The Natural Product Osthole, Known for Its Insecticidal and Antimicrobial Properties, Potentially Binds to Amidase, Offering a Novel Approach for Controlling Tomato Gray Mold for the First Time

Osthole exhibits strong inhibitory activity against phytopathogenic fungi; however, its antifungal mechanism remains unclear. This study assessed osthole's inhibitory effects on several phytopathogenic fungi, revealing a half-maximal effective concentration of 73.03 μg/ml against the hyphal growth of <i>Botrytis cinerea</i>. Micromorphological analysis showed that osthole caused abnormalities in the hyphae, including unclear organelle boundaries and organelle dissolution. Integrated transcriptomic and metabolomic assays and correlation analysis indicated that osthole induced differentially expressed genes and differentially abundant metabolites, which were enriched particularly in the pathways of glyoxylate and dicarboxylate metabolism, tyrosine metabolism, glycerophospholipid metabolism, fructose and mannose metabolism, citrate cycle, biosynthesis of unsaturated fatty acids, and ABC transporters. Molecular docking and molecular dynamics simulation assays demonstrated that osthole binds stably to amidase, a key enzyme in energy metabolism, with a relatively lower binding energy of -8.5 kcal/mol compared with osthole's analogs, suggesting that amidase may be a potential target protein in the fungus. A microscale thermophoresis assay indicated that the dissociation constant (Kd) value for osthole binding to amidase was significantly lower compared with that of osthole's analog 7-methoxycoumarin. Overall, this study demonstrates that osthole disrupts energy metabolism, nitrogen metabolism, substance transport, and the metabolism of the hyphal cell wall and cell membrane, potentially targeting the amidase of <i>B. cinerea</i>. These findings highlight the potential of osthole for controlling gray mold.