Mitochondrial loci enable specific quantitative real-time PCR detection of the pathogen causing contemporary impatiens downy mildew epidemics

Authors: Leblanc, Nicholas; Martin, Frank; CASTROAGUDIN, VANINA - Oak Ridge Institute For Science And Education (ORISE); Crouch, Jo Anne

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/4/2021
Publication Date: 9/13/2021
Citation: Leblanc, N.R., Martin, F.N., Castroagudin, V.L., Crouch, J. 2021. Mitochondrial loci enable specific quantitative real-time PCR detection of the pathogen causing contemporary impatiens downy mildew epidemics. Plant Disease. 106(1):144-150. https://doi.org/10.1094/PDIS-05-21-0933-RE.
DOI: https://doi.org/10.1094/PDIS-05-21-0933-RE

Interpretive Summary: Impatiens are a popular annual bedding plant, prized for their brilliant colors and ease of care in the landscape. After severe outbreaks of downy mildew disease in 2011, impatiens disappeared from grower shelves and garden beds worldwide. Disease symptoms typically occur only after infections are well advanced, which makes it difficult to detect the disease and reduce the movement of infected plants. To improve early detection of impatiens downy mildew, a new DNA diagnostic tool was developed. This diagnostic tool was based on a newly identified DNA target that was shown to be highly unique to the impatiens pathogens. In laboratory tests, the diagnostic tool was shown to be highly sensitive and specific, detecting tiny amounts of the downy mildew pathogen without cross-reacting with non-target DNA. The diagnostic tool was sensitive enough to detect infections prior to symptom development. This sensitive new DNA diagnostic tool will be useful for plant health practitioners, breeders and horticulture specialists for detection of the impatiens downy mildew pathogens during early stages of infection, allowing targeted, time-sensitive disease treatments before the disease causes visible damage.

Technical Abstract: Impatiens downy mildew (IDM) disease is a primary constraint on the production of Impatiens walleriana, a popular and economically important floriculture plant. IDM is caused by the biotrophic oomycete Plasmopara destructor that emerged as a pathogen of I. walleriana in the 2000s. To enable P. destructor detection and quantification, a hydrolysis probe-based quantitative PCR diagnostic assay was developed based on unique orientation and order of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and ATP synthase subunit alpha (atp1) genes in the genus Plasmopara. Nucleotide sequences and analysis of the cox1/atp1 region distinguished P. destructor and its sister-species P. obducens, consistent with prior phylogenetic analyses using cox2 and rDNA markers. Specificity for P. destructor was incorporated into a hydrolysis probe targeting the cox1 gene and flanking primers that amplified across the cox1/atp1 intergenic region. The limit of detection was 0.5 fg/µL of P. destructor DNA (~100 plasmid copies/µL), with amplification efficiency = 0.95. The assay was validated against a panel of target and non-target oomycetes, which showed that the primers were specific for Plasmopara spp., while the probe was specific for P. destructor infecting both I. walleriana and I. balsamina. Testing of Impatiens tissue collected from 23 locations across 13 states indicated all samples with IDM symptoms tested positive for P. destructor. Asymptomatic plants from two locations also tested positive for P. destructor.