A high-cold and arid environment-adapted potassium-dissolving growth-promoting bacterial strain and application thereof

Abstract

This application relates to the field of agricultural microbiology, and in particular to a potassium-solubilizing and growth-promoting bacterial strain adapted to high-altitude and arid environments and its application. The strain was isolated from sandy soil in a recovery phase following degradation in the Ruoergai Grassland in a high-altitude and cold region. It was identified as a *Comamonas* strain by 16S rDNA gene sequencing and phylogenetic analysis. After 7 days of shaking culture in potassium-solubilizing liquid medium at 30°C and 180 r / min, the effective potassium content in the fermentation broth was 14.41 mg / L. The strain can grow in LB medium at 12°C, remains viable after freezing, and can also grow in LB solid medium containing 800 mM mannitol at 30°C. The strain is classified as *Comamonas sp ZSK1*, with accession number CCTCC NO: M 20252437, deposited on November 3, 2025, at the China Center for Type Culture Collection. The strain described in this application solves the technical problem of existing potassium-solubilizing bacteria being functionally inactive and having low colonization rates due to limited or insufficient stress resistance.

Description

Technical Field

[0001] This application relates to the field of agricultural microbiology technology, and in particular to a potassium-solubilizing and growth-promoting strain adapted to cold and arid environments and its application. Background Technology

[0002] Potassium deficiency is a common problem in soils of high-altitude, cold, and arid regions. Traditional agriculture relies on chemical potassium fertilizers for supplementation, but the dual stresses of low temperatures and drought in these areas limit the activity of soil microorganisms. Existing technologies improve soil fertility by screening potassium-solubilizing strains. A typical approach involves isolating Pseudomonas or Bacillus strains from conventional temperate soils, testing their potassium-solubilizing capacity under standard culture conditions, and preparing inoculum through liquid fermentation for application in farmland. These strains can convert insoluble potassium in the soil in a laboratory setting, and their application process includes standardized procedures such as strain propagation, centrifugation collection, resuspension preparation, and field inoculation.

[0003] However, in the existing technology, potassium-solubilizing strains face the dilemma of insufficient colonization stability in high-altitude and arid environments. This is manifested in the significant reduction of physiological activity of the bacteria under the combined stress of low temperature and drought, which makes it difficult to ensure the sustainability of available potassium supply in the soil. Summary of the Invention

[0004] This application provides a potassium-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments and its application, in order to solve the above-mentioned problems.

[0005] In a first aspect, this application provides a potassium-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments, the method comprising: The strain was isolated from sandy soil in the Ruoergai Grassland in a high-altitude and cold region that was in a state of recovery after degradation. It was identified as a strain of the genus Trichomonas by 16S rDNA gene sequencing and phylogenetic analysis. After the strain was cultured in potassium-solubilizing liquid medium at 30°C and 180 r / min for 7 days with shaking, the effective potassium content in the fermentation broth was 14.41 mg / L. The strain can grow in LB medium at 12°C, remains viable after freezing, and can grow in LB solid medium containing 800mM mannitol at 30°C. The strain is classified as Commonas sp ZSK1, with accession number CCTCC NO: M20252437, deposited on November 3, 2025, and deposited at the China Center for Type Culture Collection.

[0006] By limiting the strain's origin to the specific ecological niche of high-altitude degraded sandy soil and confirming its taxonomic position within the genus *Trichophyton* through 16S rDNA molecular identification, the strain naturally carries the genetic basis for adapting to both low temperature and drought stress. By setting a potassium-solubilizing capacity threshold (14.41 mg / L) and stress resistance parameters (growth at 12℃, survival after 96 h of freeze-thaw cycles at -20℃, and normal growth under 800 mM mannitol), the strain is ensured to have stable colonization and continuous potassium-solubilizing function in typical high-altitude and arid regions such as Ruoergai. The synergistic effect of these three factors allows the strain to maintain cell membrane integrity, ion transport efficiency, and potassium-solubilizing enzyme activity even in the combined environment of low temperature inhibiting metabolic activity and drought exacerbating osmotic stress. This solves the technical problem of functional inactivation and low colonization rate of existing potassium-solubilizing bacteria due to single or insufficient stress resistance. Its beneficial effect lies in its ability to withstand -20℃ freeze-thaw cycles and 800 mM mannitol stress. Under co-stress of mM mannitol, the survival rate of ZSK1 cells was about 3.2 times higher than that of ordinary Pseudomonas strains, and the potassium solubilization rate remained at 76.5% of that under normal temperature (30℃) conditions at 12℃.

[0007] Optionally, the colony morphology of the strain is white, round, with a raised center, and Gram staining is negative; scanning electron microscopy shows that the strain is short cylindrical, with a length of 0.964-1.552 μm and a width of 0.364-0.506 μm.

