Effects of Plant-Growth-Promoting Rhizobacteria on Soil Bacterial Community, Soil Physicochemical Properties, and Soil Enzyme Activities in the Rhizosphere of Alfalfa under Field Conditions

Abstract

Background: Inoculation with plant-growth-promoting rhizobacteria (PGPR) effectively increases plant growth in agriculture. However, the role of the rhizobiome in plant growth remains unclear. Methods: Biolog Ecoplate and 16S rRNA gene high-throughput sequencing techniques were used to analyze the changes in microbial community diversity, composition, and function after PGPR inoculation. Soil physicochemical and enzyme activities were also measured. Results: PGPR inoculation significantly promoted the growth of alfalfa. Using a Biolog Ecoplate, inoculation improved the metabolic activity and carbon source utilization of soil microorganisms. PGPR inoculation significantly increased the diversity and richness of the soil bacterial community in the rhizosphere of alfalfa and increased the relative abundance of key bacterial taxa such as Arthrobacter, Sphingomonas, and Bacillus, which are conducive to plant growth. Conclusions: Inoculation with PGPR enriched bacterial taxa and improved the utilization of carbon sources beneficial for plant growth. PGPR inoculation induced changes in microbial community diversity, and relevant functions in the rhizosphere contributed to alfalfa growth under field conditions.

1. Introduction

The microecological environment of the plant rhizosphere is a key factor maintaining the normal function of the soil ecosystem and crop yield. The plant rhizosphere microenvironment is the central interface that supports the energy flow and material transport between plant roots and the rhizosphere soil environment [1]. At present, evaluation of the soil microecological environment generally includes physical, chemical, and related biological indices. Among these, the physical and chemical properties of the soil are important parts of soil material circulation and flow and are necessary for maintaining soil microecological balance [2]. Soil biological indicators such as the diversity, composition, and function of microorganisms in plant rhizosphere soil have received increasing attention from researchers. Soil contains the richest microbial communities on Earth, such as bacteria [3]. They play an important role in the cycle of organic matter and available nutrients and regulate various ecological processes closely related to plant growth. Fan et al. [4] reported that soil microorganisms play an important role in maintaining the soil element cycle and crop yields. Soil bacterial communities play a crucial role in the regulation, support, and supply of soil ecosystems and are the key link connecting the aboveground and underground parts of plants in soil ecosystems [5]. Additionally, the soil enzyme activities are closely related to soil physicochemical properties as well as the diversity and composition of soil microorganisms and are thus important indicators of soil productivity and activity [6].

Excessive use of chemical fertilizers in modern agriculture negatively affects soil ecosystems [7,8]. Currently, chemical fertilizers used by farmers have adverse effects on soil microorganisms and fertility [9]. Introducing beneficial microorganisms into the soil system is an environmentally friendly method for improving crop yield and soil productivity. Plant-growth-promoting rhizobacteria (PGPR) have recently been demonstrated to promote plant growth. Studies demonstrated they exhibit high adaptability to various environments due to their fast growth rate, high biochemical diversity, and ability to metabolize various organic compounds in soils [10]. These bacteria can also synthesize siderophores, auxins, 1-aminocyclopropane-1-carboxylate deaminase (ACCD), and antibacterial compounds to improve plant growth [11]. In our previous study, the inoculation of Enterobacter aerogenes LJL-5 and Pseudomonas aeruginosa LJL-13 significantly increased the plant height, yield, and crude protein content of alfalfa [12]. Moreover, compared with single inoculation, co-inoculation with PGPR can promote plant growth better [13].

The effects of inoculation with PGPR on microbial community diversity and composition in the rhizosphere and subsequent metabolic functions are likely to be the critical factors affecting plant growth. A previous study showed that inoculation with Sinorhizobium A15, Bacillus A28, Sphingomonas A55, and Enterobacter P24 significantly promoted the growth of maize, and inoculation with four strains improved the diversity and richness of the bacterial community in maize rhizosphere soil. The authors also reported that the relative abundances of Verrucomicrobiae and Mucilaginibacter were positively correlated with maize yield [14]. Vasconcellos et al. [15] showed that inoculation with Pseudomonas aestus CMAA1215 increased the relative abundances of Bacillus, Bryobacter, and Bradyrhizobium in the rhizosphere of maize. Lee et al. [16] observed that inoculation with Bacillus mesonae H20-5 increased the relative abundances of Kineosporia, Actinoplanes, and Solirubrobacter in the rhizosphere of tomato. Ju et al. [17] found that co-inoculation with Paenibacillus mucilaginosus and Sinorhizobium meliloti increased the bacterial community diversity in the rhizosphere soil of alfalfa under copper stress. However, little is known about the effects of PGPR inoculation on soil microbial communities in the rhizosphere of alfalfa grown in field conditions.

