Antifungal Activity of Methyl 2-Methylbutyrate Against Botrytis cinerea and the Induced Physiological, Transcriptional, and Metabolic Responses

Gray mold, caused by the necrotrophic fungus Botrytis cinerea, is a devastating disease responsible for substantial global crop losses. The escalating threat of fungicide resistance and environmental concerns necessitates the development of eco-compatible control alternatives. In this study, we investigated the antifungal efficacy of methyl 2-methylbutyrate (M2M), a microbial volatile organic compound (mVOC), against B. cinerea, and elucidated the underlying physiological, transcriptomic, and metabolomic responses. Our results demonstrated that M2M significantly inhibited mycelial growth in a dose-dependent manner, with an EC50 value of 3.49 μL/plate. Scanning electron microscopy revealed severe ultrastructural damage in M2M-treated hyphae, characterized by localized swelling, shriveling, and irregular bending. Physiological assays indicated that M2M significantly upregulated the activities of antioxidant enzymes, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), while significantly reducing malondialdehyde (MDA) content, suggesting that M2M triggers an oxidative challenge to which B. cinerea responds by activating its antioxidant defense system, thereby suppressing lipid peroxidation and the activities of key metabolic enzymes, such as succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH). These findings suggest that M2M treatment is associated with perturbations in cellular redox homeostasis and central carbon metabolism in B. cinerea. Transcriptomic profiling identified 3664 differentially expressed genes (DEGs), predominantly enriched in carbon metabolism, glyoxylate and dicarboxylate metabolism, redox pathways, and ribosomal biogenesis. Notably, M2M exposure significantly perturbed the transcriptional levels of ROS-scavenging genes (e.g., CAT, POD, GPX, PRX, and GST) and various transcription factors. Untargeted metabolomics revealed 1041 differentially accumulated metabolites (DAMs), with lipids and lipid-like molecules representing the most significantly altered class. Integrated multi-omics analysis revealed that M2M treatment was associated with coordinated changes in lipid metabolism, central carbon metabolism, and redox balance. This study provides mechanistic insights into the antifungal action of M2M and suggests that M2M warrants further investigation as a potential candidate for postharvest gray mold management.