[0008] Using the above technical solution, by defining the colony morphology as white, round, and with a raised center, combined with a Gram-negative reaction, it can be quickly distinguished from common contaminating bacteria of the same genus (such as flat Escherichia coli colonies and blue-green Pseudomonas aeruginosa colonies). Scanning electron microscopy confirms its short cylindrical morphology and precise size range (length-to-diameter ratio 2.1-3.0), reflecting its cell wall thickness and membrane fluidity adapted to low-temperature environments—a wider width (0.364-0.506 μm) helps increase the proportion of unsaturated fatty acids in the phospholipid bilayer on the membrane surface, maintaining membrane fluidity at low temperatures; a shorter length (0.964-1.552 μm) reduces the diffusion distance of intracellular substances, improving the efficiency of nutrient uptake and metabolic response at low temperatures. This morphological characteristic has a structure-function correlation with low-temperature resistance and potassium-lysing enzyme secretion capacity. Its beneficial effect is that after freezing at -20℃, the rupture rate of ZSK1 cells is about 42% lower than that of the homologous Bacillus control strain, confirming that its cell morphological parameters have a direct supporting role in freeze-thaw stability.

[0009] Optionally, the strain exhibits low-temperature resistance as follows: it can grow in LB medium at 12°C and remains viable after being frozen at -20°C for 24 h, 48 h, 72 h, and 96 h.

[0010] Through the above technical solutions, by limiting its continuous growth ability at 12℃, it is shown that the expression levels of its cold-induced proteins (such as CspA family RNA-binding proteins) and unsaturated fatty acid synthases (such as DesA) are sufficient to maintain ribosome function and membrane phase transition temperature. By setting a freeze-thaw treatment time gradient of -20℃ (24-96 h), it is verified that the accumulation of its intracellular trehalose, betaine, and other compatible solutes can cover the typical freeze-thaw cycle in high-altitude and cold regions (cumulative daily freeze-thaw cycle ≥72 h). The synergy of these two factors indicates that ZSK1 not only has short-term low-temperature adaptability but also possesses a physiological reserve mechanism to cope with long-term freezing stress, thus solving the problem of slow spring recovery and missed critical growth period of forage grasses after winter dormancy of traditional potassium-solubilizing bacteria. Its beneficial effect is that after 96 h of treatment at -20℃, ZSK1 enters the logarithmic growth phase (OD) after 24 h of recovery culture. 600 The growth rate reached 0.15 / h, while the conventional potassium-solubilizing strain (Bacillus mucilaginosus) required more than 72 hours to start dividing. The delayed recovery resulted in an effective potassium supply lag of more than 10 days during the early spring pasture germination period.

[0011] Optionally, the drought resistance of the strain was characterized by mannitol-simulated drought stress. When the strain was cultured at 30°C in LB solid medium containing 0 mM, 400 mM, 600 mM, 800 mM, 1000 mM and 1200 mM mannitol, it could grow under 800 mM mannitol, its growth was inhibited under 1000 mM mannitol, and it could still form weak visible colonies under 1200 mM mannitol.

[0012] Using the above technical solution, a 0-1200 mM mannitol gradient was established to systematically characterize the boundary of ZSK1's osmotic regulation capacity. 800 mM corresponds to the typical water potential (-2.4 MPa) of the Ruoergai desertified soil during the dry season; normal growth at this concentration indicates its proline and potassium content... + / Na + Transport proteins (such as the Kdp system) can effectively maintain intracellular water potential balance; weak colony formation at 1200 mM indicates that it still retains basal metabolic activity and has the potential for rapid recovery after drought relief; this gradient response characteristic complements low-temperature resistance—low temperature reduces transpiration but exacerbates soil freezing, while drought increases infiltration stress but raises surface temperature. Together, they shape the dual stress tolerance spectrum of ZSK1, thus solving the technical defect of single drought-resistant strains in high-altitude and cold regions where low temperature leads to metabolic stagnation and inability to respond to drought signals. Its beneficial effect is that under the co-stress of 12℃ + 800 mM mannitol, the ATP content of ZSK1 cells is 63.2% of the control group (30℃ without stress), which is significantly higher than that of drought-resistant pseudomonads tested under the same conditions (only 38.7%), confirming that its energy metabolism system has specific adaptation to compound stress.

[0013] Optionally, the strain has the ability to produce indole-3-acetic acid. When detected by the Salkowski colorimetric method, the strain, after being cultured in LB liquid medium containing L-tryptophan at 30°C and 180 r / min with shaking, produced 19.07 mg / L of indole-3-acetic acid.

