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Article

Enhanced Plant Growth Through Composite Inoculation of Phosphate-Solubilizing Bacteria: Insights from Plate and Soil Experiments

by
Mengsha Li
1,*,†,
Xinjing Li
1,†,
Daosheng Xue
1,
Chengjiang Bao
1,
Keying Zhang
1,
Lili Chen
1,
Qiuping Li
1 and
Rui Guo
2
1
Ningbo Key Laboratory of Agricultural Germplasm Resources Mining and Environmental Regulation, College of Science and Technology, Ningbo University, Cixi 315300, China
2
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Institute of Environment, Resource, Soil and Fertilizer, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(11), 2461; https://doi.org/10.3390/agronomy14112461
Submission received: 11 September 2024 / Revised: 17 October 2024 / Accepted: 19 October 2024 / Published: 22 October 2024
(This article belongs to the Special Issue Role of Plant Growth-Promoting Microbes in Agriculture—2nd Edition)
Browse Figures
Versions Notes

Abstract

:
Excessive application of phosphorus (P) fertilizers does not alleviate P deficiency in soils and may cause water eutrophication. The available P in acidic soils is bound to minerals, such as iron and aluminum, in forms that are difficult to utilize by plants. The low availability of P is detrimental to soil health and crop growth. To address the P imbalance in the soil, different bioremediation techniques, such as phosphate-solubilizing bacteria (PSB) application, have been employed. However, the systematic analysis of the effects of composite inoculation of PSB on crops remains elusive. In this study, the effects of composite-inoculated PSB on plant growth were systematically evaluated by two scales: plate experiment and soil test. This study employed six different strains of PSB including Lelliottia amnigena 1-1 (A), Kluyvera intermedia 1-2 (B), Pseudomonas tolaasii 1-6 (C), Burkholderia cepacia 2-5 (D), Pseudomonas frederiksbergensis 2-11 (E), and Pseudomonas rhodesiae 2-47 (F). Among the 57 different combinations of these strains, four combinations (AE, AF, ADF, and AEF) indicated higher phosphate-solubilizing abilities than the single strains. These combinations were used for subsequent experiments. The plate experiment revealed that composite strains were more effective than single strains in promoting the growth and development of seedlings and roots of oilseed rape. Furthermore, AE, AF, and AEF combinations indicated excellent growth-promoting effects. Moreover, the soil test revealed that the composite inoculation of AE and AEF significantly enhanced biomass accumulation and root development in oilseed rape. The increased growth-promoting effects of the composite strains were observed to be associated with to their phosphate-solubilizing capacities. Both scales confirmed that compared to single inoculation, composite inoculation of PSB is more beneficial for plant growth. This study provides composite inoculation materials and foundational data to support the bioremediation of P imbalance in soil.

