Fusarium graminearum is a primary causal agent of Fusarium head blight (FHB) on wheat and barley in North America. The fungus produces trichothecene mycotoxins that render grains unsuitable for food, feed, or malt. Strains of F. graminearum can differ in trichothecene production phenotypes (chemotypes), with single isolates producing predominantly one of the 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 to predict the four F. graminearum chemotypes. Conserved functional regions of three trichothecene biosynthetic genes (TRI1, TRI8, and TRI13) that impact chemotype were targeted to develop a high-resolution melting (HRM) assay. Multiplex HRM analysis produced unique melting profiles for each chemotype.  The assay was validated on a panel of diverse 80 fungal isolates. We applied machine-learning-based linear discriminant analysis (LDA) to automate the classification of chemotypes from HRM data, achieving a prediction accuracy of 99.7%. The assay exhibited good analytical sensitivity, with a limit of detection below 0.02 ng of fungal DNA. Together, our results demonstrate that this simple, rapid, and accurate assay can be applied to F. graminearum molecular diagnostics and population surveillance programs.

Acknowledgements and disclaimer

We are thankful to Karen Broz for excellent technical support. Funding was provided by United States Department of Agriculture-Agricultural Research Service project 5062-21220-024-000D. Mention of trade names or commercial products in this article solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture (USDA). USDA is an equal opportunity provider and employer