Microbial-Plant Interaction: Bacillus subtilis–Driven Gravel Soil Improvement and Growth Promotion of Festuca arundinacea

Abstract

The rapid expansion of tunnel engineering in China has led to extensive excavation of gravelly soils, resulting in significant land occupation that threatens the ecological environment and surrounding biota. As a result, there is an increasing need for effective ecological restoration of nutrient-poor gravelly soils, where challenges in vegetation establishment and sustainable soil management persist. This study evaluates the potential of Bacillus subtilis to promote the growth of Festuca arundinacea in engineered gravel soils through a controlled greenhouse experiment, examining its effects on plant growth, soil nutrient dynamics, and microbial community structure. The results showed that, compared to the control group (CK), neither the Bacillus subtilis treatment group (Bs) nor the nutrient application treatment group (LB) significantly altered the soil bacterial species composition at the phylum level. However, at the genus level, Azotobacter dominated the LB group, while Sphingomonas was the predominant genus in both the CK and Bs groups. Additionally, Bacillus subtilis significantly increased bacterial diversity relative to the nutrient application treatment, leading to substantial changes in microbial community composition. Furthermore, Bacillus subtilis notably enhanced both aboveground and belowground biomass, improved nutrient uptake, and increased the availability of phosphorus and potassium. It also stimulated soil enzymatic activities involved in carbon, nitrogen, and phosphorus cycling, emphasizing its critical role in nutrient cycling. Thus, Bacillus subtilis–driven soil enhancement offers a promising solution for ecological restoration in nutrient-poor gravelly soils, where conventional amendments are often ineffective. These findings underscore the potential of microbial-plant synergies to improve soil fertility and support sustainable vegetation restoration.

Introduction

Tall fescue, also known as Festuca arundinacea, is a perennial herbaceous species in the genus Festuca of the Poaceae family [1]. It is recognized for its strong resistance, excellent cold tolerance, drought resistance, broad adaptability, and well-developed root system [2, 3]. The root system of tall fescue secretes organic compounds that provide carbon sources and energy to soil microorganisms and influence nutrient cycling by modulating soil enzyme activity [4]. Due to these soil-enhancing properties, along with its superior slope stabilization and soil and water conservation capabilities, tall fescue has become widely used in the ecological restoration of disturbed areas [5]. It has been successfully applied in various high-altitude and cold regions, such as the Tibetan Plateau [6].

With the rapid expansion of infrastructure in China, the number of tunnel projects in high-altitude and cold regions has significantly increased, accompanied by considerable technological success [7]. However, during construction, particularly when using drilling and blasting methods to tunnel through mountains, substantial amounts of engineering debris and gravel are inevitably produced [8]. These waste materials predominantly consist of mixed rock types, and due to their high transportation costs, loose structure, lack of particle cohesion, nutrient deficiencies, and low microbial diversity, they are often piled nearby to form large waste dumps [9]. As a result, landscape fragmentation and ecological imbalance occur in the affected areas. This situation is further exacerbated by the infertile nature of the gravel and the harsh climatic conditions of high-altitude and cold environments, which severely restrict plant establishment, thereby hindering the natural ecological restoration process [10, 11]. Therefore, there is an urgent need to develop long-term ecological restoration technologies that facilitate stable vegetation establishment and sustainable community succession through artificial intervention in waste dump sites.

Microbe-plant synergistic remediation is a key strategy for improving nutrient-poor soils [12]. Microorganisms enhance soil fertility and optimize the rhizosphere microenvironment through various physiological and ecological functions, including nitrogen fixation, phosphorus solubilization, biocontrol, soil structure improvement, and driving nutrient cycling [13–15]. Plants, in turn, secrete root exudates (such as organic acids and sugars) and chemical signals mediated by plant hormones, establishing a mutualistic symbiotic relationship with microorganisms to jointly improve adaptation to environmental stresses [16, 17]. Functional microorganisms, plant growth-promoting rhizobacteria (PGPR), play a significant role in this process [18]. Common PGPR groups include species from the genera Bacillus, Pseudomonas, and Rhizobium [19, 20]. Among them, Bacillus subtilis, a model strain of Bacillus widely distributed in soil environments, has attracted considerable attention due to its potential applications [21]. Studies have shown that Bacillus subtilis enhances soil organic matter decomposition by secreting extracellular enzymes and organic acids, releasing plant-available nutrients such as phosphorus and potassium [22]. Its metabolic activities also contribute to the formation of soil aggregates, thereby improving soil aeration and water retention [23]. Additionally, this strain synthesizes plant growth regulators such as indole-3-acetic acid (IAA), which directly stimulate root development, enhance nutrient absorption efficiency, and promote plant growth [24].

