Salt-tolerant squirrel mammalian coccobacillus and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a salt and alkali tolerant squirrel mammalian coccus and application thereof. Mammaliicoccus sciuri ​ STM10, which is a gram-positive coccus, has been preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.38450. The strain has salt and alkali tolerance, can play the promoting functions of nitrogen fixation, phosphorus dissolution and resolution, iron carrier production and protease production, and can be prepared into microbial fertilizer. Application of the microbial fertilizer can effectively relieve salt and alkali stress, improve the seed germination and growth ability of crops, improve the physical and chemical properties of saline-alkali soil, optimize the ion environment, promote the nutrient absorption of crops, and provide support for agricultural utilization of saline-alkali land.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a salt-tolerant squirrel mammal cocci and its applications. Background Technology

[0002] Soil salinization refers to the accumulation of soluble salts in the upper layers of the soil profile. Excessive soil salinity can affect crop production, soil health, and ecosystem function, leading to land degradation and loss of soil ecosystem function. Millet, as a preferred crop for soil improvement and ecological restoration in saline-alkali areas, can maintain a certain germination rate and growth vigor even under severe saline-alkali stress, demonstrating excellent stress resistance potential.

[0003] Microbial improvement methods have become one of the core technologies for saline-alkali land management due to their advantages such as environmental friendliness, strong sustainability, and long-lasting improvement effects. Plant rhizosphere growth-promoting bacteria (PGPR), as a core resource for biological improvement of saline-alkali land, can improve the physicochemical properties of saline-alkali soil, enhance soil nutrient availability, and promote crop root development through physiological and metabolic functions such as phosphorus solubilization, iron production, and secretion of extracellular enzymes, thereby alleviating the damage of saline-alkali stress to crops. Currently, various microbial strains with salt and alkali tolerance and growth-promoting functions have been screened both domestically and internationally. Based on microorganisms such as Halomonas, Bacillus, and Pseudomonas, corresponding technologies for saline-alkali land improvement and crop growth promotion have been developed. Related strains have been applied to various crops such as Arabidopsis, maize, and tomato. Related technical solutions have been widely disclosed. For example, different types of salt and alkali tolerant strains such as Bacillus violaceus (CN121518276A), Bacillus amyloliquefaciens (CN120905084A), and Pseudomonas pseudoarthritis (CN121592541A), as well as compound microbial agents composed of multiple functional strains, can alleviate the inhibitory effect of salt and alkali stress on crops to a certain extent.

[0004] Squirrel mammal cocci ( Mammaliicoccus sciuri (formerly classified as *Staphylococcus squirrelans*) is a facultative anaerobic Gram-positive coccus, widely distributed in wild animals, livestock, humans, and various environmental media. Current research on this species largely focuses on the spread of drug-resistant genes, opportunistic pathogenicity, and basic physiological and biochemical characteristics. Some studies have found that this species can secrete active substances such as lysozyme and chitinase, showing potential applications in antifungal activity and fermentation degradation of soybean protein. However, no research has yet publicly disclosed its salt-alkali tolerance or its application value in saline-alkali land agriculture. Its potential applications in rhizosphere growth promotion and saline-alkali soil improvement remain unexplored.

[0005] Meanwhile, current research and application of salt-tolerant and growth-promoting bacteria mainly focus on Bacillus species, resulting in relatively limited strain types. Most existing functional strains are screened from ordinary soils or the rhizosphere of mainstream crops, exhibiting limited tolerance to saline-alkali environments and unstable colonization and growth-promoting effects under high salt stress. Millet, as a typical stress-resistant crop characteristic of saline-alkali lands, has a rhizosphere that has long adapted to high-salt environments, containing abundant salt-tolerant and highly active functional microbial resources with the potential to be developed into microbial agents for saline-alkali lands. However, research on the exploration and application of these resources remains relatively scarce.

[0006] The reasons for the above problems are that existing research on *Squirrelella* has long been limited to its drug resistance-related characteristics. Researchers have not paid attention to the functional potential of this species in agricultural environments, resulting in the failure to discover its salt-alkali tolerance and growth-promoting properties. At the same time, as a regional specialty crop, millet has received less attention in related research than mainstream food crops. Its rhizosphere microbial resources adapted to saline-alkali environments have not been fully explored and developed. This has also resulted in a relatively limited variety of salt-alkali tolerant functional microbial resources, making it difficult to meet the improvement needs of different saline-alkali regions and different crops. Summary of the Invention

[0007] To address the shortcomings of existing technologies, such as the limited variety of salt-tolerant growth-promoting bacterial strains and the unstable colonization and growth-promoting effects of commonly sourced functional strains in high-salt environments, and to fill the research gap in the salt-tolerant growth-promoting function of *Squirrel Lactococcus*, this invention aims to fully explore the functional microbial resources of millet rhizosphere adapted to saline-alkali environments. It seeks to enrich the functional microbial resource bank for saline-alkali lands, meet the improvement needs of different saline-alkali regions and crops, and enhance the growth performance of crops under salt-alkali stress.

[0008] The core functional strain of this invention is *Squirrelella* STM10, and its preservation information is as follows:

[0009] Strain name: Squirrel mammal cocci Category Naming: Mammaliicoccus sciuri Strain number: STM10 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address of depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: April 27, 2026 Accession number: CGMCC No. 38450.

[0010] Furthermore, this strain is a Gram-positive coccus with pale yellow colonies that are smooth, moist, and glossy with neat edges and no obvious pigment diffusion. Single colonies are round or nearly round, uniform in size, with obvious protrusions and uniform texture, and a colony diameter of about 1-2 mm. Its 16S rRNA gene sequence is shown in SEQ ID NO.1. It can stably colonize and promote growth in saline-alkali soils with a pH of 9.0-9.5.

[0011] Furthermore, this strain was isolated from the rhizosphere soil of millet in saline-alkali land and possesses excellent salt and alkali tolerance. It also has multiple crop growth-promoting functions, including nitrogen fixation, phosphorus solubilization, phosphorus hydrolysis, iron carrier production, and protease production.

[0012] Based on this strain, a second aspect of the present invention provides a microbial fertilizer, wherein the microbial fertilizer uses live bacteria of the squirrel mammal cocci STM10 as the core functional component.

[0013] Furthermore, the viable count of *Squirrel Mammal Cocci* STM10 in the microbial fertilizer is not less than 1 × 10⁻⁶. 8 CFU / g.

[0014] Furthermore, the fermentation substrate for the microbial fertilizer can be brown sugar and organic cake fertilizer, with the mass ratio of microbial suspension, brown sugar, and organic cake fertilizer being 1:0.3~0.7:4~6.

[0015] Furthermore, the prepared microbial fertilizer has a miscellaneous bacteria rate of no more than 5% and a pH of 6.5~7.5, which can effectively ensure the activity and storage stability of the strains.

[0016] The third aspect of this invention provides the application of the microbial fertilizer in the planting of crops in saline-alkali land: the microbial fertilizer can be applied to the planting of grain crops such as millet in saline-alkali land. It can be applied by rhizosphere trenching during the flowering period of the crop, which can effectively improve the nutrient availability of saline-alkali soil, regulate the soil microbial community, alleviate the damage of salt and alkali stress to crops, and enhance the germination and growth capacity of crops.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: This invention is the first to explore the salt-alkali tolerance and growth-promoting function of Squirrel Mammal Cocci, filling the research gap in the application of this species in saline-alkali land agriculture, enriching the resource types of salt-alkali tolerant functional microorganisms, breaking the limitation of the single type of salt-alkali tolerant growth-promoting bacteria mainly consisting of Bacillus in the existing technology, and providing a brand-new selection of functional microorganisms for saline-alkali land improvement.

