长春花内生细菌多样性与柑橘黄龙病菌的相关性  

Endophytic bacterial community analysis of Catharanthus roseus and its association with huanglongbing pathogen

作　　者：李佳[1] 王中康[1] 谢攀[1] 吴东 殷幼平[1] 

机构地区：[1]重庆大学生物工程学院,重庆市杀虫真菌生物农药工程技术中心,重庆市基因功能与调控重点实验室,重庆400030 [2]亚洲果业广西利添柑橘生物技术公司,合浦536128

出　　处：《微生物学报》2012年第4期489-497,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30971875);国家公益性行业(农业)科研专项(201003067);公益性行业科研专项“柑橘黄龙病和溃疡病综合防控技术研究与示范”(201003067-02-5);农业部“农作物病虫鼠害疫情监测与防治项目”(农财发[2009]137号)~~

摘　　要：【目的】分析感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构变化,为柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】本研究利用兼性厌氧可培养技术、16S rDNA限制性片段长度多态性分析(Restriction fragment length polymorphism,RFLP)以及16S rDNA序列分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。通过RFLP方法分析,感病株得到16个、健株得到23个操作分类单元(Operational TaxonomicUnits,OTUs),感病植株中除柑橘黄龙病菌Candidatus Liberibacter asiaticus外,还有丰富的CandidatusLiberibacter sp.存在。【结论】感病与健康长春花植株中均含有丰富的内生细菌,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构,且菌群多样性下降。可见长春花内生细菌在一定程度上受到柑橘黄龙病菌的抑制。[ Objective ] To analyze the microbial diversity in healthy and HLBaffected Catharanthus roseus under manualgrafting conditions and to find the association between the endophytic bacteria and the HLB pathogen. [ Methods] The endophytic bacterial communities were delineated by using the traditional culturable approach and cultivationindependent techniques based on 16S rRNA gene. The endophytic bacteria were isolated from surfacesterilized C. roseus midribs of leaves and phloem of stems and roots by plating and restriction fragment length polymorphism （RFLP）. [ Results] By anaerobic culture, we obtained 67 strains that were identified 29 genus by GenBank. Curtobacterium sp. , Erwinia sp. , Bacillus cereus and Brevundimonas sp. , Bacillus sp. were the dominant bacterial population in diseased and healthy C. roseus. However, Staphylococcus equorum was the common dominant isolate in both C. roseus. We picked 188 and 182 positive clones in 16S rDNA libraries of diseased and heahhy C. roseus that were respectively restricted by the HaeIII, MspI, RsaI restriction endonuclease. Based on the similarity of the RFLP banding profiles in diseased and healthy C. roseus, we obtained 16, 23 OTUs （Operational Taxonomic Units, OTUs） respectively. In addition to Candidatus Liberibaeter asiaticus, Candidatus Liberibacter sp. was the dominant bacterial population only in diseased C. roseus. [ Conclusions] With the infection of ＇Ca. L. asiaticus＇, the diversity in diseased C. roseus decreased. The endophytic bacteria isolated from diseased and healthy C. roseus are abundant and have remarkable differences in the composition and function, suggesting that its endophytic bacteria might be inhibited by the HLB pathogen.

关 键 词：长春花 柑橘黄龙病菌 16S rDNA 可培养 限制性片段长度多态性分析(Restriction FRAGMENT length polymorphism RFLP) 相关性 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]