瓜类果斑病菌群体感应系统luxR/luxI功能研究
摘要
瓜类果斑病是葫芦科作物上的一种种传病害，在世界多国和我国多个省份均有发生，给当地的西甜瓜种植业造成了严重的经济损失。其病原为西瓜嗜酸菌(Acidovorax citrulli,Schaad et al.2009)，革兰氏阴性菌。与大多数革兰氏阴性菌一样，该病原菌具有luxR/luxI群体感应系统，调节细菌相关生物学功能。
本论文运用同源标记置换法，获得了瓜类果斑病菌(Acidovorax citrulli)群体感应信号系统功能相关基因3个突变菌株：△luxI单基因缺失突变株、△luxR单基因缺失突变株以及△luxR/luxI双基因缺失突变株。通过比较野生菌株和突变菌株间的生物学性状差异研究了luxR/luxI群体感应系统的功能。主要获得以下结论：
(1)群体感应信号检测结果：与野生菌株相比，突变菌株△luxI和△luxR/luxI中检测不到信号物质的存在，△luxR菌株中仍有信号物质的蓝色显色反应。该实验结果进一步证明了luxI基因是自诱导物合成酶的基因，而luxR是合成信号接受载体的基因。
(2)致病性测定结果显示：突变菌株与野生型相比，致病力明显减弱，说明群体感应系统的功能基因在细菌的致病力方面起着极其重要的作用。
(3)生理生化测定结果：稳定期前，突变菌株与野生型菌株在KB培养基中生长速度一致，到稳定期，突变菌株生长能力稍微变弱；运动能力及胞外多糖的产生量与野生菌株没有明显差异。
(4) Real-Time qPCR定量检测果斑病菌5个相关毒性基因及2个群体感应相关基因的表达情况。结果显示：与野生型相比，所选取的7个基因在突变菌株中的表达量均下调，说明群体感应系统对毒性基因的表达为正调控关系。并且，群体感应系统功能基因luxI在△luxR突变株中表达量降低；luxR基因在△luxI突变株中表达量降低。
(5)萃取果斑病菌的群体感应信号物质，利用超高效液相色谱与质谱联用(UPLC-MS/MS)法鉴定，信号物质主要为N-(3-Oxo-octanoyl)-L-homoserine lactone（OOHL）；将该物质按照检测浓度与△luxI单基因缺失突变株混合接种，突变株的致病力恢复，实验结果表明群体感应信号物质与果斑病菌的致病性相关，与致病性测定实验结果一致。
Bacterial fruit blotch of melons, caused by Acidovorax citrulli, is a seed borne disease that causesserious damages to the plants of cucurbits in worldwide. As most gram-negative bacteria, the pathogenhas luxR/luxI quorum sensing system to regulate its biological functions.
Application the method of homologous recombination to construct three mutants related to the QSsignal systemic functional genes, containing luxR and luxI single gene deletion mutants and luxR/luxIdouble gene deletion mutant. luxR/luxI QS system function was studied by comparing the differnecebetween the wild type and mutant type strains. The results are as follows:
(1) Results of AHLs detection：Compared with wide-type strain signal substances cannot bedetected in△luxI and△luxR/luxI because of the deletion of luxI, while△luxR can still display ablue chromogenic reaction due to the existence of luxI. This result is consistent with the conclusion thatluxI is the gene producing signal substances and luxR is the gene generating the carrier of the signalsubstances.
(2) Results of pathogenicity determination：the mutants displayed the reduced in virulence, theresult showed that QS system played an important role in pathogenicity of Acidovorax citrulli.
(3) Results of physiology and biochemistry bioasaay: the mutant strains are not attenuated ingrowth rate until stationary phase.There is no obvious difference in motility ability extracellularpolysaccharide secretion.
(4) Results of Real-Time PCR：The expression of five genes related to virulence including luxI andluxR genes were detected quantitatively by Real-Time PCR, and the result revealed that the expressionof the seven genes we selected decreased in mutant strains, displayed negative regulation, similar withthe result of pathogenicity of the mutation-type strains. Results of Real-Time qPCR：The expression offive genes related to virulence and two gene including luxI and luxR related to quorum sensing werequantitatively detected by Real-Time qPCR. Results showed that the expression of the seven genesdecreased in mutant strains, displayed positive regulation. And the quorum sensing system related genesluxI was expressed at a lower level in△luxR and luxR was expressed at a lower level in△luxIrespectively.
(5) The AHLs of bacterial fruit blotch of melons were extracted, and identified quantitatively byUPLC-MS/MS as OOHL. The pathogenicity of△luxI to melon seedlings was recovered after addingthe OOHL.
