Rapid Isolation And Identification Of A Pathogen Causing Mulberry Bacterial Blight And A Method For Quantification Of Its Viable Cells

Mulberry bacterial blight caused by Pseudomonas syringae pv. mori is a serious disease, causing huge economic losses to each year in our country due to the lack of effective prevention and control measures. Among recent years, the early diagnosis, used as an important means of prevention and control of the phytopathogens is becoming popular. Through the early detection of pathogens, the severity of disease outbreaks is informed to in advance so that the reasonable and effective control measures were developed.A method was established for rapid isolation of pathogenic P. syringae pv. mori strain. Firstly, based on the stable fluorescent effect produced by P. syringae pv. mori strain processed under low temperatures, prescreening of the target strains was done. Secondary screening was done based on detecting pathogenic hrpZ gene from prescreening strains. After pathogenic tests, the observation of cell morphology and sequence analysis of 16S-rDNA, four pathogenic isolates including Psm13-7, Psm14-1, Psm14-7, Psm15-2 causing typical symptom of mulberry bacterial blight were obtained. Screening of target strains spent 15 days, compared to traditional method of screening, screening time shortens 60 days, screening efficiency increases 70%. This developed method is providing reliable referential criterion for isolation and screening of other pathogenic Pseudomonas syringae strains.Real-time quantitative PCR method was established for detecting and quantifying pathogenic P. syringae pv. mori. By ananlyzing the different fagments between P. syringae pv. mori and other P. syringae pathovars, a specific primer pair, Psm240F/Psm434 R is selected. Agarose gel electrophoresis and DNA sequencing verify that the primer pair can specifically amplify the sized 195 bp DNA fragment from P. syringae pv. mori, but can amplify non-specific fragments from the other pathogenic P. syringae pathovars and other species of the pathogens. The genomic DNA and bacterial suspension of P. syringae pv. mori are used as the templates for real-time quantitative PCR respectively, in 6 × 10^-5 6 ng/?L of genomic DNA, a good linear relationship is showed between Log DNA concentration and Ct values and the equation is y =-3.224 69 x + 14.034 45, R² = 0.997 09, and the lowest limit of detection was 60 fg/?L. In 10² 10⁸ CFU/mL of bacterial suspension, a good linear relationship is showed between Log bacterial concentration and Ct values, and the linear equation y =-4.56215 x + 46.91167, R² = 0.98872, and the lowest limit of detection was 102 CFU/mL. This method provides reliable technique supports for early diagose of mulberry bacterial blight.Pathogenic viable P. syringae pv. mori cells was firstly dectected based on PMA-qPCR. PMA?Propidium Monoazide? is used to pretreatment bacterial cells of P. syringae pv. mori, using 20 ng/uL concentration of PMA, incubating PMA for 10 min, exposure of PMA for 10 min, and completely inhibit 108 CFU/mL of dead cells, and have no influence on the DNA amplification of viable cells in 10¹ 10⁸ CFU/mL. A good linear relationship is showed for PMA-qPCR between Ct value and Log bacterial cells concentration, and the linear equation is y =-3.57193 + 46.04443, R² = 0.99721, the lowest limit of detection was 101 CFU/mL. This improved method avoided error evaluation of mulberry bacterial blight largely.In the present study, a method is firstly established to detect and quantify viable cells of P. syringae pv. mori, and provides a new technical support for the early dignose and effective prevention and control of mulberry bacterial blight, and meanwhile, has important practical significance for reducing the losses and protecting sustainable development of sericulture industry.