[0018] The core strain of this invention was isolated from the rhizosphere of millet in saline-alkali land and has been adapted to high salt stress environment for a long time. Compared with functional strains from ordinary soil or the rhizosphere of mainstream crops, this strain has a stronger colonization ability in high salt environment and can stably survive and function in severely saline-alkali soil with pH of 9.0~9.5, effectively solving the problems of insufficient activity and unstable growth promotion effect of existing strains under high salt stress.

[0019] This strain possesses multiple growth-promoting functions, including nitrogen fixation, phosphorus solubilization, phosphorus hydrolysis, iron carrier production, and protease production. It can simultaneously achieve soil nitrogen fixation, activation of insoluble inorganic and organic phosphorus, activation of soil iron, and decomposition of protein-based organic nutrients. It can improve the nutrient availability of saline-alkali soil in multiple dimensions. Compared with single-function microbial agents, it can more comprehensively enhance soil fertility, promote crop nutrient absorption, and avoid the limitations of single-function strains.

[0020] The microbial fertilizer developed based on this strain effectively ensures the activity and storage stability of the strain. The prepared fertilizer has a sufficient number of live bacteria and a low rate of contaminants, making it suitable for the planting needs of various grain crops. Its application is simple; it can be effectively applied through rhizosphere furrow application during the crop flowering period, making it suitable for large-scale field improvement of saline-alkali land. Experimental verification shows that this microbial fertilizer can effectively alleviate the damage to crops caused by saline-alkali stress, significantly improve growth indicators such as seed germination rate, plant height, and root length under saline-alkali stress, and simultaneously regulate the soil microbial community structure, improve the micro-ecological environment of saline-alkali soil, and achieve sustainable improvement of saline-alkali land, providing reliable technical support for the efficient agricultural utilization of saline-alkali areas. Attached Figure Description

[0021] Figure 1 This is a streak image of STM10, a type of squirrel mammal coccus.

[0022] Figure 2 Morphological image of STM10, a squirrel mammal coccus, after Gram staining.

[0023] Figure 3 This is a scanning electron microscope image of the morphology of STM10, a mammalian coccus of squirrels.

[0024] Figure 4 The image shows the electrophoresis results of PCR amplification of the 16S rRNA gene of STM10 from Squirrel Mammococcus, where M is the DNA Marker and lane 6 is the PCR amplification product of STM10.

[0025] Figure 5 Phylogenetic tree of STM10, a mammalian coccus of squirrels.

[0026] Figure 6The image shows the qualitative detection results of nitrogen fixation, phosphorus solubilization, phosphorus hydrolysis, siderophore production, and protease production capabilities of STM10, a mammalian coccus of squirrels.

[0027] Figure 7 This is a comparison chart of growth indicators (root length, shoot length, root fresh weight, and shoot fresh weight) of millet seed germination in different treatment groups. CK is the control group, MS is the salt-alkali stress group, and STM10 is the salt-alkali stress + microbial fertilizer treatment group.

[0028] Figure 8 This is a graph showing the germination status of millet seeds in different treatment groups on day 7.

[0029] Figure 9 Comparison of the growth status of millet seedlings at 50 days old in different treatment groups.

[0030] Figure 10 This image shows the finished microbial fertilizer prepared based on STM10 of Squirrel Mammal Cocci.

[0031] Figure 11 The graph shows the effects of the microbial fertilizer on the physicochemical properties of the soil in each group, with available potassium (AK), available phosphorus (AP), electrical conductivity (EC), and ammonium nitrogen (NH4) labeled. + -N), nitrate nitrogen (NO3) - Comparison results of indicators including -N), organic carbon (OC), total potassium (TK), total nitrogen (TN), total phosphorus (TP), water content (MC), pH, and total salt content (TS).

[0032] Figure 12 The diagram shows the effect of the microbial fertilizer on the soil enzyme activities of each group, with comparative results of soil enzyme activities such as catalase (CAT), phosphatase (PHO), sucrase (SUC), β-glucosidase (BG), and urease (URE) marked.

[0033] Figure 13 This is a graph showing the effect of the microbial fertilizer on the ionic composition of each soil group, with calcium ions (Ca) marked. 2 + ), magnesium ions (Mg 2+ Sodium ions (Na) + ), potassium ions (K) + ), chloride ions (Cl) - ), carbonate ions (CO3) 2- ), bicarbonate ions (HCO3) - ), sulfate ions (SO4) 2- The comparison results are as follows.

[0034] Figure 14The graph shows the effect of the microbial fertilizer on the plant height and stem diameter of each group of millet plants.

[0035] Figure 15 The graph shows the effects of the microbial fertilizer on the nutrient and ion content of millet plants in each group, with the calcium ion content (P-Ca) of the plants marked. 2+ ), plant sodium ions (P-Na) + The comparison results of total potassium (P-TK), total phosphorus (P-TP), and total nitrogen (P-TN) in plants. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] The experimental materials, culture media, and instruments used in the examples are summarized below: I. Experimental Materials 1. Soil samples: Rhizosphere soil of millet in saline-alkali land, with a sampling depth of 10-20cm from the main distribution layer of millet roots. After sampling, the samples were aseptically sealed and stored at low temperature for later use.

[0038] 2. Test strain: *Squirrel mammal cocci* isolated in this invention (… Mammaliicoccus sciuri STM10 is deposited at the China General Microbiological Culture Collection Center of the China Association for the Preservation and Management of Microbial Cultures.

[0039] 3. Test reagents: sterile phosphate buffer (pH 7.0), 30% sterile glycerol, 10% sodium hypochlorite solution, 75% ethanol, 0.05% NaOH solution, PCR reaction mixture (containing Taq DNA polymerase, dNTPs, and buffer), 16S rRNA gene amplification primers (27F, 1492R), 6× Loading Buffer, nucleic acid dye, 1× TAE buffer, DNA Marker, Salkowski test solution, CAS (chrome azure S) staining solution, and molybdenum antimony chromogenic agent.

[0040] II. Culture Medium 1.10% LB solid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 100g / L, agar 16g / L, bring to volume with deionized water, adjust pH to 7.0, autoclave at 121℃ for 20min and use.

[0041] 2.10% LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 100 g / L NaCl, bring to volume with deionized water, adjust pH to 7.0, autoclave at 121°C for 20 min and use.

[0042] 3. Functional identification media: including amylase production medium, cellulase production medium, protease production medium, IAA (auxin) production detection medium, siderophore production detection medium (CAS medium), EPS (Extracellular Polymeric Substances) production detection medium, Monkina organic phosphorus liquid medium (phosphorus solubilizing medium), phosphorus solubilizing medium, potassium solubilizing medium, and nitrogen fixation medium. Each medium is prepared according to the conventional formula for microbial functional identification and sterilized by autoclaving at 121℃ for 20 minutes before use.

[0043] 4. MKB medium: Casein amino acids 5g / L, glycerol 15mL / L, K2HPO4 2.5g / L, MgSO4·7H2O 2.5g / L, pH 7.2, autoclaved at 121℃ for 20min before use.

[0044] 5. High-salt mannitol agar medium (with added penicillin G): Peptone 5.0 g / L, tryptone 5.0 g / L, beef extract 1.0 g / L, mannitol 10.0 g / L, NaCl 75.0 g / L, phenol red 0.025 g / L, agar 16.0 g / L. Autoclave at 121℃ for 20 min. When the medium cools to 55~60℃, aseptically add penicillin G stock solution to a final concentration of 0.006 g / L. Mix well and set aside.

[0045] 6. Plate count agar (PCA) medium: peptone 5.0 g / L, yeast extract 2.5 g / L, glucose 1.0 g / L, agar 16.0 g / L, pH 7.0, autoclaved at 121℃ for 20 min before use.

[0046] 7. Martin medium: peptone 5.0 g / L, glucose 10.0 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.5 g / L, Bengal red 0.033 g / L, agar 16.0 g / L. Autoclave at 121℃ for 20 min. When the medium cools to 55~60℃, aseptically add 0.1 g / L chloramphenicol, mix well and set aside.

