<i>Pseudoperonospora cubensis</i> and <i>P. humuli</i> detection using species-specific probes and high definition melt curve analysis

<div><p></p><p>Real-time PCR assays using locked nucleic acid (LNA) probes and high resolution melt (HRM) analysis were developed for molecular differentiation of <i>Pseudoperonospora cubensis</i> and <i>P. humuli</i>, causal agents of cucurbit and hop downy mildew, respectively. The assays were based on a previously identified single nucleotide polymorphism (SNP) in the cytochrome oxidase subunit II (<i>cox2</i>) gene that differentiates the two species. Sequencing of the same region from 15 <i>P. cubensis</i> isolates collected in New York State for the current study confirmed that all isolates shared the conserved SNP. LNA probes were specific and sensitive, detecting as few as 10 sporangia for both species and as little as 1 fg <i>P. cubensis</i> total DNA and 10 fg <i>P. humuli</i> total DNA. The LNA assay detected both pathogens from air sampled using spore traps placed in vegetable fields and a hop yard during the summers of 2013 and 2014 and correctly diagnosed symptomatic leaf tissue. High resolution melt analysis (HRM) correctly identified all tested isolates as well as those isolates from symptomatic plants collected in the field. The LNA and HRM assays correctly identified both organisms when tested independently in a second laboratory. The results confirm that the LNA and HRM assays developed can provide reliable identification of both species despite the high molecular similarity of the <i>cox2</i> gene.</p>