【作者】 许明双；

【导师】 郭顺堂； 申琳；

【作者基本信息】 中国农业大学 ， 农产品加工及贮藏工程， 2014， 博士

【摘要】 植物内生菌是生活在植物体内部、不引起植物病害的、并对植物生长、抗病、抗逆及植物修复等具有促进作用的一类微生物,多功能性内生菌与植物体的内共生关系成为近年来研究的热点。本文以番茄(Lycopersicum esculentum Mill.)和水稻(Oryza sativa L.)种子为材料,分析并比较了不同品种番茄和水稻种子中内生细菌的菌群结构。16S rRNA基因PCR-RFLP分析显示,从红樱桃、粉美人、黄天使和绿珍珠四个番茄品种种子中分离得到的84株内生细菌分别隶属于芽孢杆菌属(Bacillus)5个不同的种B. altitudinis、B. amyloliquefaciens、B. atrophaeus、B. pumilus和B. subtilis,其中,B. subtilis和B. amyloliquefaciens是共有的优势种；而分离自郑稻18号、徐稻6号、绥粳4号和上育397四个水稻品种种子的48株内生细菌则分别隶属于γ-变形菌门、β-变形菌门、放线菌门和厚壁菌门的10个属,γ-变形菌门(Enterobacter、Pantoea、Xanthomonas和Pseudomonas)的菌株为优势菌群,其中,泛菌属(Pantoeα)是共有的优势菌属。固氮、溶磷、分泌生长素(IAA)和嗜铁素以及具有ACC(1-氨基环丙烷-1-羧酸)脱氨酶活性等是植物促生菌所具备的一些特性。从番茄和水稻种子中分离得到的内生细菌几乎都具有潜在的固氮能力,近50%的菌株可以分泌IAA或具有溶磷作用,大多数种子内生菌株同时具备两种或两种以上的促生特性。其中,六株内生菌具有ACC脱氨酶活性,HYT-12-1和NA-397-SYM-1活性较高,ACC脱氨酶活性分别为112.02和131.88nmolα-酮丁酸/(mg·h)。HYT-15-1、LZZ-133和LB-Z-FJ-1菌株产IAA能力较强,分别为5.12、5.49和6.53μg/mL。通过鉴定,HYT-12-1、 HYT-151-1和LZZ-133属于枯草芽孢杆菌(Bacillus subtilis), LB-Z-FJ-1属于肠杆菌属(Enterobacter), NA-397-SYM-1属于藤黄短小杆菌(Curtobacterium luteum),这是首次发现短小杆菌属菌株(Curtobacterium)具有ACC脱氨酶活性,并且这五株菌都具有自生固氮能力。两株产ACC脱氨酶菌株HYT-12-1和NA-397-SYM-1以及三株产IAA菌株LB-Z-FJ-1、HYT-151-1和LZZ-133在番茄、油菜、萝卜和水稻等植株的根、茎、叶中都能较好的定殖,同时对植物幼苗的生长具有不同程度的促进作用,幼苗根和茎的长度、重量、叶绿素含量、根系活力及氮、磷等元素含量都有显著提高,在番茄植株中,属于种子内生优势菌属Bacillus的HYT-12-1和LZZ-133菌株的促生效果更为显著。同时,这五株促生内生菌浸种处理番茄种子,可有效提高番茄幼苗对番茄灰霉病原菌Botrytis cinerea Pers的抗病防御能力,引起植株产生诱导系统抗性反应(induced systemic resistance, ISR)。不同菌株诱导植物ISR反应时参与的信号转导途径可能不同,内生促生枯草芽孢杆菌(B. subtilis)菌株HYT-12-1、HYT-151-1和LZZ-133可能通过激活JA信号转导途径引起番茄幼苗的ISR反应,C. lutuem NA-397-SYM-1和Enterobacter sp. LB-Z-FJ-1诱导番茄幼苗ISR反应则可能同时与SA和JA信号转导途径有关。综上所述,本研究表明,植物种子内生细菌菌群结构具有丰富的多样性,种子内生菌株对番茄等植株表现出明显的促生功能,并可增强番茄幼苗的抗病性。更多还原

