A compound microbial agent containing Bacillus halophilus HMF09 and its application

Abstract

This invention relates to a compound microbial agent comprising *Bacillus halophilus* HMF09 and *Bacillus subtilis*, wherein the preservation number of *Bacillus halophilus* HMF09 is CGMCC No. 30475. This compound microbial agent can effectively reduce soil pH and salinity, improve crop emergence rate and salt tolerance, and increase crop yield. Furthermore, it can degrade the insecticide carbaryl residue, which is significant for the development of green agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound microbial agent containing Bacillus halophilus HMF09 and its application. Background Technology

[0002] Saline-alkali soil is a general term for both saline and alkaline soils, a type of obstacle soil widely distributed throughout the world. Based on the severity of salinization, it is generally classified as mild, moderate, or severe. Due to its unfavorable properties, saline-alkali soil is detrimental to normal crop growth, and a large amount of saline-alkali soil cannot be effectively utilized. How to utilize this portion of saline-alkali soil is an urgent problem to be solved.

[0003] Methods for improving saline-alkali soil can be categorized into water conservancy, agricultural, biological, and chemical measures. Flood irrigation for salt leaching is the most common method, but it requires a large amount of water, making it unsuitable for arid regions. Agricultural measures often involve deep plowing, applying organic fertilizer, and returning straw to the field, which can increase soil organic matter content. Chemical measures primarily use amendments such as phosphate, gypsum, and wood vinegar to improve soil quality, but their use can cause secondary pollution. Biological measures include increasing vegetation cover to improve the local microclimate and reduce surface salt accumulation, such as by using salt-tolerant crops, and employing microbial methods for improvement.

[0004] Rhizosphere microorganisms have been shown to promote plant growth in various saline-alkali soil environments. Therefore, the screening of salt-tolerant and growth-promoting strains and their application in improving saline-alkali soils are of great importance.

[0005] Cutworms and grubs are deadly threats to corn seedlings, biting off stems and gnawing at roots, leading to large-scale seedling loss. In particular, clay soils have high water retention and humidity, which is conducive to cutworm larvae activity; similarly, grubs prefer moist, heavy soil environments. Therefore, the risk of cutworms and grubs is higher when planting corn in clay soils. Carbaryl, also known as carbaryl chloride, is a broad-spectrum carbamate insecticide. Carbaryl (such as 5% carbaryl powder) can be used to control cutworms, grubs, and other underground pests. For example, mixing 1.5–2.5 kg of 5% carbaryl powder with 15–25 kg of fine soil per acre and incorporating it into the soil before sowing can effectively suppress the activity of grubs and cutworm larvae.

[0006] Sevin belongs to the carbamate pesticide class. Although it degrades more readily than organochlorine pesticides, its residual time is greatly influenced by environmental conditions. Studies have shown that its bound residue can persist for several months in clay soils, while complete mineralization requires even longer. With the increasingly widespread use of sevin, its toxic effects are becoming increasingly apparent, particularly its recent discovery of endocrine disruption, classifying it as an endocrine disruptor (EDS) that can damage the reproductive system and affect human health. Domestic research on sevin degradation is relatively limited. Degradation methods mainly include physical, chemical, and biological approaches. Due to the potential dangers of physical and chemical methods, finding safe and effective biodegradation pathways is essential. Summary of the Invention

[0007] The purpose of this invention is to provide a compound microbial inoculant that can effectively reduce soil pH, decrease soil salinity, improve crop emergence rate and salt-alkali resistance, and increase crop yield. It also provides a microbial inoculant that can degrade carbaryl.

[0008] The present invention adopts the following technical solution:

[0009] A compound microbial agent comprising Bacillus halophilus HMF09 and Bacillus subtilis.

[0010] Furthermore, the halophilic Bacillus ( Halobacillus sp. HMF09 has the accession number CGMCCNo.30475 and is deposited at the China General Microbiological Culture Collection Center on April 30, 2024.

[0011] Furthermore, the Bacillus subtilis was purchased from Tianjin Kunhe Biotechnology Co., Ltd., with an effective viable count of 100 billion / g and product number MES814.

[0012] Furthermore, the ratio of viable Bacillus halophilus HMF09 to Bacillus subtilis is 1 to 10:2.

