Detection of <i>Phytophthora melonis</i> in samples of soil, water, and plant tissue with polymerase chain reaction

Abstract

A polymerase chain reaction (PCR) assay for the detection of Phytophthora melonis, the causal agent of phytophthora blight of cucumber, was developed. PCR primers specific to P. melonis (Pm1 and Pm2) were designed using sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA. More than 115 isolates representing 26 species of Phytophthora and 29 other species of pathogens were used to test the specificity of the primers. PCR amplification with these primers resulted in a product of ca 545 base pairs exclusively for isolates of P. melonis. The detection sensitivity with P. melonis primers was 100 fg of genomic DNA. A nested PCR procedure with DC6 and ITS4 as first-round primers, followed by Pm1 and Pm2 primers, increased detection sensitivity 1000-fold to 100 ag. With nested PCR, the detection sensitivity for the soil pathogens was 10 zoospores in 0.5 g of artificially inoculated soil. PCR with Pm1 and Pm2 was also used to detect P. melonis in naturally infected cucumber tissue, soil, and irrigation water. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogen in plant samples. The PCR-based methods developed could simplify both plant disease diagnosis and pathogen monitoring as well as help guide plant disease management.