Ammonium and Nitrate Shift the Spatial Distribution of Soil Bacterial Communities and Association Networks Along a Distance from Maize Roots in an Acidic Red Soil

Background: Ammonium (NH 4+ ) and nitrate (NO 3− ) are two major inorganic nitrogen (N) forms available for plant growth. Soil microbes affect the availability and transformation of these N forms in the rhizosphere, and this affects the N-use eciency of plants. However, little is known about the responses of the rhizosphere bacterial community structure to NH 4+ and NO 3− . Here, a rhizobox containing a root zone (root growing area) and various soil compartments (0–0.5 cm, 0.5–1 cm, 1–2 cm, 2–4 cm, and 4–9 cm from the root zone) was designed to investigate the spatial distribution of bacterial diversity, community structure, and co-occurrence patterns along a distance from maize (Zea mays L.) roots with the addition of 15 N-labeled NH 4+ or NO 3− in an acidic red soil. Results: Addition of NH 4+ and NO 3− reduced soil bacterial diversity in the maize root zone. The structures of soil bacterial communities differed between NH 4+ and NO 3− in the root zone and 0.5 cm away from the root zone. Soil pH was the major driver of bacterial community assembly during plant uptake of N. Maize roots recruited potentially benecial acidophilic bacteria (e.g. Acidibacter, Burkholderia, and Catenulispora) under NH 4+ treatment, and recruited growth-promoting bacteria that prefer higher pH (e.g. Sphingomonas, Sphingobium, Azospirillum, and Novosphingobium) under NO 3− treatment. In the N-fertilization treatments, the soil bacterial networks were more complex in the root zone and its adjacent 0.5–1 cm zone than in other soil compartments. The soil bacterial networks were more complex under NH 4+ treatment than under NO 3− . More bacterial taxa in the networks responded positively and negatively to soil residual NH 4+ than to NO 3− in all zones in the rhizobox. Conclusions: The combined effects of the N form and the rhizosphere inuenced the spatial patterns and co-occurrence network of soil bacterial communities at different distances from the maize root zone, mainly because of changes in soil pH during the uptake of NH 4+ and NO 3− by maize roots. Regulating microbial communities by adjusting soil pH through NH 4+ and NO 3− supply may be an environmentally friendly option for promoting soil microbial functions in intensively managed agro-ecosystems.


