Phosphorus solubilizing bacterial strain adapted to high-cold and arid environment and application thereof

Abstract

This application relates to the field of agricultural microbiology, and in particular to a phosphorus-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments and its application. The strain was isolated from soil in cold regions and identified as *Pantoea agglomerans* by 16S rDNA gene sequencing and phylogenetic analysis. It can grow in LB medium at 12°C, form visible colonies in LB solid medium containing 1200 mM mannitol, and exhibits phosphorus-solubilizing ability in NBRIP liquid medium. The strain is classified as *Pantoea agglomerans* ZSP3, with accession number CCTCC NO: M 20252438, deposited on November 3, 2025, at the China Center for Type Culture Collection. This application demonstrates the stable survival of Pantoea agglomerans strain isolated from soil in high-altitude and cold regions under the dual stress of low temperature and drought. This not only solves the problem of low colonization rate of strains under the dual stress of low temperature and drought, but also effectively improves the biomass and stress resistance of forage grasses in high-altitude and cold and arid environments by integrating phosphorus solubilization function and growth promotion mechanism.

Description

Technical Field

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

[0002] High-altitude and cold regions, especially high-altitude grasslands and degraded plateau grasslands, are typically characterized by low temperatures, large diurnal temperature variations, low precipitation, high evaporation, and poor soil development. Phosphorus in the soils of these areas usually exists in insoluble forms, such as calcium phosphate, which is difficult for plant roots to directly absorb and utilize. This leads to a shortage of available phosphorus in local core forage grasses, especially species like crested wheatgrass, resulting in slow growth, low biomass, and poor stress resistance.

[0003] To address these issues, current technologies typically employ the application of chemical phosphate fertilizers to replenish soil phosphorus. However, high-altitude, arid regions generally suffer from poor water and fertilizer retention, leading to low utilization rates of chemical phosphate fertilizers. These fertilizers are prone to fixation, loss, or soil compaction, making it difficult to sustainably meet the dual demands of pasture grasses for phosphorus supply and environmental adaptability. Furthermore, while existing commercial phosphorus-solubilizing bacteria can convert insoluble phosphorus to some extent, most strains lack adaptability to low temperatures or drought, making it difficult for them to survive and establish stable colonies in high-altitude, arid regions, thus limiting their effectiveness.

[0004] Therefore, in response to the practical problems of "soil phosphorus deficiency, insufficient stress resistance of conventional phosphorus-solubilizing bacteria, and unstable effects of traditional fertilization methods" in high-altitude and arid regions, it is urgent to develop a functional strain that simultaneously possesses strong low-temperature resistance, high drought tolerance, efficient phosphorus solubilization capacity, and plant growth-promoting ability, in order to adapt to the special ecological conditions of high-altitude and arid regions and improve the growth of pasture and the utilization of soil nutrients. Summary of the Invention

[0005] This application provides a phosphorus-solubilizing and growth-promoting strain adapted to cold and arid environments and its application, in order to solve the above-mentioned problems.

[0006] Firstly, this application provides a phosphorus-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments, comprising: The strain was isolated from soil in a high-altitude and cold region. It was identified as a Pantoea agglomerans strain by 16S rDNA gene sequencing and phylogenetic analysis. It can grow in LB medium at 12°C, form visible colonies in LB solid medium containing 1200 mM mannitol, and has phosphorus solubilization ability in NBRIP liquid medium. The strain is classified as Pantoea agglomerans ZSP3, with accession number CCTCC NO: M20252438, deposited on November 3, 2025, and deposited at the China Center for Type Culture Collection.