【Abstract】 Endophytic bacteria are the microorganisms that colonize the interior of plants and do no harm to the plant tissues, and they may show beneficial effects on plant growth, disease-resistant, stress-resistant and phytoremediation. The symbiotic relationship between plant and versatile endophytes becomes a hot research topic recently.In this study, seeds of different tomato (Lycopersicum esculent.um Mill.) and rice (Oiyza sativa L.) cultivars were used to analyze and compare the bacterial community compositions of endophytic bacteria. On basis of the16S rRNA PCR-RFLP analysis,84strains isolated from seeds of four tomato cultivars (Red cherry, Pink lady, Yellow angel and Green pearl) showed similarity to five Bacillus species: B. altitudinis, B. amyloliquefaciens, B. atrophaeus, B. pumilus and B. subtilis. B. subtilis and B. amyloliquefaciens were the mutual dominant speices. And48strains isolated from seeds of four rice cultivars (Zhengdao18, Xudao16, Suijing4and Shangyu397) were grouped into10genera, which were closely matched to y-Proteobacteria, α-Proteobacteria, Actinobacteria and Firmicutes. y-Proteobacteria (Enterobacter, Pantoea, Xanthomonas and Pseudomonas) was predominant in the community, and Pantoea was the mutual dominant genus.Nitrogen fixation, phosphate solubilization, production of IAA, siderophore and ACC (1-aminocyclopropane-l-carboxylic acid) deaminase are the characteristics of plant growth-promoting bacteria. Most of strains obtained from tomato and rice seeds were able to grow in nitrogen-free culture medium. Almost50%of the isolates showed the capability of IAA production or phosphate solubilization. Majority of strains exhibited two or more plant growth-promoting traits. Among six ACC deaminase producing bacteria, the enzyme activities of HYT-12-1and NA-397-SYM-1were higher than others, and their ACC deaminase activities were quantified as112.02and131.88nmol a-ketobutyrate mg-1protein h-1. HYT-151-1, LZZ-133and LB-Z-FJ-1had better ability of IAA production, and the IAA contents were5.12,5.49and6.53μg/mL. HYT-12-1, HYT-151-1and LZZ-133were identified as Bacillus subtilis, LB-Z-FJ-1was belonged to Enterobacter, and NA-397-SYM-1was affiliated to Curtobacterium luteum. This is the first report that Curtobacterium bacterium could produce ACC deaminase. These five isolates had self-generated nitrogen-fixing ability.ACC deaminase producing strains (HYT-12-1and NA-397-SYM-1) and IAA producing strains (LB-Z-FJ-1, HYT-151-1and LZZ-133) could colonize in roots, stems and leaves of tomato, rape, radish and rice seedlings, and promoted the growth of seedlings. Treatments with strains significantly improved the length and weight of roots and stems, chlorophyll content, root activity and element content. The plant growth promoting effects were more conspicuous, when treated with HYT-12-1and LZZ-133on tomato seedlings.Besides, these five strains could enhance the disease-resistance and defense function against Botrytis cinerea Pers. and cause the induced systemic resistance (ISR) response by soaking seeds treatments. The elicitation of ISR response by different strains may involve different signal transduction pathway. In Bacillus (HYT-12-1, HYT-151-1and LZZ-133) strains-treated tomato seedlings, resistance was likely to associate with JA signalling, but C. lutuem NA-397-SYM-1and Enterobacter sp. LB-Z-FJ-1treatments maybe induced both SA-and JA-dependent pathways.In conclusion, this study indicated the diversity of endophytic bacterial community compositions of plant seeds, and demonstrated the plant growth-promoting effects of endophytic bacteria on several plants and enhancement of disease resistance on tomato seedlings.更多还原