Azotobacter strain with strong stress resistance and growth promoting function and application of azotobacter strain

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to an azotobacter strain with strong stress resistance and a growth promoting function and application thereof. The strain is characterized by comprising: a pseudomonas strain ZSN2; the pseudomonas strain ZSN2 is cell suspension in a sterile distilled water-based carrier; the sterile distilled water-based carrier contains glycerol; the classification name of the pseudomonas strain ZSN2 is Pseudomonas sp. ZSN2, the preservation number of the pseudomonas strain ZSN2 is CCTCC NO: M 20252439, the preservation time is November 3, 2025, and the preservation unit is China Center for Type Culture Collection. The nitrogen-fixing bacterial strain shows remarkable stress resistance and growth promoting potential in a low-temperature environment, the bacterial strain still has considerable growth ability and metabolic activity at a low temperature, meanwhile, it is proved that a glycerin carrier effectively protects cell activity, the bacterial strain can effectively promote plant root development and nitrogen fixation in the application process, and the nitrogen-fixing bacterial strain has a good application prospect. The crop yield and the soil fertility are improved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, and in particular relates to a nitrogen-fixing strain with strong stress resistance and growth-promoting function and its application. Background Technology

[0002] In high-altitude and cold regions (such as high-altitude grasslands and plateaus), the harsh climate (low temperatures all year round, large temperature differences between day and night), low soil development, and lack of key nutrients such as nitrogen result in slow growth and low biomass of local core forage grasses (such as crested wheatgrass), which not only affects the ecological stability of grasslands but also restricts the development of animal husbandry.

[0003] To address these issues, traditional methods often rely on the application of chemical nitrogen fertilizers. However, in high-altitude and cold regions, soil fertility retention is poor, fertilizer utilization is low, nutrient loss and soil compaction are common, and these methods are difficult to adapt to the "continuous nutrient supply" requirements of forage grasses. In addition, while existing commercial nitrogen-fixing bacteria can supplement soil nitrogen, most strains have weak stress resistance (poor survival ability at low temperatures), limiting their function in high-altitude and cold environments and failing to significantly improve forage grass growth performance.

[0004] In summary, forage cultivation in high-altitude and cold regions currently faces a triple dilemma: "soil nitrogen deficiency, lack of stress-resistant strains, and inefficient and polluting traditional fertilization." There is an urgent need to develop a specialized strain that combines strong stress resistance, efficient nitrogen fixation, and significant growth-promoting effects to meet the special needs of high-altitude and cold environments. Summary of the Invention

[0005] This application provides a nitrogen-fixing strain with strong stress resistance and growth-promoting function, and its application, to solve the above-mentioned problems.

[0006] In a first aspect, this application provides a nitrogen-fixing strain with strong stress resistance and growth-promoting functions, including: Pseudomonas strain ZSN2; The Pseudomonas strain ZSN2 was a cell suspension in a sterile distilled water-based carrier. The sterile distilled water-based carrier contains glycerol; The strain ZSN2 of the Pseudomonas genus is classified as Pseudomonas sp. ZSN2, with accession number CCTCC NO: M 20252439, deposited on November 3, 2025, and deposited at the China Center for Type Culture Collection.

[0007] Through the above technical solution, the nitrogen-fixing strain configured in this embodiment exhibits significant stress resistance and growth-promoting potential under low-temperature conditions. Specifically, after culturing at 12℃ for 48 hours, visible colonies with a diameter of not less than 1 mm can be formed, indicating that the strain still possesses considerable growth capacity and metabolic activity at low temperatures. Simultaneously, after freezing at -20℃ for 24 to 96 hours, the survival rate, measured by CFU counting, is not less than 80%, demonstrating the effective protection of cell viability by the glycerol carrier. Furthermore, this strain can effectively promote plant root development and nitrogen fixation during application, thereby increasing crop yield and soil fertility.

[0008] Optionally, the Pseudomonas strain ZSN2 is a Gram-negative spindle cell with a length of 1.0 to 1.5 μm and a width of 0.5 to 0.7 μm.

[0009] Through the above technical solution and by strictly defining the morphological parameters of the strain, this embodiment achieves the following advantages: First, the spindle-shaped structure has been experimentally proven to be positively correlated with low-temperature tolerance, maintaining a survival rate of over 80% after freezing at -20℃; second, the standardized morphological characteristics ensure consistency between batches of strains, providing a basis for quality control in industrial production; in addition, this morphological characteristic also promotes the adhesion ability of the strain to the root surface, and tests show that its colonization density on the root surface of *Leymus chinensis* is about 30% higher than that of irregularly shaped strains.

[0010] Optionally, the nitrogen-fixing strain forms visible colonies with a diameter of not less than 1 mm after being cultured in a medium at 12°C for 48 hours.

[0011] Through the above technical solution and verification in this embodiment, the ZSN2 strain exhibits excellent environmental adaptability under low temperature conditions of 12℃: the diameter of the colonies formed within 48 hours reaches 1.0-1.8mm, proving that it still maintains an intact metabolic network and the ability to divide and proliferate at low temperatures; this characteristic ensures that the strain can colonize first in early spring or in high-altitude and cold regions, providing early nitrogen nutrition for plants; comparative experiments show that strains with this characteristic can increase the root biomass of crested wheatgrass seedlings by 25-40% and increase the chlorophyll content by 15-25%.

[0012] Optionally, after the nitrogen-fixing strain is frozen at 20°C for 24 to 96 hours, the survival rate is not less than 80% as determined by the CFU counting method.

[0013] Through the above technical solution, and verified in this embodiment, the ZSN2 strain exhibits excellent antifreeze performance: when frozen at -20℃ for 24-96 hours, the survival rate remains stable between 82% and 89%, significantly higher than that of conventional nitrogen-fixing strains (usually below 50%). This characteristic ensures the activity stability of the bacterial agent during winter storage and long-distance transportation, extending the effective shelf life of the product. In practical applications, the strain treated with 96 hours of freezing can still successfully colonize the rhizosphere of Leymus chinensis, with a colonization efficiency of over 85% of that of fresh strains.

[0014] Optionally, the cell concentration is 1×10⁻⁶. 8 Up to 5×10 8 CFU / mL.

[0015] Using the above technical solution, this cell concentration range exhibited excellent stability and reproducibility in experiments, maintaining a viable bacterial count of no less than 1×10⁻⁶ even after low-temperature storage (e.g., refrigeration at 0-5℃ for 30 days). 8 The concentration of CFU / mL ensures that nitrogen fixation efficiency is not affected during field application. At the same time, this concentration optimizes the adhesion between the strain and plant seeds, forming a uniform microbial cover on the surface of crested wheatgrass seeds, promoting rhizosphere colonization, and improving plant stress resistance and growth rate. In addition, this range reduces production costs and storage burden, and avoids inoculation failure due to concentration fluctuations.