However, when applying the Bacillus subtilis–tall fescue joint remediation system in engineered gravel soils, a critical and underexplored scientific question remains: whether Bacillus subtilis can influence the structure of the rhizosphere microbial community through its interaction with tall fescue, and whether these changes in microbial community composition affect soil nutrient availability, enzyme activities, and plant growth. Understanding these interactions is crucial for assessing the ecological role of Bacillus subtilis in the rhizosphere and its potential for enhancing soil fertility and plant productivity in engineered soils. This remains a topic that requires further investigation.

In light of this, the present study aims to investigate the effects of Bacillus subtilis on the growth of tall fescue in engineered gravelly soils, as well as the underlying mechanisms. A controlled greenhouse pot experiment was conducted, with treatments involving the application of nutrient solutions and Bacillus subtilis inoculum. The study focuses on analyzing the growth performance of tall fescue, changes in soil nutrient levels, and alterations in enzymatic activity under different nutrient supply strategies. The objective is to reveal the interrelationship between soil nutrient dynamics and tall fescue growth in environments dominated by Bacillus subtilis as the primary microbial community. This research aims to provide both theoretical and practical insights for improving the growth of tall fescue in gravelly soils, propose feasible approaches to enhancing its growth in nutrient-poor soils, and offer scientific support for expanding the collaborative interactions between functional microorganisms and plants in ecological restoration and agricultural applications.

Materials and Methods

Experimental Materials and Experimental Design

The gravelly soil used in this study was sourced from the spoil heap resulting from drilling and blasting operations in a tunnel construction project located in a high-altitude region of China. The soil is nutrient-poor (Table 1); however, it is free from heavy metal contamination and other harmful substances. The tall fescue seeds used in this experiment were procured from the Prairie Research Institute in Chengdu, while Bacillus subtilis (ATCC6633) was obtained from the Shanghai Microbial Culture Collection Center. To obtain Bacillus subtilis cultures with a final concentration of 10⁸ CFU/mL, a lyophilized strain stored at − 80 °C was initially revived. The revived strain was inoculated into 10 mL of LB medium and incubated at 37 °C with shaking at 220 rpm for 12 h to promote bacterial growth. The pre-culture was then transferred to 250 mL of fresh LB medium at an inoculum volume of 1% (v/v) and further incubated at 37 °C with shaking at 220 rpm for 24 h. During incubation, samples were periodically collected, and bacterial concentration was monitored using the plate count method to ensure that the culture reached the desired concentration of 10⁸ CFU/mL. After incubation, the bacterial culture was centrifuged to remove residual medium components, and the pellet was resuspended in physiological saline to achieve the final desired concentration.

Table 1.

Basic nutrient composition of the gravelly soil

Indicators	pH	Soil organic carbon	Ammonium nitrogen	Available phosphorus	Available potassium	Nitrate nitrogen	Total nitrogen	Total phosphorus	Total potassium
Results	9.8 ± 0.8	0.42 ± 0.05	0.74 ± 0.09	7.84 ± 0.43	23.94 ± 1.15	2.43 ± 0.16	0.15 ± 0.01	0.28 ± 0.05	24.16 ± 0.73

The concentrations of organic carbon, total nitrogen, total phosphorus, and total potassium are expressed in g/kg, whereas ammonium nitrogen, nitrate nitrogen, available phosphorus, and available potassium are quantified in mg/kg.

