应用DTBIA技术快速检测柑橘黄龙病菌研究
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摘要
柑橘黄龙病病原菌为韧皮部限制性寄生菌Candidatus Liberibacter,迄今为止尚未成功获得离体纯培养。因此基于特异性抗体的高通量血清学检测技术迄今尚未开发。本实验室前期研究利用异源表达重组黄龙病菌外膜蛋白Omp_(CLas)免疫动物,制备并获得了特异性多克隆抗体(Anti-Omp_(CLas)-Pab)。本研究利用直接组织印迹免疫法(Direct tissue blot immunoassay,DTBIA)技术对获得的多克隆抗体的检测效果进行了初步评价,证实该抗体可以特异性识别中国不同地理来源的柑橘黄龙病菌分离物;柑橘叶柄、叶片中脉、枝条、根部、果梗和种子中均存在黄龙病菌,其中根部和种子中的病菌含量相对较低,枝条和叶片中脉中的含量较高。该多克隆抗体的获得为后续快速检测试剂盒或试纸条的研发奠定了基础。
Citrus Huanglongbing(HLB)is caused by phloem-limited bacteria named Candidatus Liberibacters. Up to date,despite of all kinds of efforts to get pure culture,HLB bacteria still resist in vitro growth. Fast detection especially the high-throughput methods based on the specificity of serological methods has not yet been developed. In our previous work,we constructed fusion OmpCLas protein and heterogonous expressed to get antigen to produce corresponding polyclonal antibody(Pab). In the present study,we carried out experiment on the assessment of the detection efficiency of Anti-OmpCLas-Pab antibody with direct tissue blot immunoassay( DTIBA). Citrus samples collected from different geographical regions were tested. Different organs of petiole,midrib,stem,root,peduncale and seeds were all analyzed. Detection efficiency of both DTBIA and PCR were compared. Our result indicated:AntiOmpCLas-Pab could specifically recognize HLB bacteria in different isolates originated from different geographical areas in China,including samples from Jiangxi,Fujian,Yunnan,Guangdong and Hainan provinces. Tests with different tissues of petiole,leaf mid rib,stem,root,peduncale and seeds revealed the presence of HLB bacteria. However,less HLB bacteria were found in roots and seeds;higher titer was found in leaf mid-ribs and stems. Our results indicated that Anti-OmpCLas-Pab is a promising antibody for the fast detection of HLB bacteria which laid a basic foundation for the product development on the fast detection kits and dipsticks.
Citrus Huanglongbing(HLB)is caused by phloem-limited bacteria named Candidatus Liberibacters. Up to date,despite of all kinds of efforts to get pure culture,HLB bacteria still resist in vitro growth. Fast detection especially the high-throughput methods based on the specificity of serological methods has not yet been developed. In our previous work,we constructed fusion OmpCLas protein and heterogonous expressed to get antigen to produce corresponding polyclonal antibody(Pab). In the present study,we carried out experiment on the assessment of the detection efficiency of Anti-OmpCLas-Pab antibody with direct tissue blot immunoassay( DTIBA). Citrus samples collected from different geographical regions were tested. Different organs of petiole,midrib,stem,root,peduncale and seeds were all analyzed. Detection efficiency of both DTBIA and PCR were compared. Our result indicated:AntiOmpCLas-Pab could specifically recognize HLB bacteria in different isolates originated from different geographical areas in China,including samples from Jiangxi,Fujian,Yunnan,Guangdong and Hainan provinces. Tests with different tissues of petiole,leaf mid rib,stem,root,peduncale and seeds revealed the presence of HLB bacteria. However,less HLB bacteria were found in roots and seeds;higher titer was found in leaf mid-ribs and stems. Our results indicated that Anti-OmpCLas-Pab is a promising antibody for the fast detection of HLB bacteria which laid a basic foundation for the product development on the fast detection kits and dipsticks.
引文
BovéJ M.2006.Huanglongbing:a destructive,newly-emerging,century-old disease of citrus.J Plant Pathol,88:7–37.
Bertolini E,Felipe R T A,Sauer AV,Lopes S A,Arilla A,Vidal E,Mour?o Filho F A A,Nunes W M C,BovéJ M,López M M,Cambra M.2014.Tissue-print and squash real-time PCR for direct detection of‘Candidatus Liberibacter’species in citrus plants and psyllid vectors.Plant Pathology,63:1149–1158
Cen Y J,Zhang L N,Xia Y L,Guo J,Deng X L,Zhou W J,Sequeira R,Gao J Y,Wang Z R,Yue J Q,Gao Y Q.2012.Detection of‘Candidatus Liberibacter Asiaticus’in Cacopsylla(Psylla)citrisuga(Hemiptera:Psyllidae).Florida Entomologist,95:304–311.
