotial Viability of S. sclerotiorum
Sclerotial survibility of S. sclerotiorum after being subjected to carrier based formulation at different days of
storage was carried out in pot condition. Among the different formulation sclerotial survibility was lowest in methyl
cellulose amended talc based formulation of mutant of T. harzianum at 30 days of storage (Table 7). With the increase days
of storage the survibility of sclerotia of pathogen was increased and found maximum in formulation stored upto 180 days.
Field Efficacy of Mutant of T. harzianum against White Mold of French Bean
Highest seed germination (97.0%) was found highest when seeds were treated with carbendazim (@ 0.2%).
This was followed by seed treated with 0.05% carbendazim followed by talc based formulation of mutant T. harzianum.
But there was no significant difference among the treatments tested (Table 8). Disease incidence of white mold of French
bean was reduced in all the treatments when seeds were treated with wild strains or mutant of T. harzianum alone or in
combination with carbendazim. Significantly, lowest diseases incidence (9 %) was recorded when seeds were treated with
0.05% carbendazim followed by formulation of mutant T. harzianum. This was followed by carbendazim @ 0.2% w/w and
T. harzianum (X106 spore ml-1) + methyl cellulose @ 2%w/v treated seeds without any significant difference between
them. Comparative higher disease incidence was recorded when seeds were treated with wild strain of T. harzianum either
alone or in combination with carbendazim. Highest diseases incidence (93%) was recorded in inoculated control. But in
comparison to inoculated control significantly lower disease incidence was recorded when seeds were treated with talc
based formulations of wild strains of T. harzianum.
Significantly increased pod yield was recorded in all the treatments as compared to inoculated control. Maximum
pod yield (79.0 q/ ha) was recorded when the seeds were treated with carbendazim (0.2%) (Table 8). This was followed by
seed treatment with 0.05% carbendazim followed by talc based formulation of mutant T.harzianum, but without any
significant difference with the former. Lowest pod yield (42.0 q/ha) was recorded when pathogen was inoculated alone
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Pranab Dutta, Himadri Kaushik & B. C. Das

without any treatment. Per cent increase yield when calculated, was recorded highest increase of yield (88%) over
inoculated control was recorded in seeds treated with 0.2% carbendazim. This was followed by seed treatment with
carbendazim 0.05% followed by talc based formulation of mutant strains of T. harzianum (Table 8). Seed treated with talc
based formulation of mutant of T. harzianum was the next best to the best yielder. This was followed by seed treated with
talc based formulation of wild strains of T. harzianum alone or in combination with carbendazim at (0.05%).

DISCUSSIONS
Mutants of T. harzianum were resistant to higher concentrations of carbendazim than non-mutants and continued
to grow even up to 0.2%. This level of carbendazim resistance expressed by the mutants was about seven times higher than
that reported by Viji et al.(1993). Mutants capable of tolerating wider dosage of the fungicide would ideally have a better
chance of survival than those with low tolerance. Therefore, induction of mutants in T. harzianum paves the way to
develop strains with enhanced survival in the rhizosphere and enhanced capacity to control soil borne diseases under field
condition.
Characteristics of mutants were all conducive to the successful use in practice. Dual culturing of the mutants of
T. harzianum with S. sclerotiorum caused successful inhibition of the latter as compared to non mutant. All the mutants
have improved growth response with higher inhibitory ability to the pathogen. The production of chitinases, and cellulases
and a consortium of antifungal compounds namely fungitoxic metabolites have been proved to be the most important
mechanisms of biocontrol by Trichoderma spp. (Papavizas 1987). We found that the mutants evolved from this study were
capable of producing chitinases, cellulases and fungitoxic metabolites at relatively comparable amounts to that of the wild
type. These results proved that the mutants did not lose their essential antagonistic qualities and they were as good as the
wild type to all of these inherent attributes.
Greater efficacy of T. harzianum to suppress the sclerotial survibility was observed when its inoculation was done
with 30 day storage formulation. Decrease survibility of S. sclerotiorum might be due to concomitant increase of dried
conidia with increase days of storage. Higher sclerotial survivability of the pathogen observed with increased storage
period may be due to the loss of virulence resulted because of deformed spore in shape and size as observed by Puzari et
al, (2003) in Beauveria bassiana. The deformed shape and size may be due to loss of endogenous reserves like
carbohydrates including glycogen and lipids, which ultimately resulted in loss of ability to form germ tubes and to infect
hosts (Lane et al, 1991).
In the study of performance of mutants T. harzianum, we found, mutant of T. harzianum in combination with
carbendazim as highly effective for the management of the disease. Maximum reduction of disease incidence and increased
yield were observed when seeds were treated with mutant T. harzianum and carbendazim (0.05%). This indicated the
possibility of combining carbendazim with bioagents which augments the synergistic effect resulting from interaction
between T. harzianum and sublethal doses of carbendazim. This synergism is apparently due to partial suppression of soil
microflora, enabling a more effective activity of the biocontrol agent. Reduction of disease incidence by mutant
T. harzianum along with carbendazim might have resulted may also be due to the rapid multiplication of the mutant
T. harzianum resistance to carbendazim and their rapid aggressive colonization in the roots of French bean seedling, which
might have prevented the establishment of S. sclerotiorum in crop rhizosphere. To enable us to understand the mechanism
better we need to study the interaction of the mutant with the crop rhizosphere. Furthermore, increased growth parameter
was observed when mutant strain of T. harzianum was used as seed treating agent. It may be due to direct effect of
Impact Factor (JCC): 4.3594

