Construction of Drought-Resistant Microbial Consortium and Effect on Alfalfa Growth Under Drought Stress

TL;DR

This study creates a microbial consortium to help alfalfa grow better under drought conditions by improving stress resistance and plant health.

Contribution

A drought-resistant microbial consortium combining two selected PGPR strains is developed and tested for enhancing alfalfa growth under drought.

Findings

The microbial consortium increased osmotic adjustment and reduced oxidative damage in alfalfa under drought.

GAU-93 improved photosynthesis, while Y1 enhanced root development and protected against oxidative stress.

The consortium shows potential as a tool for improving drought resistance in alfalfa production.

Abstract

Alfalfa (Medicago sativa L.) is an important perennial leguminous crop whose growth and yield are frequently limited by drought stress because the main planting areas are concentrated in arid and semi-arid regions. Plant growth-promoting rhizobacteria (PGPR) are crucial for enhancing plant stress resistance and constitute an attractive supplementary strategy for alfalfa production, but this has mainly been based on the use of single-strain inoculants in rhizobia. Here, we designed a microbial consortium to alleviate drought stress in alfalfa. Seven PGPR strains isolated from the rhizosphere and five rhizobial strains with in vitro growth-promoting properties obtained from alfalfa nodules were chosen. Based on a comprehensive evaluation of drought tolerance, growth-promoting traits, and metabolite-feeding experiments, we selected Sinorhizobium meliloti GAU-93 and Bacillus mycoides Y1 to construct a drought-resistant microbial consortium (DR-MC). A pot experiment indicated that inoculation with the microbial consortium enhanced drought resistance by increasing osmotic adjustment substance levels and reducing malondialdehyde content, promoting alfalfa growth. Separately, GAU-93 promoted aboveground growth by increasing photosynthetic pigment content under different water potential conditions, whereas Y1 enhanced root development and protected the plant from drought-induced oxidative damage. The DR-MC selected in this study is a valuable tool for further development to improve drought resistance in alfalfa.

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Full text

1. Introduction

Global climate warming has increased the frequency of drought stress, which is regarded as one of the most serious abiotic stresses faced by plant growth and development, particularly in arid and semi-arid regions [1]. Drought stress disrupts osmotic metabolic processes in plants, thereby inducing oxidative damage that inhibits growth and ultimately results in plant death. It is predicted that by 2050, 50% of farmland used for crop production will be adversely affected by drought [2,3]. Strategies to enhance the drought resistance of plants include cultivating tolerant cultivars, modifying planting systems, optimizing irrigation, and adopting soil water conservation measures; however, these are constrained by intensifying climate change and the increasing demand for high yields and quality in dryland environments. Therefore, the development of effective, non-polluting, and environmentally adaptable methods for enhancing plant drought resistance is imperative.

Alfalfa (Medicago sativa L.) is one of the most widely cultivated forage legumes in the world, characterized by environmental adaptability, high crude protein content, and low fiber, and is primarily grown in arid and semi-arid regions but is highly susceptible to drought stress [4,5]. In China, the continued increase in demand for dairy products has led to a rapid increase in alfalfa imports since 2008 [6]. Drought significantly inhibits alfalfa growth and accelerates flowering, thereby seriously affecting yield and forage quality [7]. Therefore, exploring methods to enhance alfalfa drought resistance and elucidating the underlying mechanisms are of great significance for improving alfalfa production in arid and semi-arid regions. The application of plant growth-promoting rhizobacteria (PGPR), which can establish beneficial symbiotic relationships with plants to enhance tolerance to abiotic stresses, has received extensive attention in agricultural and environmental research [8]. The alleviation of drought stress in plants by PGPR inoculation is a multifaceted process involving root system development, increased aboveground biomass, enhanced photosynthesis, and reduced oxidative damage [9,10,11,12]. Several strains have been shown to enhance plant drought resistance [13,14,15], particularly through the promoting effect of Pseudomonas and Bacillus strains on root growth [16] and the enhancing capacity of rhizobia for nodule formation and nitrogen fixation in legumes, which are considered potential pathways for alleviating drought stress in plants [17]. Often based on the application of single strains, plant drought stress alleviation has been limited by inconsistent results due to insufficient compatibility and functional singularity [18]. Recently, a trend toward utilizing microbial consortia or synthetic microbial communities (SynComs) has provided an efficient method for the discovery and application of PGPR, with the aim of achieving higher effectiveness, multifunctionality, and environmental stability [19]. A microbial consortium is a low-complexity artificial assemblage composed of multiple microorganisms. Based on optimization principles and biotechnological purposes, much research has focused specifically on several aspects, including principles for community design, optimization of strain combinations for improved functionality, compatibility among strains, and host matching [19,20,21]. An increasing number of studies have shown that microbial consortia comprising diverse functional strains have a more substantial growth-promoting effect than single-strain inoculants. For example, Yang et al. [22] found that four-component SynComs of PGPRs improved drought tolerance in Arabidopsis by increasing chlorophyll and abscisic acid levels. Pakar et al. [23] used polyethylene glycol (PEG-6000) to simulate drought conditions and found that PGPR inoculated with Pseudomonas aeruginosa, Bacillus cereus, and Bacillus sp. effectively alleviated drought stress in Cicer arietinum L., significantly enhancing the drought resistance of plants by increasing the expression of reactive oxygen species (ROS)-neutralizing genes of antioxidant enzymes. Consequently, in-depth research is required on the strains included in a specific consortium, with the aim of achieving additive or synergistic positive effects and avoiding cross-inhibition between strains that could have negative impacts on plants.

