西瓜噬酸菌鞭毛基因flgM的功能分析
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摘要
西瓜(Citrullus lanatus)细菌性果斑病(bacterial fruit blotch, BFB)是世界范围内的检疫性病害,该病害的病原菌为西瓜噬酸菌(Acidovorax citrulli, Ac),自发现以来给西瓜种植业造成严重的经济损失,为了有效控制其危害,需要弄清该病菌的致病机制。细菌的鞭毛是细菌的运动器官之一,在细菌的侵染过程中也起着重要的作用。在多种不同细菌中已经证实,反σ因子flgM是调控鞭毛基因转录的必要因子,但是其在西瓜噬酸菌中的功能仍然未知,本研究旨在探讨该基因在鞭毛形成和致病性等生物学特性中的作用。根据同源重组的原理,借助自杀质粒p K8mobsacB,构建flgM基因缺失突变菌株,并对其鞭毛的形态特征、致病性、致敏性、游动性、群体感应、菌膜形成、生长速率及细菌颤泳性等生物学特性进行测定;以谷胱酰胺合成酶基因(glutathiamide synthetase gene, glnA)为参照基因,采用qRT-PCR方法,比较野生菌株、敲除菌株和互补菌株部分鞭毛蛋白基因flhD、fliE、fliC、flgK、flgA、fliS、fliD和fliA的表达量差异。结果表明,通过庆大霉素(gentamicin, Gm)抗性基因Gm筛选和PCR验证,成功地构建了西瓜噬酸菌鞭毛蛋白基因flgM的缺失突变菌株FJAc01-flgM及其互补菌株FJAc01-flgMhb,并对所得菌株的生物学特性进行观察,结果显示,与野生菌株相比,基因flgM缺失后不形成鞭毛、菌株的游动能力和菌膜形成能力均减弱、生长速率显著加快、对西瓜果实和西瓜苗的致病力明显减弱;基因互补后恢复了鞭毛的形成能力、游动性和菌膜形成能力、菌株的生长能力和对西瓜果实和西瓜苗的致病力也基本得到恢复,如针刺接种西瓜果实和喷雾接种西瓜苗后5 d,野生菌株处理的病情指数分别为74.67和26.39,基因缺失突变体处理的病情指数分别为46.00和2.78,而互补菌株处理的病情指数分别为63.85和20.83;光学显微镜下,可观察到在肉汁胨培养基(nutrient agar medium, NA)平板上的野生菌株菌落周围有明显的由细菌颤泳形成的特殊晕圈,而缺失突变菌株在NA平板上形成晕圈能力减弱,互补后晕圈形成能力部分恢复;但是,野生菌株、缺失突变菌株和互补菌株在烟草(Nicotiana benthamiana)过敏感应、群体感应和甜瓜(Cucumis melo)苗的致病性等方面无明显变化。qRT-PCR结果显示,基因flgM缺失突变后,基因flhD、fliC和fliS的表达量上升,而基因flgK、fliA和fliE的表达量下降,互补后这些基因的表达量基本与野生菌株相同,但是基因flgA和fliD的表达量没有变化。由此可见,鞭毛蛋白基因flgM对西瓜细菌性果斑病菌鞭毛丝的形成、致病性、菌膜形成能力、生长速率、游动性、菌落形态等均有调控作用。本研究通过定点突变研究了西瓜噬酸菌鞭毛基因flgM的生物学功能,为进一步解析该基因在西瓜噬酸菌鞭毛形成和致病过程中的作用提供理论依据。
Bacterial fruit blotch(BFB) caused by Acidovorax citrulli(Ac) is one of the most important destructive seed borne diseases on watermelon(Citrullus lanatus) and melon(Cucumis melo) in the world and the cause pathogen is a worldwide quarantine pest. This disease causes serious economic loss to watermelon production. In order to effectively control the disease, The pathogenicity mechanism of A. citrulli on watermelon is needed to know. Flagellum is a movement organ of bacteria and plays an important role in bacterial infection. Alterative anti-sigma factor flgM, required in the regulation of flagellar gene transcription,has been verified in different bacteria species, but its feature in A. citrulli is unclear. The objective of this study is to understand the function of gene flgM on flagellum formation and pathogenicity of A. citrulli. The flgM gene deletion mutant was generated by homologous recombination with the help of suicide plasmid pK8 mobsacB. Morphological characteristics have been tested of the flagella, pathogenicity, hypersensitive response, motility, quorum sensing, biofilm formation, growth rate, twitching, etc. among the wild type, the mutant and the complementary strain. Moreover, a real-time quantitative PCR(qRT-PCR) was carried out to compare the expression of genes, flhD, fliE, fliC, flgK, flgA, fliS, fliD and fliA in the wild type strain, the deletion mutant strain and the complementary strain using glutathiamide synthetase gene(glnA) as a reference.The results showed that the deletion mutant FJAc01-flgM and complementary strain FJAc01-flgMhb were generated successfully after gentamicin resistant screening and verified by PCR. Compared to wild type strain,the deletion mutant did not grow flagella and greatly attenuated in biofilm, motility, colonial morphology and the virulence on watermelon, but significantly accelerated in growth rate, which could be restored in the complementary strain. After 5 d of stabbed inoculation on watermelon fruit and spray inoculation on watermelon seedling, the disease indexes were 74.67 and 26.39 respectively inoculated by wild type strain FJAc01, but the disease indexes were 46.00 and 2.78 respectively by the deletion mutant strain FJAc01-flgM,and the disease indexes were 63.85 and 20.83 respectively by the complementary strain FJAc01-flgMhb.Whereas, there are no significant changes of mutant in hypersensitive response on tobacco(Nicotiana benthamiana), quorum sensing and pathogenicity to melon compared to the wild type strain. The wild type strain could form typical haloes obviously