赣南柑橘木虱体内黄龙病菌原噬菌体类型多样性的研究
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摘要
柑橘黄龙病是柑橘生产中的毁灭性病害,且该病病原菌中存在噬菌体/原噬菌体。原噬菌体序列是该病病原菌基因组动态变化的关键组分,对病原菌种内多样性起重要作用。为探明赣南地区柑橘木虱体内黄龙病菌原噬菌体类型的多样性,利用2对PCR引物对分别采自赣南赣州市、寻乌、于都、定南、瑞金、南康、崇义等7个不同柑橘产区的42头柑橘木虱阳性样品进行PCR检测和测序。结果表明在赣南地区柑橘木虱体内黄龙病菌株系原噬菌体类型的存在划分为4种:单一SC1类型、单一SC2类型、没有噬菌体类型和SC1-SC2噬菌体类型。其中单一的原噬菌体类型(SC1 or SC2)检测率达67%,而2种原噬菌体共存或未检测到噬菌体类型分别占12%、21%。由此可见赣南柑橘木虱体内黄龙病菌株中单一的原噬菌体占一定优势,可能是由于2种原噬菌体在寄主中存在竞争。对赣南柑橘木虱黄龙病菌株中噬菌体/原噬菌体类型检测有助于了解该区菌株噬菌体/原噬菌体的遗传多样性,也有助于进一步的研究分析柑橘木虱的传毒机制。
Citrus Huanglongbing( HLB),caused by Candidatus liberibacter,is the most devastating disease of citrus for which no effective cure has been found.Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. In order to investigate the genetic diversity among CLas strains,two primer sets,targeted hypervariable genomic regions of two prophage types( SC1 and SC2),respectively,were used for PCR detection and sequence analysis of 42 samples of Diaphorina citri from seven localities in Jiangxi,including Ganzhou,Xunwu,Yudou,Dingnan,Ruijin,Nankang and Congyi. PCR amplicon polymorphisms were observed,the prophage types were classified into four types,single SC1 type,single SC2 type,both SC1-SC2 type and no prophage types in "Candidatus Liberibacter asiaticus"strains of Diaphorina citri in Gannan.And the result suggested that most of CLas strains contained only one prophage( SC1 or SC2)( 67%),and CLas strains with two and no prophage types accounted for 12% and 21%,respectively.The prevalence of single prophages suggested competition events between prophages for CLas hosts.So the investigation on prophage type of Diaphorina citri in Gannan may help analyzing the genetic diversity of prophage in Gannan strains,and further researching the toxic mechanism of Diaphorina citri.
Citrus Huanglongbing( HLB),caused by Candidatus liberibacter,is the most devastating disease of citrus for which no effective cure has been found.Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. In order to investigate the genetic diversity among CLas strains,two primer sets,targeted hypervariable genomic regions of two prophage types( SC1 and SC2),respectively,were used for PCR detection and sequence analysis of 42 samples of Diaphorina citri from seven localities in Jiangxi,including Ganzhou,Xunwu,Yudou,Dingnan,Ruijin,Nankang and Congyi. PCR amplicon polymorphisms were observed,the prophage types were classified into four types,single SC1 type,single SC2 type,both SC1-SC2 type and no prophage types in "Candidatus Liberibacter asiaticus"strains of Diaphorina citri in Gannan.And the result suggested that most of CLas strains contained only one prophage( SC1 or SC2)( 67%),and CLas strains with two and no prophage types accounted for 12% and 21%,respectively.The prevalence of single prophages suggested competition events between prophages for CLas hosts.So the investigation on prophage type of Diaphorina citri in Gannan may help analyzing the genetic diversity of prophage in Gannan strains,and further researching the toxic mechanism of Diaphorina citri.
引文
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[15]陈娇月.柑橘黄龙病菌原噬菌体的基因区域遗传多态性研究[D].重庆:西南大学,2015.Chen J Y.Genetic diversity of prophage of‘Candidatus Liberibacter asiaticus’[D].Chongqing:Southwest University,2015.
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[18]Tomimura K,Miyata S,Furuya N,et al.Evaluation of genetic diversity among‘Candidatus liberibacter asiaticus’isolates collected in southeast Asia[J].Phytopathology,2009,99(9):1062-1069.
[19]苏华楠.中国柑橘黄龙病病原调查、种群遗传分化及其原噬菌体溶菌酶蛋白原核表达[D].重庆:西南大学,2013.Su H N.HLB etiology survey,population differentiation of‘Candidatus Liberibacter asiaticus’in China and Lysozyme protein prokaryotic expression of its prophage[D].Chongqing:Southwest University,2013.
[2]陈慈相,张倩,谢金招,等.赣南地区柑桔黄龙病发生规律研究[J].中国南方果树,2015,44(6):43-45.Chen C X,Zhang J,Xie J Z,et al.Reseach the occurrence regularity of Huanglongbing in Gannan[J].South China Fruits,2015,44(6):43-45.