The main objectives of this study were to (a) develop a microbial consortium that promotes alfalfa growth and is worthy of future field testing; (b) evaluate the alleviating effects of the consortium on drought stress in alfalfa under controlled conditions; and (c) establish a simplified design workflow for constructing a drought-resistant microbial consortium. We hypothesized that a consortium of beneficial bacteria can withstand drought stress and improve the growth and physiology of alfalfa seedlings better than single-strain inoculation. We also hypothesized that the specific symbiotic relationship between alfalfa and rhizobia confers distinct advantages for drought alleviation and environmental adaptation, and that established plant–microbe associations can confer benefits to plants. We provide evidence that manipulation of a microbial consortium with complementary and multiple growth-promoting effects to favor plant-beneficial growth can provide advantages for plants in coping with abiotic stress. Our research provides forward-looking perspectives for optimizing alfalfa production in arid and semi-arid regions.

2. Results

2.1. Identification and Growth Promoting Characteristics of Strains

This study selected five rhizobia strains isolated from alfalfa nodules and PGPR strains from the rhizosphere as the experimental materials, which were used to assess their promoting properties and drought tolerance. Phylogenetic tree results showed that the rhizobia strains of GAU-93, GAU-123, GAU-124, GAU-125, and GAU-438 were preliminarily identified as Sinorhizobium meliloti for 99.28–99.64% homology, respectively. The PGPR strains of Y1, Y3, and LY1F were identified as Bacillus mycoides (99.17%), B. subtilis (99.10%), and B. carbrialesii (99.21%), respectively; M1 was identified as Pseudomonas synxantha (99.37%); YBM17 was identified as Microbacterium algeriense (99.30%); YBM21 was identified as Achromobacter marplatensis (99.56%); and YBN5 was identified as Agrobacterium tomkonis (100.00%), respectively (Figure 1).

The growth-promoting characteristics of the strains were quantified, including nitrogenase activity, inorganic/organic phosphate solubilization, plant hormones (indole-3-acetic acid, IAA; trans-Zeatin, t-Z; and abscisic acid, ABA) secretion, siderophore production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. As shown in Table 1, all strains possessed nitrogenase ability with 23.89–226.32 nmol (C_2_H_4_)·(h·mL)^−1^, among which YBM21 exhibited the highest activity. Except for LY1F, Y1, and Y3, the solubility of inorganic and organic phosphate from strains ranged 11.83–362.93 μg·mL^−1^ and 61.26–184.95 μg·mL^−1^, respectively, with the strains of YBM17 and M1 showing the highest value of inorganic and organic phosphate solubility, respectively. The plant hormone production of IAA was in the range of 1.78–15.65 µg·mL^−1^, t-Z was in the range of 2.19–13.38 µg·mL^−1^, and ABA was in the range of 1.78 to 4.82 µg·mL^−1^. Among the strains, GAU-93 showed the highest IAA and ABA secretion, and YBN5 showed the highest t-Z secretion, respectively. In addition, all strains had siderophore production ability, but only four strains showed ACC deaminase activities with 0.17–58.67 µmol mg^−1^·h^−1^, among which GAU-125 showed the highest activity.