本论文运用同源标记置换法，获得了瓜类果斑病菌(Acidovorax citrulli)群体感应信号系统功能相关基因3个突变菌株：△luxI单基因缺失突变株、△luxR单基因缺失突变株以及△luxR/luxI双基因缺失突变株。通过比较野生菌株和突变菌株间的生物学性状差异研究了luxR/luxI群体感应系统的功能。主要获得以下结论：
(1)群体感应信号检测结果：与野生菌株相比，突变菌株△luxI和△luxR/luxI中检测不到信号物质的存在，△luxR菌株中仍有信号物质的蓝色显色反应。该实验结果进一步证明了luxI基因是自诱导物合成酶的基因，而luxR是合成信号接受载体的基因。
(2)致病性测定结果显示：突变菌株与野生型相比，致病力明显减弱，说明群体感应系统的功能基因在细菌的致病力方面起着极其重要的作用。
(3)生理生化测定结果：稳定期前，突变菌株与野生型菌株在KB培养基中生长速度一致，到稳定期，突变菌株生长能力稍微变弱；运动能力及胞外多糖的产生量与野生菌株没有明显差异。
(4) Real-Time qPCR定量检测果斑病菌5个相关毒性基因及2个群体感应相关基因的表达情况。结果显示：与野生型相比，所选取的7个基因在突变菌株中的表达量均下调，说明群体感应系统对毒性基因的表达为正调控关系。并且，群体感应系统功能基因luxI在△luxR突变株中表达量降低；luxR基因在△luxI突变株中表达量降低。
(5)萃取果斑病菌的群体感应信号物质，利用超高效液相色谱与质谱联用(UPLC-MS/MS)法鉴定，信号物质主要为N-(3-Oxo-octanoyl)-L-homoserine lactone（OOHL）；将该物质按照检测浓度与△luxI单基因缺失突变株混合接种，突变株的致病力恢复，实验结果表明群体感应信号物质与果斑病菌的致病性相关，与致病性测定实验结果一致。
Bacterial fruit blotch of melons, caused by Acidovorax citrulli, is a seed borne disease that causesserious damages to the plants of cucurbits in worldwide. As most gram-negative bacteria, the pathogenhas luxR/luxI quorum sensing system to regulate its biological functions.
Application the method of homologous recombination to construct three mutants related to the QSsignal systemic functional genes, containing luxR and luxI single gene deletion mutants and luxR/luxIdouble gene deletion mutant. luxR/luxI QS system function was studied by comparing the differnecebetween the wild type and mutant type strains. The results are as follows:
(1) Results of AHLs detection：Compared with wide-type strain signal substances cannot bedetected in△luxI and△luxR/luxI because of the deletion of luxI, while△luxR can still display ablue chromogenic reaction due to the existence of luxI. This result is consistent with the conclusion thatluxI is the gene producing signal substances and luxR is the gene generating the carrier of the signalsubstances.
(2) Results of pathogenicity determination：the mutants displayed the reduced in virulence, theresult showed that QS system played an important role in pathogenicity of Acidovorax citrulli.
(3) Results of physiology and biochemistry bioasaay: the mutant strains are not attenuated ingrowth rate until stationary phase.There is no obvious difference in motility ability extracellularpolysaccharide secretion.
(4) Results of Real-Time PCR：The expression of five genes related to virulence including luxI andluxR genes were detected quantitatively by Real-Time PCR, and the result revealed that the expressionof the seven genes we selected decreased in mutant strains, displayed negative regulation, similar withthe result of pathogenicity of the mutation-type strains. Results of Real-Time qPCR：The expression offive genes related to virulence and two gene including luxI and luxR related to quorum sensing werequantitatively detected by Real-Time qPCR. Results showed that the expression of the seven genesdecreased in mutant strains, displayed positive regulation. And the quorum sensing system related genesluxI was expressed at a lower level in△luxR and luxR was expressed at a lower level in△luxIrespectively.
(5) The AHLs of bacterial fruit blotch of melons were extracted, and identified quantitatively byUPLC-MS/MS as OOHL. The pathogenicity of△luxI to melon seedlings was recovered after addingthe OOHL.
引文
1.蔡学清,黄月英,杨建珍,等.福建省西瓜细菌性果斑病的病原鉴定[J].福建农林大学学报(自然科学版),2005,34(4):434~437.
2.蔡学清,鄢凤娇,林玉,等.西瓜细菌性果斑病拮抗内生细菌的分离和筛选[J].福建农林大学学报,2009,38(5):465~470.
3.陈涛等.哈密瓜果斑病细菌群体感应信号分子的检测及其合成基因luxI的功能（D）.硕士学位论文.南京:南京农业大学,2008
4.管文静.水稻白叶枯病菌转录调控因子Clpxoo的分子鉴定及功能分析(D).硕士学位论文.北京:中国农科院植保所,2009.
5.侯建雄,方雯霞.西瓜细菌性果腐病及其检疫对策[J].中国进出境动植检,1997,1:36~37.
6.胡俊,黄俊霞,刘双平,等.不同哈密瓜品种对细菌性果斑病抗病性及发展动态的研究[J].华北农学报,2006,21(6):107~110.
7.黄昆,王文辉.超高效液相色谱和质谱联用在药物研究领域的应用[J].光谱实验室,2009,(26):923-926
8.金岩,张俊杰,吴燕华,等.西瓜细菌性果斑病的发生与病原菌鉴定[J].吉林农业大学学报,2004,26(3):263~266.
9.刘鹏.哈密瓜果斑病菌群体感应系统相关基因的克隆及其在检测中的应用(D).硕士学位论文.北京:中国农科院植保所,2007.
