Rational Design of Novel Tetrazolinone-Pyrazine Derivatives: Synthesis, Antifungal Activity, and Mechanism of Action.

G143A mutation-driven target-site resistance severely impairs the field efficacy of traditional Quinone outside inhibitors (QoIs). To solve this agricultural crisis, novel tetrazolinone-pyrazine derivatives were rationally designed and synthesized by integrating potentially resistance-breaking tetrazolinone toxophore with a pyrazine scaffold via oxime ether linkage. Bioassays revealed that compound 19 exhibited exceptional in vitro activity against Sclerotinia sclerotiorum with an EC50 of 0.003 mg/L, greatly exceeding the commercial QoI metyltetraprole (EC50 = 0.13 mg/L). In vivo assays on Brassica napus L. leaves confirmed its excellent curative (95.4%) and protective (93.6%) efficacies at 200 mg/L. Scanning electron microscopy detected severe hyphal morphological disruption. Transcriptomic profiling and qRT-PCR verified that compound 19 markedly disturbed fungal energy cycles, redox homeostasis and secondary metabolite biosynthesis. Molecular docking and MM-GBSA calculations confirmed its strong binding affinity for wild-type and G143A-mutated cytochrome bc1 complexes. This work identifies compound 19 as a promising next-generation agrochemical for resistant phytopathogenic fungal management.