The Iturin Lipopeptides as Key Compounds in Antagonism of Bacillus subtilis UTB96 toward Aspergillus flavus

Members of the genus Bacillus are among the most promising candidates for biological control of plant pathogenic bacteria. In this study, cell free supernatants of three strains B. subtilis UTB96, B. subtilis UTB1 and B. subtilis UTB70 against Aspergillus flavus R5 were investigated using dual culture assay. Strain B. subtilis UTB96 showed the highest inhibition effect. Thin lyer chromatography in extracted lipopeptides of the bacterial cell free supernatants proved the presence of the three families of lipopeptides iturin, fengycin and surfactin. Bioautography analysis of the chromatogarams against A. flavus R5 revealed the antifungal potential of iturins in strain B. subtilis UTB96. Gene bymB (the third gene in iturin biosynthesis) was disrupted usisng targeted mutagenesis to confirm role of iturins in antifungal activity. Molecular study (PCR analysis and sequencing the amplified fragment) of the mutants revealed that the targeted gene (bmyB) was replaced by spectinomycin resistance gene, hence the gene was disrupted. The phenotypical comparison of the bmyB mutants with the parental strain demonstrated that the antifungal activity of cell free supernatants and extracted lipopeptides was decreased interestingly in a dual culture assay with A. flavus, bioautography analysis and also on pistachio nuts. To sum up, these findings allowed us to conclude that iturin-like lipopeptides are indispensable for the biocontrol ability of the strain UTB96 against A. flavus R5.