An α,α-Dihalogenated Acetophenone Derivative: A Novel Antifungal Agent with a Dual-Targeting Mechanism against <i>Colletotrichum gloeosporioides</i>

The widespread application of commercial chemical fungicides has accelerated the evolution of fungicide resistance in fungal pathogens, posing a significant threat to global food security and agricultural sustainability. Anthracnose, caused by <i>Colletotrichum</i>, is a devastating fungal disease that leads to significant economic losses worldwide. The rising fungicide resistance of species of <i>Colletotrichum</i> highlights the need to develop novel fungicides. In this study, we identified that certain α,α-dihalogenated acetophenone derivatives exhibit potent antifungal activity against <i>C. gloeosporioides</i>. Notably, compound <b>Ii</b> displayed exceptional inhibitory efficacy, effectively suppressing vegetative growth and pathogenicity at a low concentration of 18 μg·mL^-1. Using a T-DNA insertional mutant library, we identified two genes, <i>CgSFU1</i> and <i>CgRIDA</i>, which play distinct roles in the response to compound <b>Ii</b>. Deletion of <i>CgSFU1</i> enhanced sensitivity to compound <b>Ii</b>, while disruption of <i>CgRIDA</i> conferred resistance to compound <b>Ii</b> in <i>C. gloeosporioides</i>. Molecular docking analysis revealed that compound <b>Ii</b> binds to CgRidA at the TRP-40 or TYR-22 residues through hydrophilic interactions, thereby disrupting its regulatory functions. These findings provide insights into how <i>C. gloeosporioides</i> responds to compound <b>Ii</b> in two distinct manners and highlight its potential as a novel antifungal agent targeting fungal regulatory proteins.