Comparision between Bacillus subtilis RP24 and its antibiotic-defective mutants

Bacillus subtilis RP24, a promising plant growth-promoting rhizobacterium and a potent biocontrol agent isolated from pigeonpea rhizosphere was mutagenized with ethyl methanesulphonate to study the possible mechanism/s involved in the potential antagonistic properties of the strain. Over 10,000 mutants were screened against the phytopathogenic fungus Macrophomina phaseolina on potato dextrose agar plates to select ten mutants showing partial antagonism as compared to the parent strain and one negative mutant showing no antagonism. The parent strain RP24 was compared with its mutants for the presence of different possible mechanisms behind antagonism. Production of hydrogen cyanide, ammonia, siderophores, and hydrolytic enzymes like lipase, amylase, and protease were detected in all the mutants as well as the parent strain, whereas fungal cell-wall-degrading enzymes, beta-1, 3-glucanase and chitosanase were not detected in any of the mutants and the parent strain, indicating that none of these mechanisms was involved in the antagonistic trait of the strain. Two possible mechanisms detected behind the antifungal trait of the strain RP24 were production of antifungal volatiles and extra-cellular diffusible antibiotics. An attempt was made for extraction, partial characterization of the extra-cellular diffusible antifungal metabolite/s by thin layer chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The extracellular, methanol soluble, hydrophobic, ninhydrin-negative, thermostable and pH-stable antifungal metabolites were characterized as cyclic lipopeptides belonging to the iturin group of peptide antibiotics.