2.2. Effects of Water Potential on Strans Growth

PEG-6000 was used to simulate different water-potential conditions to evaluate the drought tolerance of the strains. As shown in Figure S1, significant differences in the growth features of the strains were observed as the concentration of PEG-6000 increased. The rhizobia strain of GAU-93 showed the highest growth performance under non-stress conditions, followed by GAU-438 and GAU-123. At moderate concentrations (5–10%), the growth of GAU-93 was decreased, but still better than that of other rhizobia strains. Meanwhile, all of the rhizobia strains were significantly inhibited at high concentrations of PEG-6000 (15–40%). Similarly, among PGPR strains, YBM21 exhibited the highest growth ability under non-stress conditions, followed by Y1 and YBN5. At 5–25% PEG concentrations, the growth of Y1 decreased, but was still better than that of other strains. Interestingly, the strain of LY1F showed a trend of increasing first and then decreasing, reaching the highest OD value of growth at a 20% concentration. At high concentrations (30–40%), the growth of PGPR strains was significantly decreased, but was generally greater than that of the rhizobial strains.

2.3. Construction of Drought-Resistant Microbial Consortium

The antagonistic relationship between strains was identified using the Oxford cup method, and the symbiotic relationship between candidate strains was studied through metabolite feeding experiments. As shown in Table S1, no antagonistic effect was observed between the five rhizobia strains by the genus of Sinorhizobium meliloti isolated from alfalfa for the PGPR strains of M1 and LY1F. Additionally, Y1 had no antagonistic effect on GAU-93, GAU-125, and GAU-438; Y3 had no antagonistic effect on GAU-93 and GAU-125; YBM17 had no antagonistic effect on GAU-124; YBN5 had no antagonistic effect on GAU-123 and GAU-438, respectively; and YBM21 had antagonistic effects on all rhizobia strains. Based on their growth-promoting and antagonistic properties, GAU-93 was selected as the candidate rhizobia strain, and Y1 and LY1F were selected as the candidate PGPR strains. Moreover, the metabolite feeding experiments revealed that the metabolites of LY1F had no significant effect on the growth of GAU-93, while Y1 exhibited the characteristics of delaying strain senescence to promote its growth (Figure 2). In addition, the metabolites of GAU-93 significantly promoted the growth of Y1 and had no effect on LY1F. Therefore, the drought-resistant microbial consortium (DR-MC) was constructed by combining the strains of S. meliloti GAU-93 (G) and B. mycoides Y1 (P).

2.4. Effects of Drought-Resistant Strains on Alfalfa Growth

The growth effects of drought-resistant strains on alfalfa seedlings are shown in Figure 3. Overall, inoculation with drought-resistant strains significantly increased the height and fresh weight of the plants under different drought conditions. The drought-resistant microbial consortium significantly increased the height and fresh weight of alfalfa at 0 and −0.8 MPa and was better than that of single-strain inoculation. Additionally, inoculation with GAU-93 was better than that in Y1.

The inoculation of drought-resistant strains significantly promoted alfalfa seedlings’ root development under different drought conditions (Table 2). Compared with CK, the inoculation of Y1, GAU-93, and the drought-resistant microbial consortium increased the total root length and root volume of alfalfa, but had no significant effect on surface area. Moreover, the microbial consortium significantly increased the total root length and the number of tips compared with single-strain inoculation under the same water potential condition.

The effects of drought-resistant strains on the chlorophyll contents of alfalfa seedlings are shown in Figure 4. The GAU-93, Y1, and the microbial consortium inoculation significantly increased the contents of chlorophyll a, chlorophyll b, and total chlorophyll under 0 and −0.4 MPa; GAU-93 and Y1 also increased the ratios of chlorophyll a/b and carotenoid/chlorophyll (a + b). At −0.8 MPa, GAU-93 and Y1 inoculation increased the contents of chlorophyll a and chlorophyll b, while the microbial consortium showed no significant effect on these characteristics.

