Study On Molecular Detection Techniques Of The Potato Scab Pathogens

Potato scab is an important economic disease in potato production. Several plantpathogenic Steptomyces spp may cause the disease in certain environment. Due to thediverse composition of the pathogen, it is difficult to detect the specific pathogen quicklyin disease research. With the development of a variety of PCR technology and applicationin other pathogens detection, that provides useful references for exploring the moleculardetection methods to potato scab pathogens. On the bases of typical pathogenic strainscollected from different areas in China, the qualitative detection methods of scabpathogens were established by screening specific primers and reaction system. Thequantitative detection method also formed by quantitative PCR in this work. The mainresults of experiments as follows:1. Qualitative detection of potato scab pathogens: The primers were designedaccording to the sequences of four genes txtA, txtB, txtC (P450) and txtD (nos) those comefrom pathogen-specific toxin biosynthesis gene cluster. The genomes of four typical scabpathogens CPS-1(S. scabies), CPS-2(S. galilaeus), CPS-3(S. acidiscabies) and CPS-4(S.turgidiscabies) were selected to screen detection primer for PCR amplification. The bestone (B1/B2) having a higher specificity to all test pathogenic strains was selected asgeneric detection primer. After optimized the sensitivity of reaction system is20pg/μL ofgenomic DNA. Based on the concentration quantitative conversion between nucleic acidand spore suspension, the minimum detection threshold for CPS-1, CPS-2, CPS-3andCPS-4were2.48CFU/μL,3.815CFU/μL,4.035CFU/μL and4.035CFU/μL respectively.The results showed that the method can be used to qualitative detect genome, sporesuspension, scabby tuber tissues and soil.2. Quantitative detection of potato scab pathogens: the qPCR primers were designedaccording to the nucleotide sequences amplified by primers B1/B2. The primersRTA1/RTA2were selected for qPCR detection primer because they have stableamplification results, better fragment size and no primer dimer formation. A certain ofspore suspension belong to four typical strains were taken to extract DNA. The DNA wasdiluted to a series of concentration which be used as template for qPCR amplification. Four standard curves were established by qPCR. The soil samples test results showed that it canbe quantitatively determined the samples.3. The inoculation spore suspension of CPS-1was determined through the greenhousepot test. The results showed that the new potato tubers will have scab symptoms when thespore concentration over7.35×103CFU/mL.