Index Copernicus Value (ICV): 3.0

133

A Carbendazim Tolerant Mutant of Trichoderma harzianum Showed Higher Biocontrol Activity

antagonist on plant growth and the secretion of growth regulating metabolites/hormone which in turn increased growth rate
or efficiency of nutrient uptake. But, the role of plant growth promoting substances associated with mutant strain of T.
harzianum needs further investigation because of its commercial possibilities.

CONCLUSIONS
From our study results, it appears feasible that the fungicide-resistant mutants would certainly help compliment
the integrated disease control strategies. In addition, the system suggested here is compatible with the regular package of
practices followed by farmers and involves no extra expenditure and hence would be easily adoptable. Therefore, it will be
possible to use mutant biotypes in Integrated Disease Management Programme involving fungicides. In addition,
the system suggested here is compatible with the regular package of practices followed by farmers and involves no extra
expenditure and hence would be easily adoptable.

ACKNOWLEDGEMENTS
Authors thanks Director of Research (Agri), Head, Department of Plant Pathology, AAU, Jorhat for their interest
and Dr. B. K. Sarma, Centre-Director, AAU-DBT centre, AAU, Jorhat and Prof. David Lindsay, University of Western
Australia for technical guidance for preparation of this manuscript.

REFERENCES
1.

Dutta S, Chatterjee NC, 2004. Raising of carbendazim tolerant mutant of Trichoderma and variation in their
hydrolytic enzyme activity in relation to mycoparasitic action against Rhizopus stolonifer. Zeitschrift fur Pflanzen
Krankheiten and Pflanzenschutz, 111, 557-565.

2.

Lane BS, Trinci APJ, Gillespei AT, 1991. Influence of cultural conditions on the virulence of conidia and
blastospores of Beauveria bassiana to the green leaf hopper, Nephotettix virescens. Mycological Resaerch 95,
829-833.

3.

Miller GL, 1959. Use of dinitrosalicylic acid reagent for the determination of reducing sugars. Analyt. Chem.
31, 426.

4.

Ohtakara A, 1988. Chitinase and -N-acetylhexosaminidase from Pycnoporus cinnabarainus. Meth. Enzymol.
168, 467468.

5.

Papavizas GC, 1987. Genetic manipulation to improve the effectiveness of biocontrol fungi for plant disease
control. In. Chet I, eds, Innovative approaches to plant disease control, New York, John Wiley Publication,
193212.

6.

Papavizas GC, Lewis JA, Abd-El. Moity TH, 1982. Evaluation of new biotypes of Trichoderma harzianum for
tolerance to benomyl and enhanced bio-control capabilities. Phytopathology 72, 126-132.

7.

Puzari KC, Hazarika LK, Dutta Pranab, Das P, 2003. Shelf life and enhancement of virulence of Beauveria
bassiana. Indian Journal of Agricultural Sciences 73 (10), 542-545.

8.

Shimakara K, Takiguchi Y, 1988. Preparation of crustacean chitin. Meth. Enzymol. 161, 126132,

9.

