Moss Biochar Facilitates Root Colonization of Halotolerant Halomonas salifodinae for Promoting Plant Growth Under Saline–Alkali Stress

Abstract

The utilization of the widely distributed saline–alkali lands by planting forage grasses is a hot topic. However, the promotion of plant growth remains a great challenge during the exploration of this stressful soil. While halotolerant bacteria are beneficial for plants against saline–alkali stress, their stable colonization on plant roots should be further strengthened. In this study, we investigated the effect of moss biochar on the root colonization of the exogenous halotolerant Halomonas salifodinae isolated from saline lake sediments. During the incubation with the bacteria, the biochar strongly bound the bacterium and induced biofilm formation on the biochar surface. When the biochar and the bacterium were added into the culturing soil of the forage grass Medicago sativa, the biochar remarkably assisted the root binding and biofilm formation of this bacterium on the plant roots. Under the biochar–bacterium combined treatment, the numbers of total bacteria, halotolerant bacteria, and nitrogen-fixing bacteria increased from 10^5.5 CFU/g soil to 10^7.2 CFU/g soil, from 10^4.5 CFU/g soil to 10^6.1 CFU/g soil, and from 10^4.7 CFU/g soil to 10^6.3 CFU/g soil, respectively. After 30 days of culturing, the biochar and the bacterium in combination increased the plant height from 10.3 cm to 36 cm, and enhanced the accumulation of chlorophyll a, reducing sugars, soluble proteins, and superoxide dismutase in the leaves. Moreover, the combined treatment increased the activity of soil enzymes, including peroxidase, alkaline phosphatase, and urease. Meanwhile, the levels of various cations in the rhizosphere soil were reduced by the combined treatment, e.g., Na⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, etc., indicating an improvement in the soil quality. This study developed the biochar–halotolerant bacterium joint strategy to improve the yield of forage grasses in saline–alkali soil.

1. Introduction

Saline–alkali lands are widely distributed in the world, with the area reaching up to 954 million hectares [1,2]. In China, the area of saline–alkali lands is about 99 million hectares, among which 33% of the lands could be developed as cultivated lands [3,4,5]. However, the utilization of saline–alkaline soil is frequently compromised by low plant yields [6,7]. Owing to the high-level salinity and pH value, common agricultural crops, e.g., grain crops, forage grasses, and vegetables, suffer from saline–alkali stress. This stress leads to the inhibition of nutrient adsorption, the attenuation of metabolism, and even plant death [8,9]. Saline–alkali stress, a combined abiotic stress involving high salinity (ionic toxicity) and elevated pH (alkaline toxicity), disrupts plant homeostasis through four primary mechanisms: osmotic imbalance, sodium-hyperaccumulation-induced ionic toxicity, oxidative stress, and pH-mediated metabolic dysfunction [10,11]. Notably, the synergistic effects of excessive Na⁺ and alkaline conditions amplify the overproduction of reactive oxygen species (ROS), including superoxide (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (·OH), which induce structural damage to cellular membranes, proteins, and nucleic acids. To counteract this, plants employ a dual-defense strategy integrating enzymatic (SOD, CAT, and APX) and non-enzymatic (ascorbate and glutathione) antioxidant systems with osmoprotectant synthesis. The ROS signaling cascade is mechanistically linked to calcium-mediated pathways. Experimental evidence indicates that transient Ca²⁺ flux activates the MAPK cascade and downstream transcription factors (e.g., NAC and WRKY), thereby upregulating antioxidant enzyme genes [8,10]. Osmoprotectant-mediated osmotic homeostasis utilize metabolites such as soluble sugars and proline. Notably, these solutes function not merely as osmotic balancers but also as multifunctional regulators—directly neutralizing ROS, modulating redox-sensitive signaling cascades, and preserving macromolecular integrity under oxidative damage. Emerging evidence highlights their synergistic roles in stress adaptation: proline stabilizes subcellular structures while enhancing NADPH-dependent antioxidant regeneration, whereas soluble sugars participate in ROS metabolism through the pentose phosphate pathway, concurrently buffering the cytoplasmic pH under alkaline conditions. How to promote the growth of cropping plants is becoming a hot topic during the exploration of this stressful soil.

