Staphylococcus hominis, complex microbial inoculant and application

Abstract

This invention relates to the field of agricultural technology, specifically disclosing a strain of *Neostachys*, a compound inoculant, and its applications. The *Neostachys* Z3 strain was deposited at the China Center for Type Culture Collection (CCTCC) on September 8, 2025, with accession number CCTCC NO: M 20251967. The *Neostachys* Z3 strain provided by this invention possesses phosphorus-solubilizing, nitrogen-fixing, siderophore-producing, and IAA-producing abilities, and its compound inoculant is used to promote the growth and development of alfalfa.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a strain of Neostachys, a compound microbial agent, and its application. Background Technology

[0002] Alfalfa, known as the "King of Forage," boasts advantages such as high protein content, excellent palatability, and strong regenerative capacity, making it a high-quality forage source for ruminants like dairy cows. With the rapid development of my country's livestock industry, the demand for high-quality alfalfa continues to grow, leading to a continuous expansion of alfalfa planting areas, especially in arid and semi-arid regions like Northwest, North, and Northeast China. Its cultivation is of great significance for ensuring national food security and promoting sustainable agricultural development. However, in actual cultivation, alfalfa still faces challenges such as insufficient soil fertility, low nutrient utilization rates, and strong dependence on chemical fertilizers.

[0003] In recent years, plant rhizosphere growth-promoting bacteria (PGPRs) have become a hot topic in green agriculture research due to their significant effects on promoting plant growth, improving soil microecology, and enhancing stress resistance. Among them, functional microorganisms such as phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and rhizobia have been proven to significantly improve crop yield and quality through mechanisms such as secreting plant hormones, activating soil nutrients, and inducing systemic resistance. Therefore, it is necessary to develop a microbial product that promotes alfalfa growth. Summary of the Invention

[0004] To develop a microbial product that promotes alfalfa growth, this invention provides a strain of *Neostachys*, a compound microbial agent, and its applications. The *Neostachys* strain provided by this invention possesses the abilities to solubilize phosphorus, fix nitrogen, produce siderophores, and produce IAA (inorganic acid), and its compound microbial agent is used to promote the growth and development of alfalfa.

[0005] This invention provides a strain of Neostachys Z3, which was deposited at the China Center for Type Culture Collection on September 8, 2025, with accession number CCTCC NO: M 20251967.

[0006] The new Stakiella Z3 provided by this invention has the ability to solubilize phosphorus, fix nitrogen, produce siderophores and produce IAA, and its compound inoculant is used to promote the growth and development of alfalfa.

[0007] The present invention also provides a Neostachys Z3 bacterial solution, wherein the Neostachys Z3 bacterial solution is composed solely of Neostachys Z3.

[0008] Furthermore, the viable count in the Neostachys Z3 bacterial suspension is 2 × 10⁻⁶. 8 ~2×10 9 CFU / mL.

[0009] The present invention also provides a compound microbial agent, wherein the compound microbial agent comprises the aforementioned Neostachys Z3 and psychrophilic short bacilli ( Brevibacterium frigoritolerans Composition of Hb1;

[0010] The psychrophilic short bacillus Hb1 was deposited at the China Center for Type Culture Collection on September 8, 2025, with accession number CCTCC NO: M 20251968.

[0011] Furthermore, the effective viable count ratio of Neostachys Z3 and psychrophilic short bacillus Hb1 in the compound bacterial agent is 1-2:1-2.

[0012] Furthermore, the effective viable bacteria count in the compound microbial agent is 2 × 10⁻⁶. 8 ~2×10 9 CFU / mL.

[0013] The present invention also provides any of the following applications of the aforementioned Neostachys Z3, the aforementioned Neostachys Z3 bacterial solution, or the aforementioned compound bacterial agent:

[0014] 1) Used for nitrogen fixation;

[0015] 2) Used for phosphorus dissolution;

[0016] 3) Used in the preparation of metal ion chelating agents;

[0017] 4) Used to promote plant growth and development;

[0018] 5) Used in the preparation of plant growth promoters.

[0019] Furthermore, the metal ion is iron ion, and the phosphorus is inorganic phosphorus.

[0020] Furthermore, the application is to promote the growth and development of alfalfa plants.