10.陆彩虹等.烯啶虫胺在棉花和土壤中的残留及消解动态[J].环境化学,2010,29(4):614-618
11.綦国红,董明盛,吴胜明,等. HPLC-MS法检测N-酰基-高丝氨酸内酯类信号分子[J]．分析测试学报,2007,26(3):417~419．
12.任小平,李小妮,王琳,等.广东西瓜果斑病的病原鉴定[J].华南农业大学学报,2010,31(4):40~43.
13.孙蕾.水稻白叶枯病菌群体感应信号DSF和c-di-GMP系统中重要基因的鉴定和功能分析(D).硕士学位论文.北京:中国农科院植保所,2009.
14.王建华,权春善,李新．细菌群体感应的信号转导机制及其对抗生素生产的影响[J]．中国生物工程杂志,2008,(4):87-92．
15.王晓东,孙玉宏,葛米红,等.武汉地区保护地甜瓜细菌性斑点病病原鉴定[J].湖北农业科学,2010,49(8):1883~1886.
16.王中武,吴广成.西瓜细菌性果斑病药剂防治试验[J].北方园艺,2009,(1):109~110.
17.温晓芳,黄俊生.细菌的群体感应及其信号分子[J].华南热带农业大学学报,2005,11(1):31~35．
18.薛莉,杨承德,陈秀蓉,等.甘肃省西瓜细菌性果斑病的诊断[J].植物保护,2009,53(2):53-57
19.阎莎莎，王铁霖，赵廷昌.瓜类细菌性果斑病研究进展[J].植物检疫，2011,25(3):71-75.
20.阎莎莎.瓜类细菌性果斑病菌种内遗传多样性的研究(D).硕士学位论文.北京:中国农科院植保所,2011.
21.张力群,田涛,梅贵英.群体感应淬灭-防治植物细菌病害的新策略[J].中国生物防治2010,26(3):241-247
22.张祥林,莫桂花.西瓜上的一种新病害—细菌性果斑病[J].新疆农业科学,1996,4:183~184.
23.张新忠，罗逢健，刘光明，等.超高效液相色谱-串联质谱法测定茶叶和土壤中丁醚脲及其代谢物的残留[J],分析化学，2011,39(9):1329-1335.
24.赵廷昌,孙福在,刘双平,等.哈密瓜细菌性果斑病及其防治[J].植物保护,2001a,27(1):46~47.
25.赵廷昌,孙福在,王兵万,等.哈密瓜果斑病病原菌鉴定[J].植物病理学报,2001b,31(4):357~364.
26.赵廷昌,孙福在,王建荣,等.药剂处理种子防治哈密瓜细菌性果斑病的研究[J].植物保护,2003a,29(4):50~53.
27.赵廷昌,王建荣,孙福在,等.哈密瓜细菌性果斑病综合治理指南[J].植保技术与推广,2003b,23(4):17~18.
28.赵廷昌,赵洪海,王怀松.山东省西瓜、甜瓜发生瓜类细菌性果斑病[J].植物保护,2009,35(5):170~171.
29.赵玉强等.瓜类细菌性果斑病菌luxR基因的功能分析[J],农业生物技术学报,2011,19(3):542-548.
30.周洪友,杨静,宋娟.荧光假单胞工程菌株对甜瓜细菌性果斑病的生物防治[J].中国植保导刊,2009,29(1):9~12.
31.朱晨光,孙明,喻子牛.带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达.生物工程学报[J],2003,19(4):397~401.
32.张晓兵,府伟灵.群体感应与细菌毒力.中华医院感染学杂志[J],2011,21(13):2866~2870.
33. Ansaldi M, Marolt D, Stebe T, et al. Speeific activation of the Bacillus quorum sensing systems byisoprenylated pheromone variants [J]. Mol.Microbiol,2002,44:1561-1573.
34. Antunes L C, Ferreira R B. Intercellular communication in bacteria [J]. Crit Rev Microbiol,2009,35(2):69-80.
35. Bassler HP, Fall R, Vivanco JM. Biocontrol of Bacillus subtilis against infection of Arabidopsisroots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J].Plant Physiology,2004,134:307-319
36. Black M C, Lsakeit T&Bames L W. First report of bacterial fruit blotch of watermelon in Texas[J]. Plant Disease.1994,78:831.
37. Booth M C, Bogie C P, Sahl H G, et al. Structural analysis and proteolytic activation ofEnterococcus faecalis cytolysin, a novel lantibiotic [J]. Mol.microbio,1996,21:1175-1184.
38. Brosius J, Dull T J, Noller H F. Complete nucleotide sequence of a23S ribosomal RNA gene fromEscherichia coli [J]. Proceedings of the National Academy of Sciences of the United States ofAmerica,1980,77:201~204.
39. Cataldi T R, Blanco G, Palazzo L, et al．Occurrence of N-acyl-L-homoserine lactones in extracts ofsome Gram-negative bacteria evaluated by gas chromatography-mass spectrometry [J]. AnalBiochem,2007,361(2):226-235．
40. Cheng A H, Hsu Y L, Huang T C, et al. Susceptibility of cucurbits to Acidovorax avenae subsp.citrulli and control of fruit blotch on melon [J]. Plant Pathology. Bull,2000,9:151~156.