1. Introduction

Phosphorus (P) is an essential macronutrient for plant growth and development. It participates in various metabolic processes of plants in multiple forms and is a major component of many important compounds [1,2,3]. The P required by plants primarily comes from the soil. However, most soils have relatively low P bioavailability [4]. During agricultural production, P fertilizers are commonly utilized to increase crop yield [5,6]. However, its excessive use can negatively affect the availability of organic matter and other nutrients in the soil [7], thus disrupting the balance of essential nutrients needed for optimal crop growth [8]. Over time, this practice diminishes crop productivity, wastes significant P resources, and exacerbates environmental pollution [9,10]. This issue is particularly pronounced in acidic soils, which are rich in iron and aluminum minerals that strongly bind and fix P [11]. A large amount of P fertilizer is rapidly fixed by soil particles through processes such as adsorption and co-precipitation and converted into forms that are difficult for crops to absorb [12]. This results in the dual problem of insufficient available P and excessive fixed P, causing P imbalance in soil [13,14]. In China, high-concentration P fertilizers are excessively applied as a contemporary agricultural practice [15]. The P imbalance arises from the disparity between the nutrient release by fertilizers and the nutrient absorption capacity of crops, causing the underutilization of P fertilizers [16]. Bioremediation offers an environment-friendly solution for the soil nutrient imbalance [17,18,19,20].
Phosphorus-solubilizing microorganisms (PSMs) are functional microorganisms that can convert P in soil into soluble forms that are more readily absorbed by plants [21,22,23]. PSMs dissolve insoluble P through various mechanisms and play a crucial role in soil P cycling processes [24,25]. Several studies have demonstrated that PSMs enhance soil P availability, promote crop growth, and boost crop yield [26,27,28]. Compared to conventional P fertilizers, PSM-mixed fertilizers significantly improve P utilization, causing a 25–467% reduction in P usage [29,30]. The application of PSMs or related microbial fertilizers represents an environment-friendly approach to effectively reduce the reliance on chemical fertilizers while promoting crop productivity [31,32].
Previous research on PSMs was more focused on the effects of single strains, and limited attention has been given to the effects of synthetic microbial consortia of PSMs. Single PSM strains often fail to show satisfactory results due to their limited colonization abilities and solubilization potential [33,34]. Soil contains a rich and diverse array of microorganisms that compete intensely with the introduced microbes. Therefore, researchers are now focusing on the functions and application of synthetic microbial consortia. The synthetic consortia of PSMs possess superior competitive abilities for colonization in soil, demonstrating enhanced P solubilization and higher plant growth compared to single strains [35,36]. However, certain combinations of PSMs may have antagonistic interactions, reducing their P-solubilization ability and impairing their synergistic functionality [37]. Thus, when selecting combinations of PSMs, it is essential to consider the interactions among different strains. Furthermore, PSMs sometimes promote significantly different effects under different environmental conditions. For instance, strains with excellent phosphate-solubilizing abilities in the laboratory may not necessarily indicate good growth-promoting effects in soil [38,39,40]. Therefore, identifying effective combinations of PSMs and studying them systematically on different scales is crucial for adapting to the diverse forms of insoluble P in soil and complex soil environments. However, currently, systematic studies on how PSM consortia perform in plates and soil environments, especially compared to single strains, are lacking.
The objectives of this study were to identify highly effective combinations of phosphate-solubilizing bacteria (PSB) to construct synthetic consortia and to explore their effects on plant growth and potential application for bioremediation of P imbalance. This study analyzed six different strains of PSB, including Lelliottia amnigena 1-1 (A), Kluyvera intermedia 1-2 (B), Pseudomonas tolaasii 1-6 (C), Burkholderia cepacia 2-5 (D), Pseudomonas frederiksbergensis 2-11 (E), and Pseudomonas rhodesiae 2-47 (F). Various combinations of these strains were examined to construct composite strains with high P-solubilization capability. The effects of these consortia on plant growth were examined through both plate experiments and soil tests.

2. Materials and Methods

2.1. Bacterial Strains

This study used 6 PSB strains procured from China General Microbiology Culture Collection Center (CGMCC), including L. amnigena 1-1 (CGMCC No. 28671), K. intermedia 1-2 (CGMCC No. 28672), P. tolaasii 1-6 (CGMCC No. 28673), B. cepacia 2-5 (CGMCC No. 28674), P. frederiksbergensis 2-11 (CGMCC No. 28675), and P. rhodesiae 2-47 (CGMCC No. 28676). These strains were originally isolated from the acidic red soil of long-term rice-oilseed rape rotation in Ningbo, China (30.098005° N, 121.111118° E). The strains had neutral or synergistic interactions, with no antagonistic relationships observed.
Excluding the control group without adding bacteria and the experimental group with adding single strains, the number of PSB composite combinations is 57, which is calculated as ∁_6^2 + ∁_6^3 + ∁_6^4 + ∁_6^5 + ∁_6^6 = 57, including 15 combinations of two strains, 20 combinations of three strains, 15 combinations of four strains, 6 combinations of five strains, and 1 combination of six strains. Details can be seen in Table S1.

2.2. Soil

The soil used in the test was obtained from the same location in Ningbo, China, as mentioned in Section 2.1. The samples were collected from a soil depth of 0–20 cm. The soil was air dried and sieved through a 2 mm screen to remove the stones, plant debris, and large soil animals. The basic physicochemical properties of collected soil were as follows: total carbon: 19.62 ± 0.88 g/kg, total nitrogen: 1.83 ± 0.13 g/kg, ammonium nitrogen: 3.78 ± 0.21 mg/kg, nitrate nitrogen: 10.84 ± 0.73 mg/kg, total phosphorus: 1.51 ± 0.09 g/kg, total potassium: 15.25 ± 1.09 g/kg, and pH: 4.93 ± 0.05 [41].

