柑橘木虱黄龙病菌携带量与其内生菌群相关性

doi:10.3864/j.issn.0578-1752.2014.11.009

摘要/Abstract

摘要： 【目的】分析柑橘木虱（Diaphorina citri）体内可能与黄龙病菌（Candidatus Liberibacter asiaticus）互作的内生细菌，为黄龙病菌的人工培养及其病害防控奠定基础。【方法】首先通过传统分离培养方法比较不同地理来源带黄龙病菌（带菌）和不带菌黄龙病菌（不带菌）的木虱中可培养内生细菌的差异。其次将带菌状况不同的木虱分别分为头、胸、腹3部分，经PCR扩增其16S rDNA的V6—V8区，用变性梯度凝胶电泳（PCR-DGGE）方法，比较带菌状况不同的木虱内生细菌的差异和木虱不同部位内生细菌的差异。选择3种差异的内生细菌：Bacillus sp.、Salmonella sp.、Enterobacter sp.，在8份带菌状况不同的木虱样品中通过q-PCR分别对其进行实时荧光定量分析，再以总细菌量为校正计算包括黄龙病菌在内的4种细菌的相对含量，数据经LSD检验，以各种细菌相对含量的-lg值作图，先比较同一样品中3种细菌分别与黄龙病菌的相对含量关系，再比较同种细菌在不同样品中的特性，分析3种内生细菌和黄龙病菌的互作关系。【结果】不带菌木虱中可培养内生菌菌落丰富度和菌落形成单位均大于带菌木虱中。在不带菌木虱中共获得14株形态不同的菌株，分属于芽孢杆菌属（Bacillus，3株）、欧文氏菌属（Erwinia，1株）、克雷伯氏杆菌属（Klebsiella，1株）、葡萄球菌属（Staphylococcus，2株）、节杆菌属（Arthrobacter，1株）、泛菌属（Pantoea，2株）、果胶杆菌属（Pectobacterium，1株）、沙门氏菌属等（Salmonella，1株）、链霉菌属（Streptomyces，1株）、Massilia brevitalea（1株）等10个细菌属。在带菌木虱中分得的4株细菌在不带菌木虱中均分离到，分属于克雷伯氏菌属、芽孢杆菌属、果胶杆菌属。其中Dc-11（嗜气芽孢杆菌属）在带菌木虱和不带菌木虱中分离频率均达到100%，表明其为木虱体内常驻细菌；对木虱不同部位（头、胸、腹）内生细菌16S rDNA的PCR-DGGE图谱显示，带菌与不带菌木虱中细菌种群差别明显，带菌状况相同的木虱不同部位之间差别不明显。其中优势条带10 （Wolbachia sp.）、12（Wolbachia pipientis）、13（Syncytium endosymbiont of Diaphorina citri）、14（Uncultured bacterium）、19（Serratia marcescens）、21和22（均为嗜麦芽寡养单胞菌Stenotrophomonas maltophilia）在木虱体内稳定存在，带菌木虱腹部特有优势内生细菌为Enterobacter sp.，木虱中同样也存在次级内生菌Wolbachia。q-PCR的结果验证了所选的3种细菌在前期传统分离培养和16S rDNA-PCR-DGGE中结果的可靠性，同时表明肠杆菌属在8份样品中与黄龙病菌呈正相关关系。【结论】黄龙病菌进入木虱体内会改变木虱内生细菌菌群种类和结构；嗜气芽孢杆菌（B. aerophilus）在柑橘木虱体内稳定存在，为木虱体内常驻菌群；Enterobacter sp.与黄龙病菌带菌量呈正相关，推测其可能与黄龙病菌互作。

