基于实时荧光定量PCR技术检测桑丁香假单胞菌

Detection of Pseudom onas syringae pv. mori Based on Real-time Quantitative PCR Technology

由桑丁香假单胞菌(Pseudomonas syringae pv.mori,以下简称Psm)引起的桑疫病是一种毁灭性的桑树细菌性病害,建立对病原菌的早期检测技术,有利于及时制定病害的防控技术措施。依据致病性相关基因hrp Z的片段设计的一对引物能够在Psm中特异性地扩增出195 bp大小的条带,而用于对其他致病变种的丁香假单胞菌和其他种属病原菌的扩增则没有此条带。利用Psm14-7菌株基因组DNA为模板进行实时荧光定量PCR(q PCR)扩增,模板DNA质量浓度在6×10^(-5)~6 ng/μL范围内与扩增所得Ct值呈现良好的线性关系,线性方程为y=-3.224 69x+14.034 45(R^2=0.997 09),检测的最低限为(60±0.001 2)fg/μL;以Psm14-7菌液为模板进行q PCR,菌液浓度在1×10~2~1×10~8CFU/m L范围内与Ct值呈现良好的线性关系,线性方程为y=-4.562 15x+46.911 67(R^2=0.988 72),最低检测限为(10~2±0.008 3)CFU/m L。于桑树植株人工接种Psm后不同时间,对出现各种症状植株中的菌群数量进行q PCR检测,结果显示:接种后的第4~8天可能为病原菌诱导桑树的过敏性反应和系统获得性抗性关键时期,影响到了Psm的增殖速度;能引起健康桑树暴发桑疫病的致病菌最低浓度为(1.5×10~8±0.076 8)CFU/g。建立的q PCR检测方法,对由Psm引起的桑疫病的田间早期诊断和及时防控具有一定指导意义。

Mulberry bacterial blight,caused by Pseudomonas syringae pv. mori（ Psm）,is a devastating bacterial disease of mulberry. Establishing an early detection technology against its pathogenic bacteria is beneficial to establish effective measures used for prevention and control of the disease. A band of 195 bp was specially amplified from Psm with one primer pair designed based on pathogenicity-related gene hrp Z of Psm,but no fragment of the same size was amplified from other P. syringae pathovars and bacterial species. Using the genomic DNA of Psm14-7 strain as template,the result of real-time fluorescent quantitative PCR（ q PCR） showed that the mass concentration of genomic DNA had a good linear relationship with amplified Ct value within the range of 6 × 10^（-5）to 6 ng / μL,the linear equation was y =- 3. 224 69 x ＋ 14. 034 45（ R^2= 0. 99709）,and the minimum detection limit was 60 ± 0. 001 2 fg / μL. Using the bacterial solution of Psm14-7 strain as template,q PCR showed that the mass concentration of genomic DNA had a good linear relationship with Ct value within the range of 1 × 10^2 to 1 × 10^8 CFU / m L,the linear equation was y =- 4. 562 15 x ＋ 46. 911 67（ R2= 0. 988 72）,and the minimum detection limit was（ 10^2± 0. 008 3） CFU / m L. Various symptoms occurred on mulberry trees at different days after artificial inoculation with Psm. q PCR detection of bacterial quantity in mulberry indicated that day 4 to day 8 after inoculation was the critical period of anaphylactic reaction and systemic acquired resistance of mulberry induced by pathogenic bacteria,and the proliferation rate of Psm was influenced during the period. The lowest concentration of pathogenic bacteria causing mulberry bacterial blight was（ 1. 5 × 10^8± 0. 076 8） CFU / g. The established q PCR method could provide a certain guidance for early diagnosis and prevention and control of mulberry bacterial blight caused by Psm in the fields.