which caused by bacteria migrating via twitching on NA medium,but the deletion mutant weakened this ability and the complementary strain recovered the ability partially. The results of qRT-PCR indicated that the expression of flgK, fliA, fliE genes were up regulated, whereas flhD, fliC,fliS genes were down regulated in the mutant strain, and the expressions of these genes were recovered in the complementary strain. However, the expressions of gene flgA and fliD were not significant changed in the mutant. In conclusion, the flagellar gene flgM could regulate the flagellum formation, pathogenicity, biofilm formation, motility, growth rate and colony morphology of A. citrulli.
Bacterial fruit blotch(BFB) caused by Acidovorax citrulli(Ac) is one of the most important destructive seed borne diseases on watermelon(Citrullus lanatus) and melon(Cucumis melo) in the world and the cause pathogen is a worldwide quarantine pest. This disease causes serious economic loss to watermelon production. In order to effectively control the disease, The pathogenicity mechanism of A. citrulli on watermelon is needed to know. Flagellum is a movement organ of bacteria and plays an important role in bacterial infection. Alterative anti-sigma factor flgM, required in the regulation of flagellar gene transcription,has been verified in different bacteria species, but its feature in A. citrulli is unclear. The objective of this study is to understand the function of gene flgM on flagellum formation and pathogenicity of A. citrulli. The flgM gene deletion mutant was generated by homologous recombination with the help of suicide plasmid pK8 mobsacB. Morphological characteristics have been tested of the flagella, pathogenicity, hypersensitive response, motility, quorum sensing, biofilm formation, growth rate, twitching, etc. among the wild type, the mutant and the complementary strain. Moreover, a real-time quantitative PCR(qRT-PCR) was carried out to compare the expression of genes, flhD, fliE, fliC, flgK, flgA, fliS, fliD and fliA in the wild type strain, the deletion mutant strain and the complementary strain using glutathiamide synthetase gene(glnA) as a reference.The results showed that the deletion mutant FJAc01-flgM and complementary strain FJAc01-flgMhb were generated successfully after gentamicin resistant screening and verified by PCR. Compared to wild type strain,the deletion mutant did not grow flagella and greatly attenuated in biofilm, motility, colonial morphology and the virulence on watermelon, but significantly accelerated in growth rate, which could be restored in the complementary strain. After 5 d of stabbed inoculation on watermelon fruit and spray inoculation on watermelon seedling, the disease indexes were 74.67 and 26.39 respectively inoculated by wild type strain FJAc01, but the disease indexes were 46.00 and 2.78 respectively by the deletion mutant strain FJAc01-flgM,and the disease indexes were 63.85 and 20.83 respectively by the complementary strain FJAc01-flgMhb.Whereas, there are no significant changes of mutant in hypersensitive response on tobacco(Nicotiana benthamiana), quorum sensing and pathogenicity to melon compared to the wild type strain. The wild type strain could form typical haloes obviously which caused by bacteria migrating via twitching on NA medium,but the deletion mutant weakened this ability and the complementary strain recovered the ability partially. The results of qRT-PCR indicated that the expression of flgK, fliA, fliE genes were up regulated, whereas flhD, fliC,fliS genes were down regulated in the mutant strain, and the expressions of these genes were recovered in the complementary strain. However, the expressions of gene flgA and fliD were not significant changed in the mutant. In conclusion, the flagellar gene flgM could regulate the flagellum formation, pathogenicity, biofilm formation, motility, growth rate and colony morphology of A. citrulli.