[0016] Optionally, the glycerol accounts for 5% to 15% of the volume.

[0017] Through the above technical solution, this glycerol ratio range demonstrated excellent protective efficacy in experiments, enabling nitrogen-fixing strains to maintain a survival rate of over 80% after 96 hours of freezing at -20℃, and simultaneously maintaining a stable viable count of 1×10⁻⁶ bacteria within 30 days under refrigeration conditions of 0-5℃. 8 CFU / mL or higher; this ratio also optimizes the compatibility of the strain with plant tissues, does not inhibit seed germination during seed treatment, but enhances rhizosphere colonization by maintaining strain activity; in addition, the cost of glycerol in this concentration range is moderate, suitable for industrial production, and poses no risk of environmental pollution.

[0018] Optionally, S1, the nitrogen-fixing strain described in claim 1 is used at a concentration of 1×10⁻⁶. 8 A CFU / mL suspension was mixed with crested wheatgrass seeds to form a microbial cover layer with a thickness of 0.1 to 0.5 mm on the seed surface. The mixture was then kept in a container for 2 to 10 hours. S2. Spray the suspension of the nitrogen-fixing strain onto the soil surface to allow it to penetrate into the soil; S3. After the nitrogen-fixing strain is placed in a sealed container, it is stored in a cold storage. During the storage period, samples are taken intermittently to count CFUs.

[0019] Through the above technical solutions, this application significantly improves the colonization rate and persistence of nitrogen-fixing bacteria in the rhizosphere through the synergistic effect of seed pretreatment and soil inoculation, thereby enhancing plant stress resistance (such as low temperature and drought tolerance) and promoting growth. Simultaneously, the low-temperature preservation method ensures that the strains maintain high activity for 30 days, reducing strain loss during application and improving the reliability and efficiency of agricultural operations. Furthermore, this process is simple and easy to implement, suitable for large-scale field application, and helps improve soil fertility and reduce the use of chemical nitrogen fertilizers. After inoculation, it significantly increases the biomass of *Leymus chinensis* (plant height, fresh weight, and dry weight are significantly higher than the control group (CK)). When combined with other functional strains, it can also increase the chlorophyll content and stress-resistant enzyme (SOD, CAT) activity of *Leymus chinensis* leaves (see appendix). Figure 7 , 8 ).

[0020] Optionally, after mixing the seeds and microbial suspension in step S1, the temperature of the container is controlled at 20 to 25°C and the relative humidity is controlled at 60% to 80%.

[0021] By employing the above technical solutions and precisely controlling the temperature and humidity environment, the uniformity and integrity of the microbial covering layer are significantly improved, increasing the adhesion rate of the strains to the seed surface by approximately 30%. These conditions maintain the activity of the strains and prevent premature seed germination, laying a good foundation for rhizosphere colonization after subsequent sowing. At the same time, standardized environmental parameters ensure consistency and repeatability between batches, which is beneficial for quality control in large-scale agricultural applications.

[0022] Optionally, in step S2, the microbial suspension is sprayed evenly to penetrate into the soil to a depth of 10 to 30 mm, and the spraying amount is 10 to 30 mL per square meter.

[0023] Through the above technical solution, this spraying method can increase the uniformity of nitrogen-fixing bacteria distribution in the rhizosphere by about 40%, and increase the survival rate of strains in the soil layer at a depth of 10-30 mm by more than 25%. The precisely controlled spraying parameters avoid waste of bacterial solution, saving about 30% of the bacterial solution compared with traditional spraying methods. The optimized penetration depth ensures the colonization efficiency of strains in the active area of ​​plant roots, providing favorable conditions for subsequent nitrogen fixation.

[0024] Optionally, the microbial preparation in step S3 is stored at 0 to 5°C, with samples taken every 7 days for CFU counting, and the viable count not less than 1 × 10⁻⁶. 8 CFU / mL, storage time not exceeding 30 days.

[0025] Through the above technical solutions, this preservation method can maintain the survival rate of the strains at over 85% within 30 days, significantly extending the effective use period of the formulation; the standardized monitoring system ensures the reliability and traceability of the bacterial agent quality; the low temperature combined with sealed storage conditions effectively prevents contamination by other microorganisms and maintains the purity of the strains; the clearly defined storage period provides a clear time window for field application, avoiding the use of ineffective bacterial agents.

[0026] Secondly, this application provides a method for applying a nitrogen-fixing strain with strong stress resistance and growth-promoting function, the method comprising: Optionally, the nitrogen-fixing strain described in claim 1 is used at a concentration of 1×10⁻⁶. 8 A CFU / mL suspension was mixed with crested wheatgrass seeds to form a microbial cover layer with a thickness of 0.1 to 0.5 mm on the seed surface. The mixture was then kept in a container for 2 to 10 hours. Optionally, the suspension of the nitrogen-fixing strain can be sprayed onto the soil surface to allow it to penetrate into the soil. Optionally, the nitrogen-fixing strain is placed in a sealed container and stored in a cold storage, with intermittent sampling for CFU counting during storage.

[0027] Through the above technical solutions, this application significantly improves the colonization rate and persistence of nitrogen-fixing bacteria in the rhizosphere through the synergistic effect of seed pretreatment and soil inoculation, thereby enhancing plant stress resistance (such as low temperature and drought tolerance) and promoting growth. Simultaneously, the low-temperature preservation method ensures that the strains maintain high activity for 30 days, reducing strain loss during application and improving the reliability and efficiency of agricultural operations. Furthermore, this process is simple and easy to implement, suitable for large-scale field application, and helps improve soil fertility and reduce the use of chemical nitrogen fertilizers. After inoculation, it significantly increases the biomass of *Leymus chinensis* (plant height, fresh weight, and dry weight are significantly higher than the control group (CK)). When combined with other functional strains, it can also increase the chlorophyll content and stress-resistant enzyme (SOD, CAT) activity of *Leymus chinensis* leaves (see appendix). Figure 7 , 8 ). Attached Figure Description

[0028] 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.

[0029] Appendix Figure 1 Morphological characteristics and Gram staining of strain ZSN2; Appendix Figure 2Phylogenetic tree of 16S rDNA genes of strain ZSN2; Appendix Figure 3 Nitrogenase activity of strain ZSN2; Appendix Figure 4 Cold resistance characteristics of strain ZSN2; Appendix Figure 5 Siderophore production capacity of strain ZSN2; Appendix Figure 6 Flowchart of the application method of a nitrogen-fixing strain with strong stress resistance and growth-promoting function; Appendix Figure 7 Effects of ZSN2 inoculation on Leymus chinensis biomass; Appendix Figure 8 Effects of ZSN2 inoculation on chlorophyll content and antioxidant enzyme activity in Leymus chinensis. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] The embodiments of this application will now be described in further detail with reference to the accompanying drawings.

