基于原噬菌体类型的我国柑橘黄龙病菌种群遗传结构分析
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摘要
柑橘黄龙病是柑橘生产上最严重的病害之一,由难培养的候选韧皮部杆菌亚洲种("Candidatus Liberibacter asiaticus",CLas)引起。目前,已报道的与CLas相关的原噬菌体有3种,并且不同的CLas样品中可以检测到不同类型的原噬菌体或不同类型的组合。本研究基于原噬菌体类型特异引物对从我国南方8省收集的548个黄龙病样品进行原噬菌体类型的鉴定和分析。结果表明,基于原噬菌体类型,在548个样品中共鉴定了7种类型的菌株,分别为Type 1(7.12%)、Type 2(60.22%)、Type 3(3.83%)、Type 1+Type 2(3.28%)、Type 1+Type 3(17.34%)、Type 1+Type 2+Type 3(3.47%)和None类型(不含上述3种类型原噬菌体)(4.74%),其中只携带Type 2类型原噬菌体菌株为华南地区优势种群。此外,基于遗传多样性和遗传距离对我国南方地区的CLas种群进行种群结构分析发现,我国南方地区的CLas种群可以分为3个组,即Ⅰ组(广东、江西、广西、福建)、Ⅱ组(浙江和湖南)以及Ⅲ组(云南和海南),其中Ⅰ组和Ⅱ组又可聚为一大组。该结果可为研究我国柑橘黄龙病流行规律提供一定的理论基础。
Citrus Huanglongbing( HLB),one of most destructive disease worldwide,is associated with an uncultivable bacterium in the α-Proteobacteria,‘Candidatus Liberibacter asiaticus'( CLas). Three types of prophages,Type 1( SC1-like),Type 2( SC2-like) and Type 3( P-JXGC-3),as well as different combinations of above prophages have been identified in CLas samples. In this study,a total of six type-specific primer sets( two sets for each prophage type) from our previous study were selected to perform the prophage typing using548 CLas samples collected from eight provinces in southern China. Based on the prophage typing profiles,seven combinations of CLas prophages were identified,including single Type 1( 7.12%),single Type 2( 60.22%),single Type 3( 3.83%),Type 1 + Type 2( 3.28%),Type 1 + Type 3( 17.34%),Type 1 + Type 2 + Type 3( 3.47%) and None type( 4.74%). The CLas samples only harboring the Type 2 prophage were predominant in CLas population in Southern China. Genetic structure of CLas population was evaluated by using Nei's diversity index( H value),genetic identity and genetic distance. Three major groups were identified based on the genetic distance of CLas population from eight provinces,i. e. group Ⅰ( Guangdong,Jiangxi,Guangxi and Fujian),group Ⅱ( Zhejiang and Hunan) and group Ⅲ( Yunnan and Hainan). Group Ⅰ and Group Ⅱ can be further clustered into a larger group. The results provide a theoretical basis for studying the epidemic of citrus Huanglongbing in China.
Citrus Huanglongbing( HLB),one of most destructive disease worldwide,is associated with an uncultivable bacterium in the α-Proteobacteria,‘Candidatus Liberibacter asiaticus'( CLas). Three types of prophages,Type 1( SC1-like),Type 2( SC2-like) and Type 3( P-JXGC-3),as well as different combinations of above prophages have been identified in CLas samples. In this study,a total of six type-specific primer sets( two sets for each prophage type) from our previous study were selected to perform the prophage typing using548 CLas samples collected from eight provinces in southern China. Based on the prophage typing profiles,seven combinations of CLas prophages were identified,including single Type 1( 7.12%),single Type 2( 60.22%),single Type 3( 3.83%),Type 1 + Type 2( 3.28%),Type 1 + Type 3( 17.34%),Type 1 + Type 2 + Type 3( 3.47%) and None type( 4.74%). The CLas samples only harboring the Type 2 prophage were predominant in CLas population in Southern China. Genetic structure of CLas population was evaluated by using Nei's diversity index( H value),genetic identity and genetic distance. Three major groups were identified based on the genetic distance of CLas population from eight provinces,i. e. group Ⅰ( Guangdong,Jiangxi,Guangxi and Fujian),group Ⅱ( Zhejiang and Hunan) and group Ⅲ( Yunnan and Hainan). Group Ⅰ and Group Ⅱ can be further clustered into a larger group. The results provide a theoretical basis for studying the epidemic of citrus Huanglongbing in China.
引文
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[3]Lin K H.Observations on yellow shoot of citrus(in Chinese)[J].Acta Phytopathology Sinica(植物病理学报),1956,2(1):13-42.
[4]Sichuan citrus Huanglongbing investigation group.An brief investigation on the occurrence of citrus Huanglongbing in Xichang area and Dukou city(in Chinese)[J].South China Fruits(中国南方果树),1997(Z2):21-22.
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[13]Feng Z,Zhou G,Deng X L.Cloning and sequence analysis of 16S rDNA fragment of citrus Huanglongbing(in Chinese)[J].Guangxi agricultural biological sciences(广西农业生物科学),2006,25(2):107-110.