[0013] Furthermore, the total viable count in the compound microbial agent is not less than 3 × 10⁻⁶. 10 cfu / g.

[0014] A method for preparing the above-mentioned composite microbial agent includes the following steps:

[0015] (a) Activation of HMF09 strain;

[0016] (b) Preparation of HMF09 seed culture;

[0017] (c) Preparation of HMF09 fermentation broth;

[0018] (d) The HMF09 fermentation broth and soluble starch were mixed evenly at a mass ratio of 5–15:1, and then sprayed using a freeze dryer to obtain HMF09 halophilic Bacillus powder with an effective viable count of not less than 3.0 × 10⁻⁶ cells / year. 10 cfu / g;

[0019] (e) Mix the prepared halophilic Bacillus HMF09 powder with Bacillus subtilis powder in a certain proportion.

[0020] In the preparation method, LB liquid culture medium with a pH of 7.2 to 7.4 is used in steps (a) to (c), and the medium is cultured at a constant temperature of 160 rpm and 35°C with shaking.

[0021] The present invention also provides an application of the above-mentioned composite microbial agent in improving the properties of saline-alkali soil.

[0022] Application of the above-mentioned compound microbial agent in promoting crop growth.

[0023] Application of the above-mentioned compound microbial agent in degrading the residue of the insecticide carbaryl.

[0024] The above-mentioned compound microbial agent is used by seed dressing, or by spraying the agent onto the soil surface before planting and then turning it into the soil through tillage.

[0025] The beneficial effects of this invention are as follows: The compound microbial agent of this invention combines Bacillus halophilus HMF09 and Bacillus subtilis. These two bacteria not only grow synergistically but also work together to lower soil pH, reduce soil salinity, improve crop emergence rate and salt tolerance, and increase crop yield. Furthermore, this compound microbial agent can also degrade the residue of the insecticide carbaryl, which is significant for the development of green agriculture. Attached Figure Description

[0026] Figure 1 The colony morphology of halophilic Bacillus HMF09 after 24 hours of culture on solid LB medium.

[0027] Figure 2 The image shows the morphology of Bacillus halophilus HMF09 under an optical microscope after Gram staining.

[0028] Figure 3 Phylogenetic tree of 16S rDNA of Halophilic Bacillus HMF09.

[0029] Figure 4 The results are from the synergistic experiment between strain HMF09 and Bacillus subtilis. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the reagents used in the following embodiments are all available from conventional biochemical reagent stores.

[0031] Example 1: Screening and identification of Halophilic Bacillus HMF09

[0032] The halophilic Bacillus HMF09 was isolated and purified by the applicant from a soil sample collected from a wheat experimental field (saline-alkali soil) in Emin County, Tacheng Prefecture, Xinjiang Uygur Autonomous Region, China.

[0033] (1) Morphological characteristics

[0034] The colonies are round, orange-yellow, with smooth, even edges and no bumps. Figure 1 As shown. The bacteria are rod-shaped, have spores, and are Gram-positive, as indicated. Figure 2 As shown.

[0035] (2) Physiological and biochemical characteristics

[0036] The physiological and biochemical characteristics of strain HMF09 were identified, and the results are shown in Table 1.

[0037] Table 1. Physiological and biochemical characteristics of strain HMF09

[0038] .

[0039] (3) Molecular biological characteristics

[0040] Genomic DNA was extracted from strain HMF09. Using it as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA. The amplified product was recovered and sequenced, yielding a DNA sequence of 1500 bp (as shown in SEQ ID No. 1). The sequencing results were entered into the GeneBank database for BLAST alignment analysis. Comparison with the 16S rDNA sequence in the NCBI database revealed that HMF09 shares 99% identity with *Bacillus halophilicus*. The phylogenetic tree was constructed as follows: Figure 3 As shown. Based on the morphological, sequencing analysis, and physiological and biochemical detection results, HMF09 was identified as a halophilic Bacillus (Bacillus). Halobacillus sp. ).

[0041] Halophilic Bacillus HMF09 was deposited on April 30, 2024, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 30475.

[0042] Example 2: Synergistic test between Bacillus halophilus HMF09 and Bacillus subtilis MES814.