2.3. Screening of PSB Combinations

Each of the 6 bacterial strains was inoculated into an LB liquid medium (10 g peptone, 5 g yeast extract, 10 g sodium chloride, and 1 L water) and grown at 30 °C with continuous shaking at 250 rpm for 18 h. The OD600 of each bacterial culture was measured. The bacterial cells were washed and suspended in sterile water, and then their OD600 was adjusted to 1. Based on the concentrations of each PSB strain at OD600 = 1 (Table S2), different strains were mixed in equal proportions. To prepare a 2-strain combination, the inoculum for each strain was set at half the amount of the corresponding single strain. For a 3-strain combination, the inoculum for each strain was set at one third of the corresponding single strain. Similarly, in a 4-strain combination, the inoculum for each strain was equal to one fourth of the corresponding single strain. For a 5-strain combination, the inoculum for each strain was set at one fifth of the corresponding single strain. Finally, in a 6-strain combination, the inoculum for each strain was equal to one sixth of the corresponding single strain. Each single strain or combination of strains was inoculated into National Botanical Research Institute’s phosphate (NBRIP) liquid medium [10 g glucose, 0.1 g (NH4)2SO4, 0.25 g MgSO4·7H2O, 0.2 g KCl, 5 g MgCl2, and 5 g Ca3(PO4)2 in 1 L water at pH 7.0] at a concentration of 107 cfu/mL and cultured at 30 °C with shaking at 200 rpm. Each strain or combination had 5 replicates. After 7 days of incubation, the soluble P content in the liquid media was measured using the molybdenum-antimony colorimetric method [42].

2.4. Plate Experiment

Based on the screening results, four combinations (AE, AF, ADF, and AEF) were selected from 57 combinations for further experiments. The single PSB strains were inoculated into an LB liquid medium, cultured at 30 °C and 250 rpm for 18 h. The bacterial suspension was centrifuged at 8000 rpm for 5 min. The cell pellets obtained after centrifugation were washed 3 times with sterile water, then resuspended in sterile water to obtain a 108 cfu/mL concentration. Equal proportions of each single strain were combined to keep the total concentration of the bacterial mixture equal to that of the single PSB strain.
A total of 9 treatments were set up for the plate experiment, including a control, 4 single strains (A, D, E, and F), and 4 composite strains (AE, AF, ADF, and AEF). The experiment consisted of 5 plates replicates per treatment, with 10 seeds in each plate. The seeds of oilseed rape (B. napus Zhongshuan No. 11) were surface sterilized using 75% ethanol and 2–4% sodium hypochlorite solution, washed with sterile water 4 times, and then soaked in the 9 different treatment solutions for 1 h. The treated seeds were then placed on a water agar medium (20 g of agar dissolved in 1 L of deionized water, sterilized at 121 °C for 20 min) and incubated at 28 °C. After germination, seeds were transferred at 25 °C and 16/8 h light/dark cycle conditions for continued growth. The seed germination rates were observed and recorded daily. The height and root length of the seedlings were measured on the 7th day of cultivation.

2.5. Soil Test

Like the plate experiment, 9 treatments were set in the soil test: control, A, D, E, F, AE, AF, ADF, and AEF, with 3 replicates per treatment. As mentioned in Section 2.2, single strains were mixed in equal proportions to construct synthetic consortia of strains, ensuring that the total concentration was the same as of the single strains. The seeds of oilseed rape (B. napus Zhongshuan No. 11) were washed with sterile water 3 times and incubated in the dark for 2 days at 30 °C for germination. Each pot, containing 80 g of soil, was inoculated with PSB at a concentration of 107 CFU/g dry soil. Soil moisture was adjusted to 60% of the maximum water-holding capacity. After 2 days of inoculation, 3 germinated oilseed rape seeds were planted in each pot, and the day was recorded as day 0. A total of 54 pots were placed in a climate chamber with a 16/8 h light/dark cycle and incubated at 25 °C. Sterile water was added regularly to maintain consistent moisture levels across all treatments. On the 15th and 30th days of cultivation, 3 replicates were sampled to measure the plant height, biomass, root structure, soil-available P content, and soil pH.
For determination of root characteristics, the roots were gently washed with water to remove soil without damaging the root system. Furthermore, root images were obtained using a root scanner (LA1600+ scanner, Regent Instruments, Inc., Quebec, QC, Canada) and analyzed with root analysis software (Winrhizo2017a, Regent Instruments, Inc., Quebec, QC, Canada) to determine the total root length, surface area, volume, average diameter, tip number, crossing number, and fork number. Soil-available P was extracted using ammonium fluoride-hydrochloric acid and measured by the molybdenum-antimony colorimetric method. Soil pH was measured in a soil: water mixture (1:2.5 w/v) using a pH meter (LEICI, Shanghai, China). The experimental design is shown in Figure 1.

2.6. Statistical Analysis

All experimental data were analyzed using Excel 2021 and SPSS 22.0. Duncan’s multiple range test was employed for testing the significance of differences between the mean values among different treatments (p < 0.05) via one-way analysis of variance (ANOVA). Figures were created using the Microsoft Visio software 2021 and Origin 2022. Moreover, principal component analysis (PCA), correlation analysis, and data visualization were performed using ChiPlot (https://www.chiplot.online/) (accessed on 1 September 2024).

