1. Introduction
Phosphorus is a vital element that plays an important role in the metabolic activities and various functions of plants. Phosphorus is the second most essential nutrient after nitrogen and contributes to energy transfer and storage, which is a critical material basis for plant growth and development [1]. However, more than 90% of phosphorus in soils is bound to metal ions such as calcium (Ca2+), aluminum (Al3+), and iron (Fe3+) to form insoluble phosphate forms, which cannot be directly absorbed by plants [2], resulting in low phosphorus bioavailability in soils and widespread phosphorus limitation in different types of ecosystems [3]. As a result, phosphorus deficiency severely limits crop growth and yield [4]. To achieve higher crop production, phosphorus fertilizers are often applied continuously, with more than 15 million tons of phosphorus fertilizer applied globally each year [5]. However, the majority of phosphorus fertilizers accumulate in the soil rather than being used by plants, which not only increases production costs but also poses potential environmental risks. Previous research has shown that only 20% of phosphate fertilizer is absorbed by crops [6]. Therefore, there is a need to improve the efficiency of phosphorus fertilizer availability to meet the growing demand for food and avoid phosphorus pollution.
Phosphorus-solubilizing microorganisms are a class of beneficial microorganisms that can degrade insoluble inorganic and organic phosphorus, mainly bacteria but also fungi and archaea [7]. In recent years, there has been a significant increase in research interest in phosphate-solubilizing bacteria (PSB). The use of PSB to improve the efficiency of soil phosphorus fertilizer and promote crop growth has become an effective approach to address the issue of phosphorus resource utilization [8]. PSB are ubiquitous in soils, and their abundance varies according to the soil matrix, vegetation type, and climatic conditions. It has been shown that PSB have obvious effects in rhizosphere soil. They can colonize the rhizosphere of crops and are an important part of the growth-promoting bacteria in the rhizosphere. The phosphorus they convert becomes an important source of phosphorus required for plant growth [9]. Studies have shown that they reduce the pH value of the culture medium by secreting low-molecular-weight organic acids such as citric acid and lactic acid, thereby solubilizing insoluble phosphates. Ahuja et al. (2007) found that PSB Paecilomyces marquandii produces protons through ammonia assimilation and exerts phosphorus-solubilizing action without secreting acidic substances [10]. In addition, PSB also produce phosphatases and other enzymes that participate in the soil phosphorus cycle, thereby improving the bioavailability and uptake efficiency of phosphorus [11]. Understanding the role of PSB in the biogeochemical cycle of phosphorus is crucial.
In addition to dissolving insoluble phosphate, some phosphorus-solubilizing microorganisms can also act as plant growth-promoting rhizobacteria (PGPR), which can promote crop growth by producing various plant hormones in the plant rhizosphere. A pot experiment under drought stress showed that two PGPR strains (Enterobacter aerogenes and Pseudomonas fluorescens) can reduce the adverse effects of salinity on maize growth [12]. PGPR also affect the growth of maize in polluted soil environments. For example, Ijaz et al. (2018) discovered that PGPR can improve the growth of maize irrigated with wastewater from sugar mill [13]. PSB can promote plant nutrient uptake and stimulate plant growth and development by secreting auxin indole-3-acetic acid (IAA) [14]. PSB have roles that are shared by PGPR, such as potassium dissolving, nitrogen fixing, and the production of siderophores. Moreover, the addition of PSB may alter the distribution of soil microorganisms, leading to the enrichment of microorganisms beneficial to crop growth. Some scholars inoculated the PSB Pantoea agglomerans in semi-arid soil and found that it reduced soil pH, increased the available phosphorus content of the soil, and significantly changed the structure and composition of the soil bacterial microbial community, with a significant increase in the relative abundance of Firmicutes [15]. Burkholderia sp. plays a role in the prevention and control of maize diseases [16], as well as in the study of phosphatase activity [17]. Inoculation with Acinetobacter radiasistens can increase the plant height, stem diameter, and fruit yield of cucumbers [18].
Maize is the third-largest staple crop in the world [5]. Rhizosphere microbes are thought to play a particularly important role in regulating plant nutrient supply [19]. The utilization efficiency of phosphorus fertilizer in the maize rhizosphere soil is low [20], and understanding phosphorus dynamics in the maize rhizosphere is critical for sustainable crop production. Therefore, this study aims to (1) isolate, screen, and identify highly efficient phosphorus-dissolving strains in maize rhizosphere soil and evaluate the biological and growth-promoting characteristics; (2) explore the effect of PSB on maize seedlings through pot experiments; and (3) reveal the effects of changes associated with single and mixed application of PSB on the microbial community structure of maize rhizosphere soil by sequencing.
