柑橘黄龙病病原菌的鉴定及16S rDNA序列分析

福建农业学报 ›› 2011, Vol. 26 ›› Issue (6): 1027-1033.

柑橘黄龙病病原菌的鉴定及16S rDNA序列分析

孙大光^1,2, 郑雪芳², 刘波², 阮传清², 夏育陆³, 段永平⁴

1. 福建师范大学生命科学学院,福建福州 350108;
2. 福建省农业科学院柑橘黄龙病研究中心,福建福州 350003;
3. 美国北卡州立大学有害生物综合治理研究中心,北卡罗来纳罗利 27606;
4. 美国农业部佛罗里达园艺实验室,弗罗里达皮尔斯堡 34945

收稿日期:2011-11-01 修回日期:2011-11-26 出版日期:2011-12-15 发布日期:2011-12-15
作者简介:刘波(1957-),男,研究员,博士,研究方向:微生物生物技术与农业生物药物(E-mail:fzliubo@163.com)
基金资助:
国家公益性行业(农业)科研专项 (201003067-05);中美合作项目——柑橘木虱及黄龙病野外研究(2010-1652-02)、抗虫柑橘品种研究(6618-22000-037-01S);福建省财政专项——福建省农业科学院科技创新团队建设基金(STIF-Y03)

Identification and 16S rDNA Sequence Analysis of Citrus Huanglongbing Pathogen

SUN Da-guang^1,2, ZHENG Xue-fang², LIU Bo², RUAN Chuan-qing², XIA Yu-lu³, DUAN Yong-ping⁴

1. College of Life Science Fujian Normal University,Fuzhou,Fujian 350108,China;
2. Citrus Huanglongbing Research Center,Fujian Academy of Agricultural Sciences, Fuzhou,Fujian 350003,China;
3. The Center of Integrated Pest Management, North Carolina State University,North Carolina 27606,USA;
4. Horticultural Research Laboratory (USDA/ARS),Florida 34945,USA

Received:2011-11-01 Revised:2011-11-26 Online:2011-12-15 Published:2011-12-15

摘要/Abstract

摘要： 采用Nested-PCR技术检测顺昌果园红心柚树中柑橘黄龙病病原的携带情况,通过黄龙病病原菌16S rDNA测序和构建系统发育树比较不同类型柑橘黄龙病原菌之间的进化关系,采用特异引物扩增16S rDNA片段鉴别不同黄龙病病原菌,结合限制性内切酶酶切确定该红心柚园黄龙病病原的类型,并分析不同类型柑橘黄龙病病原16S rDNA序列差异性。结果表明:所取的12株红心柚树叶片样品中有7株树的叶片中检测出黄龙病病原,黄龙病病原的阳性检出率为58.33%,其中4个为显性性状,3个为隐性性状。柑橘黄龙病病原菌16S rDNA进化分析及限制性内切酶XbaI酶切结果表明,顺昌柑橘黄龙病病原菌属于亚洲型。不同类型柑橘黄龙病病原菌16S rDNA的序列差异性分析表明,亚洲种与非洲种的差异性为2.75%,非洲种与美洲种的差异性为3.90%,亚洲种与美洲种的差异性为3.44%。

关键词: 柑橘黄龙病, 鉴定, 16S rDNA, 系统进化分析

Abstract: Nested-PCR detection of Shunchang citrus Huanglongbing pathogen was carried out in this study.Phylogenetic tree based on rDNA sequencing was built to find out the evolutionary between different parts citrus HLB pathogens.Digesting the target DNA fragment of 16S rDNA with restriction enzyme XbaI was carried out to confirm the species of Shunchang Huanglongbing pathogen.The results showed that there were 7 positive detections in 12 selected samples, and the rate of positive detections was 58.33%,and four were dominant character,three were recessive character.The results showed that Shunchang citrus Huanglongbing pathogen belong to Candidatus Liberibacter asiaticus based on the 16S rDNA phylogenetic analysis and restriction enzyme XbaI digestion analysis.And 16S rDNA sequence gap analysis showed that the difference between Ca. L.asiaticus and Ca. L.africanus, Ca. L.africanus and Ca.L.americanus, Ca.L.americanus and Ca.L.asiaticus were 2.75%,3.90%, 3.44%, respectively.

Key words: Citrus Huanglongbing, Identification, 16S rDNA, Phylogenetic analysis

中图分类号:

Q 931

孙大光, 郑雪芳, 刘波, 阮传清, 夏育陆, 段永平. 柑橘黄龙病病原菌的鉴定及16S rDNA序列分析[J]. 福建农业学报, 2011, 26(6): 1027-1033.

