Identification and Differentiation of the Fusarium graminearum NX-2 Chemotype Using High-Resolution Melting (HRM)

Fusarium head blight (FHB) causes significant yield losses in wheat and other cereals and contaminates grain products with trichothecene mycotoxins. Fusarium graminearum isolates are classified into different chemotypes depending on the dominant type of mycotoxin produced. The four major classes represent the type B trichothecenes 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, and nivalenol, as well as the type A trichothecene NX-2, which was first reported in 2014. 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-bp region containing four nonsynonymous single nucleotide polymorphisms that are specific to the NX-2 genotype. The amplicon generated two HRM profiles, one of which was specific for only NX-2. We confirmed that the assay is robust across quantitative PCR 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.