[0007] Through the above technical solution, by limiting the source of the strain to soil from high-altitude and cold regions, and combining the dual screening criteria of growth ability at 12℃ and tolerance to 1200mM mannitol, the strain naturally enriches low-temperature stress response genes (such as the homologous sequence of the cold shock protein CspA) and osmotic protection substance synthesis pathways (such as trehalose-6-phosphate synthase Tps1 and glycerol-3-phosphate dehydrogenase Gpd1). This allows for synergistic effects in maintaining cell membrane fluidity, regulating intracellular ion homeostasis, and ensuring the low-temperature conformational stability of key enzymes. This ensures that the strain can still initiate phosphorus-related metabolism (such as the expression of citrate transporter CitM and acid phosphatase PhoA) under the dual stress of low temperature and high osmotic pressure. Ultimately, this achieves stable colonization and continuous release of available phosphorus in high-altitude and arid environments, solving the functional failure problem caused by the single or missing stress resistance of existing phosphorus-solubilizing bacteria. Its beneficial effects are: in typical high-altitude and arid soils of Ruoergai (pH 8.2, organic matter 0.87g / kg, available phosphorus 4.3mg / kg), inoculation with ZSP3 30 After 1 day, the rhizosphere available phosphorus content increased to 112.6 mg / kg, which was 2.8 times higher than the blank control.

[0008] Optionally, the strain is isolated from the root soil of plants in a recovery state after degradation in high-altitude and cold regions.

[0009] Through the above technical solution, by restricting the strain to "root soil of plants in a recovery state after degradation in high-altitude cold regions," the obtained strain naturally undergoes low temperature, low nutrition, periodic wet-dry cycles, and selective pressure from plant root exudates, thereby enriching rhizosphere growth-promoting functional genes (such as the rate-limiting enzyme TnaA for IAA synthesis and the siderophore synthesis gene schA) and establishing a pre-adaptive interaction with the host plant. This source characteristic is directly related to the high colonization rate and growth-promoting effect of the strain in the rhizosphere of *Elymus sibiricum*. Its beneficial effects are: compared with the homologous *Pantoea* strain (ZSP3-NR) isolated from non-degraded high-altitude cold soil, ZSP3 has a 3.7-fold increased colonization stability in the rhizosphere of *Elymus sibiricum* (survival rate after 30 days: 92.4% vs. 24.8%), and the growth-promoting effect (plant height increase) is increased by 41.6%.

[0010] Optionally, the colony morphology of the strain is yellow, round, with a raised center, and Gram staining is negative; Under scanning electron microscopy, the strain was found to be short cylindrical, with a length of 0.889-1.361 μm and a width of 0.384-0.546 μm.

[0011] Using the above technical solution, a standardized morphological identification map of ZSP3 was constructed by defining macroscopic phenotypes such as colony color, shape, and Gram reaction, combined with the short cylindrical morphology and size range measured by SEM. The yellow colony characteristic is due to the accumulation of carotenoids (such as phytopenic acid), which not only enhances cold resistance as an oxidative stress protectant, but also endows the bacteria with light-shielding ability in shallow soil. The short cylindrical structure (length-to-diameter ratio 2.3-2.5) optimizes the migration and attachment efficiency in soil pores (average pore size 3-8 μm). Its beneficial effect is that this morphological parameter is significantly positively correlated with the penetration depth of ZSP3 in sandy degraded soil (4.2±0.6 cm) (p<0.01), which is 2.1 times higher than that of phosphate-solubilizing bacteria (Pseudomonas fluorescens P13).

[0012] Optionally, the strain remains viable after being frozen at -20°C for 24-96 hours.

[0013] Using the above technical solution, and by setting the survival rate of ZSP3 after freeze-thaw treatment at -20℃ for 24-96 h, it was confirmed that ZSP3 possesses a complete antifreeze protection mechanism, including intracellular trehalose accumulation (up to 128 mmol / g DCW), increased proportion of unsaturated fatty acids in membrane lipids (C16:1 / C16:0 increased from 0.82 to 1.97), and upregulation of the cold-induced chaperone protein Cpn60 expression, thereby inhibiting ice crystal formation, maintaining membrane fluidity, and preventing protein cold denaturation. Its beneficial effect is that after being frozen at -20℃ for 96 h and then thawed, ZSP3 showed a higher OD value in LB medium at 12℃. 600 The value recovered to 89.3% of the initial value, while the common P. agglomerans model strain (ATCC11591) had only 12.7%, indicating that it has the ability to overwinter and be rapidly activated in spring.

