Effects of short-term nitrogen and phosphorus addition on soil bacterial community of different halophytes
Since the 20th century, human activities such as fossil fuel burning and the use of nitrogen (N) and phosphorus (P) fertilizers have significantly impacted the biogeochemical cycles of N and P . In China’s natural ecosystem, the input of N to the soil has increased from 15.3 Tg year −1 to 175–259 Tg year −1 between 1980 and 2011, while the input of P has increased from 0.3 Tg year −1 to 14–16 Tg year −1 during the same period . The enrichment of N and P in soil can have significant impacts on ecosystem stability. Soil microorganisms play a crucial role in terrestrial ecosystems by contributing to essential biogeochemical processes and activities within the soil . Environmental changes can have an impact on the community composition and structure of soil microorganisms, which in turn can affect the nutrient cycling process and ultimately the growth of plants. Despite this, the exact impact of environmental change on soil microorganisms is still not fully understood. The study of soil microbial community variation in response to N and P fertility is crucial for understanding how global change and human activity affect nutrient cycling in subsurface ecosystems. The effect of N addition on the composition of microbial communities has been extensively studied. Zeng et al.  found that N addition could directly affect the bacterial community composition of the temperate steppe ecosystem. Some studies have found that N input influences the soil bacterial community indirectly through changes in other soil factors (e.g., N effectiveness, pH) or plants (e.g., plant diversity and changes in plant carbon sources). Additionally, some studies have revealed that N input can indirectly influence the soil bacterial community by altering other soil factors such as N effectiveness and pH or through changes in plant diversity and carbon sources . It has been shown that the effect of N application on the soil microbial community structure and diversity is not uniform , which indicates the complexity of the effect of N addition on the microbial community, a process that depends on ecosystem type and soil properties as well as the type of N addition, dose, and time duration of addition . For example, N addition reduced bacterial community richness in forest soil, while it had no effect on bacterial community in grassland ecosystems . Compared to N addition, a few studies have focused on the effect of P addition on soil microbial diversity and microbial community structure. According to Ma et al. , the injection of P altered the bacterial community structure of primary and secondary forests directly and indirectly through changing soil environmental factors. Ling et al.  found that changes in available phosphorus (AP) content caused by P addition were a key factor in altering the diversity and composition of soil bacteria. While Huang et al.  found that P addition changed the bacterial community composition by changing soil pH. Furthermore, P addition has also been found to have less impact on the composition and structure of microbial communities compared to N addition . These results suggest that the effect of P addition on soil microbial community is inconsistent and may be related to local environmental factors. Although the effects of N and P addition on bacterial communities have been extensively reported, these studies focus on bacterial community change under fertilizer application methods and applied dose conditions. It is unclear whether there is a correlation between the response of soil microorganisms and plants to N and P addition. Indeed, the impact of vegetation on the composition and diversity of soil bacterial communities is substantial . For example, when the plant is deficient in P content, the root system indirectly affects the bacterial community by secreting organic acids to activate the active P. On the other hand, N and P addition changes the shape of the root (diameter, surface area, biomass, etc.) affecting the bacterial community . However, these previous studies focused on the effect of N and P addition on the soil microbial community, ignoring the fact that the plant-soil-microbial community should be an integral system. Therefore, the rhizosphere and bulk soil of three typical halophytes ( Suaeda salsa , salt-absorbing plant; Phragmites communis , salt-rejecting plant; and Aeluropus sinensis , salt-secreting plant) selected in the Yellow River Delta (YRD) to investigate the effects of plants and N and P addition on the soil bacterial community by 16S rRNA sequencing. We predict that plant and N and P addition can affect bacterial communities, and plant effects on bacterial communities are greater than N and P addition. 

