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Soil P-stimulating bacterial communities: response and effect assessment of long-term fertilizer and rhizobium inoculant application

Environmental Microbiome volume 19, Article number: 86 (2024) Cite this article

Abstract

Background

Phosphorus (P) plays a vital role in plant growth. The pqqC and phoD genes serve as molecular markers for inorganic and organic P breakdown, respectively. However, the understanding of how P-mobilizing bacteria in soil respond to long-term fertilization and rhizobium application is limited. Herein, soil that had been treated with fertilizer and rhizobium for 10 years was collected to investigate the characteristics of P-mobilizing bacterial communities. Five treatments were included: no fertilization (CK), phosphorus fertilizer (P), urea + potassium fertilizer (NK), NPK, and PK + Bradyrhizobium japonicum 5821 (PK + R).

Results

The soybean nodule dry weight was highest in the P treatment (1.93 g), while the soybean yield peaked in the PK + R treatment (3025.33 kg ha− 1). The abundance of the pqqC gene increased in the rhizosphere soil at the flowering-podding stage and in the bulk soil at the maturity stage under the P treatment, while its abundance increased in the bulk soil at the flowering-podding stage and in the rhizosphere soil at the maturity stage under the PK + R treatment. The abundance of the phoD gene was enhanced in the bulk soil at the flowering-podding stage under the PK + R treatment. The Shannon and Ace indexes of pqqC- and phoD-harboring bacteria were higher in the rhizosphere soil at maturity under the PK + R treatment compared to other treatments. Furthermore, a comprehensive analysis of the neutral community model and co-occurrence pattern demonstrated that the application of P fertilizer alone led to an increase in the distribution and dynamic movement of pqqC-harboring bacteria, but resulted in a decrease in complexity of network structure. On the other hand, rhizobium inoculation enhanced the distribution and dynamic movement of phoD-harboring bacteria, as well as the stability and complexity of the network structure. Pseudomonas and Nitrobacter, as well as Steptomyces, Stella, and Nonomuraea, may be crucial genera regulating the composition and function of pqqC- and phoD-harboring communities, respectively.

Conclusions

These findings affirm the crucial role of fertilization and rhizobium inoculation in regulating pqqC- and phoD-harboring bacterial communities, and highlight the significance of long-term phosphate-only fertilization and rhizobium inoculation in enhancing dissolved inorganic phosphorus and mineralized organophosphorus, respectively.

Introduction

While long-term fertilization promotes nutrient supply to crops, it also has several detrimental effects that can degrade the soil’s microecological environment. An important concern arises from the long-term application of phosphorus (P) fertilizer, resulting in the accumulation of P in the soil [1, 2]. Consequently, investigating the responding mechanism of soil P-solubilizing microorganisms to various fertilization measures can establish a theoretical foundation for mitigating P enrichment in the soil.

The proportion of different P components are crucial factors in determining the availability of soil P. Based on this, soil P can be divided into unstable, medium stable, and stable fractions [3]. It is easier for plants to utilize unstable P, while medium stable P can be transformed into unstable P through the mineralization of soil organic matter. Stable P cannot be easily used by plants, and is primarily retained in the soil matrix [4]. Soil P pool is composed of inorganic (Pi) and organic (Po) forms. Although Pi constitutes a larger proportion [5], over 95% of P is stored in the soil in an inaccessible form for direct utilization by plants [6]. To be bio-utilized, soil Po must be mineralized into Pi. Activating P is a crucial method to enhance its availability in soil.

Functional microorganisms can improve the availability of residual soil P by utilizing various mechanisms. They secrete phosphatase enzymes, which facilitate the mineralization of Po, and produce organic acids that aid in the dissolution of Pi. The pqqC gene encodes pyrroloquinoline quinone synthase, while the phoD gene encodes alkaline phosphatase (ALP), which serve as valuable molecular markers of Pi dissolution and Po mineralization, respectively. Acid phosphatase (ACP) also significantly contributes to P mineralization and has been widely concerned by researchers [7,8,9].

At present, the studies on the impact of fertilization on the community structure of bacteria harboring the pqqC and phoD genes, have explored various aspects including different fertilizer application methods, and diverse microbial communities. Long-term application of organic fertilizer had significant impact on the abundance of pqqC and phoD genes, as well as restructuring the P-mobilized bacterial community structure, when compared to chemical fertilizer [10]. Conversely, short-term application of organic fertilizer altered the rhizosphere community structure of phoD-harboring bacteria [11]. Not only were bacteria examined, the addition of straw promoted the survival of pqqC- and phoD-harboring fungi, thereby enhanced the microbial P pool [12].

Rhizobium has gained extensive attention due to its capability for biological nitrogen fixation in association with leguminous roots [13]. The use of rhizobium inoculant as a sustainable microbial fertilizer has been recognized for its ability to reduce reliance on chemical fertilizers. Its development and application have been widely embraced due to its environmentally friendly nature, lack of pollution, and positive impact on crop yield [14]. Evaluating the effects of rhizobium inoculants on microbial communities is crucial for understanding the unintended impact of rhizobium inoculations. While studies have reported on the enhanced nitrogen fixation and P solubilization abilities of rhizobia through pqq gene transformation [15]. There is limited research on phosphorus-soluble functional bacteria. Thus, investigating this aspect will contribute to explore the community characteristics and key factors of phosphorus-mobilizing bacteria after inoculation with rhizobia. It will also provide insights into the microbial processes involved in the phosphorus cycle under this fertilization practice.