[0014] Optionally, after the strain was cultured in NBRIP liquid medium at 30°C and 180 r / min for 7 days with shaking, the available phosphorus content in the fermentation broth was 171.4 mg / L.

[0015] The above technical solution, by limiting the phosphorus solubility under standard NBRIP culture conditions to 171.4 mg / L, reflects the synergistic phosphorus-soothing efficiency of organic acids (citric acid accounting for 63.2% of the total acid content) and acid phosphatase (PhoA specific activity reaching 48.7 U / mg prot) in ZSP3 under optimal metabolic conditions. Citric acid, through chelation of Ca... 2+By disrupting the crystal structure of hydroxyapatite and hydrolyzing organophosphorus ester bonds, the combined effect of these two processes increases the dissolution rate of insoluble phosphorus by 2.4 times. The beneficial effects are: the phosphorus solubility is 67.5% higher than the average value (102.3 mg / L) reported by alpine phosphate-solubilizing bacteria in the literature, and this increase is also observed in simulated alpine soil extracts (pH 8.1, Ca...). 2+ It still maintains an effective phosphorus release of 138.6 mg / L in alkaline high-calcium soil (12.4 mmol / L), demonstrating its functional stability in alkaline high-calcium soil.

[0016] Optionally, the strain exhibits IAA production capacity of 18.99 mg / L after being cultured in LB liquid medium containing L-tryptophan, and also demonstrates siderophore production capacity as detected by the CAS plate method.

[0017] Through the above technical solution, by simultaneously limiting IAA production (18.99 mg / L) and siderophore positive phenotype, it was revealed that ZSP3 possesses a dual-pathway growth-promoting mechanism of "hormone-induced root growth + micronutrient activation": IAA increases primary root length by 32.4% by activating the expression of the cyclin gene (CyCD3;1) in the root tip meristem of *Leymus chinensis*; while siderophores (mainly rhizoferrin) maintain Fe within the pH range of 7.8-8.5. 3+ The solubility increased the iron content of Leymus chinensis leaves by 28.9%, alleviating the common iron deficiency chlorosis in alkaline soils. Its beneficial effects are: the dual function synergistically increased the root-to-shoot ratio of Leymus chinensis from 1.32 in the control group to 1.89, increased the total root length by 47.6%, and significantly enhanced water absorption capacity (transpiration rate increased by 35.2%).

[0018] Secondly, this application provides a method for applying a phosphorus-solubilizing and growth-promoting strain adapted to cold and arid environments, including: S1. The phosphorus-solubilizing and growth-promoting strain is inoculated into a culture medium and cultured with shaking until the logarithmic growth phase to obtain a culture solution; S2. The culture medium obtained in step S1 is centrifuged to collect the bacterial cells, and then resuspended in sterile water to adjust the bacterial concentration and obtain a liquid bacterial agent. S3. Inoculate the liquid bacterial agent obtained in step S2 into the pasture.

[0019] The above technical solution involves setting up an LB liquid medium amplification-log phase harvest-high-speed centrifugation concentration-resuspending in sterile water to 10. 8 The closed-loop process of direct inoculation of *Leymus chinensis* with CFU / mL ensures that the bacteria are enriched at their peak physiological activity and removes residual components from the culture medium, avoiding interference from organic nitrogen sources with the rhizosphere microecological balance. Simultaneously, it ensures that the density of functional bacteria per unit volume meets the rhizosphere colonization threshold (≥10). 6(CFU / g rhizosphere soil), thus completing root surface adhesion and initial biofilm formation within 72 hours after inoculation, solving the problem of unstable field effects caused by insufficient viable bacteria, impurity interference, and mismatched colonization windows of traditional inoculants. Its beneficial effects are: under greenhouse pot conditions, 14 days after soil drenching with ZSP3 inoculant, the ZSP3 colonization rate in the rhizosphere of *Leymus chinensis* reached (3.2±0.4)×10 7 The CFU / g dry soil concentration was significantly higher than that of the conventional phosphate-solubilizing bacteria control (Bacillus megaterium H12) at (8.5±1.2)×10⁻⁶. 5 CFU / g dry soil, and the number of plants established was significantly positively correlated with the fresh weight of the aboveground parts (R). 2 =0.93).

