Spinetoram, a Widely Used Insecticide, Demonstrates Strong Antifungal Activity and a Novel Antifungal Mechanism Against Gray Mold for the First Time

Spinetoram, a widely used insecticide, exhibits previously unexplored antifungal properties. This study provides the first evidence of its broad-spectrum antifungal activity against phytopathogens from tea plants, vegetables, and fruit trees, with efficacy against gray mold, with a half maximal effective concentration (EC 50 ) value being 40.41 μg/ml. In vivo trials showed a 60.2% reduction in disease on tomato fruits at a spinetoram concentration of 1,600 μg/ml, and a 70.7% reduction on tomato leaves at 800 μg/ml. Morphological analyses containing light microscopy, scanning electron microscopy, and transmission electron microscopy revealed concentration-dependent cellular alterations in B. cinerea hyphae following spinetoram exposure (EC 10 , EC 50 , and EC 90 ), including swelling, surface distortion, septal disruption at the exposures of 24 or 36 h, and organelle dissolution at elevated concentrations from EC 50 to EC 90 . Transcriptomic profiling identified ribosomal subunit assembly (40S/60S) and ribosome biogenesis as key enriched pathways, suggesting protein synthesis inhibition as the antifungal mechanism. Molecular docking predicted varying binding affinities, ranging from weak to strong, between spinetoram and 41 ribosomal proteins. Notably, 60S ribosomal protein L27 (RPL27) exhibited a lower binding free energy of −11.8 kcal/mol, with a stable binding process confirmed through molecular dynamics simulations. Our findings establish three novel insights: spinetoram’s fungicidal activity against multiple plant pathogens, its disruption of fungal cellular integrity through ribosomal targeting, and RPL27 as a potential binding site for protein synthesis inhibition. This dual pesticidal-fungicidal activity positions spinetoram as a promising lead compound for integrated pest/disease management strategies and fungicide development in the future.