Halotolerant/halophilic bacteria are one kind of microbial group commonly distributed in oceans and salty lakes. They could live in the environments with high-level salinity even at a sodium chloride concentration of >9% [12,13,14]. These bacteria include the members of Halomonas, Marinobacter, Salinivibrio, Salinibacter, Oceanobacillus, Desulfovibrio, Exiguobacterium, Halobacillus, etc., and constitute the core microbiomes in high-salinity environments [14,15,16]. Under the stress of high salinity, these bacteria express a series of ion pumps to efflux intracellular salt to ensure safe cytoplasmic ion levels, so that they may maintain normal metabolism and growth [17,18,19]. As revealed by recent studies, some of halotolerant/halophilic bacteria may increase the productivity of salt-sensitive plants, exhibiting a great potential to promote plant growth and the remediation of saline soil [14,20,21]. However, it remains a great challenge to regulate the colonization of these bacteria on the plant roots and in the rhizosphere regions.

Biochar is one kind of green and economic carbon material prepared by the pyrolysis of biomass or solid wastes, e.g., agricultural straws, wild plant bodies, and sludges [22,23]. Owing to its high surface areas and abundant active groups, biochar and biochar-derived composites have been developed as adsorbents to remediate the pollution of heavy metals, persistent organic matter, dyes, etc. [24,25,26,27]. Recently, our group found that the fungal-hypha-based biochar and moss biochar may promote the phytoremediation of heavy metals by regulating the rhizosphere microbiome. The mechanism is associated with the high capacity of biochar to bind functional bacteria on the plant roots and in the rhizosphere soils. The robust bacterial-binding capacity of biochar facilitates the stable colonization of exogenous STBs on the roots and in rhizosphere soils by cross-linking these inoculants with the indigenous microbiota. Notably, the biochar–STB synergy further enhances rhizospheric biofilm formation, creating a protective niche that shields microorganisms from environmental stressors while concurrently amplifying cadmium uptake in plants. This dual mechanism—microbial stabilization coupled with heavy metal mobilization—systematically elevates the phytoremediation efficiency through coordinated biotic–abiotic interactions [28,29]. These results indicated that biochar may remodel microbial compositions and functions. Meanwhile, high ionic strengths contribute to soil salinity; the biochar with the capacity of ion adsorption may attenuate the saline stress to plant roots. Therefore, it is hypothesized that specific kinds of biochar could be developed as a regulator of the root bacterial dynamic, soil salinity, and plant growth when the plants suffer from soil saline–alkaline stress.

Most recently, our group isolated the halotolerant Halomonas salifodinae strain 9106 from a saline lake. This maintains a normal growth rate even when the sodium chloride concentration of the medium reaches up to 12%. This study aims to investigate the effect of moss biochar on the root colonization of this halotolerant bacterium and plant growth during the culturing of the forage grass Medicago sativa in saline–alkaline soil. Scanning electron microscopy and biofilm biomass quantification were used to evaluate the induction of biofilm formation and the root binding of the bacteria by the biochar. A biochemical analysis of the plant tissues and the rhizosphere soils was further performed to explore the activity of the biochar and the halotolerant bacterium alone or in combination to regulate plant metabolism and soil improvement. This study sheds light on biochar-based strategies to regulate the behaviors of halotolerant bacteria during rhizosphere engineering against saline–alkali stress.

2. Materials and Methods

2.1. Materials

The halotolerant bacterium 9106 was isolated from the Saline Lake in Yuncheng, Shanxi Province, China, by using the solid Dmn medium. The compositions of this medium included the following: Dnm medium: glucose 0.5% (W/V), peptone 0.25%, KH₂PO₄ 0.025%, K₂HPO₄·3H₂O 0.1%, MgSO₄ 0.01%, NaCl 12%, agar 1.5%, and pH = 7.7. By 16S rDNA sequencing and BLASN analysis in the NCBI system, the strain was identified as the species Halomonas salifodinae. Medicago sativa seeds were grown in standard nursery trays filled with conventional nutrient-rich soil. The experimental soil was sourced from a cultivated field located in the Xiqing District, Tianjin.

