【作者】 周训军；

【导师】 高必达； 赵廷昌；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2012， 硕士

【摘要】 引起烟草青枯病的病原是青枯雷尔氏菌(Ralstonia solanacearum),该病是危害全球烟草生产的重要病害之一。烟草青枯病在我国长江流域及其以南烟区均造成危害,且危害程度和范围有逐年扩大的趋势。但是由于烟草青枯病菌存在丰富的种内遗传多样性,使其防治十分困难,到目前为止仍没有发现一种安全有效的防治药剂。因此,只有通过研究烟草青枯菌种内的遗传多样性,掌握其遗传变化规律,才能为选育抗病品种和研制新型高效的防治药剂提供理论依据。我国迄今为止只有少数产烟省区对省内的烟草烟草青枯菌进行了种内遗传多样性研究,但尚未对全国主要产烟省区烟草青枯菌进行全面的种内遗传多样性的研究。本研究采集了全国10个主要产烟省区不同烟草品种共计167株烟草青枯病疑似病样进行分离鉴定,并首次运用多位点序列分型(Multilocus Sequence Typing, MLST)对烟草青枯病菌进行种内遗传多样性分析,研究结果如下：(1)对2011年度采集自四川、云南、贵州、重庆、湖南、湖北、广东、广西、江西和安徽等十个主要产烟省区的167株烟草青枯病疑似病样进行分离鉴定,结果有67个烟草青枯菌菌株的16SrDNA序列与NCBI上已登录的Ralstonia solanacearum菌株的16SrDNA序列一致性达到99%或以上,确定为烟草青枯病菌。将分离鉴定得到的67株烟草青枯病菌刺伤接种到温室内的烟草幼苗,都能使烟草发病,但发病时间和发病程度稍有差异。(2)利用七个看家基因rfbD、efp、pstS1、dnaQ、ureG、ppa、adk对烟草青枯病菌进行MLST分析表明,参试的93株烟草青枯菌共被划分为51个序列型(sequence type, ST),运用程序eBURST V3分析,所有菌株被划分为4个亚群(group)和四个单一群(singleton):亚群1包含24个ST的50个菌株；亚群2包含19个ST的32个菌株；亚群3包含2个ST共2个菌株；亚群4包含2个ST的5个菌株；四个单一群各包含1个菌株分别采自云南、浙江、浙江、广西。从地理区域看,亚群1的核心型ST1包含了除江西省的所有采样地区菌株,虽然亚群2的核心型ST40的两个菌株都来自安徽,但亚群2中各菌株之间的联系比较松散,并没有与核心型菌株紧密联系在一起,表明我国烟草青枯病菌的地理分布极为广泛,但MLST组群的划分与菌株的地理区域没有直接的联系。从烟草品种看,亚群1的核心型ST1包含的菌株分别采自7个不同的烟草品种,亚群1中所有菌株则来自10个不同的烟草品种,亚群2的核心型ST40的两个菌株都采自同一烟草品种(云烟97),但亚群2中所有菌株分别来自6个不同的烟草品种。根据ST的划分也可以看出,不同烟草品种上分离的菌株可以被划分到同一ST,而同一烟草品种分离的不同菌株则可以被划分为不同的ST,且烟草品种与ST划分之间并没有规律。这一实验结果表明,烟草青枯菌的组群划分与烟草品种无关。更多还原

【Abstract】 Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases all over the world. Occurrence of Tobacco bacterial wilt in the Yangtze river basin and south of this area increasingly damaged and spread in recent years. However, the disease is hardly controlled for lack of efficient pesticide due to its rich variations. Therefore, to breed resistant varieties and develop new efficient pesticide, the study on genetic diversity of R. solanacearum should be carried out adequately.The study on genetic diversity of R. solanacearum in nationwide has not been carried out although some of the researches have been done in several provinces.167suspected samples from10main tobacco producing provinces were isolated, identified and typed by using MLST (Multilocus Sequence Typing) firstly to phylogenetic analysis. The results are as follows:(1)67strains isolated from167suspected infected samples that collected from Sichuan, Yunnan, Guizhou, Chongqing, Hunan, Hubei, Guangdong, Guangxi, Jiangxi and Anhui provinces, was compared to16SrDNA of R. solanacearum strain with results of identities up to99%by using NCBI to confirm the identification. All of the67strains in greenhouse were inoculated to tobacco seedlings by wounding and then showed symptoms although differ in period and severity.(2)The MLST analysis on seven housekeeping genes, rfbD, efp, pstSl, dnaQ, ureG, ppa, adk of R. solanacearum, showed that there was51sequence types (STs). All the strains were classified into4subgroups and4singletons:Subgroup1includes50stains with24STs; Subgroup2includes32stains with19STs; Subgroup3includes2stains with2STs; Subgroup4includes5stains with2STs. The4singletons include1strain isolated from Yunnan, Zhejiang, Zhejiang, Guangxi respectively. In geography, the ST1, which is the main ST in subgroup1, includes all the strains isolated from all provinces except Jiangxi, and strains in subgroup2were not closed to each other especially to ST40, which is their main ST, although2strains of ST40were from Anhui. This indicated that the MLST groups in this study are not related to geographical origin. In tobacco varieties, strains belonging to ST1in Subgroup1were isolated from7different tobacco species, and all strains in Subgroup1were from10different tobacco species;2strains of ST40in subgroup2were isolated from the same tobacco (Yunyan97), but all the strains in subgroup2were from6different tobaccos. From STs divisions, it is can be seen that the strains from different tobaccos could be divided into one ST, while strains isolated from same tobacco could be into different STs. It could not find out any law in relationship between tobaccos and STs. This result indicated that this pathogen was divided into subgroups irrespectively with tobacco varieties.更多还原