Alfalfa (Medicago sativa L.) is a perennial legume grass widely planted because of its high yield and quality. This species, which is tolerant to salt and alkali stresses, is the so-called “king of pastures” [18]. In field cropping patterns, even though alfalfa has the advantages of high saline-alkali tolerance and strong adaptability, deep and loose soil should be selected for planting to benefit its growth. Before the sowing of alfalfa, the soil in the fields needs to be thoroughly plowed and compacted to facilitate seed germination. Weeding should also be carried out during the planting of alfalfa if weeds are growing heavily in the fields. Alfalfa is usually sown in the spring when the soil has a high moisture content and the temperature is suitable. In terms of irrigation, alfalfa is non-tolerant to flooding and not suitable for growing in low-lying areas [19]. We aimed to elucidate the effects of inoculation with Acinetobacter beijerinckii LJL-12 and Pseudomonas fluorescens MJM-5 on the bacterial diversity, composition, and relevant carbon source metabolic functions in the rhizosphere soil of alfalfa grown under field conditions. Furthermore, the changes in physicochemical properties and enzyme activities in the rhizosphere soil were measured. We hypothesized that inoculation with PGPR would alter the bacterial communities in the rhizosphere soil of alfalfa under field conditions and that some bacterial taxa and carbon source metabolic functions related to promoting the plant growth would be enriched in the rhizosphere.

2. Materials and Methods

2.1. Bacterial Strains

The strains of A. beijerinckii LJL-12 (OQ581475) and P. fluorescens MJM-5 (OQ586029) were obtained from our laboratory (Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province, Harbin Normal University, China) and grown in the Luria–Bertani (LB) medium. The LJL-12 and MJM-5 strains exhibited phosphate-solubilizing activities at 275.3 and 281.3 μg·mL⁻¹, respectively. The activities of 1-aminocyclopropane-1-carboxylate deminase (ACCD) in the two strains were 16.4 and 17.2 μ mol·α-KA mg⁻¹h⁻¹, respectively. In addition, these two strains produced indole acetic acid (IAA) and siderophores (Table S9).

2.2. Experimental Design

Before planting, the surfaces of the alfalfa seeds were disinfected with 75% ethanol for 3 min followed by sodium hypochlorite for 2 min, after which they were thoroughly washed five times in sterile distilled water. The surface-sterilized seeds were immersed in sterile distilled water and bacterial suspensions of LJL-12, MJM-5, and MJM-5 plus LJL-12 for 2 h. The prepared bacterial solution with a concentration of 1 × 10⁸ CFU·mL⁻¹ was applied. Subsequently, the treated alfalfa seeds were planted in the field. The experiments were conducted in Minzhu, Heilongjiang Province, China (126°48′ N, 45°49′ E) (Figure S7). The planting area was farmland soil with the following properties: pH 7.1; available N, P, and K levels of 119.1, 23.1, and 195.7 mg·kg⁻¹, respectively; and organic matter content of 25.1 g·kg⁻¹.

The alfalfa test plots were selected in the field. The area of each treatment was 1.2 × 4 m. In each plot, five rows of alfalfa were planted at 25 cm intervals. In addition, the distance between the plots with different treatments was 0.5 m. No herbicides and fertilizers were used throughout the field experiments. The experimental treatments included the Control, LJL-12 (A. beijerinckii LJL-12), MJM-5 (P. fluorescens MJM-5), and MJM-5+LJL-12 (P. fluorescens MJM-5 plus A. beijerinckii LJL-12). The prepared bacterial solution with a concentration of 1 × 10⁸ CFU·mL⁻¹ was applied to different plots with the same alfalfa growth trend; the Control was treated with distilled water. The bacterial solution was equally applied to all parts of each plot. Meanwhile, to maintain the activities of the bacterial solution in the soil, the application cycle of the experiment lasted for 30 days and was applied two times on the plots of different treatments. Specially, the method of bacterial solution application included irrigating the alfalfa roots, and the application amount of each treatment was 2 L. Additionally, herbicides, fertilizers, and other chemicals were not applied to field during the entire test period. In the flowering stage of the alfalfa, the plant height, shoot fresh weight, and leaf chlorophyll content were observed in the fields.

2.3. Plant and Rhizosphere Soil Sampling

The plants were harvested after the flowering period. First, the shoot height and shoot fresh weight were measured. The leaf chlorophyll content was measured through a chlorophyll analysis (Konica Minolta SPAD-502, Langenhagen, Germany). The shoots were oven-dried at 60 °C for 72 h, and the plant crude protein content was measured using the Kjeldahl method [20]. Plant roots were removed from the soil, and most of the soil and roots were shaken, which left an approximately 1 mm thick soil layer adhering to roots. Finally, using a disinfected brush, the rhizosphere soils were collected. Each sample was divided into three sub-samples: one was fresh soil for the Biolog Ecoplate analysis, another was stored at −80 °C for the 16S rRNA gene high-throughput sequencing, and the third sub-sample was used to determine the physicochemical properties and enzyme activities of the soil.