[0020] Optionally, the culture medium is LB liquid medium; The conditions for the shaking culture were 30℃ and 180 r / min.

[0021] The above technical solution, by limiting the LB liquid culture medium and the conditions of 30℃ and 180 r / min shaking, ensures that ZSP3 achieves rapid proliferation (doubling time 47 min) and accumulation of metabolites under optimal nutrient and oxygen supply. In this solution, yeast extract in LB provides tryptophan precursors to promote IAA synthesis, tryptone provides a nitrogen source to support siderophore assembly, and the 180 r / min shaking speed maintains the dissolved oxygen concentration above 6.8 mg / L, ensuring efficient expression of aerobic phospholysinases (such as PhoA). The beneficial effect is that under these conditions, culture to OD... 600 At a concentration of 1.1, the bacterial IAA yield reached a peak of 18.99 mg / L, and the siderophore CAS value reached 86.3%, which were 2.1 times and 1.7 times higher than those of static culture, respectively.

[0022] Optionally, the OD600 corresponding to the logarithmic growth phase is 1.0-1.2; The centrifugation conditions were 12000 r / min for 10 min; The concentration of strain ZSP3 in the liquid bacterial agent is 10^8 CFU / mL.

[0023] Through the above technical solution, by controlling OD 600 Harvest cells in the 1.0-1.2 range to ensure the cells are in a state of vigorous metabolism, intact cell walls, and moderate activation of stress-responsive genes (such as rpoS); centrifugation at 12000 r / min for 10 min can achieve a 99.2% cell weight recovery rate without damaging the cell membrane; resuspend to 10 8 CFU / mL meets the minimum threshold for rhizosphere colonization (generally accepted in the literature as ≥10). 6The beneficial effect of this CFU / g rhizosphere soil inoculant is that after soil drenching with this concentration of inoculant, the colonization rate of Leymus chinensis rhizosphere ZSP3 reaches (1.05±0.13)×10⁻⁶ within 7 days. 7 CFU / g dry soil, compared to 10 7 The CFU / mL concentration group increased 3.8-fold, and showed a linear relationship with the increase in biomass (y=0.42x+1.87, R). 2 =0.96).

[0024] Optionally, the forage grass is crested wheatgrass, and the liquid microbial agent is inoculated by seed soaking or soil drenching; When applying via soil drenching, apply once a week, 50 mL each time.

[0025] Through the above technical solutions, targeting *Leymus chinensis* as the target crop, and establishing two agronomic adaptation methods—seed soaking (2 h) and soil drenching (50 mL / time / week)—ZSP3 was precisely colonized at different growth stages. Seed soaking allowed the bacteria to colonize the radicle at the early germination stage, forming a "rhizosphere primary barrier." Soil drenching transported the bacteria to the root hair zone through water transport, and weekly application maintained the density of rhizosphere functional flora (>10). 6 The beneficial effects of this method (CFU / g) are as follows: In the field test site in Ruoergai (average annual temperature 1.3℃, annual precipitation 650 mm), after inoculating ZSP3 with this method, the greening period of Leymus chinensis was advanced by 5.2 days, the biomass during the flowering period reached 1423 kg / ha, which is 58.7% higher than that of CK, and the water consumption was reduced by 19.3% (due to the deep root system reducing surface evaporation). Attached Figure Description

[0026] 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.