2. Materials and Methods
2.1. Isolation, Purification, and Identification of PSB
Maize rhizosphere soil was collected from Lanzhou City, Gansu Province, China. The soil sampling process followed the method described by Luo et al. (2021) [21]. Soil samples at 0–15 cm depth in each plot were collected by five sampling stations, thoroughly mixed to form a composite sample, then shipped to the laboratory immediately. Ten grams of fresh soil were added to a 100 mL Erlenmeyer flask, and 90 mL of sterile water was added. The flask was then placed on a shaker for 30 min to obtain a 10-fold diluted soil suspension. Subsequently, a series of soil suspensions at different dilution concentrations (10−2, 10−3, 10−4, 10−5, and 10−6) was prepared and spread on the National Botanical Research Institute’s Phosphate (NBRIP) agar medium to study the phosphorus-solubilizing capacity of the strains. The composition of the NBRIP agar medium was as follows: to 1 L added 10 g glucose, 5 g Ca3(PO4)2, 5 g MgCl2, 0.25 g MgSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, and 15 g agar. The strains were incubated at 30 °C for 4 days, and those showing a transparent circle were selected for streak purification. The diameter of the transparent circle (D) and the diameter of the colony (d) were measured, where D/d indicates the phosphate-dissolving effect of the strain. Additionally, the molybdenum blue colorimetric method described by Meador et al. (2020) was used to make a phosphorus standard curve with 5 mg/L phosphorus (KH2PO4) standard solution [22]. The available phosphorus content and pH of the screened strains were determined on the second and fifth days.
The screened strains were inoculated into LB medium and incubated at 30 °C. Morphological characteristics such as colony size, color, and shape were observed. The physiological and biochemical characteristics of the screened strains were investigated by using a micro-biochemical test reaction plate. Afterwards, the strains were sent to Gene Denovo Biotechnology (Guangzhou, China) for identification. The obtained DNA sequences were submitted to the NCBI database for BLAST homology searches (
2.2. Activity Determination of PSB
The phosphorus-solubilizing ability, IAA production, nitrogen-fixing capacity, potassium-solubilizing capacity, and siderophore production of the isolated PSB Z-7, J-1, and B-6 were determined.
2.2.1. Determination of Phosphorus-Solubilizing Capacity of PSB on Various Insoluble Phosphorus Sources
An insoluble phosphorus source medium was made by replacing calcium phosphate with the same mass of aluminum phosphate and ferric phosphate in the NBRIP medium. Strains B-6, Z-7, and J-1 were cultured and inoculated into the phosphate (lecithin) medium, aluminum phosphate medium, and ferric phosphate medium and cultured in a shaker at 180 r/min and 30 °C for 5 days. The available phosphorus content was determined on the second and fifth days. Sterile distilled water was used as a blank control.
2.2.2. Determination of IAA Production
To study plant-promoting properties such as IAA [23], the strain was inoculated in LB liquid medium (containing 200 mg/L L-tryptophan) and incubated at 180 r/min for 48 h at 30 °C on a shaker. Then, the bacterial solution was centrifuged at 10,000 r/min for 10 min. The supernatant was mixed with 500 μL of Salkowcki’s reagent in equal volume and left to stand for 30 min in the dark. The color turned red, indicating the generation of IAA. The absorbance value was measured at 530 nm using a UV-2102 spectrophotometer (Unico Instrument Co., Ltd., Shanghai, China), then substituted into the IAA standard curve to calculate the IAA production of the strain.
2.2.3. Determination of Nitrogen-Fixing Capacity, Potassium-Solubilizing Capacity, and Siderophore Production
The strains were activated by streaking and inoculated into LB liquid medium for overnight incubation until they reached the logarithmic phase, and the bacterial suspensions were prepared. Afterward, 2.5 μL of the suspension was spotted on nitrogen-free Ashby medium [24], fermentation medium [25], and Chrome Azurol S (CAS) agar [26]. The growth and size of the hydrolysis circle were observed to determine the strains’ abilities in nitrogen fixation, potassium solubilization, and siderophore production.
2.3. The Plant Growth-Promoting Ability of PSB—A Pot Experiment
The test soil was taken from Qilihe District, Lanzhou City, Gansu Province (36°1′37″ N, 103°41′53″ E). The test maize seed was “Zhengdan 958”. The antagonism test of PSB was performed first. Strains B-6, Z-7, and J-1 were cross-lined on LB solid medium after activation to observe colony formation. There was no antagonism between the strains (Figure S1).