41. Davey M, Caiazza N, Toole G, et al. Rhamnolipid surfactant production affects biofilm architecturein Pseudomonas aeruginosa PAO1[J]. Journal of Bacteriology,2003,185(3):1027-1036.
42. Demir G.A. Disease and pest outbreak: Turkey: bacterial fruit blotch of watermelon Arab andNear East [J]. Plant protection Newsletter1996,23:33.
43. Deng W L, Huang T C, Tsai Y C. First Report of Acidovorax avenae subsp. citrulli as the CausalAgent of Bacterial Leaf Blight of Betelvine in Taiwan [J]. Plant Disease,2010,94:1065.
44. Dong Y H, GustiA R, Zhang Q, et al. Identification of Quorum-Quenching N-acyl homoserinelactonases from Bacillus species[J]. Appl EnvironMicrobiol,2002,68(4):1754-1759.
45. Dow JM, Crossman L, Findlay K, et al. Biofilm dispersal in Xanthomonas campestris is controlledby cell-cell signaling and is required for full virulence to plants [J]. Proc Natl Acad Sci USA,2003,100:10995-11000
46. Dutta B, Genzlinger L L, Walcott R R. Localization of Acidovorax avenae subsp. citrulli (Aac), thebacterial fruit blotch pathogen in naturally infested watermelon seed [J]. Phytopathology,2008,98(6): suppl, p. S49.
47. Elvers KT, Park SF. Quorum sensing in Campylobacter jejuni: detection of a luxS encodedsignaling molecule [J]. Microbiology,2002,148:1475-1481.
48. Evans T A&Mulrooney R.P. First report of watermelon fruit blotch in Delawre [J]. PlantDisease,1991,75:1074.
49. Federle M J, Bassler, B.L. Interspecies communication in baeteria[J]. Clnin Ivest,2003,112(9):1291-1299
50. Fuqua C, Parsk M R, Greenbreg E P. Regulation of gene expression by cell-to-cell communication:acyl-homoserine lactone quorum sensing [J]. Annu.Rev. Genet,2001,35:439-468
51. Fuqua C. Listening inon bacteria: acyl-homoserine lactone signaling [J]. Nat.Rev.Mol.Cell Biol,2002,3(9):685-695
52. Genin, S., Brito, B., Denny, T.P. et al. Control of the Ralstonia solanacearum Type III secretionsystem (Hrp) genes by the global virulence regulator PhcA [J]. FEBS (Fed. Eur. Biochem. Soc),2005,579:2077-2081.
53. George EA, Muir TW. Molecular m echanisms of agr quorum sensing in virulent staphylococci [J].Chem BioChem,2007,8,(8):847~855.
54. Hamm P B, Spink D S, Clough G.H, et al. First report of bacterial fruit blotch of watermelon inOregon. Plant Disease,1997,81:113.
55. He Y W, Alvin Y J, Xu M, et al. Xanthomonas campestris cell-cell communication involves aputative nucleotide receptor protein Clp and a hierarchical signalling network [J]. Mol.Micro,2007,64(2):281~292.
56. Henke J, Bassler B. Bacterial social engagements [J]. Trends Cell Bio,2004,14(11):648-656.
57. Henke J M, Bassler B L.Three parallel quorum-sensing systems regulate gene expression inVibrioharvey [J]. J Bacteriol,2004,186(20):6902-6914.
58. Hopkins D L, Thompson C M, Elmstrom G W. Resistance of watermelon seedlings and fruit to thefruit blotch bacterium [J]．HortScience,1993,28(2):122~123．
59. Jacobs J L, Damicone J P&McCraw B D. First report of bacterial fruit blotch of watermelon inOklahoma [J]. Plant Disease,1992,76:1185.
60. Jec H I. Bacterial spot caused by Pseudoumonas sp. of watermelon [J]. Korean J Pathol,
1990.6:237-244.
61. Kaplan H B, Greenberg E P. Diffusion of autoinducer is involved in regulation of the Vibriofischeri luminescence system [J]. Bactero,1985,163:1210-1124.
62. Kucharek T, Perez Y, Hodge C. Transmission of watermelon fruit blotch bacterium from infestedseed to seedlings [J]. Phytopathology,1993,83:467.
63. Latin R.X&Rane KK. Bacterial fruit blotch of watermelon in Indiana [J]. Plant Disease,1990,74:331.
64. Lazazzera B A, Grossman A D. The ins and outs of peptide signaling [J]. Trends Microbiol,1998,6:288-294
65. Mayville P, Ji G, Beavis R, Yang H, Goger M, et al. Structure-activity analysis of syntheticautoinducing thiolactone peptides from Stamphylococus aureus responsible for virulence [J].Proc.Natl.Acad.Scl.USA,1999,96:1218-1223.
