Study And Application Of Molecular Detection Technique Of Phytophthora Sojae

Phytophthora root rot caused by phytophthora sojae kaufmann & Gerdemann, Is one of the most destructive disease in soybean and also the quarantine pathogens For P.R.China. Phytophthora sojae could be spread as oospores carried in soybean Seed, as well as oospores existed in soil and disease soybean residues. since more and more soybean was imported into China, to establish rapid and accurate molecular detection methods to Phytophthora sojae are very important to soybean trade between China and other countries,also is necessary to control the propagation of Phytophthora root rot.In this study ,p sojae and other fungusgenome DNA were amplified by the polymerase chain reaction (PCR) with the universal primers ITS1 and ITS4 in the internal transcribed spacer regions of the 18S—28S.products were sequenced and were aligned with other phytophthora and homogeneous species in GeneBank. A phylogenetic tree was constructed by DNAMAN software. A specific primer to p. sojae was designed:BJF and BJR. PCR amplification with the primers results in the amplification of a product of approximately 650 bp ,exclusively from isolates of p. sojae. Tests with p sojae total genomic DNA determined that the sensitivity of primers BJF and BJR is about 0.1 fg.We designed a specific probe :PS-probe and primers Ps-1 and Ps-2 according to difference of ITS sequences between P. sojae and homogeneous fungi .The Real-time fluorescence PCR method that can specifically detect p.sojae was established by optimizing reaction system and condition .The method sensitivity which is about 0.01 fg is 100 times higher than regular PCR.According to solution protein specific of P. sojae. SDS-PAGE detection method is established. Using the method Phytophthora root rot can be identified directly. The method provide a rapid and accurate way for plant quarantine department.In the research we established three molecular detection methods which can rapidly detect p.sojae from plants,seeds and soil. These methods provide theoretic base and technical assurance for port, as well as technical support for soybean production and trade.