Development of ralstonia solanacearum quantification method and its application in peanut with bacterial wilt disease | 青枯菌定量检测方法的建立及其在花生中的应用

中国人. 【目的】建立快速、准确的青枯菌定量检测方法,研究青枯菌与寄主植物的互作。【方法】以青枯菌hrpB为靶基因,利用实时荧光定量PCR(RTQ-PCR)方法建立青枯菌的定量检测方法,并利用该方法检测花生接种青枯菌后细菌数量的变化动态。【结果】以提取的细菌DNA为模板和以青枯菌的全细胞为模板均能对青枯菌准确定量,在未富集细菌的前提下,利用全细胞细菌定量的最低限为103 CFU/mL,检测的线性范围为103―108 CFU/mL,计数细菌的方法能准确反应青枯菌数量的差异,但该数值约是细菌平板计数法计数的1.5倍。利用RTQ-PCR计数细菌,发现接种后的3―5 d,是花生与青枯菌互作的关键时期。花生节对抑制青枯菌的扩展具有重要的作用。【结论】RTQ-PCR计数细菌是一种简单、快速、高通量的青枯菌定量方法。

英语. 【Objective】The aim of this study was to build rapid and accurate detection and quantification of R. solanacearum and study the interaction between bacterial and its host. 【Method】 In this study, real time quantitative PCR (RTQ-PCR) targeting the hrpB was developed for the quantification of bacteria load and the method was used to analyze the bacterial population dynamics in peanut after R. solanacearum inoculation.【Result】The results demonstrated that both the extracted DNA and whole cell of R. solanacearum could be used as template for bacterial quantification. Without enrichment step, the detection limit of the method using whole cell as template was 103 CFU/mL, the liner range was in 103-108 CFU/mL, the established RTQ-PCR method could be used for R. solanacearum quantification but the calculated number was about 1.5-fold compared with the traditional plating method. The application of the method in peanut suggested that 3-5 d post inoculation is of vital importance for peanut and R. solanacearum interaction and the node site in peanut may play a role in restriction of bacterial proliferation.【Conclusion】 RTQ-PCR is a simple, rapid and high throughput method developed for R. solanacearum quantification.