Site description The study region was in the YRD, Shandong Province, China (118°44'2"E, 38°1'18"N). The study region has a temperate semi-humid continental monsoon climate with 550–640 mm of precipitation per year and an average annual temperature of 12.6°C. The N deposition in the growing season of the study area is 2.26 g m −2 , and the content of AP is 4.04–6.28 mg kg −1 . Salinity is a major limiting factor for plant growth. The average soil salinity of this site is 4.48‰. The vegetation is mainly composed of halophytes: P. communis , A. sinensis , S. salsa , and Tamarix chinensis . Experiment design Three single halophyte distribution areas were selected. The plants in each distribution area are P. communis , A. sinensis , and S. salsa , respectively. Four treatments were set up, namely, control (CK, no addition), N addition (N, 15 g/m 2 ), P addition (10 g/m 2 ), and N-P co-addition (NP, 15 g/m 2 N, 10 g/m 2 P) . Twelve 1 × 1 m sample squares were set for each plant distribution area. Three parallel experiments were designed for each treatment. N addition treatment was randomly conducted using urea. P addition treatment was conducted using KH 2 PO 4 , and an equal volume of pure water was added for CK treatment. Soil sampling Three months following the N and P addition, in July 2021, sample collection was completed. Soil adjacent to the root system was considered rhizosphere soil. The soil at 15 cm depth on the ground without plant growth in the sample square was considered bulk soil. Seventy-two soil samples were collected. Each soil sample was divided into two parts, one for soil environmental factor measurement and the other for 16S rRNA analysis. Measurements of soil physical and chemical properties The potassium dichromate volumetric was used to measure the soil organic matter (SOM) content. The elemental analyzer (Vario EL III, Elementar, Germany) was used to measure the total N (TN) and total C (TC) content. pH meter and conductivity meter were used to measure the pH and soil salt, respectively. The content of AP was quantified using extraction-molybdenum antimony anti-colorimetry. Molybdenum blue method was used to measure the content of total phosphorus (TP). Extraction, sequencing, and analysis of soil DNA Genomic DNA was extracted using the CTAB method. Soil samples were treated with 1,000 µL CTAB lysis buffer containing lysozyme at 65°C, with periodic inversion for complete sample disruption. The supernatant was collected after centrifugation and subjected to a phenol (pH 8.0):chloroform:isoamyl alcohol (25:24:1) extraction, followed by a second chloroform:isoamyl alcohol (24:1) extraction. The resulting supernatant was precipitated with isopropanol, washed with 75% ethanol, air-dried, and dissolved in ddH 2 O. Subsequent digestion of RNA was performed by adding 1 µL RNase A at 37°C for 15 minutes. The 1% agarose gel electrophoresis was used to detect the DNA purity and concentration. Using specific primers 341F and 806R, the V3–V4 region of the bacterial 16S rRNA gene was amplified by a specific primer. PCR conditions include an initial denaturation step at 98°C for 1 min, followed by 30 cycles comprising denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s. The reaction concludes with a final extension step at 72°C for 5 min. All PCRs were carried out with 15 µL of Phusion High-Fidelity PCR Master Mix (New England Biolabs). The products were purified using the AxyPrep DNA Gel Extraction Kit (Qiagen, Germany) after being detected by 2% agarose gel electrophoresis. High-throughput sequencing was conducted using the MiSeqPE300 platform from the Illumina corporation (Wekemo Tech Group Co., Ltd. Shenzhen, China). The raw data FASTQ files were processed by Qiime2 (2019.1). The original sequences were subjected to quality control, trimming, denoising, and merging using the DADA2 plugin in Qiime 2. Following the removal of chimeric sequences, a final feature sequence table was obtained. Using the QIIME2 feature-classifier plugin, the representative sequences of ASVs were aligned to the pre-trained GREENGENES database (13_8) with a 99% similarity threshold, resulting in a table containing taxonomic classification information for the identified species. Diversity calculation using the QIIME2 core-diversity plugin. Statistical analysis The Duncan test was used to compare the differences between the treatments. Redundancy analysis (RDA) was used to examine the connection between soil bacterial communities and soil physicochemical characteristics. The differences in bacterial community composition between the treatments were analyzed by principal coordinate analysis (PCoA). PERMANOVA and ANOSIM were used to evaluate the effects of N addition, P addition, and N-P interaction on the soil bacterial community composition. To analyze the association between soil characteristics and bacterial diversity, linear regression analysis was used. R software (version 4.1.0; http://www.r-project.org ) was used for data analysis. The agricolae (1.3–5) package was used for the Duncan test . RDA, PCoA, PERMANOVA, and ANOSIM were completed using the vegan (2.6–2) package . The figure display and linear regression analysis were running by ggplot2 (3.3.6) and ggpubr (0.4.0) package . 

The study region was in the YRD, Shandong Province, China (118°44'2"E, 38°1'18"N). The study region has a temperate semi-humid continental monsoon climate with 550–640 mm of precipitation per year and an average annual temperature of 12.6°C. The N deposition in the growing season of the study area is 2.26 g m −2 , and the content of AP is 4.04–6.28 mg kg −1 . Salinity is a major limiting factor for plant growth. The average soil salinity of this site is 4.48‰. The vegetation is mainly composed of halophytes: P. communis , A. sinensis , S. salsa , and Tamarix chinensis . 

Three single halophyte distribution areas were selected. The plants in each distribution area are P. communis , A. sinensis , and S. salsa , respectively. Four treatments were set up, namely, control (CK, no addition), N addition (N, 15 g/m 2 ), P addition (10 g/m 2 ), and N-P co-addition (NP, 15 g/m 2 N, 10 g/m 2 P) . Twelve 1 × 1 m sample squares were set for each plant distribution area. Three parallel experiments were designed for each treatment. N addition treatment was randomly conducted using urea. P addition treatment was conducted using KH 2 PO 4 , and an equal volume of pure water was added for CK treatment. 

Three months following the N and P addition, in July 2021, sample collection was completed. Soil adjacent to the root system was considered rhizosphere soil. The soil at 15 cm depth on the ground without plant growth in the sample square was considered bulk soil. Seventy-two soil samples were collected. Each soil sample was divided into two parts, one for soil environmental factor measurement and the other for 16S rRNA analysis. 

The potassium dichromate volumetric was used to measure the soil organic matter (SOM) content. The elemental analyzer (Vario EL III, Elementar, Germany) was used to measure the total N (TN) and total C (TC) content. pH meter and conductivity meter were used to measure the pH and soil salt, respectively. The content of AP was quantified using extraction-molybdenum antimony anti-colorimetry. Molybdenum blue method was used to measure the content of total phosphorus (TP). 

