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

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。

Abstract:

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.