2.2. Synthesis and Characterization of the Moss Biochar

The moss biochar was prepared by our previous study [28]. Briefly, the Brachythecium plumosum plants were selected as the primary raw material. Fresh moss plants were subjected to a baking process at 80 °C for a duration of 6 h, resulting in the acquisition of dried moss. The dried moss was subsequently placed in a Muffle furnace (Tianjin Muffle Technology Co., Ltd., Tianjin, China) and heated under nitrogen flow at 500 °C for 1 h. Following this process, the cooled sample was ground to yield moss biochar, which was then utilized in subsequent experiments. The morphology of the prepared biochar was characterized through scanning electron microscopy (TESCAN MIRA LMS, Brno, Czech Republic).

2.3. The Morphology of 9106, Biochar, and the 9106 + Biochar Assembly

Commonly, the 9106 cells were cultivated in a liquid Dnm medium. When the OD value reached 1.0, the culture solution was transferred onto copper plates to facilitate the colonization of 9106 on the surface. The prepared moss biochar alone, 9106 alone, and 9106 plus biochar were suspended in the Dnm medium, with an initial bacterial cell number of 1 × 10⁸ cells/mL and a biochar concentration of 100 mg/L. The suspensions were added into polystyrene plates, and incubated at 28 °C for 24 h. The morphological characteristics of these three samples were analyzed using scanning electron microscopy (SEM).

2.4. Plant Culturing, Treatments, and Sampling

The Medicago sativa seeds were grown in standard nursery trays filled with conventional nutrient-rich soil. Upon reaching a uniform height of approximately 7–8 cm, the young plants were selected to be transplanted into the experimental soil. This soil was sourced from a cultivated field in the Xiqing District, Tianjin, with addition of 1% NaCl and 0.1% NaHCO₃ to create saline–alkali stress (pH = 8.2). After 24 h cultivation in liquid Dmn medium, 9106 cells were centrifuged at 12,000 rpm for 2 min. The harvested cellular material was then resuspended in distilled water to achieve a density of 1×10⁸ cells/mL. In parallel preparation, biochar aqueous suspension was prepared by dispersing the material in distilled water at a concentration of 1 mg/mL.

Four treatment groups were established for the saline–alkali stress experiment: (1) control group (CK1 to CK3), with no addition of 9106 and biochar; (2) 9106 group (9106-1 to 9106-3), with the addition of 9106 cells to 1 × 10⁸ cells/kg soil; (3) biochar group (biochar1 to biochar3), with the addition of biochar to the final concentration of 100 mg/kg soil; and (4) 106 + biochar (9106 + biochar 1 to 9106 + biochar 3), with the addition of 9106 (1 × 10⁸ cells/kg soil) and biochar (100 mg/kg soil). The plants were cultured under room temperature and free sunlight irradiation for 30 days at the branching stage. During this process, to induce saline–alkali stress, irrigation water was substituted with saline–alkali solution (10 mL/week, pH = 8.2, NaCl 1%, NaHCO₃ 0.1%, W/V) a week, and this regimen was continued until harvest, followed by plant sampling (collecting the leaves, stems, and roots of each plant, respectively) for further analysis.

2.5. Interaction Between 9106, Biochar, and Plant Roots

The fresh roots were sampled from the young Medicago sativa plants, and washed by the flowing water to remove the soil particles adhered on the roots. The clean roots were then suspended in PBS buffer, and, then, 9106 and biochar alone or in combination were added into the suspensions. The final concentrations of 9106 and biochar were 1 × 10⁷ cells/mL and 100 μg/mL, respectively. The suspension was then incubated at 28 °C for 24 h. The roots were then sampled and washed twice with deionized water. The obtained clean roots were immersed in 4% formalin solution for 12 h. Subsequently, the tissues were sequentially immersed in ethanol solutions of 30%, 50%, 70%, and 90% concentrations for one hour each to dehydrate the samples. The tissue samples were subsequently lyophilized in preparation for SEM observation.

