Improvement in biocontrol activity of Bacillus subtilis UTB1 against Aspergillus flavus using gamma-irradiation

Bacillus subtilis UTB1, a biocontrol bacterium isolated from Iranian pistachio nuts, has revealed to have antagonistic activity against aflatoxin-producing Aspergillus flavus R5. The strain UTB1 produces lipopeptide compounds and is able to degrade aflatoxin B1. In this study, a random mutagenesis generated using different doses of gamma irradiation (0.1e3 KGy) was applied on B. subtilis UTB1 to improve its antagonistic activity against A. flavus R5. Five hundred bacterial colonies were selected randomly after irradiation, and their effects against A. flavus R5 were assessed in a plate assay. Forty-five colonies (9%) exhibited higher inhibition activity as compared to the non-irradiated wild type. Eight colonies out of the 45 were selected based on different polymorphism patterns obtained by repetitive element sequence polymorphism-PCR (ERIC and BOX) analyses; six of which could significantly inhibit the fungal growth utilizing washed cells and cell-free supernatants as compared to the parental strain. According to thinlayer chromatograms, the production of lipopeptides including surfactin, fengycin and iturin families increased in these six mutants. A considerable inhibition of the fungal growth was observed using bioautography analysis, which associated with iturins production. A. flavus sporulation and aflatoxin content decreased significantly in pistachio nuts treated with mutants M419 and M464 as compared to the strain UTB1. These results suggest that both mutants M419 and M464 could be promising biocontrol candidates against A. flavus in pistachio nuts.