Related Experiment Video
Updated: May 14, 2026
On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System
Published on: February 23, 2018
Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR
J A J Clément1, T K Baldwin, H Magalon
1INRA, UMR1349 IGEPP, Le Rheu, France. jajclement@gmail.com
Abstract:
Molecular tools that allow intraspecific quantification and discrimination of pathogen isolates are useful to assess fitness of competitors during mixed infections. However, methods that were developed for quantifying Phytophthora infestans are only specific at the species level. Here, we reported a TaqMan-based real-time PCR assay allowing, according to the specificity of the used probes, an accurate quantification of different proportions of two genetically distinct clones of P. infestans in mixed fractions. Indeed, in addition to a primer specific to P. infestans, two primers and two TaqMan(®) probes that target single-nucleotide polymorphisms located in the Avr3a/avr3a virulence gene sequence were designed. The reliability of the method was tested on serially diluted fractions containing plasmid DNA with either the Avr3a or the avr3a sequences at concentrations ranging from 10(2) to 10(8) copies per μl. Based on its specificity, sensitivity and repeatability, the proposed assay allowed a quantification of the targeted DNA sequence in fractions with a Avr3a/avr3a ratio in the range 1/99 to 99/1. The reliability of the test was also checked for counting zoospores. Applications for future research in P. infestans/host quantitative interactions were also discussed.
Insights
A new real-time PCR assay accurately quantifies distinct clones of Phytophthora infestans during mixed infections. This molecular tool enhances understanding of pathogen competition and fitness in plant diseases.
Area of Science:
- Plant Pathology
- Molecular Biology
- Genetics
Background:
- Accurate quantification of pathogen isolates is crucial for understanding mixed infections and competitor fitness.
- Existing methods for Phytophthora infestans quantification lack specificity at the clonal level.
Purpose of the Study:
- To develop a highly specific TaqMan-based real-time PCR assay for discriminating and quantifying genetically distinct clones of Phytophthora infestans.
- To enable accurate assessment of clonal proportions in mixed infections.
Main Methods:
- Designed primers and TaqMan probes targeting single-nucleotide polymorphisms in the Avr3a/avr3a virulence gene.
- Validated the assay using serially diluted plasmid DNA with known Avr3a/avr3a ratios (10^2 to 10^8 copies/μl).
- Tested the assay's reliability for quantifying DNA in mixed fractions and counting zoospores.
Main Results:
- The developed assay accurately quantifies different proportions of two P. infestans clones in mixed samples.
- Demonstrated specificity, sensitivity, and repeatability across a wide range of DNA concentrations (Avr3a/avr3a ratios from 1/99 to 99/1).
- Confirmed reliability for quantifying pathogen DNA and counting zoospores.
Conclusions:
- The novel real-time PCR assay provides precise intraspecific quantification and discrimination of Phytophthora infestans clones.
- This tool facilitates future research on quantitative interactions between P. infestans and its hosts.
- Advances the study of pathogen population dynamics and competitive fitness.