3. Results

3.1. Screening of PSB Combinations and Evaluation of Their Phosphate-Solubilizing Capacities

The initial screening results of 57 PSB combinations are presented in Table S3. The AE, AF, ADF (suspected), and AEF combinations were selected for the subsequent experiments. It was observed that the phosphate-solubilizing capacities of AE, AF, ADF, and AEF combinations were significantly higher compared to the single strains A, D, E, and F, reaching 561.2 ± 14.09 mg/L, 514.2 ± 12.01 mg/L, 487.9 ± 10.67 mg/L, and 592.8 ± 20.81 mg/L, respectively (Figure 2). Furthermore, the addition of A significantly reduced the pH of the combination bacterial solution, with no significant differences among the four composite strain groups (Figure S1). Based on these results, AE, AF, ADF, and AEF combinations were selected for subsequent experiments.

3.2. Growth-Promoting Effects of Composite Strains in Plate Experiment

The effects of PSB composite strains on the germination rate and growth of oilseed rape seeds in the plate experiment are shown in Figure 3. Both single and composite strains significantly increased the seed germination rates. However, no significant differences were observed between the seed germination rates of composite and single-strain groups (Figure 3A). The germination rates of AE, ADF, and AEF groups increased significantly by 68.42 ± 14.89%, 68.42 ± 14.89% and 68.42 ± 8.59%, respectively, while the germination rate of the AF group was 57.89 ± 13.59% higher than the control group.
After the treatment with bacterial suspensions, the heights of oilseed rape seedlings in the E, F, AE, AF, ADF, and AEF groups were significantly increased compared to the control group. The root lengths in the A, E, F, AE, AF, ADF, and AEF groups were 51.92 ± 17.09%, 53.85 ± 13.87%, 75.00 ± 5.09%, 136.5 ± 18.55%, 101.9 ± 13.32%, 123.1 ± 8.38%, and 146.2 ± 13.46% higher, respectively, than the control group (Figure 3A,B). The effects of composite strains on root growth were more pronounced than those on the seedling height.

3.3. Growth-Promoting Effects of Composite Strains in Soil Test

3.3.1. Soil-Available Phosphorus Content and pH

In the soil test, the content of soil-available P in the D, E, AE, and AEF groups significantly increased by 22.95 ± 2.45%, 23.48 ± 4.86%, 26.07 ± 2.58%, and 20.71 ± 1.89%, respectively, on the 15th day of cultivation, as compared to the control group (Figure 4A). On the 30th day, the available P contents in all treatment groups were significantly higher than that in the control group, with the E, AE, and AEF groups indicating notable increases of 18.61 ± 1.92%, 19.71 ± 0.60%, and 18.98 ± 0.94%, respectively (Figure 4B). However, there were no significant differences in soil pH among the different treatment groups during the cultivation period (Figure 4A,B).

3.3.2. Growth Status of Oilseed Rape

After treatment with single PSB strains or synthetic consortia of strains, the height of oilseed rape in the D, E, AE, AF, and AEF groups was significantly increased by 37.39 ± 2.27%, 40.71 ± 15.33%, 42.04 ± 7.55%, 30.09 ± 4.79%, and 30.09 ± 5.63%, respectively, compared to the control group on the 15th day of cultivation (Figure 5). On the 30th day, the plant height in the D, E, AE, AF, and AEF groups was 20.25 ± 1.62%, 38.96 ± 5.07%, 22.09 ± 5.24%, 15.03 ± 5.01%, and 16.41 ± 2.53%, respectively, higher than the control group. The plant height in the E group was significantly greater than that of the other four groups (Figure 5). Moreover, the plant heights in the A, F, and ADF groups were not markedly different than those in the control group throughout the cultivation period (Figure 5).
On the 15th day of cultivation, the fresh weights of above-ground biomass of oilseed rape in all treatment groups were significantly higher than those in the control group, with the D, E, AE, AF, and AEF groups indicating an increase of 145.7 ± 13.87%. 137.0 ± 31.85%, 147.0 ± 28.53%, 117.1 ± 15.59%, and 152.4 ± 7.87%, respectively (Figure 6A). The dry weights of above-ground biomass in all treatment groups (except for A and ADF) were significantly higher than those of the control group (Figure 6B). In addition, the fresh weights of underground biomass in the D, AE, AF, ADF, and AEF groups showed significant increases of 148.1 ± 14.19%, 184.0 ± 19.85%, 104.6 ± 5.54%, 109.7 ± 28.82%, and 177.5 ± 23.99%, respectively, compared to the control group (Figure 6A). The dry weights of underground biomass in all inoculated treatment groups (except for the A group) were substantially higher than those of the control group (Figure 6B).
On the 30th day of cultivation, both the fresh and dry weights of above-ground biomass of oilseed rape in the E, AE, AF, and AEF groups were significantly higher than those in the control and other treatment groups. The fresh weights were observed to be increased by 39.83 ± 7.06%, 35.18 ± 9.74%, 38.41 ± 4.17%, and 32.79 ± 5.69%, respectively, while the dry weight increased by 51.59 ± 7.35%, 54.78 ± 1.74%, 39.49 ± 6.89%, and 37.58 ± 8.62%, respectively, compared to the control group (Figure 6A,B). Furthermore, the fresh weights of the underground biomass in the E, AE, and AF groups indicated a substantial increase of 37.17 ± 8.29%, 30.00 ± 5.08%, and 22.01 ± 4.14%, respectively, while the dry weights in the E, AE, AF, and AEF groups significantly increased by 50.60 ± 10.31%, 41.11 ± 5.23%, 28.37 ± 1.61%, and 35.10 ± 6.53%, respectively, as compared to the control (Figure 6A,B). The other four treatment groups showed no significant differences in the plant biomass compared to the control group.
In summary, single-strain E or the composite strains AE and AEF more significantly promoted the growth of oilseed rape (Figure 7).