[0043] LB solid medium formula: 5g yeast extract, 10g peptone, 10g sodium chloride, and 18g agar are placed in a 1000mL beaker, 900mL distilled water is added and heated to dissolve, the pH is adjusted to 7.2~7.4, and the volume is brought to 1L with distilled water. The mixture is then sterilized at 121℃ for 30 minutes and set aside for later use.

[0044] The strains to be tested were: Halophilic Bacillus HMF09 and Bacillus subtilis. Among them, Bacillus subtilis powder (purchased from Tianjin Kunhe Biotechnology Co., Ltd., product number: MES814, effective viable count 100 billion / g).

[0045] Bacillus subtilis bacterial powder was isolated and purified using LB medium to obtain Bacillus subtilis strain MES814, which was stored at -4℃ for later use. Halophilic Bacillus HMF09 was then activated using LB medium.

[0046] A colony of *Bacillus subtilis* was picked up using an inoculation loop and placed in 1 mL of sterile water. The mixture was repeatedly pipetted and stirred to prepare a bacterial suspension. This suspension was then added to LB medium that had not yet solidified but was not too hot to handle. After shaking well, the suspension was immediately poured into plates. A *Bacillus halophilicus* HMF09 bacterial cake was placed in the center of the cooled and solidified plate, and labeled. The process was repeated three times. The plates were incubated at 37 °C for 3 days. The results are as follows: *Bacillus subtilis* and *Bacillus halophilicus* HMF09 grew together, and no inhibition zone was formed, indicating that *Bacillus subtilis* and *Bacillus halophilicus* HMF09 do not inhibit each other. See the results below. Figure 4 .

[0047] Example 3: Degradation test of carbaryl by Bacillus halophilus HMF09 and Bacillus subtilis MES814

[0048] LB liquid medium: Place 5g yeast extract, 10g peptone, and 10g sodium chloride in a 1000mL beaker, add 900mL distilled water and heat to dissolve. Adjust the pH to 7.2-7.4, bring the volume to 1L with distilled water, and sterilize at 121℃ for 30 minutes.

[0049] Inorganic salt culture medium formula: K2HPO4 0.1g, (NH4)2HPO4 0.1g, MgSO4·7H2O 0.02g, FeCl3 0.01g, CaCl2·2H2O 0.1g, NaCl2 0.1g, distilled water 1000mL, pH 7.0, sterilized at 121℃ for 20 min.

[0050] Sevin (purity: 98%, product of Hubei Hengjingrui Chemical Co., Ltd.), purchased by the applicant from the market.

[0051] Preparation method of culture medium containing carbaryl: Sterilize 10 g / L carbaryl methanol solution by passing it through a 0.22 μm filter membrane, take a certain amount and place it in a sterile Erlenmeyer flask. After the methanol has completely evaporated, add sterile inorganic salt culture medium to make the final concentration of carbaryl 50 mg / L.

[0052] One loopful was taken from the activated Bacillus subtilis MES814 culture medium and the halophilic Bacillus HMF09 culture medium in Example 2 and inoculated into 50 mL of LB liquid medium. After incubation at 35°C and 160 rpm for 48 h on a shaker, a bacterial suspension was prepared, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 cfu / mL, for later use.

[0053] This experiment included the following 6 treatments:

[0054] 1 mL of halophilic Bacillus HMF09 suspension + 50 mL of inorganic salt culture medium + 2.5 mg of carbaryl;

[0055] 1 mL of Halophilic Bacillus HMF09 bacterial suspension + 50 mL of inorganic salt culture medium;

[0056] 1 mL of Bacillus subtilis MES814 bacterial suspension + 50 mL of inorganic salt culture medium + 2.5 mg of carbaryl;

[0057] 1 mL of Bacillus subtilis MES814 bacterial suspension + 50 mL of inorganic salt culture medium;

[0058] 0.5 mL of Bacillus halophilus HMF09 suspension + 0.5 mL of Bacillus subtilis MES814 suspension + 50 mL of inorganic salt culture medium + 2.5 mg of carbaryl;

[0059] 0.5 mL of Bacillus halophilus HMF09 suspension + 0.5 mL of Bacillus subtilis MES814 suspension + 50 mL of inorganic salt culture medium.