[14]Garnier M,Jagoueixeveillard S,Cronje P R,et al.Genomic characterization of a liberibacter present in an ornamental rutaceous tree,Calodendrum capense,in the Western Cape Province of South Africa.Proposal of‘Candidatus Liberibacter africanus subsp.Capensis’[J].International Journal of Systematic&Evolutionary Microbiology,2000,50(6):2119-2125.
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[16]Duan Y P,Zhou L J,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.
[17]Chen J,Deng X,Sun X,et al.Guangdong and Florida populations of‘Candidatus Liberibacter asiaticus’distinguished by a genomic locus with short tandem repeats[J].Phytopathology,2010,100(6):567-72.
[18]Zhou L,Powell C A,Hoffman M T,et al.Diversity and plasticity of the intracellular plant pathogen and insect symbiont‘Candidatus Liberibacter asiaticus’as revealed by hypervariable prophage genes with intragenic tandem repeats[J].Applied&Environmental Microbiology,2011,77(18):6663-73.
[19]Liu R,Zhang P,Pu X,et al.Analysis of a prophage gene frequency revealed population variation of‘Candidatus Liberibacter asiaticus’from two citrus-growing provinces in China[J].Plant Disease,2011,95(4):431-435.
[20]Wang X,Zhou C,Deng X,et al.Molecular characterization of a mosaic locus in the genome of‘Candidatus Liberibacter asiaticus’[J].BM C Microbiology,2012,12(1):18.
[21]Ma W,Liang M,Guan L,et al.Population structures of‘Candidatus Liberibacter asiaticus’in Southern China[J].Phytopathology,2014,104(2):158-62.
[22]Boyd E F,Brüssow H.Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved[J].Trends in Microbiology,2002,10(11):521-9.
[23]Zhang S,Florescruz 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].Molecular Plant-microbe Interactions,2011,24(4):735-737.
[24]Zheng Z,Bao M,Wu F,et al.A Type 3 prophage of‘Candidatus Liberibacter asiaticus’carrying a restriction-modification system[J].Phytopathology,2018,108(4):454-461.
[25]Zheng Z,Bao M,Wu F,et al.Predominance of single prophage carrying a CRISPR/cas system in‘Candidatus Liberibacter asiaticus’strains in Southern China[J].PLoS One,2016,11(1):e0146422.
[26]Zheng Z,Wu F,Kumagai L B,et al.Two‘Candidatus Liberibacter asiaticus’strains recently found in California harbor different prophages[J].Phytopathology,2017,107(6):662-668.
[27]Li W,Hartung J S,Levy L.Quantitative real-time PCR for detection and identification of‘Candidatus Liberibacter’species associated with citrus Huanglongbing[J].Journal of Microbiological M ethods,2006,66:104-115.
[28]Jagoueix S,BovéJ M,Garnier M.PCR detection of the two‘Candidatus Liberobacter’species associated with greening disease of citrus[J].Molecular and Cellular Probes,1996,10(1):43-50.
[29]Tamura K,Stecher G,Peterson D,et al.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30:2725-2729.
[30]Fang G C,Liu B,Wu R J,et al.30 years of citrus Huanglongbing research in China(in Chinese)[J].Fujian Journal of Agricultural Sciences(福建农业学报),2009,24(2):183-190.
[31]Huang L Q.A review of citrus Huanglongbing in Ganzhou area(in Chinese)[J].Gannan agricultural technology(赣南农业科技),1992(2):7-9.
[32]Ma W,Liang M,Guan L,et al.Population structures of“Candidatus Liberibacter asiaticus”in Southern China[J].Phytopathology,2014,104(2):158-62.
[33]Zeng L.Analysis of the epdimic of citrus Huanglongbing(in Chinese)[J].Journal of yunnan agricultural university(云南农业大学学报自然科学),1993,V8(3):286-287.
[2]Chen Q B.A report of a study on yellow shoot disease of citrus in Chaoshan(in Chinese)[J].New Agriculture Quarterly Bullrtin(新农季刊),1943,3(3-4):142-177.
[3]Lin K H.Observations on yellow shoot of citrus(in Chinese)[J].Acta Phytopathology Sinica(植物病理学报),1956,2(1):13-42.
[4]Sichuan citrus Huanglongbing investigation group.An brief investigation on the occurrence of citrus Huanglongbing in Xichang area and Dukou city(in Chinese)[J].South China Fruits(中国南方果树),1997(Z2):21-22.
[5]Ke C,Chen H,Lin Z X.Summary of citrus Huanglongbing research(in Chinese)[J].Fujian Fruits(福建柑橘),1980(03):1-4.
[6]Yao W S.Current situation of citrus Huanglongbing in Yunnan,Guizhou and Sichuan province(in Chinese)[J].Plant ptotection(植物保护),1985(02):47-48.
[7]Yunnan citrus Huanglongbing investigation group.Investigation report on citrus yellowing in Yunnan province(in Chinese)[J].China Citrus(中国柑橘),1981(04):14-16.
[8]Wu S P,Li J S.Preliminary observation of pathogens of citrus Huanglongbing and yellowing disease(in Chinese)[J].Scientia Agricultura Sinica(中国农业科学),1986(01):73-76.
