Styela clava-Derived Peptide Cla-H Is a Promising Green Biofungicide for Crop Protection

Background: Crop fungal diseases cause huge yield losses globally, and the abuse of chemical fungicides has led to severe resistance and environmental risks. Antimicrobial peptides (AMPs) represent ideal green alternatives, but poor salt tolerance and high production costs limit their field application. Results: In this study, we developed a His-tag engineering strategy to enhance salt tolerance of antifungal peptides, and identified a novel peptide Cla-H derived from Styela clava. Cla-H was efficiently expressed via secretory fermentation in Pichia pastoris, and its crude fermentation supernatant could be directly applied to plants. Cla-H exhibited strong and rapid fungicidal activity against Fusarium graminearum and Botrytis cinerea, accompanied by irreversible inhibition of spore germination and observable membrane permeabilization phenotypes. While SYTOX green and PI staining indicated membrane-damaging events, adequate control experiments are still required to firmly establish membrane permeabilization as its primary mode of action. Meanwhile, it showed outstanding thermal and storage stability. Most importantly, His-tag modification significantly improved its salt tolerance, enabling stable activity under high-salt conditions that completely inactivated the parental peptide. In planta assays demonstrated that crude Cla-H fermentation supernatant effectively controlled wheat scab and tomato gray mold. Conclusions: This study provides a promising salt-tolerant antifungal peptide for crop protection and establishes a simple and low-cost technical route for developing next-generation green biofungicides.