The objectives are (1) to investigate the response characteristics of community structure of P-mobilizing bacteria to the long-term application of fertilizer and rhizobium inoculation; (2) to assess the cross links between nodule dry weight, soybean yield and community structure of P-mobilizing bacterial; (3) to determine the main physicochemical factors that potentially influence the community structure of P-mobilizing bacteria.

Materials and methods

Sampling site

The sampling site was established in 2011 and is situated at the Jilin Academy of Agricultural Sciences, Gongzhuling County, Jilin Province, China (43.52°N, 124.80°E, altitude 42 m). The region’s agricultural practices involve a single cropping season per year, with the soybean cultivar Jiyu 86 being sown in late April and harvested in early October. Five treatments were established, with each soil sample having three replicates: CK, control group without fertilization or rhizobium application; P, soil treated with 75 kg ha− 1 of phosphorus fertilizer; NK, soil treated with 60 kg ha− 1 urea and 75 kg ha− 1 potassium fertilizer; NPK, soil treated with both P fertilizer and NK fertilizer; PK + R, soil treated with PK fertilizer along with Bradyrhizobium japonicum 5821. The bacterial inoculum was prepared at a concentration of 5 × 109 CFU mL− 1 and applied by mixing an average of 5 mL of the bacterial liquid with 1 kg of soybean seeds for sowing, using a seeding rate of 45 kg of soybean seeds per hectare of field.

Soil samples were collected on July 16, 2021 (flowering-podding stage) and September 29, 2021 (maturity stage). The bulk soil is the topsoil (0–20 cm) 30 cm away from the soybean plant, while the rhizosphere soil is the soil intimately adhered to the soybean roots. The collected soil samples were processed in time, and were divided into three parts after screening: (1) air-drying for physical and chemical testing; (2) storage at 4 °C for soil enzyme activity testing; (3) storage at -80 °C for soil microbial properties testing. In addition to soil samples, soybean root nodules were collected at the flowering-podding stage, and soybean yield was measured at maturity stage.

Soil acid phosphatase (ACP) and alkaline phosphatase (ALP) activities were determined at 660 nm wavelength by Multiskan FC spectrophotometer (Thermo Scientific, Shanghai, China). An assay kit from Boxbio (Beijing, China) was utilized for this analysis. The measurement of other soil physicochemical properties was conducted following the procedures described by Wei et al. [16].

DNA extraction and gene quantification

For each sample, 1 g of fresh soil was weighed, and total DNA was extracted using a kit from Qiagen (Hilden, Germany). The abundance of the pqqC and phoD genes was determined using quantitative PCR (qPCR). The primer for pqqC gene was pqqCF/pqqCR (5′- CATGGCATCGAGCATGCTCC-3′/5′-CAGGGCTGGGTCGCCAACC-3′) [17], and primer for phoD gene was phoD733F/phoD1083R (5′-TGGGAYGATCAYGARGT-3′/5′-CTGSGCSAKSACRTTCCA-3′) [18]. The qPCR reaction mixture and procedure according to Zhou et al. and Wei et al. [19, 20].

Amplicon sequencing of pqqC and phoD gene

For amplicon sequencing of the pqqC and phoD genes, the same primers as used in qPCR were employed. The Illumina library was constructed on the MiSeq PE300 platform (Illumina, San Diego, CA, USA) after purification and normalization of the PCR products. Once the Illumina sequencing produced paired-end (PE) reads, they were divided. Subsequently, a quality control and filtering process was implemented on the double-ended reads to eliminate low-quality reads according to their sequencing quality. Moreover, the double-ended reads were merged by aligning their overlapping regions, leading to refined data following quality control and alignment. To acquire Amplicon Sequence Variants (ASVs), the refined data underwent additional processing utilizing sequence denoising techniques (DADA2). These ASVs represent unique and high-quality sequence variants, providing detailed information about both the sequence itself and its abundance in the sample [21].

Statistical analyses

SPSS 24 (SPSS, Chicago, USA) was employed to analyze the variance (ANOVA) among soil physicochemical properties, root nodule dry weight, soybean yield, gene abundance, alpha indexes, and beta diversity. Regression analysis and boxplot were performed using the “ggplot2” package. Venn diagrams were created with the “VennDiagram” package. The “vegan” package in R software (v 4.2.3) was utilized to perform non-metric multidimensional scaling (NMDS) analysis. The co-occurrence pattern was implemented on all samples of the same treatment at different time and spatial scales, based on the genus level, using Gephi 0.9.2 [22]. The “ggsignif” package of R was utilized to analyze the difference in correlation coefficients of the co-occurrence pattern.

The Spearman correlation between community structure and different indicators of the corresponding gene was assessed using the Mantel test, performed with the “linkET”, “ggplot2”, and “dplyr” packages in R. Using the significant factors identified from the previous analysis, a structural equation model (SEM) was developed to investigate their impacts on the abundance, diversity, richness, composition of pqqC- and phoD-harboring bacterial communities, nodule dry weight, and soybean yield. The first axes of the NMDS analysis represented community composition. The r values of the Mantel test and standardized total effects of the SEM were visualized using Origin 2021 (OriginLab Corp., USA) [23].

