A Study On Synchronous Molecular Detection For Late Bright,Bacterial Ring Rot And Bacterial Wilt In Potato

Ralstonia solanacearum, Phytophthora infestans and Clavibacter michiganensis subsp.sepedonicus are bacterial and fungal diseases in potato production. Early detection ofpathogen has important significance to prevent and control these diseases.It is particularlyimportant that carrier state of pathogens was detected in potato tuber in early-onset disease.There are numerous approaches to detect pathogens in potato which have undergone atransition from the macroscopic to the molecular. The initial approaches, observation ofphytomorphology and symptom, physiological and biochemical analysis, serology andimmunology tests have evolved into PCR detection methods. Traditional pathogen detectionapproaches, symptom and pathogen culture can identify pathogens by naked eye ormicroscope to determine disease types. Early infected leaves and tubers cannot displayobvious symptoms onset, sprouts are also difficult to detect. There were several problemsthat have longer period time and low accuracy. Practical application of immune techniquewas restricted because pathogen have many strains and serotyping contains many types, it isdifficult to avoid undetected problems and people shouldn’t be satisfied with specificity ofserological analysis. PCR technologies and its rapid, sensitive and accurate advantages werewidely applied to the detection of pathogens. To achieve rapid detection of pathogens,several pathogens need to be detect simultaneously in the same PCR cycle. Multiplexpolymerase chain reaction (mPCR) is a modification technology of conventional PCR. It notonly retains the specificity and sensitivity of convential PCR, but also reduce the number ofsteps and reagents amount. Eventually, detection of numerous target pathogens is essential inone PCR cycle. Currently, with regard to molecular detection of mPCR in potato bacteriaand fungi diseases, molecular detection of two diseases has been reported. But simultaneousdetection of three or more diseases was not reported. So these major diseases were examinedin our research, conserved sequence and species-specific sequences of pathogen genomicsDNA were selected and cloned. Specificity and sensitivity of the primers of pathogens weretested. Then, multiplex PCR system was constructed and PCR optimize parameters wereoptimized. So molecular detection system was established in three potato bacteria and fungi.The main results as follows:1. DNA molecular of three pathogens (Ralstonia solanacearum, Phytophthora infestansand Clavibacter michiganensis subsp. Sepedonicus) were verified and cloned. According to three potato pathogens and its DNA molecular in PCR detection, related pathogens sequencewas searched in NCBI website. Sequences were compared among species, thenevolutionarily conserved specific sequences were screened. Finally, target sequences werevalidated in rDNA-ITS of Ralstonia solanacearum, cellulase gene A of pSC1vector ofPhytophthora infestans and Hrp cluster(Hrpc gene) of Clavibacter michiganensis subsp.sepedonicu vector, respectively. Genomic DNA or vector DNA of three pathogens wasapplied to DNA template. Primers were designed and synthesized to run a PCR amplification.rDNA-ITS region was cloned in Ralstonia solanacearum, full-length of cellulase gene A wascloned in pSC1vector of Phytophthora infestans, and Hrpc coding sequence were cloned inClavibacter michiganensis subsp. sepedonicu. Then amplification fragments were ligased topMD-19T vector respectively and the targeted fragments clone was obtained.2. Seed sequence and primers were selected and verified for three pathogens (Ralstoniasolanacearum, Phytophthora infestans and Clavibacter michiganensis subsp. Sepedonicus).According to the sequecing result and alignment information, homology were analysized andsequence differences sites were screened by DNAMan, Oligo, Primer software andNCBI-BLAST. Three specific-primers(PHY1/PHY2, CMS1/CMS2, RSO1/RSO2) weredesigned to detect pathogens of Ralstonia solanacearum, Phytophthora infestans andClavibacter michiganensis subsp. Sepedonicus. Meanwhile, sensitivity of three primers wasdetected between Pathogenic strain and other strains. Finally, seed sequence and primerswere verified for detection of three pathogens (Ralsonia solanacearum, Phytophthorainfestans and Clavibacter michiganensis subsp. Sepedonicus).3. A mPCR system of three pathogens (Ralstonia solanacearum, Phytophthorainfestans and Clavibacter michiganensis subsp. Sepedonicus detection was constructed byone-step method. Parameters were optimized in this mPCR system that contain annealingtemperature, cycle, dNTP concentration, enzyme concentration and ratio among threeprimers concentration. Electrophoretic bands are clear that showed there were highspecificity and sensitivity to detect three pathogens in mPCR system. So three pathogenscould be detected by a thermal cycler. Accuracy and reliability of the detection system wasconfirmed repeatedly by artificial inoculation on potato plant and field samples.81seedtubers were detected by the detection technology which produced in Gansu potato seedindustry. And further, it is proved that the mPCR technology is reliable.