【作者】 李颜方；

【导师】 王中康；

【作者基本信息】 重庆大学 ， 生物学， 2011， 硕士

【摘要】 柑橘黄龙病(Citrus Huanglongbing,HLB)是世界柑橘生产上最具毁灭性的病害,由寄生于柑橘韧皮部的韧皮杆菌Cndidatus Liberibacter spp.引起。目前该病原菌还没有得到能够稳定传代并完成科赫氏验证的纯培养。最近的报道发现感黄龙病植株内存在与病原菌伴生的内生菌,这些内生菌对病原菌的生长可能起促进或抑制作用。本研究以感亚洲韧皮杆菌的柑橘与健康柑橘植株组织中的内生细菌为研究对象,采用定向分离培养和分子生物学手段测序的方法,利用基于16S rRNA的PCR-DGGE(Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis)宏基因组学方法,比较分析柑橘发病株与健株内生菌群的差异,并获得19株可培养的兼性厌氧型内生细菌。通过分离来自广东、贵州、福建、云南、浙江、广西等柑橘黄龙病区的56份柑橘样品,利用常规生理生化鉴定与基于16S rDNA的分子鉴定相结合对19株兼性厌氧型内生菌进行分析,结果表明其属于12个细菌属。其中菌株GX21、GX34为克吕沃尔氏菌属Kluyvera sp.;菌株GX22、GX24、GX35、GX36为芽孢杆菌属Bacillus sp.;菌株GX23为短芽孢杆菌属Brevibacillus Shida;GX25属于动性球菌属Planococcus sp.;GX26为刺黄色假杆菌Pseudoclavibacter helvolus;GX27为微杆菌属Microbacterium sp.;GX28为塔特姆菌属Tatumella sp.;GX29、GX39为假单胞菌属Pseudomonas sp.;GX30、GX31、GX33为短小杆菌属Curtobacterium sp.;GX32属于泛菌属Pantoea sp.;GX37属于克雷伯菌属Klebsiella sp.;GX38属于肠杆菌属Enterobacter sp.。其中Curtobacterium sp.(IF: 29.07%),Bacillus sp.(IF: 23.12%),Microbacterium sp(.IF: 21.09%)为感亚洲韧皮杆菌植株的优势菌,Bacillus sp.(IF: 21.03%),Planococcus sp.(IF: 20.69%),Pseudomonas sp.(IF: 17.44%)为健康无症植株的优势菌;病株中Curtobacterium sp.的丰富度比健株明显增大。利用PCR-DGGE方法分析柑橘内生细菌多样性,以发病、健康柑橘不同组织的内生细菌基因组DNA为模板,选择799F-1492R这对特异性引物,巢氏PCR扩增内生菌的16S rDNA片段,经DGGE分离后将各样品DGGE图谱中50条明显的条带分别割胶回收测序,序列经BLAST比对发现,共有9个属15种不同的细菌类群,包括沙雷氏菌属Serratia sp.(28%)、泛菌属Pantoea sp.(14%)、不动杆菌属Acinetobacter sp(.10%)、节杆菌属Arthrobacter sp(. 8%)、诺卡氏菌属Nocardia sp. (10%)、黄单胞菌属Xanthomonas sp.(10%)、假单胞菌属Pseudomonas sp.(8%)、果胶杆菌属Pectobacterium betavasculorum(6%)、亚洲韧皮杆菌Candidatus Liberibacter asiatic(2%)、Uncultured bacterium clone (4%)。其中Serratia sp.是优势菌属,Pantoea sp.是次优势菌属;病果桔络中黄龙病菌含量最高(>1%),而发病植株其他部位的黄龙病菌丰度远远低于病果,因此发病果实组织可以作为黄龙病菌分离培养的最佳试验材料。PCR-DGGE图谱显示Nocardia sp.和Arthrobacter sp.在病、健植株中稳定存在,而Xanthomonas sp.和Pseudomonas sp.只在病株中存在。利用定向培养方法与PCR-DGGE方法综合分析柑橘内生细菌的多样性,得到的细菌类型差别比较大。定向分离方法得到的内生菌优势菌属为Bacillus sp.和Curtobacterium sp.,而PCR-DGGE分析未发现这两个菌属。PCR-DGGE方法显示Serratia sp.和Pantoea sp.为柑橘内生细菌的优势菌属。研究结果表明定向培养方法获得的优势菌属并不一定是样品中真正的优势种,但是定向分离培养可以获得纯培养菌株,能够进一步研究优势菌株在实际生产中的应用价值。定向分离培养方法与PCR-DGGE方法相结合,可以更全面的分析生态系统中微生物多样性及确定优势菌群,获得可培养的微生物,对于柑橘内生细菌多样性和内生菌功能的研究起到重要的作用。更多还原