2.6. Biochemical Analysis

To detect biofilm formation of 9106, the bacterium was cultured in 96-well polystyrene microplates containing the liquid Dnm medium. After 24 h of incubation at 30 °C, the wells were gently washed twice with stilled water, and stained by 0.1% crystal violet (Dingguo, Beijing, China) for 5 min. The dye adsorbed by the formed biofilms was extracted by 10% acetic acid. The absorbance of the dye extracts at 595 nm was measured by a UV–Vis spectrometer (Bio-Rad, Hercules, CA, USA). The polysaccharides produced by the bacterium was quantified by the phenol sulphuric acid method [30].

For soil enzyme analysis, root-associated soil fractions were collected from individual plants and stabilized through ambient drying protocols. Processed substrates were mechanically homogenized using ceramic mortars followed by gravimetric standardization. Enzymatic profiles were quantified, respectively, using commercial assay kits (Solarbio, Beijing) for soil peroxidase (S-POD), soil alkaline phosphatase (S-AKP), soil urease (S-UE), and soil sucrase (S-SC) activities through spectrophotometric determination at characteristic wavelengths.

To measure the content of chlorophyll in plants, triplicate foliar specimens from each experimental unit were gravimetrically standardized. Following collection, tissues were subjected to cryogenic homogenization in pre-chilled ceramic mortars containing ethanol extraction buffer (95% v/v, 3 mL) supplemented with 1% (w/v) calcium carbonate. The resultant slurry underwent vacuum-assisted filtration through 0.45 μm nylon membranes after 10 min centrifugation at 4 °C (3000× g). The absorbance of the extracts at 645 nm (A₆₄₅) and 663 nm (A₆₆₃) was measured by a UV–Vis spectrophotometer (SmartSpec Plus, Bio-Rad, Hercules, CA, USA). The content of chlorophyll a/(mg/g leaves) = (12.71A₆₆₃ − 2.59A₆₄₅) V/(m × 1000); the content of chlorophyll b/(mg/g leaves) = (22.88A₆₄₅ − 4.76A₆₆₃) V/(m × 1000); and the content of total chlorophyll/(mg/g leaves) = (20.29A₆₄₅ + 7.95A₆₆₃) V/(m × 1000) (V is the extract liquid volume, and m is the fresh leaves weight).

The antioxidant enzyme activity of the leaves, which reflects the capacity of the plants against oxidant stress, was assayed by using commercial kits. The bicinchoninic acid assays were employed to determine the protein content in the leaves. The root tissues of each plant were sampled from each treatment. The proline and malomdialdehvde (MDA) levels of the roots, which indicate the stress degree of the roots in the saline–alkaline soil, were determined by using the proline content detection kits and the MDA detection kits (Beijing Solarbio, Beijing, China), respectively.

2.7. Cation Quantification of the Rhizosphere Soils

The rhizosphere soil was sampled and dried for physical and chemical properties assays, in including soil ion content, soil carbon and nitrogen percentage, and soil available phosphorus. To detect soil ion contents, the samples underwent mechanical homogenization using Dounce tissue grinders (tight-clearance, 50 μm) prior to acid digestion in 30% (v/v) nitric acid at 95 ± 1 °C under reflux conditions for 30 min intervals. Digestates were processed through gravimetric dilution protocol (1:10 v/v) with ultrapure water before elemental quantification via high-resolution ICP-MS (Elan DRC-II, PerkinElmer Inc., Waltham, Massachusetts, USA) with method blank verification.