The standard seed germination test was then carried out. Maize seeds of uniform size were selected, first rinsed clean of surface dirt with tap water and rinsed 3–4 times with sterile water, then soaked in 75% ethanol for 5 min. The seeds were then sterilized by soaking in 5% sodium hypochlorite solution for 5 min and, finally, washed with sterile water for later use. Maize seeds were separately placed in the bacterial suspension (B-6, Z-7, and J-1) with a concentration of 1 × 108 CFU/mL and soaked for 1 h, and the blank control was sterile water. After soaking, the seeds were transferred to petri dishes with sterile filter paper at the bottom with a regent. Then, 10 mL of sterile water was added to each petri dish to wet the filter paper, and 15 maize seeds were evenly placed into the petri dishes. Then, the samples were put into an artificial climate chamber with the temperature set to 28 °C and the humidity at 60% for dark cultures, during which sterile water was added as appropriate to keep the petri dishes moist. Maize seeds were picked up after 7 days.
Finally, the PSB pot experiment was carried out using a completely randomized design. A total of six groups were set up, namely CK (control treatment), P (phosphorus fertilizer), B-6 (strain B-6 added), Z-7 (strain Z-7 added), J-1 (strain J-1 added), and consortium (B-6 + Z-7 + J-1 added) treatments, each with three replicates. Nitrogen and potassium fertilizers were applied to each treatment at N 0.15 g/kg soil and K2O 0.15 g/kg soil, and phosphorus fertilizer was applied to the P treatment alone at P2O5 0.1 g/kg soil as a positive control. Sterilized maize seeds were germinated in sterile water for 2 days. Ten seeds with equal germination effect were selected and soaked in bacterial suspension (sterile water for CK and P treatments) for 2 h before sowing in pots (upper aperture 18 cm × lower aperture 13 cm × height 16 cm) with 2 kg of soil per pot. The seeds were watered regularly after sowing. Ten days after sowing, 5 maize seedlings with similar growth were left in each pot, 10 mL of the corresponding bacterial solution (OD600 ≈ 1) was applied to each maize plant on the 10th and 20th days by root irrigation (sterile water was used for the CK and P groups), and the physiological indices of the maize plants were measured after harvesting the maize plants on day 30.
The harvested maize roots were cleaned with tap water and dried naturally; the fresh weight, plant height, and root length were measured; the number of roots was countered; and the stem thickness of the maize was determined using a caliper. Morphological metrology data consist of nine replicates calculated through standard error. Chlorophyll content was determined using the ethanol acetone method [27]. Malondialdehyde (MDA) content and soluble sugar content were determined using a malondialdehyde assay kit and a plant soluble sugar content kit (Solarbio, Beijing, China). The leaf catalase activity (CAT) was determined by the method corresponding to the catalase detection kit (Beyotime, Shanghai, China). Soluble protein content was measured using Coomassie brilliant blue staining. The physical and chemical properties of the collected maize rhizosphere soil samples were analyzed. Total phosphorus (TP) and total potassium (TK) were measured by an elemental analyzer (Analyzer, Puchheim, Germany). Available potassium (AK) was measured by flame photometry [28]. Available phosphorus (AP) was determined by the molybdenum-antimony colorimetric method [29]. Soil ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) contents were determined using an automatic analyzer (SEAL-AA3, Ludwigshafen, Germany). Alkaline phosphatase activities were determined by p-nitrophenyl phosphate disodium as the substrate [30]. Urease activity (UE) was measured by sodium phenolate sodium hypochlorite colorimetry [31]. The soil catalase activity was determined by potassium permanganate titration [32].
2.4. High-Throughput Sequencing and Data Analysis
Soil DNA was extracted according to the FastDNA® SPIN Soil Kit (MP Biomedicals, Irvine, CA, USA) operating instructions. Subsequently, DNA concentration and quality were assessed using a Nanodrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA). Primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′) with a V3-V4 hypervariable zone were performed by Guangzhou Kidio Biotechnology Co., Ltd. (Guangzhou, China). Sequencing and data processing procedures were the same as previously described [33]. Experimental data were collated and analyzed using Excel 2019 (Microsoft, Redmond, WA, USA), and ANOVA was performed using IBM SPSS 26.0 with a confidence level of 95% (α = 0.05). The least significant difference (LSD) method was then used for means comparison. Plotting was performed using Origin 9.1 software (OriginLab, Northampton, MA, USA).
