魔芋软腐病病原菌TaqMan荧光探针PCR技术的建立及应用

Establishment and application of TaqMan fluorescence probe real-time PCR detection for the pathogen of konjac soft rot

为实现对田间土壤软腐病病原菌的定量检测,基于魔芋软腐病优势病原菌胡萝卜软腐果胶杆菌胡萝卜亚种Pectobacterium carotovorum subsp. carotovorum的FyuA基因序列,设计特异性引物PCC1/PCC2/PCC3,建立TaqMan荧光探针实时荧光定量PCR技术,并对魔芋根系土壤中软腐病病原菌进行动态监测。结果显示:基于FyuA基因序列设计的引物特异性好,仅能特异性检出胡萝卜软腐果胶杆菌胡萝卜亚种;当模拟带菌土壤中病原菌浓度低至1.88 CFU/g时也能检出,灵敏度高;发病魔芋根际土壤中软腐病病原菌检出率为100.00%,病原菌DNA浓度最高达到了7.52×10~7ng/μL,健康魔芋根际土壤中也存在病原菌,检出率为40.00%;不同种植模式中,林下魔芋土壤中软腐病病原菌数量较少;连作时间与病原菌数量、病情指数存在正相关关系,连作时间越长,病原菌积累越多,魔芋病情指数也越高,魔芋连作4年土壤中病原菌DNA浓度最高达到4.03×10~4ng/μL;对魔芋土壤软腐病病原菌进行全年监测,病原菌数量随着月份增长逐渐上升,在8-10月达到峰值543.20 ng/μL后下降,病原菌数量与魔芋病情指数变化规律一致,但田间魔芋软腐病的发生相对滞后。表明建立的TaqMan荧光探针实时荧光定量PCR技术可用于田间魔芋软腐病的监测。

In order to quantify the pathogen of konjac soft rot in field soils, a TaqMan fluorescent probe PCR detection system was established. The specific primers PCC1/PCC2/PCC3 were designed based on FyuA gene sequence of Pectobacterium carotovorum subsp. carotovorum. The results showed that the dominant pathogen of konjac soft rot P. carotovorum subsp. carotovorum could be detected specifically and sensitively, with a threshold as low as 1.88 CFU/g. The detection rate of the pathogen of soft rot in the rhizosphere soil of konjac was 100.00% by real-time fluorescence quantitative PCR, and the DNA concentration of the pathogen was up to 7.52×10~7 ng/μL. Moreover, the pathogen was found in the rhizosphere soil of healthy konjac, with a detection rate of 40.00%. The number of soft rot pathogen was lower when konjac was planted under the trees. There were positive correlations between the continuous cropping time and the disease index as well as the number of pathogen. Longer cropping period caused more pathogenic bacteria accumulated and higher incidence rate. Furthermore, the pathogen DNA concentration was as high as 4.03×10~4 ng/μL in the soil where konjac had been continuously cropped for four years. The pathogen of soft rot in konjac soils was monitored in the whole year, showing that the pathogen gradually increased from January, reached the peak(543.20 ng/μL) from August to October, and then decreased after October. The number of pathogen was consistent with the disease index, but the incidence of the disease was relatively delayed in the field. It indicated that this method was suitable for monitoring and preventing the konjac soft rot in the field.