2.8. Statistical Analysis

Experimental data were acquired through triplicate biological replicates, with quantitative parameters expressed as mean ± SEM. Statistical comparisons were conducted using one-way ANOVA (p < 0.05) in SPSS 22.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. The Moss Biochar Induces Biofilm Formation of the Halotolerant Halomonas salifodinae

The biochars were fabricated via the pyrolysis of the moss Brachythecium plumosum at 500 °C. Scanning electron microscopy (SEM) revealed the irregular sheet morphology of the biochars with a diameter ranging from 10 to 60 μm, and with moss-derived particles distributing on the surfaces (Figure 1a). Moreover, while the 9106 cells alone adhered on the plates with a random distribution and low density, the co-incubation of 9106 and biochar led to the formation of dense bacterial biofilm layers on the biochar surface (Figure 1a). The biofilm quantification revealed that the 9106 + biochar group exhibited a four-fold-higher level of biofilm biomass than the 9106 group (Figure 1b). Consistently, a polysaccharide analysis demonstrated that the biochar significantly increased the polysaccharide production of 9106 from 29 mg/L to 142 mg/L (Figure 1c).

3.2. The Biochar Enhances Root Colonization of the Halotolerant Bacterium

As shown in the SEM images of Medicago sativa roots, the 9106 group, similar to the biochar group and the control (CK) group, exhibited the ineffective colonization of bacterial cells on the roots. This finding indicates that the colonization efficiency of 9106 alone was low (Figure 2a). In contrast, the 9106 + biochar group had roots with the bacterial biofilm densely surrounding the roots, suggesting the biochar facilitated the bacterial biofilm formation on the root surface (Figure 2a).

Furthermore, the CFU assays revealed that the biochar + 9106 group exhibited significantly higher bacterial numbers on the roots compared to the other three groups (Figure 2b). In detail, under the 9106 + biochar treatment, the numbers of total bacteria, halotolerant bacteria (STB), and nitrogen-fixing bacteria (NFB) increased from 10^5.5 CFU/g soil to 10^7.2 CFU/g soil, from 10^4.5 CFU/g soil to 10^6.1 CFU/g soil, and from 10^4.7 CFU/g soil to 10^6.3 CFU/g soil, respectively (Figure 2b–d). Consequently, the moss biochar promoted the colonization of 9106 on the plant roots and also enhanced the colonization of NFBs, which was not impaired by biofilm formation.

3.3. The Biochar and Halotolerant Bacterium in Combination Promote Plant Growth

The plant heights of Medicago sativa were measured after 30 days of culturing in saline–alkali soil (Figure 3a,b). The results demonstrated that the 9106 and biochar in combination led to a remarkable increase in the heights of Medicago sativa plants from 10.3 cm to 36.0 cm after 30 days of saline–alkali stress, while 9106 or biochar alone only partially increased the heights. A plant weight quantification further demonstrated that the addition of the prepared biochar and 9106 significantly increased the weights of the plants (Figure S1). For instance, after 30 days of saline–alkali stress, the 9106 and biochar in combination led to a remarkable increase in the weights of Medicago sativa plants from 0.26 g to 1.46 g, which was 4.6-fold higher than the control. In contrast, 9106 and biochar alone only increased the weights to 1.02 g and 0.65 g, respectively.

A comprehensive analysis was further conducted on the plant leaves to ascertain the status of plant metabolism. The results demonstrated that the 9106 + biochar group exhibited higher levels of chlorophyll a, reducing sugar, and protein content in the leaves compared to the control group (CK), biochar, and 9106 group (Figure 3c–e). For example, while the chlorophyll a content in the CK and 9106 group remained quite low at <1.9 mg/g leaves, the contents in the 9106 + biochar increased to 2.6 mg/g leaves (Figure 3c). Similarly, the contents of reducing sugar and soluble protein in the leaves in the 9106 + biochar group were higher than that in the other three groups. In detail, the leaf reducing sugar contents of the 9106 + biochar group reached up to 0.71%, while those of the other groups were lower than 0.65% (Figure 3d). For the leaf soluble proteins, the 9106 + biochar group had a concentration of 2.7 mg/g leaves, whereas the other groups only had a concentration of <2.3 mg/g leaves (Figure 3e).

The antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), play critical roles in scavenging reactive oxygen species under various stresses. The activity of these enzymes in the leaves was then measured. As compared to the CK group, the SOD activity of the 9106 + biochar group significantly increased from 150.0 U/g leaves to 324.3 U/g leaves (Figure 3f). On the contrary, the CAT and POD activities of the 9106 + biochar group decreased from 1361 U/g leaves to 736.8 U/g leaves, and 79,557 U/g leaves to 64,530 U/g leaves, respectively (Figure 3g,h).

3.4. The Biochar and Halotolerant Bacterium in Combination Attenuate Root Stress

After 30 days of culturing, the proline content in plant roots markedly increased. The experimental results are presented in Figure 4a. Oxygen free radicals react with the unsaturated fatty acids in lipids to generate lipid peroxides, which subsequently decompose into a variety of complex compounds, including malondialdehyde (MDA). The extent of lipid oxidation can be assessed by measuring the levels of MDA. As shown in Figure 4b, the root MDA levels of the 9106 + biochar group was lower than that of the 9106 group and the biochar group (58 nmol/g roots versus 80–95 nmol/g roots).

3.5. The Biochar and Halotolerant Bacterium in Combination Alter Soil Enzyme Activity

The rhizosphere soil of the treated plants was further collected for enzyme activity assays. The results are shown in Figure 5a–d. The 9106 + biochar group exhibited enzyme activities of 7.5 U/g soil for S-POD, 14,366.1 U/g soil for S-AKP, and 1654.9 U/g soil for S-UE, while the control group only had the enzyme activity of 2.7 U/g soil, 11,570.8 U/g soil, and 1492.9 U/g soil, respectively.

3.6. The Biochar and Halotolerant Bacterium in Combination Reduced Soil Cation Levels

Cations are the main components contributing to the high salinity of soil. In order to further evaluate the impact of the biochar and 9106 on soil quality, the contents of various cations in the rhizosphere soil of Medicago sativa, i.e., Na⁺, K⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Mn²⁺, was measured. As shown in Figure 6a–f, the 9106 or biochar alone did not reduce the levels of the most detected cations. In contrast, the 9106 + biochar treatment led to a significant decrease in the levels of Na⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Mn²⁺. For instance, while the other three groups had the levels of Na⁺ at >1200 mg/kg soil, the 9106 + biochar treatment reduced the level to 1050 mg/kg soil (Figure 6a). An abnormal result is that 9106 and biochar alone or in combination resulted in an increase in K⁺ levels from 1100 mg/kg soil to >2700 mg/kg soil (Figure 6b), which may be attributed to the accumulation of phosphorus-solubilizing microbes in the rhizosphere soils induced by these treatments. The aforementioned results suggest that the application of 9106 in conjunction with biochar can mitigate salt stress and augment plant stress tolerance.

4. Discussion

4.1. The Moss Biochar Promotes Biofilm Formation and Mitigates Salinity Stress

In this study, the moss-derived biochar significantly stimulated biofilm formation in the halotolerant bacterium Halomonas salifodinae strain 9106 by enhancing extracellular polymeric substance (EPS) biosynthesis. A biochemical quantification revealed a 4.9-fold increase in the EPS produced by the strain 9106 under biochar amendment, which consequently promoted EPS matrix accumulation and facilitated the assembly of biofilm structures. Therefore, this kind of biochar could be used for the regulation of biofilm formation during microbial remediation.

