Location: Forage Seed and Cereal ResearchTitle: Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis
|SUMMERS, CARLY - Cornell University - New York|
|Gent, David - Dave|
|MCGRATH, MARGARET - Cornell University - New York|
|SMART, CHRISTINE - Cornell University - New York|
Submitted to: Canadian Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2015
Publication Date: 6/5/2015
Citation: Summers, C.F., Adair, N.L., Gent, D.H., Mcgrath, M.T., Smart, C.D. 2015. Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis. Canadian Journal of Plant Pathology. 37(3):315-330.
Interpretive Summary: The downy mildew pathogens affecting hop and cucurbit crops are closely related. It would be useful to develop molecular diagnostic assays to differentiate the organisms when confirming disease in symptomatic specimens and when sampling environmental samples for the pathogens. In this research, we developed three approaches to identify the organisms based on differences in the a gene called cox2. The assays were successful in identifying the organisms when tested on a diverse set of samples in multiple labs. Only in the case of two isolates of the cucurbit downy mildew pathogen on squash did the assays not correctly identify each organism. Thus, the assays developed can provide reliable identification of both species despite the high molecular similarity between the pathogens.
Technical Abstract: Three assays were developed for molecular differentiation of Pseudoperonospora cubensis and P. humuli, causal agents of cucurbit and hop downy mildew, respectively, for detection of airborne sporangia and diagnosis of symptomatic leaf tissue. The assays were based on previously identified single nucleotide polymorphisms (SNPs) in the cytochrome oxidase subunit II (cox2) gene. Sequencing of the same region from 15 P. cubensis isolates collected in NY for the current study revealed that all isolates shared one conserved SNP that differs consistently from the sequence of P. humuli. Locked nucleic acid (LNA) probes developed to detect both species based on the conserved SNP were specific and sensitive, detecting as few as 10 sporangia for both species and as little as 1 fg P. cubensis total DNA and 10 fg P. humuli total DNA. High resolution melt analysis (HRM) correctly diagnosed all tested isolates as well as symptomatic plants collected in the field. The LNA and HRM assays correctly identified both organisms when tested independently in a second laboratory. A nested conventional PCR was accurate for 13 of 15 P. cubensis isolates, with two isolates collected from squash hosts inaccurately identified as P. humuli due to non-conserved SNPs lying within the reverse primer sequences. Results confirm that the assays developed can provide reliable identification of both species despite the high molecular similarity between the pathogens.