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Title: Coupling spore traps and quantitative PCR assays for detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (Peronospora schachtii)

Author
item Klosterman, Steven
item Anchieta, Amy
item MCROBERTS, NEIL - University Of California
item KOIKE, STEVEN - University Of California - Cooperative Extension Service
item SUBBARAO, KRISHNA - University Of California
item VOGLMAYR, HERMANN - University Of Vienna
item CHOI, YOUNG-JOON - Biodiversity And Climate Research Centre (BIK-F)
item THINES, MARCO - Biodiversity And Climate Research Centre (BIK-F)
item Martin, Frank

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2014
Publication Date: 6/25/2014
Citation: Klosterman, S.J., Anchieta, A.G., Mcroberts, N., Koike, S.T., Subbarao, K.V., Voglmayr, H., Choi, Y., Thines, M., Martin, F.N. 2014. Coupling spore traps and quantitative PCR assays for detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (Peronospora schachtii). Phytopathology. 104:1349-1359.

Interpretive Summary: Downy mildew on spinach, caused by the oomycete pathogen Peronospora effusa, is the major disease constraint on spinach production in California and worldwide. The pathogen is disseminated by windborne asexual spores that land on the plant and initiate primary infections. This study describes a method to detect and quantify airborne inoculum of P. effusa, as well as the closely related downy mildew pathogen on beet and Swiss chard, Peronospora schachtii. Swiss chard is often grown in close proximity to spinach, and therefore it is important to be able to differentiate the Swiss chard downy mildew pathogen from the spinach downy mildew pathogen. Using the method developed in this study, the frequency of the detected DNA target is calculated for each of the two downy mildew pathogens present on impaction spore trap samples. The method will be useful to determine optimal environmental conditions that favor the airborne dispersal of the pathogen and therefore may be useful to forecast downy mildew disease outbreaks for disease management.

Technical Abstract: Downy mildew of spinach (Spinacia oleracea L.), caused by Peronospora effusa, is a disease constraint on production worldwide, including in California where the majority of United States spinach is grown. The aim of this study was to develop a real-time quantitative PCR (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 Peronospora schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris L. subsp. vulgaris). Single nucleotide polymorphisms (SNPs) were detected between P. effusa and P. schachtii 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 ~3400-fold higher from trap samples collected near an infected field as compared to 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.