This study employed an indoor pot experiment in which gravelly soil samples from the spoil heap were transported to the greenhouse at the School of Ecological Environment, Chengdu University of Technology, for experimental treatment. Circular plastic pots, each with a diameter of 12 cm and a height of 20 cm, were used, with 4 kg of gravelly soil evenly distributed in each pot. Viable tall fescue seeds were selected using a flotation method, and a seeding rate of 0.8 g per pot was uniformly applied to the surface of the soil. The seeds were then gently pressed into the soil to a depth of approximately 2 cm, after which the pots were left to allow natural seed germination. The experiment included three treatment groups: a control group (CK), a Bacillus subtilis (Bs) treatment group, and an LB medium (LB) treatment group, with 10 pots per group, totaling 30 pots. The control group (CK) was planted in the basic gravelly soil substrate without any additives. In the Bacillus subtilis (Bs) treatment group, 10 mL of a Bacillus subtilis suspension cultured in LB liquid medium at a concentration of 10⁸ CFU/mL was applied to pots containing tall fescue seeds. Given the nutrient deficiency of the basic substrate and the potential impact of the nutrients in the microbial suspension on plant growth, the LB treatment group was designed to eliminate this confounding factor by adding 10 mL of sterile LB liquid medium directly to the pots, without microbial inoculation, to evaluate the nutrient effects of the LB medium itself. It is worth noting that, to maintain experimental consistency, 10 mL of sterile water was added to the CK treatment group to ensure that the liquid volume applied was equivalent to that in the other treatment groups. After sowing, 30 mL of water was applied daily to maintain soil moisture. During the cultivation period, 10 mL of the corresponding solution was added to each treatment group every 30 days. The LB treatment group received an addition of Luria–Bertani liquid medium, while the Bs treatment group was supplemented with Bacillus subtilis culture at a concentration of 10⁸ CFU/mL. The greenhouse temperature was maintained between 20 and 25 °C, with relative humidity ranging from 20 to 50%, and a light/dark cycle of 8 to 12 h per day. The experiment was conducted over a period of 120 days.

Plant and Soil Sampling

After 120 days of cultivation, five pots were randomly selected from each treatment group (out of ten replicates) for destructive sampling. The entire plant was carefully uprooted, and any loose soil adhering to the root system was gently shaken off. A sterile brush was then used to collect rhizosphere soil samples firmly attached to the root surface, specifically within a 1 cm radius of the root system. Approximately 10 g of the collected rhizosphere soil was immediately placed into a sterile container and transported in an icebox to the laboratory. Upon arrival, the samples were stored at 4 °C for subsequent analysis of soil enzyme activities. The remaining gravelly soil in the pots was collected, sealed in plastic bags, and analyzed for its physico-chemical properties. The plants were separated into shoot and root components using scissors, and both parts were thoroughly rinsed with deionized water to remove any residual soil. Each plant part was then placed in separate envelopes. The samples were dried in a 65 °C oven until a constant weight was achieved, then weighed to determine the aboveground and belowground biomass. The root-shoot ratio, calculated from these measurements, reflects the relative growth of the root system compared to the aboveground portion of the plant [54].

Soil pH and soil organic carbon were measured using the method described by Shen et al. [55]. Total nitrogen, total phosphorus, total potassium, ammonium nitrogen, nitrate nitrogen, available phosphorus, and available potassium were determined using an AA3 continuous flow analyzer (SEAL Analytical, UK).

In this study, the activities of five soil enzymes were determined: alkaline phosphatase (ALP), sucrase (S-SC), urease (S-UE), cellulase (S-CL), and catalase (S-CAT). The enzyme activities were measured using enzyme-linked immunosorbent assay (ELISA) kits (Cominbio, Suzhou, China) following the manufacturer’s protocol.