66. McClean K H, Winson M K, Fish L, et al．Quorum sensing and Chromobacterium violaceum:exploitation of violacein production and inhibition for the detection of N-acylhomoserinelactones[J]．Microbiology,1997,143(12):3703~3711．
67. Miller M B, Bassler B L. Quorum sensing in baeteria [J]. Annu Rev Mierobiol.2001,55:165-169.
68. Molina L, Rezzonico F, Defago G, et al. Autoinduction in Erwinia amylovora: Evidence of anAcyl-Homoserine Lactone Signal in the Fire Blight Pathogen. Journal of Bacteriology [J],2005,187:3206-3213.
69. Nakayama J, Cao y, Horii T, et al. Gelatinase biosynthesis-activating pheromone: a peptide lactonethat mediates a quorum sensing in Enterococcus faecalis [J]. Mol.microbio,2001,41:145-154.
70. Nealson K H, Hastings J W. Bacterial bioluminescence: its control and ecological significance [J].Microbiol Rev,1979,43:496-518
71. Novick R P, Geisinger E. Quorum sensing ins taphylococci [J].AnnuRevGenet,2008,42:541~564.
72. O'Brien R G.&Martin H.L. Bacterial blotch of melons caused by strains of Acidovorax avenaesubsp. citrulli [J]. Aust J Exp Agric,1999,39:479-485.
73. Oger P, Kim KS, Sackett RL, et al. Octopine-type Ti plasmids code for a mannopine-inducibledominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugaltransfer[J]. Mol Microbiol,1998,27:277-288.
74. Rhodes B B, Zhang X P, Garrett J, et al. Watermelon fruit blotch infection rates in diploids andtiploids [J]. Report-Cucurbit Genetics Cooperative,1996,(19):70~72.
75. Schaad N W, Postnikova E, Sechler A, et al. Reclassification of subspecies of Acidovorax avenaeas A. Avenae(Manns1905) emend., A.cattleyae(Pavarino,1911) comb. nov., A. citrulli (Schaad etal.,1978) comb. nov., and proposal of A. oryzaesp. nov [J]. Systematic and Applied Microbiology,2008,31:434~446.
76. Schaad N W, Sowell G, Goth R W, et al. Pseudomonas Pseudolcaligenes subsp. critrulli subsp.Nov [J]. International Journal of Systematic Bacteriology,1978,28(1):117~125.
77. Schuster M, Lostroh C P, Ogi T, et al. Identification timing and signal specificity of pseudomonasaeruginosa quorum-controlled genes: a transcriptome analysis [J]. Bacleriol,2003,185:2066-2079.
78. Shirakawa T, Klkuchi S, Kato T, et al. Occurrence of watermelon bacterial fruit blotch in Japan(injapanese with English summary)[J]. Jpn J Phytopathol,2000,66:223~231.
79. Sowell G Jr, Schaad N W. Pseudomonas pseudoalcaligenes subsp.citrulli on watermelon: Seedtransmission and resistance of plant introductions [J]. Plant Disease Report,1979,63:437~441.
80. Taga M E, Miller S T, Bassler B L. Lsr-mediated transport and processing of AI-2in Salmonellatyphimurium [J]. Mol.Microbiol,2003,50(4):1411-1427.
81. Venturi V, Venuti C, Devescovi G, et al. The plant pathogen Erwinia amylovora producesacyl-homoserine lactone signal molecules in vitro and in planta [J]. FEMS Microbiology Letters,2004,241:179-183.
82. Walcott R R, Fessehaie A, Castro A C. Differences in pathogenicity between two geneticallydistinct groups of Acidovorax avenae subsp. citrulli on cucurbit hosts [J]. Phytopathology,2004,152:277~285.
83. Wall G C. Control of watermelon fruit blotch by seed heat-treatments [J]. Phytopathology1989,79:1191(Abstract).
84. Willems A, Goor M, Thielemans S, et al. Transfer of several phytopathogenic Pseudomonasspecies to Acidovorax as Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovoraxavenae subsp. citruli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci [J].International Journal of Systematic Bacteriology,1992,42:107~119.
85. Woese C R, Gutell R, Gupta R, et al. Detailed analysis of the higher-order structure of16S-likeribosomal ribonucleic acids [J]. Microbiology Review,1983,47:621~669.
86. Yarwood J, McCormick J, Paustian M, et al. Repression of the Staphylococcus aureus AccessoryGene Regulator in Serum and In Vivo[J]. Journal of Bacteriology,2002,184(4):1095~1101.
87. Yingying Tian, et al. Simultaneous determination of aminopyralid, clopyralid and picloramresidues in vegetables and fruits using ultra-performance liquid chromatography/tandem massspectrometry [J]. Journal of AOAC INTERNATIONAL,2012,95(2):554~559.
2.蔡学清,鄢凤娇,林玉,等.西瓜细菌性果斑病拮抗内生细菌的分离和筛选[J].福建农林大学学报,2009,38(5):465~470.
3.陈涛等.哈密瓜果斑病细菌群体感应信号分子的检测及其合成基因luxI的功能（D）.硕士学位论文.南京:南京农业大学,2008
4.管文静.水稻白叶枯病菌转录调控因子Clpxoo的分子鉴定及功能分析(D).硕士学位论文.北京:中国农科院植保所,2009.
5.侯建雄,方雯霞.西瓜细菌性果腐病及其检疫对策[J].中国进出境动植检,1997,1:36~37.