Genomic DNA was extracted using the CTAB method. Soil samples were treated with 1,000 µL CTAB lysis buffer containing lysozyme at 65°C, with periodic inversion for complete sample disruption. The supernatant was collected after centrifugation and subjected to a phenol (pH 8.0):chloroform:isoamyl alcohol (25:24:1) extraction, followed by a second chloroform:isoamyl alcohol (24:1) extraction. The resulting supernatant was precipitated with isopropanol, washed with 75% ethanol, air-dried, and dissolved in ddH 2 O. Subsequent digestion of RNA was performed by adding 1 µL RNase A at 37°C for 15 minutes. The 1% agarose gel electrophoresis was used to detect the DNA purity and concentration. Using specific primers 341F and 806R, the V3–V4 region of the bacterial 16S rRNA gene was amplified by a specific primer. PCR conditions include an initial denaturation step at 98°C for 1 min, followed by 30 cycles comprising denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s. The reaction concludes with a final extension step at 72°C for 5 min. All PCRs were carried out with 15 µL of Phusion High-Fidelity PCR Master Mix (New England Biolabs). The products were purified using the AxyPrep DNA Gel Extraction Kit (Qiagen, Germany) after being detected by 2% agarose gel electrophoresis. High-throughput sequencing was conducted using the MiSeqPE300 platform from the Illumina corporation (Wekemo Tech Group Co., Ltd. Shenzhen, China). The raw data FASTQ files were processed by Qiime2 (2019.1). The original sequences were subjected to quality control, trimming, denoising, and merging using the DADA2 plugin in Qiime 2. Following the removal of chimeric sequences, a final feature sequence table was obtained. Using the QIIME2 feature-classifier plugin, the representative sequences of ASVs were aligned to the pre-trained GREENGENES database (13_8) with a 99% similarity threshold, resulting in a table containing taxonomic classification information for the identified species. Diversity calculation using the QIIME2 core-diversity plugin. 

The Duncan test was used to compare the differences between the treatments. Redundancy analysis (RDA) was used to examine the connection between soil bacterial communities and soil physicochemical characteristics. The differences in bacterial community composition between the treatments were analyzed by principal coordinate analysis (PCoA). PERMANOVA and ANOSIM were used to evaluate the effects of N addition, P addition, and N-P interaction on the soil bacterial community composition. To analyze the association between soil characteristics and bacterial diversity, linear regression analysis was used. R software (version 4.1.0; http://www.r-project.org ) was used for data analysis. The agricolae (1.3–5) package was used for the Duncan test . RDA, PCoA, PERMANOVA, and ANOSIM were completed using the vegan (2.6–2) package . The figure display and linear regression analysis were running by ggplot2 (3.3.6) and ggpubr (0.4.0) package . 

