Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR assay that targets polymorphisms of the Avr3a gene

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.