[0021] Furthermore, the application is to increase the plant height, root length, and fresh weight of alfalfa plants.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The *Neostachys* and *Bacillus psychrophilus* Hb1 provided by this invention both possess phosphorus-solubilizing, nitrogen-fixing, siderophore-producing, and IAA-producing abilities. A compound microbial agent prepared by mixing *Neostachys* and *Bacillus psychrophilus* Hb1 at a 1:1 effective viable count ratio promotes alfalfa growth and development. Inoculation of alfalfa seedlings with this compound microbial agent significantly increases plant height, root length, underground fresh weight, underground dry weight, above-ground fresh weight, and above-ground dry weight. It also helps increase the total nitrogen content of alfalfa.

[0024] Information on the Preservation of Biological Materials

[0025] Z3, referred to in this application as *Neostachys Z3*, was deposited on September 8, 2025, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20251967. The address of the depository is Wuhan University, Wuhan, China, postcode: 430072. The classification name is: Starkeya novella Z3.

[0026] Hb1, referred to in this application as psychrophilic short bacillus Hb1, was deposited on September 8, 2025, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20251968. The address of the depository is Wuhan University, Wuhan, China, postcode: 430072. Its classification is as follows: Brevibacterium frigoritolerans Hb1. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Plates were used to qualitatively determine the nitrogen fixation capacity of strains Z3 and Hb1.

[0029] In the figure, A is the plate for qualitative determination of nitrogen fixation capacity of strain Z3;

[0030] B is a plate for qualitative determination of the nitrogen fixation capacity of strain Hb1.

[0031] Figure 2 Plates for qualitative determination of the phosphate-solubilizing ability of strains Z3 and Hb1;

[0032] In the figure, A is a plate for qualitative determination of the phosphate-solubilizing ability of strain Z3;

[0033] Plate B is for qualitative determination of the phosphate-solubilizing ability of strain Hb1.

[0034] Figure 3 Plate for qualitative determination of the iron-producing capacity of Z3 and Hb1;

[0035] In the figure, A is a plate for qualitative determination of the siderophore production capacity of strain Z3;

[0036] B is a plate for qualitative determination of the siderophore production capacity of strain Hb1.

[0037] Figure 4 Plates for measuring the antagonistic effect between strain Z3 and strain Hb1;

[0038] In the figure, A represents the bottom colony (Z3) and Hb1 represents the plate for measuring the antagonistic effect of co-culture of the test strain.

[0039] B is a plate for determining the antagonistic effect of co-culture of Hb1 (bottom colony) and Z3 (test strain).

[0040] Figure 5 The figure shows the effect of compound microbial agent treatment on the growth and development of alfalfa plants. CK represents the control group, and Z3+Hb1 represents the compound microbial agent treatment group.

[0041] Figure 6 This is the IAA standard curve, which can be used to determine the IAA concentrations of Z3 and Hb1. The horizontal axis corresponding to the red dots represents the specific IAA concentrations of Z3 and Hb1. Detailed Implementation

[0042] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0043] This invention provides a strain of Neostachys, a compound bacterial agent, and its applications.

[0044] The *Neostachys* Z3 was deposited at the China Center for Type Culture Collection (CCTCC) on September 8, 2025, with accession number CCTCC NO: M 20251967. The psychrophilic short bacillus Hb1 was deposited at the China Center for Type Culture Collection (CCTCC) on September 8, 2025, with accession number CCTCC NO: M 20251968.

[0045] The *Neostachys* Z3 and *Bacillus pyrenoidosa* Hb1 provided by this invention both possess phosphorus-solubilizing, nitrogen-fixing, siderophore-producing, and IAA-producing abilities. The compound microbial agent obtained by mixing these two agents promotes alfalfa growth and development. Inoculating alfalfa seedlings with the compound microbial agent significantly increases alfalfa plant height, root length, underground fresh weight, underground dry weight, above-ground fresh weight, and above-ground dry weight. It also helps increase the total nitrogen content of alfalfa.

[0046] Example 1: A strain of Neostachys, a compound bacterial agent and its application.

