【作者】 李佳；

【导师】 殷幼平；

【作者基本信息】 重庆大学 ， 生物学， 2012， 硕士

【摘要】 柑橘黄龙病(Citrus Huanglongbing，HLB)严重威胁并阻碍着我国柑橘产业的快速发展。该病害由寄生于韧皮部筛管细胞内的革兰氏阴性细菌引起，目前认为该病菌属于α-变形菌纲(Alphaproteobacteria)的候选韧皮杆菌属(CandidatusLiberibacter)，是一种难培养原核微生物。迄今为止，柑橘黄龙病菌的体外培养仍然是攻克黄龙病的难点，为克服传统的纯培养研究思路，欲建立柑橘黄龙病菌的共培养体系，本研究以获取功能型植物伴生性内生细菌为目的，对长春花病、健植株及九里香健株的内生细菌菌群结构进行多样性分析并对其功能进行初步的鉴定。以揭示九里香及黄龙病菌侵染长春花后体内内生细菌菌群结构的变化，为寻找柑橘黄龙病菌能在其寄主体内大量增殖的内在原因及其侵染机理并利用功能性内生细菌病害防治研究提供理论基础。①以显症的3年生长春花植株及健康长春花植株样品为材料，利用兼性厌氧可培养技术、限制性片段长度多态性分析(Restriction fragment length polymorphism,RFLP)及16S rDNA序列分析相结合的方法。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.)、蜡样芽孢杆菌(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.)、芽孢杆菌属(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)，感病植株中存在柑橘黄龙病菌CandidatusLiberibacter asiaticus和丰富的Candidatus Liberibacter sp.。经一系列功能鉴定，测定了29株内生细菌的功能，其中18株菌能产生吲哚-3-乙酸(IAA)；具有抗生素(phlD)合成能力的内生细菌有1株；结合nifH和NFb固氮培养基确定有4株内生细菌具有固氮能力；8株内生细菌具有ACC脱氨酶合成能力；11株内生细菌具有铁细胞合成能力；5株内生细菌具有淀粉水解能力；7株内生细菌显示强阳性的蛋白酶合成能力。6株内生细菌具有以上4种功能，分属于马胃葡萄球菌、芽孢杆菌属、短小杆菌属、摩氏摩根菌属(Morganella morganii)及溶杆菌属(Lysobacter sp.)5个属。②以生长在福州、厦门、广州、广西等黄龙病高发区的多年生九里香植株叶及茎杆为分离对象，利用平板培养法及基于16S rDNA的限制性片段长度多态性(RFLP)序列分子鉴定法，对九里香植物内生细菌进行多样性分析。在兼性厌氧的生长环境下，获得可培养内生细菌26株，属于5个不同的类群，低G+C革兰氏阳性菌(Firmicutes)为主要类群，占内生细菌总数的50%，其次为变形菌门(Proteobacteria)，包括α、γ2个类型，分别占7.7%、30.77%；拟杆细菌(Bacteroidetes)占7.7%；放线细菌(Actinobacteria)占3.85%。26株细菌分属于9个细菌属的14个种，分别为肠杆菌属(Enterobacter sp.)、芽孢杆菌属(Bacillus sp.)、金黄杆菌属(Chryseobacterium sp.)、葡萄球菌属(Staphylococcus sp.)、枯草芽孢杆菌(Bacillussubtilis)、巨大芽孢杆菌(Bacillus megaterium)、腊样芽孢杆菌(Bacillus cereus)、短小芽孢杆菌(Bacillus pumilus)、类芽孢杆菌(Paenibacillus sp.)、假单胞菌属(Pseudomonas sp.)、寡养单胞菌(Stenotrophomonas sp.)、短波单胞菌属(Brevundimonas sp.)、单胞菌属(Sphingomonas sp.)、短小杆菌属(Curtobacterium sp.)。其中肠杆菌属(Enterobacter sp.)(IF=19.23%)、芽孢杆菌属(Bacillus sp.)(IF=38.46%)为九里香可培养内生细菌的优势菌属。利本研究选用细菌通用引物799F/1492r扩增九里香细菌基因组的16S rDNA的V5V9这5个高变区域并进行RFLP分析。筛选得到179个阳性克隆，分别进行HaeIII、 MspI、 RsaI三种限制性内切酶分析，得到20个不同的操作分类单元(Operational Taxonomic Units, OTUs)，该克隆文库的覆盖度(Coverage C)为94.97%，结合Rarefaction曲线分析，表明所构建的克隆文库是相对充分的。沙雷氏菌属(Serratia sp.)为九里香不可培养内生细菌的优势菌属。利用生理生化鉴定的方法，测定了14株内生细菌的功能，其中9株菌能产生吲哚-3-乙酸(IAA)；具有抗生素(phlD)合成能力的内生细菌有4株；结合nifH和NFb固氮培养基确定有3株内生细菌具有固氮能力；1株内生细菌具有ACC脱氨酶合成能力；8株内生细菌具有铁细胞合成能力；3株内生细菌具有淀粉水解能力；2株内生细菌显示强阳性的蛋白酶合成能力。4株内生细菌具有以上4种功能，分属于Bacillusmegaterium、Bacillus pumilus、Paenibacillus sp.和Sphingomonas sp.。通过本研究分析发现柑橘黄龙病寄主植物的植株中内生细菌菌群多样性丰富及具有多样的遗传特性，黄龙病菌的存在改变了九里香及长春花原有内生细菌的菌群结构，且初步的生理生化鉴定表明九里香及长春花体内分别含有潜在的功能型植物伴生细菌。更多还原

