Mitochondrial Loci Enable Specific Quantitative Real-Time PCR Detection of the Pathogen Causing Contemporary Impatiens Downy Mildew Epidemics.

Impatiens downy mildew (IDM) disease is a primary constraint on the production of , a popular and economically important floriculture plant. IDM is caused by the biotrophic. oomycete that emerged as a pathogen of in the 2000s. To enable detection and quantification, a hydrolysis-probe-based quantitative PCR diagnostic assay was developed based on unique orientation and order of the mitochondrial cytochrome oxidase subunit1 (1) and ATP synthase subunit alpha (1) genes in the genus . Nucleotide sequences and analysis of the 1/1 region distinguished and its sister-species , consistent with prior phylogenetic analyses using 2 and rDNA markers. Specificity for was incorporated into a hydrolysis probe targeting the 1 gene and flanking primers that amplified across the 1/1 intergenic region. The limit of detection was 0.5 fg/μl of DNA (∼100 plasmid copies/μl), with amplification efficiency = 0.95. The assay was validated against a panel of target and nontarget oomycetes, which showed that the primers were specific for spp., while the probe was specific for infecting both and . Testing of tissue collected from 23 locations across 13 states indicated all samples with IDM symptoms tested positive for . Asymptomatic plants from two locations also tested positive for .