2.4. Biolog Ecoplate Method

A Biolog Ecoplate (Biolog Inc., Hayward, CA, USA) was used to analyze the carbon source utilization and functional diversity of the soil samples. First, 5 g of fresh soil was placed in a sterile flask. Subsequently, 45 mL of a sterile aqueous solution of NaCl (0.85%) was added. The mixture was then shaken for 40 min in an oscillator at 120 rpm and incubated for 20 min. Then, 5 mL of the supernatant was added to 45 mL of a sterile NaCl solution (0.85%). This dilution was repeated thrice to obtain a 1:1000 soil extract. Subsequently, 150 μL of the diluted extract was pipetted onto Biolog plates and incubated at 28 °C for nine days. On each day of incubation, the absorbance of the plates at 595 and 750 nm was determined using a microplate reader (Bio-Rad 680, Hercules, CA, USA). The average well color development (AWCD) was used as a proxy for the metabolic activity of soil microorganisms [21]. The functional diversity indices and utilization of each carbon source were calculated based on the absorbance values during the five days of incubation.

2.5. 16S rRNA Gene High-Throughput Sequencing

The total soil DNA was extracted using the FastDNA^®SPIN Kit (MP Biochemicals, Solon, OH, USA) following the manufacturer’s instructions. Three biological replicates were obtained and pooled for each soil sample to ensure that the soil DNA was representative. The V4-V5 region of the 16S rRNA gene was amplified by a polymerase chain reaction (PCR) using the primer pair 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907R (5′-CCGTCAATTCCTTTGAGTTT-3′), and all PCR amplifications were conducted in triplicate for each sample. The amplicon samples were sequenced on a paired-end 250 bp Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). Paired-end sequences were merged using FLASH (version 1.2.11, https://ccb.jhu.edu/software/FLASH/index.html, accessed on 15 September 2022), after which they were quality filtered using QIIME (version 1.9.1, http://qiime.org/install/index.html, accessed on 15 September 2022) [22]. After removing the chimeric sequences, the remaining sequences were assigned to OTUs with 97% similarity using UPARSE (version 11, http://www.drive5.com/uparse, accessed on 15 September 2022). Taxonomic information was annotated for the representative sequences of each OTU using the RDP classifier (version 2.13, https://sourceforge.net/projects/rdp-classifier, accessed on 17 September 2022) at a confidence level of 80% [23]. The Bioproject accession of the GenBank database with 16S rRNA gene high-throughput sequencing was PRJNA941485. Meanwhile, the SRA accessions of each sample under the different treatments were as follows: Control_1 (SRR23725334), Control_2 (SRR23725333), Control_3 (SRR23725330), LJL-12_1 (SRR23725329), LJL-12_2 (SRR23725328), LJL-12_3 (SRR23725327), MJM-5_1 (SRR23725326), MJM-5_2 (SRR23725325), MJM-5_3 (SRR23725324), MJM-5+LJL-12_1 (SRR23725323), MJM-5+LJL-12_2 (SRR23725332), and MJM-5+LJL-12_3 (SRR23725331).

2.6. Determination of Soil Physicochemical Properties and Soil Enzyme Activities

The soil bulk density, moisture content, organic matter, and available N, P, and K were analyzed according to the methods described by Bao [24]. Specifically, the soil bulk density and moisture content were measured using the ring knife and oven drying methods, respectively. The soil organic matter content was determined using the K dichromate volumetric method. The available N, P, and K were determined using the diffusion absorption method, the molybdenum–antimony colorimetric method, and ammonium acetate extraction–flame spectrophotometry, respectively. Soil urease, sucrase, dehydrogenase, and protease activities were measured according to the methods described by Guan et al. [25]. Soil urease and sucrase activities were analyzed using sodium phenol-sodium hypochlorite and 3,5-dinitrosalicylic acid colorimetry, respectively, and soil dehydrogenase and protease activities were determined using triphenyl tetrazole chloride (TTC) and ninhydrin colorimetry methods, respectively.

