中国柑桔主产区柑桔木虱细菌多样性及内共生菌Wolbachia系统发育研究
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摘要
柑桔木虱(Diaphorina citri Kuwayama)主要取食危害九里香、柑桔嫩梢等芸香科植物,是柑桔黄龙病(Citrus huanglongbing,HLB)传播的主要媒介昆虫。昆虫体内是一个复杂的微生态系统,存在大量的微生物。这些微生物对宿主发育,营养物质的消化吸收和防御方面都起着重要的作用。本研究利用基于16S rRNA基因作为分子标记的变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)的方法和基于16S rRNA基因的RFLP指纹图谱法对我国部分地区野外采集的柑桔木虱体内的微生物群落的多样性进行了研究,并对共生菌Wolbachia进行了检测及系统发育分析,期望找出对柑桔木虱的生长发育起着重要影响的细菌,为柑桔木虱的防治提供新的思路和理论依据。
主要研究结果如下:
采用基于细菌16SrDNA的RFLP(Restriction Fragment Length Polymorphism )方法对柑桔木虱体内细菌多样性进行了分析。使用细菌通用引物对柑桔木虱体内的细菌基因组DNA进行PCR扩增,扩增得到的16S rDNA序列连接到pMD19-T载体中,将连接产物转化到大肠杆菌JM109中,建立16S rDNA的全长克隆文库。挑取的185个阳性克隆子经过三种限制性内切酶RsaⅠ、MspⅠ和HaeⅢ消化后产生31个不同的片断长度多态性(restriction fragment length polymorphism)图谱。将31种有差异的16S rRNA酶切图谱进行测序,与GenBank中的数据进行比对,结果表明:柑桔木虱体内存在Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae, Rhizobiaceae等6个科的微生物,能够鉴定到属种的包括:Pseudomonas sp, Ralstonia sp, Pantoea sp, Serratia sp, Wolbachia sp, Stenotrophomonas sp, Carsonellia ruddii以及Candidatus Liberibacter asiaticus(柑桔黄龙病菌),Syncytium endosymbiont(胞内共生体),Secondary endosymbiont(初级共生体)。另外还有7个不同的不可培养细菌。Syncytium endosymbiont占16SrDNA克隆文库的31%,为柑桔木虱体内的优势菌群。
以基于细菌16S rRNA基因的变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)的方法对来自于不同地区及不同寄主的柑桔木虱体内细菌多样性进行了分析。提取细菌基因组DNA,经细菌16SrRNA可变区基因的通用引物PCR扩增,DGGE分离后,回收测序了17条DGGE条带。与GenBank中数据进行了比对和系统进化树分析,得到Pseudomonadaceae, Rickettsiaceae, Enterobcteriaceae, Xanthomonadaceae, Staphylococceae和Bacillaceae等科的微生物,能够鉴定到属的细菌有Bacillus sp, Staphylococcus sp, Wolbachia sp, Pantoea sp,Pseudomonas sp以及Syncytium endosymbiont (胞内共生体)。Syncytium endosymbiont(胞内共生体)在所有柑桔木虱样品中普遍存在且在DGGE图谱中条带最亮,是主要的优势菌群。DGGE图谱显示不同寄主植物及不同地区的柑桔木虱体内细菌存在差异,而DGGE图谱的聚类分析表明来自相同寄主植物的柑桔木虱体内细菌群落差异相对较小。
使用Wolbachia的16SrRNA(核糖体基因),ftsZ(细胞分裂基因)和wsp(细胞表面蛋白基因)的特异引物检测了我国5个不同柑桔主产区来源的柑桔木虱受Wolbachia的感染情况,将获得的3段基因序列克隆测序,在GenBank中比对分析建立系统发育树,结果进一步验证了5个不同来源的柑桔木虱体内都有Wolbachia感染,3段基因的序列分析和系统发育树均表明我国柑桔木虱体内感染的Wolbachia属于B大组,以wsp基因建立的系统发育树还进一步表明其Wolbachia属于B大组的Con亚组。且不同地区来源的柑桔木虱体内的Wolbachia的16SrRNA,ftsZ和wsp基因同源性都分别高达99%以上,差异非常小。表明在不同地区的柑桔木虱体内Wolbachia几乎没有差异。
本研究的结果显示了柑桔木虱体内存在着大量的微生物,且不同地区及不同寄主植物的柑桔木虱体内的细菌种类和数量存在一定的差异。我国各地柑桔木虱体内均有Wolbachia感染,其感染的Wolbachia属于B组的Con亚组并与灰飞虱和稻飞虱感染的有极高的同源性。这些结果可望能够为分析内共生菌与昆虫之间的关系,柑桔木虱体内微生物与柑桔黄龙病菌之间的相互作用提供理论基础,并为柑桔木虱的综合防治提供新的途径。
The citrus psyllids (Diaphorina citri Kuwayama) is the main insect vectors of the HLB (Citrus huanglongbing) pathogens, which mainly damage the Rutaceous plants. The internal of insects is a complex micro-ecosystem, in which a large and varied microbial community inhabits. This community plays important roles in evelopment, digestion and absorption of nutrition of their host, and colonization resistance against invasion of exotic microbes and so on. In this study, We did some research on the microbial diversity of citrus psyllids (Diaphorina citri) collected from some wild lands in my country which vectors Liberibacter sp, Using 16S rDNA-based molecular approaches of denaturing gradient gel-electrophoresis(DGGE) and restriction fragment length polymorphism(RFLP). We try to detect andanalyse phylogenetic relationship of the endosymbiont Wolbachia in Diaphorina citri, expecting to find out the bacteria which may play an important influence on the growth of citrus psyllids,and provide a new ideas and theoretical basis for the prevention and treatment of citrus psyllids.