关键词: 柑橘木虱 , 黄龙病菌 , 内生细菌 , 分离培养 , PCR-DGGE

Abstract: 【Objective】The objective of this study is to analyze the endophytic bacteria in Diaphorina citri which may affect the pathogenesis of Candidatus Liberibacter asiaticus, and to provide a basis for artificial culture of Ca. L. asiaticus, prevent and control of HLB. 【Method】 The diversity of cultivable endophytic bacteria in different samples of D. citri with or without Ca. L. Asiaticus from different geographical origins were compared by traditional isolation and culture methods. D. citri with and without Ca. L. asiaticus were divided into three parts: the head, thorax and abdomen, respectively. Then V6-V8 area of 16S rDNA were amplified after extracting DNA, the endophytic bacteria in D. citri of with or without HLB pathogen and in different parts of the same carrier state were compared by 16S rDNA-PCR-DGGE. The relative amount in 8 D. citri samples of Bacillus sp., Salmonella sp., Enterobacter sp. and Ca. L. asiaticus was determined, which have different results in traditional isolation and culture methods and PCR-DGGE, using total amount of bacteria as an amount of contrast. Relative amounts of the 3 chosen bacteria and Ca. L. asiaticus, respectively, in every D. citri sample were compared before comparing similarity of the same bacteria in different samples in order to analyze the interaction of the 3 chosen bacteria and Ca. L. asiaticus. 【Result】 Richness of the colony and colony-forming unit of cultivable endophytic bacteria in D. citri with Ca. L. asiaticus were more than in D. citri without Ca. L. asiaticus. Fourteen kinds of bacteria were isolated from D. citri without Ca. L. asiaticus, belonging to 10 genera, such as Bacillus sp.(3), Erwinia sp.(1), Klebsiella sp.(1), Staphylococcus sp.(2), Arthrobacter sp.(1), Pantoea sp.(2), Pectobacterium sp.(1), Salmonella sp.(1), Streptomyces sp.(1), and Massilia brevitalea sp.(1). While only 4 kinds of bacteria were isolated from D. citri with Ca. L. asiaticus, from 3 genera the same as healthy psyllid, such as Bacillus sp., Klebsiella sp., Pectobacterium sp.. B. aerophilus was isolated frequently from D. citri, as a resident flora in D. citri. As the electrophoretogram of 16S rDNA PCR-DGGE revealed, there were significant differences between D. citri with and without Ca. L. asiaticus in the same tissues, but no significant differences were found in different tissues from the same D. citri. Robotic bands No. 10 (Wolbachia sp.), No. 12 (Wolbachia pipientis), No. 13 (Syncytium endosymbiont of Diaphorina citri), No. 14 (Uncultured bacterium), No. 19 (Serratia marcescens), No. 21 and No. 22 (both are Stenotrophomonas maltophilia) were stable in D. citri with and without Ca. L. asiaticus. Enterobacter sp. was one of the dominant endosymbiotic bacteria of D. citri with Ca. L. asiaticus. Wolbachia also was found in D. citri. The results of q-PCR proved that the results of traditional isolation and culture methods and 16S rDNA-PCR-DGGE were correct, and demonstrated that the relative amount of Enterobacter sp. and Ca. L. asiaticus were positive correlation in the 8 chosen samples. 【Conclusion】 Suspected resident flora B. aerophilus was isolated frequently in D. citri. There may be some interactions between the Enterobacter sp. and Ca. L. asiaticus according to the result of q-PCR, which reveals the relative amount of Enterobacter sp. and Ca. L. asiaticus are positive correlation.

Key words: Diaphorina citri , Candidatus Liberibacter asiaticus , endophytic bacteria , isolation and culture , PCR-DGGE

孙丽琴, 殷幼平, 王芳, 吴晓芳, 王中康. 柑橘木虱黄龙病菌携带量与其内生菌群相关性[J]. 中国农业科学, 2014, 47(11): 2151-2161.

SUN Li-Qin, YIN You-Ping, WANG Fang, WU Xiao-Fang, WANG Zhong-Kang. Correlation of Candidatus Liberibacter asiaticus and the Endophytic Community in Diaphorina citri[J]. Scientia Agricultura Sinica, 2014, 47(11): 2151-2161.

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参考文献

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

[2]范国成, 刘波, 吴如健, 李韬, 蔡子坚, 柯冲. 中国柑橘黄龙病研究30年. 福建农业学报, 2009, 24(2): 183-190.

Fan G C, Liu B, Wu R J, Li T, Cai Z J, Ke C. Thirty years of research on citrus Huanglongbing in China. Fujian Journal of Agricultural Sciences, 2009, 24(2): 183-190. (in Chinese)

[3]Duan Y P, Zhou L J, Hall D G, Li W B, Doddapaneni H, Lin H, Liu L, Vahling C M, Gabriel D W, Williams K P, Dickerman A, Sun Y J, Gottwald T. Complete genome sequence of citrus Huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’obtained through metagenomics. Molecular Plant-Microbe Interactions, 2009, 22(8): 1011-1020.

[4]黄炳超, 刘秀琼, 陈循渊. 柑橘木虱传播柑橘黄龙病菌的研究. 华南农业大学学报: 自然科学版, 1985, 6(4): 46-50.

