Identification and differentiation of the Fusarium graminearum NX-2 chemotype using High-Resolution Melting (HRM)

Authors: SINGH, LOVEPREET - University Of Minnesota; Drott, Milton; Elmore, James

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/21/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Fusarium Head Blight (FHB) is a devastating disease of wheat, barley, and other cereals. In addition to causing losses in crop production, Fusarium graminearum also contaminates grain with toxins that are harmful to human and animal health. Based on the type of toxin produced, F. graminearum isolates are categorized into different "chemotypes" and there is a need for high-throughput and robust diagnostic assays to rapidly identify different fungal chemotypes from cultures and infected plant samples. We report a new assay to identify the NX-2 chemotype of F. graminearum. We validated the assay using DNA from 75 fungal isolates representing the four major chemotypes. We further demonstrated the utility of the assays in detecting the NX-2 chemotype directly from DNA isolated from infected plant tissue, even when the fungal DNA was present at extremely low levels. This robust HRM diagnostic assay can be used for high-throughput molecular surveillance of FHB pathogen populations in grower's fields.

Technical Abstract: Fusarium head blight causes significant yield losses in wheat and other cereals and contaminates grain products with trichothecene mycotoxins. F. graminearum isolates are classified into different chemotypes depending on the type of mycotoxin produced, including the type B trichothecenes 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV), and the recently identified type A trichothecene NX-2. Molecular tools to differentiate NX-2 producers from other chemotypes have remained relatively laborious and time consuming. In this study, we developed and validated a high-resolution melting (HRM) assay that can identify NX-2 producers quickly and cost-effectively. By analyzing TRI1 coding sequences from 183 geographically diverse isolates representing all four F. graminearum chemotypes, we selected a 75-base pair region containing four non-synonymous single nucleotide polymorphisms (SNPs) that are specific to the NX-2 genotypes. The amplicon generated two HRM profiles, one of which was specific for only NX-2. We confirmed that the assay is robust across qPCR platforms and unambiguously differentiates NX-2 from other chemotypes using a panel of 72 diverse isolates previously collected from North America. The HRM assay was also successful in identifying NX-2 producers directly from DNA extracted from infected wheat spikes with varying levels of disease severity and fungal DNA. The assay can detect as little as 0.01 ng of fungal DNA in a background of 50 ng of plant DNA. This new diagnostic assay can be used for high-throughput molecular detection of the NX-2 chemotype of F. graminearum from infected plant samples and culture collections, thus making it a valuable tool for surveys of contemporary and historical FHB pathogen populations.