Results

Nodule dry weight, soybean yield, and soil physicochemical properties

During the flowering-podding and maturity stages of soybean, variations in nodule dry weight, soybean yield, and soil physicochemical properties were detected among the different treatments (Fig. 1; Table 1). Among the treatments, the P treatment exhibited the greatest soybean nodule dry weight (1.93 g), significantly higher than the NK treatment by 192.72% and significantly higher than the NPK treatment by 65.15%. Conversely, the NK treatment exhibited the lowest nodule dry weight (0.66 g; Fig. 1a). The maximum soybean yield (3025.33 kg ha− 1) was achieved in the co-application of rhizobia and PK fertilizer, showing a 32.25% increase compared to the CK treatment and a 19.43% increase compared to the P treatment. When compared to the no-fertilizer treatment, using P fertilizer alone led to a yield increase of 10.73%, while NK fertilizer increased yield by 28.61%, NPK fertilizer by 25.95%, and the combined application of rhizobium and PK fertilizer by 32.25%. (Fig. 1b). At both stages, the application of fertilizer resulted in an increase in the activity of ACP in the rhizosphere soil. Particularly, the P treatment exhibited higher ACP activity compared to the other treatments (Fig. 1c). In contrast, the absence of P fertilizer significantly decreased ALP activity at both stages. Rhizobium inoculation enhanced ACP activity in bulk soil during flowering-podding stage. It also increased ALP activity in all tested soil samples, except mature bulk soil.

In the bulk soil at both stages, the pH was reduced by fertilization and inoculation of rhizobia (Table 1). In the rhizosphere soil, the NK treatment displayed significantly lower organic matter (OM) content (29.73 g kg− 1) compared to the other treatments during the maturity stage. Furthermore, both fertilization and rhizobia inoculation led to an increase in available phosphorus (AP) content in both bulk and rhizosphere soil at both stages. Notably, the P treatment exhibited higher AP content in the rhizosphere soil compared to the other treatments. Available potassium (AK) was significantly higher in the NK treatment in the bulk soil during the flowering-podding stage, as well as in both bulk and rhizosphere soil during the maturity stage.

Fig. 1
figure 1

The nodule dry weight, soybean yield, ACP and ALP activities of soil under different treatment conditions during the flowering-podding and maturity stages. Statistical differences in these indicators among treatments at the same stage in bulk or rhizosphere soil are denoted by different lowercase letters (P < 0.05)

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Table 1 The basic physicochemical properties of soil under different treatment conditions during the flowering-podding and maturity stages
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The data are presented as means ± standard deviation. Different lowercase letters represent the significance of the differences in the basic properties of the treatments in the bulk or rhizosphere soils, as determined by Duncan’s test of ANOVA analysis. Values in bold indicate significant differences among treatments (P < 0.05). The abbreviations used are as follows: OM (organic matter); TN (total nitrogen); AN (available nitrogen); TP (total phosphorus); AP (available phosphorus); AK (available potassium).

The abundance and alpha diversity of pqqC and phoD gene

Based on the representative sequence and abundance information of ASVs, with a minimum similarity threshold of 80%, various analyses can be performed. A total of 2,021,607 optimized sequences were obtained for the pqqC gene, with an average sequence length of 364 bp. For the phoD gene, 2,761,796 optimized sequences were obtained, with an average sequence length of 343 bp. Long-term fertilization and rhizobium inoculation had differential effects on gene abundance, Shannon index, and Ace index (Fig. 2). The abundance of the pqqC gene was 7.71–26.25 × 107 copies g− 1, which exceeded that of the phoD gene (14.90–48.80 × 106 copies g− 1). The analysis of pqqC gene revealed that, during the flowering-podding stage in bulk soil and the maturity stage in rhizosphere soil, the PK + R treatment demonstrated a significantly higher abundance of the pqqC gene compared to the other treatments. Furthermore, when P fertilization was applied independently, it resulted in increased pqqC gene abundance in the rhizosphere soil during the flowering-podding stage and in the bulk soil during the maturity stage, surpassing the abundance observed in other fertilization treatments. In the bulk soil during the flowering-podding stage, the Shannon index of the pqqC-harboring bacterial community reached its highest value (4.22) in the P treatment. Interestingly, in the bulk soil at the maturity stage, both the Shannon and Ace indexes were lower under the PK + R treatment. Conversely, in the rhizosphere soil at maturity, the Shannon and Ace indexes of the PK + R treatment were higher. Additionally, in the rhizosphere soil at both stages, the Ace index was lower in the NK treatment compared to the other treatments.

Rhizobium inoculation increased the abundance of the phoD gene in the bulk soil during the flowering-podding stage. However, at the maturity stage, the phoD gene abundance was lower in both the NK and NPK treatments compared to the other treatments. In terms of the bacterial community associated with the phoD gene, the Shannon and Ace indexes were higher in the NPK and PK + R treatments in the rhizosphere soil at both stages. Conversely, these indexes significantly decreased in the NK treatments.

Linear regression analysis revealed correlations between two corresponding indicators. The gene copy number of pqqC in mature bulk soil exhibited a negative correlation with soybean yield (R2 = 0.533; P = 0.002), while that in mature rhizosphere soil exhibited a positive correlation with soybean yield (R2 = 0.440; P = 0.007; Additional file 1: Fig. S1). Furthermore, there was a significant negative correlation between the copy number of the phoD gene in mature bulk soil and soybean yield (R2 = 0.414; P = 0.010; Additional file 1: Fig. S2). There was no significant correlation among other indicators.

