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

摘要:

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 Taxonomic Units，OTUs)，感病植株中除柑橘黄龙病菌Candidatus Liberibacter asiaticus外，还有丰富的Candidatus Liberibacter 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。

Abstract:

Abstract:［Objective］To analyze the microbial diversity in healthy and HLB-affected 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 cultivation-independent techniques based on 16S rRNA gene． The endophytic bacteria were isolated from surface-sterilized 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 healthy 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 Liberibacter 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.