[0027] Figure 1 A flowchart illustrating the application method of a phosphorus-solubilizing and growth-promoting 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 ZSP3 provided in this application; Figure 3 A schematic diagram of the phylogenetic tree of the 16S rDNA gene of strain ZSP3 provided in this application; Figure 4 A schematic diagram illustrating the phosphate-solubilizing ability of strain ZSP3 provided in this application; Figure 5 A schematic diagram illustrating the cold resistance characteristics of strain ZSP3 provided in this application; Figure 6 A schematic diagram illustrating the drought resistance characteristics of strain ZSP3 provided in this application; Figure 7 A schematic diagram illustrating the IAA production capacity of strain ZSP3 provided in this application; Figure 8 A schematic diagram illustrating the IAA production capacity of strain ZSP3 provided in this application; Figure 9 A schematic diagram illustrating the effect of ZSP3 inoculation on the biomass of Leymus chinensis provided in this application; Figure 10 This is a schematic diagram illustrating the effect of ZSP3 inoculation on the chlorophyll content and antioxidant enzyme activity of *Leymus chinensis*, as provided in this application. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] The embodiments of this application will now be described in further detail with reference to the accompanying drawings.

[0031] The strain described in this application is classified as Pantoea agglomerans ZSP3, with accession number CCTCCNO: M 20252438, deposited on November 3, 2025, and deposited at the China Center for Type Culture Collection. Example 1

[0032] This embodiment aims to prepare and verify the typical performance parameters and standard application effects of strain ZSP3. (See [link to relevant documentation]). Figure 1 The process, specifically implemented as follows: ZSP3 bacterial strain, frozen in glycerol tubes at -80℃, was streaked onto LB agar plates and incubated at 30℃ for 24 h. Single colonies were then picked and inoculated into 5 mL of LB liquid medium, and cultured at 30℃ with shaking at 180 r / min for 12 h as the seed culture. A 1% inoculum was then transferred to 100 mL of LB liquid medium and cultured under the same conditions until OD (dose expiratory volume). 600 =1.1 (approximately 18 h); Centrifuge the culture medium at 4℃ and 12000 r / min for 10 min, discard the supernatant, resuspend the precipitate in sterile water, and adjust the bacterial concentration to 10. 8 CFU / mL yields the liquid bacterial agent.

[0033] The OD of ZSP3 in this bacterial agent was measured after 24 hours in LB medium at 12℃. 600 The effective phosphorus content was 0.82; after culturing on LB solid plates containing 1200 mM mannitol at 30°C for 48 h, yellow colonies with a diameter ≥0.8 mm were visible; after culturing on NBRIP liquid medium at 30°C and 180 r / min for 7 d, the effective phosphorus content was 171.4 mg / L; after culturing on LB medium containing L-tryptophan for 72 h, the IAA yield was 18.99 mg / L; and the CAS plate method showed a distinct orange-yellow halo.

[0034] The results show that the ZSP3 bacterial agent prepared in this embodiment fully meets all the technical characteristics defined in this application and possesses all the core performance required for adaptation to high-altitude and arid environments. Example 2

[0035] This embodiment is intended to verify the technical feasibility of the lower limit condition of "being able to grow in LB medium at 12°C" in this application.

[0036] With all other culture conditions exactly the same as in Example 1, only the shaking culture temperature was adjusted from 30°C to 12°C, and the other parameters (LB liquid medium, 180 r / min, OD) remained unchanged. 600 (Monitoring points, etc.) remained unchanged. The results showed that the OD of ZSP3 decreased after 48 h of incubation at 12℃. 600 The OD value reached 0.82, and the bacterial suspension showed uniform turbidity. Microscopic examination revealed intact bacterial morphology and active motility. In contrast, the control strain P. agglomerans ATCC 11591 showed an OD value of 0.82 under the same conditions. 600 Only 0.11 and a large amount of autolysis occurred.

[0037] The results showed that ZSP3 could still maintain its basic metabolism and proliferation capacity at a low temperature of 12℃, which verified the effectiveness and feasibility of the low temperature adaptability limit in this application. Example 3

[0038] This embodiment is intended to verify the technical feasibility of the drought resistance upper limit condition in this application, which is "the ability to form visible colonies in LB solid medium containing 1200 mM mannitol".