Deng Xiao-ling,Shan Zhen-ju,Feng Zhen,Zhou Gen,Xu Jian-hua.2006.Cloning and sequencing of Huanglongbing pathogen in Foshou(Citrus medica)//Chinese Society for Plant Pathology.Proceedings of 2006 Annual Conference,Changsha,Hunan.Beijing:China Agricultural Science and Technology Press:140.(in Chinese)邓晓玲,单振菊,冯震,周根,徐建华.2006.佛手黄龙病病原片段的克隆与序列分析//中国植物病理学会2006年学术年会论文集(长沙).北京:中国农业科学技术出版社:140.
Ding F,Wang G P,Yi G J,Zhong Y,Zeng J W,Zhou B R.2005.Infection of wampee and lemon by citrus Huanglongbing pathogen(Candidatus Liberibacter asiaticus)in China.J Plant Pathol,8:207–212.
Ding F,Duan Y P,Paul C,Brlansky R H,Hartung J S.2015.Localization and distribution of‘Candidatus Liberibacter Asiaticus’in citrus and periwinkle by direct tissue blot immuno assay with an Anti-Omp A polyclonal antibody.PLo S ONE,10:e0123939.
Ding F,Paul C,Brlansky R H,Hartung J S.2017.Immune tissue print and immune capture-PCR for diagnosis and detection of Candidatus Liberibacter Asiaticus.Sci Rep,7:46467.
D’Onghia A M,Djelouah K,Frasheri D,Potere O.2001.Detection of citrus psorosis virus by direct tissue blot immunoassay.Journal of Plant Pathology,83:139–142.
Fan G C,Cai Z J,Weng Q Y,Ke C,Liu B,Zhou L J,Duan Y P.2011.First eport of a new host(Pithecellobium lucidum Benth)of the citrus Huanglongbing bacterium,Candidatus Liberibacter Asiaticus.IRCHLB:137.
Gabler J,Ka?ergius A,Jovai?ien?Z.2004.Detection of Phomopsis vaccinii on blueberry and cranberry in Europe by Direct Tissue Blot Immunoassay and Plate-Trapped Antigen ELISA.Journal of Phytopathology,152:630–632.
Garnier M,Bove J M.1983.Transmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder.Phytopathology,73:1358–1363.
Garnier M,Martin-Gros G,Bove J M.1987.Monoclonal antibodies against the baterial-like organism associtad with citrus greening disease.Ann Inst Pasteur Microbiol,138:639–650.
Garnsey S M,Permar T A,Cambra M,Henderson C T.1993.Direct tissue blot immunoassay(DTBIA)for detection of Citrus tristeza virus(CTV).Proc 12th Conf IOCV,Riverside,CA.IOCV:39–50.
Gottwald T R.2010.Current epidemiological understanding of citrus Huanglongbing.Annu Rev Phytopathol,48:19–139.
Guo Jun,Cen Yi-jing,Wang Zi-ran,Duan Hui-fen,Gao Jun-yan.2012.A study on the morphology,biology and occurrence of Cacopsylla citrisuga.Journal of South China Agricultural University,33(4):475–479.(in Chinese)郭俊,岑伊静,王自然,段惠芬,高俊燕.2012.柚喀木虱的形态、生物学特性及发生规律研究.华南农业大学学报,33(4):475–479.
He Hong,Pan Chao-mei,Wu Li-rong,Huang Hai-bo,Zhang Gui-fang.2005.Detection of Huanglongbing pathogen in Citrus media L.var.sarcodactylis(Noot.)Swingle by polymerase chain reaction analysis.Journal of Guangzhou University of Traditional Medicine,22(6):479 –481.(in Chinese)贺红,潘超美,吴立蓉,黄海波,张桂芳.2005.应用聚合酶链反应技术检测广佛手黄龙病病原的研究.广州中医药大学学报,22(6):479 –481.
Hung T H,Wu M Z,SU H J.1999.Detection of fastidious bacteria causing citrus greening disease by nonradioactive DNA probes.Ann Phytopathol Soc Jpn,65:140–146.
Jagoueix S,Bove′J M,Garnier M.1996.PCR detection of the two‘Candidatus’liberobacter species associated with greening disease of citrus.Mol Cell Probes,10:43–50.
Li W B,Hartung J S,Levy L.2006.Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing.J Microbiol Methods,66:104–115.