Soil microbial community composition was analyzed by Shanghai Paisei Neuro Company (Shanghai, China). Total genomic DNA was extracted using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions and stored at − 20 °C prior to analysis. DNA quantity and quality were assessed using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16S rRNA gene V3–V4 region was conducted using the forward primer 338 F (5′-ACTCCTACGGGAGGCAGCA-3′) and reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Sample-specific 7-bp barcodes were included for multiplex sequencing. The PCR mixture contained 5 µL of 5 × buffer, 0.25 µL of Fast Pfu DNA Polymerase (5 U/µL), 2 µL of dNTPs (2.5 mM), 1 µL of each primer (10 µM), 1 µL of DNA template, and 14.75 µL of ddH₂O. Thermal cycling involved initial denaturation at 98 °C for 5 min, followed by 25 cycles of denaturation at 98 °C for 30 s, annealing at 53 °C for 30 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 5 min. PCR amplicons were purified using Vazyme VAHTS™ DNA Clean Beads (Vazyme, Nanjing, China) and quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After quantification, amplicons were pooled in equal amounts and subjected to pair-end 2 × 250 bp sequencing on the Illumina NovaSeq platform using the NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

Statistical Analysis

The experimental data were organized using Microsoft Excel 2020. One-way analysis of variance (ANOVA) was conducted to evaluate the effects of different treatment groups on soil nutrient content, soil enzyme activity, and plant biomass, with comparisons made to the control group. Prior to performing ANOVA, the assumptions of normality and homogeneity of variance were assessed using the Shapiro–Wilk test and Levene’s test, respectively. Post-hoc comparisons were performed using Duncan’s multiple range test, with letters assigned in the figures to indicate significant differences between treatment groups. Additionally, Mantel test analysis was conducted to examine the correlations between plant biomass and soil nutrients, as well as enzyme activity indicators within each treatment group. Finally, bar plots for the results of one-way ANOVA were generated using GraphPad Prism 10.1.2, while correlation heatmaps for the Mantel test were created using R 4.5.0. The Genescloud Platform (https://www.genescloud.cn) was used to analyze the bacterial community, including α-diversity (Chao1 and Simpson indices), β-diversity (PCoA), taxonomic composition at the phylum and genus levels (stacked bar plots), and heatmap visualization of microbial taxa abundance.

Results

The Changes in Gravel Soil Nutrient Dynamics Under Different Treatments

The study found that, compared to the CK treatment group, both the LB and Bs treatment groups exhibited a decreasing trend in pH (Fig. 1a), with the LB treatment group showing a significant difference (p < 0.05). Meanwhile, the soil organic carbon content under experimental treatment was significantly higher (p < 0.05) (Fig. 1b). The ammonium nitrogen content in the LB treatment group was significantly higher than in the CK and Bs treatment groups (p < 0.05) (Fig. 1d). However, both the LB and Bs treatment groups showed a decreasing trend in nitrate nitrogen content, with the difference in the Bs treatment group being significant (p < 0.05) (Fig. 1e). The contents of available phosphorus and available potassium were significantly increased in both the LB and Bs treatment groups, with the Bs treatment group exhibiting the highest content for both elements, followed by the LB treatment group, and the CK treatment group having the lowest (Fig. 1g, i). However, there were no significant differences in the contents of total nitrogen, total phosphorus, and total potassium among the treatment groups (Fig. 1c, f, h).

Fig. 1.

Variations in soil nutrients under different treatments. a Soil pH, b soil organic carbon (SOC), c total nitrogen (TN), d ammonium nitrogen (NH₄.⁺-N), e nitrate nitrogen (NO₃^-), f total phosphorus (TP), g available phosphorus (AP), h total potassium (TK), and i available potassium (AK). The error bars represent the standard deviation (SD). Different lowercase letters indicate statistically significant differences among treatment groups (p < 0.05). (n = 5)

The Changes in Enzymatic Activity in Gravelly Soils Under Different Treatments

This study measured the enzymatic activities involved in the cycling of key elements, including sucrase and cellulase associated with carbon metabolism, urease involved in nitrogen metabolism, alkaline phosphatase related to phosphorus metabolism, and catalase, which reflects the metabolic activity of microbial communities [25, 26]. The results of the study indicated that, compared to the CK treatment group, both the LB and Bs treatment groups exhibited a significant increase in sucrase and urease activities (p < 0.05) (Fig. 2a, d). In contrast, no significant differences were observed in catalase and cellulase activities among the treatment groups (Fig. 2b, c). Regarding alkaline phosphatase activity (Fig. 2e), the LB treatment group showed a significant increase relative to the CK group (p < 0.05), whereas the Bs treatment group demonstrated a significant decrease (p < 0.05).

