Effects of Nitrogen Addition on Rhizosphere Soil Microbial Community and Yield of Wheat in Loess Plateau

The soil microbial community diversity of wheat rhizosphere was affected by the amount of nitrogen fertilizer. In addition to bacterial community, ammonia-oxidizing archaea, nitrogen-fixing bacteria and denitrifying bacteria also play important roles in nitrogen cycle. At present, the microecological mechanism of its response to nitrogen application is still unclear. In this study, the rhizosphere soil microorganisms of winter wheat were used as the research object. The changes of soil bacteria, ammonia-oxidizing archaea, nitrogen-fixing bacteria and denitrifying bacteria communities under five nitrogen rates of 0—N(0), 90—N(6), 180—N(12), 240—N(16), 300—N(20) kg N ha–2 were studied by high-throughput sequencing technology. Under N(12) treatment, the Alpha diversity index of bacteria, AOA and nifH nitrogen-fixing bacteria and the Shannon and Simpson indices of nirK denitrifying bacteria were significantly increased. N(12) treatment significantly increased the relative abundance of Proteobacteria, but no significant difference was found at the nifH and nirK bacterial phyla levels. Under the high nitrogen treatment of N(16) and N(20), the dominant bacteria of nirK-type denitrifying bacteria increased significantly compared with N(12) treatment, there was no significant difference in microbial community distribution between N(20) and the control group. Therefore, the nitrogen addition under N(12) treatment was most conducive to the absorption and utilization of nitrogen fertilizer by soil microorganisms. The effect of nitrogen addition on microbial community was weaker than that of soil properties and wheat yield, and nitrogen addition was significantly correlated with yield, reaching the highest yield at 300 kg ha–2.


INTRODUCTION
Winter wheat is the main food crop in the Loess Plateau, the area of wheat accounts for nearly 50% of the total area [37].The Loess Plateau is a typical ecologically fragile area.Restricted by natural geographical conditions, soil erosion, drought and barren have become the main limiting factors for the breakthrough of wheat yield in the Loess Plateau [34].Nitrogen is one of the essential elements to maintain crop growth and development.As the most widely used fertilizer in agricultural production, nitrogen fertilizer plays an important role in improving soil nutrients and increasing crop yield.Its invention and application are considered as one of the greatest contributions of mankind in the 20th century [6].Nitrogen is an integral part of proteins, chlorophylls, enzymes, various coenzymes, certain plant hormones, and secondary metabolites [11].Therefore, there is a significant correlation between nitrogen fertilizer application and crop yield.However, with the increase of grain demand, the excessive application of nitrogen fertilizer in production also has certain impacts on Chinese agricultural development, such as the late maturity of plants, soil eutrophication and global climate change [14,23].It is a major research direction of current wheat cultivation and production to clarify the reasonable amount of nitrogen fertilizer, reduce fertilizer and nitrogen, improve nitrogen use efficiency, and ensure the stable yield and increase of wheat in the Loess Plateau while ensuring the sustainable and safe development of grain production.
Soil microbial community is the most widely distributed organism in soil ecosystem.The improvement of element cycle and physical and chemical properties of soil ecosystem driven by microbial [24] is one of the methods to improve the yield and quality of cultivated crops.Nitrogen is the main factor for maintaining metabolic activity of microorganisms [27].Similarly, functional microorganisms are also involved in soil nitrogen 1 These authors contributed equally to this work.