引文
蔡学清,黄月英,杨建珍,等.2005.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),34(4):434-437.(Cai X Q,Huang Y Y,YANG J Z,et al.2005.Pathogen identification of bacterial fruit blotch of watermelon in Fujian[J].Journal of Fujian Agriculture and Forestry University(Natural Science Edition),34(4):434-437.)
陈娇梅.2015.西瓜嗜酸菌致病相关基因的筛选及功能分析[D].硕士学位论文,福建农林大学,导师:胡方平,蔡学清,pp.11-32.(Chen J M.2015.Screening and function analysis of pathogecity-related genes in Acidovorax citrulli[D].Thesis for M.S.,Fuzhou:Fujian Agriculture and Forestry University,suppervisor:HU F P,CAI X Q,pp.11-32.)
方中达.1998.植病研究方法[M].3版.中国农业出版社.北京.pp.47,183(Fang Z D.1998.Plant Disease Research Method.3rd ed.Beijing:China Agriculture Press.)
季苇芹,闫建培,白雪,等.2018.西瓜噬酸菌ftsH基因功能分析[J].植物病理学报,1-19.(Ji W Q,Yan J P,Bai X,et al.2018.Functional analysis of the gene ftsH in Acidovorax citrulli[J].Acta Phytopathologica Sinica,1-19.)
李明明,申初成,雷庆斌,等.2010.细菌性果斑病菌源与接菌方法对西瓜致病力的影响[J].湖南农业科学,(15):80-82.(Li M M,Shen C C,Lei Q B,et al.2010.Effects of different pathogen sources of watermelon bacterial fruit blotch and different inoculated-pathogen methods on pathogenicity of watermelon[J].Hunan Agricultural Sciences,(15):80-82.)
刘鹏,赵廷昌.2009.哈密瓜细菌性果斑病菌群体感应系统的检测[J].植物保护,35(6):43-46.(Liu P,Zhao T C.2009.Detection of the quorum-sensing of Acidovorax avenae subsp.citrulli[J].Plant Protection,35(6):43-46.)
萨姆布洛克J,拉塞尔D W.2005.分子克隆实验指南[M].科学出版社.北京.pp.96-99,387-400,611-618,628-633,1595.(Sambrook J,Russell D W.2005.The Condensed Protocols from Molecular Cloning:A Laboratory Manual[G].Beijing:Science Press,pp.96-99,387-400,611-618,628-633,1595.)
王铁霖,杨玉文,关巍.等.2016.西瓜嗜酸菌LuxR家族基因luxR4229的功能研究[J].植物病理学报,46(2):198-206.(Wang T L,Yang Y W,Guan W,et al.2016.Functional analysis of luxR4229,a LuxR family gene in Acidovorax citrulli[J].Acta Phytopathologica Sinica,46(2):198-206.)
汪新,王卫,钱国良,等.2011.瓜类细菌性果斑病菌过敏性反应和致病性(hrp)基因簇部分基因克隆和功能分析[J].农业生物技术学报,19(1):36-44.(Wang X,Wang W,Qian G L,et al.2011.Cloning and functional analysis of Acidovorax avenae subsp.citrulli partial hypersensitive response and pathogenicity(hrp)gene cluster[J]Journal of Agricultural Biotechnology,19(1):36-44.)
杨丙烨,付丹,胡方平,等.2017.西瓜细菌性果斑病菌鞭毛基因fliS的功能分析[J].中国农业科学,50(15):2946-2956.(Yang B Y,Fu D,Hu F P,et al.2017.Function analysis of flagellar gene fliS in Acidovorax citrulli[J]Scientia Agricultura Sinica,50(15):2946-2956.)
