ISOLATION, SCREENING AND APPLICATIONS OF BACILLUS SUBTILIS AS BIOPESTICIDE

Abstract

The study was accompanied to isolate and screened Bacillus subtilis from soil sample of wheat agricultural fields, Lahore and determine the anti-fungal and anti-larval activities of Bacillus subtilis as a biopesticide. Isolated strain was sub cultured and maintained, then fermentation in shaker flask was done and after centrifugation supernatant or cell free culture were collected. Cell free supernatant of Bacillus subtilis were evaluated as antifungal against a disease specimen (Aspergillus flavus) and as a larvaecidal against third instar larvae of Aedes aegypti. In-vitro antifungal activity was determined by disc diffusion method while assay proved that Bacillus subtilis effective against Aspergillus flavus and after 24 hours showed zone of inhibition were 1.5mm and 1.2mm with positive control. Readings were documented after every 6 hours and plotted a graph between time and radius of inhibition zone which indicated with passage of time antifungal activity of Bacillus subtilis was incremental with the same concentration. In larvaecidal activity Bacillus subtilis showed highly inhibitory effects towards third instar larvae of Aedes aegypti. After 24 hours’ observation there was dead larvae observed with different concentrations of culture supernatant of Bacillus subtilis with no dead larvae detected in control (distilled water) and culture medium (potato dextrose medium). Probit Regression analysis disclosed that LC50 and LC95 of fresh cell free supernatant and stored supernatant after first and second weeks were 12.384 and 29.256, 20.237 and 47.579, 28.184 and 57.481 respectively.  Readings were documented with different intervals of time and plotted graphs among culture time vs mortality and culture time vs proportion of dead larvae all represent a directly proportion relationship. Appraisal between the results of fresh and stored cell free supernatant revealed that after storage Bacillus subtilis loses its activity to some extent as a biopesticide.