Viji G, Baby U, Manibhushan RK, 1993. Induction of fungal resistance in Trichoderma spp. through

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Pranab Dutta, Himadri Kaushik & B. C. Das

UV-irradiation. Indian Journal of Microbiology 33, 125-129.

Table 1: Population of Trichoderma Harzianum (X 10 6 CFU Ml-1) inn Czepek Dox Media (CDA) Amended
with Carbendazim at Different Concentration after Exposing to UV Irradiation
Exposure of T.
harzianum to UVPopulation of T. harzianum (X 10 6 CFU ml-1) in CDA Media Amended with different
Irradiation for
Concentration of Carbendazim (%)
Different Hour
(h)
0.00
0.01
0.05
0.10
0.20
0
66.00
34.00f
18.00h
0.00i
0.00d
1
64.00
48.00b
35.00b
29.00b
2.00c
d
c
c
2
64.00
42.00
33.00
26.00
3.00b
a
a
a
4
66.00
59.00
46.00
38.00
5.00a
c
d
d
6
61.00
45.00
30.00
20.00
3.00b
cd
e
cd
8
62.00
43.00
28.00
19.00
2.00c
e
f
c
12
60.00
38.00
25.00
18.00
2.00c
f
g
e
16
64.00
34.00
22.00
13.00
2.00c
g
h
f
20
61.00
32.00
18.00
11.00
2.00c
g
i
g
24
60.00
32.00
15.00
8.00
2.00c
g
j
h
28
60.00
30.00
12.00
6.00
2.00c
SEd ()
NS
1.15
0.97
0.86
0.38
CD 0.05
NS
2.36
1.98
1.75
0.77
Data are mean of three replication.
Means within column separated by Duncans Multiple Range Test P = 0.05
Means followed by the same letter shown in subscript are not significantly different.
Table 2: Radial Growth of T. harzianum at Fungicide Free CDA Medium
after Exposing to Different Hrs of UV Irradiation
UV Irradiated
T. Harzianum
Isolates
(h)
Th1M1
Th1M2
Th2M1
Th2M2
Th2M3
Th4M1
Th4M2
Th4M3
Th4M4
Th4M5
Th6M1
Th6M2
Th6M3
Th8M1
Th8M2
Th12M1
Th12M2
Th16M1
Th16M2
Th20M1
Th20M2
Th24M1
Th24M2
Th28M1

Colony Diameter of T. harzianum (Mm)

24h
18.75
20.00
25.25
28.00
26.50
45.50
42.00
47.00
48.75
44.25
25.00
27.50
23.00
18.00
16.75
14.75
16.50
14.50
13.75
13.25
12.50
10.75
11.50
9.25

Impact Factor (JCC): 4.3594

48h
46.00
48.00
65.50
72.50
69.75
82.00
78.50
85.00
88.00
85.50
61.50
67.75
60.50
36.75
35.00
33.00
34.75
33.50
30.25
32.50
31.25
29.00
30.25
27.75

72h
64.25
65.75
79.25
82.50
79.75
88.25
85.75
89.75
90.00
89.50
80.00
84.00
79.75
63.50
60.25
63.50
60.25
53.75
46.25
49.25
47.50
38.00
40.50
35.75

Selected
Mutant

Designated As

Th1M2

ThM4

Th2M2

ThM2

Th4M4

ThM1

Th6M2

ThM3

Th8M1

ThM5

Th12M2

ThM6

Th16M1

ThM7

Th20M1

ThM8

Th24M2
Th28M1

ThM9
ThM10

Index Copernicus Value (ICV): 3.0

135

A Carbendazim Tolerant Mutant of Trichoderma harzianum Showed Higher Biocontrol Activity

