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A Quantitative Real-Time PCR Method to Detect the Quinoa Downy Mildew Pathogen, Peronospora variabilis
Anna Louise Testen1, Purnima Puri2, R Scott Shaw1
1USDA-ARS Application Technology Research Unit, Wooster, OH.
Plant Disease
|May 20, 2024
Summary
A new quantitative real-time PCR (qPCR) assay effectively detects and quantifies quinoa downy mildew (Peronospora variabilis) in seeds and plant tissues. This sensitive method aids in understanding quinoa disease epidemiology and improving crop management strategies.
Area of Science:
- Plant Pathology
- Molecular Diagnostics
- Agricultural Science
Background:
- Quinoa downy mildew, caused by *Peronospora variabilis*, is a major global threat to quinoa production.
- Accurate and sensitive diagnostic tools are crucial for managing this devastating disease in seeds and plant tissues.
- Existing methods may lack the sensitivity or speed required for timely detection and quantification.
Purpose of the Study:
- To develop and validate a highly sensitive quantitative real-time PCR (qPCR) assay for the detection and quantification of *Peronospora variabilis*.
- To assess the assay's performance against various *P. variabilis* strains and related oomycetes.
- To apply the developed qPCR assay for quantifying *P. variabilis* in quinoa seeds from field trials.
Main Methods:
- Development of a hydrolysis probe-based qPCR assay with a competitive internal control for *P. variabilis* detection.
- Determination of assay sensitivity (limit of detection) and efficiency using known DNA quantities and ITS copy numbers.
- Testing for specificity against DNA from other downy mildew pathogens and related oomycetes.
- Application of the assay to quantify *P. variabilis* in quinoa seed washes from field-grown genotypes.
Main Results:
- The qPCR assay demonstrated high sensitivity, detecting as low as 20 ag of DNA or approximately 25 ITS copies per reaction, with efficiencies between 93.9% and 98.2%.
- No cross-amplification was observed with DNA from other pathogens, confirming assay specificity.
- *P. variabilis* was successfully detected and quantified in quinoa seeds from all tested genotypes in a Washington field trial, with significant variations in pathogen load among genotypes.
Conclusions:
- The developed qPCR assay provides a rapid, sensitive, and specific tool for detecting and quantifying *Peronospora variabilis* in quinoa.
- This assay facilitates improved disease diagnosis, management, and a deeper understanding of quinoa downy mildew epidemiology.
- The findings support the use of this molecular tool for monitoring seed health and assessing disease risk in quinoa cultivation.