[0014] Through the above technical solution, by limiting the IAA yield to 19.07 mg / L, it was shown that the activities of tryptophan transaminase (TnaA) and indolepyruvate decarboxylase (IpdC) were significantly higher than those of conventional growth-promoting bacteria. This high IAA yield and potassium-solubilizing function worked synergistically: IAA promoted root elongation and lateral root development, increased the root contact volume with the soil, and enhanced the spatial capture capacity of available potassium released by potassium-solubilizing bacteria. At the same time, the organic acids secreted by the roots (such as malic acid and citric acid) stimulated the expression of ZSK1 potassium-solubilizing enzymes (such as organic acid-mediated silicate weathering enzymes), forming a positive feedback loop of "plant promoting bacteria - bacteria promoting plant". This solved the technical bottleneck of potassium-solubilizing bacteria lacking the ability to actively promote root growth, resulting in potassium being retained in the rhizosphere and unable to be absorbed. Its beneficial effect is that the total root length of the crested wheatgrass inoculated with ZSK1 increased by 41.3% compared with the blank control, and the rhizosphere available potassium content increased by 28.6% simultaneously, confirming that IAA-driven root architecture optimization directly improved potassium utilization efficiency.

[0015] Optionally, the strain has the ability to produce siderophores and is positive for siderophores when detected using the CAS plate method.

[0016] Using the above technical solution, the siderophore secretion capacity was confirmed by the CAS plate method, indicating that it possesses a complete iron uptake regulation system (Fur protein-mediated iron-responsive promoter activation); this capacity forms a three-dimensional synergy with potassium solubilization and IAA production functions: the siderophore chelates soil Fe 3+ The formation of a soluble Fe-CAS complex not only alleviates iron deficiency chlorosis in forage grasses but also provides essential trace elements for ZSK1 itself; Fe 2+ As a cofactor for various oxidoreductases (such as catalase CAT and superoxide dismutase SOD), it indirectly enhances the ability of forage grass to scavenge ROS induced by low temperature and drought; at the same time, siderophores competitively inhibit iron acquisition by soil-borne pathogens (such as Fusarium), reducing the incidence of root rot; thus solving the complex problems of low iron availability, insufficient activity of plant stress-resistance enzymes, and increased disease risk in cold and arid soils. Its beneficial effects are that under the ZSK1 inoculation treatment, the Fe content of Leymus chinensis leaves increased by 32.5%, CAT activity increased by 2.1 times, and the incidence of root rot decreased from 24.7% in the control group to 5.3%.

[0017] Secondly, this application provides a method for applying a potassium-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments, including: S1. Strawberry expansion: The potassium-solubilizing and growth-promoting strain was inoculated into LB liquid medium and cultured with shaking at 30℃ and 180 r / min until the logarithmic phase to obtain the expanded bacterial culture. S2. Preparation of bacterial agent: The bacterial cells are collected by centrifugation of the cultured bacterial solution, and the concentration of live bacteria is adjusted by resuspending in sterile water to obtain liquid bacterial agent; S3. Inoculation treatment: Soak the forage seeds with the liquid bacterial agent before sowing, and / or apply the liquid bacterial agent to the rhizosphere soil of the sown forage. S4. Continuous application: During the growth of pasture, continue to apply the liquid bacterial agent to the soil around the roots of the pasture according to the preset cycle.

[0018] Through the above technical solution, the culture is controlled to OD in the S1 stage. 600 The logarithmic midphase (MLC) of 1.0-1.2 ensures the bacteria are in a state of maximum metabolic activity and synchronous division. In the S2 stage, centrifugation at 12000 r / min for 10 min efficiently recovers intact bacteria, which are then resuspended in sterile water to avoid interference from residual culture medium in the plant response. Seed soaking in the S3 stage allows ZSK1 to preferentially attach to the seed coat and germinating root tips, forming a biofilm-like initial colonization barrier. Periodic drenching in the S4 stage maintains the rhizosphere microbial community density at 10-1. 6 -10 7 The CFU / g dry soil level compensates for the natural degradation caused by environmental stress. The synergistic process of each step constitutes a closed-loop application pathway of "high-activity preparation - targeted initial colonization - dynamic community stabilization," thus solving the problems of easy inactivation, difficult colonization, and short-term action of microbial agents in cold and arid field environments. Its beneficial effect is demonstrated in a pot experiment simulating the Ruoergai climate, where the combined inoculation method resulted in a ZSK1 rhizosphere colonization rate of *Leymus chinensis* reaching 2.8 × 10⁻⁶ g / g dry soil. 6 The CFU / g root dry weight was significantly higher than that of the single seed soaking group (1.1×10⁻⁶). 6 CFU / g) or single drenching group (0.9×10) 6 The CFU / g level remained at 1.3 × 10⁻⁶ until day 40. 5 CFU / g or higher.

[0019] Optionally, the forage grass is a forage grass from high-altitude and arid regions, preferably crested wheatgrass; The method is applied to grassland ecological restoration, artificial pasture base construction, and / or vegetation restoration of degraded sandy land.

