Eugenol Potentially Targets Oleate Delta-12 Desaturase of <i>Botrytis cinerea</i> and Controls Gray Mold as Part of a Combination of Multiple Fungicidal Components

Eugenol exhibits a broad spectrum of antimicrobial activities and fungicide efficacy against crop diseases. However, the antifungal mechanisms of eugenol remain incompletely understood. In this study, we assessed the inhibitory activity of eugenol against several phytopathogenic fungi and found it to be particularly effective against <i>Botrytis cinerea</i>. The half-maximal effective concentration for inhibiting the hyphal growth of <i>B. cinerea</i> was determined to be 72.11 µg/ml. Eugenol exhibited a significant inhibition effect of 66.72 and 43.62% against gray mold on tomato fruits and leaves at the 1,500.0 µg/ml dose. Micromorphological analysis revealed that eugenol induced abnormalities in hyphal structures, including disrupted cell membranes and unclear organelle boundaries. Transcriptomic analysis indicated that differentially expressed genes in hyphae treated with eugenol were primarily enriched in processes related to lipid metabolism, transmembrane transport, and electron transfer activity. Molecular docking studies suggested that eugenol may bind to oleate delta-12 desaturase (FAD2) with a low free energy of -9.9 kcal/mol. Assays of cell membrane permeability and hyphal staining confirmed that eugenol disrupts the cell membrane, resulting in leakage of hyphal contents. A quantitative PCR assay showed that eugenol significantly altered the expression of genes involved in lipid metabolism. Compared with the wild type, the <i>fad2</i> mutant displayed slower hyphal growth rates and became less sensitive to the effects of eugenol. This study demonstrates that eugenol targets FAD2 and disrupts cell membrane integrity, thereby inhibiting the proliferation of <i>B. cinerea</i>. Several formulations of eugenol, including feneptamidoquin or meridianin C, exhibit stronger inhibitory effects, offering promising potential for the control of gray mold.