Clotrimazole disrupts ergosterol biosynthesis and cell membrane integrity via targeting sterol 14α-demethylases to suppress virulence of Magnaporthe oryzae

Rice blast, caused by the fungal pathogen Magnaporthe oryzae ( M. oryzae ), leads to substantial global reductions in crop yield. Clotrimazole (CLZ), a synthetic imidazole derivative with broad-spectrum antifungal properties, is widely employed in clinical and veterinary applications for fungal infections treatment. However, limited information exists regarding its antifungal efficacy and mechanisms against phytopathogenic fungi. This study systematically elucidates the antifungal effects and underlying mechanisms of CLZ against M. oryzae . CLZ exhibits potent inhibitory effects on hyphal growth, conidiation, appressorium development, and pathogenicity. CLZ treatment disrupts cellular membrane integrity and significant depletion of ergosterol content. Molecular analysis reveals that the pharmacophore ring of CLZ directly interacts with the key sterol 14α-demethylase enzymes MoCyp51A and MoCyp51B in the ergosterol biosynthetic pathway. Deletion of MoCYP51A or MoCYP51B exhibits enhanced susceptibility to CLZ, confirming that CLZ effectively suppresses ergosterol biosynthesis to impair membrane integrity in M. oryzae . Additionally, CLZ treatment triggers multiple cytological alterations including enhanced cell wall deposition, mitochondrial ultrastructural defects, dysregulated mitophagy, and excessive reactive oxygen species (ROS) accumulation. These data collectively reveal that CLZ inhibits fungal virulence of M. oryzae by simultaneously targeting structural and functional damage to the cell wall, cell membrane, and organelles. Safety assessments further confirmed that CLZ exhibits minimal toxicity to both insects and crops. This evidence collectively proves the potential application of CLZ as a promising fungicide for the management of rice blast.