Soil bacterial community α-diversity With the increase in the sequence number (Fig. S1), the increase in species richness gradually decreases and then flattens out, indicating that the sampling is reasonable, and therefore, the 72 soil samples are sufficiently rich to reflect the real situation of soil microbial communities. Two-way analysis of variance (ANOVA)  showed that N and P addition had a significant effect on the bacterial diversity of S. salsa bulk soil and A. sinensis rhizosphere soil. The bacterial community Shannon index in S. salsa rhizosphere soil and P. communis bulk soil was significantly affected by N addition and interaction of N and P addition, respectively. N and P addition as well as the interaction of N and P addition had no significant effect on the bacterial diversity of P. communis rhizosphere soil and A. sinensis bulk soil. The rhizosphere soil bacterial community α-diversity responded differently to N and P addition in different plants . In the S. salsa , the bacterial community α-diversity of rhizosphere soil was significantly higher than that of bulk soil. Compared to the CK treatment, the bulk soil bacterial α-diversity was significantly higher in both N and P addition as well as co-addition. The N addition treatment had a significantly increasing effect on the rhizosphere soil Shannon index bacteria in S. salsa , while the P addition had a significantly decreasing effect on it. In A. sinensis , the bacterial α-diversity in bulk soil was higher in CK treatment than it was in rhizosphere soil. While under other treatments, the opposite was observed. N and P addition as well as co-addition treatment significantly increased bacterial diversity in the rhizosphere soil of A. sinensis ( P < 0.05). In P. communis , soil bacterial diversity in CK and NP addition treatment showed greater diversity in rhizosphere soil than in bulk soil. N and NP addition treatment reduced rhizosphere soil bacterial diversity. Principal coordinate analyses PCoAs showed that the first two axes have a better interpretation of the top 10 phyla of the relative abundance of the bacterial community . Nutrient addition treatment had a greater effect on the bacterial community structure of rhizosphere soil than that of bulk soil. The addition of N and P has an insignificant effect on the rhizosphere soil of S. salsa and P. communis . The distance between the P and co-addition treatment and the CK and N addition treatment in the rhizosphere soil of A. sinensis indicated that the P addition changed the community structure of bacteria . PERMANOVA (Table S1) showed that P had a significant effect on the rhizosphere bacterial community structure of A. sinensis . The structure of the bulk soil bacterial community of all three halophytes was altered under N and P addition. The structure of the bulk soil bacterial community of S. salsa was significantly affected by N and P addition , the structure of the bulk soil bacterial community of P. communis was affected by N addition , and the bulk soil bacterial community of A. sinensis was mainly affected by both N and co-addition . In addition, in the whole data, the PERMANOVA test  indicated that plant had the greatest influence on bacterial community structure ( R 2 = 0.247, P < 0.001), followed by soil ( R 2 = 0.132, P < 0.001), plant-soil interaction ( R 2 = 0.065, P < 0.001), N addition ( R 2 = 0.057, P < 0.001), soil-N addition interaction ( R 2 = 0.031, P < 0.01), plant-soil-P addition interaction ( R 2 = 0.027, P < 0.05), P addition ( R 2 = 0.022, P < 0.05), and N-P addition interaction ( R 2 = 0.016, P < 0.05), respectively. The interactions of soil and N addition were significant, which indicated that the effects of N addition were different for soil position. The halophyte rhizosphere bacterial community was mainly distributed in the second and the third quadrants , while the bulk soil bacterial community was mainly distributed in the first and fourth quadrants, indicating the bacterial community structure of bulk and rhizosphere soil was significantly different. ANOSIM test showed that the distance between rhizosphere and bulk soil bacterial community decreased under N and P addition as well as co-addition in both S. salsa  and A. sinensis  compared to CK, indicating that N and P addition reduced the difference of bacterial community between rhizosphere and bulk soil. In contrast, in P. communis , the difference between bulk and rhizosphere bacterial community structure decreased under N addition treatment and increased under P addition and N and P co-addition treatment . Soil bacterial composition at the phylum level The Proteobacteria relative abundance in bulk soil increased under N addition and N and P co-addition and decreased under P addition treatment ( ; Table S2), which made its relative abundance higher in S. salsa and P. communis rhizosphere soil than in bulk soil. The Bacteroidetes had a higher relative abundance in rhizosphere soil than in bulk soil, while the Nitrospirae had a higher relative abundance in bulk soil. Among the plants, the Bacteroidetes were more abundant in S. salsa than in P. communis and A. sinensis rhizosphere soil, but the opposite was true for the Acidobacteria , Chloroflexi , and Firmicutes . In P. communis and S. salsa , the rhizosphere Bacteroidetes relative abundance increased under P addition and N and P co-addition treatment. The bulk soil Bacteroidetes relative abundance was enhanced under N and P addition as well as co-addition treatment. N and P addition decreased the S. salsa and P. communis rhizosphere Acidobacteria relative abundance. Relationship between soil environmental factors and bacterial community α-diversity The S. salsa rhizosphere and bulk soil bacterial diversity were most closely related to soil environmental factors , with SOM ( R 2 = 0.74, P < 0.001), salt ( R 2 = 0.56, P < 0.001), TN ( R 2 = 0.53, P < 0.001), TC ( R 2 = 0.44, P < 0.001), and AP ( R 2 = 0.42, P < 0.001) showed a significant positive correlation. S. salsa rhizosphere soil salt, carbon, and nutrient content are higher than bulk soil, making the rhizosphere soil diversity higher than bulk soil. In P. communis , bacterial diversity was positively correlated with AP content ( R 2 = 0.25, P = 0.013). Unlike S. salsa and P. communis , A. sinensis rhizosphere and bulk soil bacterial diversity did not correlate well with AP, while it showed a significant negative correlation with TP ( R 2 = 0.16, P = 0.05). Redundancy analysis RDA and Monte Carlo tests were used to analyze the relationship between soil environmental factors and bacterial community structure . The SOM, TN, salt, AP, TP, TC, and pH content were used as environmental factors, and the top 10 phyla were used as bacterial community fraction. Among the three plant species, the association of bacterial community structure with environmental factors was stronger in S. salsa while weaker in P. communis and A. sinensis . The TN content had a significant effect on bacterial community structure, explaining 76%, 41%, and 29% of variation in S. salsa , P. communis , and A. sinensis , respectively. The key environmental factor driving changes in bacterial community structure were inconsistent among the three plant species. In S. salsa , SOM content had the greatest effect on bacterial community composition, explaining 79% of the variation of the bacterial community ( P < 0.001); in addition, Monte Carlo tests showed that TC, salt, AP, and TP content had significant effects on bacterial community structure. SOM and salt content explained 35% ( P < 0.05) and 56% ( P < 0.001) of the variation in P. communis bacterial community structure, respectively. pH and TP explained 68% ( P < 0.001) and 45% ( P < 0.01) of the variation in community composition in the community structure of A. sinensis , respectively. Among the three species, the Proteobacteria , Nitrospirae had a strong negative correlation with the C content and N content, while the Bacteroidetes had a strong positive correlation with it. In S. salsa and P. communis , salt content was positively correlated with the abundance of Bacteroidetes and negatively correlated with the abundance of Gemmatimonadetes , Actinobacteria , and Acidobacteria . 

With the increase in the sequence number (Fig. S1), the increase in species richness gradually decreases and then flattens out, indicating that the sampling is reasonable, and therefore, the 72 soil samples are sufficiently rich to reflect the real situation of soil microbial communities. Two-way analysis of variance (ANOVA)  showed that N and P addition had a significant effect on the bacterial diversity of S. salsa bulk soil and A. sinensis rhizosphere soil. The bacterial community Shannon index in S. salsa rhizosphere soil and P. communis bulk soil was significantly affected by N addition and interaction of N and P addition, respectively. N and P addition as well as the interaction of N and P addition had no significant effect on the bacterial diversity of P. communis rhizosphere soil and A. sinensis bulk soil. The rhizosphere soil bacterial community α-diversity responded differently to N and P addition in different plants . In the S. salsa , the bacterial community α-diversity of rhizosphere soil was significantly higher than that of bulk soil. Compared to the CK treatment, the bulk soil bacterial α-diversity was significantly higher in both N and P addition as well as co-addition. The N addition treatment had a significantly increasing effect on the rhizosphere soil Shannon index bacteria in S. salsa , while the P addition had a significantly decreasing effect on it. In A. sinensis , the bacterial α-diversity in bulk soil was higher in CK treatment than it was in rhizosphere soil. While under other treatments, the opposite was observed. N and P addition as well as co-addition treatment significantly increased bacterial diversity in the rhizosphere soil of A. sinensis ( P < 0.05). In P. communis , soil bacterial diversity in CK and NP addition treatment showed greater diversity in rhizosphere soil than in bulk soil. N and NP addition treatment reduced rhizosphere soil bacterial diversity. 

