Poster: Biology & Disease Mgmt: Integrated Pest Mgmt
Rapid differentiation of Claviceps species occurring in Oregon and Washington using high resolution melting curve analysis
N. KAUR (1), R. Cating (1) (1) Oregon State University, Hermiston, OR, U.S.A.
Ergot disease of grass seed crops can be caused by Claviceps purpurea and C. humidiphila, a species recently reported to occur in certain regions of OR and WA. Routine identification of Claviceps species involves conventional PCR amplification and sequencing of ITS, β-tubulin or EF-1α genes. In this study, a high resolution melting (HRM) assay was developed to rapidly identify and differentiate C. purpurea and C. humidiphila based on the melting temperatures (Tm) of partial β-tubulin amplicons. PCR primers were designed to target and amplify a 110 bp region of the β-tubulin gene containing multiple single nucleotide polymorphisms generating amplicons with distinct melting profiles for each species. Forty-six fungal cultures, previously classified based on RAPD profiles, and ITS, β-tubulin, and EF-1α gene sequences, were used to validate the HRM assay. The Tm for C. purpurea and C. humidiphila ranged between 79.8 to 80.3 and 78.9 to 79.3, respectively. Classification of Claviceps species based on Tm using linear contrasts (P < 0.05) correctly grouped 96% (N=46) of samples previously classified using sequence data. In two cases, C. purpurea was misclassified as a mixture of the two Claviceps species. Therefore HRM analysis allows rapid detection and differentiation of these two Claviceps species and will be useful to study their distributions within OR and WA cool-season grass seed growing regions.