Discovery of a Novel Small-Molecule Inhibitor Targeting Myosin I to Control Colletotrichum siamense Anthracnose

Anthracnose caused by Colletotrichum siamense severely impacts global crop and forest productivity. Here, we identify A22, a novel small-molecule inhibitor that targets C. siamense myosin I (CsMyoI), and this represents a previously untapped antifungal mechanism. Phenotypic screening, ATPase inhibition, and microscale thermophoresis (MST) confirmed that A22 binds to CsMyoI and disrupts its motor activity. Molecular dynamics simulations revealed stable interactions with the conserved phenamacril binding pocket, with a critical K379 substitution explaining species-specific differences in inhibitor sensitivity. Notably, A22 exhibited no cross-resistance with widely used fungicides, such as carbendazim or pyraclostrobin. In greenhouse trials, A22 outperformed the commercial fungicide prochloraz in controlling anthracnose on pepper, mango, and strawberry, even at low concentrations, and also promoted strawberry growth. These findings establish myosin I as a novel antifungal target and present A22 as a potent agent with a unique mode of action, offering a promising strategy for managing anthracnose and fungicide-resistant fungal strains.