Mechanisms of methyl 2-methylbutyrate suppression on Aspergillus flavus growth and aflatoxin B1 biosynthesis

Aspergillus flavus and subsequently produced carcinogenic aflatoxins frequently contaminate postharvest food crops, resulting in a threat to global food safety. Chemical preservatives are currently the main antifungal agents. However, fungal resistance effect, biological toxicity, and environmental contamination limit their practical applications. The application of natural volatile organic compounds has great potential for controlling fungal and mycotoxin contamination of postharvest food crops. This study therefore investigated the antifungal and anti-aflatoxigenic activities of the volatile compound, methyl 2-methylbutyrate (M2M), against Aspergillus flavus and its potential mechanisms. M2M effectively inhibited A. flavus mycelia growth, with a minimum inhibitory concentration of 2.0 μL/mL. Moreover, M2M also suppressed aflatoxin production, sclerotia production, and the pathogenicity on peanut and corn flour. RNA-Seq results showed that 2899 differentially expressed genes (DEGs), and DEGs involved in ergosterol synthesis, cell wall structure, glycolysis, citric acid cycle, mitogen activated protein kinase signaling pathway, DNA replication, and aflatoxin biosynthesis, were down-regulated in A. flavus. Further studies showed that M2M strongly damaged the cell membrane and cell wall integrity, reduced ATP levels, and induced reactive oxygen species (ROS) accumulation and DNA damage. Notably, a GATA type zinc finger transcription factor, AfSreA (AFLA_132440), which is essential for A. flavus growth and aflatoxin production, was identified. The growth and aflatoxin yield in the ΔAfSreA strain decreased by 94.94 % and 71.82 %, respectively. Additionally, deletion of AfSreA destroyed cell wall integrity and decreased expressions of genes involved in aflatoxin biosynthesis. Taken together, our results identified the antifungal and anti-aflatoxigenic mechanisms of M2M against A. flavus, and confirmed the potential of M2M in protecting peanut and corn from fungal contamination.