[0033] Currently, forage grass cultivation in high-altitude and cold regions faces a triple dilemma: "soil nitrogen deficiency, lack of stress-resistant strains, and inefficient and polluting traditional fertilization." There is an urgent need to develop a special strain that combines strong stress resistance, efficient nitrogen fixation, and significant growth-promoting effects to meet the special needs of high-altitude and cold environments.

[0034] Based on this, this application provides a nitrogen-fixing strain with strong stress resistance and growth-promoting function, and its application. The nitrogen-fixing strain exhibits significant stress resistance and growth-promoting potential under low-temperature conditions. Specifically, after culturing at 12℃ for 48 hours, visible colonies with a diameter of not less than 1 mm can be formed, indicating that the strain still possesses considerable growth capacity and metabolic activity at low temperatures. Simultaneously, after freezing at -20℃ for 24 to 96 hours, the survival rate, measured by CFU counting, is not less than 80%, demonstrating the effective protection of cell viability by the glycerol carrier. Furthermore, this strain can effectively promote plant root development and nitrogen fixation during application, thereby increasing crop yield and soil fertility.

[0035] For specific implementation details, please refer to the following examples.

[0036] Figure 1 The morphological characteristics and Gram staining of a nitrogen-fixing strain with strong stress resistance and growth-promoting function provided in an embodiment of this application are as follows: Figure 1 As shown, the characteristics of this strain include: Pseudomonas strain ZSN2; The Pseudomonas strain ZSN2 was a cell suspension in a sterile distilled water-based carrier. The sterile distilled water-based carrier contains glycerol; The strain ZSN2 of the Pseudomonas genus is classified as Pseudomonas sp. ZSN2, with accession number CCTCC NO: M 20252439, deposited on November 3, 2025, and deposited at the China Center for Type Culture Collection.

[0037] Technical Background and Working Principle: In high-altitude or low-temperature environments, soil temperatures are often below 12℃. Conventional nitrogen-fixing strains are easily inhibited by low temperatures under these conditions, leading to slow growth and reduced metabolic activity, thus affecting nitrogen fixation efficiency and plant growth promotion effects. To address this issue, this embodiment uses the Pseudomonas strain ZSN2, which possesses natural low-temperature adaptability. However, its long-term survival and function depend on the protective effect of the carrier. Glycerol, as an osmotic protectant, effectively maintains cell osmotic pressure balance at low temperatures, preventing cell membrane damage due to ice crystal formation. Simultaneously, by reducing the extracellular osmotic pressure gradient, it enhances the integrity and stability of the cell membrane, thereby ensuring the strain maintains metabolic activity and nitrogen-fixing capacity in low-temperature environments. Furthermore, the sterile distilled water-based carrier provides a neutral and impurity-free suspension environment, avoiding interference from external factors on the strain.

[0038] Technical Solution and Component Functions: In this embodiment, the *Pseudomonas* strain ZSN2 is first isolated from the culture medium by centrifugation, and then resuspended in a sterile distilled water-based carrier. Glycerol is added to this carrier at a volume ratio of 5% to 15%, forming a suspension with a cell concentration of 1×10^8 to 5×10^8 CFU / mL. The sterile distilled water-based carrier provides a pure suspension medium, preventing the introduction of impurities that could affect the strain's activity. Glycerol, as a key additive, provides osmotic protection at low temperatures, preventing cell dehydration and ice crystal damage. Strain ZSN2, as the active ingredient, is responsible for nitrogen fixation and growth promotion. This suspension can be directly used for seed treatment, soil inoculation, or cryopreservation, and is simple to operate and highly stable.

[0039] Strain characteristics: Taxonomic position: Strain ZSN2 was isolated from degraded soil in a recovering state in a high-altitude cold region. 16S rDNA gene sequencing and phylogenetic analysis were performed (see attached). Figure 2 It was identified as a species of Pseudomonas sp.

[0040] Morphological and physiological characteristics: Gram staining was negative; scanning electron microscopy (SEM) showed that the strain was spindle-shaped (1.046-1.452 μm in diameter and 0.474-0.666 μm in width), with a smooth surface, exhibiting typical morphological characteristics of Pseudomonas spp. (see attached image) Figure 1 ).

[0041] Core features: Nitrogen fixation function: Detected using the acetylene reduction method, its nitrogenase activity was higher than that of other nitrogen-fixing strains screened at the same time (see appendix). Figure 3 It can effectively convert nitrogen in the air into nitrogen that plants can use; Stress resistance: It can grow normally at a low temperature of 12℃, and still maintains a high survival rate after being frozen at -20℃ for 24-96 hours (see attached). Figure 4 ); Growth-promoting auxiliary function: Detected by the CAS plate method, it has the ability to produce siderophores and can promote plant growth through nutrient chelation and other methods (see attached). Figure 5 ).

[0042] The nitrogen-fixing strain described in this embodiment exhibits significant stress resistance and growth-promoting potential under low-temperature conditions. Specifically, after culturing at 12°C for 48 hours, visible colonies with a diameter of not less than 1 mm were formed, indicating that the strain still possesses considerable growth capacity and metabolic activity at low temperatures. Simultaneously, after freezing at -20°C for 24 to 96 hours, the survival rate, measured by CFU counting, was not less than 80%, demonstrating the effective protection of cell viability by the glycerol carrier. Furthermore, this strain effectively promotes plant root development and nitrogen fixation during application, thereby increasing crop yield and soil fertility.

[0043] Alternative or modified implementation methods: Without departing from the core principles of this embodiment, various substitutions or modifications can be made: the volume percentage of glycerol can be maintained within the range of 5% to 15%, but it can be replaced with other permeation protectants such as sucrose or sorbitol, still achieving a similar cryoprotection effect; the sterile distilled water-based carrier can be replaced with autoclaved deionized water to provide a suspension environment of equivalent purity; the cell concentration can be adjusted to 1×10⁻⁶ according to actual application requirements. 7 Up to 1×10 9 CFU / mL can be achieved by changing centrifugation parameters or dilution ratios while maintaining the same viability and functional properties. Furthermore, suspension preparation can be integrated with automated equipment, such as using stirrers to ensure uniform mixing, thereby improving production efficiency and consistency.

[0044] In some embodiments, the Pseudomonas strain ZSN2 is a Gram-negative spindle cell with a length of 1.0 to 1.5 μm and a width of 0.5 to 0.7 μm.

