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

摘要:

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。

Abstract:

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 loca-tions 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 popula-tions. 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. Can-didatus Liberibacter asiaticus was only found in tangerine pith of deformed orange fruit, which sug-gested 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 re-markable difference between infected and healthy citrus plant from the PCR-DGGE profiles.