Fig. 2.

Variations in enzymatic activity under different treatments. a Soil urease (S-UE), b soil cellulose (S-CL), c soil catalase (S-CAT), d soil sucrase (S-SC), and e soil alkaline phosphatase (ALP). U/g represents the enzyme activity per gram of soil, indicating the catalytic capacity of the enzyme in relation to the soil weight. The error bars represent the standard deviation (SD). Different lowercase letters indicate statistically significant differences among treatment groups (p < 0.05). (n = 5)

The Changes of Plant Growth Variations Under Different Treatments

The study shows that the application of either nutrients or Bacillus subtilis inoculum can promote the accumulation of tall fescue biomass (Fig. 3a, b). The aboveground and underground biomass in the Bs treatment group was significantly higher than those in the CK and LB treatment groups (p < 0.05). Notably (Fig. 3c), the LB treatment group showed an increasing trend in the root-shoot ratio, whereas the Bs treatment group exhibited a significant decrease (p < 0.05).

Fig. 3.

Variations in biomass under different treatments. a Aboveground biomass dry weight of the plants. b Belowground biomass dry weight of the plants. c Root-shoot ratio of the plants. The error bars represent the standard deviation (SD). Different lowercase letters indicate statistically significant differences among treatment groups (p < 0.05). (n = 5)

The Changes in Soil Microbial Community Characteristics Under Different Treatments

This study revealed, through α-diversity analysis, that the diversity and richness of the soil bacterial community in the Bs treatment group were significantly greater than those in the LB treatment group (p < 0.05). In terms of community richness (Chao1 index), the Bs treatment group exhibited a slightly lower value than the control group (CK), although this difference was not statistically significant (Fig. 4a). The Simpson index was significantly lower in the LB group compared to CK, while the Bs group showed a higher index, similar to CK, and significantly different from LB, indicating that Bs preserved microbial diversity. Principal coordinates analysis (PCoA) based on Bray–Curtis distances indicated significant differences in the soil bacterial community composition (Fig. 4b). Additionally, 16S rRNA sequencing of rhizosphere soil bacteria from each treatment group identified the top 10 most abundant species at both the phylum and genus levels for further analysis (Fig. 4c). The results showed that Proteobacteria was the predominant phylum across all treatment groups, with the highest relative abundance observed in the LB treatment group (48.02%). At the genus level, Azotobacter was the dominant genus in the LB treatment group, with a relative abundance of 8.99%. In contrast, no significant dominant genus was observed in the CK and Bs treatment groups, with Sphingomonas being the most abundant genus in both, with relative abundances of 2.52% and 3.14%, respectively. The species composition heatmap analysis (Fig. 4d) further demonstrates that the microbial community composition is significantly shaped by the different treatments, leading to notable differences.

Fig. 4.

Changes in bacterial communities under different treatments. a α-Diversity of bacterial communities under different treatments. b Principal coordinates analysis (PCoA) of bacterial communities under different treatments. c Relative abundance of bacterial communities at the phylum and genus levels under different treatments (n = 5). d Heatmap of the species composition of bacterial communities at the phylum and genus levels under different treatments

Plant–Soil–Microbe Relationships

As shown in Fig. 5, compared with the control (CK) treatment, both the application of Bacillus subtilis inoculum (Bs) and the liquid culture medium (LB) treatment significantly enhanced the positive correlation between soil microbial community composition and soil sucrase activity. By modulating the microbial community structure, both treatments more effectively promoted sucrase activity, which plays a crucial role in soil carbon cycling. Notably, the LB treatment not only demonstrated a stronger association between the microbial community and sucrase activity but also further increased the positive correlation between microbial community composition and plant aboveground biomass, highlighting its unique potential to simultaneously enhance soil biochemical processes and plant productivity.

Fig. 5.