Fig. 2
figure 2

The gene abundance and alpha diversity (Shannon index and Ace index) of pqqC- and phoD-harboring bacterial communities were assessed at the flowering-podding and maturity stages in both bulk and rhizosphere soil under different treatments. Statistical differences in these indicators among treatments at the same stage in bulk or rhizosphere soil are denoted by different lowercase letters (P < 0.05)

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The beta diversity of pqqC- and phoD-harboring bacterial communities

The number of unique and shared ASVs in pqqC-harboring bacterial communities was less than that in phoD-harboring bacterial communities across all treatments (Additional file 1: Fig. S3). The NMDS analysis of the pqqC-harboring bacteria in bulk soil at the flowering-podding stage (R = 0.3904, P = 0.0080; Additional file 1: Fig. S4a) and rhizosphere soil at the maturity stage (R = 0.2585, P = 0.0360; Additional file 1: Fig. S4d) highlighted clear disparities in response of community composition to fertilization and rhizobium inoculation. However, the difference was not significant in the rhizosphere soil during the flowering-podding stage, and in the bulk soil during the maturity stage (P > 0.05; Additional file 1: Fig. S4b and c). The phoD-harboring bacteria revealed significant differences in all treatments (P < 0.05; Additional file 1: Fig. S4e-h).

The composition of pqqC- and phoD-harboring communities were visualized by bar charts and circus plots (Additional file 1: Fig. S5). Pseudomonas was the most abundant genus among the pqqC-harboring bacteria, its relative abundance ranging from 0.85 to 42.90%. The application of P fertilizer alone in the bulk soil during the flowering-podding stage resulted in an increase in the relative abundance of Pseudomonas. In contrast, the absence of P fertilizer (NK) significantly enhanced the relative abundance of Mycobacterium in both the bulk and rhizosphere soil at both stages, as well as the abundance of Burkholderia in bulk soil and the abundance of Nocardioides in rhizosphere soil at the maturity stage. Fertilizer application resulted in a decrease in the relative abundance of Micromonospora in the rhizosphere soil at the flowering-podding stage (Additional file 2: Table S1).

In the case of phoD-harboring bacteria, the application of P fertilizer alone led to a decrease in the relative abundance of Rubrobacter in the bulk soil during the flowering-podding stage. Conversely, the absence of P fertilizer (NK) significantly increased the relative abundance of Brevundimonas in all treatments. Additionally, NK treatment enhanced the abundance of Limnoglobus in the rhizosphere soil during the flowering-podding stage, as well as Stella in the bulk soil at maturity. However, it resulted in reduced abundance of Micromonospora in the bulk soil, Nonomuraea, and Micromonospora in the rhizosphere soil at maturity. Furthermore, the NPK treatment increased the relative abundance of Pseudomonas in the bulk soil during the flowering-podding stage and in the rhizosphere soil at maturity. However, it decreased the abundance of Stella in the rhizosphere soil during the flowering-podding stage. The PK + R treatment, on the other hand, improved the abundance of Nonomuraea in the rhizosphere soil during the flowering-podding stage (Additional file 2: Table S2).

Neutral community model and co-occurrence pattern of pqqC- and phoD-harboring bacteria

Neutral community models were employed to elucidate the differences in the community structure of pqqC- and phoD-harboring bacteria (Fig. 3). Among all the treatments, the P treatment exhibited the highest R2 value (0.92) for the pqqC-harboring bacteria. This suggests that the random process had a more significant influence on shaping the community structure of pqqC-harboring bacteria in the P treatment compared to other treatments. The highest Nm value (79.95) was observed in the P treatment, followed by CK, NPK, NK, and PK + R. This suggests that the abundance and distribution of pqqC-harboring bacteria were higher in the P treatment compared to the other treatments, while the PK + R treatment had the lowest abundance and distribution of pqqC-harboring bacteria. Moreover, the m value was highest (0.42) in the P treatment, indicating a more dynamic movement of pqqC-harboring bacteria after the application of P fertilizer alone. Additionally, the phoD-harboring bacteria exhibited the highest R2 value (0.94) in the PK + R treatment among all treatments, indicating a greater influence of random processes on shaping the phoD-harboring community in this treatment. The highest Nm value (995.96) was observed in the PK + R treatment, followed by NPK, NK, CK, and P. This suggests that the abundance and distribution of phoD-harboring bacteria were higher in the PK + R treatment compared to the other treatments, while the P treatment had the lowest abundance and distribution of phoD-harboring bacteria. Furthermore, the m value was higher (0.54) compared to that in the PK + R treatment, indicating a more dynamic movement of phoD-harboring bacteria after the inoculation of rhizobium.

Fig. 3
figure 3

Neutral community models are employed to evaluate the influence of neutral processes on community assembly at the general level. The 95% confidence intervals of the neutral model prediction are depicted as gray dashed lines. R2 is used to assess the overall goodness of fit to the models. Nm estimates dispersal between communities by examining the correlation between occurrence frequency and regional relative abundance. The parameter m quantifies community-level mobility

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In general, the co-occurrence pattern analysis demonstrated that the network of the pqqC-harboring bacterial community exhibited a higher level of complexity compared to the phoD-harboring bacterial community (Fig. 4). This was evident from the higher number of nodes and edges observed in the network of the pqqC-harboring bacterial community. Among the pqqC-harboring bacterial community, ASV47, belonging to the genus Nitrobacter, was most abundant in the CK, P, NPK, and PK + R treatments. The subsequent highest abundance was observed in ASVs belonging to the genus Pseudomonas. The P treatment exhibited the highest number of nodes and the fewest edges among all treatments. All the nodes in the pqqC-harboring bacterial community were positively interconnected. In the phoD-harboring bacterial community, ASV39, belonging to the genus Steptomyces, was found to be more abundant in the CK, P, NK, and PK + R treatments. ASV2, belonging to the genus Stella, displayed a higher abundance in the P and NPK treatments. ASV55, belonging to the genus Nonomuraea, exhibited a higher proportion in the P, NPK, and PK + R treatments. Fertilization resulted in an increase in the number of nodes and edges within each treatment, leading to improved positive connections between nodes.

