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

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  • Real-time PCR assays using locked nucleic acid (LNA) probes and high resolution melt (HRM) analysis were developed for molecular differentiation of Pseudoperonospora cubensis and P. humuli, 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 (cox2) gene that differentiates the two species. Sequencing of the same region from 15 P. cubensis 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 P. cubensis total DNA and 10 fg P. humuli 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 cox2 gene.
  • © 2015 This Article is a collaborative work. The contributions of Nanci L. Adair and David H. Gent were conducted as part of these persons’ official duties as employees of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105 no copyright protection is available for such works under U.S. law. Carly F. Summers, Margaret T. McGrath and Christine D. Smart waive their own assertion of copyright but not their status as co-Authors. The article is published by Taylor & Francis and can be found at: http://www.tandfonline.com/toc/tcjp20/current
  • Keywords: Pseudoperonospora humuli, hop downy mildew, Pseudoperonospora cubensis, detection, cucurbit downy mildew
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  • 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. doi:10.1080/07060661.2015.1053989
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  • 37
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  • 3
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  • This work was supported by the USDA's Organic Research and Extension Initiative (NIFA contract no. 2012-51300-20006), and USDA-ARS CRIS 2072-21000-046-00. Funding for C.F. Summers was also provided through an NSF Graduate Research Fellowship (DGE-1144153).
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