A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes
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 indiv...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2024-12-01
|
| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-024-81131-5 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1841559482135478272 |
|---|---|
| author | Lovepreet Singh Milton T. Drott Hye-Seon Kim Robert H. Proctor Susan P. McCormick J. Mitch Elmore |
| author_facet | Lovepreet Singh Milton T. Drott Hye-Seon Kim Robert H. Proctor Susan P. McCormick J. Mitch Elmore |
| author_sort | Lovepreet Singh |
| collection | DOAJ |
| description | 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 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. |
| format | Article |
| id | doaj-art-05a8eea7ed904cdea78646cf2a912c8d |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-05a8eea7ed904cdea78646cf2a912c8d2025-01-05T12:26:26ZengNature PortfolioScientific Reports2045-23222024-12-0114111210.1038/s41598-024-81131-5A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypesLovepreet Singh0Milton T. Drott1Hye-Seon Kim2Robert H. Proctor3Susan P. McCormick4J. Mitch Elmore5Department of Agronomy and Plant Genetics, University of MinnesotaCereal Disease Laboratory, Agricultural Research Service, US Department of AgricultureMycotoxin Prevention and Applied Microbiology, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of AgricultureMycotoxin Prevention and Applied Microbiology, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of AgricultureMycotoxin Prevention and Applied Microbiology, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of AgricultureCereal Disease Laboratory, Agricultural Research Service, US Department of AgricultureAbstract 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.https://doi.org/10.1038/s41598-024-81131-5Fusarium head blightFusarium GraminearumMycotoxinsChemotypeHigh-resolution meltingDiagnostics |
| spellingShingle | Lovepreet Singh Milton T. Drott Hye-Seon Kim Robert H. Proctor Susan P. McCormick J. Mitch Elmore A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes Scientific Reports Fusarium head blight Fusarium Graminearum Mycotoxins Chemotype High-resolution melting Diagnostics |
| title | A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes |
| title_full | A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes |
| title_fullStr | A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes |
| title_full_unstemmed | A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes |
| title_short | A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes |
| title_sort | multiplex high resolution melting hrm assay to differentiate fusarium graminearum chemotypes |
| topic | Fusarium head blight Fusarium Graminearum Mycotoxins Chemotype High-resolution melting Diagnostics |
| url | https://doi.org/10.1038/s41598-024-81131-5 |
| work_keys_str_mv | AT lovepreetsingh amultiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT miltontdrott amultiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT hyeseonkim amultiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT roberthproctor amultiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT susanpmccormick amultiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT jmitchelmore amultiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT lovepreetsingh multiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT miltontdrott multiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT hyeseonkim multiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT roberthproctor multiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT susanpmccormick multiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes AT jmitchelmore multiplexhighresolutionmeltinghrmassaytodifferentiatefusariumgraminearumchemotypes |