张爱萍,张晓晓,吴林娜,等.2017.西瓜噬酸菌趋化性及鞭毛素基因ΔcheAΔfliC双突变体构建及功能[J].农业生物技术学报,25(11):1838-1850.(Zhang A P,Zhang XX,Wu L N,et al.2017.Construction of chemotaxis and flagella gene double mutantΔcheAΔfliC and its functional analysis in Acidovorax citrulli[J].Journal of Agricultural Biotechnology,25(11):1838-1850.)
Bahar O,Goffer T,Burdman S.2009.Type IV pili are required for virulence,twitching motility,and biofilm formation of Acidovorax avenae subsp.citrulli[J].Molecular Plant-Microbe Interactions,22(8):909-920.
David A.Wilkinson,Sarah J.Chacko,Catherine ve′NIen-bryan et al.2011.Regulation of flagellum number by flia and flgm and role in biofilm formation by rhodobacter sphaeroides[J].Journal of Bacteriology,193(15):4010-4014.
Frankle W.1993.Ingress of the watermelon fruit blotch bacterium into fruit[J].Plant Disease,77:1090-1092.
Frisk A,Jyot J,Arora S K,et al.2002.Identification and functional characterization of flgM,a gene encoding the antisigma 28 factor in Pseudomonas aeruginosa[J].Journal of Bacteriology,184(6):1514-1521.
Gavin R,Rabaan A A,Merino S,et al.2002.Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation[J].Molecular Microbiology,43(2):383-397.
Kessler B,Lorenzo L,Timmisk N.1992.A general system to integrate lac Z fusions into the chromosomes of Gram-negative eubacteria:regulation of the Pm promoter of the TOLplasmid studied with all controlling elements in monocopy[J].Molecular and General Genetics,233:293-301.
Kutsukake K,Iino T.1994.Role of the FliA-FlgM regulatory system on the transcriptional control of the flagellar regulon and flagellar formation in Salmonella typhimurium[J].Journal of Bacteriology,176(12):3598-3605.
Luo J Y,Qiu W,Chen L,et al.2015.Identification of pathogenicity-related genes in biofilm-defective Acidovorax citrulli by transposon Tn5 mutagenesis[J].International Journal of Molecular Sciences,16(12):28050-28062.
Muller V,Jones C J,Kawagishi I,et al.1992.Characterization of the fliE genes of Escherichia coli and Salmonella typhimurium and identification of the FliE protein as a component of the flagellar hook-basal body complex[J].Journal of Bacteriology,174(7):2298-23004.
Osorio-valeriano M,Morra J D L,Camarena L,et al.2015.Biochemical characterization of the flagellar rod components of Rhodobacter sphaeroides:Properties and interactions[J].Journal of Bacteriology,198(3):544-552.
Petrocelli S,Arana M R,Cabrini M N,et al.2016.Deletion of pilA,a minor pilin-like gene,from Xanthomonas citri subsp.citri influences bacterial physiology and pathogenesis[J].Current Microbiology,73:904-914.
Song W Y,Kim H M,Schaad N W.2000.PCR Primers for detection and indentification of plant pathogenic species,subspecies and strains of Acidovorax[P].United States Patent 6423499B1.
Tian Y L,Zhao Y Q,Wu X R,et al.2015.The type VI protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon[J].Molecular Plant Pathology,16(1):38-47.
Tremblay J,Déziel E.2010.Gene expression in Pseudomonas aeruginosa warming motility[J].BioMed Central Genomics,11:587-602.
Walcott R R,Fessehaie A,Castro A C.2004.Differences in pathogenicity between two genetically distinct groups of Acidovorax avenae subsp.citrulli on cucurbit hosts[J].Journal of Phytopathology,152:277-285.
Wilkinson D A,Chacko S J,Vénien-Bryan C,et al.2011.Regulation of flagellum number by FliA and FlgM and role in biofilm formation by Rhodobacter sphaeroides[J].Journal of Bacteriology,193(15):4010-4014.
Wosten M M S M,van Dijk L,Veenendaal A K J,et al.2010.Temperature-dependent FlgM/FliA complex formation regulates Campylobacter jejuni flgella length[J].Molecular Microbiology,75(6):1577-1591.
