水稻白叶枯病菌和细菌性条斑病菌的分子标记筛选及检测  

Screening and detection of diagnostic molecular markers to distinguish Xanthomonas oryzae pv.oryzae from Xanthomonas oryzae pv.oryzicola

作　　者：冯雯杰 常清乐 杨龙[1] 丁新华[1] 储昭辉[1] 

机构地区：[1]作物生物学国家重点实验室/山东省农业微生物重点实验室,山东农业大学植物保护学院,泰安271018

出　　处：《植物病理学报》2013年第6期581-589,共9页Acta Phytopathologica Sinica

基　　金：国家自然科学基金(31071381;30900050);高等学校博士学科点专项科研基金(20093702120013);山东省优秀中青年科学家科研奖励基金(BS2010NY017)

摘　　要：水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)和细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola)是水稻种子产地检疫中最重要的两种检疫对象,且同属于水稻黄单胞杆菌。本研究基于生物信息学技术构建比较基因组学算法对两种病原的全基因组序列比对分析,得到一系列能够区分两种病原的特异性PCR引物。结合简单的PCR技术及全自动DNA分析系统,我们选取了12对引物分别对23株水稻白叶枯病菌和5株水稻细菌性条斑病菌及其它相关菌株进行验证。结果获得了2对显性标记(Xoo-Hpa1和Xoc-ORF2)以及3对共显性分子标记(M568、M897和M1575)可以达到理想的区分检测两种病原的效果。分子标记的检测灵敏度从5×104到5×107cfu·mL-1不等,且从水稻种子浸提液中也能成功地检测水稻白叶枯病菌和细菌性条斑病菌。本研究丰富了检测标记的靶位点,并有效的结合了高通量检测的手段对多位点联合分析,增强了检测的可靠性,有望在今后的植物检疫及病原鉴定中发挥着重要的作用。Xanthomonas oryzae pv. oryzae （ Xoo ） and Xanthomonas oryzae pv. oryzicola （ Xoc ） that cause rice bacterial blight and bacterial leaf streak respectively are the two most important targets of rice seed quarantine, and belong to Xanthomonas genus. In this study, a series of specific PCR primers were designed to distinguish Xanthomonas oryzae pv. oryzae from Xanthomonas oryzae pv. oryzicola based on computational analysis of their whole genomic sequence. Twelve pairs of primers were selected for PCR test in total 33 strains, including 23 Xoo, 5 Xoc and 5 other reference strains. The PCR products were separated with more rapid and accurate detec- tion system, either the QIAxcel system or the LabChip GX system. As a result, five molecular markers were i- dentified to remarkably differentiate between Xoo and Xoc, which included two dominant markers （Xoo-Hpal and Xoc-ORF2） and three codominant markers （M568, M897 and M1575）. The detection sensitivity ranged from 5x104 to 5 x 107 cfu · mL1. In addition, both pathogens could be detected from rice seed suspension with all polymorphic markers. As well as developing five new markers, the result is also particularly prospected to play an important role to serve in rice quarantine because the reliability and effectiveness of diagnosis has been increased by using conjoint analysis with multiple markers and high-throughput DNA quality assessment.

关 键 词：水稻白叶枯病菌 水稻细菌性条斑病菌 分子标记 植物检疫 

分 类 号：S435.111[农业科学—农业昆虫与害虫防治]