A Multiplex taqman qPCR assay for detection and quantification of clade 1 and clade2 isolates of pseudoperonospora cubensis and pseudoperonospora humuli

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemi-ological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudo-peronospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve man-agement options for controlling the diseases they cause.