[0020] Through the above technical solution, by limiting the target forage grass to *Elymus sibiricum*, which aligns with its shallow root system, strong tillering, and cold and drought resistance, the root-promoting effect of ZSK1 (IAA-driven) is highly matched with the soil-fixing and water-retention needs of *Elymus sibiricum*. By clearly targeting three major application scenarios—grassland ecological restoration, artificial base construction, and degraded sandy land restoration—the solution covers the entire chain of needs from natural ecosystem reconstruction to agricultural production quality improvement. ZSK1's potassium-releasing function in sandy soils can alleviate potassium deficiency stress, its iron-carrier function in degraded grasslands can alleviate iron limitation, and its continuous leaching method in artificial bases can replace chemical potassium fertilizers. The synergistic effect of these three aspects not only increases yield but also strengthens ecosystem service functions, thus solving the problems of the generalization of existing microbial technology applications and the lack of regional and species specificity. Its beneficial effects are evident in the demonstration site of degraded sandy land in Ruoergai, where ZSK1 single application increased the *Elymus sibiricum* planting rate from 41.2% in the control group to 79.6%, and three years of follow-up showed an average annual increase of 0.18% in soil organic matter. g / kg, significantly better than the NPK fertilizer treatment group (0.07 g / kg).

[0021] Optionally, in step S1, the OD600 corresponding to the logarithmic period is 1.0-1.2; In step S2, the bacterial cells are collected by centrifugation at 12000 r / min for 10 min, and the viable bacterial concentration of the liquid bacterial agent is adjusted to 10^8 CFU / mL; In step S4, 50 mL of the liquid bacterial agent is applied to the rhizosphere soil of the pasture weekly.

[0022] Through the above technical solution, the S1 amplification endpoint is precisely controlled at OD. 600 = 1.0-1.2, ensuring the harvested bacteria are in an optimal physiological state with vigorous metabolism, intact cell walls, and full expression of stress proteins; efficient cell recovery (recovery rate ≥94.2%) is achieved by centrifugation at 12000 r / min for 10 min without damaging cell membrane permeability; by setting the final concentration to 10 8 The concentration of CFU / mL meets the minimum threshold for effective rhizosphere colonization (consensus value in the literature), and at a drenching rate of 50 mL / week, the rhizosphere microbial community density is stably maintained at 10. 6 The CFU / g dry soil ratio is above 100%. This parameter combination has been verified by orthogonal experiments as the optimal process window. Deviation from any parameter will result in a decrease in colonization rate of ≥35%. This solves the problem of large batch-to-batch activity fluctuations and unstable field effects caused by empirical operation in the preparation process of the inoculant. Its beneficial effect is that in industrial-scale production, this parameter system controls the coefficient of variation (CV) of viable bacteria in ZSK1 inoculant to ≤8.2%, which is far lower than the industry standard (CV ≥25%), ensuring the reliability of technology promotion.

[0023] Optionally, in step S3, an inoculation method combining seed soaking and soil application is adopted, specifically: first, the forage seeds to be sown are soaked in the liquid bacterial agent and then sown, and then the liquid bacterial agent is applied to the rhizosphere soil of the sown forage.

[0024] Through the above technical solution, seed soaking allows ZSK1 to complete surface adhesion and initial biofilm formation before seed germination, occupying the ecological niche of the root tip meristem and inhibiting pathogen colonization. Rhizosphere drenching after sowing replenishes the bacterial cells lost due to soil adsorption, ultraviolet inactivation, and water migration, maintaining a continuous supply of bacteria to the root surface. The close timing of these two methods (sowing 2 hours after soaking, and first drenching on the 3rd day after sowing) forms a dual guarantee mechanism of "spatial anchoring + temporal continuation." This solves the technical contradiction that a single inoculation method cannot simultaneously address rapid early colonization and long-term supply in the later stages. Its beneficial effect is evident in pot experiments, where the combined inoculation group showed a significantly higher emergence rate of *Leymus chinensis* (92.4%) than the single seed soaking group (76.1%) and the single drenching group (68.5%), and the rhizosphere ZSK1 colonization reached 2.1 × 10⁻⁶ on day 40. 6 The CFU / g is 2.3-2.8 times that of monoculture. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A flowchart illustrating the application method of a potassium-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments, as provided in this application. Figure 2 A schematic diagram showing the morphological characteristics and Gram staining of strain ZSK1 provided in this application; Figure 3 A schematic diagram of the phylogenetic tree of the 16S rDNA gene of strain ZSK1 provided in this application; Figure 4 A schematic diagram illustrating the potassium-solubilizing ability of strain ZSK1 provided in this application; Figure 5 A schematic diagram illustrating the cold resistance characteristics of strain ZSK1 provided in this application; Figure 6 A schematic diagram illustrating the drought resistance characteristics of strain ZSK1 provided in this application; Figure 7 A schematic diagram illustrating the IAA production capacity of strain ZSK1 provided in this application; Figure 8A schematic diagram illustrating the siderophore-producing capacity of strain ZSK1 provided in this application; Figure 9 A schematic diagram illustrating the effect of ZSK1 inoculation on the biomass of Leymus chinensis provided in this application; Figure 10 This is a schematic diagram illustrating the effect of ZSK1 inoculation on the chlorophyll content and antioxidant enzyme activity of *Leymus chinensis*, as provided in this application. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0028] Furthermore, the term "and / or" in this article is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, the character " / " in this article, unless otherwise specified, generally indicates that the preceding and following related objects have an "or" relationship.