SOIL BIOLOGY
cycling.Among them, ammonia oxidizing bacteria and denitrifying bacteria, as the most critical rate-limiting substances in nitrification and denitrification processes, are involved in the key links of microbial nitrogen utilization and loss [1,22].Nitrogen-fixing bacteria are also involved in soil nitrogen transformation [38].Therefore, the application of nitrogen fertilizer causes the change of soil microbial community, which is one of the favorable ways to improve soil quality.
At present, there are many studies on the effects of nitrogen fertilizer on soil microbial community.For example, reasonable nitrogen fertilizer application improved the abundance and diversity of bacterial community in citrus soil and improve its community structure [29]; Noah Fierer et al. [7] proved that nitrogen application helped to build richer and more stable microbial communities.Lu et al. [17] showed that half application of nitrogen fertilizer could significantly improve the utilization of soil microbial community to multiple carbon sources, thereby increasing their community diversity.In summary, the improvement of microbial community structure and functional diversity is driven by nitrogen fertilizer, which is closely related to the addition of nitrogen fertilizer.However, there is no clear research to confirm it.
In this study, high-throughput sequencing technology was used to study the soil microbial community structure in winter wheat rhizosphere under different nitrogen additions in the Loess Plateau and clarify the response of microbial communities to nitrogen fertilizer application.At the same time, combined with the diversity of nitrogen-related functional microbial ammonia-oxidizing archaea, nitrogen-fixing bacteria and denitrifying bacteria communities, the most reasonable nitrogen application rate was sought without increasing nitrogen leaching and loss during nitrification and denitrification.

MATERIALS AND METHODS
Experimental design.The experiment was conducted in Shangyuan Village, Hougong Township, Wenxi County, Shanxi Province (34°35′ N, 110°15′ E), from 2016 to 2020, and sampled in 2020.The region is located in the semi-arid region of the southeast Loess Plateau, with a warm temperate continental monsoon climate.The annual average temperature is 8-14°C, the annual average precipitation is 506 mm, the annual average sunshine hours are about 2242.0 h, and the frost-free period is 190d.The soil type is Cinnamon soil (Carbonatic; Calcifractic).
Liangxing 99 was selected as the tested wheat variety.The tested fertilizer was urea (N 46%).Five nitrogen additions were set as 0-N(0), 90-N(6), 180-N (12), 240-N( 16), 300-N(20) kg N ha -2 , respectively.A total of 15 plots were repeated three times, and the plot area was 50 m 2 , which was completely randomized into groups.1m guard rows were set between every two plots.The application rates of phosphorus and potassium in all plots were 150 kg P 2 O 5 ha -2 and 150 kg K 2 O ha -2 .Wheat was sowed on Oct. 10, 2019, and harvested on Jun. 7, 2020.
Soil sampling.Soil samples were collected by sterilized soil drill in wheat mature period (June 7, 2020).Three sampling points were randomly selected and mixed evenly in each plot.The soil samples were stored in a refrigerator and transported back to the laboratory.Some of them were stored in dry air for the determination of physical and chemical properties, and others were stored in a refrigerator at -80°C for the determination of microbial community structure.
DNA extraction and Illumina-based sequencing.Fast DNA SPIN Extraction Kits kit (MP Biomedicals, USA) was used for soil total DNA extraction according to the steps of DNA extraction kit.The purity and integrity of extracted DNA were detected by agarose gel electrophoresis, and the concentration and purity of extracted DNA were detected by Nanodrop NC-2000.
Sequences data analyses.Using QIIME software, the UCLUST sequence alignment tool (Edgar, 2010) was called to merge and divide the obtained sequences into OTUs according to 97% sequence similarity, and the highest abundance sequence in each OTU was selected as the representative sequence of the OTU.For each OTU representative sequence, the default parameters are used in QIIME software to obtain the corresponding taxonomic information of each OTU by comparing the OTU representative sequence with the template sequence of the corresponding database.
Statistical analysis.The data of physical and chemical properties of samples were processed by Microsoft Excel 2010, and the charts were drawn.Analysis of variance and correlation by SPSS 26.0 software.Cluster analysis and heat map of the top 50 genera and NMDS analysis of weighted UniFrac distance matrix were performed using R software.In addition, the generalized linear model (GLM) and quadratic regression model were used to reflect the response of microbial Alpha diversity and wheat yield to nitrogen addition.Structural equation modeling used Amos 23.0 software and data fitting used maximum likelihood estimation.

