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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Publications at this Location » Publication #420135
Research Project: Disease Management and Improved Detection Systems for Control of Pathogens of Vegetables and Strawberries

Location: Crop Improvement and Protection Research

Title: Multiplexed real-time and digital PCR tools to differentiate clades of Plasmopara viticola causing downy mildew in grapes

Author
item HEGER, LEXI - Michigan State University
item SHARMA, NANCY - Michigan State University
item MCCOY, AUSTIN - Michigan State University
item Martin, Frank
item MILES, LAURA - Michigan State University
item CHILVERS, MARTY - Michigan State University
item Naegele, Rachel
item MILES, TIMOTHY - Michigan State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/11/2025
Publication Date: 3/28/2025
Citation: Heger, L., Sharma, N., McCoy, A.G., Martin, F.N., Miles, L.A., Chilvers, M.I., Naegele, R.P., Miles, T.D. 2025. Multiplexed real-time and digital PCR tools to differentiate clades of Plasmopara viticola causing downy mildew in grapes. Plant Disease. https://doi.org/10.1094/PDIS-01-25-0173-SR.
DOI: https://doi.org/10.1094/PDIS-01-25-0173-SR

Interpretive Summary: This submission describes the development of a diagnostic assay specific for detection of three lineages of the grape downy mildew pathogen Plasmopara viticola using traditional real time PCR and digital PCR technologies. The multiplexed assay was designed around a unique gene order for the genus Plasmopara to improve specificity and can detect lineages aestivalis, riparia, and vinifera, the most commonly encountered taxa in Michigan. Assay validation included tests with spores, infected leaves and air samples collected on greased rods.

Technical Abstract: In vineyards, downy mildew of grapes, caused by the oomycete pathogen Plasmopara viticola, can cause significant economic losses when left unmanaged. P. viticola is a species complex, made up of at least four clades, or cryptic species, causing disease on at least eight plant species within the family Vitaceae. In the United States, clades aestivalis, riparia, and vinifera, have been identified as being present in the Great Lakes region on cultivated grapes. Within this study, a multiplexed TaqMan qPCR assay system capable of differentiating among these three taxa was developed using a mitochondrial gene order difference unique to Plasmopara species (cox1-atp1). The assay is needed to clearly differentiate among the closely related species as research investigates relationships between the clades and their varying hosts as well as fungicide resistance development. The multiplexed assay was validated using a panel of target and non-target samples of varying types, including leaves, ToughSpot® ® stickers, and air sampling rods. The assay was also transferred to and optimized on a digital PCR (dPCR) platform. Air sampling rods and artificially inoculated mixed samples were tested using both qPCR and the dPCR assays to gauge utility of each. The multiplexed assays for each clade showed varying sensitivity of 10 to 1,000 fg of DNA and efficiency of 70-85% in qPCR. The dPCR sensitivity was the same, except for cl. riparia, which showed a tenfold increase. These results suggest that the dPCR can serve as a more sensitive option than qPCR when trying to diagnose plant pathogens, but it is dependent on the assay. This assay system provides detection of the pathogen and classification of P. viticola clades allowing discrimination in areas growing multiple cultivated or wild grape species. This will continue to be relevant as wild hosts can potentially harbor different P. viticola clades and downy mildew is intensely managed in commercial vineyards.