烟草青枯病菌遗传多样性研究
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。
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.
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。
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.
引文
1. Hayward AC. Biology and epidemiology of bacterial wilt caused by Pseudomonas Solanacearum[J]. Annual Review of Phytopathology,1991,29(1):65～87.
2.孔凡玉.烟草青枯病的综合防治[J].烟草科技,2003(4)：42-43.
3.郑继法,丁爱云,王智发.烟草细菌性病害识别及其综合防治技术[J].烟草研究与管理,2000(5)：26-27.
4.霍沁建,张深,王若焱.烟草青枯病研究进展[J].中国农学通报,2007,23(8)：364-368.
5.郑继法,丁爱云,张建华,等.烟草青枯病研究进展[J].山东农业大学学报,1998(4)：527-531.
6.徐树德,尚志强,秦西云.烟草青枯病研究进展[J].天津农业科学,2010,16(4)：49-53.
7.孙光军,林代福,刘圭义,等.烟草根结线虫病与黑胫病、青枯病的发生关系及品种抗性研究初报[J].烟草科技,1999,(5)：48.
8.朱贤朝,王彦亭,王智发.中国烟草病害[M].北京：中国农业出版社,2002：152-162.
9. Schell MA. Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory network[J]. Annual Review of Phytopathology, 2000,38:263～292.
10. Araud-Razou IJ, Vasse H, Montrozier C, et al. Detection and visualization of the major acidic exopolysaccharide of Ralstonia solanacearum and its role in tomato root infection and vascular colonization[J]. European Journal of Plant Pathology,1998, 104,795～809.
11. Huang Q, Allen C. Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants[J]. Physiological and Molecular Plant Pathology,2000,57,77～83.
12. McGarvey JA, Denny TP, Schell MA. Spatial-temporal and quantitative analysis of growth and EPS I production by Ralstonia solanacearum in resistant and susceptible tomato cultivars[J]. Phytopathology,1999,89,1233～1239.
13.潘哲超.植物青枯菌遗传多样性及致病力分化研究[D].北京：中国农业科学院,2010.
14. Poueymiro M, Genin S. Secreted proteins from Ralstonia solanacearum:a hundred tricks to kill a plant[J]. Current Opinion in Microbiology,2009,12(1):44～ 52.
15. Liu H, Denny TP. Protein analysis indicates that Ralstonia solanacearum has a distinctive type Ⅱ secretion system[J]. Phytopathology,2007(97):66～67.
16. Liu H, Zhang SP, Schell MA, et al. Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribution to virulence[J]. Molecular Plant-Micro Interactions,2005, 18(12):1296～1305.
17. Shintaro T, Kazuhiro N, Masanori A, et al. Contribution of the type II secretion system in systemic infectivity of Ralstonia solanacearum through xylem vessels[J]. Journal of General Plant Pathology,2008,74(1):71～75.
18. Turner M, Jauneau A, Genin S. et al. Dissection of bacterial wilt on Medicago truncatula revealed two type III secretion system effectors acting on root infection process and disease development [J]. Plant Physiology,2009,150:1713～1722.
19. Macho AP, Guidot A, Barberis P, et al. A Competitive Index Assay Identifies Several Ralstonia solanacearum Type III Effector Mutant Strains with Reduced Fitness in Host Plants[J]. Molecular Plant-Microbe Interactions,2010,23(9):1197～ 1205.
20.王军.青枯菌对植物的致病机制及其调节[J].林业科学,2005,41(3)：142-147.
21.王胜坤.桉树青枯菌菌株致病力分化、吸附识别及PCR快速检测研究[D].北京：中国林业科学研究院,2007.
22. Kang Y, Liu H, Genin S, et al. Ralstonia solanacearum requires type IV pili to adhere to multiple surfaces and for natural transformation and virulence[J]. Molecular Microbiology,2002,46,427～437.
23. Salanoubat M, Genin S, Artiguenave F, et al. Genome sequence of the plant pathogen Ralstonia solanacearum[J]. Nature,2002,415,497～502.
24. Wairuri CK, van der Waals JE, van Schalkwyk A, et al. Ralstonia solanacearum needs Flp pili for virulence on potato[J]. Molecular Plant-Microbe Interactions,2012, 25(4):546～556.