The rhizosphere microbiome, comprising the microorganisms residing within the soil surrounding plant roots, plays a pivotal role in maintaining plant health and nutrient availability. Most microbes could not only live freely as dispersed cells, but also interact with each other to form biofilms. These microbial cells within biofilms reside in a self-produced matrix of EPS, which are predominantly composed of polysaccharides and endow the cells with a high tolerance to environmental stresses [31]. Salt stress has been observed to reduce the rate of microbial attachment to the plant rhizosphere, thereby impeding plant growth [32]. Moss biochar, characterized by its porous structure, possesses a substantial specific surface area, which provides an ample attachment area for rhizosphere microbes [28]. When biochar is attached to roots, it can promote the secretion of polysaccharides and the formation of biofilms by 9106, which results in the colonization of 9106 on the root surface. The EPS of bacterial biofilm have diverse functions, e.g., adhesion, the retention of water, and the sorption of inorganic ions [33]. The biofilm thus functions as a protective barrier against the external environment, thereby creating a superior growth environment for plant root tissues and functional bacteria compared to the external space. The formation of biofilm promotes the colonization and growth of functional rhizoplane bacteria and reduces the salinity stress suffered by the plant root system. Our findings are consistent with the results of previous studies. For instance, the biochar facilitates the rhizosphere colonization of the functional bacteria, including Bacillus, Azospirillum, and Rhizobium [34]. In saline–alkali soil, the biochar has an impact on the relative abundance of bacterial communities, modifies the bacterial community structure, and further stimulates the growth of plants [35]. In short, the moss biochar facilitated the formation of biofilm and the colonization of the functional bacterium 9106, while concurrently isolating the plant root system from the external environment. This, in turn, contributes to a reduction in the salt stress experienced by the plants (Figure 7). The interaction between the root-surface-colonized bacterium and the plants indicates the mutualistic exosymbiosis.

4.2. The Moss Biochar Improves Plant Physiological and Biochemical Indicators

Plant height is one of the most important morphological characteristics of plants, which reflects the growth rate and health status of the plants [36]. The results of this study indicated that the addition of the prepared biochar significantly increased the heights of the plants. Consequently, the combination of 9106 and biochar enhanced plant growth under the stress. Both biochar alone and 9106 + biochar increased the content of chlorophyll a and soluble sugars in plant leaves, which indicated that the two treatments promoted the synthesis of chlorophyll a, and thus enhanced the photosynthetic efficiency of the plants for plant growth under the saline–alkali condition [37].

The plant root is the first organ suffering from saline–alkali stress in the soil. The proline and malondialdehyde contents reflect the stress degree [38,39]. After 30 days of culturing, the proline content in plant roots markedly increased, serving as an indicator of stress tolerance. The experimental results demonstrated that the proline concentration in the three experimental groups was significantly higher compared to the control group. The root malondialdehyde levels of the 9106 + biochar group was lower than that of the 9106 group and the biochar group. This confirmed that the biochar and 9106 in combination drastically alleviated the oxidative stress caused by saline–alkali stress. This is consistent with the existing results of Nielsen’s research, who demonstrated that the Halomonas strain stimulates the growth of Alfalfa in salty soil [40]. Moreover, CFU assays revealed that the biochar increased the root colonization of NFBs. The root colonization of NFBs may further alleviate salt stress and promote the plants to tolerate high-level NaCl.

However, the single 9106 treatment led to a decline in photosynthetic efficiency; likely, biochar has enhanced the tolerance of 9106 to high salt. This phenomenon further constrained the viability of other functional bacteria within the saline soil, exerting a deleterious effect on plant growth. However, 9106 increased SOD activity in plant leaves and increased proline levels in roots, thus mitigating the negative effects of salt stress on plants and reducing the energy consumed by plants in response to salt stress. Interestingly, the 9106 + biochar group had a lower activity of CAT and POD, suggesting that the oxidative stress induced by H₂O₂ and peroxide was attenuated by the combined treatment. Consequently, the plants were able to accumulate more nutrients after a single 9106 treatment, which showed better growth than the CK group. From the final results, both 9106 and biochar enhance plant growth under salt stress conditions; however, the combination of 9106 and biochar appears to optimize this growth.

4.3. Effects of 9106 and Biochar on Soil Enzyme Activity

Soil enzymes, such as soil peroxidase (S-POD), soil alkaline phosphatase (S-AKP), soil urease (S-UE), and soil sucrase (S-SC), represent a category of biologically active factors that can function as indicators of soil fertility and quality [41,42,43]. Normally, the activity of enzymes in soil are attenuated by salt and alkali stress. This decline can be attributed to the inactivation of microbes by heightened salinity stress [44]. In this study, the results demonstrated that the combined 9106 and biochar treatment led to a substantial increase in the activity of S-POD, S-AKP, and S-UE. These findings suggest a positive influence of 9106 and biochar in combination on soil enzyme activity. This phenomenon is likely attributed to the rich nutrients (e.g., organic carbon, nitrogen, and phosphorus), large specific surface area (SSA), and porous architecture. These components collectively furnish soil microorganisms with ample habitats, moisture, and nutrients. These favorable conditions subsequently enhance the activity of S-POD, S-AKP, and S-UE in microbial communities [45,46]. Consistent with the existing results, our research suggests that biochar application can enhance soil enzyme activities.