[0047] I. Isolation, purification and identification of bacterial strains

[0048] 1. Isolation, purification and identification of strain Z3

[0049] Z3 strain of surface-sterilized Amorpha fruticosa ( Amorphafruticosa Separate from L.) root nodules. The specific procedure is as follows:

[0050] Freshly collected *Amorpha fruticosa* root nodule samples were surface-sterilized, then gently crushed and evenly spread on YMA solid medium using sterile forceps, and subsequently incubated at 28°C for 48 h. After incubation, typical single colonies were selected based on colony morphology and isolated and purified using the three-zone streak method. This purification process was repeated three times. Finally, the purified strain underwent 16S rRNA gene sequencing, and sequence alignment was performed using the EZ Biocloud database. The results showed that the strain was *Neostachys*. Starkeyanovella ).

[0051] The YMA medium formula is as follows: mannitol 10.0 g, yeast powder 3.0 g, K2HPO4 0.25 g, KH2PO4 0.25 g, MgSO4•7H2O 0.20 g, NaCl 0.10 g, agar 18 g, add distilled water to make up to 1 L, adjust the pH to 7.0, and autoclave at 121℃ for 30 min.

[0052] 2. Isolation, purification and identification of Hb1 strain

[0053] Hb1 strain of surface-sterilized Lespedeza ( Lespedeza bicolor Isolate from root nodules of Turcz. The specific isolation procedure is the same as that for strain Z3.

[0054] The purified Hb1 strain was identified as a psychrophilic short bacillus by 16S rRNA sequencing and EZ Biocloud comparison. Brevibacterium frigoritolerans ).

[0055] II. Determination of the growth-promoting characteristics of the strain

[0056] 1. Preparation of Z3 and Hb1 bacterial cultures

[0057] Z3 bacterial culture preparation: Z3 strain was inoculated into YMA solid medium and activated at 28℃ for 48 h, then inoculated into TY liquid medium and cultured on a shaker at 180 rpm / min at 28℃ for 48 h to obtain Z3 bacterial culture.

[0058] Preparation of Hb1 bacterial culture: Hb1 strain was inoculated into YMA solid medium and activated at 28℃ for 48 h, then inoculated into TY liquid medium and cultured on a shaker at 180 rpm / min at 28℃ for 48 h to obtain Hb1 bacterial culture.

[0059] 2. Phosphorus solubility test

[0060] 50 μL of bacterial suspension was inoculated onto YMA solid medium and cultured at 28℃ for 48 h. Then, it was transferred to solid phosphate-solubilizing medium by spot inoculation. Four replicates were set up for each plate and cultured at 28℃ for 5 days. The size of the phosphate-solubilizing ring was observed and graded as follows: Grade 0 (no halo), Grade 1 (halo thickness ≤ 2.5 mm), Grade 2 (halo thickness 2.5 mm to 5 mm), and Grade 3 (halo thickness > 5 mm).

[0061] 3. Qualitative determination of iron-producing capacity

[0062] Take 50 μL of bacterial suspension and inoculate it into YMA medium. After incubation at 28℃ for 48 h, spot it onto CAS test plates. Each plate has 4 replicates. Incubate at 28℃ for 5 days. The siderophore secretion capacity is assessed according to the size of the orange-yellow halo. The grading criteria are the same as those for phosphorus solubility determination.

[0063] 4. IAA production capacity determination

[0064] 50 μL of bacterial suspension was inoculated into TY liquid medium and cultured with shaking at 28℃ for 72 h. The culture was then centrifuged at 10000 rpm for 10 min at 4℃. The supernatant was collected and mixed with an equal volume of Salkowski's reagent. The mixture was incubated at room temperature in the dark for 30 min, and the absorbance was measured at 530 nm. Deionized water was used as a blank control instead of the supernatant. The IAA concentration in the samples was calculated using an IAA standard curve ranging from 0 μg / mL to 100 μg / mL.

[0065] 5. Qualitative determination of nitrogen fixation capacity

[0066] Take 50 μL of bacterial suspension and inoculate it onto YMA plates. After incubating at 28°C for 48 h, spot-inoculate it onto nitrogen-fixing medium containing staining agent. Repeat the process 4 times per plate and incubate at 28°C for 5 days. Gradation is performed according to the size of the clear zone, using the same criteria as above.