6.胡俊,黄俊霞,刘双平,等.不同哈密瓜品种对细菌性果斑病抗病性及发展动态的研究[J].华北农学报,2006,21(6):107~110.
7.黄昆,王文辉.超高效液相色谱和质谱联用在药物研究领域的应用[J].光谱实验室,2009,(26):923-926
8.金岩,张俊杰,吴燕华,等.西瓜细菌性果斑病的发生与病原菌鉴定[J].吉林农业大学学报,2004,26(3):263~266.
9.刘鹏.哈密瓜果斑病菌群体感应系统相关基因的克隆及其在检测中的应用(D).硕士学位论文.北京:中国农科院植保所,2007.
10.陆彩虹等.烯啶虫胺在棉花和土壤中的残留及消解动态[J].环境化学,2010,29(4):614-618
11.綦国红,董明盛,吴胜明,等. HPLC-MS法检测N-酰基-高丝氨酸内酯类信号分子[J]．分析测试学报,2007,26(3):417~419．
12.任小平,李小妮,王琳,等.广东西瓜果斑病的病原鉴定[J].华南农业大学学报,2010,31(4):40~43.
13.孙蕾.水稻白叶枯病菌群体感应信号DSF和c-di-GMP系统中重要基因的鉴定和功能分析(D).硕士学位论文.北京:中国农科院植保所,2009.
14.王建华,权春善,李新．细菌群体感应的信号转导机制及其对抗生素生产的影响[J]．中国生物工程杂志,2008,(4):87-92．
15.王晓东,孙玉宏,葛米红,等.武汉地区保护地甜瓜细菌性斑点病病原鉴定[J].湖北农业科学,2010,49(8):1883~1886.
16.王中武,吴广成.西瓜细菌性果斑病药剂防治试验[J].北方园艺,2009,(1):109~110.
17.温晓芳,黄俊生.细菌的群体感应及其信号分子[J].华南热带农业大学学报,2005,11(1):31~35．
18.薛莉,杨承德,陈秀蓉,等.甘肃省西瓜细菌性果斑病的诊断[J].植物保护,2009,53(2):53-57
19.阎莎莎，王铁霖，赵廷昌.瓜类细菌性果斑病研究进展[J].植物检疫，2011,25(3):71-75.
20.阎莎莎.瓜类细菌性果斑病菌种内遗传多样性的研究(D).硕士学位论文.北京:中国农科院植保所,2011.
21.张力群,田涛,梅贵英.群体感应淬灭-防治植物细菌病害的新策略[J].中国生物防治2010,26(3):241-247
22.张祥林,莫桂花.西瓜上的一种新病害—细菌性果斑病[J].新疆农业科学,1996,4:183~184.
23.张新忠，罗逢健，刘光明，等.超高效液相色谱-串联质谱法测定茶叶和土壤中丁醚脲及其代谢物的残留[J],分析化学，2011,39(9):1329-1335.
24.赵廷昌,孙福在,刘双平,等.哈密瓜细菌性果斑病及其防治[J].植物保护,2001a,27(1):46~47.
25.赵廷昌,孙福在,王兵万,等.哈密瓜果斑病病原菌鉴定[J].植物病理学报,2001b,31(4):357~364.
26.赵廷昌,孙福在,王建荣,等.药剂处理种子防治哈密瓜细菌性果斑病的研究[J].植物保护,2003a,29(4):50~53.
27.赵廷昌,王建荣,孙福在,等.哈密瓜细菌性果斑病综合治理指南[J].植保技术与推广,2003b,23(4):17~18.
28.赵廷昌,赵洪海,王怀松.山东省西瓜、甜瓜发生瓜类细菌性果斑病[J].植物保护,2009,35(5):170~171.
29.赵玉强等.瓜类细菌性果斑病菌luxR基因的功能分析[J],农业生物技术学报,2011,19(3):542-548.
30.周洪友,杨静,宋娟.荧光假单胞工程菌株对甜瓜细菌性果斑病的生物防治[J].中国植保导刊,2009,29(1):9~12.
31.朱晨光,孙明,喻子牛.带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达.生物工程学报[J],2003,19(4):397~401.
32.张晓兵,府伟灵.群体感应与细菌毒力.中华医院感染学杂志[J],2011,21(13):2866~2870.
33. Ansaldi M, Marolt D, Stebe T, et al. Speeific activation of the Bacillus quorum sensing systems byisoprenylated pheromone variants [J]. Mol.Microbiol,2002,44:1561-1573.
34. Antunes L C, Ferreira R B. Intercellular communication in bacteria [J]. Crit Rev Microbiol,2009,35(2):69-80.
35. Bassler HP, Fall R, Vivanco JM. Biocontrol of Bacillus subtilis against infection of Arabidopsisroots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J].Plant Physiology,2004,134:307-319
36. Black M C, Lsakeit T&Bames L W. First report of bacterial fruit blotch of watermelon in Texas[J]. Plant Disease.1994,78:831.
37. Booth M C, Bogie C P, Sahl H G, et al. Structural analysis and proteolytic activation ofEnterococcus faecalis cytolysin, a novel lantibiotic [J]. Mol.microbio,1996,21:1175-1184.