Introduction
The rhizosphere is an important biological hotspot where dramatic nutrient processes involving microbes occur [1]. The chemical, physical, and biological properties of soil in the rhizosphere are fundamentally affected by the presence and activity of plant roots [2]. Soil moisture and nutrients form gradients from the root surface to bulk soil due to the uptake of water and nutrients by plant roots [3,4]. The release of protons and ions from roots into the surrounding soil changes the pH of soil within 2 to 5 mm of the roots, depending on the soil's buffering capacity and the amount of ions released [4]. These microhabitat properties vary along a horizontal distance from the roots, and are major drivers of microbial community assembly [5,6]. Thus, the horizontal variations in these microhabitat properties may lead to gradients of microbial diversity from the root surface to bulk soil. Moreover, plant roots can recuit speci c microbiota from surrouding soil by releasing root exudates [7,8], leading to differences in microbiomes among the endosphere (root interior), rhizoplane (root surface), and rhizosphere (soil close to the root surface) [9].
The strength of the rhizosphere effect depends on the plant genotype, plant developmental stage, root morphology, abiotic stresses, and soil properties [10]. Investigating the shifts in microbial communities along a lateral gradient from the root surface to bulk soil can provide insights into the function of plant rhizosphere microbiomes. Although microbial community structures and co-occurrence patterns are known to differ between root-adhered soil and bulk soil [11,12], less is known about continuous changes in microbial communities with increasing distance from plant roots.
Acidic soils (de ned as pH < 5.5) are present at more than 50% of the cultivated and cultivable land area worldwide [13]. The cultivation of crops in acidic soils is important for agricultural production and food security. However, agricultural productivity in acidic soils is limited by a combination of stress factors [14]. Nitrogen (N) is a major limiting essential nutrient for crop growth and productivity in acidic soils [15].
Large amounts of N fertilizers are applied to meet the growing food demands of the increasing population. However, the low N-use e ciency of crops means that a large proportion of applied N escapes into the environment, causing negative effects such as water pollution, greenhouse gas emissions, and further soil acidi cation [16,17]. In this context, there is an urgent need to improve the Nuse e ciency of crops in acidic soils.
Ammonium (NH 4 + ) and nitrate (NO 3 − ) are the two main inorganic N sources available for plant growth.
Their availability and transformation in the plant rhizosphere play important roles in improving N-use e ciency and are mainly regulated by soil microbes [18]. Thus, investigating the assemblies of microbial groups along a lateral gradient from the plant root surface to bulk soil after the addition of NH 4 + and NO 3 − is key to understanding how the functions of soil microbes improve N-use e ciency. The uptake of these two N forms by plants and their transformation in soils result in gradients of various environmental factors (such as nutrient concentrations and soil pH, porosity, and moisture) with increasing distance from the root surface [19]. Inputs of NH 4 + and NO 3 − may also lead to differences in plant growth and root morphology [20]. Thus, NH 4 + and NO 3 − may exert different effects on the assemblies of microbial groups along a gradient from the plant root surface to bulk soil because of the combination of changes in soil properties and rhizosphere effects. However, few studies have tried to quantify changes in microbial community composition and structure with increasing distance from roots of plants fertilized with NH 4 + and NO 3 − .
Among modern crops, maize (Zea mays, L.) has the highest production and the greatest demands for N nutrition [21]. However, the annual maize yield continues to decrease because of N-fertilizer-induced soil acidi cation in regions with acidic red soil in southern China [22,23]. Improving the N-use e ciency of maize can reduce the amount of N fertilizer required, thereby alleviating soil acidi cation. There is increasing interest in rhizosphere microbial communities because of their potential to improve maize growth and N-use e ciency. Previous studies have shown that microbial communities in the maize rhizosphere are affected by soil properties [24], plant genotype and age [25], and agricultural management methods [26]. For example, previous studies have shown that the addition of urea fertilizer leads to the enrichment of Bacillales, Nitrosomonadales, and Rhodocyclales in the maize rhizosphere because these orders are able to use urea as carbon or N sources [27], while Myxococcales and Burkholderiales are indicator orders in the maize rhizosphere under organic fertilization [26]. Although many studies have explored the interactive effect of agricultural management and plant selection on the microbial communities associated with maize roots [26], it is not enough to compare microbial communities only between rhizosphere soil and bulk soil under the same fertilization conditions. It is much more informative to investigate the continuous changes in soil microbial communities with increasing distance from maize roots under different N-fertilizer treatments.
In this study, we used a rhizobox system to investigate the gradients in soil chemical properties, bacterial communities, and co-occurrence patterns with increasing distance from maize roots in an acidic red soil supplied with 15

Rhizobox design and sampling
A rhizobox (length × width × depth, 19 × 12 × 18 cm) was used to divide the soil fractions at different distances from maize roots (Fig. 1a). The rhizobox was divided into 11 compartments, which were separated by nylon cloth with a pore radius of less than 28 µm. This nylon mesh prevented the penetration of the root system, but allowed water, nutrients, and root secretions to pass through. Seeds were planted in the central compartment, which was designated as the root zone. There were ve compartments on each side of the root zone, with widths of 0.5 cm, 0.5 cm, 1 cm, 2 cm, and 5 cm, respectively; these soil fractions were de ned as 0-0.5 cm, 0.5-1 cm, 1-2 cm, 2-4 cm and 4-9 cm soil compartments from the root zone (Fig. 1a).
The experiment consisted of a control (CK, no N fertilizer) and two treatments: (1)  dried plant materials were ground to pass a 0.149-mm sieve for determination of total N content and 15 N atom % enrichment. Soil samples were immediately collected after the maize roots were removed from the soil. Soil fractions in each compartment were sampled separately. Soils in each compartment were sampled using a stainless steel blade, as described by Wang et al. [28]. Two soil samples collected at the same distance from the root zone (root growing area) at the right and left were mixed well to produce a composite sample. The collected soil samples were divided into two parts. One part was frozen immediately and stored at − 20 °C until soil DNA exaction and soil NH 4 + -N and NO 3 − -N analyses. The other part was air-dried, then ground to pass through a 2-mm sieve for analyses of soil pH, available P, and available K content, and to pass a 0.149-mm sieve for determination of soil total N content and 15 N atom % enrichment.

