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Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii)

December 2014 , Volume 104 , Number  12
Pages  1,349 - 1,359

Steven J. Klosterman, Amy Anchieta, Neil McRoberts, Steven T. Koike, Krishna V. Subbarao, Hermann Voglmayr, Young-Joon Choi, Marco Thines, and Frank N. Martin

First, second, and ninth authors: United States Department of Agriculture–Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA; third author: University of California, Davis; fourth author: University of California Cooperative Extension, Salinas; fifth author: University of California, Davis, 1636 E. Alisal St., Salinas; sixth author: University of Vienna, Vienna; seventh and eighth authors: Goethe University Frankfurt am Main, Faculty of Biosciences, Institute of Ecology, Evolution and Diversity, D-60438 Frankfurt am Main, and Biodiversity and Climate Research Center (BiK-F), D-60325 Frankfurt am Main, Germany; and eighth author: BiK-F, Germany.

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Accepted for publication 12 June 2014.

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2014.