3. Results and Discussion
3.1. Screening and Identification of PSB
Three strains (named J-1, Z-7, and B-6) with a good phosphorus-solubilizing effect were screened by NBRIP screening medium. Strains J-1, Z-7, and B-6 exhibited varying degrees of phosphorus-solubilizing transparent circles after 4 days of cultivation on the medium, with D/d values of 1.24 ± 0.01, 3.90 ± 0.11, and 3.16 ± 0.17, respectively. The strains were activated to determine the content of organic phosphorus and pH (Table S1). The results show that B-6 has higher organic phosphorus content than J-1 and Z-7. The pH of the culture solution decreased to different degrees as cultivation progressed, and the decrease was more pronounced in the culture solution of strain Z-7. This may be due to the production of low-molecular-weight organic acids such as acetic acid and gluconic acid by PSB, leading to a decrease in pH [34]. These three strains are all Gram-negative bacteria, and the physiological and biochemical characteristics are shown in Table S2. The 16S rDNA sequence results of strains J-1, Z-7, and B-6 were analyzed using the NCBI database for BLAST homology comparison. The results showed that strains J-1, Z-7, and B-6 were identified to Pantoea sp. (100% homology), Burkholderia cepacia (99% homology), and Acinetobacter baumannii (99% homology), respectively. The GenBank accession numbers of strains J-1, Z-7, and B-6 are OR976177, OR987444, and OR975656, respectively. This is similar to the studies of Son et al. (2006) [35] and You et al. (2020) [36], who found that strains of Pantoea cenocepacia and Burkholderia contaminans were capable of generating phosphorus solubilization circles in the rhizosphere of soybean and maize, respectively. The phosphorus solubilization indices of Burkholderia cenocepacia and Burkholderia contaminans isolated from maize rhizosphere soils in South Korea were 1.9 and 1.6, respectively [16]. Additionally, the strain Burkholderia cenocepacia isolated from tobacco rhizosphere, had a solubilization index of 2.6 [37]. The phosphorus solubilization index of the strain Burkholderia cepacia screened in this study was 3.9, which is higher than that reported in previous studies of the same type. In addition, the phosphorus solubility of Burkholderia cepacia isolated from the rhizosphere of maize in the Brazilian Cerrado Biome was 52.7 and 92.8 mg/L after 10 days [38]. These values are similar to the phosphorus solubility values of Burkholderia cepacia screened in our study.
3.2. Phosphorus-Solubilizing Characteristics and Growth-Promoting Function of PSB
The solubilization abilities of strains B-6, Z-7, and J-1 for insoluble phosphorus sources such as organophosphate (lecithin), aluminum phosphate, and ferric phosphate are shown in Table S3. The results show that the three strains had no significant effect on organophosphate. This could be because soil organic phosphorus has a weaker solid-phase affinity than inorganic phosphorus, making it more prone to leaching [39]. However, they had obvious solubilization effects on aluminum phosphate and ferric phosphate, and strain Z-7 works most efficiently in phosphorus solubilization, followed by J-1 and B-6. Phosphorus in the soil is easily converted into insoluble and inaccessible phosphates of iron (Fe) and aluminum (Al), which are not readily available to plants [40]. The phosphorus-solubilizing capacity of PSB depends on the nature of the microorganism itself but is also related to the type of phosphorus source. Aluminum phosphates and ferric phosphates are less soluble compared to calcium phosphates [5], but they still show significant solubility. The greater solubility of Ca-P compared to Al-P and Fe-P may be due to the combined effect of proton release and the secretion of organic acids.
The IAA production, nitrogen-fixing capacity, potassium-solubilizing capacity, and siderophore production of the PSB strains were determined to initially explore their growth-promoting capacity (Figure 1). The ability of the three strains to produce IAA was calculated based on the IAA standard curve (Figure S2). The IAA production of the three PSB strains in test tubes showed that strain J-1 had the highest IAA production ability (Figure 1d). There was a significant difference (p < 0.05) in the IAA production of strains B-6, Z-7, and J-1, which were 0.46 ± 0.11 mg/L, 0.16 ± 0.06 mg/L, and 5.23 ± 0.11 mg/L, respectively (Figure 1e). IAA is an important indicator for promoting plant growth. You et al. (2020) screened highly efficient growth-promoting bacteria Burkholderia cenocepacia CR318 in maize soil, with an IAA yield of 29.8 μg/L [36]. IAA production of 0.5 mg/L has also been reported for Pantoea sp, namely YR343 isolated from the rhizosphere of American Populus deltoides [41]. Of course, IAA production is affected by multiple factors, such as temperature and incubation time, which makes it challenging to compare IAA production across studies. Certain genes in nitrogen-fixing [42] and potassium-solubilizing bacteria [43] play important roles in enhancing plant nutrient uptake.