25. He LY, Sequeira L, Kelman A. Characteristics of strains of Pseudomonas Solanacearum from China [J]. Plant Disease,1983,67:1357～1361.
26.郑雪芳,车建美,林营志,等.基于ITS序列和RAMS标记分析青枯雷尔氏菌的遗传多样性[J].农学生物技术学报,2008,16(4)：694-700.
27.陈永芳,何礼远,徐进.我国植物青枯菌菌株的遗传多样性和组群划分[J].植物病理学报,2003,33(6)：503-508.
28. Qing YX, Yan NY, Wei Y, et al. Genetic diversity of Ralstonia solanacearum strains from China assessed by PCR-based fingerprints to unravel host plant-and site-dependent distribution patterns[J]. Microbiology Ecology,2011,75:507～519.
29. Yi TK, Kim JH, Kang SK, et al.Classification of Pseudomonsa solanacearum isolates from tobacco plants in korea[J]. Korean Journal of Plant Protection,1982, 21(3):123～127.
30.郑向华,邓海滨,刘琼光,等.广东省烟草青枯菌的菌系和遗传多样性[J].华中农业大学学报,2007,26(4)：463-468.
31.徐进,顾刚,潘哲超,等.福建烟草青枯菌演化型及生化变种鉴定研究[J].中国烟草学报,2010,16(6)：66-71.
32.李柱刚,崔崇士,马荣才,等.遗传标记在植物上的发展与应用[J].东北农业大学学报,2001,32(4)：313-319.
33. Jarvis BDW, Tighe SW. Rapid identification of Rhizobium species based on cellular fatty acid analysis [J]. Plant and Soil,1994,161:31～41.
34. Jarvis BDW, Sivakumaran S, Tighe S W, et al. Identification of Agrobacterium and Rhizobium species based on cellular fatty acid composition [J]. Plant and Soil, 1996,184:143～158.
35. Milkman R. Electrophoretic variation in Escherichia coli from natural sources[J]. Science,1973,182:1024～1026.
36. Selander RK, Caugant DA, Howard O, et al. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics [J]. Applied and Environmental Microbiology,1986,51(5):873～884.
37. Schwartz DC, Cantor CR. Separation of yeast chromosome sized DNAs by pulsed field gradient gel electrophoresis [J]. Cell,1984,37(1):67～75.
38. Fakhr MK, Nolan LK, Logue CM. Multilocus sequence typing lacks the discriminatory ability of pulsed-field gel electrophoresis for typing Salmonella enterica serovar typhimurium [J]. Journal of Clinical Microbiology,2005,43(5): 2215～2219.
39. Swaminathan B, Barrett TJ, Hunter SB, et al. PulseNet:The molecular subtyping network for foodborne bacterial disease surveillance, United States [J]. Synopses, 2001,7(3):382～389.
40. Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers [J]. Nucleic Acids Research,1990,18(24):7213～7218.
41. Williams JGK, Kubelik AR, Livak KJ, et al. DNA polymorphism amplified by arbitrary primers are useful as genetic markers [J]. Nucleic Acids Research,1990, 18(22):6531～6535.
42. Neilan BA. Identification and plylogenetic analysisof toxic cyanobacteria by multiplex randomly amplified polymorphic DNA PCR [J]. Applied and Environmental Microbiology,1995,61:2286～2291.
43. Hu J, Van Eysden J, Quiros CF. Generation of DNA-based markers in specific genome regions by two-primer RAPD reactions [J]. PCR methods and Application, 1995,4:346～351.
44. Higgins CF, Ames GF, Barnes WM, et al. A noval intercistronic regulatory element of prokaryotic operons [J]. Nature (London),1982,298:760-762.
45. Sharples GJ, Lloyd RG. A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes [J]. Nucleic Acids Research,1990,18(22):6503～ 6508.
46. Martin B, Humbert O, Camara M, et al. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae [J]. Nucleic Acids Research,1992,20(13):3479～3483.
47. Enright MC,Spratt BG. A multilocus sequence typing scheme for Streptococcus pneumoniae:identification of clones associated with serious invasive disease [J]. Microbiology,1998,144:3049～3060.
48. Scally M, Schuenzel EL, Ctouthamer R, et al. Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diverisity [J]. Applied and Environmental Microbiology, 2005,71(12):8491～8499.