The primary results were as follows:
The analysis of microbial diversity of citrus psyllids was conducted using 16S rDNA-based of restriction fragment length polymorphism (RFLP) method with bacterial universal primers.And ligation of the 16SrDNA fragement to the pMD19-T, then tranfor it to E.coli strain JM109 competent cell.The 16S rDNA clone librarys were constructed. The 16SrDNA fingerprints of the microorganisms were analyzed by isolated plasmid and digested with HaeⅢ, MspI and RsaI enzymes respectively. A total of 185 postive clones were analyzed by RFLP, and 31 unique RFLP patterns were obtained with three restriction enzyme (HaeⅢ, MspⅠand RsaⅠ).Sequenced the 31 clones, and aligned the sequences with GenBank database. It showed that they were belonged to six different families of bacteria, including Pseudomonadaceae, Enterobact -eriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae, Rhizobiaceae. The microorganisms can be identified to bacterial genera bacteria including Pseudomonas sp, Ralstonia sp, Pantoea sp, Serratia sp, Wolbachia sp, Stenotrophomonas sp, and Carsonellia ruddii, Candidatus Liberibacter asiaticus, Syncytium endosymbiont, Secondary endosymbiont. There are also seven different uncultured bacterias. The dominant bacteria in the citrus psyllids belonged to Syncytium endosymbiont and accounted for 31 % of the total 16S rRNA clones.
The molecular method of PCR of denaturing gradient gel electrophoresis (PCR-DGGE) analysis based on the sequence of 16S rRNA V3 region gene was used to the microflora analysis of the psyllids citrus from different geography and different host plants. By 16S rRNA V3 region gene DGGE method, seventeen distinct bands were obtained from 16S rDNA amplificons. The bands were purified and sequenced.The sequences were aligned with GenBank database and the result showed that they were Pseudomonadaceae, Rickettsiaceae, Enterobcteriaceae,Xanthomonadaceae, Staphylo- cocceae and Bacillaceae family microorganisms. The microorganisms can be identified to genera bacteria including Bacillus sp, Staphylococcus sp, Wolbachia sp, Pantoea sp, Pseudomonas sp and Syncytium endosymbiont. Syncytium endosymbiont is the dominant bacteria group based on the strength of the bands from DGGE, existing in all citrus psyllids samples in this study. Wolbachia and Pantoea sp also exist in all the samples. The Cluster dendrogram analysis of DGGE bands showed that the difference of the bacterial community is relatively small in the citrus psyllids come from the same host plants.