[0047] III. Main Experimental Instruments Clean bench, constant temperature incubator (accuracy ±1℃), high-speed centrifuge, -80℃ ultra-low temperature freezer, pipette, optical microscope, scanning electron microscope, vacuum freeze dryer, PCR instrument, nucleic acid electrophoresis instrument, gel imaging analyzer, vortex mixer, flame spectrophotometer, gas chromatograph, enzyme-linked immunosorbent assay (ELISA) reader, pH meter, UV-Vis spectrophotometer, electronic balance (accuracy 0.01g), vernier caliper, measuring tape, autoclave, aseptic fermenter, colony counter, constant temperature and light incubator.

[0048] Example 1 This embodiment describes the isolation and screening of STM10, a type of squirrel mammal coccus.

[0049] I. Experimental Materials Soil samples: collected from the rhizosphere soil of millet in saline-alkali land, at a depth of 10-20 cm, where the main distribution layer of millet roots is located. After sampling, the samples were placed in sterile sealed bags, stored at low temperature, and brought back to the laboratory for processing as soon as possible.

[0050] Culture media: 10% LB solid medium, 10% LB liquid medium.

[0051] Reagents and instruments: sterile phosphate buffer (pH 7.0), 30% sterile glycerol, 10% sodium hypochlorite solution, 75% ethanol; clean bench, constant temperature incubator (accuracy ±1℃), high-speed centrifuge, -80℃ ultra-low temperature freezer, pipettes (10μL, 100μL, 1000μL), sterile culture dishes (90mm in diameter), sterile conical flasks (250mL).

[0052] II. Experimental Procedure Soil suspension preparation: In a clean bench, accurately weigh 5g of the above-mentioned saline-alkali land millet rhizosphere soil sample and place it in a 250mL sterile Erlenmeyer flask containing 45mL of sterile phosphate buffer (pH 7.0). After sealing, place the flask on a shaker and shake at 150rpm / min for 30min to obtain a concentration of 10. -1 Soil suspension. Subsequently, using a serial dilution method, 10 liters were sequentially diluted with sterile phosphate buffer. -1 Soil suspension diluted to 10 -2 10 -3 10 -4 10 -5 10 -6 Concentration gradients were used, and each dilution process was performed under strict aseptic conditions.

[0053] Spread culture: from 10 -2 10 -4 10 -6200 μL of each of the three concentration gradients of soil dilution was taken and evenly spread onto 10% LB agar plates. Three parallel petri dishes were set up for each concentration gradient to ensure the repeatability of the experiment. The spread agar plates were placed upside down in a 30°C incubator and incubated in the dark for 3-4 days, during which time the colony growth was observed regularly.

[0054] Strain purification: After obvious colony growth appears on the culture medium plates, based on the differences in colony morphology, single colonies of different morphologies are picked using a sterile inoculation loop and inoculated separately onto fresh 10% LB medium plates. Purification is then performed using the four-zone streak method. The streaked plates are then incubated at 30°C for 3-4 days. If contaminating bacteria (with morphology different from the target colonies) appear on the plates, the four-zone streak procedure is repeated until the colonies on the plates have completely identical morphologies, at which point the strain is considered purified.

[0055] Strain preservation: Pick a purified single colony and inoculate it into a sterile Erlenmeyer flask containing 10 mL of LB liquid medium. Incubate at 30 °C and 150 rpm / min for 2 days to obtain bacterial suspension. Take 400 μL of bacterial suspension and mix it thoroughly with 600 μL of sterile 30% glycerol. Transfer it to a sterile cryovial, label it with the strain number, and store it in an ultra-low temperature freezer at -80 °C for long-term storage.

[0056] Gram staining: Freshly purified bacterial colonies were collected, and a small amount of cells were picked up using a sterile inoculation loop. Sterile water was added to a clean glass slide, and the mixture was evenly spread to form a bacterial film. After air drying, the film was lightly fixed with a flame. Gram staining was then performed sequentially: first, crystal violet staining was added for about 1 minute, followed by slow rinsing with water; then iodine mordanting was added for about 1 minute, followed by rinsing with water; then 95% ethanol was added for destaining, generally for 10-20 seconds, until the effluent showed almost no obvious purple color, and destaining was immediately stopped with water; finally, safranin counterstaining was added for about 30-60 seconds, followed by rinsing with water and drying. After staining, the staining and cell morphology of the bacteria were observed under an optical microscope. Purple cells were considered Gram-positive, and red or pink cells were considered Gram-negative. The preliminary taxonomic classification was determined based on the morphological characteristics of the bacteria.

[0057] Scanning electron microscopy (SEM) observation: Purified bacterial cell samples were collected by centrifugation and fixed with 2.5% glutaraldehyde fixative at 4°C for 12 h. The samples were then washed 2-3 times with 0.1 mol / L phosphate buffer to remove residual fixative. The fixed samples were then dehydrated sequentially with different concentration gradients (30%, 50%, 70%, 80%, 90%, and 100%) of ethanol solution, each dehydration stage lasting 10-15 min, with two treatments using anhydrous ethanol. After dehydration, the samples were freeze-dried under vacuum, then fixed on a sample stage and sputter-coated with gold to improve conductivity. Finally, the samples were observed and images were captured under a scanning electron microscope to record the ultrastructural characteristics of the bacterial cell surface morphology, cell outline, size, and arrangement.

[0058] III. Test Results Through the above isolation and screening process, a total of 174 purified bacterial strains were obtained from the rhizosphere soil samples of *Eriobotrya japonica* in saline-alkali land. Among them, strain STM10 exhibited characteristic morphology on 10% LB agar plates: colonies were milky white to pale yellow, with a smooth, moist, and glossy surface, neat edges, no obvious pigment diffusion, and single colonies were round or nearly round, uniform in size, with obvious protrusions, and uniform texture, with a colony diameter of approximately 1-2 mm (see...). Figure 1 (STM10 strain streak morphology image). Gram staining and scanning electron microscopy confirmed that this strain is a Gram-positive coccus, arranged in grape-like clusters or short chains. Figure 2 The image shows the morphology of the STM10 strain after Gram staining, and... Figure 3 The image shown is a scanning electron microscope image of the strain.

[0059] Example 2 This embodiment describes the phylogenetic identification of STM10, a type of squirrel mammal coccus.

[0060] I. Experimental Materials Strain sample: STM10 purified and preserved in Example 1.

[0061] Reagents: 0.05% NaOH solution (filtered and sterilized), PCR reaction mixture (containing Taq DNA polymerase, dNTPs, and buffer), 16S rRNA gene amplification primers (forward primer 27F, reverse primer 1492R), 6× Loading Buffer, nucleic acid dye, 1× TAE buffer, and DNA Marker.

[0062] Instruments: PCR instrument, electrophoresis instrument, gel imaging analyzer, clean bench, vortex mixer, pipettes (10μL, 100μL).

[0063] II. Experimental Procedure (a) Extraction of bacterial DNA Inside the clean bench, use a sterile inoculation loop to pick up an appropriate amount of STM10 single colony and place it in a centrifuge tube containing 100 μL of filtered and sterilized 0.05% NaOH solution. Use a vortex mixer to shake thoroughly to ensure that the bacteria and alkali solution are evenly mixed.

[0064] Place the centrifuge tubes into a PCR instrument and process them according to the DNA extraction procedure shown in Table 1 to break the bacterial cell wall and release DNA.

[0065] Table 1: DNA Extraction Procedure

[0066] (II) Bacterial 16S rRNA gene amplification Prepare the PCR reaction system according to the ratio shown in Table 2. Add each component to a sterile PCR tube in sequence, mix gently, and then centrifuge briefly (1-2 seconds) to concentrate the liquid at the bottom of the tube.

[0067] Table 2: PCR reaction system

[0068] Place the PCR tube containing the PCR reaction system into the PCR instrument and run the reaction according to the 16S rRNA gene amplification program shown in Table 3.