Mantel test analysis of the relationship between plants, soil nutrients, soil enzyme activity, and microbial composition. The plot shows the results of the Mantel test, highlighting the relationship between various soil properties, enzyme activity, and microbial composition. Significant correlations are indicated by asterisks, with the level of significance marked as p < 0.01, p < 0.05, and p > 0.05. The plot also includes Mantel’s p-value significance testing for spatial autocorrelation between the variables. pH, soil pH; SOC, soil organic carbon; TN, total nitrogen; NH₄⁺-N, ammonium nitrogen; NO₃^-, nitrate nitrogen; AP, available phosphorus; TP, total phosphorus; TK, total potassium; AK, available potassium; S-UE, soil urease; S-CL, soil cellulase; S-CAT, soil catalase; S-SC, soil sucrase; ALP, soil alkaline phosphatase; AB, aboveground biomass; BB, belowground biomass; R/S, root-shoot ratio

Discussion

Impact of Treatments on Soil Nutrients and Enzyme Activity

Soil nutrients are essential elements absorbed from the soil during plant growth and are crucial for evaluating soil fertility [27, 28]. In this study, both the Bs and LB treatment groups caused significant changes in soil nutrients and enzyme activities. Additionally, we observed that, due to the nutrient-deficient nature of gravelly soil, root exudates in the CK treatment may interact with soil minerals, thereby facilitating nutrient release. This interaction resulted in increased organic matter and ammonium nitrogen content in the soil compared to the initial soil conditions [56]. Compared to the CK group, the LB treatment group lowered the pH by 0.80% through ammonium nitrogen input, driving the soil acidification process, while the Bs treatment group reduced the pH by 0.52% through the production of various organic acids during its metabolic processes, thus facilitating soil acidification [29, 30]. Additionally, the LB treatment group directly supplemented organic carbon, increasing the organic carbon content by 5.06%, whereas the Bs treatment group raised the organic carbon content by 28.60%, possibly due to Bacillus subtilis breaking down minerals and producing an extracellular capsule with adsorption properties that accumulate organic carbon in the soil [31, 32]. In contrast, no significant differences in total nitrogen content were observed between the treatment groups. Meanwhile, nitrate nitrogen content in the LB and Bs treatment groups decreased by 29.05% and 48.35%, respectively. This reduction is hypothesized to be due to the poor water and nutrient retention capacity of the engineered gravel soil, where nutrients leach away quickly under continuous precipitation, diminishing nitrogen fixation or nitrification, which aligns with the notion that increased gravel content leads to higher runoff and sediment loss, thereby promoting nitrogen and phosphorus leaching [33, 34]. In this study, phosphorus and potassium in the gravel soil were primarily present as insoluble compounds, making them difficult for plants to directly absorb and utilize. Despite no significant differences in total phosphorus and total potassium content between the treatment groups, Bacillus subtilis significantly increased the contents of available phosphorus and available potassium by 60.89% and 28.60%, respectively, through the secretion of organic acids and other metabolic products [22]. The LB treatment group also increased the contents of available phosphorus and available potassium by 18.59% and 5.06%, respectively, by supplementing soluble phosphorus and potassium compounds.

Sucrase activity is an indicator of the carbon cycling potential in soil and microbial activity, while urease activity is a crucial indicator for evaluating soil nitrogen cycling and the utilization efficiency of urea-based fertilizers [35, 36]. Compared to the CK group, both the LB and Bs treatment groups significantly increased soil sucrase and urease activities, with the Bs group showing a greater increase than the LB group. Alkaline phosphatase is a key enzyme in the soil phosphorus cycle, involved in the mineralization of organic phosphorus to release plant-available inorganic phosphorus [37]. In this study, the LB treatment group significantly increased alkaline phosphatase activity by 25.82%, while the Bs treatment group significantly decreased alkaline phosphatase activity by 67.44%. This decrease in alkaline phosphatase activity in the Bs treatment group is hypothesized to result from the recruitment of microorganisms in the soil that inhibit alkaline phosphatase activity [38, 39]. In contrast, the LB treatment group stimulated microbial growth and reproduction by providing nutrients to the soil microorganisms, thereby increasing alkaline phosphatase activity.