The endosymbiont Wolbachia infection of Diaphorina citri from five regions was detected with PCR amplification using primer pairs designed from the 16S rDNA, ftsZ and the wsp gene of Wolbachia,three genes were cloned and sequenced, aligned the sequences with GenBank database.Then phylogenetic analysised the three genes information of Wolbachia.The result confirmed the five regions citrus psyllids were infected by Wolbachia.The three genes phylogenetic trees show that the Wolbachia infecting citrus psyllids in our country belong to the supergroup B.The wsp gene phylogenetic further showed that the Wolbachia belong to the Con strain. The homology of 16SrRNA, ftsZ and wsp gene sequences in different geography populations is up to 99%, which indicated Wolbachia in different geography Diaphorina citri almost have no remarkable difference.
The result showed that the rich microbiota diversity existed in the citrus psyllids and the species and amount of the bacteria are different in the citrus psyllids of different host plants and different geography.The result confirmed citrus psyllids were infected by Wolbachia, and which belong to the Con strain of supergroup B. The result expected to provide a theoretical foundation about the relationship between endosymbiont and insect, the interaction between the microorgnism and candidatus Liberibacter in citrus psyllids.And expected to provide a new ideas about control of citrus psyllids.
主要研究结果如下:
采用基于细菌16SrDNA的RFLP(Restriction Fragment Length Polymorphism )方法对柑桔木虱体内细菌多样性进行了分析。使用细菌通用引物对柑桔木虱体内的细菌基因组DNA进行PCR扩增,扩增得到的16S rDNA序列连接到pMD19-T载体中,将连接产物转化到大肠杆菌JM109中,建立16S rDNA的全长克隆文库。挑取的185个阳性克隆子经过三种限制性内切酶RsaⅠ、MspⅠ和HaeⅢ消化后产生31个不同的片断长度多态性(restriction fragment length polymorphism)图谱。将31种有差异的16S rRNA酶切图谱进行测序,与GenBank中的数据进行比对,结果表明:柑桔木虱体内存在Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae, Rhizobiaceae等6个科的微生物,能够鉴定到属种的包括:Pseudomonas sp, Ralstonia sp, Pantoea sp, Serratia sp, Wolbachia sp, Stenotrophomonas sp, Carsonellia ruddii以及Candidatus Liberibacter asiaticus(柑桔黄龙病菌),Syncytium endosymbiont(胞内共生体),Secondary endosymbiont(初级共生体)。另外还有7个不同的不可培养细菌。Syncytium endosymbiont占16SrDNA克隆文库的31%,为柑桔木虱体内的优势菌群。
以基于细菌16S rRNA基因的变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)的方法对来自于不同地区及不同寄主的柑桔木虱体内细菌多样性进行了分析。提取细菌基因组DNA,经细菌16SrRNA可变区基因的通用引物PCR扩增,DGGE分离后,回收测序了17条DGGE条带。与GenBank中数据进行了比对和系统进化树分析,得到Pseudomonadaceae, Rickettsiaceae, Enterobcteriaceae, Xanthomonadaceae, Staphylococceae和Bacillaceae等科的微生物,能够鉴定到属的细菌有Bacillus sp, Staphylococcus sp, Wolbachia sp, Pantoea sp,Pseudomonas sp以及Syncytium endosymbiont (胞内共生体)。Syncytium endosymbiont(胞内共生体)在所有柑桔木虱样品中普遍存在且在DGGE图谱中条带最亮,是主要的优势菌群。DGGE图谱显示不同寄主植物及不同地区的柑桔木虱体内细菌存在差异,而DGGE图谱的聚类分析表明来自相同寄主植物的柑桔木虱体内细菌群落差异相对较小。
使用Wolbachia的16SrRNA(核糖体基因),ftsZ(细胞分裂基因)和wsp(细胞表面蛋白基因)的特异引物检测了我国5个不同柑桔主产区来源的柑桔木虱受Wolbachia的感染情况,将获得的3段基因序列克隆测序,在GenBank中比对分析建立系统发育树,结果进一步验证了5个不同来源的柑桔木虱体内都有Wolbachia感染,3段基因的序列分析和系统发育树均表明我国柑桔木虱体内感染的Wolbachia属于B大组,以wsp基因建立的系统发育树还进一步表明其Wolbachia属于B大组的Con亚组。且不同地区来源的柑桔木虱体内的Wolbachia的16SrRNA,ftsZ和wsp基因同源性都分别高达99%以上,差异非常小。表明在不同地区的柑桔木虱体内Wolbachia几乎没有差异。
本研究的结果显示了柑桔木虱体内存在着大量的微生物,且不同地区及不同寄主植物的柑桔木虱体内的细菌种类和数量存在一定的差异。我国各地柑桔木虱体内均有Wolbachia感染,其感染的Wolbachia属于B组的Con亚组并与灰飞虱和稻飞虱感染的有极高的同源性。这些结果可望能够为分析内共生菌与昆虫之间的关系,柑桔木虱体内微生物与柑桔黄龙病菌之间的相互作用提供理论基础,并为柑桔木虱的综合防治提供新的途径。