[0069] Table 3: 16S rRNA gene amplification procedure

[0070] (III) Electrophoresis detection and sequencing After the PCR amplification reaction is completed, take 5 μL of PCR product and mix it with 1 μL of 6×Loading Buffer and 0.5 μL of nucleic acid dye to obtain an electrophoresis sample.

[0071] Prepare a 1% agarose gel (containing nucleic acid dye), add the electrophoresis sample to the gel wells, and add DNA marker as a molecular weight reference. Electrophore in 1×TAE buffer at 120V for 30 min.

[0072] After electrophoresis, the agarose gel is placed in a gel imaging analyzer and observed and recorded under ultraviolet light. If the PCR product bands are single and bright, the amplification is considered successful.

[0073] The amplified PCR products were sent to a sequencing institution (Beijing Novogene Technology Co., Ltd.). First-generation sequencing was performed using forward primer 27F (CGGTGAATACGTTCYCGG, SEQ ID NO.2) and reverse primer 1492R (AAGGAGGTGATCCRGCCGCA, SEQ ID NO.3). Poor-quality sequences in the sequencing peaks were removed to obtain the 16S rRNA gene sequence of STM10 of Squirrel Mammococcus, which is about 1500 bp in length.

[0074] (iv) Phylogenetic analysis The obtained 16S rRNA gene sequence of STM10 was uploaded to the NCBI database. The Blast tool was used to perform homology comparison in the Nucleotide collection (nr / nt) database. The taxonomic position of the strain was preliminarily determined based on the sequence similarity (similarity ≥ 97.00% was judged as the same species).

[0075] Sequences of related strains with high homology to STM10 were selected from the database, and phylogenetic analysis was performed using MEGA11 software: First, multiple sequences were aligned using the Cluster W tool, and then the Kimura2-parameter model was used to construct a neighbor-joining (NJ) phylogenetic tree through Bootstrap analysis (1000 replicates).

[0076] III. Test Results Electrophoresis results show (see) Figure 4 The PCR product of the 16S rRNA gene from STM10 showed a single bright band at approximately 1500 bp, consistent with the expected fragment size, indicating successful amplification.

[0077] Sequencing revealed that the 16S rRNA gene sequence is shown in SEQ ID NO.1. Blast homology alignment results showed that this sequence is related to *Mammalian cocci* (…). Mammaliicoccus The sequence similarity of *Squirrelella* strains was ≥97.00%; combined with the NJ phylogenetic tree ( Figure 5 It can be seen that strain STM10 clustered with *Squirrel-mammal cocci* strain. Therefore, strain STM10 can be identified as *Squirrel-mammal cocci* of the *Mammalococcus* genus, and its classification name is: *Squirrel-mammal cocci*. Mammaliicoccus sciuri Squirrel mammal cocci STM10 is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38450.

[0078] Example 3 This embodiment describes the identification of the growth-promoting function of STM10 of Squirrel Mammal Cocci.

[0079] I. Experimental Materials Strain sample: The STM10 strain purified and preserved in Example 1 was identified as Squirrel Mammal Cocci by Example 2.

[0080] Identification media: Prepare amylase production medium, cellulase production medium, protease production medium, IAA (auxin) production detection medium, siderophore production detection medium (CAS medium), EPS production detection medium, Monkina organic phosphorus liquid medium (phosphorus solubilizing medium), phosphorus soluble medium, potassium solubilizing medium, and nitrogen fixation medium according to Table 4. All media are sterilized by autoclaving at 121℃ for 20 min before use.

[0081] Table 4: Components of Identification Culture Media

[0082] Reagents and instruments: Salkowski test solution (for IAA quantification), CAS (chrome azure S) staining solution (for siderophore quantification), molybdenum anti-chromogenic reagent (for phosphorus content determination); flame spectrophotometer (for potassium content determination), gas chromatograph (for acetylene reduction method for nitrogen fixation quantification), clean bench, constant temperature incubator, microplate reader, pH meter.

[0083] II. Experimental Procedure (a) Qualitative determination of reproductive function Enzyme production capacity assay: Single colonies of *S. squirrel-forming mammalian cocci* STM10 were picked and inoculated onto cellulase-producing, amylase-producing, and protease-producing agar plates, respectively, and incubated at 30℃ for 5–7 days. After incubation, the clear zone was observed by adding trichloroacetic acid solution to the protease-producing medium, Congo red solution to the cellulase-producing medium, and iodine solution to the amylase-producing medium. The presence of a clear zone indicated that the strain possessed the corresponding enzyme production capacity.

[0084] Determination of phosphorus solubilization, phosphorus solubilization, potassium solubilization, and nitrogen fixation capabilities: STM10 of *Squirrelella* was inoculated into phosphorus solubilization, phosphorus solubilization, potassium solubilization, and nitrogen fixation media and cultured at 30℃ for 7-10 days. Observe whether a clear zone appears around the colony in the phosphorus solubilization and phosphorus solubilization media (indicating phosphorus solubilization and phosphorus solubilization capabilities), whether there are signs of potassium salt dissolution in the potassium solubilization media, and whether the strain grows normally in the nitrogen fixation media (indicating nitrogen fixation capabilities).

[0085] IAA, siderophore, and EPS production capacity assays: STM10 of *S. squirrel mammalian cocci* was inoculated into IAA production detection medium (containing tryptophan), siderophore production detection medium (CAS medium), and EPS production detection medium. The cultures were incubated at 30°C for 5-7 days. The color changes of the IAA production medium, whether the blue color in the siderophore production medium turned orange, and whether the colonies in the EPS production medium contained mucus-like substances (indicating EPS production) were observed.

[0086] (II) Quantitative determination of reproductive function Nitrogen fixation capacity determination: When identifying the nitrogen fixation capacity of nitrogen-fixing bacteria using the acetylene reduction method, the nitrogen-fixing bacteria to be tested are first activated and cultured in a nitrogen-free medium until the logarithmic growth phase. An appropriate amount of bacterial solution is placed in a sealed serum bottle, and some air is extracted with a syringe before high-purity acetylene gas is injected to make the acetylene concentration in the system reach a suitable level. The system is then cultured in the dark at a suitable temperature for a certain period of time, allowing nitrogenase to catalyze the reduction of acetylene to ethylene. After the culture is completed, the headspace gas in the bottle is extracted, and the amount of ethylene produced is detected using a gas chromatograph. The amount is then quantitatively calculated using a standard ethylene curve. Blank and negative controls are set up to eliminate interference. Finally, the amount of ethylene produced per unit of bacterial cell or protein per unit time is used to characterize nitrogenase activity and reflect the nitrogen fixation capacity of the strain.