38. Brosius J, Dull T J, Noller H F. Complete nucleotide sequence of a23S ribosomal RNA gene fromEscherichia coli [J]. Proceedings of the National Academy of Sciences of the United States ofAmerica,1980,77:201~204.
39. Cataldi T R, Blanco G, Palazzo L, et al．Occurrence of N-acyl-L-homoserine lactones in extracts ofsome Gram-negative bacteria evaluated by gas chromatography-mass spectrometry [J]. AnalBiochem,2007,361(2):226-235．
40. Cheng A H, Hsu Y L, Huang T C, et al. Susceptibility of cucurbits to Acidovorax avenae subsp.citrulli and control of fruit blotch on melon [J]. Plant Pathology. Bull,2000,9:151~156.
41. Davey M, Caiazza N, Toole G, et al. Rhamnolipid surfactant production affects biofilm architecturein Pseudomonas aeruginosa PAO1[J]. Journal of Bacteriology,2003,185(3):1027-1036.
42. Demir G.A. Disease and pest outbreak: Turkey: bacterial fruit blotch of watermelon Arab andNear East [J]. Plant protection Newsletter1996,23:33.
43. Deng W L, Huang T C, Tsai Y C. First Report of Acidovorax avenae subsp. citrulli as the CausalAgent of Bacterial Leaf Blight of Betelvine in Taiwan [J]. Plant Disease,2010,94:1065.
44. Dong Y H, GustiA R, Zhang Q, et al. Identification of Quorum-Quenching N-acyl homoserinelactonases from Bacillus species[J]. Appl EnvironMicrobiol,2002,68(4):1754-1759.
45. Dow JM, Crossman L, Findlay K, et al. Biofilm dispersal in Xanthomonas campestris is controlledby cell-cell signaling and is required for full virulence to plants [J]. Proc Natl Acad Sci USA,2003,100:10995-11000
46. Dutta B, Genzlinger L L, Walcott R R. Localization of Acidovorax avenae subsp. citrulli (Aac), thebacterial fruit blotch pathogen in naturally infested watermelon seed [J]. Phytopathology,2008,98(6): suppl, p. S49.
47. Elvers KT, Park SF. Quorum sensing in Campylobacter jejuni: detection of a luxS encodedsignaling molecule [J]. Microbiology,2002,148:1475-1481.
48. Evans T A&Mulrooney R.P. First report of watermelon fruit blotch in Delawre [J]. PlantDisease,1991,75:1074.
49. Federle M J, Bassler, B.L. Interspecies communication in baeteria[J]. Clnin Ivest,2003,112(9):1291-1299
50. Fuqua C, Parsk M R, Greenbreg E P. Regulation of gene expression by cell-to-cell communication:acyl-homoserine lactone quorum sensing [J]. Annu.Rev. Genet,2001,35:439-468
51. Fuqua C. Listening inon bacteria: acyl-homoserine lactone signaling [J]. Nat.Rev.Mol.Cell Biol,2002,3(9):685-695
52. Genin, S., Brito, B., Denny, T.P. et al. Control of the Ralstonia solanacearum Type III secretionsystem (Hrp) genes by the global virulence regulator PhcA [J]. FEBS (Fed. Eur. Biochem. Soc),2005,579:2077-2081.
53. George EA, Muir TW. Molecular m echanisms of agr quorum sensing in virulent staphylococci [J].Chem BioChem,2007,8,(8):847~855.
54. Hamm P B, Spink D S, Clough G.H, et al. First report of bacterial fruit blotch of watermelon inOregon. Plant Disease,1997,81:113.
55. He Y W, Alvin Y J, Xu M, et al. Xanthomonas campestris cell-cell communication involves aputative nucleotide receptor protein Clp and a hierarchical signalling network [J]. Mol.Micro,2007,64(2):281~292.
56. Henke J, Bassler B. Bacterial social engagements [J]. Trends Cell Bio,2004,14(11):648-656.
57. Henke J M, Bassler B L.Three parallel quorum-sensing systems regulate gene expression inVibrioharvey [J]. J Bacteriol,2004,186(20):6902-6914.
58. Hopkins D L, Thompson C M, Elmstrom G W. Resistance of watermelon seedlings and fruit to thefruit blotch bacterium [J]．HortScience,1993,28(2):122~123．
59. Jacobs J L, Damicone J P&McCraw B D. First report of bacterial fruit blotch of watermelon inOklahoma [J]. Plant Disease,1992,76:1185.
60. Jec H I. Bacterial spot caused by Pseudoumonas sp. of watermelon [J]. Korean J Pathol,
1990.6:237-244.
61. Kaplan H B, Greenberg E P. Diffusion of autoinducer is involved in regulation of the Vibriofischeri luminescence system [J]. Bactero,1985,163:1210-1124.
62. Kucharek T, Perez Y, Hodge C. Transmission of watermelon fruit blotch bacterium from infestedseed to seedlings [J]. Phytopathology,1993,83:467.
