Abstract
Chemical fungicides act by targeting specific proteins or pathways, known as modes of action (MoAs). In this study, we established a systematic screening platform using Fusarium graminearum gene knockout mutants to evaluate the efficacy of fungicides and identify novel antifungal compounds. Target genes corresponding to major fungicide classes were deleted by homologous recombination, and the resulting mutants exhibited altered sensitivity to the corresponding fungicides compared with the wild-type strain. Deletion of genes encoding known fungicide targets revealed distinct sensitivity profiles, supporting MoA prediction. Using the platform that detects sensitivity differences through OD(₆₀₀) changes, we examined a chemical library of 2,704 small molecules and identified 8 compounds with activity likely involved in the osmotic signal transduction pathway. Several of these compounds exhibited previously unreported antifungal activity, underscoring their potential as novel scaffolds for future development. Collectively, this platform provides a robust, mechanism-informed system for antifungal discovery, with broad applicability to synthetic agents, natural products, and biocontrol metabolites. IMPORTANCE: This study provides a perspective on developing strategies to manage fungal pathogens with implications for agriculture and food security. The effectiveness of current fungicides is increasingly limited by resistance, narrow target ranges, and environmental concerns. By combining genetic approaches with chemical screening, this work offers a more systematic way to identify candidate antifungal agents and better understand their possible mechanisms. The framework may also be adapted for use in other areas, including medical and veterinary applications, suggesting potential broader applicability. In addition, the emphasis on exploring new molecular scaffolds supports efforts to develop more sustainable and long-term disease management strategies. Overall, this study contributes useful knowledge and tools for advancing antifungal research and improving approaches to pathogen control.