[0087] Phosphate solubilization capacity determination: Squirrel mammalian cocci STM10 was inoculated into 100 mL of Monkina organophosphate liquid medium. The medium was cultured with shaking at 28℃ and 180 r / min, with the medium without Squirrel mammalian cocci STM10 serving as a control. Each treatment was repeated three times. After 4 days of shaking culture, the fermentation product was filtered through a 0.22 μm microporous membrane to remove the bacterial cells, and the filtrate sample was stored at 4℃. The phosphorus content was determined using the molybdenum-antimony alkali method. The specific steps are as follows: (1) Preparation of molybdenum-antimony alkali reagent: Weigh 5g of potassium antimony tartrate, dissolve it in 1L of deionized water to prepare a 5mg / L potassium antimony tartrate solution; weigh 5g of ammonium molybdate, dissolve it in 225mL of deionized water, and then slowly add 76.5mL of concentrated sulfuric acid while stirring. After the liquid temperature drops to room temperature, add 100mL of 5g / L potassium antimony tartrate solution, and dilute to 500ml with deionized water. Shake well and store in a color bottle for later use to prepare an ammonium molybdate-sulfuric acid solution. Before use, add 1.5g / L ascorbic acid to every 100mL of ammonium molybdate-sulfuric acid solution (molybdenum-antimony alkali reagent). (2) Phosphorus standard curve preparation: Pipette 0, 1.0, 2.0, 3.0, 4.0, and 5.0 ml of 5 mg / L KHPO4 standard solution into 50 mL volumetric flasks, and simultaneously add an equal volume of blank solution to the sample solution used for colorimetric determination. Add 2-3 drops of dinitrophenol indicator, and then adjust the solution to a slightly yellow color with 0.5 mol / L NaHCO3 solution. Finally, accurately add 5 mL of molybdenum antimony reagent, shake well, and dilute to volume with deionized water. Measure the absorbance at a wavelength of 700 nm. Plot the absorbance value on the ordinate (Y) and the phosphorus standard solution concentration on the abscissa (X), obtaining Y = 0.0011X + 0.009(R² / X²) ... 2 =0.9922). (3) Take 1 mL of the phosphorus filtrate sample stored at 4℃, add it to a 50 mL volumetric flask, dilute it with deionized water to about 3 / 5 of the total volume, add 1~2 drops of dinitrophenol indicator, and adjust the solution to just turn slightly yellow with 0.5 mol / L NaHCO3 solution. Accurately add 5 mL of molybdenum antimony reagent, make up to volume and shake well. After standing for 30 min, perform colorimetric analysis at a wavelength of 700 nm using an ultraviolet spectrophotometer. Use the uninoculated Monkina organic phosphorus culture medium as the reference solution, adjust the absorbance of the reference solution to 0, perform colorimetric analysis, and read the absorbance value at 700 nm. Substitute the absorbance value into the phosphorus standard curve equation to obtain the increased soluble phosphorus content in the liquid culture medium.

[0088] Phosphorus solubility determination: *S. squirrel mammalian cocci* STM10 was inoculated into 100 mL of phosphate-solubilizing liquid medium. The medium was cultured with shaking at 28℃ and 180 rpm, with an uninoculated medium serving as a control. Each treatment was repeated three times. After 4 days of shaking culture, the fermentation product was filtered through a 0.22 μm microporous membrane to remove bacterial cells, yielding a phosphate-solubilizing filtrate sample, which was stored at 4℃. Phosphorus content was determined using the molybdenum-antimony assay, with the uninoculated phosphate-solubilizing culture medium as a reference solution. The absorbance of the reference solution was adjusted to 0, and colorimetric measurements were performed. The absorbance value at 700 nm was read, and the absorbance was substituted into the phosphorus standard curve equation to calculate the phosphorus solubility determination results.

[0089] Siderophore production capacity determination: STM10 of *Squirrelella* was inoculated into MKB medium at a 5% inoculum and cultured at 30°C and 180 r / min for 48 h. The fermentation broth of the strain was centrifuged at 3500 r / min for 15 min to remove the bacterial cells. 3 mL of supernatant was mixed with an equal volume of CAS reagent and reacted at room temperature for 1 h. The absorbance (As) of the solution at 630 nm was then measured. 3 mL of blank medium was mixed with an equal volume of CAS reagent and its absorbance was measured as a reference value (Ar) according to the above method. The relative content of siderophores (SU) was calculated as [(Ar-As) / Ar] × 100%.

[0090] Protease production capacity determination: The protease production capacity of *S. squirrel mammalian cocci* STM10 was determined using the Folin-Ciocalteu method. Casein was used as the substrate. The fermentation broth was centrifuged to obtain a crude enzyme solution, which was reacted with the casein substrate in a suitable temperature and pH buffer system for a certain period of time. Trichloroacetic acid was added to terminate the reaction and precipitate unhydrolyzed protein. After centrifugation, the supernatant was collected and sodium carbonate solution and Folin-Ciocalteu reagent were added for color development. The absorbance was measured at 660 nm using a UV-Vis spectrophotometer. A standard curve was plotted using tyrosine, and the tyrosine content generated in the reaction was calculated. The amount of enzyme required to hydrolyze the substrate to produce 1 μg of tyrosine per unit volume of enzyme solution per unit time was defined as one enzyme activity unit (U), thereby quantitatively determining the protease activity.

[0091] III. Test Results Qualitative test results: such as Figure 6As shown, *S. squirrel-mammal cocci* STM10 grows normally on Assumption medium, indicating that *S. squirrel-mammal cocci* STM10 has nitrogen-fixing ability; the appearance of a clear zone in phosphate-solubilizing medium indicates that *S. squirrel-mammal cocci* STM10 has the ability to dissolve inorganic phosphorus; the appearance of a clear zone in Monkina organic phosphorus medium indicates that *S. squirrel-mammal cocci* STM10 has the ability to decompose organic phosphorus; the production of a distinct orange-yellow siderophore chelation zone on CAS detection plates indicates that *S. squirrel-mammal cocci* STM10 has the ability to produce siderophores; the appearance of a degradation zone in protease-producing medium indicates that *S. squirrel-mammal cocci* STM10 has the ability to produce protease; however, no corresponding positive reactions were observed on EPS production detection medium, amylase production medium, cellulase production medium, IAA production medium, and potassium-solubilizing medium. In summary, *S. squirrel-mammal cocci* STM10 possesses nitrogen-fixing ability, phosphate-solubilizing ability, phosphate-solubilizing ability, siderophore production ability, and protease production ability.

[0092] Quantitative determination results: The nitrogen fixation, phosphorus solubilization, phosphorus hydrolysis, siderophore production, and protease production capabilities of *S. squirrel-mammal cocci* STM10 were further quantitatively determined, and the results are as follows: Nitrogenase activity was determined by acetylene reduction method, and the nitrogenase activity of this strain was calculated to be 283.64 pmol / (mL·h); the increase in soluble organic phosphorus content in *Montagna spp.* organic phosphorus culture medium was 43 mg / L, and the increase in soluble inorganic phosphorus content in phosphorus solubilization culture medium was 171 mg / L, determined by molybdenum antimony colorimetric method; after mixing the sterile filtrate of *S. squirrel-mammal cocci* STM10 with CAS detection solution and reacting in the dark for 1 h, the relative ironophile yield was calculated to be 40.72%; the protease production capability of *S. squirrel-mammal cocci* STM10 was quantitatively determined by Folin-Ciocalteu method, and the protease activity of its crude fermentation enzyme solution was calculated to be 28.78 U / mL.

[0093] Quantitative analysis showed that the relative contents of nitrogen fixation, phosphorus solubilization, phosphorus hydrolysis, siderophore production, and protease production of STM10 all reached the effective growth-promoting threshold. Its ability to produce enzymes and synthesize growth-promoting substances can meet the needs of crop growth under saline-alkali conditions, further verifying the growth-promoting functional characteristics of this strain.

[0094] Example 4 I. Experimental Materials This example describes the germination test of Squirrel Mammal Cocci STM10.

[0095] Strains and seeds: STM10 of Squirrel Mammal Cocci isolated and purified in Example 1; millet seeds (the variety is Yixuan Dahongmi, the main variety cultivated in local saline-alkali land, with plump grains and free from diseases and pests).

[0096] Reagents and culture media: 10% sodium hypochlorite solution, 75% ethanol, sterile distilled water, LB liquid medium, mixed saline solution (formulation: Na2CO3:NaHCO3:NaCl:Na2SO4=1:9:1:9, concentration 80mmol / L, prepared with sterile distilled water).

[0097] Instruments: Clean bench, constant temperature incubator (accuracy ±1℃), shaker, centrifuge, sterile culture dishes (90mm in diameter), sterile filter paper, pipettes (1mL, 5mL).

[0098] II. Experimental Procedure (I) Preparation of bacterial suspension Squirrel mammal cocci STM10 was taken out from the -80℃ ultra-low temperature freezer and streaked onto LB agar plates in a clean bench. The strain was activated by constant temperature incubation at 28℃ for 2 days. This process was repeated twice to ensure the activity of the strain.