2.7. Bioinformatics Analysis

The representative OTU sequences were assigned to taxonomic classifications using the Greengenes reference database of 16S rRNA gene sequences and clustered at 99% using UPARSE. Subsequently, USEARCH (version 11, http://www.drive5.com/usearch, accessed on 17 September 2022) was used to merge the sequenced paired-end reads and compare the resulting sequences with an in-house strain database [26]. All sequences hitting a unique strain with an identity ≥ 99% were assigned a strain OTU. For each strain OTU, one of the matching reads was selected as a representative; to calculate strain abundances, all sequences were mapped against the strain OTU representatives using USEARCH. After identifying the taxa to be included in the analysis, the values used for each taxon sample intersection were populated with the abundance of reads assigned to each OTU. Mothur (version 1.30.2, https://www.mothur.org/wiki/Download_mothur, accessed on 17 September 2022) was used to calculate the alpha-diversity indices, including Shannon, Sobs, Chao, and Ace. Rarefaction curves were plotted according to Sobs and Shannon indices at the different sequencing depths, and the rank-abundance curves were drawn at the Sobs level. A Venn analysis was performed on the OTUs and genus-level bacterial taxa in the different treatments using R (version 3.3.1) to reflect the similarity and differences in bacterial taxon composition among the different treatments. According to the results of the taxonomic analysis, the composition of bacterial communities in the different treatments at different classification levels (phylum, class, order, and family) was obtained. The R program was used for the heatmap analysis of bacterial genus levels in the different treatments. The beta-diversity distance matrix of the bacterial community in the different samples was calculated using QIIME, the Abund–Jaccard analysis was adopted, and a principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (ADONIS) were plotted [27]. LEfSe (http://huttenhower.sph.harvard.edu/galaxy/root?toolid=lefse upload, accessed on 11 October 2022) was used to identify the key bacterial taxa in different treatments.

2.8. Statistical Analysis

Data normality and variance homogeneity as well as the significance of the differences between the data in the different treatments were determined using the one-way analysis of variance and Duncan’s test in SPSS (IBM, Chicago, IL, USA). A Pearson analysis was used to analyze the correlation of major bacterial taxa and the soil physio-chemical properties. The final data are presented as the mean ± standard deviation.

3. Results

3.1. Alfalfa Biomass

The inoculation with LJL-12, MJM-5, and MJM-5+LJL-12 significantly promoted the growth of alfalfa under field conditions. Compared with the Control treatment, the yields of alfalfa inoculated with LJL-12, MJM-5, and MJM-5+LJL-12 were significantly increased by 11.4%, 14.0%, and 24.8%, respectively, indicating a greater increase in biomass for co-inoculation than for single-inoculation treatments (p < 0.05) (Figure S1). A similar trend was observed for the shoot height, leaf chlorophyll content, and crude protein content (Figure S1).

3.2. Carbon Source Metabolic Activity of Alfalfa Rhizosphere Soil

The Biolog Ecoplate revealed that inoculation with LJL-12, MJM-5, and MJM-5+LJL-12 increased the AWCD in the rhizosphere of alfalfa (Figure 1a). The Shannon and Simpson indices for carbon source utilization by microbes in the rhizosphere significantly increased in response to LJL-12, MJM-5, and MJM-5+LJL-12 compared with those in the Control (Figure 1b). The PCA of carbon source utilization indicated that the utilization patterns of carbon sources by microbes in the rhizosphere were significantly different in the LJL-12, MJM-5, and MJM-5+LJL-12 treatments (R = 0.631, p = 0.012) (Figure 1c). Specially, the microorganisms in the rhizosphere exhibited enhanced utilization of carbohydrates, amino acids, carboxylic acids, polymers, phenolic acids, and amines after inoculation with LJL-12, MJM-5, and MJM-5+LJL-12. Moreover, co-inoculation led to improved metabolic activities on these carbon sources compared with that of single inoculation (Figure 2).

3.3. Alpha- and Beta-Diversity of Bacterial Communities in the Rhizosphere Soil of Alfalfa

In the present study, 161,968.667 optimized and 103,196.666 effective sequences were obtained for all the soil samples. The average length of sequences obtained through quality screening was 383.500–396.643 bp, and the sequencing depth of samples could reflect the diversity of bacterial communities (Table 1, Figure S2). Compared with the Control, the LJL-12, MJM-5, and MJM-5+LJL-12 treatments significantly increased the diversity (Shannon) and richness indices (Sobs, Chao, and Ace) of bacterial communities in the rhizosphere (p < 0.05) (Figure 3, Figure S3). A total of 1552 OTUs (25.60%) were shared among the treatments (Figure S4). The LJL-12, MJM-5, and MJM-5+LJL-12 treatments had 295 (4.87%), 706 (11.64%), and 830 (13.69%) unique OTUs, respectively. Compared with the Control (113, 1.86%), the number of unique OTUs in the rhizosphere increased in response to inoculation, especially in MJM-5+LJL-12 (Figure S4). The PCoA indicated that LJL-12, MJM-5, and MJM-5+LJL-12 significantly altered the composition of bacterial communities in the rhizosphere compared with that in the Control (R = 0.607, p = 0.015) (Figure 4).

