Effects Different Nitrogen Concentractions Growth And Metabolism Of Three Nitrogen-Fixing Bacteria In Rice Rhizosphere

Nitrogen is one of the elements necessary for the growth and development of animals and plants,and it is also an important nutrient element for the growth of microorganisms.However,the excessive use of chemical fertilizers left fertilizers in the soil,leading to environmental pollution and eutrophication of water.How to improve the nitrogen absorption and utilization efficiency of plants to increase crop yield is an urgent problem to be solved in the development of green sustainable agriculture.Biological nitrogen fixation has proven to be an effective method.Exploring the regulation mechanism of microbial nitrogen fixation and growth promotion is of great significance for guiding the application of nitrogen fertilizer and the development of green agriculture.In this study,three different types of rice root nitrogen-fixing bacteria were selected to study the utilization of their carbon and nitrogen sources.Liquid chromatography was used to analyze the biosynthesis of bacterial organic acids and plant hormones under different nitrogen concentrations,and combined with whole-genome sequencing to reveal The genomics mechanism of the biosynthesis of three nitrogen-fixing bacteria.The main findings are as follows:1.Observation of two different culture mediums showed that the strains showed differences in colony morphology on LB medium containing nitrogen source and Ashby medium without nitrogen source.All three strains can grow in nitrogen-free medium containing different carbon sources,but the available carbon source materials are inconsistent.The strain Raoultella terrigena NC01 can utilize glycerol,glucose,sucrose,serine,proline and pyruvate.Strain Sinorhizobium sp.NC02 can utilize pyruvate,ethanol,mannitol,sorbitol,glycerol,serine,proline,cysteine,glucose,sucrose,lactose and ethyl acetate.The strain Herbaspirillum sp.ACC01 can utilize glycerol,pyruvate,serine,proline and lactose.When a nitrogen source is added to the culture medium,all three strains can utilize the five carbon sources of citric acid,succinic acid,acetic acid,α-ketoglutarate and malic acid,which are involved in the tricarboxylic acid cycle.Increased,the growth of the strain is promoted.2.Through the phosphorus solubilization test of the strains,it was found that all three strains had the ability to solubilize phosphorus.Strain R.terrigena NC01 has strong phosphorus solubilization,and the amount of phosphorus solubilized has a maximum when the nitrogen concentration is 0 m M,which is 603.4 mg·L^-1·OD^-1.Both the strain Sinorhizobium sp.NC02 and the strain Herbaspirillum sp.ACC01 had the largest amount of phosphate dissolved at a nitrogen concentration of 0 m M,which were 125.9 mg·L^-1·OD^-1and 31.3 mg·L^-1·OD^-1,respectively.With the increase of nitrogen concentration,the three strains all showed the characteristic of gradually weakening the ability of dissolving phosphorus.3.Use liquid chromatography to detect the secretion of plant hormones of the three strains under different carbon sources and different nitrogen concentrations.The results show that adding a nitrogen source promotes the secretion of plant hormones,but an excessively high nitrogen concentration（2.5 m M）inhibits the secretion of hormones.The three strains can use different carbon sources to synthesize and secrete growth hormone（IAA）,cytokinin（6-BA）,abscisic acid（ABA）,jasmonic acid（JA）and gibberellin（GA3,GA7）,but they secrete auxin（IAA）,cytokinin（6-BA）,abscisic acid（ABA）,jasmonic acid（JA）and gibberellin（GA3,GA7）.There are differences in concentration and type.Mannitol significantly improves the biosynthesis of IAA,ABA and GA3 by R.terrigena NC01 and Sinorhizobium sp.NC02.Mannitol is the carbon source and when the nitrogen concentration is 1m M,R.terrigena NC01biosynthesizes 0.21μg/ml IAA,0.09μg/ml ABA and 5.67μg/ml GA3,respectively,and Sinorhizobium sp.NC02 biosynthesizes 0.14μg/ml IAA,0.11μg/ml ABA And3.66μg/ml GA3.Mannitol significantly improves Herbaspirillum sp.ACC01biosynthesis of IAA,ABA,JA and 6-BA.When mannitol is a carbon source and a nitrogen concentration of 1 m M,Herbaspirillum sp.ACC01 biosynthesizes 4.73μg/ml IAA,0.84μg/ml ABA,5.56μg/ml JA and 3.44μg/ml 6-BA,respectively.4.Use liquid chromatography to detect the secretion of organic acids of the three strains under different carbon sources and nitrogen concentrations.It was found that all three strains could use different carbon sources to synthesize and secrete tartaric acid,formic acid,malic acid,acetic acid,pyruvate,citric acid and fumaric acid,but only part of the carbon source could be used to synthesize and secrete oxalic acid.The three nitrogen-fixing strains have different optimal carbon sources for biosynthesis of organic acids.The secretion of different organic acids has inconsistent responses to nitrogen concentration.Overall,as the nitrogen concentration increases,the secretion of organic acids increases.5.The whole genome sequencing analysis and functional annotation of the three strains showed that the three strains all have more active carbohydrate metabolism,amino acid metabolism and secondary metabolite synthesis abilities.The results of pathway annotation of carbohydrate metabolism and amino acid metabolism are consistent with the results of carbon source utilization.The strains can encode pathways such as glycolysis,TCA cycle,glucuronide mutual conversion,serine metabolism,proline metabolism,and phosphoinositide metabolism.In addition,all three strains can encode a synthetic PTS transport system to regulate carbon and nitrogen balance.In summary,different nitrogen concentrations can regulate the growth of rice root nitrogen-fixing bacteria,phosphorus solubilization,and the secretion of plant hormones and organic acids.