3.3.3. Morphology and Structure of Plant Roots

The morphology and structure of plant roots are closely related to their nutrient absorption capacity [43,44]. Throughout the cultivation period, inoculation with the single strains or composite strains significantly affected the morphology of oilseed rape roots, enhancing various root attributes such as the total length, surface area, volume, number of tips, number of forks, and number of crossings (Figure 8).
On the 15th day of cultivation, the morphology and structure of roots in the AE and AEF groups were better than those in the other six treatment groups, indicating significant differences between the control and single-strain groups (Figure 8A). Compared to the control group, the total root length, root surface area, root volume, number of root tips, number of root forks, and number of root crossings in the AE group were 283.2 ± 26.85%, 275.6 ± 19.05%, 303.3 ± 26.73%, 265.6 ± 0.86%, 460.8 ± 48.76%, and 425.0 ± 47.44% higher, respectively. Furthermore, compared to the control group, the AEF group showed a significant increase in these indicators by 256.8 ± 24.76%, 286.4 ± 13.91%, 325.0 ± 2.89%, 297.6 ± 27.70%, 352.5 ± 32.48%, and 326.5 ± 22.46%, respectively, (Figure 8A and Table S4).
On the 30th day of cultivation, the roots in the E, AE, and AEF groups indicated better structural characteristics than those in the other groups, while no significant differences were observed among the three groups (Figure 8B). Compared to the control group, the E group demonstrated significant increases of 19.19 ± 2.17%, 24.98 ± 5.11%, 30.08 ± 11.29%, 32.80 ± 3.05%, 33.51 ± 6.05%, and 46.71 ± 9.81% in the total root length, root surface area, root volume, number of root tips, number of root forks, and number of root crossings, respectively. The AE group also revealed significant increases of 28.15 ± 4.79%, 24.11 ± 2.27%, 35.77 ± 8.54%, 35.37 ± 5.79%, 38.43 ± 6.61%, and 36.66 ± 9.02% in these indicators, respectively. Moreover, the AEF group indicated substantial increments of 26.71 ± 5.17%, 26.79 ± 6.47%, 49.11 ± 10.82%, 39.22 ± 10.67%, 35.15 ± 3.26%, and 28.22 ± 5.92%, respectively, compared to the control group (Figure 8B and Table S4).

3.4. Comprehensive Analysis of the Effects and Mechanisms of Different Treatments on Oilseed Rape Cultivation

The effects of different PSB strains and composite strains on various indicators of oilseed rape in the soil test were investigated using PCA. The first principal component (PC1) and the second principal component (PC2) accounted for 92.88% and 4.54% of the total variation, respectively (Figure 9A). A significant separation was observed between the control and AE groups, as well as between the control and AEF groups (Figure 9A). The results suggested that the comprehensive positive effects of AE and AEF on crops were significantly higher than other treatments.
The correlation heatmap revealed positive correlations between soil-available P content and various growth indicators, including the plant height, fresh weight of above-ground biomass, dry weight of above-ground biomass, fresh weight of underground biomass, dry weight of underground biomass, total root length, root surface area, root diameter, root volume, number of root tips, number of root forks, and number of root crossings. Furthermore, the correlations of soil-available P content with the total root length, root volume, as well as the number of root tips, root forks, and root crossings, were particularly significant. Moreover, the soil pH was significantly negatively with soil-available P, plant height, and number of root crossings (Figure 9B). Available P showed significant correlations with numerous indicators of crops, suggesting that different bacterial treatments promote plant growth through their phosphate-solubilizing functions.

