Methods
Culture: Trichoderma virens (Gv-A and Gv-V) used in the study were isolated from agricultural field soils, collected from various parts of Tamil Nadu, India. An alien isolate Gv was obtained as 'gratis' from Prof. Turhan, Ege University, Turkey. The isolates were maintained on potato dextrose agar at 28 Β± 2 ΒΊC.
Substrates: Natural materials such as sugarcane bagasse, green gram hull, bengal gram hull, wheat bran, rice bran, soya meal, soya meal + tapioca, tapioca powder, tapioca peel and gingelly cake were chosen for evaluating the growth and the production of gliotoxin by T. virens.
Chemicals:
All chemicals used in the study: gliotoxin (SIGMA), chlorothalonil (NOVARTIS), methanol and dichloromethane (MERCK) were purchased.
Differentiation of 'P' and 'Q' strain:
The isolates of Trichoderma virens were differentiated into 'P' and 'Q' strains by inoculating discs of the isolates (Gv, Gv-A and Gv-V) on PDA medium amended with 50 Β΅g/ml of gliotoxin (SIGMA) and chlorothalonil (NOVARTIS). The plates were maintained at 28 Β± 2 ΒΊC for 5 days and observations were recorded.
Gliotoxin production:
The isolates (Gv, Gv-A and Gv-V) of T. virens were inoculated into 2% of respective natural substrates (bengal gram hull, gingelly cake, greengram hull, ricebran, soya meal, sugarcane bagasse, soya meal + tapioca, tapioca powder, tapioca peel and wheat bran) in water. The fungus was incubated at 28 Β± 2 ΒΊC for a period of 15 days as still cultures and the production of gliotoxin was monitored every alternate day. The growth (dry weight) and the pH of the culture filtrate were recorded simultaneously.
Production of gliotoxin under varied light condition:
The substrate which supported good growth and gliotoxin production was chosen for the experiment. Sugarcane bagasse (2%) in distilled water was inoculated with the isolates of T. virens and incubated under continuous dark and 12 hrs of alternative illumination as still cultures at room temperature. The suspension was extracted every alternate day up to 15 days of incubation and gliotoxin was quantified.