Incubation of three PSB strains on nitrogen-free Ashby medium for 4 days showed that strain Z-7 grew well, while the other two strains grew poorly, indicating that strain Z-7 has significant nitrogen-fixation capacity, whereas strains B-6 and J-1 do not (Figure 1a). Similar to our results, Burkholderia caballeronis was screened in tomato soil and found to induce effective nitrogen-fixing nodules on the roots of Phaseolus vulgaris [44]. Following incubation of three PSB strains on a fermentation medium for 4 days, strain Z-7 grew well, indicating its ability to solubilize potassium. In contrast, the other two strains did not show the same ability (Figure 1b). Following incubation of three PSB strains on CAS agar for 4 days, strain Z-7 grew well, followed by B-6, while J-1 was unable to grow normally, indicating that strain Z-7 had a significant ability to produce siderophore, while the ability of B-6 was slightly weaker and J-1 had no effect (Figure 1c). Siderophores are a class of low-molecular-weight metal ion chelates produced by microorganisms in the plant rhizosphere that mediate the transmembrane translocation and bioavailability of iron ions, which, in turn, regulate microbial metabolism and impact plant growth [45]. Therefore, it is of great significance to screen strains with high siderophore-producing functions. Burkholderia is known to produce iron siderophores (pyochelin and pyoverdine) [46]. Other bacteria that produce siderophores, such as Pseudomonas koreensis and Bacillus subtilis, have also been reported to be used as biocontrol agents [47].
3.3. Effect of PSB on Maize Seed Germination and Seedling Growth Promotion
A pot experiment was conducted to investigate the effects of adding PSB on the physiological and biochemical indices of maize leaves, as well as on maize growth. The purpose of the experiment was to determine the growth-promoting effects of the screened strains on maize. The antagonism test showed that there was no antagonism among the three strains. The germinated maize seeds were tested in pots, and the growth of maize in different treatments is shown in Figure 2e,f. The application of PSB promoted maize growth. Specifically, the fresh weight of maize under the consortium treatment, Z-7 treatment, and J-1 treatment were 30.07 ± 2.76 g, 30.00 ± 1.92 g, and 27.66 ± 0.75 g, corresponsing to increases of 23.04, 22.75, and 13.18%, respectively, compared with the CK (Figure 2a). However, the B-6 treatment did not have a significant promotion effect on the fresh weight of maize. In addition, the consortium treatment and Z-7 treatment promoted maize fresh weight equivalent to 86.55% and 86.35% of the P treatment, respectively (Figure 2a). The heights of the maize plants in the consortium treatment and Z-7 treatment were 58.38 ± 0.98 cm and 57.97 ± 1.64 cm, corresponsing to increases of 7.57% and 6.81%, respectively, compared to the CK, and the promotion effect was equivalent to 93.47% and 92.81% of the P treatment (Figure 2a), respectively. There was no significant difference in the plant height of maize between the J-1 and B-6 treatments compared with the CK.
The consortium treatment had the most significant promotion effect on the root length of maize, measuring 26.37 ± 1.21 cm. This length was 30.09% higher than that of the CK, followed by the B-6 treatment, with a root length of 21.38 ± 0.82 cm, which was 5.48% higher than that of the CK (Figure 2c). It should be noted that the promotion effects of the consortium and B-6 treatments on root length were equivalent to 149.49% and 121.20% of the P treatment, respectively. The consortium and Z-7 treatments had the largest stem thickness, but there were no significant differences compared to the other groups. In addition, the number of roots in all bacteria treatments increased to different degrees compared to the CK. The J-1, B-6, Z-7, and consortium treatments showed 16.95%, 9.69%, 4.84%, and 2.42% longer root lengths than the blank control, respectively. PSB can meet the phosphorus demand for plant growth by secreting organic acids, proton exchange, and enzymatic degradation to enhance phosphorus effectiveness and uptake efficiency [48]. Several physicochemical indicators of plant growth-promoting properties were identified and validated in pot tests. The results showed that our screened PSB with growth characteristics such as IAA promoted maize plant growth in terms of fresh weight, plant height, root length, and stem thickness. Acetobacter Pasturianus AJK-9 isolated from wheat rhizosphere soil in Pakistan significantly increased root length and shoot biomass [49]. Similarly, some researchers found that IAA-producing strains Sphingobium sp. SX14 and Paenibacillus sp. B1 significantly contributed to the root length and dry weight of the crop. It was found that the highly efficient PSB B1 mainly colonized the root surface and epidermal tissue by confocal laser scanning microscopy (CLSM) and survived by forming spores, which is a good alternative for biofertilizers [50]. Although the growth-promoting effects of the three PSB strains investigated in this study have been confirmed, further research is needed to explore their mechanisms of promoting maize growth and determine their relevance as biofertilizers.
