水稻细菌性条斑病原菌室内快速分离与鉴定方法

A Rapid Laboratory Method for Isolation and Identification of Pathogen Causing Rice Bacterial Leaf Streak

本研究以海南海口罗牛山基地、屯昌水稻品种区试基地采集的感染细菌性条斑病(细条病)的水稻叶片为研究对象,探索细条病菌室内分离、鉴定与致病性测定方法。结果表明,70%酒精消毒1 min,10~4~10~5稀释倍数,培养3 d是有效分离细条病菌的优化因素;随机挑取的12个单克隆菌株经分子鉴定及致病性鉴定均为细菌性条斑病菌,且16S rDNA PCR产物测序序列与细条病菌模式菌株RS105一致性为100%。在所有的接种方法中均显示,浸染后保湿培养是影响致病效果的关键因子,在发病率方面,菌液浸泡抽真空、针刺法是最有效的方法,致病率均为100%。本研究结果为细条病菌的室内分离鉴定提供标准方法,有助于抗细条病种质资源的大规模筛选。

In this study, the leaves infected by bacterium leaf streak from Luoniushan and Tunchang in Hainan province were taken as research object to explore a rapid laboratory method for isolation and identification of pathogen causing rice bacterial leaf streak. The results revealed that the optimal factors to separate pathogen isolates were 70% ethanol disinfect, 10^4-10^5 dilution times and cultured for 3 days. Total twelve isolates were identified as Xanthomonas oryzae pave. oryzicola （Xoc） based on their molecular biology identification and patho- genic determination. The sequence of 16S rDNA PCR isolates shared 100% similarity with the model strain RS 105. At last, keeping humidity was the key factor of pathogenic determination in all of inoculation methods. The pathogenic incidence of vacuum pumping and that of acupuncture were 100%, both of them were the most effective inoculation method. These results provided a standard protocol for isolation and identification of Xoc, which contributed to high-throughput screen resistance resource of rice.