| Alfalfa is a high quality forage resource and is widely grown in China.In the process of soil nutrient transformation,the effects of different forms of nitrogen on the microbial community in the rhizosphere soil of alfalfa is still unclear.This study set up two experiments including pot planting and plot cultivation to study the effects of adding different forms of nitrogen on the community structure and diversity of alfalfa rhizosphere soil microorganisms.Pot experiment was set up with six treatments,Con(control),NN(add nitrate-nitrogen alone),AN(add ammonium-nitrogen alone),NN-AN(add ammonium-nitrate with the ratio of 1:1),3NN-7AN(add ammonium-nitrate with the ratio of 3:7)and 7NN-3AN(add ammonium-nitrate with the ratio of 7:3).Plot cultivation experiment was set up with four treatments:Con(control),NN(add nitrate-nitrogen alone),AN(add ammonium-nitrogen alone)and NN-AN(add ammonium-nitrate with the ratio of 1:1).The main results were as follows:(1)In the pot experiment,high throughput sequencing revealed that the mean number of OTUs in the mixed forms of nitrogen treatment was higher than that in the single forms of nitrogen treatment,but the difference was not significant(P>0.05).At the phylum level,the soil microbial communities were mainly distributed among Proteobacteria,Actinobacteria,Bacteroidetes and Acidobacteria.The relative abundance of Actinobacteria under the mixed nitrogen treatment was significantly higher than that under the single nitrogen treatment(P<0.05),and the relative abundance of Bacteroidetes under the mixed nitrogen treatment was significantly lower than that under the single nitrogen treatment(P<0.05).(2)The ?-diversity analysis of the soil microbial community in the pot experiment showed that the application of mixed forms of nitrogen increased the diversity of the soil microbial community more than the application of nitrogen alone,but the difference was not significant.In contrast,? diversity analysis of the soil microbial community showed a significant difference(P<0.05)between the treatment with mixed forms of nitrogen and the treatment with nitrogen alone.LefSe analysis showed that Actinobacteria were the dominant group of soil microbial communities in the 7NN-3 AN treatment was the dominant group in the AN treatment and Cytophagales was the dominant group in the NN treatment.(3)RDA was used to analyse the relationship between the dominant soil microbial communities and the physiological indicators and environmental factors of alfalfa in the pot experiment.The results showed that the nitrate-nitrogen content of alfalfa roots,soil amylase and soil available potassium were important factors affecting the structure and diversity of its rhizosphere soil microbial community.They more effectively promoted nutrient uptake and utilisation by plants and soil under the ammonium nitrate iso-ratio mix treatment,which indirectly influenced nitrogen uptake and utilisation by alfalfa by altering the competition between plants and microbes for soil nutrients through changes in microbial abundance,increasing plant nitrogen content,above-ground biomass and crude protein content.(4)A total of three stubbles of alfalfa were mown in the plot cultivation experiment.The results showed that OTU numbers were the highest in all stubbles under the NN-AN treatment.The dominant microbial communities were more abundant under each treatment in the plot cultivation compared to the pot experiment.At the phylum level,the soil microbial communities were mainly distributed among Proteobacteria,Actinobacteria,Firmicutes,Bacteroidetes,Acidobacteria,Bacillariophyta,Gemmatimonadetes and Chloroflexi.The relative abundance of Proteobacteria was significantly lower than that of other treatments in the first stubble(P<0.05),and the relative abundance of the second and third stubble was also lower than that of other treatments,but the difference was not significant.The relative abundance of Firmicutes and Bacteroidetes were significantly higher than those of other treatments under the mixed nitrogen treatment in the first and second stubbles,respecitively(P<0.05),but not in the third stubble.The relative abundance of Lysobacter which belong to the was the highest in the second stubble.(5)To analysis the a diversity of the soil microbial community in the plot cultivation experiment,the results showed that the abundance and diversity of soil microorganisms under the NN treatment in the third stubble were significantly higher(P<0.05)than those under the NN treatment and AN treatment in the first stubble.However,there are no significant differences among different treatments in the same stubbles.Moreover,the ? diversity of soil microbial community also had no significant differences among different treatments in the same stubbles.(6)RDA analysis of the dominant soil microbial community and alfalfa growth and physiological indicators as well as environmental factors in the plot cultivation experiments showed that the nitrate nitrogen content of the first alfalfa stubble was closely related to the soil microbial community structure,while the ammonium nitrogen content of the second and third alfalfa stubbles was closely related to the soil microbial community structure,indicating that the nitrate nitrogen content of the soil was gradually enriched with time going by.Under conditions of nitrogen sufficiency,the demand for whole soil nitrogen by plant inter-root soil microorganisms gradually changed to demand for soil available nutrients over time.This previous study found that nitrogen utilisation,above-ground biomass and crude protein content of alfalfa were all higher under the treatment of NN-AN than the treatments of NN?AN and Con.The relative abundance of dominant bacterial groups played an important role in promoting nitrogen uptake and utilization in alfalfa.In summary,the application of different forms of nitrogen indirectly changed the structure and diversity of the inter-root soil microbial community by altering soil physicochemical properties as well as soil enzyme activity,which in turn affected the uptake and utilisation of nitrogen by alfalfa and altered the nitrogen content of the plant.The plot cultivation experiment had a richer soil microbial taxon than the soil pot experiment,suggesting that in an open soil ecosystem,more microbial taxa would be involved in the nutrient transformation process.Both experiments showed that the application of mixed forms of nitrogen,particularly a 1:1 mixture of ammonium nitrate,increased the relative abundance and diversity of the soil microbial community. |
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