亚洲韧皮杆菌兼性厌氧型伴生细菌鉴定及优势菌群分析

Isolation of facultative anaerobic entophytic bacterial companioned Ca Las infected citrus tissues and evaluation of dominant bacterial populations

以定向分离培养和基于16S rDNA的PCR-DGGE(Denaturing gradient gel electrophoresis,DGGE)方法,分析感黄龙病柑橘与健康柑橘植株不同部位的内生细菌多样性,分离柑橘组织共获得19株可培养的兼性厌氧型内生细菌,经形态、生理生化结合16S rDNA分子方法鉴定其隶属于12个属,其中短小杆菌属Curtobacterium sp.(IF:29.07%)、芽孢杆菌属Bacillus sp.(IF:23.12%)和微杆菌属Microbacterium sp.(IF:21.09%)为罹病植株的优势菌群,芽孢杆菌属Bacillus sp.(IF:21.03%)、动性球菌属Planococcus sp.(IF:20.69%)和假单胞菌属Pseudomonas sp.(IF:17.44%)为无症健株的优势菌群。对DGGE方法得到的50条16S rDNA目标条带进行序列比对,共鉴定出9个属的细菌,结果表明沙雷氏菌属Serrations sp.(IF:28%)是优势菌属,泛菌属Pantoea sp.(IF:14%)是次优势菌属;病果桔络中黄龙病菌含量最高(>1%),而罹病植株其他部位的黄龙病菌丰度较低。PCR-DGGE图谱也显示感病和健康柑橘组织的内生细菌存在差异。

To analysis the entophytic bacterial diversity of citrus and find the companion bacteria populations associated with Huanglongbing pathogen-infected and healthy citrus plant tissues for decipher the co-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（Denaturing gradient gel electrophoresis） analysis based on the sequence of 16S rRNA V6-V8 fragment gene.By the directional isolation of the facultative anaerobic entophytic bacteria and 16S rDNA amplification,total 12 genera of bacteria were identified from 19 cultivable bacterial populations.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%）.From 50 target bands obtainded by the DGGE approach,9 genera of cultivable bacteria were recognized.The dominant bacterium population belonged to Serratia sp.（IF： 28%） and Pantoea sp.（IF： 14%） followed by it.Candidatus Liberibacter asiaticus was only found 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 PCR-DGGE profiles.