Aspergillus flavus is an airborne fungus that can contaminate agricultural commodities very rapidly, including grains and nuts. This pathogen causes aflatoxin contamination in peanuts, which leads to significant crop losses and their quality, particularly in storage conditions. Accurately identifying toxin and non-toxin producing A. flavus isolates is important in terms of effective management because traditional approaches have limitations such as being less effective and time-consuming. To address this problem, we developed molecular tools to differentiate toxigenic and non-toxigenic A. flavus isolates. We identified A. flavus isolates through morphological and species-specific primer (FLA1/FLA2). Also, to characterize toxigenic and non-toxigenic A. flavus isolates, we employed a combination of approaches such as RT-PCR, RT-qPCR, and aflatoxin measurement, focusing on four genes (AflD, AflQ, AflP, AflR) from the aflatoxin biosynthesis gene cluster with primer optimization. In addition, research on the co-infection pattern of aflatoxin contamination on peanut seeds during storage conditions is limited. To address this, we collected peanut isolates from different seed lots and performed ITS sequencing to understand the pattern of microbial communities on peanut seeds during aflatoxin contamination. Regarding aflatoxigenic and non-toxigenic A. flavus detection, different gene expression patterns were observed among four AFs biosynthesis genes. However, by combining gene expression patterns, quantification and AFs production, we differentiated toxin and non-toxin producing A. flavus isolates. These results indicated the efficacy and specificity of these molecular tools, which could be helpful for developing good molecular markers for distinguishing toxigenic and atoxigenic isolates and to manage A. flavus contamination in peanut seed lots. Our co-infection results from peanut seed lots during multiple years observed the infection of diverse microbiota along with A. flavus, which indicates other microbes co-infect with A. flavus infection. This co-infection could trigger aflatoxin production in peanut seeds. Therefore, this approach offers novel insights into peanut seed-associated microbiomes, co-infection and aflatoxin production, shedding light on the correlation between the role of microbial communities and aflatoxin contamination.