[0045] Technical Background and Working Principle: The morphological characteristics of microorganisms are closely related to their environmental adaptability, especially under stress conditions such as low temperature and high osmotic pressure. Conventional nitrogen-fixing strains are prone to losing activity in low-temperature environments due to damage to cell membrane integrity. The Gram-negative spindle-shaped cell structure of Pseudomonas strain ZSN2, through its specific aspect ratio (length 1.0 to 1.5 μm, width 0.5 to 0.7 μm), physically reduces the ratio of cell surface area to volume, thereby reducing the risk of cell rupture caused by membrane lipid phase transition and osmotic pressure imbalance under low-temperature conditions. This morphological characteristic is also related to cell division and material transport efficiency, helping to maintain basic metabolic activities under low-temperature environments.

[0046] Technical Solution and Component Functions: This embodiment uses an optical microscope to observe the morphology of ZSN2 strain cultured to the logarithmic growth phase. Specifically, Gram staining is used to prepare samples, and the length and width of no fewer than 100 cells are measured under a 1000x oil immersion microscope, ensuring that all measurements conform to the size range specified in claim 2 (length 1.0-1.5 μm, width 0.5-0.7 μm). This morphological identification system consists of a sample preparation module, an optical magnification module, and a digital measurement module working collaboratively. The sample preparation module ensures the uniformity of cell distribution, the optical magnification module provides a clear imaging basis, and the digital measurement module automatically records and statistically analyzes cell size data using image analysis software, ultimately generating a morphological examination report.

[0047] By strictly defining the morphological parameters of the strains, this embodiment achieves the following advantages: First, the spindle-shaped structure has been experimentally proven to be positively correlated with low-temperature tolerance, maintaining a survival rate of over 80% after freezing at -20°C; second, standardized morphological characteristics ensure consistency between batches of strains, providing a basis for quality control in industrial production; furthermore, this morphological characteristic also promotes the adhesion of the strains to the root surface, with tests showing that its colonization density on the root surface of *Leymus chinensis* is approximately 30% higher than that of strains with irregular morphology.

[0048] Alternative or modified implementation methods: While maintaining the core morphological characteristics, this embodiment can be modified as follows: The morphological observation equipment can be replaced with a scanning electron microscope (SEM) or an atomic force microscope (AFM), where SEM can provide higher resolution surface structure information, and AFM can achieve nanoscale three-dimensional morphological reconstruction; the sample preparation method can use fluorescent staining instead of Gram staining, such as using DAPI for cell nuclear staining; the measurement standards can be appropriately adjusted, allowing the length range to be extended to 0.8-1.8 μm and the width range to be adjusted to 0.4-0.9 μm, as long as the aspect ratio is maintained between 2:1 and 3:1, the same stress resistance characteristics can still be maintained; the automated measurement system can be upgraded to a fully automated microscope equipped with an artificial intelligence image recognition module to improve detection efficiency and accuracy.

[0049] In some embodiments, the nitrogen-fixing strain forms visible colonies with a diameter of not less than 1 mm after being cultured in a medium at 12°C for 48 hours.

[0050] Technical Background and Working Principle: At low temperatures (e.g., 12°C), the metabolic activity of most microorganisms slows significantly, leading to growth restriction. Nitrogenase activity in nitrogen-fixing strains is particularly sensitive to temperature changes, and their nitrogen fixation efficiency at low temperatures directly affects the nitrogen supply to plants. This embodiment verifies the mechanism by which nitrogen-fixing strains maintain metabolic activity under low-temperature stress, based on the important indicator of colony-forming ability. When the strain can still form visible colonies with a diameter ≥1 mm at 12°C, it indicates that its ribosome function, protein synthesis system, and energy metabolism pathways remain active at this temperature, especially the nitrogen-fixing-related enzyme system, which can maintain basic functions.

[0051] Technical Solution and Component Functions: This embodiment uses the standard plate culture method for verification: ZSN2 strain in the logarithmic growth phase is inoculated onto the surface of a nitrogen-free solid culture medium and placed in a precision temperature-controlled incubator (CCI) for continuous incubation at 12±0.5℃ for 48 hours. After incubation, the colony diameter is automatically measured using a colony counter, supplemented by manual verification using vernier calipers. The nitrogen-free culture medium provides selective pressure to ensure that the observed colonies indeed possess nitrogen-fixing capabilities; the temperature-controlled incubator precisely maintains a low-temperature environment; the colony counter is equipped with an image acquisition and analysis module, which can automatically identify and record the number of colonies with a diameter ≥1mm, ensuring the objectivity and accuracy of the data.

[0052] Through the methods provided in this embodiment, and through verification in this embodiment, the ZSN2 strain exhibits excellent environmental adaptability under low temperature conditions of 12℃: the diameter of the colonies formed within 48 hours reaches 1.0-1.8mm, proving that it still maintains an intact metabolic network and the ability to divide and proliferate at low temperatures; this characteristic ensures that the strain can be the first to colonize in early spring or in high-altitude and cold regions, providing early nitrogen nutrition for plants; through comparative experiments, strains with this characteristic can increase the root biomass of crested wheatgrass seedlings by 25-40% and increase the chlorophyll content by 15-25%.

[0053] Alternative or modified implementation methods: This embodiment can be adjusted as follows according to specific experimental conditions: The culture medium can be replaced with a low-temperature adaptation culture medium containing trace amounts of organic nitrogen sources to accelerate the formation of initial colonies; the culture temperature can fluctuate within the range of 10-15℃, and the culture time can be adjusted accordingly to 36-72 hours, while still obtaining colonies with a diameter ≥1mm; the observation equipment can be upgraded to a fully automated colony analysis system, integrating temperature control, imaging, and data analysis functions; the criteria for judging colony diameter can be appropriately relaxed to 0.8-2.0mm, as long as colonies that can be clearly seen with the naked eye can be formed under 12℃ conditions, they are considered to have low-temperature activity.

[0054] In some embodiments, the survival rate of the nitrogen-fixing strain, measured by CFU counting method, is not less than 80% after being frozen at 20°C for 24 to 96 hours.

[0055] Technical Background and Working Principle: Low-temperature freezing is a common preservation method in the production, transportation, and storage of microbial preparations, but this process can cause multiple stresses on cells. When the temperature drops to -20°C, the formation of ice crystals inside and outside the cell directly damages the cell membrane structure, while simultaneously leading to an increase in intracellular solute concentration and osmotic pressure stress. Glycerol, as an osmotic protectant, works by forming hydrogen bonds to replace water molecules, lowering the freezing point and inhibiting ice crystal growth; it also regulates the osmotic pressure balance inside and outside the cell, maintaining membrane protein conformation and fluidity, thereby protecting the integrity of the cell structure. This embodiment systematically verifies the survival rate of strains after freezing at -20°C for different durations, evaluating their antifreeze performance in practical applications.

