In vitro and greenhouse assays demonstrate that a novel phenamacril derivative inhibits myosin I in Botrytis cinerea, highlighting a potent alternative antifungal strategy for crop protection.
Gray mold caused by Botrytis cinerea severely impacts global crop productivity. In this study, we identified B33 [(Z)-3-amino-N-(3-chloro-4-fluorobenzyl)-2-cyano-3-phenylacrylamide], a novel phenamacril derivative bearing a distinctive substitution pattern where the ester group is replaced by an amino group. As an unprecedented small-molecule inhibitor targeting B. cinerea myosin I (BcMyoI), it represents an unexplored antifungal mode of action for gray mold disease management. Combined assays including phenotypic screening, ATPase inhibition, and microscale thermophoresis (MST) confirmed that B33 binds BcMyoI, and molecular dynamics simulations revealed stable interactions with the conserved phenamacril-binding pocket. In the greenhouse experiment, the protective effect of B33 on strawberry leaves was comparable to that of the commercial fungicide boscalid. These findings establish BcMyoI as a novel antifungal target and present B33 as a potent agent with a unique mode of action, thus providing a promising alternative strategy to control gray mold and diverse phytopathogens
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