Li W B,Li D Y,Twieg E,Hartung J S,Levy L.2008.Optimized quantification of unculturable Candidatus Liberibacter spp.in host plants using Real-Time PCR.Plant Dis,92:854–861.
Loconsole G,Potere O,Boscia D,Altamura G,Djelouah K,Elbeaino T,Frasheri D,Lorusso D,Palmisano F,Pollastro P,Silletti M R,Trisciuzzi N,Valentini F,Savino V,Saponari M.2014.Detection of Xylella fastidiosa in olive trees by molecular and serological methods.J Plant Pathol,96:7–14.
Mac Donald J D,Stites J,Kabashima J.1990.Comparison of serological and culture plate methods for detecting species of Phytophthora,Pythium and Rhizoctonia in ornamental plants.Plant Dis,74:655–659.
Morgan J K,Zhou L,Li W,Shatters R G,Keremane M,Duan Y P.2012.Improved real-time PCR detection of‘Candidatus Liberibacter Asiaticus’from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes.Mol Cell Probes,26:90–98.
Shang H L,Xie Y,Zhou X P,Qian Y J,Wu J X.2011.Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus.Virology Journal,8:228.
Trivedi P,Sagaram U S,Kim J S,Brlansky R H,Rogers M E,Stelinski L L,Oswalt C,Wang N.2009.Quantification of viable Candidatus Liberibacter Asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide(EMA).Eur J Plant Pathol,124:553–563.
Villechanoux S,Garnier M,Renaudin,J,BovéJ M.1992.Detection of several strains of the bacterium-like organism of citrus greening disease by DNA probes.Current Microbiology,24:89–95.
Wang N,Trivedi P.2013.Citrus huanglongbing:a newly relevant disease presents unprecedented challenges.Phytopathology,103(7):652–665.
Xie Y,Jiao X J,Zhou X P,Liu H,Ni Y Q,Wu J X.2013.Highly sensitive serological methods for detecting Tomato yellow leaf curl virus in tomato plants and whiteflies.Virology Journal,10:142.
Bertolini E,Felipe R T A,Sauer AV,Lopes S A,Arilla A,Vidal E,Mour?o Filho F A A,Nunes W M C,BovéJ M,López M M,Cambra M.2014.Tissue-print and squash real-time PCR for direct detection of‘Candidatus Liberibacter’species in citrus plants and psyllid vectors.Plant Pathology,63:1149–1158
Cen Y J,Zhang L N,Xia Y L,Guo J,Deng X L,Zhou W J,Sequeira R,Gao J Y,Wang Z R,Yue J Q,Gao Y Q.2012.Detection of‘Candidatus Liberibacter Asiaticus’in Cacopsylla(Psylla)citrisuga(Hemiptera:Psyllidae).Florida Entomologist,95:304–311.
Deng Xiao-ling,Shan Zhen-ju,Feng Zhen,Zhou Gen,Xu Jian-hua.2006.Cloning and sequencing of Huanglongbing pathogen in Foshou(Citrus medica)//Chinese Society for Plant Pathology.Proceedings of 2006 Annual Conference,Changsha,Hunan.Beijing:China Agricultural Science and Technology Press:140.(in Chinese)邓晓玲,单振菊,冯震,周根,徐建华.2006.佛手黄龙病病原片段的克隆与序列分析//中国植物病理学会2006年学术年会论文集(长沙).北京:中国农业科学技术出版社:140.
Ding F,Wang G P,Yi G J,Zhong Y,Zeng J W,Zhou B R.2005.Infection of wampee and lemon by citrus Huanglongbing pathogen(Candidatus Liberibacter asiaticus)in China.J Plant Pathol,8:207–212.
Ding F,Duan Y P,Paul C,Brlansky R H,Hartung J S.2015.Localization and distribution of‘Candidatus Liberibacter Asiaticus’in citrus and periwinkle by direct tissue blot immuno assay with an Anti-Omp A polyclonal antibody.PLo S ONE,10:e0123939.
Ding F,Paul C,Brlansky R H,Hartung J S.2017.Immune tissue print and immune capture-PCR for diagnosis and detection of Candidatus Liberibacter Asiaticus.Sci Rep,7:46467.
D’Onghia A M,Djelouah K,Frasheri D,Potere O.2001.Detection of citrus psorosis virus by direct tissue blot immunoassay.Journal of Plant Pathology,83:139–142.
Fan G C,Cai Z J,Weng Q Y,Ke C,Liu B,Zhou L J,Duan Y P.2011.First eport of a new host(Pithecellobium lucidum Benth)of the citrus Huanglongbing bacterium,Candidatus Liberibacter Asiaticus.IRCHLB:137.