[0056] Technical solution and component function: This embodiment adopts a method combining programmed freezing and viable cell counting: the ZSN2 strain suspension (cell concentration 1×10⁻⁶) is used to count the cells. 8 (CFU / mL, containing 10% glycerol) was dispensed into 2mL cryovials, 1.5mL per tube. The temperature was lowered to -20°C at a rate of 1°C / min using a programmed cooling system, and the tubes were frozen for 24, 48, 72, and 96 hours. After the set time, the tubes were rapidly thawed in a 37°C water bath, and CFU were counted using the plate plating method. The programmed cooling system ensured the standardization of the freezing process; the cryovials prevented container breakage; and the plate counting system accurately calculated the viability using an automated colony counter, with the formula: (CFU after freezing / initial CFU) × 100%.

[0057] Through the method provided in this embodiment, and verified by this embodiment, the ZSN2 strain exhibits excellent antifreeze performance: when frozen at -20℃ for 24-96 hours, the survival rate remains stable between 82% and 89%, significantly higher than that of conventional nitrogen-fixing strains (usually below 50%). This characteristic ensures the activity stability of the bacterial agent during winter storage and long-distance transportation, extending the effective shelf life of the product. In practical applications, the strain treated with 96 hours of freezing can still successfully colonize the rhizosphere of Leymus chinensis, with a colonization efficiency of over 85% of that of fresh strains.

[0058] Alternative or modified implementation methods: This embodiment can be adjusted as follows according to actual needs: the freezing temperature can fluctuate within the range of -15℃ to -25℃ while maintaining a survival rate of not less than 80%; the glycerol concentration can be adjusted to 5%-15%, or replaced with cryoprotectants such as dimethyl sulfoxide (DMSO) or trehalose; the thawing method can be slow thawing at room temperature or low-temperature thawing at 4℃; the survival rate detection can use fluorescent staining method (such as FDA-PI double staining) instead of plate counting method to achieve rapid detection; the freezing container can be replaced with cryopreservation bags or ampoules to suit different production scales.

[0059] In some embodiments, the cell concentration is 1×10⁻⁶. 8 Up to 5×10 8 CFU / mL.

[0060] Technical Background and Working Principle: Cell concentration is a key factor affecting the colonization efficiency and nitrogen fixation capacity of nitrogen-fixing strains in soil. In agricultural applications, too low an inoculation concentration will prevent the strains from effectively competing with indigenous microorganisms, thereby reducing rhizosphere colonization density and nitrogen fixation activity; while too high a concentration may lead to resource waste or cause unnecessary metabolic burden, affecting strain survival. This embodiment selects a 1×10⁻⁶ cell concentration. 8 Up to 5×10 8The CFU / mL concentration range, optimized experimentally, maintains cell membrane stability under adverse conditions such as low temperature and high salinity, and ensures the formation of a stable microbial community in the soil through sufficient initial bacterial load, thereby improving nitrogen fixation efficiency and plant growth promotion effects. Its working principle lies in the fact that this concentration range balances the growth rate of the strain with environmental adaptability, enabling the strain to rapidly adapt to the rhizosphere microenvironment after inoculation, and utilizing the protective effect of the glycerol carrier to maintain a high viability rate during storage and application.

[0061] Technical Solution and Component Functions: In this embodiment, the culture medium of Pseudomonas spp. strain ZSN2 is centrifuged at 8000 rpm for 10 minutes at 4°C using a centrifuge. The bacterial pellet is collected and then resuspended in a sterile distilled water-based carrier (containing 5%-15% glycerol). The pellet is then thoroughly mixed using a vortex mixer to form an initial suspension. Subsequently, the absorbance value is measured at a wavelength of 600 nm using an automated spectrophotometer. The cell density is calibrated according to a pre-established absorbance-CFU standard curve. The final cell concentration is precisely adjusted to 1×10⁻⁶ cells / mL by adding sterile distilled water or concentrated bacterial solution dropwise. 8 Up to 5×10 8 Within the CFU / mL range; this suspension can be used directly for seed treatment or soil spraying, with centrifugation for efficient cell separation, oscillator to ensure uniform suspension, and spectrophotometer to provide rapid and accurate density monitoring, thereby ensuring consistent activity and reliability of the strain in subsequent applications.

[0062] The method provided in this embodiment demonstrates excellent stability and reproducibility of this cell concentration range in experiments, maintaining a viable bacterial count of no less than 1×10⁻⁶ even after low-temperature storage (e.g., refrigeration at 0-5°C for 30 days). 8 The concentration of CFU / mL ensures that nitrogen fixation efficiency is not affected during field application. At the same time, this concentration optimizes the adhesion between the strain and plant seeds, forming a uniform microbial cover on the surface of crested wheatgrass seeds, promoting rhizosphere colonization, and improving plant stress resistance and growth rate. In addition, this range reduces production costs and storage burden, and avoids inoculation failure due to concentration fluctuations.

[0063] Alternative or modified implementation methods: The cell concentration can be adjusted to 5×10⁻⁶ depending on the specific application scenario. 7 Up to 1×10 9 CFU / mL, for example, a higher concentration (e.g., 1×10⁻⁶) can be used in low-nutrient soils. 9 (CFU / mL) to enhance competitiveness, while lower concentrations (e.g., 5 × 10⁻⁶) can be used in mild environments. 7(CFU / mL) to save resources; the concentration adjustment method can be replaced by flow cytometry or plate counting for calibration, replacing the spectrophotometer; the centrifugation step can be adjusted to 5000-10000 rpm or time to 5-15 minutes, while still achieving the target concentration range; the glycerol in the carrier can be partially replaced by other protective agents such as sucrose or trehalose, but the concentration needs to be optimized accordingly to maintain cell viability.

[0064] In some embodiments, the glycerol accounts for 5% to 15% of the volume.

[0065] Technical Background and Working Principle: Glycerol plays a crucial role as an osmotic protectant in microbial preparations, especially in handling cryopreservation and freezing. At low temperatures, ice crystals easily form inside and outside cells, leading to cell membrane damage and osmotic pressure imbalance, ultimately causing cell death. Glycerol effectively prevents mechanical damage to cells from ice crystals by lowering the freezing point of the solution, creating a vitrified state, and regulating the osmotic pressure balance inside and outside the cell. A glycerol concentration range of 5%-15% by volume has been systematically optimized to achieve the best balance between protective effect and biocompatibility—too low a concentration provides insufficient protection, while too high a concentration may inhibit cellular metabolic activity. This ratio is particularly suitable for the physiological characteristics of Pseudomonas strain ZSN2, maintaining cell membrane integrity and enzyme activity within a temperature range of -20℃ to 12℃, providing a reliable protective barrier for the strain during cold storage and field application.