Huang B C, Liu X Q, Chen X Y. Research of HLB spread by citrus psyllid. Journal of South China Agricultural University: Natural Science Edition, 1985, 6(4): 46-50. (in Chinese)

[5]许长藩, 李开本, 柯冲, 廖锦泽. 木虱传播柑橘黄龙病及电镜检验报告. 植物病理学报, 1985, 15(4): 241-245.

Xu C F, Li K B, Ke C, Liao J Z. Transmission of Candidatus Liberibacter asiaticus by citrus psyllid and report of electron microscope inspection. Acta Phytopathologica Sinica, 1985, 15(4): 241-245. (in Chinese)

[6]许长藩, 夏雨华, 李开本, 柯冲. 柑橘木虱传播黄龙病的规律及病原在虫体内分布的研究. 福建农业学报, 1988, 3(2): 57-62.

Xu C F, Xia Y H, Li K B, Ke C. Research of the spreading pattern of Candidatus Liberibacter asiaticus by citrus psyllid and the distribution in citrus psyllid. Fujian Journal of Agricultural Sciences, 1988, 3(2): 57-62. (in Chinese)

[7]姜林蕙. 柑橘黄龙病在柑橘木虱体内的增殖与分布[D]. 台北: 台湾大学, 2005: 1-43.

Jiang L H. Multiplication and distribution of Candidatus Liberibacter asiaticus, the bacterium causing citrus Huanglongbing, in the Asian citrus psyllid (Diaphorina citri Kuwayama)[D]. Taibei: Taiwan University, 2005: 1-43. (in Chinese)

[8]Subandiyah S, Nikoh N, Tsuyumu S, Somowiyarjo S, Fukatsu T. Complex endosymbiotic microbiota of the citrus psyllid Diaphorina citri (Homoptera: Psylloidea). Zoological Science, 2000, 17(7): 983-989.

[9]Hert M, Hunter W, Dowd S, Hall D. Endosymbiotic microbiota of Asian citrus psyllid//International Research Conference on Huanglongbing, Orlando, Florida, 2008: 1-3.

[10]殷幼平, 刘婷婷, 田圣超, 胡修峰, 吴东, 王中康. 基于16S rDNA序列的柑橘木虱体内共生菌多样性研究. 昆虫学报, 2011, 54(6): 664-674.

Yin Y P, Liu T T, Tian S C, Hu X F, Wu D, Wang Z K. Microbial diversity in Diaphorina citri (Homoptera: Psyllidae) estimated by 16S rDNA analysis using DGGE and RFLP. Acta Entomologica Sinica, 2011, 54(6): 664-674. (in Chinese)

[11]Saha S, Hunter W B, Reese J, Morgan J K, Marutani-HertM, Huang H, Lindeberg M. Survey of endosymbionts in the Diaphorina citri Metagenome and assembly of a Wolbachia wDi draft genome. Plos One, 2012, 7(11): e50067.

[12]Fagen J R, Giongo A, Brown C T, Davis-Richardson A G, Gano K A, Triplett E W. Characterization of the relative abundance of the citrus pathogen Ca. Liberibacter asiaticus in the microbiome of its insect vector, Diaphorina citri, using high throughput 16S rRNA sequencing. The Open Microbiology Journal, 2012, 6: 29-33.

[13]Wang Z K ,Yin Y P, Hu H, Yuan Q, Peng G X, Xia Y X. Development and application of molecular-based diagnosis for ‘Candidatus Liberibacter asiaticus’, the causal pathogen of citrus Huanglongbing. Plant Pathology, 2006, 55(5): 630-638.

[14]Sagaram U S, DeAngelis K M, Trivedi P, Sagaram U S, DeAngelis K M, Trivedi P, Andersen G L, Lu S E, Wang N. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip arrays and 16S rRNA gene clone library sequencing. Applied and Environmental Microbiology, 2009, 75(6): 1566-1574.

[15]王爱华, 殷幼平, 熊红利, 李颜方, 李佳, 贤家旭, 王中康. 广西柑橘黄龙病植株韧皮部内生细菌多样性分析. 中国农业科学, 2010, 43(23): 4823-4833.

Wang A H, Yin Y P, Xiong H L, Li Y F, Li J, Xian J X, Wang Z K. Endophytic bacterial diversity analysis of Huanglongbing pathogen- infected citrus phloem tissue in Guangxi. Scientia Agricultura Sinica, 2010, 43(23): 4823-4833 (in Chinese)

[16]东秀珠, 蔡妙英. 常见细菌系统鉴定手册. 北京: 科学出版社, 2001: 39-418.

