Mutations and Metabolic Factors Affecting Interaction of Toxigenic and Atoxigenic Aspergillus flavus

Aflatoxin B₁ (AFB₁) produced by <i>Aspergillus flavus</i> poses severe food safety risks. Competitive exclusion using atoxigenic <i>A. flavus</i> strains offers a promising biological control approach to managing agricultural contamination by reducing populations of toxigenic strains and aflatoxin levels. However, reliable identification of atoxigenic strains remains challenging, and the mechanisms underlying competitive interactions between toxigenic and atoxigenic strains require clarification for effective implementation. Therefore, this study systematically analysed <i>A. flavus</i> strains for aflatoxin gene clusters and AFB₁ production to address these critical gaps. Our analysis revealed that atoxigenic strains had intron losses and high-impact mutations in several genes, particularly <i>aflL</i> and <i>aflLa</i>, which affect aflatoxin biosynthesis. Key genes <i>norA</i>/<i>aflE</i>, <i>verA</i>/<i>aflN</i>, and <i>omtA</i>/<i>aflP</i> emerged as mutation hotspots, sometimes causing false-negative PCR results that complicate strain identification. Also, AFB₁ production was inversely related to spore concentration on MEA medium, with fewer spores resulting in higher toxin levels. Interaction tests demonstrated that toxigenic and atoxigenic strains exhibited morphological changes only when co-cultured without physical separation, suggesting that this was mediated by diffusible molecules. Furthermore, differences in the levels of linoleic acid reduction products distinguished toxigenic from atoxigenic strains. These findings thus illuminate the complex genetic and metabolic factors influencing aflatoxin production and fungal interactions.