49. Karaolis DK, Lan R, Reeves PR. The sixth and seventh clolera pandemics are duo to independent clones separately derived from environmental nontoxigenic, non-O1 Vibrio cholerae [J]. Journal of Bacteriology,1995,177(11):3191～3198.
50. Dingle KE, Colles FM, Wareing DRA, et al. Multilocus Sequence Typing System for Campylobacter jejuni [J]. Journal of Clinical Microbiology,2001,37(1):14～23.
51. McCombie RL, Finkelstein RA, Woods DE. Multilocus sequence typing of historical Burkholderia pseudomallei isolates collected in southeast Asia from 1964 to 1967 provides insight into the epidemiology of melioidosis [J]. Journal of Clinical Microbiology,2006,44(8):2951～2962.
52. Maiden MCJ, Bygraves JA, Feil E, et al. Multilocus sequence typing:Aprotable approach to the identification of clones within populations of pathogenic microorganisms [J]. Proceedings of the National Academy of Sciences of the United States of America,1998,95:3140～3145.
53. Feil EJ, Li BC, Aanensen DM, et al. eBURST:Inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data [J]. Journal of Bacteriology,2004,186(5):1518～1530.
54. Urwin R, Maiden MCJ. Multi-locus sequence typing:a tool for global epidemiology [J]. Trends in Microbiology,2003,11(10):479～487.
55.番华彩,唐嘉义,秦小萍.烟草青枯病防治研究进展[J].云南大学学报,2008,30(S1)：31-35.
56.方树民,顾刚,纪成灿,等.烟草青枯菌致病型及分布的研究[J].中国烟草学报,2002,8(3)：40-43.
57.顾刚,赖荣泉,张绍升,等.防治烟草青枯病杀菌剂筛选[J].海峡科学,2009,36(12)：28-31.
58.陈泽鹏,王涛,陈伟明,等.烟草抗青枯病的药剂诱导效应与抑菌增效作用[J].烟草科技,2011,282(1)：74-78.
59.陈勇,金晨钟.植物青枯病的生物防治研究进展[J].湖南农业科学,2009,(7)：82-85.
60. Anan Wang, Zhifeng Zhao, Zhenzhen Liu, et al. Effect of K1, K2 Anti-Bacterial Agents on Tobacco Ralstonia Solanacearum[J]. Engineering,2010,2,930～934.
61.吕建林,刘二明,柏连阳,等.烟草青枯病生防菌混合接种对其定殖及防效的影响[J].中国生物防治,2010,26(2)：200-205.
62.曾维爱,龙世平,李宏光,等.苗期接种不同丛枝菌根真菌对烟草青枯病防治效果的影响[J].南方农业学报,2011,42(6)：612-615.
63. Woese CR, Gutell R, Gupta R, et al. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids [J]. Microbiology Review,1983,47: 621～669.
64. Brosius J, Dull TJ, Noller HF. Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia coli [J]. Proceedings of the National Academy of Sciences of the United States of America,1980,77:201～204.
65. Feng JJ, Schuenzel EL, Li JQ, et al. Multilocus sequence typing reveals two evolutionary lineages of Acidovorax avenae subsp. citrulli [J]. Phytopathology,2009, 99:913～920.
66.刘雯静,邱少富,刘雪林,等.沙门氏菌的分子分型方法[J].现代生物医学进展,2010,20(10)：3948-3950.
67.邓小玲,管大伟,黎薇,等MLST分型技术应用于脑膜奈瑟菌的分子流行病学研究[J].华南预防医学,2008,34(3)：10-14.
68.廖亚玲,邹全明,张卫军,等.对中国4个不同地区幽门螺杆菌菌株的多位点序列分析研究[J].第三军医大学学报,2008,30(11)：1049-1051.
69.冯建军.西瓜细菌性果斑病菌分子检测及其遗传多样性分析[D].北京：中国农业大学,2008.
70.陈国强,陈扬,薛峰,等.多位点序列分型分析华东地区动物源产品中肠道沙门氏菌分离株[J].扬州大学学报,2010,31(3)：13-16.
71.王新,周婷,孟洪江.禽肉空肠弯曲杆菌的多位点序列分型技术研究[J].中国食品学报,2010,10(2)：180-186.