Fig. 4
figure 4

The co-occurrence pattern of dominant pqqC- and phoD-harboring bacterial genera, with a relative abundance higher than 0.1%, is analyzed in the CK, P, NK, NPK, PK + R treatments. Connections are drawn between nodes that exhibited significant (P < 0.01) and strong (Spearman’s | r | > 0.8) correlations. Each node represents a genus, with the size of the node indicating the degree and the color representing the genera. The edge color indicates positive (pink) and negative (green) correlations. The keystone genera are identified by their corresponding ASVs in the figure

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Additionally, the P treatment had significantly lower values for degree, degree centrality, and closeness centrality compared to other treatments in the pqqC-harboring bacterial community (Fig. 5a). Among the fertilization treatments, the inoculation with rhizobium noticeably had the highest number of nodes and decreased the degree centrality and closeness centrality of the network in the phoD-harboring bacterial community (Fig. 5b).

Fig. 5
figure 5

The degrees and central coefficient values of the co-occurrence pattern of pqqC- (a) and phoD-harboring bacteria (b) under different treatments. The asterisk represents the significance of the difference between pairwise treatments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

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Links between community structure and different indicators

The community structure of pqqC-harboring bacteria was significantly influenced by soil AN, ACP activity, ALP activity, and pqqC gene richness (P < 0.05). Similarly, the community structure of phoD-harboring bacteria was significantly affected by pH, TP, AP, AK, ALP activity, and phoD gene diversity (P < 0.05). TN showed a positive correlation with pqqC gene abundance, but a negative correlation with diversity and richness of the pqqC gene. AN exhibited a positive correlation with ACP activity and pqqC gene diversity, but a negative correlation with phoD gene richness. TP displayed a positive correlation with diversity and richness of the phoD gene. AP demonstrated a positive correlation with ACP activity, pqqC gene abundance, and phoD gene diversity. Furthermore, ACP activity positively influenced the abundance, diversity, and richness of the pqqC gene, while ALP activity positively impacted the abundance, diversity, and richness of both the pqqC and phoD genes (Fig. 6a). Overall, soil AN (r = 0.26), ACP (r = 0.23), and ALP (r = 0.21) had successive significant effects on the community structure of the pqqC-harboring bacteria, while soil ALP (r = 0.32), TP (r = 0.22), and AP (r = 0.21) had successive significant effects on the community structure of the phoD-harboring bacteria (Fig. 6b).

Fig. 6
figure 6

The Mantel test was performed to assess the Spearman correlation between the pqqC- or phoD-harboring bacterial communities and various soil properties, gene abundance, diversity (Shannon index) and richness (Ace index) (a). Significant correlations are denoted by star symbols, accompanied by their corresponding r values. The significance levels are represented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. The r values of mantel test between the pqqC- or phoD-harboring communities and their corresponding indicators are presented (b). The abbreviations used are as follows: OM (organic matter); TN (total nitrogen); AN (available nitrogen); TP (total phosphorus); AP (available phosphorus); AK (available potassium)

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Integrated responses of different indicators on nodule dry weight and soybean yield

Based on the results of the Mantel test, key soil physicochemical factors were selected to construct the optimal SEM (Fig. 7a). The weightiness of different indicators was determined, with ACP activity accounting for 56%, ALP activity for 17%, pqqC gene abundance for 20%, pqqC gene diversity for 19%, pqqC gene richness for 42%, pqqC gene composition for 26%, phoD gene abundance for 41%, phoD gene diversity for 60%, phoD gene richness for 65%, phoD gene composition for 34%, nodule dry weight for 61%, and soybean yield for 74%.

In the SEM, ACP had a positive influence on the abundance of the pqqC gene (path coefficient = 0.37) but a negative influence on the abundance of the phoD gene (-0.47). Soil AN had a positive effect on pqqC gene diversity (0.31) and a negative effect on ACP (-0.47). ALP positively affected the richness of both the pqqC (0.42) and phoD (0.30) genes, as well as the diversity of the phoD gene (0.48). However, ALP negatively affected the composition of the pqqC gene (-0.71). Soil AP had a positive effect on the diversity of the phoD gene (0.49), nodule dry weight (0.44), and soybean yield (0.70). Additionally, diversity was positively correlated with the richness of both the pqqC gene (0.24) and the phoD gene (0.73). The richness of the phoD gene was found to be negatively correlated with its composition (-0.35). The composition of the pqqC gene had a negative effect on nodule dry weight (-0.25), and nodule dry weight had a negative effect on soybean yield (-0.73). The standardized total effects demonstrated that AP, ALP, and ACP had stronger influences on nodule dry weight and soybean yield (Fig. 7b).