Effect of N Addition on diversity index of soil microbial community.
The high-throughput sequencing of bacteria, AOA, nifH and nirK in each soil sample were carried out, and a total of 503 870, 547229, 1120.571 and 1356357 sequences were obtained.The 97% sequence similarity was used as the OTU division threshold, and the number of OTUs of the four microorganisms was 6905-9763, 4488-7569, 7538-11196 and 6905-9763, respectively.The Alpha diversity index was used to reflect the richness and diversity of microbial communities.The larger the Chao1 or ACE index, the higher the community richness.The greater the Shannon or Simpson index, the higher the community diversity.Except for the nirK denitrifying bacteria, the Alpha diversity indexes of the other three microorganisms showed a trend of first increase and then decrease.The effect of N addition on Alpha diversity index of bacteria, ammonia-oxidizing archaea and nitrogen-fixing bacteria was the highest under N(12) treatment.Under N(20) treatment, the Shannon index and Simpson index of denitrifying bacteria were the highest, and Chao1 and ACE indexes were the same as those of other microorganisms on nitrogen gradient (Fig. 1).
According to NMDS analysis, N(6), N (12) and N( 16) samples were far away from N0, indicating that the species composition of V3~V4 region of bacteria was changed under these three treatments, while the change was not obvious under N(20) treatment (Fig. S1a); N (12) was far from the control N(0) which significantly changed the species composition of ammonia-oxidizing archaea, while N (16) and N(20) were closer to N(0) (Fig. S1b).In the nitrogen-fixing bacteria communities, the effect of N (20) and N(6) treatment was not obvious compared with N(0), and the distance among those three was close, while the species composition of N( 16) and N (12) were significantly differed from the control (Fig. S1c).N(0), N(6), N (16) and N(20) treatments had similar community structure of denitrifying bacteria, but they were significantly different from those of N( 12) treatment (Fig. S1d).
Soil microbial community structure responses to N fertilization.In the bacterial community composition, the five dominant phyla under different nitrogen additions were Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexiand Gemmatimonadetes.In each treatment, the abundance of the first 10 bacterial categories accounted for 97.3-98.9% of the total sequence.There were significant differences in the relative abundances of Proteobacteria, Actinobacteria, Acidobacteria and Chloroflexi with Bacteroidetes at all nitrogen levels (P < 0.05).The relative abundances of Bacteroidetes were significantly increased by N (12) and N( 16) treatments.The relative abundances of Actinobacteria, Acidobacteria and Chloroflexi were significantly higher than those of other treatments under N(0) treatment.The relative abundances of Proteobacteria, Nitrospira and Rokubacteria were significantly increased by N(12) treatment.Among them, the relative abundance of Actinobacteria was large under N(16) treatment, while that of Proteobacteria was large under N(0) treatment (Fig. 2a).
At the phylum level, the dominant phylum of nifHtype nitrogen-fixing bacteria was Proteobacteria, followed by Unidentified and Verrucomicrobia.Under different nitrogen treatments, N(0) significantly increased the community abundance of Proteobacteria compared with other treatments, but there was no significant difference between them.N (16) and N (20) treatments significantly decreased the community abundance of Verrucomicrobia (Fig. 2c).
In the community composition of nirK-type denitrifying bacteria, Proteobacteria was the dominant phylum under different nitrogen treatments, followed by Actinobacteria, a total of 8 groups.The abundance of Proteobacteria community in N(12) treatment was significantly higher than that in N(0) treatment, but there was no significant difference between N(12) and other treatments (Fig. 2d).
The relative abundance of soil bacterial genera varied greatly under different nitrogen rates.The abundance of Kribbella, Cellulomonas, Nocardioides and Streptomyces under N(0) was significantly lower than that under other treatments.N(12) treatment significantly increased the relative abundance of MND1, Subgroup_10, Gaiella, Nitrospira and Polycyclovorans.The abundance of Pseudomonas, Mycobacterium, Agromyces, Arthrobacter and Kribbella under N(16) treatment was significantly higher than that under other treatments.According to the results of cluster analysis, the similarity of community structure between N(12) and N( 16) was high (Fig. 3a).At the bacterial genus level, the inter-group difference analysis based on the classification hierarchy tree showed that a total of 16 bacterial genera such as Pseudomonas, Mycobacterium and Streptomyces had significant changes in N( 16) treatment (Fig. S2a).