[0039] The method is the same as in Example 1, except that the mannitol concentration in the LB solid medium is adjusted from 0 mM to 1200 mM, the medium is poured into plates and sterilized by UV irradiation for 30 min, and after cooling, ZSP3 bacterial suspension is spread on the plates (10 mM). 6 (CFU / plate), incubated at 30℃ for 48 h. The results showed that ZSP3 formed yellow circular colonies with a diameter of 0.8-1.2 mm on 1200 mM mannitol plates, with clear edges and smooth surfaces; while no colonies were observed to grow on 1400 mM mannitol plates.

[0040] The results show that the drought tolerance of ZSP3 can indeed cover a concentration of 1200 mM mannitol, supporting the rationality of the parameter boundary in this application. Example 4

[0041] This embodiment is intended to verify the technical feasibility of the antifreeze time window described in this application, which states that the organism "remains viable after being frozen at -20°C for 24-96 hours".

[0042] Take the ZSP3 liquid bacterial agent prepared in Example 1 (10 8 (CFU / mL) was aliquoted into sterile cryovials and frozen at -20°C for 24 h, 48 h, 72 h, and 96 h, respectively. Immediately after freezing, the samples were rapidly thawed in a 30°C water bath (≤2 min). 100 μL of the thawed solution was spread onto LB agar plates and incubated at 30°C for 24 h before counting. The results showed that after treatment at -20°C for 24 h, 48 h, 72 h, and 96 h, the survival rates of ZSP3 were 96.3%, 92.7%, 89.5%, and 85.1%, respectively. All treatment groups formed typical yellow colonies, and the colony morphology was consistent with the untreated group.

[0043] The results showed that ZSP3 maintained a high survival rate during freezing at -20℃ for 24-96 h, fully supporting the parameter range of the examples. Example 5

[0044] This embodiment aims to verify that "the concentration of strain ZSP3 in the liquid bacterial agent is 10" in this application. 8 The technical feasibility of setting a lower limit for concentration "CFU / mL".

[0045] Based on the process of Example 1, in OD 600 Collect bacterial cells at a concentration of 1.1 and resuspend them in sterile water to a final concentration of 10⁻⁶. 7 CFU / mL, 10 8CFU / mL, 10 9 Three concentration gradients of CFU / mL were used; 50 mL of each concentration of inoculant was applied to each potted *Leymus chinensis* plant via soil drenching, once a week for four consecutive weeks; on day 28, the plant height, fresh weight, and rhizosphere ZSP3 colonization of *Leymus chinensis* were measured. The results showed that 10 7 The CFU / mL group had a plant height of 32.4 cm, a fresh weight of 4.2 g / plant, and a rhizosphere colonization of (1.8±0.3)×10⁻⁶. 6 CFU / g dry soil; 10 8 The CFU / mL group had a plant height of 48.7 cm, a fresh weight of 7.9 g / plant, and a rhizosphere colonization of (1.05±0.13)×10⁻⁶. 7 CFU / g dry soil; 10 9 CFU / mL group and 10 8 There was no significant difference between the groups (p>0.05).

[0046] The results showed that 10 8 CFU / mL represents the minimum effective concentration required to achieve a significant growth-promoting effect, supporting the necessity and rationality of this concentration limit in this application. Example 6

[0047] This embodiment is intended to verify the technical feasibility of the application parameter "soil leaching method, application once a week, 50 mL each time" in this application.

[0048] ZSP3 liquid bacterial agent (10) was prepared according to Example 1. 8 The dosage was determined using CFU / mL. Three application frequencies and dosages were set up: Group A (50 mL once a week), Group B (100 mL every two weeks), and Group C (25 mL twice a week). Each group was replicated three times, and other conditions were the same as in Example 5. Indicators were measured on day 28. The results showed that the plant height of *Leymus chinensis* in Group A was 48.7 cm and the fresh weight was 7.9 g / plant; the plant height in Group B was 38.2 cm and the fresh weight was 5.3 g / plant; and the plant height in Group C was 46.5 cm and the fresh weight was 7.1 g / plant. The root colonization rate in Group A was 1.05 × 10⁻⁶ CFU / mL. 7 The CFU / g ratio was significantly higher in group B than in group B (3.2×10). 6 CFU / g) and Group C (6.8×10) 6 CFU / g)(p<0.05).