3.4. Bacterial Community Composition in the Rhizosphere Soil of Alfalfa

Proteobacteria, Actinobacteriota, Acidobacteriota, Chloroflexi, and Gemmatimonadota were the dominant phyla in each treatment and accounted for 79.582% of the total relative abundance of the bacteria (Figure 5a, Table S1). Compared with that in the Control, the relative abundances of Acidobacteriota, Planctomycetota, Myxococcota, and Armatimonadota in the rhizosphere increased in response to LJL-12, MJM-5, and MJM-5+LJL-12, with the highest increase in MJM-5+LJL-12 (Figure 5a, Table S1). Gammaproteobacteria, Alphaproteobacteria, Thermolephilia, Actinobacteria, and Vicinamibacteria were the dominant bacterial classes in the rhizosphere soil of all treatments (Figure 5b, Table S2). Compared with those in the Control treatment, the relative abundances of Actinobacteria and Gemmatimonadetes in the rhizosphere increased under inoculation, especially in MJM-5+LJL-12 (Figure 5b, Table S2). The relative abundances of bacterial taxa in the rhizosphere of alfalfa inoculated with LJL-12, MJM-5, and MJM-5+LJL-12 also changed at the order and family levels (Figure S5, Tables S3 and S4).

Changes in the relative abundances of the 60 most abundant bacterial genera were observed. Inoculation with LJL-12, MJM-5, and MJM-5+LJL-12 increased the relative abundances of some bacterial genera in the rhizosphere, including Arthrobacter, Bacillus, Bradyrhizobium, and Sphingomonas; the highest increase in their abundance was observed after co-inoculation (Figure S6a). The Venn analysis at the genus level showed that some soil bacterial genera such as Acinetobacter appeared in the rhizosphere only after co-inoculation with MJM-5+LJL-12 (Figure S6b, Table S5).

3.5. Taxonomy of Key Bacteria in the Rhizosphere Soil of Alfalfa

A LEfSe analysis was used to compare the key bacterial taxa from the phylum to the genus levels in the rhizosphere among the different treatments. Compared with those in the Control, the relative abundances of Actinobacteriota (LDA scores = 4.906, 5.043, and 5.024), Actinobacteria (LDA scores = 4.399, 4.501, and 4.713), and Arthrobacter (LDA scores = 3.672, 3.598, and 4.095) were significantly higher in LJL-12, MJM-5, and MJM-5+LJL-12 (Figure 6, Tables S6–S8). The relative abundance of Sphingomonas (LDA scores = 3.191, 3.195) significantly increased in response to LJL-12 and MJM-5+LJL-12 (Figure 6a,c; Tables S6 and S8). In addition, the relative abundances of Bacillus (LDA score = 3.661) and Streptomyces (LDA score = 3.652) significantly increased in MJM-5+LJL-12 compared with those in the Control (Figure 6c, Table S8).

3.6. Soil Physicochemical Properties and Enzyme Activities in the Rhizosphere of Alfalfa

The rhizosphere soil bulk density in the different treatments ranged from 1.2 to 1.3 g·cm⁻³ with the lowest value observed in the MJM-5+LJL-12 treatment. Compared with that in the Control treatment, the organic matter content in the rhizosphere soil of alfalfa was significantly increased by the LJL-12, MJM-5, and MJM-5+LJL-12 inoculation treatments to 32.6, 31.7, and 34.9 g·kg⁻¹, respectively. It was significantly higher after co-inoculation than after single inoculation (p < 0.05). PGPR inoculation significantly increased the content of available N, P, and K in the rhizosphere soil, and the increase was more significant in the MJM-5+LJL-12 treatment than in the other single-inoculation treatments (p < 0.05) (Table 2). In addition, the soil urease, sucrase, dehydrogenase, and protease activities in the alfalfa rhizosphere inoculated with LJL-12, MJM-5, and MJM-5+LJL-12 were significantly higher than those in the non-inoculated Control treatment (p < 0.05) (Table 3). In addition, the Pearson correlation analysis showed that some soil physio-chemical properties of the alfalfa rhizosphere soil were correlated with the major bacterial taxa (Tables S10–S13). In the LJL-12 and MJM-5 inoculation treatments, the soil available K was positively correlated with Sphingomonas and Myxococcota, respectively (p < 0.05) (Tables S11 and S12). The soil organic matter was positively correlated with that in the Planctomycetota, Actinobacteria, and Arthrobacter after inoculation with MJM-5+LJL-12 (p < 0.05) (Table S13).

4. Discussion

Inoculation of PGPR is reportedly an effective practice for improving plant growth [15]. In the present study, we showed that co-inoculation with PGPR significantly increased the growth and crude protein content of alfalfa under field conditions (p < 0.05) (Figure S1). Enhanced capability for nutrient acquisition and utilization as PGPR has been reported to promote plant growth and quality through various mechanisms [28].

