建立以lipA和purH为靶基因的RTQ-PCR方法对水稻白叶枯病菌侵染过程进行定量分析

Development of a Real-Time Quantitative PCR Targeting lipA and purH for Quantification of Bacterial Infection Process of Rice by Xanthomonas oryzae pv. oryzae

【目的】建立水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)的分子定量法,测定Xoo侵染水稻植株后不同时间的种群量及其变化【。方法】选用以lipA和purH为靶基因序列的引物P4和P5,用SYBR Green I实时定量PCR(RTQ-PCR)分析在水稻接种植株内的病菌种群量。【结果】与细菌平板计数法相比,RTQ-PCR法定量结果基本相近或略高(≤10倍),变化趋势基本相似,用2对引物P4和P5进行RTQ-PCR定量无显著差异。接种3d的植株内己有菌量积累,但不显症;从接种5d开始,细菌明显增加,植株显症并逐渐加重;接种9～14d菌量增加到峰值,并进入稳定平台期,植株显症严重。病菌种群量与植株显症间的关系可能与细菌群体感应机制有关。【结论】用RTQ-PCR分子定量法可以直接对水稻植株内的Xoo进行动态定量研究;提出了水稻病害分子定量"病菌靶基因拷贝数-DNA总量-种群量-植物病症"的研究模式。

[Objective] To establish the novel molecular quantitative assays for quantification of bacterial population of Xanthomonas oryzae pv. oryzae （Xoo） in planta. [ Method ] Real-time quantitative polymerase chain reaction （RTQ-PCR） assay based on SYBR Green I technology was developed to target lipA and purH for the quantification of in planta growth of Xoo. [Result] The changes in bacterial population density in planta measured by RTQ-PCR assay is similar to those assessed by bacterial plate counting There is no significant difference between the two primer sets evaluated for RTQ-PCR. Bacterial accumulation within rice showing no disease symptoms was observed at 3 d post-inoculation （dpi）, then the bacterial density within rice increased significantly 5 dpi with rising bacterial leaf blight, and bacterial numbers reached a peak and maintained a high population 9-14 dpi when the plants displayed severe disease symptoms. Such a relation between bacterial population density in planta and host plant disease progression might be associated with quorum sensing of the pathogen. [Conclusion] The results of this study illustrate that RTQ-PCR can be successfully used to directly and accurately quantify Xoo within leaf tissues of rice. Furthermore, ＂bacterial target gene copies-total DNA amount-bacterial population-host disease progression＂, a hypothetical model of the pathogen assessment, has been proposed for accurate monitoring of bacterial infection process of rice by Xoo, which might be applicable to the molecular quantification of other bacterial and fungal diseases of rice.