The citrus psyllids (Diaphorina citri Kuwayama) is the main insect vectors of the HLB (Citrus huanglongbing) pathogens, which mainly damage the Rutaceous plants. The internal of insects is a complex micro-ecosystem, in which a large and varied microbial community inhabits. This community plays important roles in evelopment, digestion and absorption of nutrition of their host, and colonization resistance against invasion of exotic microbes and so on. In this study, We did some research on the microbial diversity of citrus psyllids (Diaphorina citri) collected from some wild lands in my country which vectors Liberibacter sp, Using 16S rDNA-based molecular approaches of denaturing gradient gel-electrophoresis(DGGE) and restriction fragment length polymorphism(RFLP). We try to detect andanalyse phylogenetic relationship of the endosymbiont Wolbachia in Diaphorina citri, expecting to find out the bacteria which may play an important influence on the growth of citrus psyllids,and provide a new ideas and theoretical basis for the prevention and treatment of citrus psyllids.
The primary results were as follows:
The analysis of microbial diversity of citrus psyllids was conducted using 16S rDNA-based of restriction fragment length polymorphism (RFLP) method with bacterial universal primers.And ligation of the 16SrDNA fragement to the pMD19-T, then tranfor it to E.coli strain JM109 competent cell.The 16S rDNA clone librarys were constructed. The 16SrDNA fingerprints of the microorganisms were analyzed by isolated plasmid and digested with HaeⅢ, MspI and RsaI enzymes respectively. A total of 185 postive clones were analyzed by RFLP, and 31 unique RFLP patterns were obtained with three restriction enzyme (HaeⅢ, MspⅠand RsaⅠ).Sequenced the 31 clones, and aligned the sequences with GenBank database. It showed that they were belonged to six different families of bacteria, including Pseudomonadaceae, Enterobact -eriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae, Rhizobiaceae. The microorganisms can be identified to bacterial genera bacteria including Pseudomonas sp, Ralstonia sp, Pantoea sp, Serratia sp, Wolbachia sp, Stenotrophomonas sp, and Carsonellia ruddii, Candidatus Liberibacter asiaticus, Syncytium endosymbiont, Secondary endosymbiont. There are also seven different uncultured bacterias. The dominant bacteria in the citrus psyllids belonged to Syncytium endosymbiont and accounted for 31 % of the total 16S rRNA clones.
The molecular method of PCR of denaturing gradient gel electrophoresis (PCR-DGGE) analysis based on the sequence of 16S rRNA V3 region gene was used to the microflora analysis of the psyllids citrus from different geography and different host plants. By 16S rRNA V3 region gene DGGE method, seventeen distinct bands were obtained from 16S rDNA amplificons. The bands were purified and sequenced.The sequences were aligned with GenBank database and the result showed that they were Pseudomonadaceae, Rickettsiaceae, Enterobcteriaceae,Xanthomonadaceae, Staphylo- cocceae and Bacillaceae family microorganisms. The microorganisms can be identified to genera bacteria including Bacillus sp, Staphylococcus sp, Wolbachia sp, Pantoea sp, Pseudomonas sp and Syncytium endosymbiont. Syncytium endosymbiont is the dominant bacteria group based on the strength of the bands from DGGE, existing in all citrus psyllids samples in this study. Wolbachia and Pantoea sp also exist in all the samples. The Cluster dendrogram analysis of DGGE bands showed that the difference of the bacterial community is relatively small in the citrus psyllids come from the same host plants.