Soil and plant analyses
Soil pH was measured using a pH meter (PB-21, Sartorius, Göttingen, Germany) in a soil:water solution

Processing of 16S rRNA gene data
The high-throughput sequencing data were analyzed according to Chen et al. [29]. Brie y, after sequencing, paired-end reads were processed using Trimmomatic software [30] to detect and remove ambiguous bases and sequences with an average quality score below 20. After assembling the preprocessed reads using FLASH software [31], sequences were further denoised using QIIME software (version 1.8.0) [32]. Reads with ambiguous sequences, homologous sequences, or chimera, and reads shorter than 200 bp were excluded from further analyses. After quality ltering, 1,527,237 high-quality sequences with 10,410 to 50,658 sequences per sample were obtained. The sequences were analyzed after resampling to the same depth (8328, 80% of minimum sequencing depth). Operational taxonomic units (OTUs) were generated using Vsearch software with a 97% similarity cut-off. The representative read for each OTU was selected using the QIIME package and annotated with reference to the Silva database (Version 123).

Data analysis
Two-way ANOVAs were carried out to evaluate the effect of the interaction between the N fertilizer form and distance from the root zone on soil properties and network topological features. One-way ANOVAs were performed to compare differences among soil compartments and N-fertilizer treatments. Duncan's test was used for multiple comparisons (p < 0.05). The Kruskal-Wallis test was used for pair-wise comparisons to detect signi cant differences in the relative abundance of soil bacteria between CK and NH 4 + , CK and NO 3 − , and the NH 4 + and NO 3 − treatments (p < 0.05). Pearson's correlation coe cients were used to test the relationship between soil chemical properties and the relative abundance of speci c genera. All the above analyses were performed using SPSS v.19 (SPSS Inc. Chicago, IL, USA) and R software v.3.6.1. The N-recovery e ciency and amount of soil N derived from 15 N-labeled fertilizer (fertilizer-N) were calculated according to Zhang et al. [33].
Bacterial alpha diversity was calculated as estimated community diversity based on Shannon's index using QIIME software v.1.8.0. Bray-Curtis distances were calculated using the vegan package in R software v.3.6.1, and this package was also used for non-metric multidimensional scaling (NMDS) based on Bray-Curtis distance matrices. These analyses illustrated the clustering of different samples and further indicated bacterial community structure. Signi cant differences in bacterial community structure were tested by Permutational Multivariate Analysis of Variance (PERMANOVA) using the "adonis" function in the vegan package in R software. Signi cant correlations between community structures and soil properties were tested using Mantel tests. A redundancy analysis (RDA) was performed using R software to detect relationships between community structures and soil properties.

Association network analyses of bacterial communities
The co-occurrence patterns of bacterial taxa were explored by network analysis. To reduce the complexity of the networks and to avoid bias due to the differences in OTU numbers, only the 500 most abundant OTUs in CK and the NH 4 + and NO 3 − treatments were included. Spearman's correlations between selected bacterial OTUs were calculated and the matrix was constructed using R software. A Spearman's correlation between two bacterial OTUs was considered statistically signi cant if the correlation coe cient (r) was > 0.6 and the p-value was < 0.05. The p-values were adjusted using the Benjamini and Hochberg false discovery rate (FDR) test. All the signi cant correlations identi ed from pair-wise comparisons of bacterial OTUs were visualized in a correlation network, where each node represents one OTU, and each edge stands for a signi cant correlation between nodes. The topological features of bacterial networks were calculated and assigned to each sample using the igraph package in R software.
The network complexity was calculated as the number of links per node according to Wagg et al. [34]. The networks were visualized using the Gephi platform.

