A multiplex TaqMan qPCR assay for detection and quantification of clade 1 and clade 2 isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli

Authors: CRANDALL, SHARIFA - Pennsylvania State University; RAMON, MARINA - Former ARS Employee; BURKHARDT, ALYSSA - Former ARS Employee; BELLO, J - Michigan State University; Adair, Nanci; Gent, David - Dave; HAUSBECK, MARY - Michigan State University; QUESADA-OCAMPO, LINA - North Carolina State University; Martin, Frank

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2021
Publication Date: 2/16/2021
Citation: Crandall, S.G., Ramon, M.L., Burkhardt, A.K., Bello, J.C., Adair, N., Gent, D.H., Hausbeck, M.K., Quesada-Ocampo, L.M., Martin, F.N. 2021. A multiplex TaqMan qPCR assay for detection and quantification of clade 1 and clade 2 isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli. Plant Disease. https://doi.org/10.1094/PDIS-11-20-2339-RE.
DOI: https://doi.org/10.1094/PDIS-11-20-2339-RE

Interpretive Summary: The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing of control measures. This need is acute for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (P. cubensis clade 1 and 2 isolates and P. humuli, respectively). Molecular detection techniques were developed that could identify and quantify these pathogens in a single amplification, which should provide growers the information they need for improving disease management.

Technical Abstract: The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing of control measures. This need is acute for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (P. cubensis clade 1 and 2 isolates and P. humuli, respectively). A highly specific multiplex TaqMan qPCR assay targeting unique sequences in the pathogen’s mitochondrial genome was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluate if results were influenced by PCR inhibitors that made it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was validated with DNA extracted from purified sporangia, infected plant tissue and environmental samples collected on impaction spore traps samplers.