[0060] Under aseptic conditions, each treatment was repeated three times, and cultured at 35°C and 160 rpm for 48 hours on a shaker to obtain the first-generation bacterial culture. Following the same procedure, using 1 mL of the first-generation culture, the second-generation bacterial culture was prepared sequentially using the same design of six treatments, and then the third-generation bacterial culture was prepared. If the strain in the third-generation culture could grow and the OD... 600 A value greater than or equal to the blank control indicates that the strain can be determined to have the function of degrading carbaryl using carbaryl as the sole carbon source; if the strain fails to grow in the third generation of bacterial culture, it is determined that the strain does not have the function of degrading carbaryl.

[0061] Table 2. OD of the first generation strain600 Value statistics results

[0062] .

[0063] Table 3. Reproduction OD of the second-generation strain 600 Value statistics results

[0064] .

[0065] Table 4. Reproduction OD of the third-generation strain 600 Value statistics results

[0066] .

[0067] The results showed that the combination of halophilic Bacillus HMF09 and Bacillus subtilis MES814 could effectively degrade carbaryl, and the degradation effect was better than that of a single strain.

[0068] Example 4: Preparation of Bacillus halophilicus HMF09 bacterial powder

[0069] (1) LB liquid culture medium: 5g yeast extract, 10g peptone and 10g sodium chloride are placed in a 1000mL beaker, 900mL distilled water is added and heated to dissolve, pH is adjusted to 7.2-7.4, and the volume is adjusted to 1L with distilled water. Sterilize at 121℃ for 30 minutes and set aside.

[0070] (2) Activation of strain: Pick one loop of halophilic Bacillus HMF09 colony, inoculate it into a test tube containing 10 mL LB liquid medium, and activate it by constant temperature shaking at 160 rpm and 35 ℃ for 24 h.

[0071] (3) Preparation of seed culture: Take 5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid culture medium. Incubate at 160 rpm and 35 °C for 24 h to obtain seed culture.

[0072] (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.6 L of LB liquid medium and cultured at 160 rpm and 35 °C for 48 h with constant temperature shaking to obtain the fermentation broth of Bacillus halophilus HMF09, with an effective viable count of 3.31 × 10⁻⁶ cells / mL. 9 cfu / mL.

[0073] (5) Preparation of microbial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed by freeze dryer to obtain Bacillus halophilicus HMF09 powder. The effective viable count was tested to be 3.15 × 10⁻⁶. 10 cfu / g.

[0074] Example 5 Preparation of compound microbial inoculant

[0075] The *Bacillus halophilus* HMF09 bacterial powder prepared in Example 4 was mixed with *Bacillus subtilis* inoculum (purchased from Tianjin Kunhe Biotechnology Co., Ltd., product number: MES814, effective viable count 100 billion / g) at viable count ratios of 1:2, 5:2, and 5:1, respectively, to obtain compound microbial inoculum 1, compound microbial inoculum 2, and compound microbial inoculum 3. Testing showed that the total viable count of compound microbial inoculum 1 was 60.19 billion / g, compound microbial inoculum 2 was 39.57 billion / g, and compound microbial inoculum 3 was 35.75 billion / g.

[0076] Examples 6-8: Pot experiments of seed dressing with three compound microbial agents under sodium chloride stress

[0077] 1. Test materials

[0078] Wheat variety: Cangmai 6002.

[0079] The original soil physicochemical indicators were as follows: organic matter 8.76 g / kg, available nitrogen 30.96 mg / kg, available phosphorus 15.98 mg / kg, available potassium 34.28 mg / kg, salt content 0.22 g / kg, and pH 6.58.

[0080] Flower pot: 10cm in diameter.

[0081] 2. Test methods

[0082] (1) Preparation of test soil: Local soil was selected as the test subject. This soil has low organic matter content and is prone to compaction after watering. It was thoroughly watered with 1.2% NaCl and 1% Na2CO3 solution and then dried to constant weight. The soil was sieved through a 2mm sieve and used as the test soil. The pH of the soil was measured to be 7.82 by potentiometric method and the salt content of soluble salts in the soil was measured to be 6.33 g / kg of dried soil by drying oven method.

