Influence Of Long Term Nitrogen Fertilization On Microorganisms And Major N-cycling Related Communities In Black Soil In Northeast China

Nitrogen nutrient is one of the main factors influencing soil microbial organisms,soil properties and crop yields.Long term application of nitrogen fertilizer will significantly affect not only soil microbial community structure and their functions,but also the soil production capacity.Based on long term fertilization experiment in black soil in northeast China,we applied quantitative PCR,454-pyrosequencing and Illumina Miseq platform to study the influence of 34 and 35 years?the cultivation of wheat and soybean,respectively?nitorgen fertilization on soil properties and nitrogen agronomic efficiency.On this basis,we would like to explore changes of soil microbial community and major N-cycling related communities?ammonia oxidizing organisim and nitrogen fixing bacteria?.The results will provide a theoretical basis to reveal the ecological effects of long term application of nitrogen and soil plant microbial interactions.The main achievements are listed as follows:1.Soil acidification was obvious after 34 and 35 years application of nitrogen fertilizer,with a lowest pH value of 4.6 observed in high nitrogen addition treatment?N₂?.Compared to the control group,agronomic efficiency of nitrogen in high nitrogen input treatments decreased by 65.6%and 41.2%,respectively...The concentration of nitrogen nutrients was increased,including ammonium,nitrate and total nitrogen.2.After 34 and 35 years of continuous application of nitrogen fertilizer,bacterial abundance decreased and fungal abundance increased,which led to the decrease of the ratio of bacterial/fungal abundance,ranging from 2.1�105 to 4.6�104 and 8.4�104 to 3.8�104 in wheat and soybean seasons,respectively.Long term application of nitrogen fertilizer resulted in the change of black soil from"bacterial type" to "fungal type",and the a-diversity of the bacterial and fungal communities in black soil significantly decreased in four fertilization treatments.3.Long term application of nitrogen fertilizer increased the relative abundances of actinomycetes and proteobacteria,while decreased the acidobacteria,verrucomicrobia and nitrospirae abundances.Nitrogen fertilizer increased the relative abundances of sordariomycetes and Eurotiomycetes,decreased dothideomycetes abundances.The bacterial and fungal community structures in each fertilizer treatment with the same urea inputs were similar with each other,while those in fertilizer treatments with different nitrogen inputs was significantly different with each other?P<0.05?.The soil properties and crop seasons explained 25.5%and 12.2%of total bacterial variance,respectively.And the two respectively explained 29.3%and 23.9%of total fungal variance.pH and NO₃^-concentration played important roles in shaping bacerial and fungal community structures.4.Ammonia oxidizing organism?AOA and AOB?was significantly affected by nitrogen fertilizer level.With the increase of nitrogen level,AOA Arch-amoA copy number decreased from 2.6�107 to 8.3�105/g soil,while AOB Bacteria-amoA copy number increased from 1.3�104 to 1.1�105g soil.The AOA and AOB community structures in each fertilizer treatment with the same urea inputs were similar with each other,while those in fertilizer treatments with different nitrogen addition were significantly different with each other.The AOA in black soil mainly belongs to Nitrososphaera and Nitrosotalea,and AOB belongs to Nitrosospira and Nitrosomonas.5.The abundance and community composition of nitrogen-fixing bacteria were significantly affected by nitrogen fertilizer levels and crops seasons.nifH copy number in soybean season was higher than those in wheat season.In both seasons,four fertilizer treatment significantly decreased the nifH gene abundance.In wheat season,fertilization significantly affected nitrogen-fixing bacteria OTU number and diversity,while had no influence in soybean season.The dominant nitrogen fixing bacteria was Bradyrhizobium in black soil,with a proportion of 53.9?95.8%in wheat season,and 87.8?97.9%in soybean season,respectively.The concentration of NO₃^--N,NH₄⁺-N,available phosphorus and soil pH were significantly correlated with the nitrogen-fixing bacteria community structure?P<0.05?.6.Culturable bacteria and fungi were isolated under long term nitrogen fertilizer regimes from black soil,and indentified by the 16S rRNA and ITS gene,respectively.With the increase of nitrogen level,the number of culturable fungi increased significantly,while the number of culturable bacteria did not changed.The dominant groups in different nitrogen levels were also different.The above results showed that long-term nitrogen fertilization reduced the a diversity of bacteria and fungi in black soil in Northeast China,and changed the black soil from "bacterial type" to "fungal type".The community structure changes of bacteria,fungi,AOA,AOB and nitrogen-fixing bacteria in black soil were related to nitrogen fertilizer levels.The results indicating that long-term nitrogen application had a significant effect on the microbial community of black soil in northeast China.