PCoAs showed that the first two axes have a better interpretation of the top 10 phyla of the relative abundance of the bacterial community . Nutrient addition treatment had a greater effect on the bacterial community structure of rhizosphere soil than that of bulk soil. The addition of N and P has an insignificant effect on the rhizosphere soil of S. salsa and P. communis . The distance between the P and co-addition treatment and the CK and N addition treatment in the rhizosphere soil of A. sinensis indicated that the P addition changed the community structure of bacteria . PERMANOVA (Table S1) showed that P had a significant effect on the rhizosphere bacterial community structure of A. sinensis . The structure of the bulk soil bacterial community of all three halophytes was altered under N and P addition. The structure of the bulk soil bacterial community of S. salsa was significantly affected by N and P addition , the structure of the bulk soil bacterial community of P. communis was affected by N addition , and the bulk soil bacterial community of A. sinensis was mainly affected by both N and co-addition . In addition, in the whole data, the PERMANOVA test  indicated that plant had the greatest influence on bacterial community structure ( R 2 = 0.247, P < 0.001), followed by soil ( R 2 = 0.132, P < 0.001), plant-soil interaction ( R 2 = 0.065, P < 0.001), N addition ( R 2 = 0.057, P < 0.001), soil-N addition interaction ( R 2 = 0.031, P < 0.01), plant-soil-P addition interaction ( R 2 = 0.027, P < 0.05), P addition ( R 2 = 0.022, P < 0.05), and N-P addition interaction ( R 2 = 0.016, P < 0.05), respectively. The interactions of soil and N addition were significant, which indicated that the effects of N addition were different for soil position. The halophyte rhizosphere bacterial community was mainly distributed in the second and the third quadrants , while the bulk soil bacterial community was mainly distributed in the first and fourth quadrants, indicating the bacterial community structure of bulk and rhizosphere soil was significantly different. ANOSIM test showed that the distance between rhizosphere and bulk soil bacterial community decreased under N and P addition as well as co-addition in both S. salsa  and A. sinensis  compared to CK, indicating that N and P addition reduced the difference of bacterial community between rhizosphere and bulk soil. In contrast, in P. communis , the difference between bulk and rhizosphere bacterial community structure decreased under N addition treatment and increased under P addition and N and P co-addition treatment . 

The Proteobacteria relative abundance in bulk soil increased under N addition and N and P co-addition and decreased under P addition treatment ( ; Table S2), which made its relative abundance higher in S. salsa and P. communis rhizosphere soil than in bulk soil. The Bacteroidetes had a higher relative abundance in rhizosphere soil than in bulk soil, while the Nitrospirae had a higher relative abundance in bulk soil. Among the plants, the Bacteroidetes were more abundant in S. salsa than in P. communis and A. sinensis rhizosphere soil, but the opposite was true for the Acidobacteria , Chloroflexi , and Firmicutes . In P. communis and S. salsa , the rhizosphere Bacteroidetes relative abundance increased under P addition and N and P co-addition treatment. The bulk soil Bacteroidetes relative abundance was enhanced under N and P addition as well as co-addition treatment. N and P addition decreased the S. salsa and P. communis rhizosphere Acidobacteria relative abundance. 

The S. salsa rhizosphere and bulk soil bacterial diversity were most closely related to soil environmental factors , with SOM ( R 2 = 0.74, P < 0.001), salt ( R 2 = 0.56, P < 0.001), TN ( R 2 = 0.53, P < 0.001), TC ( R 2 = 0.44, P < 0.001), and AP ( R 2 = 0.42, P < 0.001) showed a significant positive correlation. S. salsa rhizosphere soil salt, carbon, and nutrient content are higher than bulk soil, making the rhizosphere soil diversity higher than bulk soil. In P. communis , bacterial diversity was positively correlated with AP content ( R 2 = 0.25, P = 0.013). Unlike S. salsa and P. communis , A. sinensis rhizosphere and bulk soil bacterial diversity did not correlate well with AP, while it showed a significant negative correlation with TP ( R 2 = 0.16, P = 0.05). 

RDA and Monte Carlo tests were used to analyze the relationship between soil environmental factors and bacterial community structure . The SOM, TN, salt, AP, TP, TC, and pH content were used as environmental factors, and the top 10 phyla were used as bacterial community fraction. Among the three plant species, the association of bacterial community structure with environmental factors was stronger in S. salsa while weaker in P. communis and A. sinensis . The TN content had a significant effect on bacterial community structure, explaining 76%, 41%, and 29% of variation in S. salsa , P. communis , and A. sinensis , respectively. The key environmental factor driving changes in bacterial community structure were inconsistent among the three plant species. In S. salsa , SOM content had the greatest effect on bacterial community composition, explaining 79% of the variation of the bacterial community ( P < 0.001); in addition, Monte Carlo tests showed that TC, salt, AP, and TP content had significant effects on bacterial community structure. SOM and salt content explained 35% ( P < 0.05) and 56% ( P < 0.001) of the variation in P. communis bacterial community structure, respectively. pH and TP explained 68% ( P < 0.001) and 45% ( P < 0.01) of the variation in community composition in the community structure of A. sinensis , respectively. Among the three species, the Proteobacteria , Nitrospirae had a strong negative correlation with the C content and N content, while the Bacteroidetes had a strong positive correlation with it. In S. salsa and P. communis , salt content was positively correlated with the abundance of Bacteroidetes and negatively correlated with the abundance of Gemmatimonadetes , Actinobacteria , and Acidobacteria . 