[0066] Technical Solution and Component Functions: In this embodiment, a specified volume of sterile distilled water-based carrier is first measured using a precision pipette. Then, analytical grade glycerol is added at a ratio of 5%-15% of the total volume. The mixture is stirred at 200-400 rpm for 10-15 minutes using a magnetic stirrer until completely mixed, forming a homogeneous and transparent protective agent solution. Subsequently, Pseudomonas spp. strain ZSN2, collected by centrifugation, is added to this solution and resuspended using a vortex mixer to ensure that glycerol is evenly distributed in the cell environment. The precision pipette ensures the accuracy of the glycerol addition volume, while the magnetic stirrer ensures thorough mixing of the solution, avoiding excessively high local concentrations that could stress the cells. The resulting suspension can be directly used for subsequent cold storage or field application, ensuring that the strain maintains stable biological activity during storage and application.

[0067] The method provided in this embodiment demonstrates that this glycerol ratio range exhibits excellent protective efficacy in experiments, enabling nitrogen-fixing strains to maintain a survival rate of over 80% after 96 hours of freezing at -20°C, while maintaining a stable viable count of 1×10⁻⁶ bacteria within 30 days under refrigeration conditions of 0-5°C. 8CFU / mL or higher; this ratio also optimizes the compatibility of the strain with plant tissues, does not inhibit seed germination during seed treatment, but enhances rhizosphere colonization by maintaining strain activity; in addition, the cost of glycerol in this concentration range is moderate, suitable for industrial production, and poses no risk of environmental pollution.

[0068] Alternative or modified implementation methods: Glycerin can be replaced with other osmotic protectants such as sucrose, sorbitol, or trehalose, with a volume percentage maintained in the range of 3%-20%, of which sucrose is recommended at a ratio of 8%-12% and sorbitol at a ratio of 5%-10%; under extreme low temperature conditions (below -30°C), the glycerin ratio can be increased to 15%-25% to enhance the protective effect; the mixing method can be replaced with an ultrasonic homogenizer or a high-speed shear emulsifier, suitable for large-scale production scenarios; for specific sensitive strains, a gradient addition method can be used to gradually increase the glycerin concentration to reduce osmotic stress damage to cells.

[0069] Figure 2 A flowchart illustrating the application method of a nitrogen-fixing strain with strong stress resistance and growth-promoting function, as provided in an embodiment of this application, is shown below. Figure 2 The process, as shown, includes the following steps: S1. The nitrogen-fixing strain described in claim 1 is used at a concentration of 1×10⁻⁶. 8 A CFU / mL suspension was mixed with crested wheatgrass seeds to form a microbial cover layer with a thickness of 0.1 to 0.5 mm on the seed surface. The mixture was then kept in a container for 2 to 10 hours. S2. Spray the suspension of the nitrogen-fixing strain onto the soil surface to allow it to penetrate into the soil; S3. After the nitrogen-fixing strain is placed in a sealed container, it is stored in a cold storage. During the storage period, samples are taken intermittently to count CFUs.

[0070] Technical Background and Working Principle: In harsh environments such as high altitudes or droughts, plant seed germination and seedling growth are often limited by nitrogen deficiency. Conventional nitrogen-fixing bacteria are easily inactivated under low temperature or dry conditions, resulting in low rhizosphere colonization rates. This application improves the colonization efficiency of nitrogen-fixing bacteria in the rhizosphere by combining seed pretreatment and soil inoculation. The working principle is as follows: forming a microbial covering layer on the seed surface allows the strains to be directly introduced into the rhizosphere during the early stages of seed germination, utilizing the nitrogenase activity of the strains to provide a nitrogen source for the plant; soil spraying ensures the strains penetrate evenly around the roots, enhancing soil microbial community diversity; low-temperature preservation utilizes the osmotic protection of glycerol to inhibit cell metabolism and reduce energy consumption, thereby maintaining strain activity. Furthermore, intermittent CFU counting monitoring ensures the stability of the strains during preservation, preventing a decline in viable cell counts from affecting the application effect.

[0071] Technical Solution and Component Functions: The technical solution of this embodiment includes three main steps: First, the *Pseudomonas* strain ZSN2 cultured to the logarithmic growth phase is concentrated by centrifugation and resuspended in a sterile distilled water-based carrier containing 5%–15% glycerol, adjusting the cell concentration to 1×10^8 CFU / mL to form a suspension; then, this suspension is mixed with *Leymus chinensis* seeds in a mixing container (such as an incubator with temperature and humidity control), maintaining a temperature of 20–25℃ and a relative humidity of 60%–80% for 2–10 hours to form a 0.1–0.5 mm thick microbial cover layer on the seed surface; next, the suspension is evenly sprayed onto the soil surface using an adjustable flow rate atomizer at a spraying rate of 10–30 mL / m², ensuring a penetration depth of 10–30 mm. mm; Finally, the remaining suspension was placed in a polypropylene sealed bag (component 70) and stored in a 0–5℃ cold storage (component 80). Samples were taken every 7 days using a pipette (component 90) for CFU counting to ensure a viable count of at least 1 × 10⁻⁶. 8 CFU / mL, shelf life not exceeding 30 days. The functions of each component are as follows: the suspension provides a high concentration of live bacteria for inoculation; the mixing container maintains suitable temperature and humidity to optimize capping formation; the sprayer ensures uniform penetration; the sealed container and cold storage provide a stable low-temperature environment to extend shelf life; and the pipette facilitates accurate sampling and monitoring.

[0072] Through the method provided in this embodiment, this application significantly improves the colonization rate and persistence of nitrogen-fixing bacteria in the rhizosphere through the synergistic effect of seed pretreatment and soil inoculation, thereby enhancing plant stress resistance (such as low temperature and drought tolerance) and promoting growth. Simultaneously, the low-temperature preservation scheme ensures that the strains maintain high activity for 30 days, reducing strain loss during application and improving the reliability and efficiency of agricultural operations. Furthermore, this process is simple and easy to implement, suitable for large-scale field application, and helps improve soil fertility and reduce the use of chemical nitrogen fertilizers. After inoculation, it can significantly increase the biomass of *Leymus chinensis* (plant height, fresh weight, and dry weight are significantly higher than the control group (CK)). When combined with other functional strains, it can also increase the chlorophyll content and stress-resistant enzyme (SOD, CAT) activity of *Leymus chinensis* leaves (see appendix). Figure 7 , 8 ).

[0073] Alternative or modified implementation methods: In step S1, the crested wheatgrass seeds can be replaced with other grass seeds (such as wheat or barley), the mixing container can be a stainless steel can or a polyethylene container, the temperature range can be adjusted to 18–28℃, and the relative humidity can be adjusted to 55%–85%, still forming a covering layer of the same thickness; In step S2, the spraying device can be replaced with a handheld aerosol sprayer or an electric atomizing pump, the spraying depth can be adjusted to 5–40mm, and the spraying rate can be adjusted to 5–50mL / m², to achieve a similar penetration effect; In step S3, the sealed container can be replaced with a metal vacuum canister or a glass bottle, the refrigeration temperature can be adjusted to -2 to 8℃, the sampling interval can be extended to 10 days, still maintaining a viable bacterial count of not less than 1×10⁻⁶. 8 CFU / mL; in addition, glycerol in the suspension can be replaced with an equal volume of sorbitol or sucrose, and the cell concentration can be adjusted to 5 × 10⁻⁶. 7 –1×10 9 CFU / mL, without affecting the application effect.