[3]刘登全,涂怀妹,李霖,等.江西省大余县柑橘黄龙病菌PCR检测[J].生物灾害科学,2012,35(3):261-263.Liu D Q,Tu H M,Li L,et al.PCR detection of Citrus Huanglongbing pathogen in Dayu County of Jiangxi Province[J].Biological Disaster Science,2012,35(3):261-263.
[4]刘登全,崔朝宇,蒋军喜,等.不同柑橘品种对黄龙病的抗性鉴定[J].江西农业大学学报,2014,36(1):97-101.Liu D Q,Cui C Y,Jiang J X,et al.Resistance identification of different citrus cultivars against Huanglongbing[J].Acta Agriculturae Universitatis Jiangxiensis,2014,36(1):97-101.
[5]卢占军,刘映雪,丁鹏,等.基于SSR标记的赣南柑桔黄龙病菌遗传多样性分析[J].中国南方果树,2016,45(5):1-6.Lu Z J,Liu Y X,Ding P,et al.Genetic diversity of Candidatus liberibacter asiaticus in Gannan using SSR markers[J].South China Fruits,2016,45(5):1-6.
[6]金丹凤,熊大维,黄国昌,等.赣南脐橙柑橘黄龙病株内生细菌群落多样性研究[J].江西科学,2016,34(4):434-437.Jin D F,Xiong D W,Huang G C,et al.Comparison of cultured and uncultured endophytic bacterial community in Huanglongbing affected Gannan navel oranges[J].Jiangxi Science,2016,34(4):434-437.
[7]熊大维,金丹凤,黄国昌,等.柑橘黄龙病赣南脐橙内生菌种群结构分析[J].微生物学通报,2017,44(3):631-638.Xiong D W,Jin D F,Huang G C,et al.Endophytic bacterial community in Gannan navel orange affected by huanglong disease[J].Microbiology China,2017,44(3):631-638.
[8]Duan Y,Zhou L,Hall D G,et al.Complete genome sequence of citrus Huanglongbing bacterium,‘Candidatus Liberibacter asiaticus’obtained through metagenomics[J].Molecular Plant-Microbe Interactions,2009,22(8):1011-1020.
[9]刘蕤.柑橘黄龙病菌在九里香中消长变化及原噬菌体序列分析[D].广州:华南农业大学,2010.Liu R.The change of‘Candidatus Liberibacter asiaticus’and analysis of prophage sequence in Murraya exotica[D].Guangzhou:South China Agricultural University,2010.
[10]Zhou L,Powell C A,Hoffman M T,et al.Diversity and plasticity of the intracellular plant pathogen and insect symbiont‘Candidatus Liberibacter asiaticus’revealed by hypervariable prophage genes with intragenic tandem repeats[J].Appled and Environmenal Microbiology,2011,77(18):6663-6673.
[11]Zhang S,Flores C Z,Zhou L,et al.‘Ca.Liberibacter asiaticus’carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections[J].Proteomics,2011,24(4):458-468.
[12]Zhou L,Powell C A,Wenbin L,et al.Prophage-mediated dynamics of‘Candidatus Liberibacter asiaticus'populations,the destructive bacterial pathogens of citrus huanglongbing[J].Plos One,2013,8(12):e82248.
[13]Lin H,Han C S,Liu B,et al.Complete genome sequence of a Chinese strain of‘Candidatus Liberibacter asiaticus’[J].Genome Announcements,2013,1(2):e00184-13.
[14]谭锦.中国柑橘黄龙病病原菌原噬菌体遗传多样性研究[D].重庆:西南大学,2013.Tan J.Genetic diversity of prophage region from‘Candidatus Liberibacter asiaticus’in China[D].Chongqing:Southwest University,2013.
[15]陈娇月.柑橘黄龙病菌原噬菌体的基因区域遗传多态性研究[D].重庆:西南大学,2015.Chen J Y.Genetic diversity of prophage of‘Candidatus Liberibacter asiaticus’[D].Chongqing:Southwest University,2015.
[16]Zheng Z,Zeng Y B,Bao M L,et al.Diversity analysis of‘Candidatus Liberibacter asiaticus’associated phage type in China[C].2015.
[17]Jagoueix S,Bove J M,Garnier M.The phloem-limited bacterium of greening disease of citrus is a member of theαsubdivision of the Proteobacteria[J].International Journal of Systematic Bacteriology,1994,44(3):379-386.
[18]Tomimura K,Miyata S,Furuya N,et al.Evaluation of genetic diversity among‘Candidatus liberibacter asiaticus’isolates collected in southeast Asia[J].Phytopathology,2009,99(9):1062-1069.
[19]苏华楠.中国柑橘黄龙病病原调查、种群遗传分化及其原噬菌体溶菌酶蛋白原核表达[D].重庆:西南大学,2013.Su H N.HLB etiology survey,population differentiation of‘Candidatus Liberibacter asiaticus’in China and Lysozyme protein prokaryotic expression of its prophage[D].Chongqing:Southwest University,2013.