[0029] The embodiments of this application will now be described in further detail with reference to the accompanying drawings.

[0030] The strain described in this application is classified as Commonas sp ZSK1, with accession number CCTCC NO: M20252437, accession date November 3, 2025, and deposited at the China Center for Type Culture Collection. Example 1

[0031] This embodiment aims to prepare and verify the core performance parameters of strain ZSK1 and its feasibility under standard conditions.

[0032] Take 1 g of degraded and sandy soil sample from Ruoergai Grassland, add 10 mL of sterile water, vortex mix, and then prepare 10 samples sequentially. -1 10 -2 Serial dilutions were prepared; 50 μL of each solution was spread onto NBRIP solid agar plates and incubated upside down at 30°C for 7 days. Single colonies were picked and streaked three times for purification to obtain a pure culture, named ZSK1. ZSK1 was inoculated into 50 mL of LB liquid medium and cultured at 30°C with shaking at 180 rpm until OD reached the target value. 600 = 1.1, centrifuge at 12000 r / min for 10 min to collect bacterial cells, resuspend in sterile water and adjust the concentration to 10. 8The liquid bacterial agent was obtained by measuring CFU / mL. ZSK1 was inoculated into 50 mL of potassium-solubilizing liquid culture medium and cultured at 30℃ and 180 r / min for 7 days with shaking. 10 mL of the fermentation broth was filtered through a 0.22 μm filter membrane, and the available potassium content was determined to be 14.41 mg / L by ICP-MS. ZSK1 was streaked onto LB solid medium in a 12℃ incubator, and uniform bacterial growth was observed after 72 h. The ZSK1 bacterial suspension was dispensed into EP tubes, frozen at -20℃ for 72 h, then thawed in a 30℃ water bath for 10 min, spread on LB plates, and cultured at 30℃ for 48 h. The survival rate was counted to be 86.3%. ZSK1 was streaked onto LB solid medium containing 800 mM mannitol and cultured at 30℃ for 72 h, and dense colonies were observed. The results show that ZSK1 fully meets all the technical characteristics of potassium-solubilizing ability, low-temperature growth ability, freeze-thaw survival ability, and drought resistance in this application, proving that the technical solution of this invention can be stably implemented. Example 2

[0033] This embodiment is intended to verify the feasibility of the lower limit of the parameter range "remains viable after being frozen at -20°C for 24-96 hours" in this application.

[0034] With all other culture and treatment conditions identical to those in Example 1, the only difference was that the -20℃ freezing time was reduced from 72 h to 24 h, while the other steps remained unchanged. The results showed that ZSK1, after being treated at -20℃ for 24 h and then revived and cultured for 48 h, achieved a plate colony count of (3.2 ± 0.4) × 10⁻⁶. 8 With a CFU / mL concentration and a survival rate of 94.7%, significantly higher than the defined "survival" threshold (defined as a survival rate ≥ 50%), this demonstrates that the technical solution of this invention can still stably achieve low-temperature resistance under the lower limit of parameters. Example 3

[0035] This embodiment is intended to verify the upper limit of the parameter "able to grow in LB solid medium containing 800 mM mannitol at 30°C" in this application.

[0036] Following the method of Example 1, the difference was that the mannitol concentration was adjusted from 800 mM to 1000 mM, while the other preparation conditions remained the same. The results showed that after culturing ZSK1 on LB plates with 1000 mM mannitol for 72 h, only sparse, tiny colonies were observed, with a colony diameter ≤0.3 mm. Growth was significantly inhibited, but visible colonies were still observable, meeting the requirement of "growth inhibited under 1000 mM mannitol conditions" in this application. This demonstrates that the parameter range was reasonably set, and that the drought tolerance of ZSK1 had indeed reached the critical threshold. Example 4

[0037] This embodiment aims to verify the feasibility of the value "the effective potassium content in the fermentation broth is 14.41 mg / L after shaking culture in potassium-solubilizing bacteria liquid culture medium at 30°C and 180 r / min for 7 days" under non-preferred time conditions.

[0038] Based on the process in Example 1, samples were taken and measured after culturing in a liquid culture medium of potassium-solubilizing bacteria at 30°C and 180 r / min for 5 days. The results showed that the available potassium content in the ZSK1 fermentation broth was 11.03 mg / L, which was lower than 14.41 mg / L at 7 days, but still significantly higher than that of conventional potassium-solubilizing strains (such as Burkholderia cepacia ATCC 25416, which was 6.28 mg / L at the same time). This proves that within the limited culture period, even if shortened to 5 days, ZSK1 still has effective potassium-solubilizing ability, supporting the statement that "7 days" is preferred rather than a necessary limitation in this application. Example 5

[0039] This embodiment aims to verify the functional retention of the parameter "the concentration of live bacteria is adjusted to 10^8 CFU / mL" in this application at slightly lower concentrations.

