Study On Molecular Detection Of Pseudomonas Syringae Pv. Actinidiae

Bacterial canker of kiwifiruit is a kind of devastating bacterial disease, which has become a great threat and one of the most serious factors that limit the cultivation of kiwifruit around the world. The disease has been included in the national forest plant quarantine objects, so it is necessary to strengthen the quarantine of the disease. Because of the latent infection, it’s very urgent to develop a rapid and precise method to detect the disease at its early infection stage for the early diagnosis of disease. In this study, a specific fragment of pathogenic bacteria was obtained from the strain of P. syringae pv. actinidiae and its similar strains by RAPD analysis. The fragment was cloned and sequenced. Based on the sequences of the specific fragment, a pair of specific primers was designed and synthesized to convert the RAPD markers into the SCAR markers. Then, optimized the amplification conditions of the specific primers and applied it into the detection of kiwifruit bacterial canker. The result of this study as follows:1. Using the single factor variable method to optimize the RAPD-PCR reaction conditions from aspects of template DNA dosage, primer dosage, dNTPs dosage, Mg2+dosage, Taq DNA polymerase dosage and annealing temperature. The optimized system is as followed:a total volume of25μL containing1μtemplate DNA (100ng/μL);2μL Mg2+(25mmol/L);1μL primer (12.5μmol/L);2μL dNTPs (10mmol/L);0.2μL Taq DNA polymerase (5U/μL);10×Buffer2.5μL and16.3μL sterilized distilled water. Besides, PCR was carried out in a thermo cycler programmed for40cycles consisting of a denaturing at94℃for60s, and annealing step at36℃for60s, and an extension step at72℃for120s. These cycles were preceded by an initial denaturing step at95℃for5min, and ended with a final extension at72℃for10min.2. The specific random primer P7(5’-CGCAGCCGAGAT-3’) was screened from120random primers, which can only detect P. syringae pv. actinidiae from all the tested strains, and obtained a1300bp specific fragment of P. syringae pv. actinidiae.3. Based on the sequences of the specific fragment, a pair of specific primers (F7:5’-CGATTCGCAGCCGAGATG-3’; R7:S’-CTACGAGGTTAGGTTCAGAGT-3’) was designed, which convert the RAPD marker into SCAR marker successfully. For verification of the designed primer set, the result indicated that it has strong specificity for detection the P. syringae pv. actinidiae, and the sensitivity of the primer set for genomic DNA of Pseudomonas syringae pv. actinidiae was100fg/μL.4. The specific primer, used in disease detection, not only can detect the Pseudomonas syringae pv. actinidiae from the tissues of kiwifruit branches which have been inoculated the pathogenic bacteria, but also can detect the Pseudomonas syringae pv. actinidiae from infected tissues collected from kiwifruit orchard. Meanwhile, regardless of whether infected branches showed symptom or not, using SCAR specific primer can detect the pathogens. In addition, the designed primers have been compared with four pairs of published primers. The comparison concluded that only the primers designed in this study was specific to detect bacterial canker of kiwifruit in Sichuan.In a word, using the specific primer F7/R7under the optimized PCR reaction conditions, combined with the simple method of reagent kit for DNA extraction, the molecular detection for the pathogen of kiwifruit canker can be completed in a short time.