Development and validation of a diagnostic high resolution melting curve assay for the NX-2 chemotype of Fusarium graminearum

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

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/20/2023
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 (FHB) is a devastating disease that can result in significant yield losses in wheat and other cereal crops. Fusarium graminearum, a major causal agent of FHB, poses food safety risks by contaminating kernels with trichothecene mycotoxins. Based on the type of mycotoxin produced, F. graminearum isolates are categorized into different chemotypes including 3-acetyl DON (3-ADON), 15-acetyl DON (15-ADON), nivalenol (NIV), and the recently identified NX-2. Fungal chemotypes can be identified through biochemical or molecular genetic analyses. In this study, we developed and validated a high-resolution melting (HRM) curve assay to identify NX-2 isolates. By analyzing TRI1 coding sequence from 183 geographically diverse isolates, representing the four major chemotypes, we identified and selected four clustered, non-synonymous single nucleotide polymorphisms (SNPs) that were specific to NX-2 genotypes. Primers annealing to conserved sequences flanking the SNP region were designed to amplify a 75bp region for the HRM assay. Melting analysis of the amplicon generated two HRM curve profiles, one specific for NX-2 and the other for the non-NX-2 chemotypes. Using a panel of 75 pure culture isolates collected from southern Canada and northern USA, the NX-2 HRM assay unambiguously differentiated NX-2 from other chemotypes. In addition to pure culture DNA, the HRM assay was also successful in identifying NX-2 isolates directly from DNA extracted from infected wheat spikes with varying levels of disease severity and fungal biomass. The HRM assay was highly sensitive with detection limit of as low as 0.01 ng DNA template. In conclusion, this new HRM assay can be used for high-throughput molecular surveillance of the NX-2 chemotype of F. graminearum.