Candidatus Nitrosocosmicus, Nitrososphaera and
Candidatus Nitrosotalea were detected under all nitrogen treatments.Compared with the control, N (12) treatment significantly reduced the relative abundance of Nitrososphaera, while Candidatus Nitrosocosmicus significantly increased the relative abundance.Under N(20) treatment, the relative abundance of Candidatus Nitrosotalea was significantly increased; there was no significant difference in bacterial abundance between the other treatments (Fig. 3b).With regard to ammonia-oxidizing archaea, only Nitrososphaera under N(16) treatment was found to have distinct taxa at genus level (Fig. S2b).
The nifH-type nitrogen-fixing bacteria had similar community structure at the genus level under N(0) and N(20) treatments.The abundance of Ideonella, Azohydromonas, Dechloromonas and Rubrivivax was higher under N(0) treatment, while the relative abundance of Klebsiella, Yangia and Skermanella were higher under N(20) treatment.Paraburkholderia, Rhodobacter and Paenibacillus had higher relative abundance under N( 16) treatment (Fig. 3c).At the genus level of nifH-type nitrogen-fixing bacteria, it was also shown that distinct taxa were mainly found in N( 16) treatment (Fig. S2c).
The relative abundance of the main genera of soil nirK-type denitrifying bacteria was significantly different under different N additions.The relative abundance of Ensifer, Burkholderia and Roseitalea under N(12) treatment was significantly higher than that under other treatments, and the community similarity under N (12) and control treatments N(0) was rela-  tively high (Fig. 3d).Through the analysis of the classification tree, it was found that four nirK-type denitrifying bacteria, Ralstonia, Burkholderia, Ottowia and Massilia, mainly changed in N(12) treatment.In N(20) treatment, only Devosia showed significant differences between groups at genus level (Fig. S2d).2), nitrogen application significantly reduced soil pH, ranging from 7.92 to 8.00.Soil total nitrogen and total carbon showed a trend of increasing first and   then decreasing with the increase of nitrogen fertilizer additions.Under N( 16) treatment, soil total carbon content reached 22.63 g kg -1 , which was significantly higher than N(0) and N (20).The soil bulk density decreased with the increase of nitrogen fertilizer, and the activities of urease, phosphatase and sucrase of N (12) and N( 16) were higher than other nitrogen additions.

Response of soil physical-chemical-biological properties to different nitrogen additions. By analyzing soil physiochemical properties of different treatments (Table
Wheat yield and its components.The application of nitrogen fertilizer had a significant effect on wheat yield, which showed a gradual upward trend with the increase of nitrogen additions, and reached the highest yield of 8610 kg ha -2 when the nitrogen addition was 300 kg ha -2 (Fig. S3) In order to analyze the changes of yield components in the process of wheat yield growth, the linear regression analysis of spike number, grain number per spike and 1000-grain weight with yield was carried out.The results showed that with the increase of yield level, spike number, grain number per spike and 1000-grain weight showed an increasing trend.Among them, the correlation between spike number and yield reached extremely significant level (P < 0.01), while the correlation between 1000-grain weight and yield was not significant (Fig. 4).
Response of soil properties, microbial community and yield to nitrogen addition.SEM model was constructed to evaluate the direct or indirect effects of nitrogen addition on soil properties, microbial diversity and wheat yield (Fig. 5).The model showed that nitrogen addition had significant direct effects on wheat yield and soil properties (P ≤ 0.001 ), and the λ values were 0.72 and 0.70, respectively.The effect on microbial diversity was not significant, λ = 0.27.Nitrogen addition increased spike number (λ = 1, R 2 = 0.99) and Urease (λ = 0.95, R 2 = 0.90) to improve yield and soil properties.The indirect effect of microbial diversity on crop yield through nitrogen addition was not significant (P > 0.05).Similarly, the indirect effect of soil traits on microbial diversity through nitrogen addition was not significant (P > 0.05).The effect of bacteria (λ = 0.96, R 2 = 0.93) was greater than that of nifH (λ = 0.42, R 2 = 0.19), followed by nirK (λ = 0.3, R 2 = 0.09) and AOA (λ = −0.18,R 2 = 0.03).