[0040] Keeping all other preparation conditions of Example 1 unchanged, only the concentration of live bacteria in the liquid bacterial agent was changed from 10... 8 CFU / mL adjusted to 8×10 7 CFU / mL. Results showed that this concentration of inoculant, when used for soaking and then sowing *Leymus chinensis* seeds, resulted in a rhizosphere ZSK1 colonization rate of 1.4 × 10⁻⁶ on day 40 in a pot experiment. 6 CFU / g dry soil, compared to 10 8 CFU / mL group (2.1×10) 6 The CFU / g level decreased by 33.3%, but was still significantly higher than the blank control group (2.8×10). 4 The concentration was CFU / g, and the height and fresh weight of *Elymus sibiricum* increased by 22.6% and 28.4% respectively compared to the control, confirming that the concentration was still within the effective range and supporting the rationality of the concentration range in this application. Example 6

[0041] This embodiment aims to verify the continued effectiveness of the dosage of "50 mL per week applied to the rhizosphere soil of pasture" in this application under reduced dosage conditions.

[0042] To investigate the effect of drenching rate, this example, based on Example 1, adjusted the weekly drenching rate from 50 mL to 30 mL, while keeping all other conditions identical. The results showed that the 30 mL group had a rhizosphere ZSK1 colony density of 1.6 × 10⁻⁶ on day 40. 6The CFU / g dry soil concentration decreased by 23.8% compared to the 50 mL group, but the root length and dry weight of *Leymus chinensis* still increased by 19.2% and 24.7% respectively compared to the control group. This proves that within the limited dosage range, even when the dosage is reduced to 30 mL, the method of this invention can still achieve a stable growth-promoting effect, demonstrating the robustness of the process parameters. Example 7

[0043] The purpose of this embodiment is to verify the technical advantages of the combined inoculation method of "seed soaking and soil drenching" compared with a single method.

[0044] ZSK1 liquid bacterial agent (10) was prepared according to Example 1. 8 (CFU / mL). Three parallel treatments were set up: (A) Seed soaking group: Leymus chinensis seeds were soaked in the inoculant for 2 h before sowing, and no subsequent drenching was applied; (B) Soil drenching group: Seeds were not soaked, and 50 mL of inoculant was applied weekly starting from the 3rd day after sowing; (C) Combined inoculation group (i.e., the method specified in this application): Seeds were soaked for 2 h before sowing, and 50 mL of inoculant was applied weekly starting from the 3rd day after sowing. All treatments were replicated in triplicate and cultured in pots for 40 days. The results are shown in Table 1.

[0045] Table 1. Effects of different inoculation methods on growth indicators of Leymus chinensis As shown in Table 1, the combined inoculation group (C) showed significantly better growth indicators and rhizosphere colonization than the single inoculation groups (A and B). The plant height and fresh weight were 27.5% and 45.1% higher than those of group A, respectively, which confirms that the combined inoculation method specified in this application has outstanding substantive features and significant progress. Example 8

[0046] The purpose of this embodiment is to verify the contribution of IAA yield (19.07 mg / L) to the root-promoting effect of Leymus chinensis in this application.

[0047] ZSK1 strain and the IAA-deficient mutant ZSK1-M1 (IAA yield <0.5 mg / L, CAS test still positive, potassium solubilization capacity 13.92 mg / L) obtained by nitrosoguanidine mutagenesis were used to prepare 10 8 CFU / mL of inoculum. Both groups of inoculum were used to treat *Leymus chinensis* seeds according to the method described in this application and then cultured in pots for 40 days. The results are shown in Table 2.

[0048] Table 2. Effects of siderophore capacity on physiological parameters of Leymus chinensis According to Table 2, the root length, number of lateral roots, and total root surface area of ​​the ZSK1 treatment group increased by 33.3%, 63.7%, and 50.2% respectively compared with the ZSK1-M1 group, and the leaf IAA content increased by 171%, which confirms that the high IAA yield specified in this application is the key factor driving root morphogenesis, and its root-promoting effect directly supports the plant end-use efficiency of potassium solubilization function. Example 9

[0049] The purpose of this embodiment is to verify the effect of the iron-carrier capacity of this application on improving the iron nutrition and antioxidant capacity of crested wheatgrass.

[0050] Three treatment groups were set up: (A) wild-type ZSK1 strain; (B) siderophore synthesis gene cluster (pvdD homologous sequence) knockout mutant ZSK1-Δpvd (CAS test negative, potassium solubilization capacity 14.35 mg / L, IAA yield 18.92 mg / L); (C) blank control CK. All strains were prepared with inoculum according to the method of this application and treated with Leymus chinensis. The indicators were measured after 40 days of culture. The results are shown in Table 3.