72.张少敏,徐建国.多位点序列分型及其应用[J].疾病监测,2008,23(10)：648-650.
2.孔凡玉.烟草青枯病的综合防治[J].烟草科技,2003(4)：42-43.
3.郑继法,丁爱云,王智发.烟草细菌性病害识别及其综合防治技术[J].烟草研究与管理,2000(5)：26-27.
4.霍沁建,张深,王若焱.烟草青枯病研究进展[J].中国农学通报,2007,23(8)：364-368.
5.郑继法,丁爱云,张建华,等.烟草青枯病研究进展[J].山东农业大学学报,1998(4)：527-531.
6.徐树德,尚志强,秦西云.烟草青枯病研究进展[J].天津农业科学,2010,16(4)：49-53.
7.孙光军,林代福,刘圭义,等.烟草根结线虫病与黑胫病、青枯病的发生关系及品种抗性研究初报[J].烟草科技,1999,(5)：48.
8.朱贤朝,王彦亭,王智发.中国烟草病害[M].北京：中国农业出版社,2002：152-162.
9. Schell MA. Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory network[J]. Annual Review of Phytopathology, 2000,38:263～292.
10. Araud-Razou IJ, Vasse H, Montrozier C, et al. Detection and visualization of the major acidic exopolysaccharide of Ralstonia solanacearum and its role in tomato root infection and vascular colonization[J]. European Journal of Plant Pathology,1998, 104,795～809.
11. Huang Q, Allen C. Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants[J]. Physiological and Molecular Plant Pathology,2000,57,77～83.
12. McGarvey JA, Denny TP, Schell MA. Spatial-temporal and quantitative analysis of growth and EPS I production by Ralstonia solanacearum in resistant and susceptible tomato cultivars[J]. Phytopathology,1999,89,1233～1239.
13.潘哲超.植物青枯菌遗传多样性及致病力分化研究[D].北京：中国农业科学院,2010.
14. Poueymiro M, Genin S. Secreted proteins from Ralstonia solanacearum:a hundred tricks to kill a plant[J]. Current Opinion in Microbiology,2009,12(1):44～ 52.
15. Liu H, Denny TP. Protein analysis indicates that Ralstonia solanacearum has a distinctive type Ⅱ secretion system[J]. Phytopathology,2007(97):66～67.
16. Liu H, Zhang SP, Schell MA, et al. Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribution to virulence[J]. Molecular Plant-Micro Interactions,2005, 18(12):1296～1305.
17. Shintaro T, Kazuhiro N, Masanori A, et al. Contribution of the type II secretion system in systemic infectivity of Ralstonia solanacearum through xylem vessels[J]. Journal of General Plant Pathology,2008,74(1):71～75.
18. Turner M, Jauneau A, Genin S. et al. Dissection of bacterial wilt on Medicago truncatula revealed two type III secretion system effectors acting on root infection process and disease development [J]. Plant Physiology,2009,150:1713～1722.
19. Macho AP, Guidot A, Barberis P, et al. A Competitive Index Assay Identifies Several Ralstonia solanacearum Type III Effector Mutant Strains with Reduced Fitness in Host Plants[J]. Molecular Plant-Microbe Interactions,2010,23(9):1197～ 1205.
20.王军.青枯菌对植物的致病机制及其调节[J].林业科学,2005,41(3)：142-147.
21.王胜坤.桉树青枯菌菌株致病力分化、吸附识别及PCR快速检测研究[D].北京：中国林业科学研究院,2007.
22. Kang Y, Liu H, Genin S, et al. Ralstonia solanacearum requires type IV pili to adhere to multiple surfaces and for natural transformation and virulence[J]. Molecular Microbiology,2002,46,427～437.
23. Salanoubat M, Genin S, Artiguenave F, et al. Genome sequence of the plant pathogen Ralstonia solanacearum[J]. Nature,2002,415,497～502.
24. Wairuri CK, van der Waals JE, van Schalkwyk A, et al. Ralstonia solanacearum needs Flp pili for virulence on potato[J]. Molecular Plant-Microbe Interactions,2012, 25(4):546～556.
25. He LY, Sequeira L, Kelman A. Characteristics of strains of Pseudomonas Solanacearum from China [J]. Plant Disease,1983,67:1357～1361.
