Action Mechanism and Efficacy of Pyrimethanil Against Corynespora cassiicola , Causing Cucumber Target Spot

Cucumber Corynespora leaf spot, caused by Corynespora cassiicola, poses a significant threat to crop yield and quality. Currently, the application of fungicides is a convenient and effective strategy for managing the disease. This study evaluated the antifungal activity of pyrimethanil against various phytopathogenic fungi, yielding a half-maximal effective concentration (EC 50 ) of 25.55 μg/ml against C. cassiicola hyphae. Microscopic observations revealed that pyrimethanil caused restricted growth of newly formed hyphae, swelling of hyphal diameter, and abnormal accumulation of cellular contents. Integrated transcriptomic and proteomic analyses identified the differentially expressed genes and differentially abundant proteins that influence the cysteine and methionine metabolic pathway. Molecular docking assay demonstrated that pyrimethanil binds to homocysteine S-methyltransferase (HMTs), a crucial enzyme in the pathway, with a binding free energy of −9.4 kcal/mol, which is more favorable than that of the other two analogues, cyprodinil and mepanipyrim. In particular, cyprodinil and mepanipyrim exhibited comparatively lower binding free energies of −7.8 and −7.4 kcal/mol, respectively, when interacting with cystathionine γ-lyase-like protein (CGL-like protein) and spermidine synthase (SpdS) in the pathway. Microscale thermophoresis and surface plasmon resonance assays indicated that pyrimethanil forms a relatively stable binding with HMTs, a candidate target, yielding the dissociation constant values of 0.7 ± 0.28 μM and 29.48 μM. Gene knockout and transient overexpression of mmuM gene encoding HMTs further demonstrated that HMTs is associated with the pathogenicity in C. cassiicola. The formulations of pyrimethanil and berberine demonstrated stronger inhibitory effects, underscoring their potential in the management of the disease caused by C. cassiicola.