The impact of screened strains on the physiological indices of maize plants was evaluated. The chlorophyll content of maize in the Z-7 and consortium treatments was 22.54% and 16.43% higher than that of the CK (Figure 2d). The MDA content of both the consortium and B-6 treatments was 0.016 μmol/L, which is 38.46% lower than that of the CK. The soluble sugar content of the consortium and B-6 treatments was 0.071 μmol/g and 0.074 μmol/g, respectively—23.66% and 20.43% lower than the CK, respectively. The consortium treatment significantly increased the soluble protein content and catalase activity, which were 17.35% and 18.55% higher than those of the CK, respectively, and the other treatments showed lower levels compared to the blank control (Figure 2b). Moreover, P treatment resulted in fewer soluble proteins compared to other treatments. Moderate use of phosphorus fertilizer can increase the soluble protein content in plants. However, excessive use of phosphorus fertilizer may lead to an excess of insoluble phosphorus, which is not conducive to the process of plant protein synthesis. Further research is needed on the concentration of phosphorus fertilizer that is beneficial for protein synthesis in maize growth. It is clear that the addition of the screened PSB altered the physiological and biochemical indices of maize. Strain Z-7 (Burkholderia cepacia) had the highest phosphorus content (Table S3) and the highest chlorophyll content in this study, consistent with the idea that chlorophyll content is positively correlated with phosphate-solubilizing activity [51]. It was previously shown that a gene of Burkholderia cepacia IS-16 encodes a functional portion of a phosphate transporter protein when expressed in Escherichia coli, thereby increasing phosphatase activity [17]. Furthermore, we observed a negative correlation between chlorophyll and AP, which may be due to phosphorus limiting chlorophyll synthesis and accumulation.
The addition of PSB had a significant effect on the physical and chemical properties of the maize rhizosphere soil (Table S4). Soil pH was significantly higher (p < 0.05) in both the bacteria and P treatments, and the soil TP content was significantly higher (p < 0.05) in the bacteria treatments compared to the CK. There was no significant difference in soil TK level among all groups. Soil AP content was enhanced in the Z-7, J-1, and consortium treatments but significantly decreased (p < 0.05) in the B-6 treatment compared to the CK. The consortium treatment significantly increased soil phosphatase activity by 12.64% compared to the CK. Moreover, Pearson correlation analysis was conducted on the physiological and biochemical indicators of maize plants and the physical and chemical properties of the rhizosphere soil (Figure 2g). The physiology and biochemistry of maize plants were more affected by soil pH, TP, TK, AP, NH4+-N, and NO3−-N. Plant height, fresh weight, and chlorophyll content were significantly positively correlated (p < 0.05) with soil pH. MDA content was significantly negatively correlated (p < 0.05) with soil pH, TK, and NO3−-N content and significantly positively correlated (p < 0.05) with TP, AP, and NH4+-N. The abundance and type of PSB were related to soil physicochemical properties [52]. PSB promoted peanut growth and improved soil quality in saline soils, including elevating the levels of available nitrogen (AN), AP, and AK, in addition to significantly increasing the activities of alkaline phosphomonoesterase, urease, and dehydrogenase [53]. In this study, the consortium treatment had the most pronounced effect on soil physicochemical properties and enzyme activities. Indeed, some PSB can secrete organic acids to dissolve phosphate. PSB ensure their own metabolism by absorbing nutrients from the rhizosphere, thereby executing PGPR effects [54]. Soil pH was significantly higher in both PSB and P treatments compared to the control, which may be because the screened PSB promote plant growth by enhancing plant utilization and uptake of nutrients rather than by secreting organic acids.
