梧县药用野生稻内生固氮菌分离鉴定与系统发育分析  

Isolation and phylogenetic analysis of endophytic nitrogen-fixing bacteria from Oryza officinalis in Wuxian

作　　者：谭泽文 谭志远[1] 黄慧灵[1] 张新梅[1] 刘丽辉[1] 彭桂香[2] 

机构地区：[1]华南农业大学农学院,广州510642 [2]华南农业大学资源与环境学院,广州510642

出　　处：《应用与环境生物学报》2017年第4期622-627,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31370 052);广东省科技项目(2014A050503058;2014A030313459);广东省烟草专卖局(公司)科技项目(粤烟科[2014]02)资助~~

摘　　要：以广西梧县药用野生稻为材料,采用两种无氮培养基及改变其碳源的无氮培养基分离纯化野生稻内生固氮菌,用乙炔还原法检测固氮酶活性;并结合插入序列指纹图谱进行聚类分析,测定各类群代表菌株的16S r RNA序列分析其系统发育;进一步扩增nif H基因片段以验证固氮菌,并通过生理生化试验筛选出具有较好功能的内生固氮菌.共分离得到27株内生固氮菌,固氮酶活性范围0.872-103.077 nmol m L^(-1) h^(-1);聚类分析6个类群中,类群Ⅰ代表菌株111与嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia MTCC 434T)相似性为99.61%,类群Ⅱ代表菌株与肺炎克雷伯氏肺炎亚种(Klebsiella pneumoniae subsp.pneumoniae DSM 30104T)相似性为99.48%,类群Ⅲ代表菌株23与Kosaconia sacchari SP1T相似性为99.74%,类群Ⅳ代表菌株JX41′与Enterobacter oryzendophytics LMG 26432T相似性为98.51%,类群V代表菌株a32N与肺炎克雷伯氏鼻硬结亚种(Klebsiella pneumoniae subsp.rhinoscleromatis ATCC 13884T)相似性为99.74%,类群Ⅵ代表菌株15C3与变栖克雷伯氏(Klebsiella variicola DSM 15968T)相似性为99.74%.nif H基因片段检测进一步验证分离的细菌为固氮菌,发现类群I未扩增得到nif H片段,有可能代表一类新的固氮体系,有待进一步研究.类群VI 15C3具有产脲酶、较强分泌生长素的能力,且同时具有溶磷解硅、产铁载体和分泌硫化氢的能力,具有较强促生作用,有很好的应用前景.本研究表明梧县药用野生稻内生固氮菌资源具有遗传多样性,并筛选得到一类具有促生特性内生固氮菌,应用前景广泛.This study aimed to investigate the diversity of endophytic nitrogen-fixing bacteria and to screen for some strong growth-promoting endophytic nitrogen-fixing bacteria from Oryza officinalis in Wuxian, China. Two selective media with modified carbon sources but without nitrogen sources were used to isolate the endophytic nitrogen-fixing bacteria. The nitrogenase activity of the bacteria was determined by acetylene reduction assay （ARA）. The IS-PCR DNA fingerprinting patterns were used to cluster the isolates; thereafter, the 16S rRNA gene from the representative bacteria were amplified and the sequences were aligned for phylogenetic analysis. The nifH gene fragment was amplified to verify the presence of nitrogenase, while growth-promoting endophytic nitrogen-fixing bacteria were screened through physiological and biochemical tests. Twenty-seven endophytic diazotrophs were isolated and assigned to six groups. The nitrogenase activity of those isolates was between 0.872-103.077 nmol mL-1 h-1. The homology of 16S rRNA gene between Group I and Stenotrophomonas maltophilia MTCC 434T was 99.61%, 99.48% between Group II and Klebsiella pneumoniae subsp. pneumoniae DSM 30104T, 99.74% between Group III and Kosaconia sacchari SP1T, 98.51% between Group IV and Enterobacter oryzendophytics LMG 26432T, 99.74% between Group V and K. pneumoniae subsp. rhinoscleromatis ATCC 13884T, 99.74% between Group VI and K. variicola DSM 15968T. Group I had nitrogenase activity, but did not yield nifH PCR products, which may indicate a new system of nitrogen-fixation, and requires further research. The results of physiological and biochemical tests showed that the group VI 15C3 not only had the ability to secreting urease and produce auxin, but also had the ability to solubilize phosphorus and silicate, produce siderophore and hydrogen sulfide. The results suggested that endophytic nitrogen-fixing bacteria of O. officinalis have genetic diversity, and a new kind of endophytic bacteria was obtained, which has great application prospects.

关 键 词：药用野生稻 内生固氮菌 遗传多样性 系统发育分析 促生 

分 类 号：Q93[生物学—微生物学] S511.9[农业科学—作物学]