Maize growth and distribution of 15 N-labeled fertilizer in plant-soil system
Maize shoot dry weight was higher in the NH 4 + and NO 3 − treatments than in CK, but did not differ signi cantly between the NH 4 + and NO 3 − treatments (Fig. 1b). The root dry weight did not differ signi cantly among treatments. The N-recovery e ciency did not differ signi cantly between the NH 4 + and NO 3 − treatments (Fig. 1c). The amount of soil residual fertilizer N showed a U-shaped pattern with increasing distance from roots in both the NH 4 + and NO 3 − treatments, and was higher in the NH 4 + treatment than in the NO 3 − treatments across the soil compartments from 0.5-cm to 4-cm from the root zone (Fig. 1d).

Soil chemical properties
Soil residual fertilizer N, soil pH, total N, NH 4 + -N, and NO 3 − -N contents were signi cantly affected by both the N form and the distance from the roots. Soil available K content was signi cantly affected by only distance from the roots and available P was signi cantly affected by only the N form (Table S1). The interaction between N form and distance from the roots (D × N) signi cantly affected soil residual fertilizer N, soil pH, NH 4 + -N, and NO 3 − -N, but did not affect total N, available K, and available P.
In the NO 3 − treatment, soil pH was the highest in the root zone and the 0-0.5 cm soil compartment, and then decreased to similar levels to that in CK with increasing distance from the roots (Fig. 2a). Soil pH was the lowest in the NH 4 + treatment in all compartments of the rhizobox. Under both N treatments, the variations in soil total N were similar to those in soil residual fertilizer N, and there was no signi cant difference in soil total N between the NH 4 + and NO 3 − treatments (Fig. 2b). The soil NH 4 + -N contents were very low in all compartments of the rhizobox in CK and the NO 3 − treatments, but increased dramatically with increasing distance from the roots in the NH 4 + treatment (Fig. 2c). Soil NO 3 − -N contents were similar between the root zone and 0-4 cm soil compartments in the NH 4 + and NO 3 − treatments, but were dramatically increased in the 4-9 cm compartment in the NO 3 − treatment (Fig. 2d). The soil NO 3 − -N contents were low in all compartments of the rhizobox in CK. In all treatments, the soil available K contents slightly decreased from the root zone to the 0-0.5 cm compartment and then increased with increasing distance from the roots (Fig. 2e). The soil available P contents were similar in all compartments in all treatments, and were generally higher in the NH 4 + treatment than in the NO 3 − treatment (Fig. 2f).

Bacterial alpha and beta diversity
Compared with CK, both the NH 4 + and NO 3 − treatments reduced the bacterial alpha diversity (Shannon's index) in root zone soil, but not in other soil compartments (Fig. 3a). With increasing distance from the roots, the bacterial alpha diversity tended to increase in the NH 4 + and NO 3 − treatments but not in CK.
These results suggested that bacterial alpha diversity was inhibited by the combination of N fertilization and a rhizosphere effect.
The Bray-Curtis distance is used to indicate differences in microbial community structure between two treatments. The Bray-Curtis distances between CK and NH 4 + , CK and NO 3 − , and NH 4 + and NO 3 − were signi cantly higher in the root zone and 0-0.5 cm compartment than in the other soil compartments (Fig. 3b). This indicated a more pronounced effect of the N form on the formation of bacterial community structures within 0.5 cm of the root zone. The Bray-Curtis distance between the root zone and other soil compartments increased with increasing distance from the root zone, and it was higher in the NH 4 + and NO 3 − treatments than in CK (Fig. 3c). The rhizosphere effect was detected within 1 cm and 0.5 cm of the root zone under NH 4 + and NO 3 − treatment, respectively (Table S2). There were no signi cant variations in bacterial communities among different soil compartments in CK, indicating that the rhizosphere effect on the soil bacterial community was stimulated by N fertilizer. Therefore, in the NMDS analysis, two distinct groups formed as a result of the N form and rhizosphere effect (Fig. 3d).
The bacterial community composition was also in uenced by the N form and rhizosphere effect (Fig. 3e).
Gemmatimonadales was the dominant bacterial order in all soil compartments in CK. Under NH 4 + treatment, Burkholderiales became the dominant bacterial order in the root zone, and its abundance decreased with increasing distance from the roots. Under NO 3 − treatment, Sphingomonadales became the dominant bacterial order in the root zone, and its abundance also decreased with increasing distance from the roots.