In the preceding discussion on changes in soil nutrients and enzyme activities, both the LB and Bs treatment groups exhibited significant effects compared to the CK group. To further elucidate the microbial mechanisms underlying these changes, it is crucial to examine the roles of the dominant microbial genera in the soil. In this study, Azotobacter was the dominant genus in the LB treatment group, while Sphingomonas predominated in the Bs treatment group. The nitrogen fixation activity of Azotobacter in the LB group is likely the primary factor contributing to the substantial increase in soil urease activity [57]. Urease activity is closely linked to nitrogen cycling in the soil, and Azotobacter provides abundant nitrogen sources through its nitrogen fixation capabilities, thereby facilitating effective nitrogen transformation. In contrast, the role of Sphingomonas in the Bs group is more intricate. Although this genus did not significantly enhance urease activity, it demonstrated stronger activity in promoting carbohydrate degradation and the conversion of sugar compounds [58]. As a result, the Bs group exhibited significantly higher sucrase activity than the LB group. Furthermore, Azotobacter in the LB group increased alkaline phosphatase activity by directly supplementing organic carbon and stimulating soil microbial growth, a process that plays a key role in phosphorus mineralization and release [59]. In contrast, Sphingomonas in the Bs group may have inhibited the growth of beneficial microorganisms through resource competition or the production of antagonistic substances, leading to a significant reduction in alkaline phosphatase activity [60].

Impact of Treatments on Variations in Plant Growth

Biomass reflects the accumulation of material in plants and is a key indicator of plant growth [40]. Under nutrient-limited stress in engineered gravelly soils, the LB treatment group provided Festuca arundinacea with a significant amount of readily available nutrients. Compared to the CK treatment group, the LB treatment resulted in a 22.41% increase in underground biomass and an 18.72% increase in aboveground biomass, although these differences were not statistically significant. In contrast, the Bs treatment exhibited statistically significant increases in both underground and aboveground biomass. The Bs treatment group notably enhanced the growth of Festuca arundinacea by improving the root environment and nutrient absorption capacity. In the presence of Bacillus subtilis, the aboveground biomass of Festuca arundinacea increased by 102.75%, while underground biomass rose by 77.52%. The root-to-shoot ratio is an indicator of plant adaptability to various environmental conditions [41]. In the CK treatment group, the root-to-shoot ratio was close to 1, reflecting a balanced distribution of biomass between the aboveground and underground portions of Festuca arundinacea. After experimental treatment, the root-shoot ratio in the LB treatment group increased by 3.20% relative to the CK group. In contrast, the Bs treatment group showed a 12.39% reduction in the root-shoot ratio.

Bacillus subtilis, through the secretion of organic acids and enzymes, can dissociate fixed phosphorus and potassium in the soil, transforming them into forms that are available for plant uptake [22]. In this study, the application of Bacillus subtilis (Bs treatment group) indirectly enhanced the rhizosphere microenvironment of Festuca arundinacea through microbial activity, thereby improving nutrient absorption efficiency by the roots and promoting greater aboveground growth than underground growth, which ultimately led to a significant reduction in the root-shoot ratio [42, 43]. In the nutrient application treatment (LB treatment group), key soil nutrients rapidly reached sufficient levels; however, high concentrations of ammonium nitrogen presented potential toxicity to plant roots. Given the elevated ammonium nitrogen content in the LB treatment group, it is hypothesized that Festuca arundinacea, in an effort to maintain ion balance and alleviate ammonium nitrogen stress, allocated more resources to underground biomass [44]. Additionally, the poor water and nutrient retention capacity of the gravelly soil matrix hindered the long-term effectiveness of nutrients, leaving the soil environment in a state of nutrient stress [9]. As a pioneer species, Festuca arundinacea adopts an adaptive strategy of prioritizing root development during nutrient acquisition, enhancing absorption capacity and establishing a foundation for subsequent growth [45]. Therefore, under nutrient stress conditions, it may exhibit preferential root growth, resulting in an elevated root-shoot ratio.