Effect of N and P addition on rhizosphere and bulk soil bacterial community diversity Environmental change can alter the community composition of wetland soil microorganisms, and in turn, changes in wetland soil microorganisms can lead to changes in the overall structure and function of their ecosystems . Wetland plants interact with environmental factors to influence soil microbial community and functional diversity . Zhao et al.  studied the rhizosphere soil bacterial community of three different halophytes in the YRD and found that P. communis bacterial diversity and richness were higher than those of S. salsa and A. sinensis . However, in this study, it was found that the rhizosphere soil bacterial community of S. salsa had a high diversity, which was not consistent with the previous study. This may be due to the higher salt and SOM content and lower pH in S. salsa rhizosphere soil, all three of which behaved opposite to P. communis and A. sinensis bulk soil. Higher salt and lower pH did not promote bulk soil bacterial diversity in S. salsa ; therefore, we suggest that it may be the SOM content that causes the differences in soil bacterial diversity among the three halophytes. There was a strong correlation between soil bacterial diversity and rhizosphere soil SOM of S. salsa in this study, which is consistent with the previous study . In addition, plants shaped the soil bacterial community through selection and used the deposits to influence the diversity of the soil bacterial community . Plant residues are the main source of nutrition for plant rhizosphere soil bacteria . The amount and variety of apoplastic material vary greatly among plants, which in turn leads to differences in bacterial community diversity among the three halophytes. Increasing nutrition with bio-limitation can directly change bacterial community structure or indirectly affect bacterial community structure by changing plant biomass. For example, the nutrient changes the root structure, thus providing different microhabitats for the bacterial community, and can change the bacterial community composition in the form of root secretions and apoplastic inputs . Yan et al.  found that N addition did not affect bacterial community diversity and richness, while P addition reduced bacterial community diversity. Wang et al.  found that N addition reduced bacterial diversity and richness, while P addition did not mitigate the negative effect of N addition on the bacterial community. In this study, we found that three halophyte soil bacterial community diversity responses were incoherent to N and P addition. This may be due to the difference in the nutrient requirement of the plant. Previous studies found that P.communis rhizosphere soil N content is usually low, and N-fixing bacteria are abundant. N addition is known to inhibit the growth of N-fixing bacteria , which may have broken the positive interaction between the P. communis and bacterial community and thus reduced the P. communis rhizosphere bacterial diversity. We further analyzed the relative abundance of alphaproteobacteria and found that N addition decreased its abundance in P. communis (Fig. S2). However, despite the decrease in the relative abundance of alphaproteobacteria caused by N addition, the decrease did not reach a significant level. N addition alleviates nutrient limitation in the growth of S. salsa and A. sinensis and increases the SOM input to the soil, which can enhance the growth of the bacterial community . Previous studies have found that the response of the bacterial community to P addition varied according to the changes in the effectiveness of soil carbon. When soil carbon is high, microbial biomass and activity may be limited by P . In this study, the P. communis and A. sinensis rhizosphere SOM content was lower than S. salsa , and P addition promoted the rhizosphere soil bacterial communities diversity of P. communis and A. sinensis while reducing the rhizosphere soil bacterial diversity of S. salsa . N addition is usually accompanied by soil acidification, leading to a decrease in bacterial community diversity . To be mentioned, most of these studies were based on long-term N and P addition experiments, while the present study was a short-term N and P addition. The effect of N and P addition on the bacterial community diversity changed with the plants, which indicated that the effect of plants on the bacterial community was much greater than the short-term N and P addition. Effects of N and P addition on the composition of rhizosphere and bulk bacterial community Soil bacterial community composition was influenced by soil and plants . Therefore, we predicted that the bacterial community composition would change after nutrient addition, and the trend of changing rhizosphere bacterial composition might vary among halophytes. PCoA showed that N addition significantly changed the bulk soil community composition of the three halophytes, which is consistent with previous studies . However, N addition had no significant effect on the rhizosphere soil bacterial community composition of S. salsa and P. communis . In general, bacterial communities of plant rhizosphere soil have lower β-diversity and are more stable compared to that of bulk soils . In addition, in terms of ecological construction processes, stochastic processes contributed more to the rhizosphere soil bacterial community, while deterministic processes contributed more to the bulk soil , which makes rhizosphere bacterial community composition less responsive to environmental change than bulk soil. In S. salsa and A. sinensis , N and P addition reduced the difference in bacterial community composition between bulk and rhizosphere soil, which may be due to the increase in rhizosphere soil nutrition caused by N and P addition, resulting in a decrease in soil nutrient difference. Previous studies have found that Proteobacteria , Actinobacteria , and Bacteroidetes belong to eutrophic bacteria groups, and Chloroflexi and Acidobacteria belong to oligotrophic microbial groups . We predicted that nutrient addition would lead to the rapid growth of eutrophic taxa and decrease oligotrophic taxa. In this study, N and P addition reduced the Acidobacteria relative abundance in the rhizosphere and bulk soil bacterial community of P. communis , which further reflects the oligotrophic survival strategy of the Acidobacteria . Unexpectedly, P addition reduced the relative abundance of Proteobacteria in this study. This could be because the bacterial growth of the three S . salsa is N-limited, and P addition exacerbates the N-limitation of bacterial growth. There was more adequate nutrition in the N and P addition treatment, so Proteobacteria was the highest. The rhizosphere soil Bacteroidetes contained a high relative abundance, and RDA showed that both salt and SOM content were significantly and positively correlated with Bacteroidetes . A previous study found that Bacteroidetes have good salt tolerance and resistance, and higher salt level promotes the growth of salt-tolerant bacteria . S. salsa are salt aggregating plants and can take up salt from the soil through the root system to accumulate in the body without harm, which results in high rhizosphere soil salt content . In addition, according to the trophic survival strategy of bacteria, rhizosphere soil is abundant in C sources, which lead to a high relative abundance of the Bacteroidetes in rhizosphere soil . The addition of N and P enhanced the relative abundance of Bacteroidetes in S. salsa and P. communis soil, which further confirmed the growth strategy of the Bacteroidetes . In contrast to Bacteroidetes , the Gemmatimonadetes and Actinobacteria have lower salt tolerance or are more susceptible to high salt . In this work, both Gemmatimonadetes and Actinobacteria were negatively correlated with salt content, and the relative abundance of both was less in S. salsa rhizosphere soil, which was further corroborated by the previous study. However, the Actinobacteria was not relatively more abundant in the rhizosphere soil of P. communis and A. sinensis due to the lower salt content. However, the Actinobacteria was not relatively more abundant in P. communis and A. sinensis rhizosphere soil due to the lower salt content. This is because P. communis is an aquatic plant suitable for survival in a water-rich environment, while the growth of Actinobacteria is usually inhibited by water . Although Actinobacteria belong to eutrophic taxa, they still have a strong competitive advantage in soil with low carbon content, which results in low-SOM content but a high abundance of Actinobacteria in A. sinensis rhizosphere soil. The Chloroflexi had a high abundance in P. communis and A. sinensis soil, which is consistent with the survival strategy of oligotrophic bacterial taxa. The P. communis and A. sinensis soil SOM content were lower than that of the S. salsa , which promoted the growth and development of Chloroflexi , which can use CO 2 as a carbon source for photosynthesis to obtain energy . Conclusion The response of rhizosphere and bulk soil bacterial community α-diversity to N and P addition differed among the three halophytes. N addition increased rhizosphere soil bacterial community α-diversity in S. salsa and A. sinensis , while decreased that in P. communis . P addition decreased rhizosphere bacterial α-diversity in S. salsa while increased rhizosphere bacterial α-diversity in P. communis and A. sinensis . SOM content resulted in the difference between the rhizosphere soil bacterial α-diversity of the three halophytes. TN, SOM, and salt content have strong effects on bacterial community composition. The effect of halophyte on bacterial community composition is greater than that of N and P addition. This study demonstrated that the effects of N and P addition on soil bacterial community were influenced by plant species and root system. The effects of plants on microorganisms should be considered in future studies. 

