Fusarium 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 for identification and differentiation of NX-2 chemotype. 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 NX-2 genotypes. The amplicon generated two HRM profiles, one of which was specific for only NX-2. We validated the assay using a panel of 72 diverse pure-culture isolates collected from North America and it unambiguously differentiates NX-2 from other chemotypes. 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 biomass. The assay is very sensitive and can detect as little as 0.01 ng of fungal DNA in a background of 50 ng of plant DNA. This new diagnostic tool can be used for high-throughput molecular surveillance of the NX-2 chemotype of F. graminearum from plant samples and culture collections.
Acknowledgements and disclaimer
We are thankful to Dr. H. Corby Kistler and Karen Broz for their very helpful discussions. We would like to thank Karen Broz for excellent technical support. Funding was provided by United States Department of 370 Agriculture-Agricultural Research Service project 5062-21220-024-000D. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.