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

Authors: SINGH, LOVEPREET - University Of Minnesota; Drott, Milton; Kim, Hye-Seon; Proctor, Robert; McCormick, Susan; Elmore, James

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/25/2024
Publication Date: 12/30/2024
Citation: Singh, L., Drott, M.T., Kim, H., Proctor, R., Mccormick, S.P., Elmore, J.M. 2024. A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes. Scientific Reports. https://doi.org/10.1038/s41598-024-81131-5.
DOI: https://doi.org/10.1038/s41598-024-81131-5

Interpretive Summary: Fusarium head blight (FHB) is a fungal disease that is among the greatest threats to cereal production worldwide. F. graminearum is the major causal pathogen of FHB and can contaminate infected grain with mycotoxins that render it unsafe for human and animal consumption. Isolates of the fungus are classified into different “chemotypes” based on the type of mycotoxin they produce. Recent studies have reported shifts in pathogen population and chemotype distribution in North America and abroad, and regular monitoring is necessary to understand the current mycotoxin threats on a regional basis. To support pathogen surveillance efforts in the field, there is a need for a rapid and robust diagnostic assay that can be used to differentiate chemotypes. This study developed a new molecular tool, based on high-resolution melting (HRM), that can identify all known chemotypes of F. graminearum in a simple, single-tube format. The assay was validated on a geographically diverse set of 80 fungal isolates, and we applied a machine-learning approach to automatically classify isolates from the HRM data. We demonstrated that the assay is sensitive and extremely accurate on the isolates tested. This is the first report of a single assay that can differentiate all four chemotypes of F. graminearum, and it reduces the hands-on time needed to collect and analyze results. Thus, we anticipate this work will improve molecular diagnostics and accelerate large-scale field surveys of this devastating disease.

Technical Abstract: 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 Fusarium gerlachii, Fusarium asiaticum, and Fusarium 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.