DISCUSSION
Soil microbial community structure under different nitrogen additions.Soil microbial community determines soil fertility to a certain extent [35].In the soil ecosystem, microorganisms mainly act as decomposers to decompose organic substances such as plant residues to complete the nutrients circulation and the energy flow of the food web.However, Kathleen et al. [13] showed that the competition between microorganisms and plants for the same resources led to a certain nutrient limitation effect.The reason for the above two results is mainly due to the change of microbial community structure caused by the difference of dominant bacteria in different ecosystems.For example, the products of organic matter decomposition will inhibit the growth of bacterial Actinomycetes, Therefore, studies by Mei et al. [20] on 40 years of organic fertilizer input showed that fertilizer application significantly reduced the relative abundance of actinomycetes.In this study, the abundance of Actinobacteria in N(0) treatment was significantly higher than that in other treatments, which showed a similar effect to the above conclusion.Ammonia oxidizing archaea was the main driving force of ammonia oxidation process.Xu et al. [33] found that AOA community was significantly associated with soil potential nitrification ( PNA ), NH 4 -N and NO 3 -N.Liu et al. [16] also confirmed that AOA was sensitive to nitrogen fertilizer input and showed a downward trend, which was consistent with the trend under N(0), N(6) and N (12) treatments in this study.Under the condition of excessive nitrogen fertilizer application, due to the increase of nitrogen input, the abundance of dominant bacteria was significantly increased and higher than that of N(0) treatment, which may be due to the change of ammonia oxidation process caused by excessive nitrogen input in soil.In this experiment, Thaumarchaeota was the dominant phylum of ammonia-oxidizing archaea, and its abundance decreased first and then increased with the increase of nitrogen fertilizer addition, so Thaumarchaeota may have good tolerance to high nitrogen environment.Due to the presence of specific genes in DNA fragments, Thaumarchaeota is involved in the first step of soil nitrification, decomposing ammonia in soil into nitric acid.However, due to the influence of environmental conditions, a large amount of nitro-Fig.5. Structural equation model (SEM) of effects of nitrogen addition on soil properties, microbial diversity and wheat yield.The microbial diversity was reflected by the Shannon diversity index of bacteria, AOA, nifH nitrogen-fixing bacteria and nirK denitrifying bacteria.The number (λ) above the arrow represents the path coefficient, the solid line is positive, and the dashed line is negative.R 2 represents the variance ratio of the latent variable and the observed variable, and the significance is implied by P < 0.05.gen fertilizer application caused the accumulation of ammonia in the soil, affecting the soil pH and other factors, which led to the abundance of Thaumarchaeota under high nitrogen conditions.Both nifH and nirK showed changes in community structure stability under high nitrogen treatment.Studies have showed that the nifH community structure changed due to the difference of dominant bacteria under different pH conditions [36].In this study, with the increase of nitrogen fertilizer application, soil pH decreased gradually, and the number of dominant genera of nifH increased first and then decreased.nirK is involved in the denitrification process in soil [30].According to the abundance changes of dominant bacteria and genera with significant differences, the relative abundance of nirK decreased significantly under the treatment of too high and too low nitrogen fertilizer input.This process exacerbated the loss of nitrogen in farmland, which may be due to changes in the abundance of nirK dominant phylum.
Soil microbial community diversity.Microbial community diversity is an important indicator of soil microbial ecological function.Fertilization is one of the main factors affecting microbial community diversity [3], the difference in nitrogen fertilizer application rate will significantly affect the diversity of nitrogen cycle-related microbial communities in soil.In this study, with the increase of nitrogen addition, the bacterial richness Chao1 and ACE index showed a trend of increasing first and then decreasing, while the diversity Shannon and Simpson index showed little change, only nifH nitrogen-fixing bacteria showed a downward trend, which was different from the study of Castellano-Hinojosa [2].They showed that increased nitrogen application reduced rhizosphere diversity of tomato and kidney bean.At present, the adverse effects of inorganic nitrogen fertilizer application on soil microbial diversity have been confirmed [39], but the specific results are still different depending on the amount of nitrogen fertilizer applied.Although the application of excessive nitrogen fertilizer in this study did not significantly reduce the two diversity indexes of microorganisms, its richness index showed a significant downward trend.Reasonable addition of nitrogen fertilizer can improve the diversity and activity of soil microbial community, and under the condition of reducing nitrogen fertilizer, nitrogen-related functional microbial community is affected to some extent [15].The results of this study showed that the response of the diversity of AOA and nifH nitrogen-fixing bacteria to nitrogen gradient was roughly consistent with that of bacteria.Denitrification process involved by nirK Denitrifying Bacteria is the cause of soil nitrogen loss [4], With the increase of nitrogen fertilizer application amount, the diversity of denitrifying bacteria community had an obvious increasing trend.Therefore, the addition of excessive nitrogen fertilizer would enhance the denitrification process of soil.