Gabler J,Ka?ergius A,Jovai?ien?Z.2004.Detection of Phomopsis vaccinii on blueberry and cranberry in Europe by Direct Tissue Blot Immunoassay and Plate-Trapped Antigen ELISA.Journal of Phytopathology,152:630–632.
Garnier M,Bove J M.1983.Transmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder.Phytopathology,73:1358–1363.
Garnier M,Martin-Gros G,Bove J M.1987.Monoclonal antibodies against the baterial-like organism associtad with citrus greening disease.Ann Inst Pasteur Microbiol,138:639–650.
Garnsey S M,Permar T A,Cambra M,Henderson C T.1993.Direct tissue blot immunoassay(DTBIA)for detection of Citrus tristeza virus(CTV).Proc 12th Conf IOCV,Riverside,CA.IOCV:39–50.
Gottwald T R.2010.Current epidemiological understanding of citrus Huanglongbing.Annu Rev Phytopathol,48:19–139.
Guo Jun,Cen Yi-jing,Wang Zi-ran,Duan Hui-fen,Gao Jun-yan.2012.A study on the morphology,biology and occurrence of Cacopsylla citrisuga.Journal of South China Agricultural University,33(4):475–479.(in Chinese)郭俊,岑伊静,王自然,段惠芬,高俊燕.2012.柚喀木虱的形态、生物学特性及发生规律研究.华南农业大学学报,33(4):475–479.
He Hong,Pan Chao-mei,Wu Li-rong,Huang Hai-bo,Zhang Gui-fang.2005.Detection of Huanglongbing pathogen in Citrus media L.var.sarcodactylis(Noot.)Swingle by polymerase chain reaction analysis.Journal of Guangzhou University of Traditional Medicine,22(6):479 –481.(in Chinese)贺红,潘超美,吴立蓉,黄海波,张桂芳.2005.应用聚合酶链反应技术检测广佛手黄龙病病原的研究.广州中医药大学学报,22(6):479 –481.
Hung T H,Wu M Z,SU H J.1999.Detection of fastidious bacteria causing citrus greening disease by nonradioactive DNA probes.Ann Phytopathol Soc Jpn,65:140–146.
Jagoueix S,Bove′J M,Garnier M.1996.PCR detection of the two‘Candidatus’liberobacter species associated with greening disease of citrus.Mol Cell Probes,10:43–50.
Li W B,Hartung J S,Levy L.2006.Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing.J Microbiol Methods,66:104–115.
Li W B,Li D Y,Twieg E,Hartung J S,Levy L.2008.Optimized quantification of unculturable Candidatus Liberibacter spp.in host plants using Real-Time PCR.Plant Dis,92:854–861.
Loconsole G,Potere O,Boscia D,Altamura G,Djelouah K,Elbeaino T,Frasheri D,Lorusso D,Palmisano F,Pollastro P,Silletti M R,Trisciuzzi N,Valentini F,Savino V,Saponari M.2014.Detection of Xylella fastidiosa in olive trees by molecular and serological methods.J Plant Pathol,96:7–14.
Mac Donald J D,Stites J,Kabashima J.1990.Comparison of serological and culture plate methods for detecting species of Phytophthora,Pythium and Rhizoctonia in ornamental plants.Plant Dis,74:655–659.
Morgan J K,Zhou L,Li W,Shatters R G,Keremane M,Duan Y P.2012.Improved real-time PCR detection of‘Candidatus Liberibacter Asiaticus’from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes.Mol Cell Probes,26:90–98.
Shang H L,Xie Y,Zhou X P,Qian Y J,Wu J X.2011.Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus.Virology Journal,8:228.
Trivedi P,Sagaram U S,Kim J S,Brlansky R H,Rogers M E,Stelinski L L,Oswalt C,Wang N.2009.Quantification of viable Candidatus Liberibacter Asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide(EMA).Eur J Plant Pathol,124:553–563.
Villechanoux S,Garnier M,Renaudin,J,BovéJ M.1992.Detection of several strains of the bacterium-like organism of citrus greening disease by DNA probes.Current Microbiology,24:89–95.
Wang N,Trivedi P.2013.Citrus huanglongbing:a newly relevant disease presents unprecedented challenges.Phytopathology,103(7):652–665.
Xie Y,Jiao X J,Zhou X P,Liu H,Ni Y Q,Wu J X.2013.Highly sensitive serological methods for detecting Tomato yellow leaf curl virus in tomato plants and whiteflies.Virology Journal,10:142.