极耐盐碱固氮菌的分离鉴定及固氮特性研究  

Isolation and identification of extremely salt-tolerant azotobacter and its nitrogen-fixing characteristics

作　　者：曹晶晶[1] 熊悯梓 钞亚鹏[2,3] 赵盼 汪志琴[2,3] 仲乃琴 Jingjing Cao;Minzi Xiong;Yapeng Chao;Pan Zhao;Zhiqin Wang;Naiqin Zhong(School of Agriculture,Ningxia University,Yinchuan 750021,Ningxia Hui Autonomous Region,China;Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;The Enterprise Key Laboratory of Advanced Technology for Potato Fertilizer and Pesticide,Hulunbuir 021000,Inner Mongolia Autonomous Region,China;Engineering Laboratory for Advanced Microbial Technology of Agriculture,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]宁夏大学农学院,宁夏银川750021 [2]中国科学院微生物研究所,北京100101 [3]内蒙古自治区马铃薯肥料农药高效利用技术企业重点实验室,内蒙古呼伦贝尔021000 [4]中国科学院农业微生物先进技术工程实验室,北京100101

出　　处：《微生物学报》2021年第11期3483-3495,共13页Acta Microbiologica Sinica

基　　金：中国科学院战略性光导科技专项(XDA13020601);山东省重大科技创新项目(2018CXCC0303);广东省重点领域研发计划项目(2020B0202010005);中国科学院科技扶贫项目(KFJ-FP-201905,KFJ-FP-202001);中国科学院科技服务网络计划(KFJ-STS-QYZD-199)。

摘　　要：我国盐碱土面积约9913万hm2,其中pH值高于9、盐含量大于0.6%的重度盐碱地每年以1.4%的速率增长。利用固氮微生物改善植物根际环境,提高作物产量,是盐碱地改良的重要方法。【目的】从来自海南省三沙市热带珊瑚岛礁的土壤中,分离鉴定自生固氮菌,为极端盐碱地改良提供候选菌株。【方法】通过形态学观察、生理生化特征分析和16S rRNA序列测定等方法进行菌种鉴定,分析其固氮、耐盐碱和促生长特性,盆栽试验验证其对玉米主要农艺性状的影响。【结果】获得1株极端耐盐碱的固氮细菌DJ-1,其菌落呈圆形,菌体杆状,大小(0.5–1.3)μm×(0.3–0.5)μm,革兰氏染色阴性,与根癌土壤杆菌(Agrobacterium tumefaciens)的16S rRNA序列高度同源,确定其为根癌土壤杆菌。DJ-1在pH 9、NaCl含量为1%–4%的培养基上可正常生长,能耐受pH 12、NaCl含量8%的环境。从中克隆到固氮酶基因nifH。盆栽试验结果表明,DJ-1可显著促进玉米生长。【结论】菌株DJ-1能耐受极端盐碱条件,且具有较强的固氮和促生长能力,有可能作为贫瘠盐碱耕地改良功能菌剂的候选菌株。In China,the saline-alkali soil covers an area of approximately 99.13 million hm2,of which the severe saline-alkali soil with pH value above 9 and salinity greater than 0.6%represents a growth rate of 1.4%per year.Improving plant rhizosphere environment by utilization of nitrogen-fixing microorganisms to increase crop yield is an important method for saline-alkali soil amelioration.[Objective]Candidate strains were selected for the amelioration of extreme saline-alkali soils by isolating the autogenous azotobacter from the soil sample collected from the coral reefs in Sansha City,Hainan Province.[Methods]The strains were identified through morphological observation,physiological and biochemical characteristics analysis and 16S rRNA sequencing,followed by the analysis of their nitrogen-fixing,salt-tolerant and growth-promoting characteristics,and verification of their effects on the main agronomic traits of corn.[Results]One strain of extremely salt-tolerant nitrogen-fixing bacteria named DJ-1 was obtained.The colony of the strain is round in shape,The individual bacterium is rod-shaped,sizing(0.3–0.5)μm×(0.5–1.3)μm.Negative in Gram Staining,the strain is highly homologous with the 16S rRNA sequence of Agrobacterium tumefaciens,hence it is determined to be Agrobacterium tumefaciens.Under medium culture condition,DJ-1 presents normal growth at pH value of 9 with NaCl concentration of 1%–4%,and tolerance at pH value of 12 and NaCl concentration of 8%.The nitrogenase gene nifH was cloned from DJ-1.It has been indicated in the pot experiment results that the growth of corn was significantly accelerated by DJ-1.[Conclusion]The strain DJ-1 has the tolerance to extreme saline-alkali conditions,coupled with high nitrogen fixation and growth-promoting capabilities.It may be a potential candidate strain of functional bacteria for poor saline-alkali farmland amelioration.

关 键 词：盐碱土 固氮菌 根癌土壤杆菌 促生长 

分 类 号：Q939.113[生物学—微生物学]