[0067] The culture medium and reagent formulations used in the above tests are as follows:

[0068] Solid phosphate-solubilizing medium (g / L): glucose 10.0, (NH4)2SO4 0.5, yeast extract 0.5, NaCl 0.3, KCl 0.3, MgSO4•7H2O 0.3, Fe2SO4 0.03, MnSO4 0.03, Ca3(PO4)2 5.0, agar 20.0.

[0069] SM liquid medium (g / L): glucose 5.0, NH4NO3 1.0, KH2PO4 0.5, Na2HPO4 1.5, NaCl 1.0, MgSO4•7H2O 0.2, pH 7.2.

[0070] CAS detection culture medium:

[0071] Solution A: 0.012 g chrome azurol sulphonate (CAS) + 10 mL deionized water + 2 mL 1 mmol / L FeCl3;

[0072] Solution B: 0.015 g hexadecy-trimethyl-ammonium bromide (HDTMA) + 8 mL deionized water;

[0073] Dye solution C: Slowly add solution A to solution B, mix well, and sterilize at 121°C for 20 min;

[0074] Culture medium D: 20 mL MM9 salt solution + 6.04 g anhydrous piperazine (dissolved in 150 mL water, pH adjusted to 6.8) + 3.2 g agar, sterilized at 121℃ for 20 min;

[0075] When using the medium, add 0.2 mL of 1 mmol / L CaCl2, 4 mL of 1 mmol / L MgSO4•7H2O, 2 mL of 20% glucose, 6 mL of 10% acid-hydrolyzed casein, and the above-mentioned staining solution C to medium D, mix well to obtain the CAS detection medium.

[0076] Salkowski's reagent: 2 mL 0.5 mol / L FeCl3•6H2O + 98 mL 35% HClO4.

[0077] TY liquid medium (g / L): tryptone 5.0, yeast extract 3.0, CaCl2•6H2O 0.7.

[0078] MM9 salt solution (g / 500 mL): KH2PO4 15.0, NaCl 2.5, NH4Cl 5.0, Na2HPO4 30.0.

[0079] Nitrogen-fixing medium (g / L): mannitol 10.0, KH2PO4 0.2, MgSO4•7H2O 0.2, NaCl 0.2, CaSO4•7H2O 0.1, CaCO3 5.0, agar 18, plus 10 mL of 0.5% Congo red solution.

[0080] 6. Test Results

[0081] The results are as follows Figure 1 As shown, the nitrogen fixation capacity of strain Z3 is graded as level 1, and that of strain Hb1 is graded as level 2.

[0082] The results are as follows Figure 2As shown, the phosphorus solubility of strain Z3 is graded as level 2, and the phosphorus solubility of strain Hb1 is graded as level 2.

[0083] The results are as follows Figure 3 As shown, the siderophore production capacity of strain Z3 is graded as level 2, and that of strain Hb1 is graded as level 3.

[0084] The results are as follows Figure 6 As shown, the IAA concentration of Z3 was 2.060 mg / L, and the IAA concentration of Hb1 was 9.097 mg / L.

[0085] III. Preparation and Application of Compound Microbial Agents

[0086] 1. Determination of antagonistic effects between strain Z3 and strain Hb1

[0087] (1) Spread the Z3 bacterial suspension evenly on YMA solid medium plates and incubate at 28℃ for 48 h. Take a colony of Hb1 strain (the test strain) about the size of a soybean and spot it onto a YMA plate pre-spread with Z3 strain, and continue to incubate at 28℃ for 6 days. After incubation, observe the growth status of the test strain and whether an inhibition zone is formed. If the growth of the test strain is inhibited and a clear inhibition zone appears between it and the bottom colony, it is determined that there is an antagonistic effect between the two strains. If no inhibition zone is formed, it indicates that there is no antagonistic effect between the two.

[0088] (2) The Hb1 bacterial suspension was evenly spread on YMA solid medium plates and cultured at 28℃ for 48 h. Subsequently, a piece of Z3 strain (the test strain) about the size of a soybean was taken and inoculated onto a YMA plate pre-spread with Hb1 strain, and cultured at a constant temperature of 28℃ for another 6 days. After the culture was completed, the growth status of the test strain and whether an inhibition zone was formed were observed. If the growth of the test strain was inhibited and a clear inhibition zone appeared between it and the bottom colony, it was determined that there was an antagonistic effect between the two strains. If no inhibition zone was formed, it was indicated that there was no antagonistic effect between the two.

