Detection of Pseudoperonspora cubensis and P. humuli using 5’ nuclease probes, specific primers and high resolution melt curve analysis
C. F. SUMMERS (1), D. H. Gent (2), C. D. Smart (1). (1) Cornell NYSEAS, Geneva, NY, U.S.A.; (2) USDA-ARS, Corvallis, OR, U.S.A.
<i>Pseudoperonospora cubensis</i>, the causal agent of cucurbit downy mildew, results in much economic loss worldwide. The closely related pathogen, <i>P. humuli</i>, causes hop downy mildew, one of the most serious diseases of hop. Both pathogens produce wind-blown sporangia capable of spreading disease rapidly. Thus, detection of sporangia would be useful in early management of the disease. However, the genetic similarity between the two pathogens has prevented differentiation, and consequently successful detection, of either pathogen. Our goal was to utilize conserved single nucleotide polymorphisms in the <i>cox2</i> gene of each pathogen to develop methods that allow for their differentiation and detection. 5’ nuclease assay probes were developed that permit concurrent detection of both pathogens from one reaction with great specificity. Specific primers developed for conventional PCR offer a lower cost option for detection. High resolution melt curve analysis was a third approach developed that allowed for differentiation and diagnosis of these pathogens. These three methods have been tested for use in diagnosis of diseased plants from the field as well as detection of DNA extracted from air samples collected using a roto-rod spore trap.
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