2.5. Effects of Drought-Resistant Strains on Alfalfa Antioxidant and Osmotic Regulator Systems

The enzymatic antioxidant system plays an important role in mitigating damage caused by reactive oxygen species (ROS) under drought stress. As shown in Figure 5, the regulatory effects of different drought-resistant strains were varied intensity with drought conditions. Compared to CK without inoculation, GAU-93, Y1, and microbial consortium inoculation significantly enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) of alfalfa under 0 MPa conditions. Furthermore, the microbial consortium outperformed single-strain inoculations in SOD and POD activities under −0.4 MPa conditions, while showing no significant difference in CAT activity. At −0.8 MPa conditions, the microbial consortium significantly increased the activities of SOD, POD, and CAT compared to CK, and the activities of POD and CAT were not significantly different for the GAU-93 inoculation.

Malondialdehyde (MDA) reflects the degree of lipid peroxidation in plants, for which excessive content causes damage to the cell membrane. As shown in Figure 5D, there was no significant difference between the different inoculation treatments under 0 MPa conditions. Meanwhile, at −0.4 and −0.8 MPa conditions, the GAU-93, Y1, and microbial consortium inoculation significantly reduced the content of MDA, with the effect observed as microbial consortium > GAU-93 > Y1.

Soluble sugars (SS), soluble proteins (SP) and proline (Pro) are key osmotic regulatory substances in plants. As shown in Figure 6, Y1 and microbial consortium inoculation significantly increased the contents of SS, SP and Pro in alfalfa seedlings under different water potential conditions, and GAU-93 increased the contents of SS and Pro. In addition, the microbial consortium inoculation showed the highest concentrations of these osmolytes, which peaked at −0.4 MPa conditions, followed by Y1.

2.6. Principal Component (PCA) and Cluster Analysis

PCA revealed distinct trait associations under different water potential conditions (Figure 7). Under non-stress conditions (Figure 7A), PC1 (63.4%) was influenced by the activites of CAT, SOD, POD and the contents of total chlorophyll, chlorophyll a, and SP, mainly reflecting antioxidant and photosynthetic characteristics; PC2 (19.1%) was influenced by root length, root volume, plant height, and the ratio of carotenoid/chlorophyll (a + b), mainly reflecting growth characteristics. Under −0.4 MPa conditions (Figure 7B), PC1 (45.1%) was influenced by the activities of SOD and POD, the contents of SP and Pro, and plant height, mainly reflecting osmotic regulation and antioxidant characteristics. PC2 (26.0%) was influenced by root traits and the ratio of carotenoid/chlorophyll (a + b), mainly reflecting plant growth and photosynthetic pigment. Under −0.8 MPa conditions (Figure 7C), PC1 (43.1%) was influenced by fresh weight, plant height, root surface area, and the contents of SS, SP, and Pro, mainly reflecting osmotic regulation and plant growth. PC2 (32.4%) was influenced by the contents of total chlorophyll, chlorophyll b, and carotenoids; the ratio of chlorophyll a/b and carotenoid/chlorophyll (a + b); and the activity of CAT, chlorophyll a/b ratio, mainly reflecting photosynthetic pigment characteristics. In addition, the indicators with the highest weights were Pro (0.0696), SS (0.0674), and POD activity (0.0662) under non-stress conditions; POD (0.0668), Pro (0.0650), and SP (0.0627) under −0.4 MPa conditions; and POD activity (0.0671), root surface area (0.0644), and Car concentration (0.0633) under −0.8 MPa conditions. PC1 still showed strong contributions from osmotic substances, while PC2 was mainly associated with root growth characteristics (Table 3).

Cluster analysis revealed that the drought-resistant microbial consortium exerted different effects on the growth and physiological indices of alfalfa under different water potentials (Figure 8). Under severe drought stress (−0.8 MPa), the microbial consortium significantly affected the activity of POD, while it had a significant influence on the activity of SOD under moderate drought stress (−0.4 MPa). In addition, the microbial consortium still exhibited a positive effect on alfalfa growth under non-stress conditions (0 MPa). These results indicated that the drought-resistant microbial consortium mainly alleviated membrane lipid peroxidation induced by drought by increasing SOD activity and decreasing malondialdehyde (MDA) content. It maintained intracellular water balance through osmotic regulation, thereby ensuring the normal physiological functions of alfalfa.