63. Latin R.X&Rane KK. Bacterial fruit blotch of watermelon in Indiana [J]. Plant Disease,1990,74:331.
64. Lazazzera B A, Grossman A D. The ins and outs of peptide signaling [J]. Trends Microbiol,1998,6:288-294
65. Mayville P, Ji G, Beavis R, Yang H, Goger M, et al. Structure-activity analysis of syntheticautoinducing thiolactone peptides from Stamphylococus aureus responsible for virulence [J].Proc.Natl.Acad.Scl.USA,1999,96:1218-1223.
66. McClean K H, Winson M K, Fish L, et al．Quorum sensing and Chromobacterium violaceum:exploitation of violacein production and inhibition for the detection of N-acylhomoserinelactones[J]．Microbiology,1997,143(12):3703~3711．
67. Miller M B, Bassler B L. Quorum sensing in baeteria [J]. Annu Rev Mierobiol.2001,55:165-169.
68. Molina L, Rezzonico F, Defago G, et al. Autoinduction in Erwinia amylovora: Evidence of anAcyl-Homoserine Lactone Signal in the Fire Blight Pathogen. Journal of Bacteriology [J],2005,187:3206-3213.
69. Nakayama J, Cao y, Horii T, et al. Gelatinase biosynthesis-activating pheromone: a peptide lactonethat mediates a quorum sensing in Enterococcus faecalis [J]. Mol.microbio,2001,41:145-154.
70. Nealson K H, Hastings J W. Bacterial bioluminescence: its control and ecological significance [J].Microbiol Rev,1979,43:496-518
71. Novick R P, Geisinger E. Quorum sensing ins taphylococci [J].AnnuRevGenet,2008,42:541~564.
72. O'Brien R G.&Martin H.L. Bacterial blotch of melons caused by strains of Acidovorax avenaesubsp. citrulli [J]. Aust J Exp Agric,1999,39:479-485.
73. Oger P, Kim KS, Sackett RL, et al. Octopine-type Ti plasmids code for a mannopine-inducibledominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugaltransfer[J]. Mol Microbiol,1998,27:277-288.
74. Rhodes B B, Zhang X P, Garrett J, et al. Watermelon fruit blotch infection rates in diploids andtiploids [J]. Report-Cucurbit Genetics Cooperative,1996,(19):70~72.
75. Schaad N W, Postnikova E, Sechler A, et al. Reclassification of subspecies of Acidovorax avenaeas A. Avenae(Manns1905) emend., A.cattleyae(Pavarino,1911) comb. nov., A. citrulli (Schaad etal.,1978) comb. nov., and proposal of A. oryzaesp. nov [J]. Systematic and Applied Microbiology,2008,31:434~446.
76. Schaad N W, Sowell G, Goth R W, et al. Pseudomonas Pseudolcaligenes subsp. critrulli subsp.Nov [J]. International Journal of Systematic Bacteriology,1978,28(1):117~125.
77. Schuster M, Lostroh C P, Ogi T, et al. Identification timing and signal specificity of pseudomonasaeruginosa quorum-controlled genes: a transcriptome analysis [J]. Bacleriol,2003,185:2066-2079.
78. Shirakawa T, Klkuchi S, Kato T, et al. Occurrence of watermelon bacterial fruit blotch in Japan(injapanese with English summary)[J]. Jpn J Phytopathol,2000,66:223~231.
79. Sowell G Jr, Schaad N W. Pseudomonas pseudoalcaligenes subsp.citrulli on watermelon: Seedtransmission and resistance of plant introductions [J]. Plant Disease Report,1979,63:437~441.
80. Taga M E, Miller S T, Bassler B L. Lsr-mediated transport and processing of AI-2in Salmonellatyphimurium [J]. Mol.Microbiol,2003,50(4):1411-1427.
81. Venturi V, Venuti C, Devescovi G, et al. The plant pathogen Erwinia amylovora producesacyl-homoserine lactone signal molecules in vitro and in planta [J]. FEMS Microbiology Letters,2004,241:179-183.
82. Walcott R R, Fessehaie A, Castro A C. Differences in pathogenicity between two geneticallydistinct groups of Acidovorax avenae subsp. citrulli on cucurbit hosts [J]. Phytopathology,2004,152:277~285.
83. Wall G C. Control of watermelon fruit blotch by seed heat-treatments [J]. Phytopathology1989,79:1191(Abstract).
84. Willems A, Goor M, Thielemans S, et al. Transfer of several phytopathogenic Pseudomonasspecies to Acidovorax as Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovoraxavenae subsp. citruli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci [J].International Journal of Systematic Bacteriology,1992,42:107~119.
85. Woese C R, Gutell R, Gupta R, et al. Detailed analysis of the higher-order structure of16S-likeribosomal ribonucleic acids [J]. Microbiology Review,1983,47:621~669.
86. Yarwood J, McCormick J, Paustian M, et al. Repression of the Staphylococcus aureus AccessoryGene Regulator in Serum and In Vivo[J]. Journal of Bacteriology,2002,184(4):1095~1101.
87. Yingying Tian, et al. Simultaneous determination of aminopyralid, clopyralid and picloramresidues in vegetables and fruits using ultra-performance liquid chromatography/tandem massspectrometry [J]. Journal of AOAC INTERNATIONAL,2012,95(2):554~559.