[0083] (2) Pretreatment: Transfer the prepared saline-alkali soil into flower pots with a diameter of 10 cm, 350 g per pot; select wheat seeds with plump grains and uniform size, soak them in 50℃ warm water for 30 minutes for disinfection, and set aside.

[0084] (3) Experimental design: The experiment set up 6 treatments, with 8 pots for each treatment and 10 wheat seeds sown in each pot. Among them, treatment 1 was treated with compound microbial agent 1, treatment 2 was treated with compound microbial agent 2, treatment 3 was treated with compound microbial agent 3, treatment 4 was not treated with microbial agents and served as a blank control (CK), treatment 5 was treated with a single halophilic Bacillus HMF09 agent, and treatment 6 was treated with a single Bacillus subtilis agent. The dosage of each treatment was 10g / kg of seeds. After sowing, all wheat seeds were transferred to a light incubator for further cultivation.

[0085] (4) Detection indicators: After 30 days of cultivation, the growth indicators (plant height, longest root length, number of roots) of wheat seedlings in the blank control group, single bacterial agent treatment group and compound microbial treatment group under salt and alkali stress were detected. Four soil samples were randomly collected from each treatment, mixed and then 200 g were taken by quartering and dried. The pH of the dried soil and the content of soluble salts in the soil were detected.

[0086] 3. Results and Analysis

[0087] As shown in Table 5, the compound microbial agents 1-3 prepared in Example 5 can significantly reduce the soluble salt content of the soil, lower the soil pH, and alleviate the inhibitory effect of salt-alkali stress on wheat growth. The effects of compound microbial agents 1-3 are significantly better than those of single agents Bacillus subtilis and Bacillus halophilus HMF09, and HMF09 and Bacillus subtilis MES814 have a synergistic effect.

[0088] Table 5 Statistical Results of Each Treatment Survey Indicator

[0089] .

[0090] Examples 9-11: Field trials of compound microbial agents for improving saline-alkali land

[0091] Experimental Site Overview: The experimental site is located in Huanghua City. It is a dryland saline-alkali wheat field where the applicant is experimenting with planting wheat, and the next crop will be corn after the wheat is harvested. The soil texture of this site is loam. The soil sample taken before planting showed the following data: pH 7.62, available nitrogen 114.1 mg / kg, available phosphorus 30.18 mg / kg, available potassium 86 mg / kg, organic matter 9.97 g / kg, and salt content 2.93 g / kg, classifying it as moderately saline-alkali soil.

[0092] Experimental Design: The experiment consisted of 6 treatments, each covering 0.5 mu (approximately 0.067 hectares), with 3 replicates and randomized arrangements. One day before planting, treatments 1-3 received 250g each of the three compound microbial agents prepared in Example 5 per mu; treatment 4 received 250g of a single Bacillus halophilus HMF09 agent per mu; treatment 5 received 250g of a single Bacillus subtilis agent per mu; and treatment 6 was a blank control (CK) with water instead of compound microbial agents. All agents were diluted 150 times and sprayed evenly on the soil surface. Immediately after spraying, tillage machinery was used to incorporate the diluted agent solution into the soil.

[0093] Experimental site management: Land preparation was carried out on June 10, 2023. 40 kg of compound fertilizer (N:P2O5:K2O=28:6:7) and 160 kg of organic fertilizer were applied per mu as base fertilizer, and sowing was carried out the following day. Planting depth: 5-8 cm; planting density: 5000 plants / mu; plant spacing: 30 cm; row spacing: 33 cm; planted variety: Weike 985. Other production management measures were carried out as usual.

[0094] Survey methods: The emergence and plant height of maize in each treatment plot were investigated 20 days after sowing; at maturity, the yield of each treatment plot was measured.

[0095] Table 6. Statistical table of corn emergence rate, plant height and yield for each treatment

[0096] .

[0097] Table 6 shows that the effects of compound microbial agents 1-3 are significantly better than those of single agents Bacillus subtilis and Bacillus halophilus HMF09. HMF09 and Bacillus subtilis MES814 have a synergistic effect. Compound microbial agents 1-3 prepared in Example 5 can all alleviate the inhibition of maize growth by salt-alkali stress, improve maize emergence rate, and increase maize yield.