Table 2: Contd,
Th28M2
7.00
23.25
38.50
Th wild
22.00
58.00
90.00
SEd ()
0.78
0.58
0.48
CD 0.05
1.47
1.19
0.99
Numerical in subscript in Th denote hours of irradiation
Table 3: Radial Growth Selected Isolates of T. harzianum after Exposing to Different H of
UV Irradiation at Carbendazim (0.2%) Amended CDA Medium
UV Irradiated T. harzianum Isolates Colony Diameter (mm) in Presence of Carbendazim (0.2%)
Th-M1
46.50a
Th-M2
44.50b
Th-M3
43.00b
Th-M4
42.80bc
Th-M5
42.60c
Th-M6
40.00d
Th-M7
39.60e
Th-M8
37.70f
Th-M9
34.20g
Th-M10
34.00h
Th-wild
0.00
SEd ()
0.86
CD 0.05
1.80
Data are mean of three replication, Observation taken after 6d of incubation.
Means within column separated by Duncans Multiple Range Test P= 0.05
Means followed by the same letter shown in subscript are not significantly different

Table 4: Phenotypic Characters Observed after Exposing Trichoderma

harzianum to EMS and UV Radiation for Different h
Mutants
ThM1
ThM2
ThM3
ThM4
ThM5
ThM6
ThM7
ThM8
ThM9
Thwild

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Phenotypic Characteristics
Highly sporulating dark green colony where spores were densely formed appeared heavily
throughout the surface of plates. Colony with heavy yellow pigmentation aerial mycelium.
Initially colony with whitish mycelium less number of spores appeared. Yellow pigment
appears late.
Colony with few sporulation and sporeless marked zonation was also visible. No prominent
yellow pigmentation.
Colony fluffy and green coloured, initially whitish cottony growth on the surface of the
medium, sporulation at the periphery that appear greenish white colour.
Greenish white colony, no sporulation at 4 day of incubation, no yellow pigmentation.
Colony with sparse to fluffy cottony whitish green colour, sporulation at the periphery that
appear green, clear non sporulating zonation is also observed.
Faded green mycelia and compact growth, colony fluffy and faded green, very less sporulation,
but sporulation appeared in the older regions.
Colony fluffy whitish green with very less sporulation, sporeless zonation was also visible.
Yellow pigment appeared lately.
Green coloured sporulation in every alternate region, no yellow pigmentation, and colony fluffy
and green coloured, absence of yellow pigment was prominent.ThM10Off green mycelial
growth with less sporulation, tint of yellow pigmentation appeared on media.
Greenish white colony, uniformly distributed in the whole petriplate, sporulation was sparsely
distributed.

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136

Pranab Dutta, Himadri Kaushik & B. C. Das

Table 5: Effect of Wild and UV Exposed Isolates of Trichoderma harzianum on

Radial Growth Inhibition (%) of Sclerotinia sclerotiorum
UV Irradiated T. harzianum
Radial Growth of S.
Growth Inhibition Over Control (%)
Isolates
sclerotiorum(mm)
a
Th-M1+ S. sclerotiorum
30.48
53.53(47.02)
Th-M2 + S. sclerotiorum
33.98b
48.19(43.96)
Th-M3 + S. sclerotiorum
35.18b
46.36(42.89)
Th-M4 + S. sclerotiorum
38.32c
41.59(40.15)
Th-M5+ S. sclerotiorum
43.85d
33.16(35.15)
Th-M6 + S. sclerotiorum
52.32e
20.24(26.71)
Th-M7 + S. sclerotiorum
56.44f
13.96(21.91)
Th-M8 + S. sclerotiorum
57.81g
11.88(20.11)
Th-M9 + S. sclerotiorum
60.73h
7.42(15.80)
Th-M10 + S. sclerotiorum
63.86hi
2.66(5.30)
Th-wild + S. sclerotiorum
44.15d
32.70 (37.58)
Control (S. sclerotiorum alone)
65.60i
SEd()
1.11
CD 0.05
2.30
Means within coloumn separated by Duncans Multiple Range Test P= 0. 05
Means followed by the same letter shown in subscript are not significantly different
Data in parentheses are arc sine transformed value.
Table 6: Enzyme Activity of Wild and Mutant T. harzianum
Antagonist
Th-Wild
ThM1
Th M2
ThM3
ThM4
ThM5
ThM6
ThM7
ThM8
ThM9
ThM10
Results are mean of five replications.