4.4. 9106 and Biochar Change Plant Ion Uptake Strategy

Na⁺ is one of the main cations contributing to soil salinity. Herein, we found that the rhizosphere soils treated with 9106 and biochar had remarkable lower levels of Na⁺ than the control rhizosphere soil. The possible explanation is that the exogenous 9106 and the endogenous halotolerant bacteria recruited by the biochar may form a protection layer, which may prevent the inclusion of Na⁺ to the rhizosphere soil, and even excluded this cation out of this region. Therefore, the combination of 9106 and biochar may mitigate the toxic effects of excess Na⁺ on plants under salt stress.

Metal elements play a crucial role as cofactors or activators of numerous enzymes involved in photosynthesis. Magnesium, for instance, is the core element of the chlorophyll molecule and functions as the activator of various enzymes associated with photosynthesis. Magnesium plays a pivotal role in the photosynthetic machinery of the chloroplast [47]. Iron is an essential element for activating δ-amino-levulinic acid synthase, which is responsible for the formation of chlorophyll [48]. Manganese was also found to promote photosynthesis. The combined application of 9106 and biochar increased plant uptake of iron, magnesium, and manganese from the soil, which may promote the capacity of photosynthesis in the plants.

SOD prevents oxidative chain reactions that cause extensive damage and prevent the formation of a cascade of harmful reactive oxygen species. Cu²⁺, Fe²⁺, and Mn²⁺ act as cofactors for a variety of SODs, which facilitate the scavenging of root reactive oxygen species in plants [49]. When subjected to salt stress, plants have been observed to produce reactive oxygen species in their root tissues. Plants in the 9106 + biochar group exhibited an increased uptake of Fe²⁺, Cu²⁺, and Mn²⁺, and the accumulated ions activated SOD and the scavenging of tissue reactive oxygen species, which improved the salinity stress tolerance of the plants.

The protective effect of proline is thought to be related to its ability to detoxify reactive oxygen species and inhibit lipid peroxidation [50]. The MDA content can reflect the degree of peroxidation of the plant plasma membrane. The plant roots in the 9106 + biochar group contained less MDA and a lower proline content. This demonstrates the greater reactive oxygen species scavenging capacity of the root system of plants in this group.

Generally, the findings indicate that 9106 and biochar promote the plant excretion of Na⁺ and uptake of Fe²⁺, Cu²⁺, Mg²⁺, and Mn²⁺. This process reduces the Na⁺ toxicity from salt stress and activates SOD to scavenge reactive oxygen species. Additionally, it increases the photosynthetic level of the plant and promotes plant growth under salt stress.

5. Conclusions

In summary, this study revealed that the moss biochar may promote the root colonization of the exogenous halotolerant Halomonas salifodinae 9106 on the forage grass Medicago sativa. During the incubation with the bacteria, the biochar strongly bound the bacteria and induced biofilm formation on the biochar surface. When the biochar and the bacteria were added into the culturing soil, the biochar assisted in the root binding and biofilm formation on the plant roots. After 30 days of culturing, the biochar and the bacteria in combination remarkably increased plant growth, with the enhanced accumulation of chlorophyll a, soluble sugar, and proteins, together with increased POD activity in the leaves. Moreover, the combined treatment increased the activity of soil enzymes, and reduced the levels of various cations, e.g., Na⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, etc., indicating an improvement in soil quality. This study demonstrates a novel biochar–microbe integrated approach, synergistically enhancing forage crop productivity in saline–alkaline soils through rhizosphere microbiome modulation and stress mitigation mechanisms.