[0049] The results showed that the application method of "once a week, 50 mL each time" can achieve the best growth-promoting effect while ensuring the homeostasis of the rhizosphere microbiota, supporting the scientific validity of the agronomic parameters in this application. Example 7

[0050] To systematically evaluate the overall performance of ZSP3, the following comparative experiments were conducted: ① Comparative Example 1 (blank control, CK): No bacterial agent was inoculated, and 50 mL of sterile water was applied weekly; ② Comparative Example 2 (samples outside the parameter range): The wild strain ZSP3-W of P. agglomerans (isolated from low-altitude farmland soil) was used, which was homologous but not screened for high-altitude cold and drought conditions. It did not grow in LB at 12℃ and had a colony diameter of <0.3 mm on 800 mM mannitol LB plates. ③ Comparative Example 3 (commercially available product): A domestically produced phosphorus-solubilizing bacterial fertilizer (containing Burkholderia cepacia, with a nominal phosphorus-solubilizing capacity of 120 mg / L); ④ Comparative Example 4 (closest to existing technology): The survival rate of the cold-resistant phosphate-solubilizing bacterium Bacillus megaterium H12 (CN110564562A) reported in the literature was only 18.3% after freezing at -20℃ for 48 h; ⑤ Comparative Example 5 (missing functional component): ZSP3 ΔphoA mutant (acid phosphatase gene knockout), with an available phosphorus content of <5 mg / L in NBRIP. All treatments were conducted under the same potting conditions (alpine degraded sandy soil, available phosphorus 4.3 mg / kg, organic matter 0.87 g / kg), with three replicates per group. The ZSP3 treatment group was treated according to the application methods of the inoculant in Example 1 and Example 10. After 40 days of cultivation, various indicators were measured, and the results are shown in Table 1.

[0051] Table 1. Comparison of overall performance between ZSP3 and the comparative example. As shown in Table 1, ZSP3 significantly outperformed all comparative studies in four core indicators: low-temperature growth, high-osmotic tolerance, phosphorus solubilization capacity, and growth-promoting effect (p<0.01). It demonstrated irreplaceable superiority, particularly in the stress resistance indicators of 12℃ growth and 1200 mM mannitol tolerance. Its phosphorus solubilization capacity (171.4 mg / L) was 73.9% higher than the closest existing technology (H12), and this improvement was not linearly additive but rather stemmed from the synergistic activation of low-temperature stress response pathways (such as CspA) and phosphorus solubilization metabolic pathways (such as Pho regulon). qRT-PCR showed that ZSP3's phoA expression level at 12℃ was 2.8 times that of H12, confirming its integrated "stress resistance-function" mechanism. This effect cannot be achieved by stress resistance or phosphorus solubilization capacity alone, representing a synergistic effect that would be difficult for those skilled in the art to anticipate. Example 8

[0052] In this application example, the test samples included ZSP3 inoculants prepared according to each of the embodiments in Examples 1 to 6, and their application effects were verified one by one in the planting of Leymus chinensis. Specifically: Seed soaking treatment (2 h) was performed using inoculant from Embodiment 1 (standard process); soil drenching was performed using inoculant from Embodiment 2 (cultured at 12℃) (50 mL / time / week); foliar spraying was performed using inoculant from Embodiment 3 (prepared under 1200 mM mannitol stress) (100 mL / time / week); root irrigation was performed using inoculant from Embodiment 4 (frozen at -20℃ for 96 h and then thawed) (30 mL / time / week); and inoculant from Embodiment 5 (10... 8 Both the CFU / mL and the inoculant from Implementation Method 6 (50 mL per week) were applied to the soil by drenching. All treatment groups were conducted at the Ruoergai field test site (altitude 3450 m, average annual temperature 1.3℃), with each treatment replicated three times, and a control (CK) was included. After 40 days, the plant height, fresh weight, root length, and chlorophyll SPAD value of *Leymus chinensis* were measured. The results are shown in Table 2.

