Discovery of WML-01, a Spirocyclic Butenolide Derivative Featuring a Rhodanine Motif, Exhibiting High Fungicidal Activity against <i>Fusarium</i> Species

Controlling the Fusarium head blight (FHB) epidemic and deoxynivalenol (DON) contamination, predominantly caused by <i>Fusarium graminearum</i>, remains challenging. In this study, rhodanine was introduced into a spirocyclic butenolide-based chemical library to design novel fungicidal agents. <i>In vitro</i> assays revealed that target compounds exhibited excellent activities against <i>F. graminearum</i>, <i>F. fujikuroi,</i> and <i>Sclerotinia sclerotiorum</i>. The EC₅₀ of the representative compound <b>WML-01</b> was 0.079 μg/mL against <i>F. graminearum</i> and 0.72 μg/mL against <i>F. fujikuroi</i>. Transmission electron microscopy showed that <b>WML-01</b>, like phenamacril, disrupted the cell membrane of <i>F. graminearum</i>, causing deposition of lipid droplets. <b>WML-01</b> inhibited DON biosynthesis as effectively as phenamacril but showed no cross-resistance, indicating a different mode of action. Transcriptome analysis indicated that <b>WML-01</b> strongly suppresses ribosomal subunit assembly, impairing protein synthesis. Field trials demonstrated that <b>WML-01</b> performs comparably to phenamacril in controlling FHB. Overall, <b>WML-01</b> is a promising candidate for managing FHB and DON contamination.