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Int. J. LifeSc. Bt & Pharm. Res. 2014 B Avanti et al., 2014

DEVELOPMENT OF HIGHER TEMPERATURE

TOLERANT MUTANT OF BEAUVERIA BASSIANA

AND VERTICILLUM LECANII

B Avanti1*, K Balaraman2 and R Gopinath2

Research Paper

The natural populations of insects are attacked by using fungal pathogen of Beauveria bassianaand Verticillium lecanii. Thermotolerant mutants of B. bassiana and V. lecanii were yielded afterexposing to the moist heat and UV treatment can offer effective control in all season. The mutantsof B. bassiana (BBMh1) and V. lecanii (VLMh1) were yielded maximum biomass and blastosporeat 27oC and 35oC as compare to wild type after moist heat treatment. The mutants obtained at40oC were yields very less biomass and blastospore. Fungal spore suspension were taken forrespective treatment at varying time interval of 5, 10, 15, 20 and 30 min. The eight mutants wereobtained on the basis of thermotolerant characteristics.

Keywords: Beauveria bassiana, Verticillium lecanii, Thermotolerant, Biomass, Blastospores

*Corresponding Author: B Avanti � [email protected]

ISSN 2250-3137 www.ijlbpr.com

Vol. 3, No. 3, July 2014

© 2014 IJLBPR. All Rights Reserved

Int. J. LifeSc. Bt & Pharm. Res. 2014

1 Mahtama Gandhi Missions Institute of Biosciences and Technology, Aurangabad, Maharashtra, India 431003.

2 Gokulam Biotech, Kannikoil, Pondichery, India 607402.

INTRODUCTION

The natural population of insects are commonly

controlled by fungal pathogen. There has been

only a limited success in using these organism

as bio-control agent. The fungal mycelium usually

invades all body tissues and eventually causes

suffocation by blocking the tracheal system of

insects (Clarson, 2007). When these fungi are

applied in the agricultural field they exhibit poor

survival due to higher temperature in the field this

results in lower control of the target pests. Simple

and expensive method for obtaining large

quantities of entomopathogenic fungus, free from

substrate contamination by culturing

entomopathogen yields 1010/g biomass for

Tolypocladium cylindrosporium, Verticillium

lecanii, and Beauveria bassiana and 109 for

Culicinomyces clavisporus (Batist et al., 1988).

The conidial germination in three strains each of

M. anisopliae and B. bassiana at 25oC and 30oC.

The germination rate was reported to be species

dependent with M. anisopliae strain having faster

than that of B. bassiana (Hywel-Jones and

Gillespie, 1990). The influence of UV light on the

pathogenicity of B. bassiana was observed

significantly influenced the mortality effect. The

variant SK99C showed the highest level of

infectivity. Optimized conidial production of M.

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anisopliae var. acridium in mycosed cadavers of

Schistocercea gregaria during 10 days incubation

at 96oC relative humidity and temperature

between 20oC-30oC reported producing 109

conidia per cadaver. Condial yield was reported

to be maximal at 25oC, cadaver remain in contact

with the dump substrate. Little sporulation occurs

at temperature of 15oC and 40oC regardless of

RH and there was no sporulation occurred at

10oC and 45oC (Arthur and Thomas, 2001). The

report of high variability in conidial thermotolerance

was found among the Beauveria spp. isolates

after exposure to 45oC for 2 h has evidenced by

low (0-20%), medium (20-60%), or high

germination (60-80%). The thermal death point

(0% germination) for three rather thermotolerant

B. bassiana isolates (CG 138, GHA and ARSEF

252) was 46oC for 6 h (Kumar et al , 2007). The

investigated effect of temperature on P. lilacinus

growth at different temperature (15oC-35oC) were

analyzed (Fang et al., 2007). From the above it

has been proved that no work found on the

development of thermotolerant from B. bassiana

and V. lecanii. Isolate of Beauveria and Verticillium

that have higher thermotolerance if developed it

will be more effective in the field even during

summer when temperatures are as high as 35oC-

40oC. A thermotolerant isolate can offer effective

control in all season.