Relationships between bacterial community structures and environmental variables
The results of Mantel tests indicated that pH was the major driver of bacterial community assembly (Table S3). The RDA analysis revealed that the bacterial communities in the root zone and the 0-0.5 cm soil compartment were positively correlated with soil pH under NO 3 − treatment, and those in the 2-9 cm soil compartments were negatively correlated with soil pH under NH 4 + treatment (Fig. 4).
We identi ed the bacterial genera showing signi cant differences (Kruskal-Wallis test, p < 0.05) in abundance between CK and NH 4 + , CK and NO 3 − , and the NH 4 + and NO 3 − treatments (Fig. 5). Compared with CK, the NH 4 + treatment resulted in speci c enrichment of four genera, Asticcacaulis, Acidibacter, Granulicella and Sinomonas within 0.5 cm of the maize root zone (Fig. 5a). The abundance of Acidibacter was negatively correlated with soil pH. In the NO 3 − treatment, four genera were speci cally enriched within 0.5 cm of the maize root zone: Sphingomonas, Sphingobium, Pseudolabrys, and Azospirillum (Fig. 5b). The abundance of Sphingobium was positively correlated with soil pH. Comparing the NH 4 + and NO 3 − treatments, the speci cally enriched genera within 0.5 cm of the root zone under NH 4 + treatment were Burkholderia, Mucilaginibacter, Acidibacter, Leifsonia, Catenulispora, and Asticcacaulis, and their abundance was negatively correlated with soil pH (Fig. 5c). The speci cally enriched genera within 0.5 cm of the root zone under NO 3 − treatment were Sphingomonas, Sphingobium, Pseudolabrys, Azospirillum, and Novosphingobium, and their abundance was positively correlated with soil pH.

Bacterial co-occurrence patterns
Multiple network topological features consistently showed that bacterial co-occurrence pattern was greatly affected by the N form and distance from the roots ( Table 1 and Table S4). Compared with CK, the NH 4 + treatment resulted in higher values for edge density, complexity, average clustering coe cient (agvCC), and degree of centralization of bacterial association networks in all soil compartments, and the NO 3 − treatment resulted in lower values for all of these topological features (Table 1). This indicated that the NH 4 + treatment resulted in the formation of more complex bacterial association networks, regardless of the distance from the maize roots. Under NH 4 + treatment, the edge density, degree of centralization, average clustering coe cient, and complexity had similar values in the root zone, 0-0.5 cm, and 0.5-1 cm soil compartments, and the values of these topological features were lower in outer soil compartments (Table 1). Under NO 3 − treatment, the values of these topological features were similar between the root zone and its adjacent 0.5 cm soil compartment, and were lower in the compartments further from the roots. Under CK, the topological features showed no signi cant differences among the different soil compartments. This indicated that the bacterial association networks were more complex in the rhizosphere than in the bulk soil under both of the N-fertilization treatments, but not in CK.