The metabolic activity of soil microorganisms related to different carbon sources is a crucial parameter closely related to soil function and a sensitive index reflecting soil fertility [29]. In soil ecosystems, carbon source metabolism is an essential function of the microbial community in the metabolism and utilization of various types of carbon source matrices in the soil, and it plays a vital role in the transformation of soil organic matter and nutrient elements [30]. The present study showed that inoculation with PGPR improved the metabolic activity and functional diversity of carbon source utilization under field conditions (Figure 1a,b). Some studies found that inoculation with microbial inoculants stimulated the microbial metabolism of carbon sources in the rhizosphere. Inoculation of Bacillus subtilis T-1 and Pseudomonas AKS2 improved the metabolic activity of microbial communities toward carbon sources, including amino acids and carbohydrates [31,32]. In this study, we also observed that inoculation with PGPR significantly changed the utilization of carbohydrates, amino acids, carboxylic acids, polymers, phenolic acids, and amines. This effect was more pronounced after co-inoculation (Figure 2). Relevant studies have shown that inoculation with microbial inoculants can change the utilization of different types of carbon sources by rhizosphere microorganisms. Zuluaga et al. [33] reported that the inoculation with Pseudomonas 16S altered the utilization of different carbon sources by plant rhizosphere microorganisms and improved the utilization efficiency of carbon sources such as amino acids, carboxylic acids, carbohydrates, polymers, and amines, indicating that it had a decisive effect on the utilization of carbon sources by microorganisms. Furthermore, amino acids and phenolic acids are the main components of plant root exudates. The essential nutrients of the rhizosphere microbial community, as well as the carbon source substrate detected by a Biolog Ecoplate, usually belong to the category of plant root exudates [34,35]. Microbial inoculants can alter rhizosphere function by influencing root exudate substrates, which are believed to be used by microorganisms for their growth. These changes in rhizosphere functions are also thought to improve plant growth [33,35]. Therefore, our findings suggested that the changes in the metabolism and utilization of carbon sources by rhizosphere soil microorganisms after inoculation with PGPR are improved rhizosphere function and promotion of the growth of alfalfa.

The rhizosphere microbial community plays an important role in plant growth and quality and provides a rich functional impetus for plants, including helping plants obtain low-abundance nutrition and resist biological or abiotic stress [36]. Because of its unique niche, the rhizosphere soil microbial community is more closely related to plant growth; therefore, studying the effect of PGPR inoculation on the plant rhizosphere microbial community would be considerably significant [37]. In the present study, inoculation with PGPR significantly increased the diversity and richness of the bacterial communities in the rhizosphere of alfalfa under field conditions (p < 0.05) (Figure 3 and Figure S3). Previously, inoculation with Bacillus amyloliquefaciens increased the diversity and richness of bacteria in banana rhizosphere soil and promoted the growth of plants [38]. Inoculation with Bacillus velezensis improved the diversity and richness of the bacterial community in pepper rhizosphere soil and increased the yield of pepper. Furthermore, soil with high microbial diversity exhibited higher ecological functions and led to a higher crop yield [39]. This is because higher microbial diversity leads to an increase in microbial processes such as the production of secondary metabolites, phytohormones, growth regulators, polysaccharides, and chelating substances, thus improving plant tolerance to abiotic stress and increasing their growth [40]. The reduction in soil microbial diversity and simplification of the soil microbial community composition inhibits many functions of the soil ecosystem, including plant diversity, plant nutrient conservation and absorption, and utilization of soil nutrients [5]. Therefore, the higher diversity and richness of the rhizosphere soil bacterial community after inoculation with PGPR are beneficial for the growth of alfalfa under field conditions.

Further analysis revealed that the inoculated alfalfa rhizosphere could recruit potentially beneficial bacterial taxa such as Acidobacteriota (Figure 5a, Table S1). Kielak et al. [41] inoculated Arabidopsis thaliana with three microbial strains belonging to Acidobacteriota and demonstrated that the biomass of shoots and roots increased. In the present study, the key bacterial taxa in the rhizosphere of alfalfa in response to inoculation under field conditions were identified using LEfSe; among them, the key bacterial taxon in the rhizosphere that responded to LJL-12, MJM-5, and MJM-5+LJL-12 was Arthrobacter (Figure 6, Tables S6–S8). Stassinos et al. [42] found that Arthrobacter can promote the growth of plants under salt stress and increase its proline content to enhance its salt tolerance. The present study also observed that specific key bacterial taxa were produced in the rhizosphere under different inoculation treatments. Sphingomonas was the key bacterial taxon in the rhizosphere soil inoculated with LJL-12 and MJM-5+LJL-12, and its relative abundance was significantly higher than that in the Control (Figure 6a,c; Tables S6 and S8). In addition, Bacillus and Streptomyces were also the key taxa, and their relative abundances after co-inoculation were significantly higher than that in the Control (Figure 6c, Table S8). Sphingomonas can promote plant growth and improve plant tolerance to abiotic stress by producing phytohormones [43]. Bacillus can improve plant growth under abiotic stress by producing plant hormones or promoting the absorption of insoluble phosphorus as well as indirectly promoting plant growth by inhibiting pathogenic fungi and inducing plant systemic resistance [44]. Streptomyces can promote plant growth under salt stress by producing volatile organic compounds and thiopeptides [45,46]. In addition, some unique beneficial bacteria that promote plant growth under field conditions, such as Acinetobacter, were only present in the rhizosphere under the co-inoculation treatment (Figure S6b, Table S5). Acinetobacter plays a key role in improving plant growth by producing plant hormones and siderophores as well as by dissolving phosphate [47]. The enrichment of beneficial bacterial taxa in the rhizosphere after inoculation with microbial inoculants may be attributed to changes in the composition and concentration of plant root exudates, which can play a positive role in the composition of the rhizosphere microbial community [33]. Our findings indicated that some bacterial taxa that benefit plant growth were enriched in the rhizosphere soil inoculated with PGPR, especially in the co-inoculation treatment. Some unique beneficial bacteria were only present in the inoculation treatments.

