Isolation And Identification Of Extremely Salt-tolerant Azotobacter And Nitrogen-fixing Characteristics

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.The amelioration of saline-alkali land resources,as well as the promotion of its production capacity is regarded as effective measures on both securing food supply and suppression of soil salinization.Improving plant rhizosphere environment by utilization of nitrogen-fixing microorganisms to increase crop yield is an important method for saline-alkali soil amelioration.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.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 crop.The research obtained the following experimental results:1.One strain of extremely salt-tolerant nitrogen-fixing bacteria named DJ-1 was obtained.The colony of the strain is round in shape,white and translucent in color,with neat and smooth edge and convex middle part.The individual bacterium is rod-shaped with blunt ends at both ends,sizing 0.3?0.5?m×0.5-1.3?m and Negative in Gram Staining.The 16S rRNA sequence of this the strain is highly homologous with that of Agrobacterium tumefaciens,indicating it is determined to be Agrobacterium tumefaciens,Under medium culture condition.2.DJ-1 can grow normally on a medium with a pH of 9 and a NaCl content of 1%to 4%,and the threshold tolerant to saline environment is pH 9 to 12 and a NaCl content of 4%to 8%.3.After 24 hours of culture,9.8 mg/L of extracellular ammonium secreted was detected,while the total nitrogen content in the culture medium was 0.28%.the nitrogenase activity and the production of long hormone were 424.33 ng/L and 34.5 ?g/mL,respectively.The nitrogenase gene nifH was cloned from DJ-1.4.DJ-1 can secrete auxin.After 48 hours of culture,the concentration of auxin(IAA)in the culture medium is 34.50 ?g/mL.5.DJ-1 can significantly promote the growth of corn.The yield traits such as plant height,stem thickness,leaf width and chlorophyll content are increased by 28.55%,13.45%,27.48%and 23.63%respectively compared with the control,and dry matter accumulation in the above-ground and underground parts Compared with the control,the amount increased by 86.50%and 74.37%respectively,The difference reaches a significant level.6.DJ-1 can promote the germination of cabbage seeds,increase its germination rate and germination potential,and also has a significant promoting effect on the growth of rice plants.The strain DJ-1 has the tolerance to extreme saline-alkali conditions,coupled with high nitrogen fixation and growth-promoting capabilities.DJ-1 is a potential candidate strain of functional bacteria for poor saline-alkali farmland amelioration.