3-hydroxycoumarin effectively inhibits development and pathogenicity by targeting Pempk proteins of Penicillium expansum

Penicillium expansum, a causal agent of blue mold rot, leads to substantial postharvest losses in the fruit industry. Due to environmental pollution and the emergence of fungicide-resistant strains, the use of synthetic chemical fungicides faces severe challenges. Natural antifungal compounds derived from plants are considered promising alternatives. In this study, the antifungal activities of coumarin and five hydroxylated derivatives against P. expansum and involved mechanisms were investigated. Among the six coumarins, 3-hydroxycoumarin (3-HC) demonstrated strong inhibitory effects on mycelial growth, germ tube elongation, conidiation, patulin production, and the best control effect on blue mold in apple and jujube fruit. Moreover, 3-HC increased the sensitivity of P. expansum to the cell wall, oxidative, and osmotic stresses. In silico analyses, including target fishing, molecular docking, and molecular dynamics simulations, revealed that the mitogen-activated protein kinase (MAPK) family in P. expansum (Pempks), were the potential targets of 3-HC. Bio-layer interferometry and ATP competition assays found that 3-HC could directly bind to PempkB and PempkC, and inhibit the MAPK activity. Our results suggested that 3-HC may act as an ATP-competitive MAPK inhibitor to affect the MAPK signaling pathway and inhibit the development and pathogenicity of P. expansum.