[0089] The results are as follows Figure 4 As shown, no antagonistic effect was observed between the Z3 strain and the Hb1 strain when they were used as test strains.

[0090] 2. Preparation of compound microbial agents

[0091] Based on the growth-promoting properties and lack of antagonism between strains Z3 and Hb1, a compound microbial agent with growth-promoting and yield-increasing functions for alfalfa was prepared, specifically according to the following steps:

[0092] Z3 and Hb1 strains were inoculated into YMA solid medium and activated at 28℃ for 3 days. The activated strains were then inoculated into TY liquid medium and cultured at 28℃ with shaking at 180 rpm / min until the logarithmic growth phase, yielding Z3 and Hb1 culture media. Both Z3 and Hb1 culture media were centrifuged at 10,000 rpm for 15 min at 4℃, the supernatant was removed, and the bacterial cells were collected. The cells were washed twice with sterile distilled water by centrifugation, then resuspended in sterile distilled water, and the OD value was adjusted to 0.8 at 600 nm to obtain Z3 and Hb1 bacterial suspensions. 600 The suspensions of the two strains were mixed evenly in equal volume ratios to obtain a compound bacterial agent (the total effective viable bacteria count of the compound bacterial agent was approximately 2 × 10⁻⁶). 9 (CFU / mL).

[0093] The YMA solid culture medium contained 10.0 g mannitol, 3.0 g yeast extract, 0.25 g K2HPO4, 0.25 g KH2PO4, 0.20 g MgSO4•7H2O, 0.10 g NaCl, 1 L distilled water, and 18 g agar. After adjusting the pH to 7.0, it was sterilized at 121℃ for 30 min.

[0094] The above-mentioned TY liquid culture medium contains 5.0 g of tryptone, 3.0 g of yeast extract, 0.70 g of CaCl2•6H2O, 1 L of distilled water, and is sterilized at 121℃ for 30 min after the pH is adjusted to 7.0.

[0095] IV. Effects of Compound Microbial Agents on Alfalfa Growth and Development

[0096] 1. Plant materials

[0097] The alfalfa variety used in this experiment was WL319HQ, purchased from Beijing Zhengdao Seed Industry Co., Ltd. This variety possesses characteristics such as strong cold resistance, numerous branches, high yield, excellent quality, abundant leaves, and a long service life. Seed disinfection involved treatment with 75% ethanol for 30 seconds, followed by soaking in 5% HClO4 solution for 5 minutes, rinsing three times with sterile water, and then pre-treatment for germination on water agar plates.

[0098] 2. Test treatment

[0099] The experiment was conducted in March 2025 in the artificial climate chamber of the College of Grassland Science and Technology, Northwest A&F University. The cultivation substrate was a mixture of perlite and vermiculite at a mass ratio of 3:2. After being dispensed into cultivation bags, 300 mL of Hoagland's nutrient solution was added, and the mixture was sterilized at 121℃ for 20 min before use.

[0100] Hoagland's nutrient solution formula is as follows:

[0101] Macroelements (mg / L): Ca(NO3)2•4H2O 1417, KNO3 607, MgSO4 493, (NH4)3PO4 115; Microelements (mg / L): H3BO3 2.86, MnCl2•4H2O 1.81, ZnSO4•7H2O 0.22, CuSO4•5H2O 0.08, H2MnO4•H2O 0.02, FeSO4 50.

[0102] Hoagland's nutrient solution is prepared with sterile deionized water and the pH is adjusted to 7.0 ± 0.2 using a 1 mg / L NaOH or HCl solution.

[0103] Two pre-germinated alfalfa seeds were sown in each bag on the surface of the substrate, with ventilation holes left when sealing. The experiment was repeated 10 times, with a control group not inoculated with the inoculant. After the cotyledons of the seedlings were fully expanded, 2 mL of a compound inoculant was inoculated into the roots of the alfalfa using a sterile disposable syringe. In the third week of growth, an additional 2 mL of the same concentration of the compound inoculant was inoculated into the roots of the alfalfa using a sterile disposable syringe, along with 200 mL of Hoagland's nutrient solution. After 5 weeks of cultivation, plant height, root length, and total nitrogen content were measured.