[0099] Pick a single activated colony and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium. Incubate at 30 °C and 180 rpm with shaking for 16 h. Centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the precipitate three times with sterile water, resuspend, and adjust the bacterial suspension concentration to 1 × 10⁻⁶. 8 The STM10 bacterial suspension was obtained by measuring CFU / mL (verified by plate counting method) and stored at 4℃ for later use.

[0100] (II) Seed disinfection and soaking Select uniform and plump millet seeds, soak them in 10% sodium hypochlorite solution for 5 minutes, then soak them in 75% ethanol for 60 seconds, and rinse them three times with sterile distilled water to complete surface disinfection.

[0101] The sterilized seeds were divided into three groups for treatment, and each group underwent the following soaking treatment: Control group (CK): Seeds were soaked in sterile distilled water for 2 hours and then air-dried in a clean bench. Salt-alkali stress group (MS): seeds were soaked in a mixed salt-alkali solution for 2 hours and then air-dried in a clean bench. Salt stress + bacterial suspension treatment group (STM10): Soak in STM10 bacterial suspension for 2 hours and air dry in a clean bench.

[0102] (III) Germination Test Procedure Inside the laminar flow hood, two layers of sterile filter paper were placed on the bottom of a 9mm diameter sterile petri dish. 100 treated seeds were placed in each petri dish, and five replicates were set up.

[0103] Add 5 mL of sterile distilled water to the CK group culture dish, and add 5 mL of 80 mmol / L mixed saline solution to the MS group and STM10 group culture dishes, ensuring that the filter paper is fully moistened but without excess liquid.

[0104] Place all petri dishes in a 25°C constant temperature incubator and incubate in the dark. During this period, add a small amount of the corresponding liquid daily to keep the filter paper moist.

[0105] (iv) Indicator Measurement Germination potential was calculated on day 3 of cultivation: Germination potential = (Number of germinated seeds on day 3 / Total number of seeds tested) × 100%; Germination rate was calculated on day 7 of cultivation: Germination rate = (Number of germinated seeds on day 7 / Total number of seeds tested) × 100%.

[0106] III. Test Results Table 5: Seed germination indicators

[0107] As shown in Table 5, salt and alkali stress significantly inhibited the germination of millet seeds: compared with the CK group, the germination potential, germination rate and germination rate of the MS group decreased significantly, with the germination rate decreasing by about 54%, indicating that the 80 mmol / L mixed salt and alkali solution posed a significant stress on the germination of millet seeds.

[0108] Treatment with STM10 bacterial suspension significantly alleviated salt-alkali stress: the germination potential of the STM10 group was 121.46% higher than that of the MS group, the germination rate was 109.51% higher, and the root length, shoot length, root fresh weight, and shoot fresh weight were all significantly increased. Figure 7 This indicates that *Sacchariformis sTM10* can effectively promote the germination ability of millet seeds under salt-alkali stress (see the seed germination status of each group on day 7). Figure 8 ).

[0109] Example 5 This embodiment describes a pot experiment of Squirrel Mammal Cocci STM10.

[0110] I. Experimental Materials Strains and seeds: Squirrel mammal cocci STM10 isolated and purified in Example 1; millet seeds as in Example 4.

[0111] Soil and reagents: Nutrient soil, sterilized after passing through a 2mm sieve (121℃, 2h); mixed saline-alkali solution (same as in Example 4, 80mmol / L); STM10 bacterial suspension (concentration 1×10⁻⁶). 8 CFU / mL, prepared by the same method as in Example 4); sterile distilled water.

[0112] Instruments and apparatus: plastic flower pots (15cm in diameter), constant temperature and light incubator (16h light / 8h darkness, 26℃), measuring tape, electronic balance (0.01g accuracy), vernier calipers.

[0113] II. Test Methods Each flowerpot was filled with 400g of sterilized nutrient soil, watered several times with distilled water, and then evaporated to a suitable humidity. Millet seeds that had been sterilized in Example 4 were sown. Each treatment group was repeated in 3 pots. One week after emergence, 50 seedlings were transplanted. When the seedlings reached the three-leaf stage, each group was treated as follows: CK group: Water each pot with 50mL of sterile distilled water, and then water with 30mL of distilled water every 3 days in the later stage; MS group: Water each pot with 50mL of mixed saline-alkali solution, and then water with 30mL of mixed saline-alkali solution every 3 days in the later stage; STM10 group: Water each pot with 50mL of mixed saline-alkali solution. In the later stage, water with 30mL of mixed saline-alkali solution every 3 days. Water with 30mL of STM10 bacterial suspension the next day. Water for a total of 3 times.

[0114] The light exposure was set for 16 hours, the darkness for 8 hours, and the room temperature was 26°C. The placement of different treatment groups was changed every 5 days to eliminate environmental differences, and the culture period was 50 days.

[0115] After cultivation, carefully remove the plant, rinse the soil around the roots, and measure the following indicators: Plant height: The vertical height from the base of the stem to the top of the plant; Root length: The length of the taproot from the root tip to the base of the root; Stem diameter: diameter at the base of the stem (measured with vernier calipers); Fresh weight: Fresh weight of the above-ground and underground parts of the plant (weighed by electronic balance).

[0116] III. Test Results Table 6: Seedling Stage Growth Indicators

[0117] As shown in Table 6, salt-alkali stress significantly inhibited the growth of millet: compared with the CK group, the plant height, stem diameter, fresh weight and root length of the MS group were all lower, indicating that 80 mmol / L mixed salt-alkali solution significantly inhibited the growth of millet seedlings.

[0118] STM10 bacterial suspension can effectively alleviate salt and alkali stress: the plant height, stem diameter, and fresh weight of the STM10 group were all higher than those of the MS group, and the plant growth was significantly better than that of the MS group. Seedling growth at 50 days was as follows... Figure 9 As shown in the figure. The results confirmed that *Squirrelella* STM10 significantly promoted the growth and development of millet seedlings under salt-alkali stress.

[0119] Example 6 This embodiment describes the microbial fertilizer fermentation of Squirrel Mammal Cocci STM10.

[0120] I. Experimental Materials Strain: STM10 of Squirrel Mammal Cocci isolated and purified in Example 1.

[0121] Culture medium and fermentation substrate: LB liquid medium, peanut cake (crushed through a 40-mesh sieve), brown sugar; 0.12 mmol / L PBS buffer (pH 7.2, sterilized at 121℃ for 20 min).

[0122] Instruments and equipment: Clean bench, constant temperature shaking incubator, high-speed centrifuge, sterile fermenter (5L), pH meter, colony counter, autoclave.

[0123] II. Experimental Procedure (I) Strain activation and bacterial suspension preparation The STM10 strain was taken out from the -80℃ ultra-low temperature freezer and streaked onto a 10% LB medium plate in a clean bench. It was then activated by incubation at 28℃ for 2 days. This process was repeated twice to ensure the activity of the strain.

[0124] Select a single activated colony and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid LB medium. Incubate at 28 °C and 180 rpm for 12 h to obtain the seed culture.

[0125] Centrifuge the seed culture at 8000 rpm for 10 min, discard the supernatant, wash the bacterial cells twice with 0.12 mmol / L PBS buffer (centrifuge at 8000 rpm for 5 min each time), resuspend, and adjust the OD of the bacterial suspension using a spectrophotometer. 600 =0.8, store at 4℃ for later use.

[0126] (II) Fermentation substrate preparation Prepare the raw materials according to the mass ratio of bacterial suspension: brown sugar: peanut cake = 1:0.5:5 (on dry weight); mix the crushed peanut cake with brown sugar evenly, add the above STM10 bacterial suspension, stir for 15~20 minutes until the matrix is ​​fully moistened, and adjust the matrix moisture content to 50~60%.