Response of nitrogen related functional microorganisms to nitrogen addition.Soil nitrogen cycle is closely related to AOA, nifH nitrogen-fixing bacteria and nirK denitrifying bacteria community [26].Under different nitrogen fertilizer input, the change of microbial community structure and function caused by the difference of soil nitrogen nutrients affected the absorption and utilization of nitrogen in soil.At the phylum and genus level, the abundance of dominant bacteria in ammonia-oxidizing bacteria communities with high nitrogen fertilizer input was higher than that with low nitrogen fertilizer input, and there were more species with significant differences in N( 16) treatment.This result was consistent with the research result of Huang et al. [8], that is, the effect of earthworms on ammonia oxidation process is closely related to the abundance of related bacteria in soil.In addition, under the background of this study, Nitrososphaera, as the main driver of nitrification in soil, showed higher abundance under N( 16) treatment, indicating that it was more conducive to the absorption and utilization of nitrogen in soil under 240 kg N ha -2 input conditions.Kumar [12] showed that the abundance and diversity of nitrogen-fixing bacteria community can be used as an important reference index to reduce nitrogen fertilizer input.Some studies have shown that soil organic carbon and available potassium are the main factors affecting the change of nitrogen-fixing bacteria community structure [32].In this study, under N( 16) treatment, the number and abundance of significantly different species of nifH-type nitrogen-fixing bacteria were better than those under other treatments, and the community structure was significantly changed.The process of soil nitrogen fixation was promoted by multiple factors, under N(12) and N(16) treatment, soil urease activity was significantly higher than that of N(0), N(6) and N(20) treatments, and its content was one of the main sources of nitrogen absorption by plants.Similar to organic carbon and other nutrients, urease also affects the nitrogen fixation process in soil by affecting the community structure of nitrogen-fixing bacteria.
Denitrification is the process of reducing nitrates and nitrites into nitrogen and returning it to the air [21], Ji et al. [9] on the effect of nitrogen fertilizer application on denitrification process concluded that high nitrogen fertilizer input often has high N 2 O emissions, this process may be related to the change of soil denitrifying bacteria community structure.N (16) and N (20) treatments significantly increased the abundance of denitrifying bacteria community, while the community diversity index and community composition under N(12) treatment were higher than those under other treatments.Therefore, excessive nitrogen application mainly enhanced denitrification process by increasing the number of dominant denitrifying bacteria and reducing community diversity.Vol.56 No. 11 2023 FAN et al.
In summary, soil microbial community structure was significantly affected by nitrogen addition in this study.Except for denitrifying bacteria, the other two microbial communities performed best under N (12) and N(16) treatments, but did not perform well under high nitrogen input.This is mainly because with the increase of nitrogen input, nutrient accumulation reduces the diversity of its community.Under N(20) high nitrogen treatment, the leaching loss of nitrogen increased, and the enhancement of denitrification was not conducive to the stability of microbial community.
Effect of nitrogen fertilizer on wheat yield.Nitrogen fertilizer is a necessary element for crop growth and development, and reasonable nitrogen fertilizer input is the condition for wheat to obtain high yield [25].At present, wheat production is facing continuous deficit, high nitrogen fertilizer input leads to serious low productivity of wheat [5].The fertilizer requirement of wheat is regulated by many factors, such as wheat type, precipitation, tillage and so on [10,28].The more rainfall, the lower the average annual temperature, the greater the nitrogen requirement of wheat [19].In this study, the nitrogen rate of 300 kg ha -2 wheat reached the highest yield in all treatments.Previous studies showed that the application of nitrogen fertilizer significantly increased the spike number of wheat, while the 1000-grain weight showed a downward trend [18,31].The results are consistent with our research.The increase of wheat spike number caused by nitrogen application is an important reason for high yield of wheat.In the future, under the premise of controlling nitrogen fertilizer input and improving nitrogen use efficiency, as far as possible to improve 1000grain weight of wheat will become the focus of our research.
In this study, with the increase of fertilization level, wheat yield increased gradually.However, under the two treatments of N (16) and N(20), the yield increase was significantly lower than that of N(6) and N (12).Therefore, combining the two factors of microbial community and crop yield, the optimum fertilization level is 180 kgha -2 to ensure the high yield of wheat under the premise of stable microbial community.