【Abstract】 Huanglongbing (HLB) is as serious disease found in citrus and hinder our nationaldevelopment of citrus industry. HLB is caused by a negative phloem-restricted,nonculturable bacterium that belongs to the genus “Candidatus Liberibacter,” whichbelongs to the Alphaproteobacteria.It is by far the biggest difficult to culture the pathogen of HLB in vitro. In order toovercome the thought of the traditional pure culture and found the new method aboutco-culture of HLB, we analyze microbial diversity varied in the host plants-Catharanthus roseus and Murraya paniculata and look for the association between theendophytic bacteria and the HLB pathogen.Thus, Our objective in this study was toanalyze, under manual-grafting conditions, how the microbial diversity varied inasymptomatic and symptomatic C. roseus or M. paniculata and delineation of thecultivated endophytic bacterial isolates and characterization of their salient metabolicfeatures. We hope find the endophytic bacteria that have potential interaction with theHLB pathogen.①The healthy and HLB-affected Catharanthus roseus under manual-graftingconditions were used as research material. The endophytic bacterial communities weredelineation by using the traditional culturable approach and cultivation-independenttechniques based on16S rRNA gene. The endophytic bacteria were isolated fromsurface-sterilized C.roseus midribs of leaves and phloem of stems and roots by platingand restriction fragment length polymorphism (RFLP). By the artificial anaerobicculture, we obtained67strains that were identified29genus by GenBank.Curtobacterium sp., Erwinia sp., Bacillus cereus and Brevundimonas sp., Bacillus sp.were the dominant bacterial population in diseased and healthy C.roseus, respectively.However, Staphylococcus equorum was the common dominant isolate in both C.roseus.By the methods of physiological biochemical identification, The number ofbacterial isolates producing IAA and amylolysis activity was18and5. The frequency ofendophytic bacteria abling to produce ACC deaminase, siderophore and protein activitywas8,11and7. The screened for N-fixation ability of Bacterial isolates were4byusing N-free medium and nifH gene. The amount of endophytic bacteria producingantibiotic synthesis (phlD) was1. A total of6bacterial isolates show a minimum of four traits.6functional bacterial strains belonged to5genus that included Staphylococcusequorum、Bacillus sp.、Curtobacterium sp.、Morganella morganii and Lysobacter sp.。We picked188and182positive clones in16S rDNA libraries of diseased andhealthy C.roseus that were respectively restricted by the HaeIII, MspI, RsaI restrictionendonuclease. Based on the similarity of the RFLP banding profiles in diseased andhealthy C.roseus, we obtained16,23OTUs (Operational Taxonomic Units, OTUs)respectively. In addition to Candidatus Liberibacter asiaticus, Candidatus Liberibactersp. was the dominant bacterial population only in diseased C.roseus.②We used M. paniculata that were growth in the cirtus garden of HLB. M.paniculata were seen as huanglongbing’s hidden host plant and we analyze theendophytic bacteria in M.paniculate that have potential to suppress the HLB pathogen.The endophytic bacteria were isolated from surface-sterilized M. paniculata midribs ofleaves and phloem of stems by plating and restriction fragment length polymorphism(RFLP). By the artificial anaerobic culture, we obtained26strains that were groupedinto9genus by GenBank. Enterobacter sp.(IF=19.23%), Bacillus sp.(IF=38.46%) werethe dominant bacterial population in M. paniculata. However, the bacterial colonynumber of endophytic bacteria in the stems was higher than in the leaves.By the methods of physiological biochemical identification, The number ofbacterial isolates producing IAA and amylolysis activity was9and3. The frequency ofendophytic bacteria abling to produce ACC deaminase, siderophore and protein activitywas1,8and2. The screened for N-fixation ability of Bacterial isolates were3by usingN-free medium and nifH gene. The amount of endophytic bacteria producing antibioticsynthesis (phlD) was4. A total of4bacterial isolates show a minimum of four traits.4functional bacterial strains belonged to3genus that included Bacillus sp., Paenibacillussp. and Sphingomonas sp.We chose179positive clones in16S rRNA gene clone library of M. paniculatathat were respectively restricted by the HaeIII, MspI, RsaI restriction endonuclease.20OTUs (Operational Taxonomic Units) were observed based on the similarity of theRFLP banding profiles. Coverage C of the clone library was94.97%and the rarefactioncurve of the clone library tended to plateau, which indicate that the library are largeenough to reflect the bacterial endophytic diversity of the respective samples.63.69%ofthe observed clones were Serratia sp. in M. paniculata.The above the results in our study suggest the following two conclusion: ①With the infection of ‘Ca. L. asiaticus’, the diversity in diseased C.roseusdecreased. The endophytic bacteria isolated from diseased and healthy C.roseus areabundant and have remarkable differences in the composition and function, suggestingthat its endophytic bacteria might be inhibited by the HLB pathogen in a certain degree.②The results suggest that the endophytic bacteria isolated from M. paniculatahave abundant diversity and genetic characterization.更多还原