[0127] (III) Fermentation process control The prepared substrate is loaded into a sterile fermentation tank (filling the tank to 60% of its volume) and placed in a constant temperature fermentation chamber at 28~30℃ to ferment in the dark for 15 days.

[0128] For the first 5 days of fermentation, aerate the substrate for 30 minutes daily to promote aerobic cell growth. From day 6 to day 15, aerate every 2 days to maintain a loose substrate. Monitor the substrate temperature regularly during this period, and if it exceeds 35°C, stir and cool it down promptly to ensure a stable fermentation environment.

[0129] (iv) Quality testing of microbial fertilizer After fermentation, the following indicators of the microbial fertilizer were tested: Effective viable count determination: The dilution plating method was used. 10g of bacterial fertilizer sample was added to 90mL of sterile physiological saline and serially diluted. 0.1mL of bacterial suspension at different dilutions was then pipetted onto high-salt mannitol agar plates supplemented with penicillin G (final concentration 10U / mL). The plates were incubated at 30℃ for 48h. The effective viable count was determined by counting plates with a dilution of 20-300 colonies.

[0130] Contamination rate determination: The contamination rate of STM10 fermentation broth was determined using the serial dilution plate spread method. 10.0 mL of the fermentation broth was added to 90 mL of sterile water to prepare a bacterial suspension. After shaking and mixing, a 10-fold serial dilution was performed. Suitable dilutions were spread onto high-salt mannitol agar, PCA agar, and Martin agar supplemented with penicillin G (final concentration 10 U / mL). The high-salt mannitol agar and PCA agar were incubated at 30℃ for 24–48 h, and the viable count of the target strain and the total bacterial count were counted. The Martin agar was incubated at 25℃ for 72 h, and the number of fungi (molds, yeasts, etc.) was counted. A sterile blank control was also included in the experiment. Under the same dilution gradient, the bacterial contamination count was obtained by subtracting the viable count from the total bacterial count. Total contamination count = bacterial contamination count + fungal contamination count. The sample contamination rate is calculated using the formula: Contamination rate (%) = Total number of contaminants / (Number of viable bacteria + Total number of contaminants) × 100%.

[0131] Physicochemical index testing: Observe the appearance of the microbial fertilizer (it should be odorless and loose in texture); take 10g of microbial fertilizer, add 90mL of sterile distilled water, shake for 30min and let stand, and measure the pH of the supernatant with a pH meter (it should be 6.5~7.5).

[0132] III. Test Results After 15 days of fermentation, the viable bacteria count of the prepared STM10 microbial fertilizer reached 2.8 × 10⁻⁶. 8 CFU / g, contamination rate 3.3%, pH 7.2, appearance is a uniform yellowish-brown paste, with high overall viscosity, fine texture, a small amount of natural fermentation bubbles on the surface, no putrid odor, no layering, no large amount of clumps or sediment, and no mold spots. All indicators meet the standards for qualified microbial fertilizer (see Figure 10 This microbial fertilizer can be used directly in field trials or packaged and stored at 4°C (shelf life ≥ 6 months), providing a stable microbial inoculant for crops grown in saline-alkali land.

[0133] Example 7 This experiment describes the effects of microbial fertilizer application on soil and plant improvement.

[0134] I. Experimental Design (a) Test materials Microbial fertilizer and seeds: The microbial fertilizer prepared by solid-state fermentation in Example 6 was used, and the number of effective live bacteria in the microbial agent met the requirements for microbial fertilizer application; the millet seeds were the same as in Example 4.

[0135] (II) Test Treatment The experiment consisted of 3 treatments, with 5 biological replicates per treatment, arranged in a randomized block design: CK: Normal soil control group, local normal plot (pH 8.76), no microbial fertilizer applied; MS: Salt-alkali stress treatment group, local saline-alkali environment plot (pH 9.2), no microbial fertilizer applied; STM10: Salt-alkali stress + microbial fertilizer treatment group, STM10 microbial fertilizer was applied during the flowering period of millet.

[0136] (III) Measurement Indicators Soil parameters: available potassium (AK), available phosphorus (AP), electrical conductivity (EC), ammonium nitrogen (NH4+) + -N), nitrate nitrogen (NO3) - -N), organic carbon (OC), total potassium (TK), total nitrogen (TN), total phosphorus (TP), moisture content (MC), pH, total salt content (TS), catalase (CAT), phosphatase (PHO), sucrase (SUC), β-glucosidase (BG), urease (URE), calcium ions (Ca) 2+ ), magnesium ions (Mg 2 + Sodium ions (Na) + ), potassium ions (K) + ), chloride ions (Cl) - ), carbonate ions (CO3) 2- ), bicarbonate ions (HCO3) - ), sulfate ions (SO4) 2- ).

[0137] Plant indicators: Plant calcium ions (P-Ca) 2+ ), plant sodium ions (P-Na) + Total potassium (P-TK), total phosphorus (P-TP), and total nitrogen (P-TN) in plants.

[0138] II. Experimental Procedure (a) Cultivation and Management Measures The clean water control experiment was conducted on ordinary local plots, while the saline-alkali experiment was conducted on moderately saline-alkali plots. Land preparation, sowing, irrigation, and pest and disease control were standardized to ensure consistent light, water, and cultivation management conditions across all treatments. Millet was planted according to local high-yield cultivation practices, and management remained consistent throughout the growing season.

[0139] (II) Timing and Method of Applying Microbial Fertilizer During the flowering period of millet (a critical period for the transition from vegetative growth to reproductive growth), STM10 microbial fertilizer was applied to the concentrated distribution layer of the millet root system using the rhizosphere trench method, covered with soil and lightly watered to facilitate the colonization of the strains; the CK and MS groups underwent the same agricultural operations at the same time, without the application of microbial fertilizer.

[0140] (III) Sample Collection and Processing Seven days after the application of microbial fertilizer, samples of rhizosphere soil and aboveground plants of millet were collected from each treatment: Soil samples: Remove impurities, air dry, grind, sieve, and store at 4℃ for later use in determining soil physicochemical properties, ion content, and enzyme activity; Plant samples: washed, blanched, dried, and pulverized for the determination of total nitrogen, total phosphorus, total potassium, calcium, sodium, and other nutrients in the plants.

[0141] (iv) Methods for determining nutrient indicators Soil total nitrogen was determined using the Kjeldahl method, followed by distillation titration after digestion with concentrated sulfuric acid and a catalyst. Soil organic carbon was determined using the potassium dichromate external heating capacity method, with organic matter content calculated from organic carbon content. Soil total phosphorus and total potassium were pretreated using the sodium hydroxide alkaline fusion method and determined by the molybdenum antimony colorimetric method and flame photometry, respectively. Soil ammonium nitrogen and nitrate nitrogen were extracted using 2 mol / L potassium chloride and determined by the indophenol blue colorimetric method and ultraviolet spectrophotometry, respectively. Soil available phosphorus was determined by extraction with 0.5 mol / L sodium bicarbonate and the molybdenum antimony colorimetric method, while available potassium was determined by extraction with 1 mol / L ammonium acetate and the flame photometric method. Soil pH was determined using the potentiometry method (water-to-soil ratio 1:2.5), electrical conductivity was determined using the conductivity meter method (water-to-soil ratio 1:5), and total salt content was determined by the mass method of the dried residue from the extract. Soil cation K + Na + Ca was determined by flame photometry or atomic absorption spectrophotometry. 2+ Mg 2+ The anion Cl was determined by EDTA complexometric titration or atomic absorption spectrophotometry. - SO4 was determined by silver nitrate titration. 2- CO3 was determined by indirect EDTA titration or turbidimetric method. 2- With HCO3 -The determination was performed using a dual-indicator neutralization titration method. Soil urease was determined using the urea substrate-phenol-sodium hypochlorite colorimetric method, catalase was determined using the potassium permanganate titration method, sucrase was determined using the 3,5-dinitrosalicylic acid colorimetric method with sucrose as the substrate, β-glucosidase was determined using the p-nitrobenzene-β-D-glucosidase colorimetric method, and phosphatase was determined using the p-nitrobenzene disodium phosphate colorimetric method.