The endosymbiont Wolbachia infection of Diaphorina citri from five regions was detected with PCR amplification using primer pairs designed from the 16S rDNA, ftsZ and the wsp gene of Wolbachia,three genes were cloned and sequenced, aligned the sequences with GenBank database.Then phylogenetic analysised the three genes information of Wolbachia.The result confirmed the five regions citrus psyllids were infected by Wolbachia.The three genes phylogenetic trees show that the Wolbachia infecting citrus psyllids in our country belong to the supergroup B.The wsp gene phylogenetic further showed that the Wolbachia belong to the Con strain. The homology of 16SrRNA, ftsZ and wsp gene sequences in different geography populations is up to 99%, which indicated Wolbachia in different geography Diaphorina citri almost have no remarkable difference.
The result showed that the rich microbiota diversity existed in the citrus psyllids and the species and amount of the bacteria are different in the citrus psyllids of different host plants and different geography.The result confirmed citrus psyllids were infected by Wolbachia, and which belong to the Con strain of supergroup B. The result expected to provide a theoretical foundation about the relationship between endosymbiont and insect, the interaction between the microorgnism and candidatus Liberibacter in citrus psyllids.And expected to provide a new ideas about control of citrus psyllids.
引文
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[51] Laven H. Eradication of Culex pipiens fatigans through cytoplasmic in compatibility[J]. Nature. 1967, 261: 383—384.
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[54] Masui S, Sasaki T, Ishikawa H. groE homologous operon of Wolbachia, an intracellular symbiont of arthropods: a new approach for their phylogeny[J]. Zool Sci. 1997, 14(4): 701– 7061.
[55] Meyer JM, Hoy MA, Boucias DG. Morphological and molecular characterization of a Hirsutella species infecting the Asian citrus psyllid,Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in Florida[J]. J. Invertebr.Pathol. 2007, 95:101–109.
[56] Meyer JM, Hoy MA, Boucias DG. Isolation and characterization of an Isaria fumosorosea isolate infecting the Asian citrus psyllid in Florida[J].2008, journal homepage: www. elsevier. com/locate/yjipa
[57] Moran NA, Plague GR, Sandstrom JP, Wilcox JL. A genomic perspective on nutrient provisioning by bacterial symbionts of insects.Proc[J]. Natl . Acad. Sci. 2003, 100: 14543– 145481.
[58] Muyzer G, De Waal EC, Uitterlinden A G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Appl. Environ. Microbiol. 1993, 59: 695-700.
[59] Nakabachi A, Yamashita A, Toh H, Ishikawa H, Dunbar HE, Moran NA, Hattori M. The 160-kilobase genome of the bacterial endosymbion Carsonella[J]. Science. 2006,314(5797):267
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[62] Oliver KM, Russell JA, Moran NA, Hunter MS, Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proc[J]. Natl .Acad. Sci. 2003, 100: 1803-18071
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[64] O’Neill SL, Hoffmann AA, Werren JH (Eds.) . Influential Passengers. New York: Oxford University Press. 1997.
[65] Rae DJ, Liang WG, Waston DM, Beattie GAC, Huang MD. Evaluation of petroleum spray oils for control of the Asian citrus psylla, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae), in china[J]. International Journal of Pest Management.1997,43: 71-75.
[66] Rasgon JL, Scott TW. Phylogenetic characterization of Wolbachia symbionts infecting Cimex lectularius L.and Oeciacus vicarious Horvath (Hemiptera:Cimicidae). J.Med Entomo. 2004, 41: 1175-1178.
[67] Reinking OA. Diseases of economic plants in South China[J].Philippine Agriculture, 1919, 109～1351.
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