Yang T C,Leu Y W,Chang-chien H C,et al.2009.Flagellar biogenesis of Xanthomonas campestris requires the alternative sigma factors RpoN2 and FliA and is temporally regulated by FlhA,FlhB,and FlgM?[J].Journal of Bacteriology,191(7):2266-2275.
Zivanovic M,Walcott R.R.2017.Further characterization of genetically distinct groups of Acidovorax citrulli strains[J].Phytopathology,107(1):29-35.
陈娇梅.2015.西瓜嗜酸菌致病相关基因的筛选及功能分析[D].硕士学位论文,福建农林大学,导师:胡方平,蔡学清,pp.11-32.(Chen J M.2015.Screening and function analysis of pathogecity-related genes in Acidovorax citrulli[D].Thesis for M.S.,Fuzhou:Fujian Agriculture and Forestry University,suppervisor:HU F P,CAI X Q,pp.11-32.)
方中达.1998.植病研究方法[M].3版.中国农业出版社.北京.pp.47,183(Fang Z D.1998.Plant Disease Research Method.3rd ed.Beijing:China Agriculture Press.)
季苇芹,闫建培,白雪,等.2018.西瓜噬酸菌ftsH基因功能分析[J].植物病理学报,1-19.(Ji W Q,Yan J P,Bai X,et al.2018.Functional analysis of the gene ftsH in Acidovorax citrulli[J].Acta Phytopathologica Sinica,1-19.)
李明明,申初成,雷庆斌,等.2010.细菌性果斑病菌源与接菌方法对西瓜致病力的影响[J].湖南农业科学,(15):80-82.(Li M M,Shen C C,Lei Q B,et al.2010.Effects of different pathogen sources of watermelon bacterial fruit blotch and different inoculated-pathogen methods on pathogenicity of watermelon[J].Hunan Agricultural Sciences,(15):80-82.)
刘鹏,赵廷昌.2009.哈密瓜细菌性果斑病菌群体感应系统的检测[J].植物保护,35(6):43-46.(Liu P,Zhao T C.2009.Detection of the quorum-sensing of Acidovorax avenae subsp.citrulli[J].Plant Protection,35(6):43-46.)
萨姆布洛克J,拉塞尔D W.2005.分子克隆实验指南[M].科学出版社.北京.pp.96-99,387-400,611-618,628-633,1595.(Sambrook J,Russell D W.2005.The Condensed Protocols from Molecular Cloning:A Laboratory Manual[G].Beijing:Science Press,pp.96-99,387-400,611-618,628-633,1595.)
王铁霖,杨玉文,关巍.等.2016.西瓜嗜酸菌LuxR家族基因luxR4229的功能研究[J].植物病理学报,46(2):198-206.(Wang T L,Yang Y W,Guan W,et al.2016.Functional analysis of luxR4229,a LuxR family gene in Acidovorax citrulli[J].Acta Phytopathologica Sinica,46(2):198-206.)
汪新,王卫,钱国良,等.2011.瓜类细菌性果斑病菌过敏性反应和致病性(hrp)基因簇部分基因克隆和功能分析[J].农业生物技术学报,19(1):36-44.(Wang X,Wang W,Qian G L,et al.2011.Cloning and functional analysis of Acidovorax avenae subsp.citrulli partial hypersensitive response and pathogenicity(hrp)gene cluster[J]Journal of Agricultural Biotechnology,19(1):36-44.)
杨丙烨,付丹,胡方平,等.2017.西瓜细菌性果斑病菌鞭毛基因fliS的功能分析[J].中国农业科学,50(15):2946-2956.(Yang B Y,Fu D,Hu F P,et al.2017.Function analysis of flagellar gene fliS in Acidovorax citrulli[J]Scientia Agricultura Sinica,50(15):2946-2956.)
张爱萍,张晓晓,吴林娜,等.2017.西瓜噬酸菌趋化性及鞭毛素基因ΔcheAΔfliC双突变体构建及功能[J].农业生物技术学报,25(11):1838-1850.(Zhang A P,Zhang XX,Wu L N,et al.2017.Construction of chemotaxis and flagella gene double mutantΔcheAΔfliC and its functional analysis in Acidovorax citrulli[J].Journal of Agricultural Biotechnology,25(11):1838-1850.)
