Molecular Distinction Of Phytophthora Melonis And Detection Of P. Melonis And P. Sojae

Mophological and molecular distinctions among Phytophthora melonis and P. sinensisand P. drechsleri were studied. Phylogenetic analyses were performed on regions of nuclearand mitochondrial gene sequences. Rapid and accurate methods for specific detection of P.melonis and P. sojae were developed. The conventional, nested and real-time PCR methodwe developed could detect P. melonis and P. sojae in the oospores, zoospores, infected planttissues, infested soil and irrigation water samples.Three isolates of Phytophthora melonis were isolated from cucumber, wax gourd andgourd. These 3 isolates and 1 isolate of P. drechsleri were identified based on colonymorphology, size, pathogenicity and so on. Molecular analysis were performed on regionsof nuclear gene sequences (internal transcribed spacer; translation elongation factor 1a;β-tubulin) and mitochondrial (cytochrome c oxidase subunit 1; NADH dehydrogenasesubunit 1). Sequences from the above isolates were compared with published sequences of14 isolates of P. melonis, P. sinensis, P. sojae, P. vignae, P. drechsleri and P. cryptogea inGenbank database and phylogenetic trees were produced based on ITS,β-tubulin, EF-1a,cox1 and nadh1 sequences data by "Neighbour-joining" methods of the software Mega. Thecombined results of morphology, pathogenicity and gene sequences analysis indicate that P.melonis is synonymous with P. sinensis and it's a different species from P. drechsleri. Wewould suggest therefore that P. sinensis and similar isolates are subsumed into a re-definedP. melonis.A polymerase chain reaction (PCR) assay for the detection of Phytophthora melonis,the causal agent of Phytophthora blight of cucumber, was developed. PCR primers specificto P. melonis (Pm) were designed using nuclear ribosomal DNA internal transcribed spacer(ITS) sequences. More than 115 isolates representing 26 species of Phytophthora and 29other species of pathogens were used to test the specificity of the primers. PCRamplification with these primers resulted in a product of ca 545 bp, exclusively fromisolates of P. melonis. The detection sensitivity with Pm primers was 100 fg of genomicDNA. A nested PCR procedure using DC6/ITS4 as the first-round primers, followed by Pmprimers Pm1/Pm2 increased detection sensitivity 1000-fold to 100 ag. The detection sensitivity for the soil pathogens was 10 zoospores in 0.5g of artificially inoculated soilusing nested PCR. PCR with the Pm primers was also used to detect P. melonis in naturallyinfected cucumber tissue, soil and irrigation water. Real-time fluorescent quantitative PCR(qPCR) assays were developed to detect and monitor the pathogen in plant samples. ThePCR-based methods developed could simplify both plant disease diagnosis and pathogenmonitoring as well as help guide plant disease management.Phytophthora sojae is an ecologically important pathogen. In this study, PCR assayswere developed with primer pair PsYpt3F/PsYpt2R derived from ras-related protein genesfor rapid detection and identification of this organism. The specificity of this primer pairwas evaluated against 52 isolates of P. sojae, 58 isolates from 25 other Phytophthora spp.,and 54 isolates of Pythium spp. and true fungi. PCR with this primer pair amplified theDNA from all isolates of P. sojae regardless of origin. The PsYpt3F and PsYpt2R primersshowed adequate specificity among all other species tested. Nested PCR withYph1F/Yph2R and PsYpt3F/PsYpt2R primers detected the pathogen at levels of 10 pg ofgenomic DNA, a single oospore or 1 zoospore, respectively. The PCR assay, combinedwith a rapid NaOH lysis technique, could also be used to detect P. sojae from inoculatedsoybean tissue. In addition, real-time fluorescent quantitative PCR (qPCR) assaysconjugated with the fluorescent SYBR^? Green I dye were developed to detect this pathogen.The conventional and real-time PCR assays provide a rapid and sensitive tool for thedetection of P. sojae at port or in production fields.