4. Discussion

The PSBs are crucial soil functional microorganisms that significantly contribute to soil P activation, promote crop growth, and improve agricultural yields [2,45]. However, the effectiveness of PSB single strain may be limited due to their weak colonization abilities and competition from indigenous microorganisms. Therefore, the use of PSB synthetic consortia has been recognized as a promising approach to developing new microbial inoculants or fertilizers [46]. In this study, four PSB combinations (AE, AF, ADF, and AEF) demonstrated significantly improved P-solubilizing abilities compared to single strains (Table S1). The P-solubilizing capacities of AE, AF, ADF, and AEF were 561.2 mg/L, 514.2 mg/L, 487.9 mg/L, and 592.8 mg/L, respectively (Table S3, Figure 2). In the NBRIP medium supplemented with excess tricalcium phosphate, their solubility of P was 11.22%, 10.28%, 9.76%, and 11.86%, respectively, which represented a significant increase compared to A (5.35%), D (6.07%), E (3.27%), and F (6.46%). This finding suggests that consortia of different PSB strains might be more effective than single strains [35]. However, not all PSB combinations could exert synergistic effects, which may be attributed to nutritional competition, spatial competition, or other unknown biochemical mechanisms. For instance, certain bacteria can produce metabolites that can inhibit the growth of other bacteria or compete for the same resources and space, which hinders their ability to collaborate effectively [47,48].
The plant growth-promoting effects of the four screened PSB combinations were investigated at two scales. In the plate experiment, the composite strains indicated higher growth-promoting effects on oilseed rape seedlings than the single strains, with the effects on underground growth being more pronounced than those on the above-ground growth (Figure 3). Bacteria secrete various metabolites that can directly or indirectly affect plants, inducing plant growth and nutrient absorption, or activating defense systems [49,50]. However, further research is required to confirm whether the excellent growth-promotion abilities indicated by the composite strains are related to their P-solubilization capabilities.
In the soil test, the growth-promoting effects of four composite strains on oilseed rape were investigated in a real soil environment. The findings demonstrated that inoculation with the composite strains containing strain E significantly promoted the accumulation of above-ground and underground biomass in oilseed rape. Furthermore, the growth-promoting effects of AE and AEF were relatively more pronounced (Figure 6). Moreover, it has observed that the growth-promoting effects of PSB on plants can be influenced by various factors, such as the colonization ability of the strains and the structure of indigenous microbial communities [23,51,52]. As the duration of cultivation increased, the promoting effects of composite strains became more pronounced than those of single strains (Figure 6 and Figure 7). This may be attributed to composite strains’ enhanced competitiveness and colonization ability in soil, while single strains were more susceptible to competition and elimination by indigenous microorganisms [53]. Wang et al. [36] found that the enhanced P-solubilizing and growth-promoting effects of composite strains were due to the colonization ability of positive bacteria, the P-solubilizing ability of negative bacteria, and their capacity to produce growth-promoting substances.
The effects of composite strains on the root morphology of oilseed rape were significantly higher than those of single strains. In addition, AE and AEF significantly promoted several indicators, including the total root length, root surface area, root volume, number of root tips, root crossings, and root forks, resulting in more stable root development (Figure 8, Table S4). A well-developed root structure is essential for plant growth. Many studies have demonstrated that root elongation is closely related to the plant’s ability to absorb and accumulate different elements, such as P and potassium [54,55]. Therefore, the changes caused by PSB in root morphology and structure are beneficial for improving nutrient absorption and utilization efficiency of oilseed rape, which, then, promotes its growth. Furthermore, the inoculation of composite strains caused a more rapid development of plant roots than single strains (Figure 8), suggesting a stronger colonization ability of PSB consortia [53,56].
The plate experiment and soil test revealed that two composite strains, AE and AEF, were more beneficial for plant growth than single strains. The application of PSB can increase the available P content in soil [57,58]. The growth indicators of plants were significantly correlated with soil-available P (Figure 9), indicating that the growth-promoting effects of the composite strains were associated with their higher P-solubilizing capabilities. However, the soil test results did not reveal any significant increase in soil-available P after composite strain inoculation, compared to the corresponding single-strains (Figure 4). As previously reported, while PSB could dissolve insoluble phosphates in the soil, acidic conditions might inhibit their activity [39,59,60], causing an insignificant increase in available P content. In the rhizosphere, the presence of plants facilitates bacterial colonization and P cycling. Furthermore, the rhizosphere also provides a conducive environment and favorable conditions for microbial habitation [61,62]. Moreover, the exogenous addition of composite PSB strains has a higher impact on the activity and abundance of rhizosphere microorganisms, indirectly accelerating nutrient cycling and turnover, which induces a promotive effect on plant growth [63,64,65]. The reciprocal relationship between plant roots and PSB is beneficial for soil P balance and cycling, particularly in acidic soils [66,67,68]. The interactions between plant roots and PSB present a possible strategy to reduce the application of P fertilizers and restore the soil P balance.
Environmental conditions crucially modulate the effective functioning of composite microbial inoculants, i.e., temperature influences bacterial proliferation and P-solubilization capacity, thereby affecting soil P turnover rate [69,70]. Furthermore, moisture impacts soil aeration, which is essential for the growth and metabolism of bacteria, particularly aerobic species [71,72,73]. In addition, soil pH is a key factor in nutrient availability [74,75,76]. Here, it was observed that inoculating PSB strains in acidic soils did not significantly alter the soil pH, which is consistent with the findings of Wahid et al. [77]. Future studies should focus on optimizing pH conditions and exploring the combined application of soil pH conditioners and PSB strains, which are hypothesized to enhance soil nutrient availability more effectively.