3. Discussion

Alfalfa is one of the most important leguminous forages worldwide [24]. In China, alfalfa cultivation is primarily concentrated in the arid and semi-arid regions of the Northwest and Northeast, where water availability is the primary limiting factor for yield. Moreover, as droughts become more frequent and severe due to climate warming, alfalfa production is increasingly threatened. Drought, caused by water deficiency, significantly inhibits alfalfa growth and accelerates flowering, thereby severely reducing both yield and forage quality. Although some drought-resistant varieties (e.g., Medicago sativa ‘WL440HQ’) can maintain their vitality index by activating antioxidant enzymes, such as superoxide dismutase, or by regulating osmotic pressure balance under low-drought conditions, excessive stress may lead to an imbalance in their resistance system [25,26]. Therefore, there is an urgent need to develop novel, sustainable approaches to help alfalfa withstand drought stress. Previous studies have shown that the in vitro growth-promoting potential of PGPR plays a vital role in regulating plant growth and alleviating drought stress. Among these, the symbiotic nitrogen fixation and organic/inorganic phosphate dissolution abilities of PGPRs primarily alleviate drought-induced growth stress by enhancing plant growth and root development, thereby increasing the surface area for water and nutrient absorption [27,28]. By contrast, plant hormones (PHs) secreted by PGPR, such as abscisic acid (ABA) and indole-3-acetic acid (IAA), can induce stomatal closure to reduce water evaporation, thereby alleviating drought stress in leaves. Root-targeted PHs promote root elongation, thereby enhancing water uptake [29]. In addition, 1-aminocyclopropane-1-carboxylate (ACC) deaminase degrades the ethylene precursor ACC into α-ketobutyrate and ammonia, thereby reducing ethylene accumulation in stressed roots and improving tolerance [30]. Moreover, microbial siderophores help plants to withstand drought by enhancing iron nutrition, recruiting beneficial rhizosphere microorganisms, and balancing redox states. In this study, all strains exhibited nitrogen-fixation ability and showed lower ACC deaminase production and higher PHs secretion than those in previous studies, suggesting that the PHs secreted by the strains may play a crucial role in enhancing the drought tolerance of alfalfa. Among the rhizobia, GAU-93 exhibited the most vigorous overall growth-promoting activity and produced the highest levels of IAA and ABA, making it the most promising rhizobial candidate.

PEG-6000 is a high-molecular-weight polymer that induces a water deficit state in plants by reducing the osmotic potential of the solution and is commonly employed to simulate drought conditions under controlled settings for evaluating plant drought tolerance [31]. Using this method, we measured the drought tolerance of selected strains. We found that the growth ability of the rhizobia under drought conditions was lower than that of the promoting bacteria. LY1F and Y1 exhibited multiple growth-promoting activities and showed a slow decline in growth at 0–25% PEG (w/v). Metabolite feeding tests further revealed strong compatibility between Y1 and GAU-93, indicating that combining complementary strains into a microbial consortium as an inoculant is feasible.

The most direct response of plants subjected to abiotic stress is the inhibition of normal growth [32]. In this study, compared with the control, under both drought and non-drought stress conditions, different microbial inoculants significantly increased plant height and fresh weight in alfalfa. At the same water potential, the consortium inoculant significantly increased the number of alfalfa root tips and stimulated lateral root formation. The results indicate that the microbial consortium inoculant promotes plant growth more than single-strain inoculation and that rhizobia perform better than other bacteria under drought stress. This may be because root nodules formed by alfalfa provide a favorable environment for rhizobia to resist stress, whereas rhizobia enhance the nitrogen-fixation efficiency of nodules, converting atmospheric nitrogen into plant-available forms that better support aboveground plant growth [33], consistent with the findings of Jochum et al. [10]. Changes in chlorophyll (Chl) content reflect the intensity of photosynthesis and sensitivity of plants to drought stress, which gradually decreases under drought stress [34]. Pakar et al. [23] found that the co-inoculation of Pseudomonas and Bacillus strains increased the Chl a, Chl b, total Chl, and carotenoid content of Cicer arietinum L. under combined salt-drought stress. Similar results were obtained in the present study; all inoculation treatments increased the contents of Chl a, Chl b, total Chl, and carotenoids in alfalfa leaves under different drought stress conditions. Moreover, the microbial consortium inoculant increased both the Chla/Chlb and carotenoid/total Chl ratios, providing the strongest stimulation of photosynthesis in alfalfa seedlings. This result is consistent with the observed increase in growth characteristics, indicating that the microbial consortium supports a high net photosynthetic rate in alfalfa under drought conditions by increasing chlorophyll and carotenoid biosynthesis, thereby promoting aboveground growth [35,36].

