A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes

Fusarium graminearum is a primary cause of Fusarium head blight (FHB) on wheat and barley. The fungus produces trichothecene mycotoxins that render grain unsuitable for food, feed, or malt. Isolates of F. graminearum can differ in trichothecene production phenotypes (chemotypes), with individuals producing predominantly one of four toxins: 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, or NX-2. Molecular tools to diagnose chemotypes remain inefficient. This study aimed to develop a single-tube, multiplex molecular assay that can predict the four F. graminearum chemotypes. Conserved functional regions of three trichothecene biosynthetic genes (TRI1, TRI8, and TRI13) were targeted to develop a high-resolution melting (HRM) assay. Multiplex HRM analysis produced unique melting profiles for each chemotype, and was validated on a panel of 80 isolates. We applied machine learning-based linear discriminant analysis (LDA) to automate the classification of chemotypes from the HRM data, achieving a prediction accuracy of over 99%. The assay is sensitive, with a limit of detection below 0.02 ng of fungal DNA. The HRM analysis also differentiated chemotypes from a small sample of F. gerlachii, F. asiaticum, and F. vorosii isolates. Together, our results demonstrate that this simple, rapid, and accurate assay can be applied to F. graminearum molecular diagnostics and population surveillance programs.