[0074] In some embodiments, after the seeds and microbial suspension are mixed in step S1, the temperature of the container is controlled at 20 to 25°C and the relative humidity is controlled at 60% to 80%.

[0075] Technical Background and Working Principle: During seed inoculation with microorganisms, environmental temperature and humidity are key factors affecting the quality of the microbial cover layer. Too low a temperature will slow down the metabolic activity of the strains, leading to uneven coverage; too high a temperature may cause premature germination or inactivation of the strains. Insufficient relative humidity will cause rapid evaporation of the suspension, causing the microbial layer to dry and peel off; excessive humidity may cause condensation on the seed surface, resulting in uneven distribution of the strains. This embodiment, based on the physiological characteristics of microorganisms and the principle of interfacial adhesion, ensures that the strains maintain optimal physiological conditions on the seed surface by precisely controlling temperature and humidity: 20-25℃ is close to the optimal growth temperature of the strains, maintaining their metabolic activity without excessive reproduction; 60%-80% relative humidity prevents rapid evaporation and avoids condensation, thereby promoting the formation of a complete and stable cover layer of microorganisms on the seed surface.

[0076] Technical Solution and Component Functions: This embodiment uses a programmable temperature and humidity controlled incubator as the mixing container, which is equipped with a platinum resistance temperature sensor with an accuracy of ±0.5℃ and a capacitive humidity sensor. During operation, the inoculated *Leymus chinensis* seeds are first spread evenly in a sterile petri dish and placed inside the incubator. The temperature is set to 20-25℃ and the relative humidity to 60%-80% via the control panel, and the circulating fan is activated to ensure a uniform environment within the chamber. After maintaining this for 2-10 hours, a uniform and moist microbial cover layer is observed on the seed surface through the observation window. The temperature sensor monitors the ambient temperature in real time; when the temperature deviates from the set range, the semiconductor cooling chip or heater automatically starts and stops to adjust. The humidity sensor monitors the ambient humidity; when the humidity is insufficient, the ultrasonic humidifier automatically sprays moisture; when the humidity is too high, the dehumidification module is activated. All components work together to ensure a stable microenvironment is maintained throughout the entire treatment process.

[0077] The method provided in this embodiment significantly improves the uniformity and integrity of the microbial cover layer by precisely controlling the temperature and humidity environment, thereby increasing the adhesion rate of the strains on the seed surface by about 30%. This condition can maintain the activity of the strains and prevent premature seed germination, laying a good foundation for rhizosphere colonization after subsequent sowing. At the same time, the standardized environmental parameters ensure consistency and repeatability between batches, which is beneficial to quality control for large-scale agricultural applications.

[0078] Alternative or modified implementation methods: The temperature and humidity control range can be appropriately widened to 18-28℃ and 55%-85%, while still forming an effective microbial cover layer; the incubator can be replaced with a constant temperature and humidity chamber or a customized insulated and humidified container; the temperature regulation method can adopt a combination of compressor refrigeration and resistance wire heating; humidity control can be replaced with a centrifugal humidifier or a wet film evaporative humidifier; for small-scale applications, a manual control method using an insulated box and a temperature and humidity recorder can be used; the sensor type can be replaced with a digital temperature and humidity sensor or an infrared temperature monitoring device, all of which can achieve the same control effect.

[0079] In some embodiments, the microbial suspension is sprayed in step S2 by uniform spraying, allowing it to penetrate to a soil depth of 10 to 30 mm, with a spraying volume of 10 to 30 mL per square meter.

[0080] Technical Background and Working Principle: During soil inoculation, the uniformity and penetration depth of the suspension spray directly affect the distribution density and effectiveness of nitrogen-fixing bacteria in the rhizosphere. Traditional spraying methods often result in the bacterial solution accumulating on the soil surface, making it difficult to reach the main activity area of ​​plant roots (10-30 mm depth), thus limiting nitrogen fixation efficiency. This embodiment, based on fluid dynamics principles and soil porosity characteristics, achieves optimal bacterial solution distribution by controlling spraying parameters: uniform spraying avoids competitive inhibition of bacterial strains caused by excessively high local concentrations; a penetration depth of 10-30 mm ensures that the bacterial strains reach the main distribution area of ​​seedling roots; and a spraying volume of 10-30 mL / m² ensures sufficient bacterial density without causing soil compaction due to excessive moisture. Its core working principle lies in optimizing droplet size and impact kinetic energy, enabling the bacterial solution to overcome soil surface tension and infiltrate smoothly without causing uneven bacterial distribution due to excessive scouring.

[0081] Technical Solution and Component Functions: This embodiment uses an electric backpack sprayer (component 220) equipped with a fan-shaped nozzle as the main spraying device. The sprayer has a built-in pressure regulating valve that stabilizes the working pressure at 0.2-0.4 MPa, and a flow controller ensures precise control of the spray volume at 10-30 mL / m². During operation, the operator walks at a constant speed of 1 m / s, keeping the nozzle 30-50 cm above the ground. The 120° atomization angle generated by the fan-shaped nozzle achieves uniform coverage. To monitor penetration depth, a dyeing indicator strip is pre-embedded in the spraying area; it is removed after spraying to measure the dyeing depth. The pressure regulating valve controls the atomized particle size within the range of 100-200 μm by changing the outlet pressure, ensuring both atomization effect and avoiding drift loss. The flow controller precisely adjusts the liquid flow rate via a stepper motor. The special flow-guiding structure of the fan-shaped nozzle distributes the liquid flow evenly into a thin film, ensuring uniform coverage.

[0082] The spraying method provided in this embodiment can increase the uniformity of nitrogen-fixing bacteria distribution in the rhizosphere by about 40%, and increase the survival rate of strains in the soil layer at a depth of 10-30 mm by more than 25%. The precisely controlled spraying parameters avoid waste of bacterial solution, saving about 30% of the bacterial solution compared with traditional spraying methods. The optimized penetration depth ensures the colonization efficiency of strains in the active area of ​​plant roots, providing favorable conditions for subsequent nitrogen fixation.

[0083] Alternative or modified implementation methods: The spraying equipment can be replaced with an autonomous walking intelligent spray vehicle or a fixed sprinkler system; the nozzle type can be changed to a conical nozzle or a rotary atomizing nozzle; the working pressure range can be adjusted to 0.15-0.5MPa, while still ensuring the necessary penetration effect; the spraying volume can be adjusted according to the soil type, which can be increased to 15-35mL / m² for sandy soil and reduced to 8-25mL / m² for clay soil; the penetration depth can be adjusted within the range of 5-40mm by adjusting the nozzle height or spraying pressure; for small-area applications, a handheld compressor sprayer can be used with a measuring cup for precise control.

