Integrating Multi-Omics Data Reveals the Mechanism of Action of the Antifungal Agent Ferimzone Against the Tea Leaf Spot Pathogen Didymella segeticola

Tea leaf spot caused by Didymella segeticola is prevalent in many tea-growing regions of China and has a significant negative impact on both tea yield and quality. Ferimzone, as an uncoupler, demonstrates antimicrobial activity against various pathogens; however, its inhibitory mechanism remains poorly understood. The present study found that it effectively inhibited several phytopathogenic fungi. Specifically, the half-maximal effective concentration (EC 50 ) for D. segeticola hyphae was 58.48 μg/ml. Microstructural analyses revealed that ferimzone caused several changes in the hyphae, including shortened inter-septum distances, hyphal swelling, accumulation of hyphal contents, and inhibition of new hyphal growth. Transcriptomic, proteomic, and metabolomic analyses indicated that ferimzone affects hyphal energy production, metabolic processes, biosynthesis of biomolecules, and material transport. Molecular docking and molecular dynamics simulations demonstrated that ferimzone binds to the candidate target protein pyruvate carboxylase (PC), with a predicted binding free energy of −7.9 kcal/mol. Microscale thermophoresis and surface plasmon resonance assays confirmed that ferimzone binds directly to PC. The dissociation constant (Kd) for the ferimzone-PC interaction was significantly lower than that observed for its structural analog fluazinam, indicating that ferimzone exhibits a higher binding affinity for this fungal target. Furthermore, ferimzone inhibited the mitochondrial membrane potential of the hyphae, with this effect becoming more pronounced as the dose increased. It was likely that ferimzone targets PC in the hyphae, disrupting energy metabolism, ultimately impairing hyphal growth. Several formulations of ferimzone including osthole, exhibited enhanced inhibitory activity, suggesting promising potential for controlling tea leaf spot caused by D. segeticola.