Dong X Z, Cai M Y. Common Bacteria Manual System Identification. Bejing: Science Press, 2001: 39-418. (in Chinese)

[17]Meyer J M, Hoy M A. Removal of fungal contaminants and their DNA from the surface of Diaphorina citri (Hemiptera: Psyllidae) prior to a molecular survey of endosymbionts. Florida Entomologist, 2008, 91(4): 702-705.

[18]Lane D. 16s/23s rRNA sequencing//Nucleicacid Techniques in Bacterial Systematics. West Sussex, United Kingdom: John Wiley & Sons, 1991: 115-175.

[19]Muyzer G, De Waal E C, Uitterlinden A G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology, 1993, 59(3): 695-700.

[20]Rueckert A, Ronimus R S, Morgan H W. Development of a real-time PCR assay targeting the sporulation gene, spo0A, for the enumeration of thermophilic bacilli in milk powder. Food Microbiology, 2006, 23(3): 220-230.

[21]Rahn K, De Grandis S A, Clarke R C. Amplification of an inv-A gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Molecular and Cellular Probes, 1992, 6(4): 271-279.

[22]相辉, 黄勇平. 肠道微生物与昆虫的共生关系. 昆虫知识, 2008, 45(5): 687-693.

Xiang H, Huang Y P. Symbiosis between gut microbiota and insects. Chinese Bulletin of Entomology, 2008, 45(5): 687-692. (in Chinese)

[23]向芸庆, 王晓强, 冯伟, 周围, 谢洪霞, 万永继. 不同饲料饲养家蚕其肠道微生态优势菌群类型的组成及差异性. 生态学报, 2010, 30(14): 3875-3882.

Xiang Y Q, Wang X Q, Feng W, Zhou W, Xie H X, Wan Y J. Comparative analysis of the dominant intestinal microflora in silkworm reared with different forages. Acta Ecologica Sinica, 2010, 30(14): 3875-3882. (in Chinese)

[24]刘莉, 王中康, 俞和韦, 陈仕江, 阎光凡, 夏玉先, 殷幼平. 贡嘎蝠蛾幼虫肠道细菌多样性分析. 微生物学报, 2008, 48(5): 616-622.

Liu L, Wang Z K, Yu H W, Chen S J, Yan G F, Xia Y X, Yin Y P. Analysis of the bacterial diversity in intestines of Hepialus gonggaensis larae. Acta Microbiologica Sinica, 2008, 48(5): 616-622. (in Chinese)

[25]何正波, 殷幼平, 曹月青, 董亚敏, 张伟. 桑粒肩天牛幼虫肠道菌群的研究. 微生物学报, 2001, 41(6): 741-744.

He Z B, Yin Y P, Cao Y Q, Dong Y M, Zhang W. Study on the Apriona germari (Hope) larvae’s intestinal bacterial flora. Acta Microbiologica Sinica, 2001, 41(6): 741-744. (in Chinese)

[26]常伟. 蜜蜂消化道共生细菌及其多态性研究初探[D]. 福州: 福建农林大学, 2010.

Chang W. Diversity of symbiotic bacteria in honeybee alimentary tract[D]. Fuzhou: Fujian Agriculture and Forestry University, 2010. (in Chinese)

[27]陈虹, 梅建凤, 闵航. 白蚁肠道微生物. 微生物学杂志, 2005, 25(2): 75-79.

Chen H, Mei J F, Min H. Microorganisms in termite gut. Journal of Microbiology, 2005, 25(2): 75-79. (in Chinese)

[28]孙雪奇, 黄玉祥, 董长江, 刘智慧, 郑长文, 刘鸿莺. 家蚕肠道好氧和部份兼厌氧微生物及蚕用微生态制剂的研究. 四川蚕业, 1996, 24(1): 13-15.

Sun X Q, Huang Y X, Dong C J, Liu Z H, Zhen C W, Liu H Y. Aerobic and facultative anaerobic bacteria in silkworm intestinaltract. Sichuan Sericulture, 1996, 24(1): 13-15. (in Chinese)

[29]Davis M J, Mondal S N, Chen H, Roger M E, Brlansky R H. Co- cultivation of' ‘Candidatus Liberibacter asiaticus’ with Actinobacteria from citrus with Huanglongbing. Plant Disease, 2008, 92(11): 1547-1550.

[30]Riegler M, O’Neill S L. The genus Wolbachia. Prokaryotes, 2006, 5: 547-561.