Chitinase
160 0.64
196 0.69
180 1.58
165 0.87
120 0.67
115 0.87
106 1.22
96 0.54
80 0.65
42 1.11
32 0.66

, 1-3 Glucanse
2160 2.99
2570 1.87
2010 1.66
1870 1.11
1520 0.98
1210 2.11
989 1.32
860 1.22
545 82.0
298 0.67
186 0.76

Table 7: Viability of Sclerotia of Sclerotinia sclerotiorum after Being Subjected to Different Carrier Based
Formulation Stored for Different Days in Freeze Temperature (in Pot Condition)
Carrier Based
Formulation
T1: Inoculated
control (S.
sclerotiorum
@2% w/w alone)
T2: Th-mutant +
talc (1:3) + S.
sclerotiorum
@2% w/w
T3: Th-mutant +
talc (1:3) + methyl
cellulose @ 2%
w/v + S.
sclerotiorum
@2% w/w

Formulation Stored for (in Days)

90
120
% Survibility of Sclerotia

30

60

100.00d
(90.00)

100.00d
(90.00)

100.00d
(90.00)

100.00e
(90.00)

100.00
(90.00)

13.50b
(21.56)

21.60b
(27.69)

100.00
(90.00)

8.60a
(17.05)

13.20a
(21.30)

Impact Factor (JCC): 4.3594

150

180

100.00e
(90.00)

100.00e
(90.00)

100.00e
(90.00)

32.60 c
(34.82)

40.50c
(39.52)

48.40b
(43.97)

52.20c
(46.6)

26.40b
(30.92)

31.60b
(34.20)

39.10b
(38.70)

44.80b
(42.20)

Index Copernicus Value (ICV): 3.0

137

A Carbendazim Tolerant Mutant of Trichoderma harzianum Showed Higher Biocontrol Activity

Table 7: Contd,
T4: Th-wild + talc
(1:3) + S.
100.00
17.40c
26.62c
42.20d
46.20d
sclerotiorum
(90.00)
(24.65)
(31.06)
(40.51)
(42.82)
@2% w/w
T5: Th-wild + talc
(1:3) + methyl
cellulose @2%
100.00
15.20b
23.20b
34.60c
38.10c
(90.00)
(22.95)
(28.79)
(36.03)
(38.11)
w/v + S.
sclerotiorum
@2% w/w
T6: Soil drench
with carbendazim
100.0
14.40b
14.30a
14.40a
18.80a
@0.3% w/w + S.
(90.00)
(21.97)
(22.92)
(22.22)
(25.70)
sclerotiorum
SEd ()
0.82
1.56
1.39
2.06
CD 0.05
1.80
3.40
2.51
4.12
Means within coloumn separated by Duncans Multiple Range Test P= 0.05

50.50d
(45.29)

60.20d
(50.89)

43.60h
(41.32)

52.60c
(46.99)

21.60a
(27.70)

28.40a
(32.20)

2.44
4.88

2.84
5.68

Means followed by the same letter shown in subscript are not significantly different
Table 8: Potentiality of UV Irradiated Mutant of T. harzianum as Seed Treatment on Disease Incidence of
White Mold, Growth Parameter of French Bean Under Field Condition

Treatment
Seed treatment with carbendazim @ 0.2% w/w +
S. sclerotiorum @ 2%w/w
Seed treated with 0.05% carbendazim followed by
talc based formulation of mutant T. harzianum + S.
sclerotiorum @ 2%w/w
Seed treated with talc based formulation of mutant
T. harzianum + S. sclerotiorum @ 2%w/w
Seed treated with 0.05% carbendazim followed by
talc based formulation of wild T.. harzianum + S.
sclerotiorum @ 2%w/w
Seed treated with T. harzianum (X106 spore ml-1)+
methyl cellulose @ 2%w/v + S. sclerotiorum @
2%w/w
Seed treated with talc based formulation of wild T.
harzianum + S. sclerotiorum @ 2%w/w
Inoculated control (Sclerotinia sclerotiorum @
2%w/w alone)
SEd()
CD (P = 0.05)
Data are mean of two years experimentation.

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Seed
Germination (
%)
97.0
96.0
94.0

Disease
Incidence (%)

Yield (Q
Ha-1)

%Increase Of
Yield Over
Inoculated
Control
88.0

10.0

79.0

9.0

77.0

84.0

11.0

75.0

77.0

70.0

67.0

68.0

61.0

70.0

66.0

42.0

90.0

13.0

91.0

10.0

88.0

13.0

81.0

93.0

NS
NS

0.71
1.41

1.26
2.61

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