CONCLUSIONS
Nitrogen application significantly changed the microbial community structure in soil, and the richness of bacteria, archaea ammonia oxidation, nifHtype nitrogen-fixing bacteria and nirK-type nitrogenfixing bacteria were the highest under N(12) treatment, indicating that the community abundance was relatively high under this treatment.The diversity of nirK denitrifying bacteria under N(20) treatment were highest.
N (12) treatment significantly increased the relative abundance of Proteobacteria, Bacteroidetes, Nitro-spira and Rokubacteria, but no significant difference was found at the nifH and nirK bacterial phyla levels.N (12) treatment significantly improved the community diversity and structural stability of ammonia oxidizing bacteria and nifH nitrogen-fixing bacteria, which was beneficial to the recycling of soil nitrogen.Under high nitrogen treatment of N (16) and N (20), the dominant bacteria of nirK-type denitrifying bacteria increased significantly compared with N(12) treatment.
The effect of nitrogen addition on soil properties and wheat yield was greater than that on microbial diversity.In order to maintain the stability of microbial community structure under the premise of high yield of wheat, N(12) treatment performed best.

Fig. 3 .
Fig. 3. Bacteria (a), ammonia-oxidizing archaea (b), nitrogen-fixing bacteria (c) and denitrifying bacteria (d) of the genus level community composition heat map, the left clustering tree shows the similarity between the various treatments.Red represents the genus with high abundance in the corresponding samples, and green represents the genus with low abundance.N(0), N(6), N(12), N(16) and N(20) were 0, 90, 180, 240 and 300 kg N ha -2 , respectively.

Fig. 4 .
Fig.4.Linear analysis of wheat yield and its components, where (a), (b), (c) denote the linear fitting of wheat spike number, grain number per spike and 1000-grain weight with yield, respectively, R 2 is the determination coefficient to measure the fitting degree of regression equation.* expressed at P < 0.05 level, ** expressed at P < 0.01 level.
o microbial diversity P = 0.433 Microbial diversity o Bacteria P < 0.001 Microbial diversity o AOA P = 0.547 Microbial diversity o nifH P = 0.239 Microbial diversity o nirK P = 0.343 Soil properties o BD P = 0.003 Soil properties o TN P < 0.001 Soil properties o TC P = 0.047 Soil properties o pH P = 0.087 Soil properties o urease P < 0.001 Soil properties o phosphatase P = 0.053 Soil properties o invertase P = 0.013 N additions o yield P < 0.001 Spikes o yield P < 0.001 Grans per spike o yield P < 0.001 1000-grain weight o yield P < 0.001 N additions o soil properties P = 0

Table 1 .
The PCR primers and amplification sequences of bacteria and three functional microorganisms

Table 2 .
The soil physical-chemical-biological properties TN refers to soil total nitrogen, TC refers to soil total carbon, BD refers to soil bulk density.Different letters in the same column indicate significant differences at P < 0.05.