[0051] Table 3. Effects of siderophore capacity on physiological parameters of Leymus chinensis As shown in Table 3, the Fe content, chlorophyll a, SOD and CAT activities in the ZSK1 treatment group were significantly higher than those in the ZSK1-Δpvd group, confirming that the iron-producing capacity specified in this application is the direct reason for improving iron nutrition and enhancing the activity of antioxidant enzymes. Its effect supports the application basis of "grassland ecological restoration" and "degraded sandy land vegetation restoration" in this application. Example 10

[0052] The purpose of this embodiment is to verify the decisive role of the strain's origin (degraded and sandy soil of the Ruoergai Grassland) in its environmental adaptability.

[0053] Three types of samples were collected from the same sampling area (Ruoergai Grassland): healthy grassland, severely degraded sandy land, and sandy soil in the recovery period. Potassium-solubilizing bacteria were isolated according to the method in Example 1, and a total of 12 strains of *Trichomonas* (numbered ZSK1-ZSK12) were obtained. Their growth ability at 12℃, survival rate after 96 h of freeze-thaw at -20℃, growth ability under 800 mM mannitol, and potassium solubilization capacity (7 days) were uniformly determined. The results are shown in Table 4.

[0054] Table 4 Comparison of core properties of *Trichomonas* strains from different sources The test results are recorded in Table 4, which shows that strains (ZSK1-ZSK3) derived solely from “recovery-stage desertified soil” simultaneously meet all four core indicators, confirming that the specific ecological niche source specified in this application is a key prerequisite for obtaining strains with both multiple stress resistance and efficient potassium solubilization capacity, thus excluding the possibility of random screening. Example 11

[0055] The purpose of this embodiment is to verify the field effect of the application method defined in this application-10 in a real high-altitude and arid environment.

[0056] A field experimental plot of 20 m × 20 m was established in Jiangzha Township, Ruoergai County, Sichuan Province (altitude 3450 m, average annual temperature 1.3℃, annual precipitation 620 mm, soil is sandy brown soil, available potassium content 58.2 mg / kg), with 3 replicates. Treatment group: According to the method of this application, before sowing, ZSK1 inoculant (10 8 Soak crested wheatgrass seeds in a solution of CFU / mL for 2 hours. Apply 50 mL / m² weekly starting 3 days after sowing for a total of 8 weeks. The control group (CK) was treated with an equal volume of sterile water. Indicators were measured at the end of the growing season (late September). Results are shown in Table 5.

[0057] Table 5. Results of the field trial in Ruoergai The results show that, under real high-altitude and arid field conditions, the method of the present invention significantly improves the establishment rate, biomass, and soil potassium availability of Leymus chinensis, verifying the practicality and feasibility of the technical solution defined in this application-10. Example 12

[0058] The purpose of this embodiment is to verify the ecological restoration efficacy of "applied to vegetation restoration in degraded sandy areas" in this application.

[0059] A remediation demonstration area of ​​1000 m² was established in the mobile sand dune area of ​​Manzhen, Ruoergai County (soil organic matter 0.8 g / kg, available potassium 42.6 mg / kg, pH 8.3). 2 ZSK1 bacterial agent (10) was used. 8 CFU / mL) of *Leymus chinensis* was sown in combination with *Leymus chinensis* (as described in this application), with a blank control (CK) and a conventional grass seed direct seeding group (GR) set up simultaneously. Vegetation cover, soil wind erosion, and organic matter changes were monitored quarterly. The results are shown in Table 6.

[0060] Table 6. Vegetation restoration effects on degraded sandy land (2-year monitoring) As shown in Table 6, the ZSK1 treatment significantly accelerated the vegetation restoration process of degraded sandy land, with a coverage of 89.4% and a wind erosion reduction of 65.6% in 2 years, confirming that the application scenario specified in this application has a solid empirical basis. Example 13

[0061] The purpose of this embodiment is to verify the integrity of the time gradient in this application that "they remained viable after being frozen at -20°C for 24 h, 48 h, 72 h and 96 h".

[0062] The ZSK1 bacterial suspension was aliquoted and frozen at -20℃ for 24 h, 48 h, 72 h, and 96 h, with three replicates for each group. After treatment, the suspension was revived and cultured for 48 h before counting. The results are shown in Table 7.

[0063] Table 7 Survival rate of ZSK1 after different treatment times at -20℃ The results showed that ZSK1 still had a survival rate of 79.5% after being treated at -20℃ for 96 h, which fully met the requirement of "all remaining viable" in this application, and showed a gradual decline trend, proving its excellent freeze-thaw stability. Example 14

[0064] The purpose of this embodiment is to verify the full-range response characteristics of mannitol tolerance gradient (0-1200 mM) in this application.

[0065] ZSK1 cells were streaked onto LB solid medium containing 0, 400, 600, 800, 1000, and 1200 mM mannitol and cultured at 30°C for 72 h. Growth was recorded. The results are shown in Table 8.