[9]Zhao L,Meng Y Q,Lin Q B,et al.Current situation and control strategy of citrus Huanglongbing in Zhejiang province(in Chinese)[J].Zhejiang Citrus(浙江柑橘),2008,25(2):17-20.
[10]Jagoueix S,BovéJ M,Garnier M.The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria[J].International Journal of Systematic Bacteriology,1994,44(3):379-86.
[11]Deng X L,Tang W W.Application of PCR in detection of pathogen of citrus Huanglongbing(in Chinese)[J].Journal of South China Agricultural University(华南农业大学学报),1996(3):119-120.
[12]TianY N,Ke S,Ke C.Application of PCR in detection and quantification of pathogen of Citrus Huanglongbing(in Chinese)[J].Acta Phytopathology Sinica(植物病理学报),1996,26(3):243-250.
[13]Feng Z,Zhou G,Deng X L.Cloning and sequence analysis of 16S rDNA fragment of citrus Huanglongbing(in Chinese)[J].Guangxi agricultural biological sciences(广西农业生物科学),2006,25(2):107-110.
[14]Garnier M,Jagoueixeveillard S,Cronje P R,et al.Genomic characterization of a liberibacter present in an ornamental rutaceous tree,Calodendrum capense,in the Western Cape Province of South Africa.Proposal of‘Candidatus Liberibacter africanus subsp.Capensis’[J].International Journal of Systematic&Evolutionary Microbiology,2000,50(6):2119-2125.
[15]Bastianel C,Garniersemancik M,Renaudin J,et al.Diversity of‘Candidatus Liberibacter asiaticus’based on the omp gene sequence[J].Applied&Environmental Microbiology,2005,71(11):6473-8.
[16]Duan Y P,Zhou L J,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.
[17]Chen J,Deng X,Sun X,et al.Guangdong and Florida populations of‘Candidatus Liberibacter asiaticus’distinguished by a genomic locus with short tandem repeats[J].Phytopathology,2010,100(6):567-72.
[18]Zhou L,Powell C A,Hoffman M T,et al.Diversity and plasticity of the intracellular plant pathogen and insect symbiont‘Candidatus Liberibacter asiaticus’as revealed by hypervariable prophage genes with intragenic tandem repeats[J].Applied&Environmental Microbiology,2011,77(18):6663-73.
[19]Liu R,Zhang P,Pu X,et al.Analysis of a prophage gene frequency revealed population variation of‘Candidatus Liberibacter asiaticus’from two citrus-growing provinces in China[J].Plant Disease,2011,95(4):431-435.
[20]Wang X,Zhou C,Deng X,et al.Molecular characterization of a mosaic locus in the genome of‘Candidatus Liberibacter asiaticus’[J].BM C Microbiology,2012,12(1):18.
[21]Ma W,Liang M,Guan L,et al.Population structures of‘Candidatus Liberibacter asiaticus’in Southern China[J].Phytopathology,2014,104(2):158-62.
[22]Boyd E F,Brüssow H.Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved[J].Trends in Microbiology,2002,10(11):521-9.
[23]Zhang S,Florescruz 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].Molecular Plant-microbe Interactions,2011,24(4):735-737.
[24]Zheng Z,Bao M,Wu F,et al.A Type 3 prophage of‘Candidatus Liberibacter asiaticus’carrying a restriction-modification system[J].Phytopathology,2018,108(4):454-461.
[25]Zheng Z,Bao M,Wu F,et al.Predominance of single prophage carrying a CRISPR/cas system in‘Candidatus Liberibacter asiaticus’strains in Southern China[J].PLoS One,2016,11(1):e0146422.
[26]Zheng Z,Wu F,Kumagai L B,et al.Two‘Candidatus Liberibacter asiaticus’strains recently found in California harbor different prophages[J].Phytopathology,2017,107(6):662-668.
[27]Li W,Hartung J S,Levy L.Quantitative real-time PCR for detection and identification of‘Candidatus Liberibacter’species associated with citrus Huanglongbing[J].Journal of Microbiological M ethods,2006,66:104-115.
[28]Jagoueix S,BovéJ M,Garnier M.PCR detection of the two‘Candidatus Liberobacter’species associated with greening disease of citrus[J].Molecular and Cellular Probes,1996,10(1):43-50.
[29]Tamura K,Stecher G,Peterson D,et al.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30:2725-2729.
[30]Fang G C,Liu B,Wu R J,et al.30 years of citrus Huanglongbing research in China(in Chinese)[J].Fujian Journal of Agricultural Sciences(福建农业学报),2009,24(2):183-190.
[31]Huang L Q.A review of citrus Huanglongbing in Ganzhou area(in Chinese)[J].Gannan agricultural technology(赣南农业科技),1992(2):7-9.
[32]Ma W,Liang M,Guan L,et al.Population structures of“Candidatus Liberibacter asiaticus”in Southern China[J].Phytopathology,2014,104(2):158-62.
[33]Zeng L.Analysis of the epdimic of citrus Huanglongbing(in Chinese)[J].Journal of yunnan agricultural university(云南农业大学学报自然科学),1993,V8(3):286-287.