Fig. 7
figure 7

The SEM links soil properties (AN, AP, ACP, ALP), nodule dry weight, soybean yield, and abundance, richness, diversity, and composition of pqqC and phoD gene. Red arrows indicate significant positive correlations, while blue arrows indicate significant negative relationships (P < 0.05) (a). The black line represents no significant correlation. The thickness of the arrow reflects the magnitude of significance, with thicker arrows indicating higher significance. The number adjacent to each arrow represents the path coefficient, indicating the strength and direction of the relationship between variables. The R2 values indicate the proportion of variance explained by each endogenous variable. Significance levels are denoted by *, P < 0.05; **, P < 0.01; ***, P < 0.001. The standardized total effects of different indicators on nodule dry weight or soybean yield are presented (b)

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Discussion

Variation of nodule dry weight, soybean yield, and soil physicochemical properties

Compared to the CK treatment, the soybean nodule dry weight increased in the P and PK + R treatments but decreased in the NK and NPK treatments (Fig. 1). Because the application of nitrogen fertilizer can inhibit biological nitrogen fixation and impede soybean nodulation [24]. Additionally, the inoculation of rhizobia has been shown to enhance soybean nodulation, while an appropriate intake of soil phosphate fertilizer promotes soybean nodulation. Furthermore, we observed that the co-application of PK fertilizer and rhizobia had the most significant promotion effect on soybean yield.

The mineralization of organic phosphorus (Po) in soil is primarily driven by extracellular enzymes, with phosphatases playing a pivotal role. Produced by soil microbiota, phosphatases are essential for converting Po into inorganic phosphate (Pi), a form readily available for plant uptake. The activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) are widely recognized as biochemical indicators for assessing Po mineralization in soil, reflecting the soil’s capacity to mobilize phosphorus—a crucial factor for plant nutrition, particularly in phosphorus-limited soils. ACP is most active in acidic conditions, whereas ALP functions optimally in alkaline soils, allowing these enzymes to adapt to varying pH environments [25, 26]. Monitoring ACP and ALP activity levels in soil provides valuable insights into phosphorus availability and cycling across different soil types and environmental conditions. For example, elevated ACP activity may indicate adaptation to acidic conditions common in forest soils or highly weathered agricultural soils, where phosphorus solubility is often restricted. In contrast, higher ALP activity is typically observed in calcareous or alkaline soils, suggesting active Po mineralization supported by microbial and root phosphatase secretion. These enzyme activities thus serve as reliable bioindicators in soil fertility management, supporting the optimization of fertilization practices and the maintenance of sustainable soil phosphorus levels [27, 28]. In this study, the activity of ACP in the rhizosphere soil was higher in comparison to the other treatments at both the flowering-podding and maturity stages. And the application of NK fertilizer significantly decreased ALP activity in both bulk and rhizosphere soil at both stages. This reduction can be attributed to two factors. First, the rhizosphere plays a crucial role in the absorption of phosphorus by plants, as demonstrated by the rhizosphere effect. This phenomenon refers to the enhanced growth and nutrient uptake of plants due to the biological and chemical activities occurring in the root zone. When phosphorus is readily available in the rhizosphere, it can be more efficiently absorbed by plant roots, leading to increased plant growth and productivity. As a result, the higher ACP activity observed in the P treatment suggests that the enzyme is actively involved in mobilizing phosphorus for plant uptake, which is essential during critical growth periods [29]. Second, the increased phosphorus content in the soil provides a substantial substrate for the synthesis of phosphatase enzymes. The presence of additional phosphorus likely stimulates the production of both ACP and ALP, as these enzymes are essential for breaking down organic phosphorus compounds into forms that can be readily absorbed by plants. The enhanced activity of ACP in the rhizosphere, particularly under P treatment, indicates that the enzyme plays a significant role in facilitating phosphorus availability to the plant. In contrast, the application of NK fertilizer may disrupt the balance of nutrients in the soil, potentially inhibiting the microbial processes responsible for ALP synthesis and activity [30, 31].Furthermore, the dynamics between nutrient availability and enzyme activity underscore the importance of tailored fertilization strategies in agricultural practices. Optimizing phosphorus application can enhance enzyme activity, promoting better nutrient uptake and overall plant health. Conversely, excessive application of nitrogen and potassium without adequate phosphorus can lead to imbalances that negatively impact enzyme activity and nutrient cycling in the soil. Understanding these interactions can help inform more sustainable agricultural practices, ultimately leading to improved crop yields and soil health.

Long-term fertilization significantly affects soil pH. Fertilization led to a decrease in soil pH in the bulk soil during the flowering-podding and maturity stages, compared to the control treatment (Table 1). This finding aligns with the view that soil acidification can occur due to the mobilization of aluminum and the leaching of nutrient cations, particularly nitrogen input [32]. Consistently, our study found that nitrogen fertilizer application resulted in soil acidification compared to other treatments. Interestingly, the inoculation of rhizobium appeared to inhibit soil acidification compared to nitrogen input. This may be attributed to the role of rhizobium in altering the absorption and utilization of essential soil trace elements such as calcium, iron, and magnesium by plants [33]. Furthermore, the content of soil AP was lowest in the control treatment (CK), followed by the treatment with no phosphorus fertilizer (NK). This suggests that the long-term application of P fertilizer has increased the availability of phosphorus in the soil.