Environmental change can alter the community composition of wetland soil microorganisms, and in turn, changes in wetland soil microorganisms can lead to changes in the overall structure and function of their ecosystems . Wetland plants interact with environmental factors to influence soil microbial community and functional diversity . Zhao et al.  studied the rhizosphere soil bacterial community of three different halophytes in the YRD and found that P. communis bacterial diversity and richness were higher than those of S. salsa and A. sinensis . However, in this study, it was found that the rhizosphere soil bacterial community of S. salsa had a high diversity, which was not consistent with the previous study. This may be due to the higher salt and SOM content and lower pH in S. salsa rhizosphere soil, all three of which behaved opposite to P. communis and A. sinensis bulk soil. Higher salt and lower pH did not promote bulk soil bacterial diversity in S. salsa ; therefore, we suggest that it may be the SOM content that causes the differences in soil bacterial diversity among the three halophytes. There was a strong correlation between soil bacterial diversity and rhizosphere soil SOM of S. salsa in this study, which is consistent with the previous study . In addition, plants shaped the soil bacterial community through selection and used the deposits to influence the diversity of the soil bacterial community . Plant residues are the main source of nutrition for plant rhizosphere soil bacteria . The amount and variety of apoplastic material vary greatly among plants, which in turn leads to differences in bacterial community diversity among the three halophytes. Increasing nutrition with bio-limitation can directly change bacterial community structure or indirectly affect bacterial community structure by changing plant biomass. For example, the nutrient changes the root structure, thus providing different microhabitats for the bacterial community, and can change the bacterial community composition in the form of root secretions and apoplastic inputs . Yan et al.  found that N addition did not affect bacterial community diversity and richness, while P addition reduced bacterial community diversity. Wang et al.  found that N addition reduced bacterial diversity and richness, while P addition did not mitigate the negative effect of N addition on the bacterial community. In this study, we found that three halophyte soil bacterial community diversity responses were incoherent to N and P addition. This may be due to the difference in the nutrient requirement of the plant. Previous studies found that P.communis rhizosphere soil N content is usually low, and N-fixing bacteria are abundant. N addition is known to inhibit the growth of N-fixing bacteria , which may have broken the positive interaction between the P. communis and bacterial community and thus reduced the P. communis rhizosphere bacterial diversity. We further analyzed the relative abundance of alphaproteobacteria and found that N addition decreased its abundance in P. communis (Fig. S2). However, despite the decrease in the relative abundance of alphaproteobacteria caused by N addition, the decrease did not reach a significant level. N addition alleviates nutrient limitation in the growth of S. salsa and A. sinensis and increases the SOM input to the soil, which can enhance the growth of the bacterial community . Previous studies have found that the response of the bacterial community to P addition varied according to the changes in the effectiveness of soil carbon. When soil carbon is high, microbial biomass and activity may be limited by P . In this study, the P. communis and A. sinensis rhizosphere SOM content was lower than S. salsa , and P addition promoted the rhizosphere soil bacterial communities diversity of P. communis and A. sinensis while reducing the rhizosphere soil bacterial diversity of S. salsa . N addition is usually accompanied by soil acidification, leading to a decrease in bacterial community diversity . To be mentioned, most of these studies were based on long-term N and P addition experiments, while the present study was a short-term N and P addition. The effect of N and P addition on the bacterial community diversity changed with the plants, which indicated that the effect of plants on the bacterial community was much greater than the short-term N and P addition. 