[0104] 3. Test Results

[0105] Table 1. Effects of inoculation with compound microbial agents on the growth of alfalfa plants.

[0106]

[0107] Note: Different letters represent significant differences in growth indicators under different inoculation treatments (P<0.05).

[0108] Table 2. Effects of inoculation with compound microbial agents on total nitrogen content in alfalfa.

[0109]

[0110] Note: Different letters represent significant differences in total nitrogen content under different inoculation treatments (P<0.05).

[0111] As shown in Table 1, the plant height, root length, underground fresh weight, underground dry weight, above-ground fresh weight, and above-ground dry weight of alfalfa in the aseptic treatment group (CK) were 6.10 cm, 6.53 cm, 0.0133 g / plant, 0.0063 g / plant, 0.036 g / plant, and 0.0180 g / plant, respectively. The height of alfalfa plants in the Z3+Hb1 treatment group was 11.83 cm, a significant increase of 93.93% (p<0.05); the root length was 10.70 cm, a significant increase of 63.86% (p<0.05); the underground fresh weight was 0.0233 g / plant, an increase of 75.19%; the underground dry weight was 0.0150 g / plant, a significant increase of 138.10% (p<0.05); the aboveground fresh weight was 0.210 g / plant, a significant increase of 483.33% (p<0.05); and the aboveground dry weight was 0.0466 g / plant, a significant increase of 158.89% (p<0.05).

[0112] As shown in Table 2, the total nitrogen content of alfalfa in the aseptic treatment group (CK) was 9.39 g / kg, while that in the compound microbial agent treatment group was 10.00 g / kg, an increase of 6.50%.

[0113] It is evident that the compound microbial agent of the present invention has a significant promoting effect on alfalfa plant height, root length, underground fresh weight, underground dry weight, above-ground fresh weight, and above-ground dry weight, while also increasing the total nitrogen content of alfalfa.

[0114] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0115] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Neostachys ( Starkeya novella Z3, characterized in that, The novel staghorn strain Z3 was deposited at the China Center for Type Culture Collection on September 8, 2025, with accession number CCTCC NO: M 20251967.

2. A Neostachys Z3 bacterial suspension, characterized in that, The Neostachys Z3 bacterial solution uses Neostachys Z3 as the sole active ingredient as described in claim 1.

3. The Neostachys Z3 bacterial suspension according to claim 2, characterized in that, The viable count of the Neostachys Z3 bacterial culture was 2 × 10⁻⁶. 8 ~2×10 9 CFU / mL.

4. A compound microbial agent, characterized in that, The compound bacterial agent comprises Neostachys Z3 as described in claim 1 and psychrogenic short bacilli (… Brevibacterium frigoritolerans Composition of Hb1; The psychrophilic short bacillus Hb1 was deposited at the China Center for Type Culture Collection on September 8, 2025, with accession number CCTCC NO: M 20251968.

5. The compound microbial agent according to claim 4, characterized in that, The effective viable count ratio of Neostachys Z3 and psychrophilic short bacillus Hb1 in the compound bacterial agent is 1-2:1-2.

6. The compound microbial agent according to claim 4, characterized in that, The effective viable bacteria count in the compound microbial agent is 2 × 10⁻⁶. 8 ~2×10 9 CFU / mL.

7. Any of the following applications of the *Neostachys Z3* strain of claim 1, the *Neostachys Z3* bacterial suspension of any one of claims 2-3, or the compound bacterial agent of any one of claims 4-6: 1) Used for nitrogen fixation; 2) Used for phosphorus dissolution; 3) Used in the preparation of metal ion chelating agents; 4) Used to promote the growth and development of alfalfa; 5) Used in the preparation of alfalfa growth promoters; The metal ion is iron ion, and the phosphorus is inorganic phosphorus.

8. The application according to claim 7, characterized in that, The promotion of alfalfa growth and development is achieved by increasing the plant height, root length, and fresh weight of alfalfa plants.

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