Related Experiment Video
Updated: Aug 2, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Mitochondrial Loci Enable Specific Quantitative Real-Time PCR Detection of the Pathogen Causing Contemporary
Nicholas LeBlanc1,2, Frank Martin3, Vanina Castroagudín1,2
1United States Department of Agriculture, Agricultural Research Service (USDA-ARS), Mycology and Nematology Genetic Diversity and Biology Laboratory, Beltsville, MD 20705.
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 subunit1 (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 nontarget 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.
Insights
A new quantitative PCR assay accurately detects Impatiens downy mildew (IDM) caused by Plasmopara destructor. This diagnostic tool aids in identifying the pathogen in both symptomatic and asymptomatic Impatiens plants, crucial for disease management.
Area of Science:
- Plant Pathology
- Molecular Diagnostics
- Floriculture
Background:
- Impatiens downy mildew (IDM) significantly impacts the production of Impatiens walleriana, a key floriculture species.
- The oomycete Plasmopara destructor, identified in the 2000s, is the causal agent of IDM.
- Accurate detection and quantification methods are essential for managing this economically important plant disease.
Purpose of the Study:
- To develop a sensitive and specific quantitative PCR (qPCR) diagnostic assay for detecting Plasmopara destructor.
- To enable the quantification of P. destructor DNA in Impatiens plants.
- To differentiate P. destructor from closely related species like P. obducens.
Main Methods:
- A hydrolysis-probe-based qPCR assay was designed utilizing unique mitochondrial gene sequences (cox1 and atp1) in Plasmopara.
- Primers were designed to amplify the intergenic region between cox1 and atp1, while a probe targeted the cox1 gene for specificity.
- The assay's limit of detection, amplification efficiency, and specificity were validated against various oomycetes and Impatiens tissues.
Main Results:
- The qPCR assay demonstrated a low limit of detection (0.5 fg/μl) and high amplification efficiency (0.95).
- The assay specifically detected Plasmopara destructor in both Impatiens walleriana and Impatiens balsamina.
- Field testing confirmed the presence of P. destructor in all symptomatic Impatiens samples and in asymptomatic plants from two locations.
Conclusions:
- A novel qPCR assay provides a reliable tool for the specific detection and quantification of Plasmopara destructor.
- This diagnostic assay is effective in identifying IDM in both symptomatic and asymptomatic Impatiens plants.
- The developed assay will aid in the effective management and control of Impatiens downy mildew in commercial settings.