Bahar O,Goffer T,Burdman S.2009.Type IV pili are required for virulence,twitching motility,and biofilm formation of Acidovorax avenae subsp.citrulli[J].Molecular Plant-Microbe Interactions,22(8):909-920.
David A.Wilkinson,Sarah J.Chacko,Catherine ve′NIen-bryan et al.2011.Regulation of flagellum number by flia and flgm and role in biofilm formation by rhodobacter sphaeroides[J].Journal of Bacteriology,193(15):4010-4014.
Frankle W.1993.Ingress of the watermelon fruit blotch bacterium into fruit[J].Plant Disease,77:1090-1092.
Frisk A,Jyot J,Arora S K,et al.2002.Identification and functional characterization of flgM,a gene encoding the antisigma 28 factor in Pseudomonas aeruginosa[J].Journal of Bacteriology,184(6):1514-1521.
Gavin R,Rabaan A A,Merino S,et al.2002.Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation[J].Molecular Microbiology,43(2):383-397.
Kessler B,Lorenzo L,Timmisk N.1992.A general system to integrate lac Z fusions into the chromosomes of Gram-negative eubacteria:regulation of the Pm promoter of the TOLplasmid studied with all controlling elements in monocopy[J].Molecular and General Genetics,233:293-301.
Kutsukake K,Iino T.1994.Role of the FliA-FlgM regulatory system on the transcriptional control of the flagellar regulon and flagellar formation in Salmonella typhimurium[J].Journal of Bacteriology,176(12):3598-3605.
Luo J Y,Qiu W,Chen L,et al.2015.Identification of pathogenicity-related genes in biofilm-defective Acidovorax citrulli by transposon Tn5 mutagenesis[J].International Journal of Molecular Sciences,16(12):28050-28062.
Muller V,Jones C J,Kawagishi I,et al.1992.Characterization of the fliE genes of Escherichia coli and Salmonella typhimurium and identification of the FliE protein as a component of the flagellar hook-basal body complex[J].Journal of Bacteriology,174(7):2298-23004.
Osorio-valeriano M,Morra J D L,Camarena L,et al.2015.Biochemical characterization of the flagellar rod components of Rhodobacter sphaeroides:Properties and interactions[J].Journal of Bacteriology,198(3):544-552.
Petrocelli S,Arana M R,Cabrini M N,et al.2016.Deletion of pilA,a minor pilin-like gene,from Xanthomonas citri subsp.citri influences bacterial physiology and pathogenesis[J].Current Microbiology,73:904-914.
Song W Y,Kim H M,Schaad N W.2000.PCR Primers for detection and indentification of plant pathogenic species,subspecies and strains of Acidovorax[P].United States Patent 6423499B1.
Tian Y L,Zhao Y Q,Wu X R,et al.2015.The type VI protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon[J].Molecular Plant Pathology,16(1):38-47.
Tremblay J,Déziel E.2010.Gene expression in Pseudomonas aeruginosa warming motility[J].BioMed Central Genomics,11:587-602.
Walcott R R,Fessehaie A,Castro A C.2004.Differences in pathogenicity between two genetically distinct groups of Acidovorax avenae subsp.citrulli on cucurbit hosts[J].Journal of Phytopathology,152:277-285.
Wilkinson D A,Chacko S J,Vénien-Bryan C,et al.2011.Regulation of flagellum number by FliA and FlgM and role in biofilm formation by Rhodobacter sphaeroides[J].Journal of Bacteriology,193(15):4010-4014.
Wosten M M S M,van Dijk L,Veenendaal A K J,et al.2010.Temperature-dependent FlgM/FliA complex formation regulates Campylobacter jejuni flgella length[J].Molecular Microbiology,75(6):1577-1591.
Yang T C,Leu Y W,Chang-chien H C,et al.2009.Flagellar biogenesis of Xanthomonas campestris requires the alternative sigma factors RpoN2 and FliA and is temporally regulated by FlhA,FlhB,and FlgM?[J].Journal of Bacteriology,191(7):2266-2275.
Zivanovic M,Walcott R.R.2017.Further characterization of genetically distinct groups of Acidovorax citrulli strains[J].Phytopathology,107(1):29-35.