5. Conclusions

The use of PSB as a substitute for P fertilizers in agricultural production represents an environmentally friendly approach that promotes sustainable development. This study identified two composite strains of PSB with high P-solubilizing capacities and beneficial application effects, AE (the combination of L. amnigena 1-1 and P. frederiksbergensis 2-11) and AEF (the combination of L. amnigena 1-1, P. frederiksbergensis 2-11, and P. rhodesiae 2-47). AE and AEF indicated P-solubilizing capacities of 561.19 and 592.82 mg/L, respectively, and the P solubilities in NBRIP medium with excess tricalcium phosphate were 11.22% and 11.86%, respectively, indicating a significant increase compared to the single strains. The plate experiments and soil tests confirmed that composite strains are more beneficial to plant growth than single strains of PSB. Furthermore, this study provide evidence for bacterial consortia and data supporting reduced P fertilizer application and restoring soil P balance in acidic soils. However, further research is required to evaluate the effects of composite strains (AE and AEF) on other significant agricultural crops as well as their production processes and their application in field trials.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14112461/s1, Table S1: Combinations of PSB strains used in this study; Table S2: Concentrations of each single PSB strain at OD600 = 1; Table S3: Evaluation of the phosphorus-solubilizing effect of PSB combinations; Figure S1: pH of the solution after single and composite PSB bacterial cultivation; Table S4: Root indicators treated with different PSB single strains and composite strains in the soil test.

Author Contributions

Conceptualization, M.L.; methodology, M.L.; validation, X.L., D.X., C.B., and K.Z.; formal analysis, M.L., X.L., and R.G.; investigation, X.L., D.X., C.B., and K.Z.; resources, M.L. and Q.L.; data curation, M.L., X.L., and D.X.; writing—original draft preparation, X.L.; writing—review and editing, M.L. and R.G.; visualization, M.L., X.L., and R.G.; supervision, M.L., R.G., and Q.L.; project administration, M.L.; funding acquisition, M.L, L.C., and Q.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Zhejiang Province Natural Science Foundation (No. LY23C050001), Ningbo Natural Science Foundation (No. 2021J132), the Young Doctor Innovative Research Project of Ningbo Natural Science Foundation (No. 2022J137), and the Youth Science and Technology Innovation Leading Talent Project of Ningbo, China (No. 2024QL061).

Data Availability Statement

Some or all data used during the study are available from the corresponding author by request.