26.郑雪芳,车建美,林营志,等.基于ITS序列和RAMS标记分析青枯雷尔氏菌的遗传多样性[J].农学生物技术学报,2008,16(4)：694-700.
27.陈永芳,何礼远,徐进.我国植物青枯菌菌株的遗传多样性和组群划分[J].植物病理学报,2003,33(6)：503-508.
28. Qing YX, Yan NY, Wei Y, et al. Genetic diversity of Ralstonia solanacearum strains from China assessed by PCR-based fingerprints to unravel host plant-and site-dependent distribution patterns[J]. Microbiology Ecology,2011,75:507～519.
29. Yi TK, Kim JH, Kang SK, et al.Classification of Pseudomonsa solanacearum isolates from tobacco plants in korea[J]. Korean Journal of Plant Protection,1982, 21(3):123～127.
30.郑向华,邓海滨,刘琼光,等.广东省烟草青枯菌的菌系和遗传多样性[J].华中农业大学学报,2007,26(4)：463-468.
31.徐进,顾刚,潘哲超,等.福建烟草青枯菌演化型及生化变种鉴定研究[J].中国烟草学报,2010,16(6)：66-71.
32.李柱刚,崔崇士,马荣才,等.遗传标记在植物上的发展与应用[J].东北农业大学学报,2001,32(4)：313-319.
33. Jarvis BDW, Tighe SW. Rapid identification of Rhizobium species based on cellular fatty acid analysis [J]. Plant and Soil,1994,161:31～41.
34. Jarvis BDW, Sivakumaran S, Tighe S W, et al. Identification of Agrobacterium and Rhizobium species based on cellular fatty acid composition [J]. Plant and Soil, 1996,184:143～158.
35. Milkman R. Electrophoretic variation in Escherichia coli from natural sources[J]. Science,1973,182:1024～1026.
36. Selander RK, Caugant DA, Howard O, et al. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics [J]. Applied and Environmental Microbiology,1986,51(5):873～884.
37. Schwartz DC, Cantor CR. Separation of yeast chromosome sized DNAs by pulsed field gradient gel electrophoresis [J]. Cell,1984,37(1):67～75.
38. Fakhr MK, Nolan LK, Logue CM. Multilocus sequence typing lacks the discriminatory ability of pulsed-field gel electrophoresis for typing Salmonella enterica serovar typhimurium [J]. Journal of Clinical Microbiology,2005,43(5): 2215～2219.
39. Swaminathan B, Barrett TJ, Hunter SB, et al. PulseNet:The molecular subtyping network for foodborne bacterial disease surveillance, United States [J]. Synopses, 2001,7(3):382～389.
40. Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers [J]. Nucleic Acids Research,1990,18(24):7213～7218.
41. Williams JGK, Kubelik AR, Livak KJ, et al. DNA polymorphism amplified by arbitrary primers are useful as genetic markers [J]. Nucleic Acids Research,1990, 18(22):6531～6535.
42. Neilan BA. Identification and plylogenetic analysisof toxic cyanobacteria by multiplex randomly amplified polymorphic DNA PCR [J]. Applied and Environmental Microbiology,1995,61:2286～2291.
43. Hu J, Van Eysden J, Quiros CF. Generation of DNA-based markers in specific genome regions by two-primer RAPD reactions [J]. PCR methods and Application, 1995,4:346～351.
44. Higgins CF, Ames GF, Barnes WM, et al. A noval intercistronic regulatory element of prokaryotic operons [J]. Nature (London),1982,298:760-762.
45. Sharples GJ, Lloyd RG. A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes [J]. Nucleic Acids Research,1990,18(22):6503～ 6508.
46. Martin B, Humbert O, Camara M, et al. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae [J]. Nucleic Acids Research,1992,20(13):3479～3483.
47. Enright MC,Spratt BG. A multilocus sequence typing scheme for Streptococcus pneumoniae:identification of clones associated with serious invasive disease [J]. Microbiology,1998,144:3049～3060.
48. Scally M, Schuenzel EL, Ctouthamer R, et al. Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diverisity [J]. Applied and Environmental Microbiology, 2005,71(12):8491～8499.