3.4. Effects of PSB on the Microbial Community Structure of Maize Rhizosphere Soil
The trend of the dilution curve tends to be stable (Figure S3), indicating that the sequencing data are highly reliable and can be analyzed subsequently. Microbial community composition was analyzed, and it was found that the main phyla in the maize rhizosphere soil were Proteobacteria (18.53–31.47%) and Acidobacteria (15.37–18.90%) (Figure 3a). Compared to the CK, the microbial community in the P treatment did not show significant changes. However, the bacterial treatments exhibited significant changes. Among them, compared to CK, the relative abundance of Proteobacteria increased by 69.10%, 61.74%, 48.47%, and 16.07% in the consortium, J-1, B-6, and Z-7 treatments, respectively. Inoculation with PGPR can significantly alter the microbial community [55], and our research also supports this finding. Jiang et al. (2021) found enrichment of Acidobacteria, Chloroflexi, and Planctomycetes in peanut rhizosphere soil after the addition of PSB (Providencia rettgeri) [53]. RB41, Lelliottia, Sphingomonas, and Acinetobacter were the dominant genera (Figure 3b). Among them, compared to the CK, the relative abundance of Lelliottia significantly increased in the J-1 treatment and consortium treatment (p < 0.05). Similarly, the relative abundance of Acinetobacter significantly increased in the B-6 treatment and consortium treatment (p < 0.05). In addition, compared to the CK, the relative abundance of the RB41 genus showed varying degrees of decline, while the Sphingomonas genus exhibited varying degrees of elevation in the other groups. Principal coordinate analysis (PCoA) based on OTUs showed that the bacterial communities of the B-6, J-1, and consortium treatments were significantly separated from that of the CK. This indicates that the addition of screened PSB significantly altered the composition of the maize rhizosphere soil (Figure 4a). A Venn diagram showed that the number of OTUs shared by several groups was 1478, and the number of unique OTUs in the CK, P, Z-7, B-6, J-1, and consortium groups was 1644, 1693, 1423, 1644, 1481, and 2134, respectively (Figure 4b). Compared to the CK, the number of OTUs in the consortium increased significantly, while the number of OTUs in individual strain groups remained unchanged or declined marginally.
A correlation heatmap was generated to analyze the relationship between the top 10 dominant bacteria and soil physical and chemical properties at both the phylum and genus levels (Figure 5). The results showed that soil TK, TP, AP, NO3−-N, UE, and CAT had a strong influence on the microbial community at the bacterial phylum level. Soil AP was significantly positively correlated with the relative abundance of Gemmatimonadetes (p < 0.01). Soil urease was significantly negatively correlated with the relative abundance of Proteobacteria (p < 0.01) and significantly positively correlated with the relative abundance of Acidobacteria, Bacteroidetes, Actinobacteria, and Nitrospirae (p < 0.05). Soil catalase was significantly positively correlated with the relative abundance of Patescibacteia (p < 0.05). In addition, soil TP, TK, NH4+-N, alkaline phosphatase (ALP), and UE had a significant influence on the microbial community at the bacterial genus level. Soil TP and NH4+-N were significantly positively correlated with the relative abundance of Sphingomonas (p < 0.05). Soil ALP was significantly negatively correlated with the relative abundance of Flavisolibacter (p < 0.05). Both soil physicochemical properties and the metabolic activities of microorganisms can enhance soil enzyme activities [56,57]. In this study, the relative abundance of the Proteobacteria phylum, Lelliottia genus, and Acinetobacter genus significantly increased in the consortium treatment, which may also be a significant factor contributing to the rise in soil phosphatase levels. However, soil ALP does not correlate with these species, so soil physical and chemical properties may have a more obvious impact on soil ALP. Soil urease was significantly positively correlated with Nitospira (p < 0.001) and negatively correlated with Lelliottia (p < 0.001). Soil urease participates in the soil nitrogen cycle [58]. Nitrospira is involved in multiple nitrogen cycle processes, including complete nitrification [59]. The significant positive correlation between soil UE and Nitospira in this study (p < 0.001) is consistent with the results.
In this study, PICRUSt2 was used to predict the function of the bacterial community in the maize rhizosphere, as shown in Figure 6. The prediction system involved a total of 6 categories of metabolic pathways, including metabolic and organic systems, and 33 subfunctions, such as amino acid metabolism, energy metabolism, and membrane transport, which exhibit rich functional diversity. The results showed that compared with the CK, the Z-7 treatment increased the level of all functional categories. On the other hand, the consortium treatment decreased the level of all functional categories. There was no significant difference observed between the other treatments. Tax4Fun metabolic pathway analyses of microbial communities showed the same results (Figure S4).