[0098] Examples 12-14: Field trials of compound microbial inoculants degrading carbaryl.

[0099] Experimental site: A cornfield in Ruhe Town, Changli County, Qinhuangdao City (soil is heavy clay soil). 3 kg of 5% carbaryl granules produced by Anhui Ruite Agricultural Chemical Technology Co., Ltd. were used per mu in this field. Before sowing, the granules were mixed with fine soil at a ratio of 1:10 and evenly spread on the surface, then plowed into the soil (to a depth of 10-15 cm) to control underground pests.

[0100] Experimental design: The experiment consisted of 6 treatments, each lasting 20m. 2 The treatment was repeated three times, with each treatment arranged randomly.

[0101] Treatments 1-3: Seeds were treated with the three compound microbial agents prepared in Example 5;

[0102] Treatment 4: Seed dressing with a single halophilic Bacillus HMF09 inoculum;

[0103] Treatment 5: Seed dressing with a single Bacillus subtilis MES814 inoculum;

[0104] Treatment 6: No inoculant was used for seed treatment; this was the blank control (CK).

[0105] The dosage of inoculant for each treatment was 10g / kg of seeds.

[0106] Experimental site management: Land preparation was carried out on April 25, 2024. 50 kg of compound fertilizer (N:P2O5:K2O=17:17:17) and 80 kg of organic fertilizer were applied per mu as base fertilizer, and sowing was carried out the following day. Planting depth: 5-8 cm; planting density: 4000 plants / mu; plant spacing: 30 cm; row spacing: 35 cm; planted variety: Keyu 520. Other production management measures were carried out as usual.

[0107] Methods: Rhizosphere soil samples were collected 50 days after maize sowing using a 5-point sampling method. Soil samples from different treatments and replicates were mixed thoroughly. 10g of each mixed sample was added to 100mL of methanol and mixed for 2 hours in a shaker. After centrifugation, the supernatant was dried under nitrogen, dissolved in chromatographically pure methanol, and filtered through a 0.45μm filter. The carbaryl content in the soil samples from different treatments (average of three replicates for each treatment) was determined using high-performance liquid chromatography (HPLC).

[0108] Table 7 Statistical Tables for Each Treatment

[0109] .

[0110] Table 7 shows that the combined microbial agents 1-3 are significantly more effective than the single agents Bacillus subtilis and Bacillus halophilus HMF09 in degrading carbaryl. HMF09 and Bacillus subtilis exhibit a synergistic effect. All three combined microbial agents 1-3 prepared in Example 5 can degrade carbaryl residues in the soil.

[0111] The above description is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A compound microbial agent, characterized in that, It includes halophilic Bacillus HMF09 and Bacillus subtilis; the preservation number of halophilic Bacillus HMF09 is CGMCC No. 30475; the ratio of viable bacteria of halophilic Bacillus HMF09 to Bacillus subtilis is 1~10:

2.

2. The compound microbial agent according to claim 1, characterized in that, Its total viable count is not less than 3×10 10 cfu / g.

3. A method for preparing a composite microbial agent as described in any one of claims 1 or 2, characterized in that, It includes the following steps: (a) Activation of HMF09 strain; (b) Preparation of HMF09 seed culture; (c) Preparation of HMF09 fermentation broth; (d) The HMF09 fermentation broth and soluble starch were mixed evenly at a mass ratio of 5–15:1, and then sprayed using a freeze dryer to obtain HMF09 halophilic Bacillus powder with an effective viable count of not less than 3.0 × 10⁻⁶ cells / year. 10 cfu / g; (e) Mix the prepared halophilic Bacillus HMF09 powder with Bacillus subtilis powder in a certain proportion.

4. The preparation method according to claim 3, characterized in that, In steps (a) to (c), LB liquid culture medium with a pH of 7.2 to 7.4 is used, and the medium is cultured at a constant temperature of 160 rpm and 35°C with shaking.

5. The application of a compound microbial agent as described in any one of claims 1 or 2 in improving the properties of saline-alkali soil.

6. The application of a compound microbial agent as described in any one of claims 1 or 2 in promoting crop growth.

7. The application of a compound microbial agent as described in any one of claims 1 or 2 in the degradation of insecticide carbaryl residues.

Images