Nitrogen fertilizer-correlated OTUs and their cooccurrence patterns
According to the results described above, we de ned the root zone and its adjacent 1-cm soil compartment as the maize rhizosphere in the NH 4 + treatment, and the root zone and its adjacent 0.5-cm soil compartment as the maize rhizosphere in the NO 3 − treatment. Four new bacterial networks were constructed for the NH 4 + rhizosphere, NH 4 + bulk soil, NO 3 − rhizosphere, and NO 3 − bulk soil (Fig. S1).
Compared with the NO 3 − treatment, the NH 4 + treatment resulted in bacterial networks with more associations in both the rhizosphere and bulk soil (Fig. S1). To further explore the potential links between bacterial networks and soil residual fertilizer N in maize rhizosphere and bulk soil, we identi ed the "soil residual fertilizer N-correlated microbes" showing strong (r > 0.8, p < 0.01) correlations with soil residual fertilizer N in the networks (Table S5, Fig. 6 Solirubrobacterales). In the bulk soil, soil N derived from NO 3 − fertilizer was positively correlated with one OTU (in Enterobacteriales) and negatively correlated with two OTUs (in Rhizobiales and Gaiellales) (Table  S5). These results indicated that N fertilizer, regardless of the N form, exerted more negative than positive effects on the growth of bacterial taxa. Bacterial networks in the maize rhizosphere were more sensitive than those in bulk soil, and were more sensitive to NH 4 + than to NO 3 − .
The "N fertilizer-correlated microbes" showed different patterns of co-occurrence (Fig. 6). The OTUs that were positively correlated with soil N derived from N fertilizers had more negative edges than positive edges in the bacterial networks in the maize rhizosphere and bulk soil. In contrast, the OTUs that were negatively correlated with soil N derived from N fertilizers had more positive edges than negative edges in the bacterial networks in maize rhizosphere and bulk soil ( Fig. 6a and 6c). In the NH 4 + treatment, the rhizosphere OTUs that were positively correlated with soil N derived from NH 4 + fertilizer showed 95 positive correlations with other microbes mainly in the Frankiales, Xanthomonadales, Burkholderiales, Micrococcales, and Rhodospirillales orders, and 130 negative correlations with other microbes mainly in the Gemmatimonadales and Myxococcales orders (Fig. 6a). In bulk soil in the NH 4 + treatment, the OTUs that were positively connected with soil N derived from NH 4 + fertilizer showed three positive correlations with OTUs (in the Burkholderiales, Rhizobiales and an unknown order), and three negative correlations with the OTUs (in the Sphingomonadales, Xanthomonadales, and an unknown order) in the bacterial network (Fig. 6b). In the NO 3 − treatment, the rhizosphere OTUs that were positively correlated with soil N derived from NO 3 − fertilizer showed seven negative correlations with other microbes in the Chlorobiales, Rhizobiales, Solirubrobacterales, Rhodospirillales, Gemmatimonadales, and Sphingobacteriales (Fig. 6c).
In the bulk soil in the NO 3 − treatments, the 123 OTUs that were positively correlated with soil N derived from NO 3 − fertilizer showed only one negative correlation (with an uncultured bacteria) in the network (Fig. 6d).

Nitrogen form and maize rhizosphere strongly affected soil chemical properties
Our study showed that both the N form and the rhizosphere effect in uenced the distribution patterns of various soil chemical properties with increasing distance from the roots. Compared with soil residual NH 4 + , the soil residual NO 3 − uctuated more widely across the whole rhizobox after maize seedlings were harvested. This was mainly because NO 3 − is more mobile than NH 4 + [20]. In addition, plants were able to attract NO 3 − from outer compartments to the root surface, resulting in a relative enrichment of NO 3 − fertilizer in the root zone. However, relative NO 3 − enrichment in the root zone was also observed in the NH 4 + treatment in our study. Considering that NH 4 + is generally immobile within soils and the NH 4 + diffusion rate is lower than the crop uptake rate [20], we speculated that a portion of the NH 4 + fertilizer was nitri ed into NO 3 − , and the NO 3 − derived from NH 4 + fertilizer moved from the outer compartments to the root zone.
The pH in the root zone was decreased by application of NH 4 + but increased by application of NO 3 − , compared with that in CK. The distance of signi cant pH change under NO 3 − treatment was up to 0.5 cm from the root zone. Across the whole rhizobox, the soil pH was lower in the NH 4 + treatment than in CK. In another study, NO 3 − -based fertilizer increased soil pH in the maize rhizosphere, while NH 4 + -based fertilizer decreased soil pH in both the rhizosphere and bulk soil [35]. In the present study, the variations in soil total N content were similar to those of fertilizer-N, indicating that variations in soil total N content mainly depended on fertilizer-N. Soil available K and NH 4 + -N contents decreased sharply in the proximity of the roots in the present study, probably because both K + and NH 4 + are poorly mobile in soil [36]. In the present study, soil available P content did not vary across the rhizobox. Previous reports have shown that the depletion zone for P is usually 2-3 mm from the roots [4], so the depletion of soil available P by maize seedlings may not have been detected in the present study.