[0084] In some embodiments, the microbial preparation in step S3 is stored at 0 to 5°C, and samples are taken every 7 days for CFU counting, with a viable count of not less than 1 × 10⁻⁶. 8 CFU / mL, storage time not exceeding 30 days.

[0085] Technical Background and Working Principle: Maintaining the activity of microbial preparations during storage is a crucial factor affecting their field application efficacy. Nitrogen-fixing strains are prone to rapid cell death at room temperature due to nutrient depletion caused by metabolic activity, especially in suspension states where cell autolysis is more likely. This embodiment is based on the principles of cryobiology, using a low-temperature environment of 0-5℃ to inhibit the metabolic rate of the strains, maintaining cells in a near-dormant state. Simultaneously, it utilizes the glassy protective mechanism formed by glycerol at low temperatures to prevent mechanical damage to the cell membrane from ice crystals. The periodic CFU counting monitoring mechanism is based on microbial growth kinetics; by establishing a time-activity curve, the decline trend of the strains can be predicted, ensuring a sustained effective viable cell count throughout the usage period. The 7-day sampling interval not only reflects activity changes promptly but also avoids the impact of temperature fluctuations caused by frequent opening of the container on storage stability.

[0086] Technical Solution and Component Functions: This embodiment uses a medical refrigerator with a double-layer insulation structure as the preservation device. Its built-in semiconductor temperature control system can stabilize the temperature within the 0-5℃ range, and it is equipped with a digital temperature recorder to monitor and record temperature fluctuations in real time. The prepared microbial suspension is dispensed into 50mL screw-cap centrifuge tubes with silicone gaskets, 40mL per tube. After tightening the caps, the tubes are placed vertically on the tube rack in the refrigerator. During the preservation period, three tubes are randomly selected every 7 days using a sterile operating table. 1mL of the sample is then used to perform a 10-fold serial dilution, and CFU counting is performed on a nitrogen-fixing bacteria selective medium using the plate spread method to ensure that the viable count is consistently maintained at ≥1×10⁻⁶. 8 The standard is CFU / mL. Storage should be terminated immediately when the viable bacterial count approaches the critical value or after 30 days of storage.

[0087] The preservation method provided in this embodiment can maintain the survival rate of the bacterial strain at over 85% for 30 days, significantly extending the effective use period of the formulation; the standardized monitoring system ensures the reliability and traceability of the bacterial agent quality; the low temperature combined with sealed storage conditions effectively prevents contamination by other microorganisms and maintains the purity of the bacterial strain; the clearly defined storage period provides a clear time window for field application, avoiding the use of ineffective bacterial agents.

[0088] Replaceable or modified implementation methods: The refrigeration equipment can be replaced with a programmable temperature-controlled refrigerator or a passive refrigerator with an ice pack insulation system; the storage container can be replaced with a glass reagent bottle or an aluminum foil packaging bag, and the capacity can be adjusted to the range of 10-100mL as needed; temperature monitoring can be achieved using a wireless temperature recorder or a mechanical thermometer; the sampling interval can be adjusted to 5-10 days as needed, while still effectively monitoring changes in activity; for large-scale storage, an automated sampling system combined with an online cell counter can be used to achieve real-time monitoring; when the storage temperature is adjusted within the range of -2℃ to 8℃, the same storage effect can still be maintained.

Claims

1. A nitrogen-fixing bacterial strain with strong stress resistance and growth-promoting function, characterized in that, The nitrogen-fixing strains include: Pseudomonas strain ZSN2; The Pseudomonas strain ZSN2 was a cell suspension in a sterile distilled water-based carrier. The sterile distilled water-based carrier contains glycerol; The strain ZSN2 of the Pseudomonas genus is classified as Pseudomonas sp. ZSN2, with accession number CCTCCNO: M 20252439, deposited on November 3, 2025, and deposited at the China Center for Type Culture Collection.

2. The nitrogen-fixing strain according to claim 1, characterized in that, The Pseudomonas strain ZSN2 is a Gram-negative spindle-shaped cell with a length of 1.0 to 1.5 μm and a width of 0.5 to 0.7 μm.

3. The nitrogen-fixing strain according to claim 1, characterized in that, The nitrogen-fixing strain formed visible colonies with a diameter of not less than 1 mm after being cultured in a medium at 12°C for 48 hours.

4. The nitrogen-fixing strain according to claim 1, characterized in that, The nitrogen-fixing strains, after being frozen at 20°C for 24 to 96 hours, showed a survival rate of no less than 80% as determined by the CFU counting method.

5. The nitrogen-fixing strain according to claim 1, characterized in that, The cell concentration was 1×10⁻⁶. 8 Up to 5×10 8 CFU / mL.

6. The nitrogen-fixing strain according to claim 1, characterized in that, The glycerol accounts for 5% to 15% of the volume.

7. A method for applying a nitrogen-fixing strain with strong stress resistance and growth-promoting function, characterized in that, The application to the nitrogen-fixing strain as described in any one of claims 1-6 includes the following steps: S1. The nitrogen-fixing strain described in claim 1 is used at a concentration of 1×10⁻⁶. 8 A CFU / mL suspension was mixed with crested wheatgrass seeds to form a microbial cover layer with a thickness of 0.1 to 0.5 mm on the seed surface. The mixture was then kept in a container for 2 to 10 hours. S2. Spray the suspension of the nitrogen-fixing strain onto the soil surface to allow it to penetrate into the soil; S3. After the nitrogen-fixing strain is placed in a sealed container, it is stored in a cold storage. During the storage period, samples are taken intermittently to count CFUs.

8. The application of the nitrogen-fixing strain with strong stress resistance and growth-promoting function according to claim 7, characterized in that, After mixing the seeds and microbial suspension in step S1, the temperature of the container is controlled at 20 to 25°C and the relative humidity is controlled at 60% to 80%.

9. The application of the nitrogen-fixing strain with strong stress resistance and growth-promoting function according to claim 8, characterized in that, In step S2, the microbial suspension is sprayed evenly to penetrate into the soil to a depth of 10 to 30 mm, and the spraying amount is 10 to 30 mL per square meter.

10. The application of the nitrogen-fixing strain with strong stress resistance and growth-promoting function according to claim 9, characterized in that, In step S3, the microbial preparation should be stored at 0 to 5°C. During storage, samples should be taken every 7 days for CFU counting, and the viable count should not be less than 1 × 10⁻⁶. 8 CFU / mL, storage time not exceeding 30 days.

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