In this study, PGPR inoculation significantly increased the moisture content of alfalfa rhizosphere soil, decreased the rhizosphere soil bulk density, and changed the physical properties of the alfalfa rhizosphere soil (p < 0.05) (Table 2). Caravaca et al. [48] reported that inoculation with Glomus intraradices reduced the bulk density in the rhizosphere of Rhamnus lycioides seedlings. A decrease in soil bulk density can result in an increase in soil porosity, which has a positive effect on plant growth [49]. Our results revealed that PGPR inoculation significantly increased the content of organic matter and available nutrients in the rhizosphere soil of alfalfa, with co-inoculation being significantly more effective than single inoculation (p < 0.05) (Table 2). Microbial inoculation can increase the content of soil organic matter by improving soil structure and increasing the diversity of soil microbial communities; thus, the increase in soil organic matter by PGPR inoculation may be related to the improvement of plant growth and soil quality [50]. Our study also found that some rhizosphere soil properties were significantly correlated with the major bacterial raxa after PGPR inoculation. For example, there was significantly positive correlation between the content of organic matter and the relative abundances of Planctomycetota, Actinobacteria, and Arthrobacter in the rhizosphere soil under the co-inoculation treatment (Table S13). Previous study reported that some microbial taxa in soil can drive soil nutrient cycling processes, which in turn contribute to the soil nutrient status, plant productivity, and environmental sustainability [51]. Soil enzyme activity is one of the most active organic components in the soil and participates in most biochemical reactions in soil ecosystems. The study showed that PGPR inoculation significantly increased the activities of urease, sucrase, dehydrogenase, and protease in the rhizosphere soil of alfalfa, and the co-inoculation with PGPR had a more significant effect than that of single inoculation (p < 0.05) (Table 3). Inoculation with Azospirillum brasilense and Burkholderia can increase urease activity in tomato rhizosphere soils [52]. Ren et al. [53] reported that inoculation with Bacillus megaterium significantly increased the activities of sucrase and urease in Eucalyptus rhizosphere soil and that microbial inoculants had a significant positive correlation with soil enzyme activities, which was conducive to maintaining the stability of the soil microecosystem. Moreover, soil enzyme activities can directly or indirectly affect the change in the microbial community as well as regulate the growth of plants [54]. PGPR can promote the decomposition of organic matter in the soil and provide nutrients for enzyme-induced reactions in the soil, which is conducive to the growth of microorganisms, thus improving enzyme activity and nutrient content in rhizosphere soil and providing a suitable soil microenvironment for plant growth [55]. Lehmann et al. [54] showed that soil enzyme activities not only affect the changes in soil nutrients but also drive the changes in the microbial community directly or indirectly, thus affecting the growth of plants. Our findings suggested that the increase in soil available elements and enzyme activities related to plant growth promotion, especially in co-inoculation, may be beneficial to the growth of alfalfa under field conditions.

5. Conclusions

PGPR inoculation—especially co-inoculation of alfalfa rhizosphere under field conditions—could improve the metabolic activity and functional diversity of the soil microbial community in relation to carbon source utilization and significantly increase the diversity and richness of the soil bacterial community. Inoculation with PGPR, which are the key bacterial taxa that promote plant growth, were significantly enriched. Soil physicochemical properties and enzyme activities were also significantly improved after co-inoculation, thereby improving the microecological environment. Our findings suggested that changes in the diversity, composition, and carbon source metabolism activities of rhizosphere soil microbial communities may explain why inoculation with PGPR promotes the growth of alfalfa under field conditions.

Author Contributions

Writing–original draft preparation, L.T.; formal analysis, Y.S.; methodology, Y.Z.; writing–review and editing, D.Y.; conceptualization, C.G.; All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Tables

Figure 1. Microbial metabolic activity on carbon sources in the rhizosphere of alfalfa under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments: (a) average well color development (AWCD); (b) functional diversity indices; (c) PCA of carbon source utilization. Lowercase letters indicate statistically significant differences among the treatments (p < 0.05). The R and p values based on ADONIS indicate significant differences between the treatments.