【Abstract】 Huanglongbing (HLB) is the most destructive disease of citrus that represent a major threat to the citrus production in the world. The causative agent is phloem-restricted, non stable cultured, Gram-negative bacterium named Candidatus Liberibacter spp. Until now the‘Ca. Liberibacter spp.’has been cultivated from Huanglongbing-infected citrus but the cultivation could not be repeated or completed Koch’s postulates. Nowdays, many Biological scientific researchers devoted themselves to the study of companion microbe in Huanglongbing pathogen-infected plant tissues, and reported that some endosymbiotic bacterial associated with pathogen were present in diseased plant tissue. The companion microbe may cause intricately and multiformity symptom of disessed citrus. The aim of this study was to analyze the diversity of cultivable and nonculturable entophytic bacterial communities in Candidatus Liberibacter sp. pathogen-infected and healthy citrus plant and find the companion microbial associated citrus plant tissues for decipher the artificial cultivation of HLB pathogen. We selected varied parts of citrus tissues collected from different locations of citrus planted area; the facultative anaerobic entophytic bacteria were isolated and purified based on bacterial morphology, physiology, biochemistry characteristics and the molecular method of PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) analysis based on the sequence of 16S rRNA V6-V8 fragment.By the directional isolation of the facultative anaerobic and entophytic bacteria from Huanglongbing pathogen-infected and healthy citrus plant tissues. The samples came from different origins included Guangdong, Guizhou, Fujian, Yunnan, Zhejiang, Guangxi provinces. By the directional isolation of the facultative anaerobic entophytic bacteria on the basis of colonial morphology, mycelium differences and 16S rDNA amplification methods, total 12 genera of bacteria were identified from 19 cultivable bacterial populations. The morphological and sequence analysis showed that: GX21 and GX34 belonged to Kluyvera sp., GX22, GX24, GX35, GX36 belonged to Bacillus sp., GX23 belonged to Brevibacillus Shida, GX25 belonged to Planococcus sp., GX26 belonged to Pseudoclavibacter helvolus, GX27 belonged to Microbacterium sp.,GX28 belonged to Tatumella sp., GX29,GX39 belonged to Pseudomonas sp., GX30, GX31, GX33 belonged to Curtobacterium sp., GX32 belonged to Pantoea sp., GX37 belonged to uncultured Klebsiella sp., GX38 belonged to Enterobacter sp., The dominant bacterial population in infected citrus plants were Curtobacterium sp. (IF: 29.07%), Bacillus sp. ( IF: 23.12%), Microbacterium sp. (IF: 21.09%), while in healthy citrus tissues belonged to Bacillus sp.( IF: 21.03%), Planococcus sp.(IF: 20.69%), Pseudomonas sp.(IF: 17.44%).The molecular method of PCR-DGGE analysis based on the sequences of 16S rDNA V6-V8 region was used to the micro flora analysis of citrus tissue from Guangxi province. We selected a pair of bacterial 16S rDNA (799F-1492R) to amplify bacterial sequences directly from citrus tissues by PCR for exclusion of chloroplast DNA. The total DNA of microorganisms was extracted from the different tissue of Huanglongbing pathogen-infected and healthy citrus, and then amplified the 16S rDNA V5-V9 fragment about 730 bp. Then we selected a pair of 968FGC-1378R to amplify V6-V8 region of 16SrRNA by Nested PCR. Purified PCR amplification products were separted by PCR-DGGE method. By 16S rRNA V6-V8 region gene DGGE method, fifty 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: fifty bands were detected; included 9 genera of bacteria: Serratia sp. (28%), Pantoea sp. (14%), and Acinetobacter sp. (10%), Arthrobacter sp. (8%), and Nocardia sp. (10%), Xanthomonas sp. (10%), and Pseudomonas sp. (8%), Pectobacterium betavasculorum(6%),Candidatus Liberibacter Asiati(c2%),Uncultured bacterium clone (4%)。The dominant bacterium population belonged to Serratia sp.(IF: 28%)and Pantoea sp. (IF: 14%)followed by it. Candidates Liberibacter asiaticus was found only in tangerine pith of deformed orange fruit, which suggested that the content(>1%)of Huanglongbing was more in diseased fruits and other tissues of citrus had low abundance percentage. The density and species of entophytic bacteria were also observed in remarkable difference between infected and healthy citrus plant from the DGGE profiles. Nocardia sp.and Arthrobacter sp. exists stable in all the samples.In this study, we adopted two methods including directional isolation and PCR-DGGE to comprehensively analyze the entophytic bacterial diversity of citrus, to analyze the entophytic bacterial diversity of citrus and find the companion microbe in Huanglongbing pathogen-infected and healthy citrus plant tissues for decipher the artificial cultivation of HLB pathogen, the density and species of entophytic bacteria of the two methods were different. By the culture-dependent method, Curtobacterium sp. and Bacillus sp. were the dominant groups, but the two groups couldn’t find by PCR-DGGE method. The results indicated that many of the dominant bacterial species could not be isolated or cultured by artificial cultivation method. Nevertheless, studies on entophytic bacterial diversity by 16S rDNA cloning and sequencing have some limitations. Therefore, combining the traditional directional isolation and PCR-DGGE method, may explore the dominant populations more effectively.更多还原