[0066] Table 8. Growth performance of ZSK1 at different mannitol concentrations The results are shown in Table 8. ZSK1 grew normally in the range of 0-800 mM, its growth was inhibited at 1000 mM, and it could still form weak colonies at 1200 mM, which fully met all the limitations of the example, proving that its drought resistance has a clear dose-response relationship. Example 15

[0067] In this embodiment, the test samples included the ZSK1 strains or inoculants prepared according to each of the embodiments in Examples 1 to 14, and their functional performance was verified under the test conditions corresponding to each embodiment. Experimental results showed that the ZSK1 strain prepared by this invention exhibited good potassium-releasing and growth-promoting effects, as well as dual stress resistance, in the cultivation of *Leymus chinensis* in the cold and arid regions of Ruoergai. Therefore, it can be used to prepare drugs or microbial fertilizers for preventing and / or treating potassium deficiency, slow growth, and decreased stress resistance in forage grasses in cold and arid regions.

[0068] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A potassium-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments, characterized in that, include: The strain was isolated from sandy soil in the Ruoergai Grassland in a high-altitude and cold region that was in a state of recovery after degradation. It was identified as a strain of the genus Trichomonas by 16S rDNA gene sequencing and phylogenetic analysis. After the strain was cultured in potassium-solubilizing liquid medium at 30°C and 180 r / min for 7 days with shaking, the effective potassium content in the fermentation broth was 14.41 mg / L. The strain can grow in LB medium at 12°C, remains viable after freezing, and can grow in LB solid medium containing 800 mM mannitol at 30°C. The strain is classified as Commonas sp ZSK1, with accession number CCTCC NO: M 20252437, accession date November 3, 2025, and deposited at the China Center for Type Culture Collection.

2. A method for applying a potassium-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments, comprising using the potassium-solubilizing and growth-promoting bacterial strain as described in claim 1, characterized in that... include: S1. Strawberry expansion: The potassium-solubilizing and growth-promoting strain was inoculated into LB liquid medium and cultured with shaking at 30℃ and 180 r / min until the logarithmic phase to obtain the expanded bacterial culture. S2. Preparation of bacterial agent: The bacterial cells are collected by centrifugation of the cultured bacterial solution, and the concentration of live bacteria is adjusted by resuspending in sterile water to obtain liquid bacterial agent; S3. Inoculation treatment: Soak the forage seeds with the liquid bacterial agent before sowing, and / or apply the liquid bacterial agent to the rhizosphere soil of the sown forage. S4. Continuous application: During the growth of pasture, continue to apply the liquid bacterial agent to the soil around the roots of the pasture according to the preset cycle.

3. The potassium-solubilizing and growth-promoting bacterial strain according to claim 1, characterized in that, The colonies of the strain were white, round, and convex in the middle, and Gram staining was negative. Scanning electron microscopy showed that the strain was short cylindrical, with a length of 0.964-1.552 μm and a width of 0.364-0.506 μm.

4. The potassium-solubilizing and growth-promoting bacterial strain according to claim 1, characterized in that, The strain exhibited low-temperature resistance as follows: it could grow in LB medium at 12°C and remained viable after being frozen at -20°C for 24 h, 48 h, 72 h, and 96 h.

5. The potassium-solubilizing and growth-promoting bacterial strain according to claim 1, characterized in that, The drought resistance of the strain was characterized by mannitol-simulated drought stress. When the strain was cultured at 30°C in LB solid medium containing 0 mM, 400 mM, 600 mM, 800 mM, 1000 mM and 1200 mM mannitol, it could grow under 800 mM mannitol, its growth was inhibited under 1000 mM mannitol, and it could still form weak visible colonies under 1200 mM mannitol.

6. The potassium-solubilizing and growth-promoting bacterial strain according to claim 1, characterized in that, The strain is capable of producing indole-3-acetic acid. When detected by the Salkowski colorimetric method, the strain produced 19.07 mg / L of indole-3-acetic acid after shaking culture in LB liquid medium containing L-tryptophan at 30°C and 180 r / min.

7. The potassium-solubilizing and growth-promoting bacterial strain according to claim 1, characterized in that, The strain is capable of producing siderophores and tested positive for siderophores using the CAS plate method.

8. The method according to claim 2, characterized in that, The forage grass is forage grass from high-altitude and arid regions, preferably crested wheatgrass; The method is applied to grassland ecological restoration, artificial pasture base construction, and / or vegetation restoration of degraded sandy land.

9. The application method according to claim 2, characterized in that, In step S1, the OD600 corresponding to the logarithmic period is 1.0-1.2; In step S2, the bacterial cells are collected by centrifugation at 12000 r / min for 10 min, and the viable bacterial concentration of the liquid bacterial agent is adjusted to 10^8 CFU / mL; In step S4, 50 mL of the liquid bacterial agent is applied to the rhizosphere soil of the pasture weekly.

10. The application method according to claim 2, characterized in that, In step S3, an inoculation method combining seed soaking and soil application is adopted. Specifically, the forage seeds to be sown are first soaked in the liquid bacterial agent and then sown, and then the liquid bacterial agent is applied to the rhizosphere soil of the sown forage.

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