Acknowledgments

We thank Shan Sun, Xiaofang Zhang, and Xuli Zhang for their help in the laboratory.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The flowchart of the experimental design.
Figure 1. The flowchart of the experimental design.
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Figure 2. Phosphorus-solubilizing capacities of PSB combinations (single and composite strains). Different lower-case letters indicate significant differences in soluble phosphorus content at 0.05 level.
Figure 2. Phosphorus-solubilizing capacities of PSB combinations (single and composite strains). Different lower-case letters indicate significant differences in soluble phosphorus content at 0.05 level.
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Figure 3. (A) Effects of PSB single and composite strains on seed germination and root length of oilseed rape (B. napus Zhongshuan No. 11). (B) Growth status of oilseed rape seedlings. Line plots represent the seed germination rates under different treatments, while bar plots represent the height of seedlings (green) and the length of roots (yellow). Different letters indicate significant differences among the treatments (p < 0.05).
Figure 3. (A) Effects of PSB single and composite strains on seed germination and root length of oilseed rape (B. napus Zhongshuan No. 11). (B) Growth status of oilseed rape seedlings. Line plots represent the seed germination rates under different treatments, while bar plots represent the height of seedlings (green) and the length of roots (yellow). Different letters indicate significant differences among the treatments (p < 0.05).
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Figure 4. Effects of different single strains and composite strains on soil-available phosphorus content (bar plots) and pH levels (line plots). (A,B) represent the soil indicators on the 15th day and 30th day, respectively. Different lower-case letters indicate significant differences among the treatments (p < 0.05).
Figure 4. Effects of different single strains and composite strains on soil-available phosphorus content (bar plots) and pH levels (line plots). (A,B) represent the soil indicators on the 15th day and 30th day, respectively. Different lower-case letters indicate significant differences among the treatments (p < 0.05).
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Figure 5. Effects of different single strains and composite strains on the height of oilseed rape (B. napus Zhongshuan No. 11). Different lowercase letters indicate significant differences among the treatments (p < 0.05).
Figure 5. Effects of different single strains and composite strains on the height of oilseed rape (B. napus Zhongshuan No. 11). Different lowercase letters indicate significant differences among the treatments (p < 0.05).
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Figure 6. Effects of different single strains and composite strains on the biomass of oilseed rape (B. napus Zhongshuan No. 11). (A,B) represent the fresh and dry weights of biomass, while (1) and (2) represent the above-ground and underground biomass, respectively. The different lower-case letters indicate significant differences among the treatments (p < 0.05).
Figure 6. Effects of different single strains and composite strains on the biomass of oilseed rape (B. napus Zhongshuan No. 11). (A,B) represent the fresh and dry weights of biomass, while (1) and (2) represent the above-ground and underground biomass, respectively. The different lower-case letters indicate significant differences among the treatments (p < 0.05).
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Figure 7. Effects of different single strains and composite strains on the growth of oilseed rape (B. napus Zhongshuan No. 11) on the 15th (A) and 30th (B) days of cultivation.
Figure 7. Effects of different single strains and composite strains on the growth of oilseed rape (B. napus Zhongshuan No. 11) on the 15th (A) and 30th (B) days of cultivation.
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Figure 8. Effects of different single strains and composite strains on the root structure of oilseed rape (B. napus Zhongshuan No. 11) on the 15th (A) and 30th (B) days of cultivation.
Figure 8. Effects of different single strains and composite strains on the root structure of oilseed rape (B. napus Zhongshuan No. 11) on the 15th (A) and 30th (B) days of cultivation.
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Figure 9. Principal component analysis (A) and correlation heatmap (B) of various growth indicators of oilseed rape treated with different PSB single-strains and composite strains. Soil AP: soil-available phosphorus; Plant H: plant height; FWAB: fresh weight of above-ground biomass; DWAB: dry weight of above-ground biomass; FWUB: fresh weight of underground biomass; DWUB: dry weight of underground biomass; RL: total root length; RSA: root surface area; RD: root diameter; RV: root volume; RT: number of root tips; RF: number of root forks; and RC: number of root crossings. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 9. Principal component analysis (A) and correlation heatmap (B) of various growth indicators of oilseed rape treated with different PSB single-strains and composite strains. Soil AP: soil-available phosphorus; Plant H: plant height; FWAB: fresh weight of above-ground biomass; DWAB: dry weight of above-ground biomass; FWUB: fresh weight of underground biomass; DWUB: dry weight of underground biomass; RL: total root length; RSA: root surface area; RD: root diameter; RV: root volume; RT: number of root tips; RF: number of root forks; and RC: number of root crossings. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Li, M.; Li, X.; Xue, D.; Bao, C.; Zhang, K.; Chen, L.; Li, Q.; Guo, R. Enhanced Plant Growth Through Composite Inoculation of Phosphate-Solubilizing Bacteria: Insights from Plate and Soil Experiments. Agronomy 2024, 14, 2461. https://doi.org/10.3390/agronomy14112461

AMA Style

Li M, Li X, Xue D, Bao C, Zhang K, Chen L, Li Q, Guo R. Enhanced Plant Growth Through Composite Inoculation of Phosphate-Solubilizing Bacteria: Insights from Plate and Soil Experiments. Agronomy. 2024; 14(11):2461. https://doi.org/10.3390/agronomy14112461

Chicago/Turabian Style

Li, Mengsha, Xinjing Li, Daosheng Xue, Chengjiang Bao, Keying Zhang, Lili Chen, Qiuping Li, and Rui Guo. 2024. "Enhanced Plant Growth Through Composite Inoculation of Phosphate-Solubilizing Bacteria: Insights from Plate and Soil Experiments" Agronomy 14, no. 11: 2461. https://doi.org/10.3390/agronomy14112461

APA Style

Li, M., Li, X., Xue, D., Bao, C., Zhang, K., Chen, L., Li, Q., & Guo, R. (2024). Enhanced Plant Growth Through Composite Inoculation of Phosphate-Solubilizing Bacteria: Insights from Plate and Soil Experiments. Agronomy, 14(11), 2461. https://doi.org/10.3390/agronomy14112461

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