The use of PSB to convert insoluble phosphate into soluble phosphate for plant growth is the most common and effective solution to the problem of phosphorus deficiency in crops [60]. PSB can act as growth promoters without harming the environment [61]. The several strains screened in this study have been confirmed to have phosphorus solubilization ability and various growth-promoting properties, but it is still unknown which characteristic or several characteristics lead to the growth-promoting effects of these strains. Moreover, the plant growth-promoting effects exerted by PSB are sometimes uncertain. Laboratory culture and even the results obtained in pot experiments may not be applicable to actual agricultural production, so the preparation of bacterial fertilizers (strains J-1, Z-7, B-6, or J-1 + Z-7 + B-6) to verify their actual field effects is needed in the next step.
4. Conclusions
In this study, highly efficient PSB (Pantoea sp. strain J-1, Burkholderia cepacia strain Z-7, and Acinetobacter baumannii strain B-6) screened from the maize rhizosphere exhibited varying degrees of IAA production, nitrogen fixation, phosphorus solubilization, and siderophore production capacity. The pot experiment revealed that the addition of PSB enhanced the enzyme activity of maize rhizosphere soil and changed the physicochemical properties and bacterial community structure of the soil. It has been confirmed that the screened PSB have a growth-promoting effect on maize plants, and the effect is more pronounced when the three PSB strains are mixed. The PSB strains screened in this experiment are expected to be used as biofertilizers to improve the soil fertility of maize and increase maize production. These results provide a realistic basis for the utilization of PSB fertilizer and the development of sustainable agriculture. Further research can be carried out on the application of these strains in field crops.
Conceptualization, D.L., J.S., M.L., J.C., T.W., Q.Z., L.Y., N.Z. and Y.W.; Data curation, D.L., J.S. and M.L.; Formal analysis, Q.Z., and L.Y.; Funding acquisition, D.L., J.C., N.Z. and Y.W.; Investigation, N.Z.; Methodology, J.C., T.W., Q.Z. and L.Y.; Project administration, D.L., M.L., J.C., T.W., Q.Z., L.Y. and Y.W.; Resources, D.L., J.C. and Y.W.; Software, D.L., M.L., and N.Z.; Supervision, J.C. and Y.W.; Validation, D.L. and T.W.; Visualization, D.L. and J.S.; Writing—original draft, D.L.; Writing—review and editing, D.L., M.L., J.C., T.W., Q.Z., L.Y., N.Z. and Y.W. All authors have read and agreed to the published version of the manuscript.
The original data were uploaded to the NCBI database, and the numbers are provided in the article. Data will be made available on request.
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 1. Nitrogen-fixation, potassium-solubilizing, siderophore-producing, and IAA-producing capacity of strains B-6, Z-7, and J-1. (a) Nitrogen-free Ashby medium; (b) fermentation medium; (c) Chrome Azurol S (CAS) agar; (d) LB liquid medium containing 200 mg/L L-tryptophan; (e) IAA production of the strains.
Enlarge this image.Figure 2. Effect of different treatments on maize plant growth (e,f) and physiological and biochemical indicators. (a) Fresh weight and plant height; (b) soluble protein and leaf catalase; (c) root length, stem thickness, and number of roots; (d) malondialdehyde, soluble sugar, and chlorophyll; (g) the correlation between physiological and biochemical indicators of maize plant and soil physicochemical properties and enzyme activities. TP, total phosphorus; TK, total potassium; AP, available phosphorus; AK, available potassium; ALP, alkaline phosphatase; UE, urease activity; CAT, catalase activity. * Rrepresent p < 0.05; ** represent p < 0.01.
Enlarge this image.Figure 3. Bacterial community composition in the rhizosphere soil of maize under different treatments. (a) Phylum level; (b) genus level.
Enlarge this image.Figure 5. Pearson correlation analysis of the relative abundance and physicochemical properties and enzyme activities at the phylum level (a) and genus level (b) in maize rhizosphere soil. * Rrepresent p < 0.05; ** represent p < 0.01; *** represent p < 0.001.
Enlarge this image.Figure 6. Metabolic pathway analysis using PICRUSt2 analysis using 16S rRNA gene sequences. (a) CK; (b) P; (c) Z-7; (d) J-1; (e) B-6; (f) consortium.
Supplementary Materials
The following supporting information can be downloaded at:
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1 College of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou 730050, China;
2 School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China