[0053] Table 2. Growth-promoting effects of ZSP3 inoculants prepared according to different implementation methods on *Leymus chinensis*. The results showed that regardless of the implementation method used, the ZSP3 inoculant exhibited a significant growth-promoting effect on *Elymus sibiricum* (plant height, fresh weight, root length, and chlorophyll value increased by 32.4-58.7% compared to the control group), and there were no significant differences among the implementation methods (p>0.05), proving that the technical solution of the present invention has a stable and reproducible growth-promoting function within all defined parameter ranges. Experimental results indicate that the ZSP3 prepared by the present invention showed good growth-promoting and stress-resistance effects in *Elymus sibiricum* cultivation in the arid and cold region of Ruoergai. Therefore, it can be used to prepare microbial agents for the prevention and / or treatment of problems related to slow pasture growth, low biomass, and soil phosphorus deficiency in arid and cold regions.

[0054] 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 phosphorus-solubilizing and growth-promoting bacterial strain adapted to cold and arid environments, characterized in that, The strain was isolated from soil in a high-altitude and cold region. It was identified as a Pantoea agglomerans strain by 16S rDNA gene sequencing and phylogenetic analysis. It can grow in LB medium at 12°C, form visible colonies in LB solid medium containing 1200 mM mannitol, and has phosphorus solubilization ability in NBRIP liquid medium. The strain is classified as Pantoea agglomerans ZSP3, with accession number CCTCC NO: M20252438, deposited on November 3, 2025, and deposited at the China Center for Type Culture Collection.

2. A method for applying a phosphorus-solubilizing and growth-promoting strain adapted to cold and arid environments, comprising using the phosphorus-solubilizing and growth-promoting strain as described in claim 1, characterized in that... include: S1. The phosphorus-solubilizing and growth-promoting strain is inoculated into a culture medium and cultured with shaking until the logarithmic growth phase to obtain a culture solution; S2. The culture medium obtained in step S1 is centrifuged to collect the bacterial cells, and then resuspended in sterile water to adjust the bacterial concentration and obtain a liquid bacterial agent. S3. Inoculate the liquid bacterial agent obtained in step S2 into the pasture.

3. The phosphorus-solubilizing and growth-promoting strain according to claim 1, characterized in that, The strain was isolated from the root soil of plants in a recovery state after degradation in high-altitude and cold regions.

4. The phosphorus-solubilizing and growth-promoting strain according to claim 1, characterized in that, The colony morphology of the strain is yellow, round, with a raised center, and Gram staining reaction is negative; Under scanning electron microscopy, the strain was found to be short cylindrical, with a length of 0.889-1.361 μm and a width of 0.384-0.546 μm.

5. The phosphorus-solubilizing and growth-promoting strain according to claim 1, characterized in that, The strain remained viable after being frozen at -20°C for 24-96 hours.

6. The phosphorus-solubilizing and growth-promoting strain according to claim 1, characterized in that, After the strain was cultured in NBRIP liquid medium at 30°C and 180 r / min for 7 days with shaking, the available phosphorus content in the fermentation broth was 171.4 mg / L.

7. The phosphorus-solubilizing and growth-promoting strain according to claim 1, characterized in that, The strain exhibited IAA production capacity of 18.99 mg / L after being cultured in LB liquid medium containing L-tryptophan, and also demonstrated siderophore production capacity as detected by the CAS plate method.

8. The application method according to claim 2, characterized in that, The culture medium is LB liquid medium; The conditions for the shaking culture were 30℃ and 180 r / min.

9. The application method according to claim 2, characterized in that, The OD600 corresponding to the logarithmic growth phase is 1.0-1.2; The centrifugation conditions were 12000 r / min for 10 min; The concentration of strain ZSP3 in the liquid bacterial agent is 10^8 CFU / mL.

10. The application method according to claim 2, characterized in that, The forage grass is crested wheatgrass, and the liquid microbial agent is inoculated by seed soaking or soil drenching. When applying via soil drenching, apply once a week, 50 mL each time.

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