Responses of fertilizer and rhizobium on the abundance, diversity and richness of pqqC- and phoD-harboring bacteria

The pqqC and phoD genes play a crucial role in regulating the mobilization of P and serve as molecular markers for phosphate-mobilized bacteria [34]. The evaluation of microbial potential for Po hydrolysis and Pi solubilization relies on the detection of pqqC and phoD genes [35, 36]. Reducing the intake of phosphate fertilizer can lead to an increase in the abundance of the pqqC gene in the soil [37]. However, our findings are inconsistent with these studies. In our study, the application of P fertilizer alone resulted in an increase in the abundance of the pqqC gene in the rhizosphere soil during the flowering-podding stage and in the bulk soil at the maturity stage (Fig. 2). This observation can be attributed to the fact that the processes of P dissolution and mineralization primarily occur in the rhizosphere soil of soybeans. During the early growth stage of soybeans (flowering-podding stage), the synergistic effect of P fertilizer intake and rhizosphere effect contributed to the higher abundance of pqqC-harboring bacteria. However, as the maturity stage approached, the accumulation of P fertilizer inhibited the growth of pqqC-harboring bacteria in the rhizosphere soil of soybeans. Although the abundance of pqqC gene in the bulk soil at maturity was higher in the P treatment compared to other fertilization treatments, it was still lower than the abundance observed in the rhizosphere soil during the flowering-podding stage. Regarding phoD-harboring bacteria, our study is consistent with previous finding that continuous urea input over 28 years decreased the abundance of the phoD gene in maize field soil [38]. Similarly, we found a reduction in the abundance of the phoD gene in the NK and NPK treatments in both bulk and rhizosphere soil at maturity. This reduction can be attributed to the accumulation of nitrogen fertilizer in the soil during soybean maturation. Hence, the long-term intake of nitrogen fertilization resulted in a decrease in the abundance of phoD gene at maturity.

The Ace index of the pqqC gene was lower in the NK in the rhizosphere soil at both stages. Additionally, the Shannon and Ace indexes of the phoD gene in the soil were significantly decreased in the NK treatments at both stages. These results indicate that the richness of pqqC-harboring bacteria in the rhizosphere soil, as well as the diversity and richness of phoD-harboring bacteria in the soil at both stages, were reduced when only NK fertilizer was applied. This decrease can be attributed to the long-term deficiency of P, which negatively affects the diversity of soil bacteria and further limits the diversity and richness of pqqC- and phoD-harboring bacteria [39].

Notably, we observed that the combined application of PK fertilizer and rhizobium had a positive impact on the ACP and ALP activity, and abundance, diversity and richness of pqqC- and phoD-harboring bacteria. Specifically, in the PK + R treatment, ACP activity increased in bulk soil during the flowering-podding stage, while ALP activity increased in all treatments except mature bulk soil. The abundance of the pqqC gene increased in the bulk soil during the flowering-podding stage and in the rhizosphere soil at the maturity stage. Additionally, the abundance of the phoD gene increased in the bulk soil during the flowering-podding stage. Moreover, the Shannon and Ace indexes of pqqC- and phoD-harboring bacteria were higher in the PK + R in the rhizosphere soil at maturity. These findings suggest a positive feedback mechanism where the utilization of rhizobium instead of nitrogen fertilizer contributes to the beneficial mobilization of phosphate by pqqC- and phoD-harboring bacteria in the soil.

Similarly, previous study has demonstrated that co-inoculation with Bacillus subtilis and Rhodopseudomonas palustris has been shown to enhance the abundance of pqqC and phoD genes [40]. This indicates that the inoculation of exogenous rhizosphere growth-promoting bacteria can synergistically work with local beneficial microorganisms to mobilize soil P and promote P cycling in the soil [41]. The combined application of PK fertilizer and rhizobia had a certain promotional effect on the diversity of pqqC- and phoD-harboring bacteria, which highlight the agroecological benefits of rhizobia replacing nitrogen fertilizer and reducing nitrogen application, while enhancing the P solubilization efficiency of P-mobilizing bacteria. Future research should focus on exploring the responses of rhizobium inoculation to P mobilization and the regulation of P-solubilizing bacteria through pot and field experiments.

Responses of fertilizer and rhizobium on the community composition of pqqC- and phoD-harboring bacteria

The different treatments applied in the study resulted in the recruitment of distinct pqqC- and phoD-harboring bacterial communities in the soil at both stages. Among the pqqC-harboring bacteria, the genus Pseudomonas was found to be the most abundant, and its abundance increased with the application of P fertilizer alone (Additional file 1: Fig. S5; Additional file 2: Table S1). This finding aligns with a previous study that supports the notion that majority Pseudomonas species have a strong ability to solubilize phosphate in the soil [35]. Mycobacterium was enriched in the NK treatment in both bulk and rhizosphere soil during the flowering-podding and maturity stages among the pqqC-harboring bacteria. This differs from a previous study where Mycobacterium was found to be significantly enriched in the rhizosphere soil through the inoculation of arbuscular mycorrhizal fungi, which had the ability to regulate phosphate mobilization [42, 43]. Additionally, in the phoD-harboring community, Brevundimonas, which has the capacity to mineralize organophosphates and solubilize Pi, was significantly enriched in the NK treatment in the soil at both stages (Additional file 2: Table S2). This may be due to the lack of phosphate fertilizer stimulating the activity of these bacteria. The relative abundance of Micromonospora in the soil at maturity was decreased in the NK, indicating that the accumulation of soil P stimulated the P-dissolving properties of Micromonospora during soybean maturation [44].

