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.

黄曲霉及其产生的致癌黄曲霉毒素经常污染收获后的粮食作物，对全球食品安全造成威胁。化学防腐剂是目前主要的抗真菌剂。然而，真菌抗性效应、生物毒性和环境污染限制了它们的实际应用。天然挥发性有机化合物的应用在控制粮食作物采后真菌和霉菌毒素污染方面具有巨大潜力。因此，本研究调查了挥发性化合物 2-甲基丁酸甲酯 (M2M) 对抗黄曲霉的抗真菌和抗黄曲霉毒素活性及其潜在机制。M2M能有效抑制黄曲霉菌丝体生长，最低抑菌浓度为2.0 μL/mL。此外，M2M还抑制黄曲霉毒素的产生、菌核的产生以及对花生和玉米粉的致病性。RNA-Seq结果显示，2899个差异表达基因（DEG），以及参与麦角甾醇合成、细胞壁结构、糖酵解、柠檬酸循环、丝裂原激活蛋白激酶信号通路、DNA复制和黄曲霉毒素生物合成的DEG在黄曲霉。进一步的研究表明，M2M 强烈破坏细胞膜和细胞壁的完整性，降低 ATP 水平，并诱导活性氧 (ROS) 积累和 DNA 损伤。值得注意的是，鉴定出了GATA 型锌指转录因子 AfSreA (AFLA_132440)，它对于黄曲霉生长和黄曲霉毒素产生至关重要。Δ AfSreA菌株的生长和黄曲霉毒素产量分别下降了94.94%和71.82%。此外，AfSreA的缺失破坏了细胞壁的完整性，并减少了黄曲霉毒素生物合成相关基因的表达。总而言之，我们的结果确定了 M2M 对抗黄曲霉的抗真菌和抗黄曲霉毒素机制，并证实了 M2M 在保护花生和玉米免受真菌污染方面的潜力。