Drought stress triggers excessive ROS accumulation in plant cells, which causes oxidative damage and impairs normal plant growth and development. The plant antioxidant defense system consists of both non-enzymatic antioxidants and key antioxidant enzymes (including SOD, POD, and CAT), which play critical roles in scavenging ROS and resisting abiotic stress. The efficiency of ROS detoxification and elimination varies greatly under different drought intensities [37,38]. Inoculation with beneficial microorganisms can regulate antioxidant enzyme activities and protect plant cells from drought-induced oxidative damage, and such effects have been widely reported in various crops, such as potato and barley seedlings [39,40]. In the present study, the activities of SOD, POD, and CAT in alfalfa seedlings inoculated with single or combined microbial strains were significantly higher than those in uninoculated seedlings, and the regulatory effects varied with drought stress intensity. Under drought stress, seedlings inoculated with the microbial consortium showed the highest SOD and POD activities, whereas CAT activity exhibited no significant changes among different stress levels in uninoculated plants. Meanwhile, lower MDA content indicates alleviated lipid peroxidation and improved the integrity of cell membranes under external stress [41]. Notably, the MDA content in alfalfa seedlings inoculated with the microbial consortium was lower than that in all non-stressed treatments, suggesting that the combined inoculation effectively alleviated oxidative damage in alfalfa. These results demonstrate that the microbial consortium performs better than single-strain inoculation in enhancing the drought resistance of alfalfa seedlings [42].

Osmotic adjustment is the central strategy used by plants to cope with water deficit. Plants usually accumulate osmotic adjustment substances, such as Pro and SS, to counteract the osmotic imbalance caused by drought stress. Eswaran et al. [43] found that an increase in Pro and SS content improved the osmotic adjustment ability of Arachis hypogaea L., thereby enhancing drought tolerance when co-inoculated with Acinetobacter calcoaceticus and Bacillus amyloliquefaciens. In the present study, we found that inoculation significantly promoted the accumulation of SS, SP, and Pro in alfalfa seedlings under drought conditions. Moreover, Y1 was more effective than GAU-93 in the single-inoculation treatment, a result similar to that for antioxidant enzymes, suggesting that it may help alfalfa seedlings resist drought stress by regulating oxidative stress and osmotic adjustment systems. Therefore, the synergistic effects of Y1 on stress alleviation and GAU-93 on the nodulation and nitrogen fixation for growth promotion within the consortium were the main reasons for the superior results of co-inoculated alfalfa seedlings under drought stress.

In summary, the regulatory effects of different strains and their combinations in assisting alfalfa seedlings to resist drought stress varied with drought intensity (Figure 9). POD activity maintained consistently high weights across all stress conditions, highlighting the central role of the drought-resistant microbial consortium in the antioxidant defense system of alfalfa. The rhizobium strain GAU-93 primarily promoted aboveground growth by increasing the contents of photosynthetic pigments, whereas the Bacillus strain Y1 enhanced root development and protected plant cell membranes from drought-induced oxidative damage. The combination of these strains systematically enhanced alfalfa drought tolerance through synergistic effects across multiple targets. Specifically, the combined microbial inoculation stabilized alfalfa physiology by reinforcing the antioxidant defense system, thereby enhancing osmotic adjustment and precise water retention. Furthermore, considering the complementary functions and ecological niches among the strains, it is feasible to combine growth-promoting bacteria for root development with rhizobia to colonize and fix nitrogen in root nodules. This method enhances plant growth and improves the acquisition of both soil water and nutrients. Consequently, these multifunctional combined strains exhibited greater stability and adaptability than single strains and have potential as microbial inoculants for alfalfa production in arid and semi-arid regions.

4. Materials and Methods

4.1. Strain Identification and Growth-Promoting Properties’ Determination

The strains used in this study were provided by the Grassland Microorganisms Research Group from the College of Pratacultural Science, Gansu Agricultural University, China, and all the selected strains were non-pathogenic. The strain information is shown in Table 4. The genomic DNA of strains was extracted using the CTAB method following the manufacturer’s instructions (TIANGEN Biotech Co., Ltd., Beijing, China). Then, the universal primers 27F/1492R were used for PCR amplification of the 16S rRNA gene. The PCR products were sent to Sangon Biotech (Shanghai, China) for sequencing after qualification. The obtained sequences were submitted to EzBioCloud (https://www.ezbiocloud.net/, accessed on 20 May 2025), and the sequences of closely related strains were downloaded from the database. A phylogenetic tree was subsequently constructed using the neighbor-joining method in MEGA 7.0.