SUN Da-guang, ZHENG Xue-fang, LIU Bo, RUAN Chuan-qing, XIA Yu-lu, DUAN Yong-ping. Identification and 16S rDNA Sequence Analysis of Citrus Huanglongbing Pathogen[J]. Fujian Journal of Agricultural Sciences, 2011, 26(6): 1027-1033.

参考文献

[1] BOVE J M. Huanglongbing: a destructive,newly-emerging,century-old disease of citrus [J]. Journal of Plant Pathology,2006,88(1): 7-37.

[2] 廖晓兰,朱水芳,赵文军,等. 柑桔黄龙病病原16SrDNA克隆,测序及实时荧光PCR检测方法的建立[J]. 农业生物技术学报,2004,12(1): 80-85.

[3] DO C,TEIXEIRA D,DANET J,et al. Citrus Huanglongbing in So Paulo State,Brazil: PCR detection of the Candidatus Liberibacter species associated with the disease [J]. Molecular and Cellular Probes,2005,19(3): 173-179.

[4] 田亚南,柯冲. 柑桔黄龙病诊断法的研究进展[J]. 福建农业学报,1998,13(1): 27-35.

[5] 李德望,唐伟文. 柑桔黄龙病的血清学检测与诊断方法的初步研究[J]. 华南农业大学学报,1992,13(2):16-22.

[6] 丁芳,易干军,王国平. 应用PCR及Nested-PCR技术检测柑橘黄龙病病原研究[J]. 园艺学报,2004,31(6): 803-806.

[7] 孔维文,邓晓玲. 柑桔黄龙病病原DNA片段的克隆及序列分析[J]. 植物病理学报,2000,30(1): 71-75.

[8] LI W,HARTUNG J S,LEVY L. Evaluation of DNA amplification methods for improved detection of "Candidatus Liberibacter species" associated with citrus huanglongbing[J]. Plant Disease,2007,91(1):51-58.

[9] PLANET P,JAGOUEIX S,BOEÉ J M,et al. Detection and characterization of the African citrus greening Liberobacter by amplification,cloning,and sequencing of the rplKAJL-rpoBC operon[J]. Current Microbiology,1995,30(3):137-141.

[10] SUBANDIYAH S,IWANAMI T,TSUYUMU S,et al. Comparison of 16S rDNA and 16S/23S intergenic region sequences among citrus greening organisms in Asia[J]. Plant Disease,2000,84(1): 15-18.

[11] DING F,DENG X,HONG N,et al. Phylogenetic analysis of the citrus Huanglongbing (HLB) bacterium based on the sequences of 16S rDNA and 16S/23S rDNA intergenic regions among isolates in China [J]. European Journal of Plant Pathology,2009,124(3): 495-503.

[12] 廖晓兰,朱水芳,赵文军,等. 柑桔黄龙病病原 16SrDNA 克隆,测序及实时荧光 PCR 检测方法的建立[J]. 农业生物技术学报,2004,12(1):80-85.

[13] 刘福明,张纯胄,李妙寿,等. 温州柑橘缺素症调查,鉴别与防治技术研究[J]. 浙江柑橘,2006,23(2): 12-13.

[14] 林秀萍,柑桔黄龙病综合防控技术[J]. 果农之友,2007,(2): 29-30.

[15] 谢钟琛,李健,施清,等. 福建省柑橘黄龙病危害及其流行规律研究[J]. 中国农业科学,2009,42(11): 3888-3897.

[16] 丁芳,洪霓,钟云,等. 中国柑橘黄龙病病原16S rDNA序列研究[J]. 园艺学报,2008,35(5):649-654.

[17] SUBANDIYAH S,IWANAMI T,TSUYUMU S,et al. Comparison of 16S rDNA and 16S/23S intergenic region sequences among citrus greening organisms in Asia[J]. Plant Disease,2000,84(1):15-18.

[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] JAGOUEIX S,BOVÉ J M,GARMIER M. Techniques for the specific detection of the greening liberobacter species: DNA-DNA hybridization and DNA amplification by PCR// 13th Conf Int Organ Citrus Virol Fuzhou,China,1995:101.

[20] 董鹏,包改丽,冉志伟,等.云南柑橘黄龙病病原检测及其16S rDNA序列分析[J].云南农业大学学报,2011,26(5):607-611.

[21] COLETTA-FILHO H D,TAKITA M A,TARGON M,et al. Analysis of 16S rDNA sequences from citrus huanglongbing bacteria reveal a different 'Candidatus Liberibacter’ strain associated with citrus disease in Sao Paulo [J]. Plant Disease,2005,89(8):848-852.

[22] GARNIER M,JAGOUEIX-EVEILLARD 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 and Evolutionary Microbiology,2000,50(6):2119-2125.