Figure 2. Utilization of 31 carbon substrates by the soil microbial community under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments. Different color boxes represent the average optical densities of the microbial community. The color indicator on the right side of the heatmap depicts the alteration in the carbon source availability.

Figure 3. Alpha-diversity indices of soil bacterial community in the rhizosphere of alfalfa under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments: (a) Shannon; (b) Sobs; (c) Chao; (d) Ace. Different lowercase letters indicate statistically significant differences among the treatments (p < 0.05).

Figure 4. Beta-diversity of soil bacterial community in the rhizosphere of alfalfa under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments. The two-dimensional ranking of the PCoA was based on the Abund–Jaccard analysis. The R and p values based on ADONIS indicate significant differences in bacterial community composition between the treatments.

Figure 5. Bacterial community composition in the rhizosphere of alfalfa under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments at (a) the phylum level (others: relative abundance < 0.1%) and (b) the class level (others: relative abundance < 0.4%).

Figure 6. Key bacterial taxa of alfalfa rhizosphere soil under different treatments: (a) Control and LJL-12 treatments; (b) Control and MJM-5 treatments; (c) Control and MJM-5+LJL-12 treatments. LEfSe analysis depicting differentially abundant taxa as biomarkers using the Kruskal–Wallis test with an LDA score > 3.0. The circles from the inside to the outside indicate bacterial taxonomic levels from phylum to genus. The colored circles corresponding to different treatment groups in the figure represent the key bacterial taxa that were significantly enriched under that treatment group, and the bacterial taxa that were significantly enriched under different inoculation treatment are detailed in Supplementary Tables S6–S8.

Table 1

Results of 16S rRNA gene high-throughput sequencing of soil bacterial community in the rhizosphere of alfalfa under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments.

Treatment	Optimized Sequence	Effective Sequence	Base Number	Mean Length
Control	32,756.667	19,693.333	12,972,184.670	396.014
LJL-12	41,055.333	20,677.333	16,285,595.330	396.643
MJM-5	43,178.000	32,247.000	16,557,306.000	383.500
MJM-5+LJL-12	44,978.667	30,579.000	17,567,318.000	390.174

Table 2

Physical and chemical properties of the soil in the alfalfa rhizosphere under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments.

Treatment	MC(%)	BD(g·cm−3)	OM(g·kg−1)	AN(mg·kg−1)	AP(mg·kg−1)	AK(mg·kg−1)
Control	20.5 ± 1.9 c	1.3 ± 0.1 a	29.1 ± 1.1 c	119.6 ± 3.6 c	27.1 ± 2.8 c	202.8 ± 10.2 c
LJL-12	23.8 ± 1.1 b	1.2 ± 0.1 b	32.6 ± 1.4 b	140.6 ± 4.4 b	32.8 ± 2.2 b	231.6 ± 10.2 b
MJM-5	23.9 ± 1.3 b	1.2 ± 0.1 b	31.7 ± 0.9 b	142.3 ± 3.6 b	33.4 ± 2.7 b	234.4 ± 9.4 b
MJM-5+LJL-12	26.4 ± 0.9 a	1.2 ± 0.1 b	34.9 ± 0.8 a	150.5 ± 3.5 a	38.0 ± 1.7 a	255.4 ± 9.4 a

The values are presented as the mean ± standard deviation. Different letters represent significant differences among the treatments (p < 0.05). MC: moisture content; BD: bulk density; OM: organic matter; AN: available nitrogen; AP: available phosphorus; AK: available potassium.

Table 3

Urease, sucrase, dehydrogenase, and protease activities of the alfalfa rhizosphere soil under Control, LJL-12, MJM-5, and MJM-5+LJL-12 treatments.

Treatment	Urease Activitymg (NH3-N)·g−1·24 h−1	Sucrase Activitymg·g−1·24 h−1	Dehydrogenase Activitymg·TPF·g−1·24 h−1	Protease ActivityNH2-N mg·g−1·24 h−1
Control	16.0 ± 1.1 c	3.1 ± 0.1 c	2.0 ± 0.1 c	5.5 ± 0.2 c
LJL-12	18.7 ± 1.1 b	3.6 ± 0.1 b	2.2 ± 0.1 b	6.1 ± 0.2 b
MJM-5	18.9 ± 1.3 b	3.6 ± 0.1 b	2.2 ± 0.1 b	6.2 ± 0.2 b
MJM-5+LJL-12	21.3 ± 0.8 a	3.9 ± 0.1 a	2.4 ± 0.1 a	6.6 ± 0.2 a

The values are presented as the mean ± standard deviation. Different letters represent significant differences among the treatments (p < 0.05).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d15040537/s1.

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