The organic and inorganic phosphate solubilization abilities of the strains were determined using the molybdenum blue method [44]. The strains were inoculated into NBRIP medium (with tricalcium phosphate, Ca_3_(PO_4_)2, as an inorganic phosphorus source) and MJN medium (with calcium phytate, Ca-IHP, as an organic phosphorus source), respectively. After incubation at constant temperature for 12 days, the amount of solubilized phosphate was quantified using a standard curve. The ACC deaminase activity was measured using the colorimetric method [45]. Specifically, it is important to resuspend the strain cells in DF salts medium and induction culture with ADF medium for 36 h, then centrifuge at 4 °C and wash twice with 0.1 M Tris-HCl (pH = 7.6). Then, add 30 µL of toluene and shake to obtain the crude enzyme solution. Measure the strain’s protein content in 100 µL of the mixture to assess biomass, and use the remaining mixture to determine ACC deaminase activity. Determine the siderophore production ability using the colorimetric method after inoculating into an iron-limited SA medium. Mix the culture supernatant and the chrome azurol S (CAS) solution in equal volumes and incubate at room temperature for 1 h. Then, measure the OD_630_ value (As) and the SA medium mixed with CAS as a reference (Ar). Calculate the siderophore production value as [(Ar − As)/Ar] × 100% [46]. The amount of indole-3-acetic acid (IAA), trans-Zeatin (t-Z) and abscisic acid (ABA) secretion was determined using high-performance liquid chromatography (HPLC) method, as described by Wang et al. [47], with minor modifications. Briefly, the strains were inoculated into King’s B medium and incubated with shaking at 28 °C and 180 r·min^−1^ for 7 days. The supernatant was extracted with ethyl acetate, concentrated, and re-dissolved in anhydrous ethanol. The concentrations of secreted hormones were quantified based on the standard curve.

4.2. Selection and Construction of Drought-Resistant Microbial Consortium

Polyethylene glycol (PEG-6000) was used to simulate drought stress. The strains were inoculated into sterilized YMA medium (for rhizobia) and LB medium (for PGPR) supplemented with 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40% (w/v) PEG-6000, then incubated at 28 °C with shaking at 180 r·min^−1^ for 3 d. Subsequently, 5 mL of each bacterial suspension was centrifuged, and the cells were resuspended in sterile water. The optical density (OD) at 600 nm was measured using a spectrophotometer (TU-1901, Persee, Beijing, China) to evaluate bacterial growth under different drought stress levels.

The antagonistic effect between the strains was evaluated using the Oxford cup method, where 5 mm filter-paper disks were substituted for the cups. The strains were paired and inoculated separately on agar plates and filter-paper disks, and the formation of inhibition zones was recorded. Meanwhile, the selected non-antagonistic strains were further analyzed using the metabolic feeding method [48]. Specifically, rhizobia and PGPR were cultured in LB medium for 60 h, then the cultures were centrifuged at 10,000 rpm for 10 min, and the supernatants were filter-sterilized through a 0.45 µm membrane to obtain cell-free metabolites. A total of 180 µL of sterile LB medium containing 10% metabolites of strains was added to a 96-well plate, followed by the addition of 20 µL fresh strain cultures. The growth of the strains was measured for the OD value at 600 nm and recorded every 3 h for a total of 72 h by using the sterile LB medium as a control [49].