Nitrogen form and maize rhizosphere in uenced soil bacterial community diversity and composition
Previous studies have shown that application of N fertilizers (including NH 4 NO 3 , urea, and NO 3 − ) can inhibit soil bacterial diversity [37][38][39]. This may be because an increased inorganic N content reduces the competitiveness of microbes that are less tolerant to high osmotic potential and/or those that are able to x N 2 [40]. Consistent with those reports, the Shannon's index values were lower in the NH 4 + and NO 3 − treatments than in CK. However, this inhibitory effect was detected only in the maize root zone, indicating a dual effect of N fertilizer and rhizosphere on soil microbes. While N fertilizer inputs alter microbial niches as discussed above, plant roots preferentially recruit bene cial members of the bacterial community to colonize the rhizosphere, thereby forming a rhizosphere that is more sensitive than bulk soil to changes in the environment [7][8][9]. We detected decreased bacterial alpha diversity in the proximity of roots in the N-fertilized treatments. This "rhizosphere effect" was not detected in CK, indicating that it was stimulated by N fertilization. Nitrogen affects the belowground distribution of plant photosynthates, and rhizodeposition is positively correlated with the N fertilization rate [41]. In the present study, the enhancement of maize shoot growth by N fertilization may have signi cantly affected the soil bacterial community structure and diversity.
Although NH 4 + and NO 3 − fertilizers had similar effects on bacterial alpha diversity, they had different effects on bacterial beta diversity (bacterial community structures and composition). These differences were more pronounced in the maize root zone and its adjacent 0.5-cm soil compartment than in the outer compartments. This nding further indicated that maize roots play an essential role in selecting speci c species that adapt to niche utilization [42]. The "rhizosphere effect" to shape root-associated bacterial communities varies with different N forms. Soil pH is the major driver of soil bacterial assembly. In this study, in the NH 4 + treatment, Burkholderiales was the dominant order in the maize root zone and 0-0.5 cm soil compartment. This order is known to dominate in low-pH and carboxylate-rich rhizospheres, where it metabolizes citrate and oxalate and ameliorates the effects of aluminum stress [10,43]. The genera speci cally enriched within 0.5 cm of the maize root zone under NH 4 + compared with CK and NO 3 − were Acidibacter, Burkholderia, and Catenulispora, which are potentially bene cial acidophilic bacteria [44][45][46]. In the NO 3 − treatment, Sphingomonadales was the dominant order in the maize root zone and its adjacent 0.5-cm soil compartment. This order is known to contribute to higher crop yield [37].
Furthermore, the genera Sphingomonas, Sphingobium, Azospirillum, and Novosphingobium were speci cally enriched within 0.5 cm of the maize root zone in the NO 3 − treatment compared with CK and the NH 4 + treatment. These genera are plant growth-promoting rhizobacteria that favor nutrient-rich and higher-pH soils [24,47]. Therefore, in this study, the maize roots tended to recruit potentially bene cial bacteria with a higher tolerance to acid and aluminum to cope with decreased soil pH during NH 4 + uptake, and to recruit growth-promoting microbes that prefer higher pH during NO 3 − uptake.
The bacterial communities in the 0-1 cm soil compartment under NH 4 + treatment and in the 0-0.5 cm soil compartment under NO 3 − treatment were more similar to those in the root zone (0 cm) but signi cantly differed from those in the outer compartments. Therefore, the "rhizosphere effect" on bacterial communities was not restricted to the root zone, but extended to 1 cm from the root zone under NH 4 + treatment and 0.5 cm from the root zone under NO 3 − treatment. Root exudates are selective factors that shape the structure of the rhizosphere microbiome [10]. The range of the "rhizosphere effect" is highly dependent on the ow of root exudates [28]. Previous studies have shown that root exudates can penetrate up to 8-14 mm into most upland soils [48][49][50], and the size of this range can be affected by soil structure and compaction, water status, and the activity of soil microbes [2]. In the present study, the rhizosphere effect had a larger range in the NH 4 + treatment than in the NO 3 − treatment. This may be because of changes in pH during the uptake of NH 4 + and NO 3 − by plants. Since soil pH is known to be negatively correlated with belowground carbon allocation [41], increased pH may decrease the range of the rhizosphere effect on microbiomes [4]. Therefore, applying NH 4 + may increase the range of the rhizosphere effect due to reduced soil pH.