[0142] Plant samples were blanched at 105℃, dried and pulverized at 65℃. Total nitrogen was determined by sulfuric acid-hydrogen peroxide digestion-Kjeldahl method, total phosphorus by sulfuric acid-hydrogen peroxide digestion-molybdenum antimony colorimetric method, total potassium by sulfuric acid-hydrogen peroxide digestion-flame photometry method, and calcium and sodium content by dry ashing or wet digestion followed by atomic absorption spectrophotometry.

[0143] (v) Data Analysis Data were compiled using Excel, and one-way ANOVA was performed using SPSS. Plotting was done using Origin. Tukey's method was used for multiple comparisons, and the significance level was set at P<0.05.

[0144] III. Test Results (I) The effect of STM10 microbial fertilizer on improving soil physical and chemical properties like Figure 11 As shown, compared with the MS group, the available potassium (AK) content in the soil treated with STM10 was reduced but still significantly higher than that in the control group, indicating that the strain alleviated the K content by optimizing the soil ionic environment. + Na + The displacement effect stabilized soil potassium supply. Soil available phosphorus (AP), organic carbon (OC), and total nitrogen (TN) contents were significantly lower than in the MS group and below the CK level. This is closely related to the significant increase in nutrient uptake by millet plants during the flowering period, reflecting that STM10 promoted the translocation and utilization of soil nutrients to plants, rather than increasing nutrient accumulation in the soil. Compared with the MS group, the STM10 treatment significantly reduced soil pH, increased soil moisture content, and alleviated soil alkalization; it also regulated soil electrical conductivity (EC) and total salt content (TS), reducing the toxicity of free salts to roots. Compared with the MS group, the STM10 treatment significantly reduced ammonium nitrogen (NH4+). + The content of nitrate nitrogen (NO3-N) and the increase of nitrate nitrogen (NO3-N) content. - The content of (-N) can alleviate the inhibition of nutrient mineralization and activation by salt and alkali stress, improve the soil nutrient supply capacity, and provide sufficient nutrients for the growth of millet from flowering to grain filling.

[0145] like Figure 12As shown, soil enzyme activity reflects soil microbial activity and nutrient cycling intensity. Compared with the control (CK), the MS treatment significantly increased the activities of soil catalase (CAT), phosphatase (PHO), sucrase (SUC), β-glucosidase (BG), and urease (URE), indicating that salt-alkali stress had a significant stress disturbance on the soil ecosystem and confirming the significant existence of the salt-alkali stress effect. Compared with MS, the STM10 treatment reduced the activities of soil phosphatase (PHO), sucrase (SUC), β-glucosidase (BG), and urease (URE), with only catalase (CAT) activity further increasing. The overall enzyme activity pattern approached the physiological homeostasis under normal conditions, indicating that STM10 effectively alleviated the stress effect of salt-alkali stress on the soil system.

[0146] like Figure 13 As shown, compared with the control (CK), the soil chloride ion concentration (Cl) under MS saline-alkali stress treatment was significantly higher. - ), bicarbonate ions (HCO3) - ), potassium ions (K) + Sodium ions (Na) + ), sulfate ions (SO4) 2- The content of calcium ions (Ca) increased significantly. 2+ ), carbonate ions (CO3) 2- ), magnesium ions (Mg 2+ The content of carbonate ions (CO3-) in the soil was significantly reduced, and the ion balance was severely disrupted; compared with the MS treatment, the STM10 treatment significantly reduced the content of carbonate ions (CO3-) in the soil. 2- ), bicarbonate ions (HCO3) - ), potassium ions (K) + The content of calcium ions (Ca) decreased significantly. 2+ The content of magnesium ions (Mg) has rebounded, while the content of magnesium ions (Mg) has increased. 2+ The content of chloride ions (Cl) increased significantly, although the content of chloride ions (Cl) increased significantly. - ) and sodium ions (Na) + The content of ) remains at a high level, but the soil alkaline anion (CO3) content is high. 2- HCO3 - The significant reduction in ) and beneficial cations (Ca) 2+ Mg 2+ The addition of these substances effectively optimized the soil ion composition, alleviated the damage to the soil ion environment caused by MS-induced salinity stress, and improved the soil physicochemical properties.

[0147] (III) The effect of STM10 microbial fertilizer on improving plant nutrition Salt-alkali stress significantly reduced plant height and stem diameter, while the application of STM10 alleviated the stress, resulting in an increase in both plant height and stem diameter of millet (see...). Figure 14 ).like Figure 15 As shown, compared with the control (CK), salt-alkali stress in MS significantly inhibited the calcium absorption of millet plants. 2+ The absorption of beneficial nutrients such as nitrogen, phosphorus, and potassium is reduced, while the plant's sodium content decreases. + Excessive accumulation leads to a severe imbalance in ion balance and nutrient status; after applying STM10 microbial fertilizer, the plant's Ca2+ levels... 2+ The contents of total nitrogen, total phosphorus, and total potassium were all significantly increased compared to the MS group, while the Na content of the plants was also significantly increased. + The content was significantly reduced, which effectively improved the ion homeostasis and nutritional status of the plants, indicating that STM10 microbial fertilizer can significantly alleviate the inhibitory effect of salt and alkali stress on nutrient absorption of millet plants and enhance the adaptability of plants to salt and alkali environments.

[0148] (iv) Overall effect Under field conditions, applying STM10 salt-alkali tolerant bacterial fertilizer during the flowering period of millet can effectively improve the physical and chemical properties of saline-alkali soil, reduce soil alkalization and harmful ion content, and enhance soil enzyme activity and nutrient supply capacity. At the same time, it promotes the absorption of nitrogen, phosphorus, potassium and calcium by millet, inhibits the accumulation of sodium ions in the plant, improves plant nutrition and ion balance, and significantly alleviates the inhibitory effect of salt-alkali stress on millet growth, showing good soil improvement and plant stress resistance enhancement effects.

[0149] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A strain of *Squirrel-like Mammal Cocci*, characterized in that, The classification of *Squirrel mammal cocci* is named as follows: Mammaliicoccus sciuri It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38450.

2. The squirrel mammal cocci according to claim 1, characterized in that, The squirrel mammal cocci are Gram-positive cocci, and their 16S rRNA gene sequence is shown in SEQ ID NO.

1.

3. The squirrel mammal cocci according to claim 1, characterized in that, The squirrel mammal cocci were isolated from the rhizosphere soil of millet in saline-alkali land and have salt and alkali tolerance.

4. The squirrel mammal cocci according to claim 1, characterized in that, The squirrel mammal cocci also possess nitrogen-fixing, phosphorus-solubilizing, phosphorus-dissolving, siderophore-producing, and protease-producing abilities.

5. A microbial fertilizer, characterized in that, The microbial fertilizer contains live bacteria of *Squirrel Mammal Cocci* as described in claim 1.

6. The microbial fertilizer according to claim 5, characterized in that, The viable count of *Squirrel mammalian cocci* in the microbial fertilizer is not less than 1 × 10⁻⁶. 8 CFU / g.

7. The microbial fertilizer according to claim 5, characterized in that, The fermentation substrate of the microbial fertilizer contains brown sugar and organic cake fertilizer, and the mass ratio of bacterial suspension, brown sugar and organic cake fertilizer is 1:0.3~0.7:4~6.

8. The microbial fertilizer according to claim 5, characterized in that, The microbial fertilizer has a miscellaneous bacteria rate of no more than 5% and a pH of 6.5-7.

5.

9. The application of the microbial fertilizer according to claim 5 in the cultivation of crops in saline-alkali land.

10. The application according to claim 9, characterized in that, The crop is a grain crop, and the microbial fertilizer is applied by rhizosphere trenching during the crop's flowering period.

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