4.3. Pot Experimental Design

Medicago sativa L. cv. ‘Longdong’ was selected as the experimental material in this study, which showed a strong response to drought stress. The seeds were sterilized with 3% NaClO for 10 min, washed clean with sterile water, and then germinated in the dark at 28 °C on sterile moist filter paper for 5 days. The pot experiment used the nitrogen-free Hoagland’s solution combined with PEG-6000 to simulate drought stress. Seedlings were cultivated in three water-potential gradients (0, −0.4, and −0.8 MPa) and four inoculation treatments (control without inoculation, CK; inoculated with PGPR, P; rhizobia, G; and drought-resistant microbial consortium, H), and four replicates were conducted. The fermentation broth cultured for 3 days was centrifuged to harvest bacterial cells, which were resuspended in sterile water to an optical density at 600 nm (OD_600_) of 0.8, with a viable cell count of 10^8^ CFU/mL. The microbial consortium was formulated by blending equal volumes of the PGPR and rhizobial suspensions. Furthermore, 18 uniform alfalfa seedlings at the 5-day-old stage were transplanted into hydroponic plant boxes (10.8 cm × 11.5 cm × 8.0 cm) and grown in a chamber at 25 °C/20 °C (day/night) with a daytime light intensity of 4000 lx and a relative humidity of 45%. After one week of acclimatization, 10 mL of the prepared bacterial suspension was inoculated into each box. Seven days after inoculation, Hoagland’s solution corresponding to each water potential was replaced every three days, and the stress treatment was applied continuously for 15 days. Finally, alfalfa samples were collected for the determination of various physiological and biochemical indices. Three biological replicates were set up for each treatment, with a total of 36 hydroponic boxes used in this experiment. Finally, alfalfa samples were collected for the measurement of various indicators, and three replicates were conducted, with 36 pots in total.

4.4. Sample Collection and Analysis

Collect alfalfa seedlings to measure their height, determine fresh weight, and use a root scanner (Expression 10000XL, Epson, Suwa, Japan) to scan and analyze with WinRHIZO to obtain parameters, including the total root length, surface area, average diameter, volume, and number of tips. The chlorophyll content was determined using the ethanol method. Fresh leaves were placed in a tube containing 5 mL of 95% (v/v) ethanol and left in the dark for 24 h. Subsequently, the supernatant was centrifuged and measured at 663 nm, 645 nm, and 470 nm using a spectrophotometer (TU-1901, Persee, Beijing, China). The contents of chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoids (Car), chlorophyll a + chlorophyll b (Chl a + Chl b), and the ratios of chlorophyll a/chlorophyll b (Chl a/Chl b) and carotenoid/chlorophyll (a + b) (Car/Chl a + Chl b) were calculated [50].

Fresh leaf samples were homogenized in 4 mL of 50 mM phosphate-buffered solution (PBS, pH = 7.8) and centrifugation 12,000 rpm for 15 min at 4 °C to collect the supernatants. According to the manufacturer’s instructions, we determined the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China), respectively. Malondialdehyde (MDA) content was determined using the thiobarbituric acid method [51]. Soluble sugar (SS) content was measured using the anthrone colorimetric method [52]. Soluble protein (SP) content was determined using the Coomassie brilliant blue method [53]. Free proline (Pro) content was analyzed using the acidic ninhydrin colorimetric method [54].

4.5. Statistical Analysis

All statistical analyses were conducted using SPSS software version 22.0. The data were subjected to one-way ANOVA (Duncan’s test, p < 0.05) and two-way ANOVA (drought stress, bacteria inoculation, and interactions among them as three sources of variation). The variations in all parameters are expressed as the mean ± standard error of three replicates for each treatment. Principal component analysis (PCA) and cluster analysis were performed using the Chiplot (https://www.chiplot.online, accessed on 22 June 2025).

5. Conclusions

We systematically investigated the physiological and molecular mechanisms by which a microbial consortium comprising B. mycoides and S. meliloti enhances drought tolerance in alfalfa under varying water potential conditions. Our findings demonstrate that co-inoculation with drought-tolerant PGPR and rhizobial strains exerts synergistic effects that significantly outperform single-strain inoculation in alleviating drought-induced growth inhibition. Specifically, the microbial consortium maintained homeostasis of osmoregulatory substances (proline, soluble sugars, and soluble proteins) and enhanced antioxidant enzyme activities (SOD and POD), thereby effectively scavenging ROS and protecting cellular integrity under water-deficit conditions.

Our results provide novel insights into microbe-mediated drought adaptation strategies in leguminous plants and highlight the potential of precision-engineered microbial consortia as sustainable biofertilizers for dryland agriculture. Future research should focus on elucidating the dynamic restructuring of rhizosphere microbial communities and the underlying transcriptional regulatory networks to further unravel the molecular basis of consortium-induced stress tolerance.