Bioassay of Extracts of the Endophytic Fungi

Materials and Reagents

1. Culture of plant pathogenic fungi (Sclerotinia sclerotiorum)
   
2. Methanol (Qualigens)
   
3. Potato dextrose agar (PDA) media (HiMedia Laboratories)
   
4. Commercial disinfectant (70% ethanol)
   
5. Distilled water
   
6. PDA media (see Recipes) 
   

Equipment

1. Measuring cylinder
   
2. Inoculating needle
   
3. Spirit lamp
   
4. Cork borer
   
5. Conical flask
   
6. Measuring scale
   
7. 10 cm Petri dishes
   
8. pH meter (Eutech Instruments pH tutor)
   
9. Autoclave (Nat steel)
   
10. BOD incubator (Toshiba)
    
11. Laminar air flow hood (Toshiba)
    
12. Micropipette (Eppendorf)
    
13. Microbalances (Sartorious, model: RC210P )
    

Procedure

1. Solvent extracts from endophytic fungi were tested against Sclerotinia sclerotiorum a well known plant pathogenic fungus.
   
2. 30 mg of dried extract was dissolved in 800 μl of methanol.
   
3. 200 and 400 μl of the dissolved extract were added to 30 ml of molten PDA media, mixed and then poured equally into three 10 cm Petri plates to obtain 250 and 500 μg/ml extract concentrations, respectively.
   
4. To obtain 1,000 μg/ml concentration, 30 mg of dried extracts were dissolved in 400 μl of methanol and 400 μl were added to 30 ml of molten PDA.
   
5. Control growth plates contained 400 μl of methanol.
   
6. S.sclerotiorum was inoculated at the centre of the plate and radial growth was measured at 48 h intervals till the control plate attained the full growth.
   
7. Per cent growth inhibitions (%GI) of the extracts were calculated relative to the growth on the control plate.
   %GI = {(A-B)/A} x 100
   Where A = Growth of plant pathogenic fungus in control plate
             B = Growth of plant pathogenic fungus plates supplemented with extract from endophytic fungus
   

Recipes

1. PDA media
   39.1 g of PDA media was dissolved in 1,000 ml of distilled water and pH was adjusted to 7.0
   

Acknowledgments

This protocol was adopted from Kumar and Kaushik (2013). Authors are grateful to their host institution, The Energy and Resources Institute (TERI), New Delhi, India for funding the research. Susheel Kumar is grateful to University Grant Commission, New Delhi for a research fellowship.

References

1. Kumar, S. and Kaushik, N. (2013). Endophytic fungi isolated from oil-seed crop Jatropha curcas produces oil and exhibit antifungal activity. PLoS One 8(2): e56202.