49. Karaolis DK, Lan R, Reeves PR. The sixth and seventh clolera pandemics are duo to independent clones separately derived from environmental nontoxigenic, non-O1 Vibrio cholerae [J]. Journal of Bacteriology,1995,177(11):3191～3198.
50. Dingle KE, Colles FM, Wareing DRA, et al. Multilocus Sequence Typing System for Campylobacter jejuni [J]. Journal of Clinical Microbiology,2001,37(1):14～23.
51. McCombie RL, Finkelstein RA, Woods DE. Multilocus sequence typing of historical Burkholderia pseudomallei isolates collected in southeast Asia from 1964 to 1967 provides insight into the epidemiology of melioidosis [J]. Journal of Clinical Microbiology,2006,44(8):2951～2962.
52. Maiden MCJ, Bygraves JA, Feil E, et al. Multilocus sequence typing:Aprotable approach to the identification of clones within populations of pathogenic microorganisms [J]. Proceedings of the National Academy of Sciences of the United States of America,1998,95:3140～3145.
53. Feil EJ, Li BC, Aanensen DM, et al. eBURST:Inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data [J]. Journal of Bacteriology,2004,186(5):1518～1530.
54. Urwin R, Maiden MCJ. Multi-locus sequence typing:a tool for global epidemiology [J]. Trends in Microbiology,2003,11(10):479～487.
55.番华彩,唐嘉义,秦小萍.烟草青枯病防治研究进展[J].云南大学学报,2008,30(S1)：31-35.
56.方树民,顾刚,纪成灿,等.烟草青枯菌致病型及分布的研究[J].中国烟草学报,2002,8(3)：40-43.
57.顾刚,赖荣泉,张绍升,等.防治烟草青枯病杀菌剂筛选[J].海峡科学,2009,36(12)：28-31.
58.陈泽鹏,王涛,陈伟明,等.烟草抗青枯病的药剂诱导效应与抑菌增效作用[J].烟草科技,2011,282(1)：74-78.
59.陈勇,金晨钟.植物青枯病的生物防治研究进展[J].湖南农业科学,2009,(7)：82-85.
60. Anan Wang, Zhifeng Zhao, Zhenzhen Liu, et al. Effect of K1, K2 Anti-Bacterial Agents on Tobacco Ralstonia Solanacearum[J]. Engineering,2010,2,930～934.
61.吕建林,刘二明,柏连阳,等.烟草青枯病生防菌混合接种对其定殖及防效的影响[J].中国生物防治,2010,26(2)：200-205.
62.曾维爱,龙世平,李宏光,等.苗期接种不同丛枝菌根真菌对烟草青枯病防治效果的影响[J].南方农业学报,2011,42(6)：612-615.
63. Woese CR, Gutell R, Gupta R, et al. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids [J]. Microbiology Review,1983,47: 621～669.
64. Brosius J, Dull TJ, Noller HF. Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia coli [J]. Proceedings of the National Academy of Sciences of the United States of America,1980,77:201～204.
65. Feng JJ, Schuenzel EL, Li JQ, et al. Multilocus sequence typing reveals two evolutionary lineages of Acidovorax avenae subsp. citrulli [J]. Phytopathology,2009, 99:913～920.
66.刘雯静,邱少富,刘雪林,等.沙门氏菌的分子分型方法[J].现代生物医学进展,2010,20(10)：3948-3950.
67.邓小玲,管大伟,黎薇,等MLST分型技术应用于脑膜奈瑟菌的分子流行病学研究[J].华南预防医学,2008,34(3)：10-14.
68.廖亚玲,邹全明,张卫军,等.对中国4个不同地区幽门螺杆菌菌株的多位点序列分析研究[J].第三军医大学学报,2008,30(11)：1049-1051.
69.冯建军.西瓜细菌性果斑病菌分子检测及其遗传多样性分析[D].北京：中国农业大学,2008.
70.陈国强,陈扬,薛峰,等.多位点序列分型分析华东地区动物源产品中肠道沙门氏菌分离株[J].扬州大学学报,2010,31(3)：13-16.
71.王新,周婷,孟洪江.禽肉空肠弯曲杆菌的多位点序列分型技术研究[J].中国食品学报,2010,10(2)：180-186.
72.张少敏,徐建国.多位点序列分型及其应用[J].疾病监测,2008,23(10)：648-650.