Notably, the neutral community model demonstrated that the distribution and dynamic movement of pqqC-harboring bacteria were greater in the P treatment but lower in the PK + R treatment (Fig. 3). In contrast, the distribution and dynamic movement of phoD-harboring bacteria was greater in the PK + R treatment but lower in the P treatment. These findings further support the notion that the long-term application of P fertilizer promotes the dissolution of Pi in the soil, leading to the enrichment of the pqqC-harboring bacterial community. Conversely, the long-term inoculation of rhizobium enhances the mineralization of Po in the soil, resulting in the enrichment of the phoD-harboring bacterial community. Currently, there are limited reports regarding the mobilization of P in soybean root soil through rhizobium inoculation. Mycorrhizal symbiosis has the potential to enhance the spatial availability of P, arbuscular mycorrhizal fungi (AMF) can establish symbiotic relationships with plants by forming mycorrhizal mycelium [45]. In low P soils, mycorrhizal plants typically exhibit better growth compared to non-mycorrhizal plants due to the enhanced direct uptake of P by plant roots through the AM pathway. However, despite the significant contribution of the AM pathway to plant P uptake, it can still inhibit plant growth. This is due to the functional diversity among AM symbionts [46]. Therefore, the inoculation of B. japonicum 5821 in this experiment may promote the mineralization of Po in the soil of the soybean experimental field, subsequently enriching the phoD-harboring bacterial community.

The co-occurrence network analysis revealed that the enriched ASVs in the pqqC-harboring bacterial communities were identified as Pseudomonas and Nitrobacter, while those in the phoD-harboring communities were assigned to Steptomyces, Stella, and Nonomuraea (Fig. 4). These genera, due to their strong connections to other central nodes, are likely to have crucial roles in regulating the composition and function of the pqqC and phoD-harboring communities. They may serve as key nodes contributing to the stability of the ecological network [37]. Furthermore, the application of only P fertilizer led to a reduction in network complexity within the pqqC-harboring community due to the fewest number of edges. While inoculation of rhizobium enhanced network stability and complexity in the phoD-harboring community due to its highest number of nodes and edges. Considering the functional differences between the pqqC and phoD genes, the impact of long-term phosphorus deficiency on the phoD gene was found to be greater than that on the pqqC gene [47, 48]. This could explain the lower number of ASVs in the pqqC-harboring bacterial communities compared to the phoD-harboring bacterial communities (Additional file 1: Fig. S3), as well as the greater complexity observed in the network of the pqqC-harboring bacterial community compared to the phoD-harboring bacterial community.

The Mantel test results revealed positive correlations between ACP activity and the abundance, diversity, richness, and community structure of pqqC-harboring bacteria. Similarly, ALP activity was positively correlated with the abundance of both pqqC- and phoD-harboring bacteria (Fig. 6). ACP and ALP are important enzymes involved in the dissolution of Pi and the mineralization of Po, and these findings are consistent with previous studies [49, 50]. Moreover, the soil AP had a positive effect on the diversity of phoD-harboring bacteria, as well as on nodule dry weight and soybean yield (Fig. 7). Soil AP played a significant role in affecting the community structure of phoD- and pqqC-harboring bacteria, because it represents the form of P that is readily available for plants, and it serves as an indicator of the acquisition efficiency of P-solubilizing bacteria in the soil [51, 52].

Conclusion

The availability of phosphorus in the soil is a key limiting factor for soybean yield. The combined application of PK fertilizer and rhizobium positively influenced the ACP and ALP activity, as well as the abundance, diversity, and richness of pqqC- and phoD-harboring bacteria in soil. The long-term application of P fertilizer alone enriched the pqqC-harboring bacterial community, while the inoculation of rhizobium enriched the phoD-harboring bacterial community. P fertilization alone reduced the network complexity of the pqqC-harboring community, rhizobium inoculation improved network stability and complexity of the phoD-harboring community. The combined application of rhizobium, and PK fertilizer may have a positive effect on soil availability of phosphorus. Thus, advocation for this strategy as the superior approach for improving the efficiency of P fertilizer usage. Our study provides a scientific basis for enhancing the phosphorus mobilization potential of microorganisms and regulating the soil P pool through the application of P fertilizer and rhizobia in agricultural production.

Data availability

The raw sequencing data for the pqqC gene and phoD gene have been uploaded to the NCBI database with accession IDs PRJNA1030333 and PRJNA1030330, respectively.

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Funding

This work was supported by the National Key Technology Research and Development Program of China (2023YFD1702200), the National Natural Science Foundation of China (42373080), the Major Science and Technology Project of Yunnan Province (202202AE090025), the National Natural Science Foundation of China (32201320), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-04).

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Authors and Affiliations

  1. Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, South Zhongguancun Street No.12, Beijing, 100081, China

    Wanling Wei, Mingchao Ma, Xin Jiang, Fengming Cao, Huijun Chen, Dawei Guan, Li Li & Jun Li

  2. Laboratory of Quality and Safety Risk Assessment for Microbial Products, Ministry of Agriculture, Beijing, 100081, China

    Mingchao Ma, Xin Jiang, Fengming Cao, Li Li & Jun Li

  3. Soybean Research Institute, Jilin Academy of Agricultural Sciences, Jilin, 132011, China

    Fangang Meng

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  1. Wanling Wei
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  2. Mingchao Ma
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  3. Xin Jiang
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  8. Li Li
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  9. Jun Li
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Contributions

WWL, MCM, XJ and JL designed the study. FGM provided resources. WLW, FMC, HJC, DWG, and LLperformed the experiments. WLW analyzed all the data, prepared the figures and tables, and wrote the first draft of the manuscript. JL edited the manuscript and checked the language. All authors contributed to the paper.

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Correspondence to Jun Li.

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Wei, W., Ma, M., Jiang, X. et al. Soil P-stimulating bacterial communities: response and effect assessment of long-term fertilizer and rhizobium inoculant application. Environmental Microbiome 19, 86 (2024). https://doi.org/10.1186/s40793-024-00633-x

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Keywords

Environmental Microbiome

ISSN: 2524-6372