In the present study the conidia of both

B. bassiana and V. lecanii were subjected to

physical mutagenesis; with UV irradiation and

moist heat treatment. The survivor were tested

for their ability to grow either at higher

temperature. Development of theromotolerant

from entomopathogenic fungi were represented

best control on insect pathogen through fungal

attack.

MATERIALS AND METHODS

Culture Media

B. bassiana and V. lecanii were obtained from

Gokulam Biotech. The cultures were transferred

to Czapek-Dox Agar plates incubated at 27oC for

10-12 days. After incubation spore suspensions

were prepared in sterile distill water and tenfold

serial dilutions were prepared up to 10-3.

Test for Change in Characteristics in theNew Isolates

Three separate colonies of each fungal strain was

taken and inoculated in 10 mL of Czapek-Dox

liquid media in test tubes for 2 days at 27oC. This

was used as inoculum for 100 mL Czapek-Dox

media in duplicate and incubated at room

temperature for 4-5 days.

Treatment of B. bassiana and V. lecaniiwith Moist Heat

Fungal spore dilution 10-3 /mL were taken for moist

heat treatment approximately 2 mL of sample

exposed (100oC) for varying time interval of 5 min,

10 min, 20 min and 30 min. Treated samples

were incubated at 27oC for 15-17 days on the

same media and growth parameters were

studied.

Treatment of B. bassiana and V. lecaniiwith Ultra Violet Light

The 2 mL of spore dilution 10-3 /mL were taken in

sterile petri plates and kept for exposure under

UV light (254 nm) for same time intervals

mentioned for moist heat. Treated samples were

plated on agar plates and were incubated at room

temperature for 8 days.

Selection of Mutant Colonies with HighTemperature Tolerance

The colonies were screened individually. The

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mutant colonies of B. bassiana and V. lecanii were

taken for determination of conidial count and

biomass respectively.

RESULTS AND DISCUSSION

One mutant of each of B. bassiana (BBMh) and

V. lecanii (VLMh) was selected after treatment

with moist heat. When they grew in liquid medium

the former species produced higher biomass as

well as blastospores where as latter species

produced lesser quantity of biomass compared

with parent strains. In case of UV treatment, three

mutants of B.bassiana (BBM1, BBM2, BBM3) and

V. lecanii (VLM1, VLM2, VLM3) were selected. The

B. bassiana mutants BBM1 and BBM2 produced

higher quantity of biomass where as B. bassiana

(BBM3) produced lesser quantity of biomass at

27oC as compared to the parent strain.

Surprisingly all three mutants of V. lecanii VLM1,

VLM2, and VLM3 were produced lesser biomass

compared to the parent strain.

All the mutants of B.bassiana (BBMh) and V.

lecanii (VLMh) produced higher number of

blastospores/mL at 35oC as compared to the

parent strain. In case of VLMh mutant produced

7 times biomass as compared to the parent

strain. In UV treated mutants of B. bassiana

(BBM1) 1.2 times higher biomass as compared

to the parent strain. BBM2 and BBM3 yielded

approximately same and 1.5 times less biomass.

In the case of VLM2, mutant of V. lecanii yielded

7.02 times higher biomass compared to parent

strain. The UV treated mutants of B. bassiana

BBM1, BBM2, and BBM3 and V. lecanii VLM1,

VLM2, and VLM3 produced lower biomass

compared to the parent strain. The UV light

significantly influenced mortality effect as well as

infectivity of entomopathogenic fungus B.

bassiana. High variability in conidial

thermotolerance among B. bassiana spp.

Isolates after exposure to 45oC for 2 h as

evidenced by low (0-20%), medium (20-60%),

high germination (60-80%) (Everton et al., 2007).

Comparative account of these observations

in the present study the mutants of B. bassiana

and V. lecanii produced higher level of biomass

and blastospore at 27oC and 35oC.

Insect population controlled by using

entomopathogenic fungi.

CONCLUSION

In the present study we have focused on isolation

of thermotolerant mutants of entomopathogenic

fungi, i.e. ,Beauveria bassiana and Verticillium

lecanii. Mutants isolated by physical mutagenesis;

moist heat stress and UV treatment exhibited high

degree of control on the insert at versatile

temperature range when compared the

controlling ability of entomopathogenic fungus

between mutant and corresponding parent strain.

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