Efficacy and mode of action of antifungal peptides against the wheat pathogen Fusarium graminearum and functional analysis of its virulence genes

Fusarium graminearum is a devastating wheat pathogen causing severe yield losses and mycotoxin contamination. Conventional control strategies, mainly based on fungicides, face growing challenges due to resistance and environmental concerns. In this study, we assessed the antifungal activity against this fungal pathogen of synthetic peptides derived from the peptaibol trichogin GA IV. Among 24 analogs tested, Pep 5 and Pep 4r significantly reduced Fusarium Head Blight (FHB) symptoms and DON mycotoxin accumulation in wheat spikes. Pep 5 also decreased powdery mildew severity on wheat leaves, matching the efficacy of sulphur-based treatments. Microscopy and ultrastructural analyses on F. graminearum conidia demonstrated that effective peptides induce morphological and cellular alterations consistent with an apoptotic-like mechanism, including cytoplasmic shrinkage, mitochondrial disruption, nuclear fragmentation, and lack of reactive oxygen species production. Additionally, we used gene knockout strategies to investigate the virulence mechanisms exploited by F. graminearum, focusing in particular on the role of two fungal endo-xylanases highly expressed during wheat spikes infection. FGSG_10999 deletion markedly reduced virulence, DON production, and enzymatic activity, suggesting this gene is essential for fungal virulence. Moreover, we observed pleiotropic effects associated with its deletion, affecting other fungal traits beyond xylanase activity, while FGSG_03624 deletion showed limited impact. Double knockout mutants revealed that the order of gene deletions influences fungal phenotypes, suggesting possible compensatory mechanisms and complex genetic interactions driven by a few crucial genes. This work underscores the potential of peptide-based antifungal strategies to enhance wheat protection and food safety, and highlight the limits of the gene knock-out approach in identifying potential fungal targets to be counteracted.