Lipopeptide Extraction and Antifungal Disc Diffusion Assay

Based on the presence of lipopeptide genes in bacterial isolates, three bacterial isolates (Bacillus subtilis-KAS-2, Bacillus tequilensis-KAS3, and Bacillus velezensis-KAS7) were grown in liquid culture for lipopeptide production and extraction. Using the method described by Gond et al. (2015), lipopeptide was extracted from bacterial isolates. For that, selected bacterial isolates (KAS2, KAS3, and KAS7) were grown in nutrient broth with constant shaking with 200 rpm on a rotary shaker at 27°C. After 4 days of incubation, bacterial cultures were centrifuged at 4,500 rpm (15 min at 4°C); culture supernatants were collected and acidified (up to pH 2°C) with concentrated HCl and were incubated overnight at 4°C. Acidified culture supernatants were centrifuged at 9,000 rpm (15 min at 4°C). The pellets were collected and dissolved in methanol and then filtered through a 0.22 μm filter membrane to remove bacterial cells debris. Methanol filtrate was dried through a vacuum evaporator and stored at 4°C. Using disc diffusion assays, the antifungal activity of lipopeptide was checked against fungal phytopathogens including Fusarium sp., Curvularia sp., Alternaria sp., Rhizoctonia solani, Epicoccum sorghinum, and Exserohilum rostratum. For that, a sterile paper disc was loaded with 20 μl (200 μg) of methanolic solution of lipopeptides and the control disc contained only 20 μl methanol. Loaded discs were transferred onto potato dextrose agar (PDA) plates with a centrally placed small disc of fungal mycelia and incubated for 4 days. Stereo microscopy (Magnus, India) was used to observe the zone of inhibition between lipopeptide-loaded disc and fungal pathogens.