Soil bacterial community composition was influenced by soil and plants . Therefore, we predicted that the bacterial community composition would change after nutrient addition, and the trend of changing rhizosphere bacterial composition might vary among halophytes. PCoA showed that N addition significantly changed the bulk soil community composition of the three halophytes, which is consistent with previous studies . However, N addition had no significant effect on the rhizosphere soil bacterial community composition of S. salsa and P. communis . In general, bacterial communities of plant rhizosphere soil have lower β-diversity and are more stable compared to that of bulk soils . In addition, in terms of ecological construction processes, stochastic processes contributed more to the rhizosphere soil bacterial community, while deterministic processes contributed more to the bulk soil , which makes rhizosphere bacterial community composition less responsive to environmental change than bulk soil. In S. salsa and A. sinensis , N and P addition reduced the difference in bacterial community composition between bulk and rhizosphere soil, which may be due to the increase in rhizosphere soil nutrition caused by N and P addition, resulting in a decrease in soil nutrient difference. Previous studies have found that Proteobacteria , Actinobacteria , and Bacteroidetes belong to eutrophic bacteria groups, and Chloroflexi and Acidobacteria belong to oligotrophic microbial groups . We predicted that nutrient addition would lead to the rapid growth of eutrophic taxa and decrease oligotrophic taxa. In this study, N and P addition reduced the Acidobacteria relative abundance in the rhizosphere and bulk soil bacterial community of P. communis , which further reflects the oligotrophic survival strategy of the Acidobacteria . Unexpectedly, P addition reduced the relative abundance of Proteobacteria in this study. This could be because the bacterial growth of the three S . salsa is N-limited, and P addition exacerbates the N-limitation of bacterial growth. There was more adequate nutrition in the N and P addition treatment, so Proteobacteria was the highest. The rhizosphere soil Bacteroidetes contained a high relative abundance, and RDA showed that both salt and SOM content were significantly and positively correlated with Bacteroidetes . A previous study found that Bacteroidetes have good salt tolerance and resistance, and higher salt level promotes the growth of salt-tolerant bacteria . S. salsa are salt aggregating plants and can take up salt from the soil through the root system to accumulate in the body without harm, which results in high rhizosphere soil salt content . In addition, according to the trophic survival strategy of bacteria, rhizosphere soil is abundant in C sources, which lead to a high relative abundance of the Bacteroidetes in rhizosphere soil . The addition of N and P enhanced the relative abundance of Bacteroidetes in S. salsa and P. communis soil, which further confirmed the growth strategy of the Bacteroidetes . In contrast to Bacteroidetes , the Gemmatimonadetes and Actinobacteria have lower salt tolerance or are more susceptible to high salt . In this work, both Gemmatimonadetes and Actinobacteria were negatively correlated with salt content, and the relative abundance of both was less in S. salsa rhizosphere soil, which was further corroborated by the previous study. However, the Actinobacteria was not relatively more abundant in the rhizosphere soil of P. communis and A. sinensis due to the lower salt content. However, the Actinobacteria was not relatively more abundant in P. communis and A. sinensis rhizosphere soil due to the lower salt content. This is because P. communis is an aquatic plant suitable for survival in a water-rich environment, while the growth of Actinobacteria is usually inhibited by water . Although Actinobacteria belong to eutrophic taxa, they still have a strong competitive advantage in soil with low carbon content, which results in low-SOM content but a high abundance of Actinobacteria in A. sinensis rhizosphere soil. The Chloroflexi had a high abundance in P. communis and A. sinensis soil, which is consistent with the survival strategy of oligotrophic bacterial taxa. The P. communis and A. sinensis soil SOM content were lower than that of the S. salsa , which promoted the growth and development of Chloroflexi , which can use CO 2 as a carbon source for photosynthesis to obtain energy . 

The response of rhizosphere and bulk soil bacterial community α-diversity to N and P addition differed among the three halophytes. N addition increased rhizosphere soil bacterial community α-diversity in S. salsa and A. sinensis , while decreased that in P. communis . P addition decreased rhizosphere bacterial α-diversity in S. salsa while increased rhizosphere bacterial α-diversity in P. communis and A. sinensis . SOM content resulted in the difference between the rhizosphere soil bacterial α-diversity of the three halophytes. TN, SOM, and salt content have strong effects on bacterial community composition. The effect of halophyte on bacterial community composition is greater than that of N and P addition. This study demonstrated that the effects of N and P addition on soil bacterial community were influenced by plant species and root system. The effects of plants on microorganisms should be considered in future studies. 