Nitrogen form and maize rhizosphere in uenced bacterial co-occurrence patterns
Microbial co-occurrence patterns re ect direct and indirect interactions among microbial taxa coexisting in environmental samples, and can provide insights into bacterial communities' functional roles and/or the environmental niches occupied by microorganisms [51]. In the present study, we found that bacterial networks were more complex in the NH 4 + treatment than in CK, whereas those in the NO 3 − treatment were less complex than those in CK. Unfavorable soil environmental conditions (acidity, nutrient de ciency) promote both co-operation and competition in bacterial networks. Accordingly, microbial networks were found to be less complex under limed and N-fertilized conditions than under control conditions [52]. In this study, NO 3 − fertilization resulted in less complex bacterial networks in all soil compartments, probably because NO 3 − application provided more favorable conditions in terms of higher soil pH and N nutrition. Although the NH 4 + treatment provided su cient N in the soil environment, the soil pH was decreased to 4.5 due to NH 4 + absorption and nitri cation. This may have enhanced niche sharing and intensi ed competition and cooperation among bacterial taxa [52]. The bacterial networks were more complex in the maize root zone and its adjacent soil compartments (0.5-1 cm) than in the outer compartments in both the NH 4 + and NO 3 − treatments, indicative of more complex bacterial networks in the maize rhizosphere than in bulk soil. Since roots act as a lter during the assembly of rhizosphere bacterial communities [12] and promote the development of niches populated by dominant taxa, it is reasonable for competition and cooperation to be stronger in the rhizosphere due to shared niches [53]. In contrast, microbes in bulk soil occupy heterogeneous and disconnected habitats [53]. Whereas some long-term studies considering the rhizosphere community as a subset of the bulk soil community have detected less complex network structures in the rhizosphere than in bulk soil [12,54], our greenhouse study revealed an instant effect of plant roots on rhizosphere bacterial networks.

Soil residual fertilizer N-correlated OTUs in bacterial networks
In this study, we de ned the soil compartment 0.5 cm and 1 cm from the root zone as the rhizosphere under NO 3 − and NH 4 + treatment, respectively. We found that more bacterial OTUs were strongly correlated with soil residual N derived from NH 4 + fertilizer, and formed more complexed networks with other microbes under NH 4 + treatment compared with NO 3 − in both maize rhizosphere and bulk soil. This indicated that bacterial growth and associations were more sensitive to NH 4 + fertilizer than to NO 3 − fertilizer. The strong positive correlations between OTUs from Burkholderiales / Rhodospirillales and soil residual N derived from NH 4 + fertilizer in the present study provide insight into a carbon-N coupling mechanism, since these correlated microbes have been reported to use root exudates as carbon sources and to degrade organic materials [10,55]. We also found that the OTUs which positively correlated with soil residual N derived from NH 4 + fertilizer had the most positive correlations with OTUs from Frankiales, indicating that these OTUs interacted with members of the Frankiales to e ciently colonize the maize rhizosphere. Our results show that N fertilizer, regardless of the N form, had stronger negative effects on bacterial networks in the maize rhizosphere than in bulk soil. This may be attributed to pH changes and the accumulated NH 4 + -N and NO 3 − -N contents, as most bacterial taxa exhibit narrow tolerance to changes in pH [56], and some N-cycling microbes may be inhibited by increasing NH 4 + -N and NO 3 − -N contents [26,57]. For example, in the present study, OTUs from the Myxococcales were negatively correlated with soil residual N derived from NH 4 + fertilizer in the maize rhizosphere. Myxococcales are known to be inhibited by chemical N fertilizer due to soil acidi cation [58]. The OTUs from the Streptomycetales were negatively correlated with soil residual N derived from NO 3 − fertilizer. Members of this order participate in heterotrophic nitri cation, which is inhibited by increased NO 3 − -N content [59].

Conclusion
The maize rhizosphere and the N form affected the spatial distribution of soil chemical properties, bacterial communities, and bacterial co-occurrence patterns along a lateral distance from maize roots in an acidic red soil.