Impact of Treatments on Changes in Soil Bacterial Community

The study demonstrated that different experimental treatment methods significantly reshaped the microbial community composition in gravelly soils [46]. Using the Chao1 and Simpson indices to evaluate the evenness and richness of the bacterial community, significant differences were observed across the various treatment groups. Compared to the control group (CK), the exogenous nutrient treatment (LB) led to a notable reduction in both bacterial community richness and evenness. These findings suggest that the introduction of exogenous nutrients disrupted the balance of soil organic-mineral complexes, potentially decreasing the stability of soil aggregates and reducing the diversity of soil pore structures [47]. This process of environmental homogenization significantly weakened the spatial niche differentiation of microorganisms, limiting the coexistence of different functional microbial groups at the microscale, which ultimately resulted in a comprehensive decline in bacterial community α-diversity [48].

In contrast, the treatment with Bacillus subtilis exhibited a more complex community response. As a dominant alien species introduced artificially, Bacillus subtilis suppressed the colonization and growth of certain indigenous bacterial groups through intense resource competition or antagonistic interactions, although the decrease in overall community richness was not statistically significant [49, 50]. The introduction of Bacillus subtilis modified the composition of native bacterial populations, suppressing certain groups that were more abundant in the CK treatment. However, no clear dominance was observed at the genus level in either the CK or Bs groups. At the same time, the ecological niche pressure and resource space released by Bacillus subtilis provided opportunities for the development of rare or disadvantaged species that were previously at a competitive disadvantage [51]. This process of community restructuring resulted in a relatively more balanced distribution of remaining species within the community, thereby increasing the evenness of the bacterial community.

Implications and Limitations

Despite the beneficial effects of Bacillus subtilis on the growth of Festuca arundinacea observed in this study, its application still faces several challenges. Firstly, the characteristics of gravel soils, such as their loose structure, nutrient deficiency, and low microbial diversity, complicate the establishment of symbiotic relationships between microorganisms and plants. While Bacillus subtilis performs well under controlled experimental conditions, ensuring its stability and activity in real-world gravel soils remains an area requiring further investigation [43, 52]. Additionally, environmental factors such as temperature and humidity may significantly affect the functional efficacy of Bacillus subtilis [53]. Therefore, future research should focus on optimizing the application conditions for microbial inoculants, as well as exploring their stability and effectiveness across diverse ecological environments. Furthermore, we plan to conduct follow-up studies that will include the analysis of metabolites secreted by Bacillus subtilis. This will allow us to gain deeper insights into the metabolic pathways involved in microbial community dynamics and the mechanisms by which Bacillus subtilis influences its surrounding environment. We will also explore the interaction between Bacillus subtilis and other soil microorganisms to further elucidate the role of secretion in microbial community structure and function.

Overall, microbe-plant synergistic remediation technologies show great promise for improving nutrient-poor soils, particularly in ecological restoration efforts in high-altitude and cold regions. As a soil amendment, Bacillus subtilis can effectively promote plant growth and accelerate ecosystem recovery. With a deeper understanding of the mechanisms underlying plant–microbe interactions, the application of plant–microbe synergistic remediation is expected to expand, providing novel technical support for ecological restoration.

Conclusions

In conclusion, this study demonstrates that both the application of exogenous nutrients and the inoculation of Bacillus subtilis significantly impacted the soil environment and the growth of Festuca arundinacea in nutrient-deficient engineered gravel soil matrices. Bacillus subtilis, through its metabolic activities, effectively promoted the decomposition and transformation of insoluble elements in the matrix, particularly enhancing the accumulation of available phosphorus and potassium. This, in turn, led to a significant increase in the aboveground biomass of Festuca arundinacea. In contrast, while the sole application of exogenous nutrients rapidly supplied nutritional support to the plants, it also posed potential risks of physiological stress and insufficient long-term nutrient availability in the soil. In terms of microbial community dynamics, Bacillus subtilis inhibited the growth of specific indigenous microbial populations through resource competition and antagonistic interactions, leading to a decrease in community richness, but a slight increase in evenness. In contrast, the addition of exogenous nutrients facilitated the overgrowth of certain microbial groups, resulting in a reduction in both community balance and diversity. Therefore, compared to traditional nutrient addition methods, Bacillus subtilis offers a more comprehensive advantage in improving the soil fertility of engineered gravel soils and promoting plant growth, providing a cost-effective and feasible solution for the ecological restoration of gravel soils in similar projects, such as tunnel engineering.

Data Availability

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.