Composite microbial agent containing halophilous bacillus HMF09 and application thereof

Abstract

The invention relates to a compound microorganism bacterium agent which comprises halophilous bacillus HMF09 and bacillus subtilis, and the preservation number of the halophilous bacillus HMF09 is CGMCC (China General Microbiological Culture Collection Center) No.30475. The complex microbial inoculant can effectively reduce the pH value of soil, reduce the salt content of the soil, improve the emergence rate and salt and alkali resistance of crops and increase the yield of the crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a composite microbial agent comprising halobacillus sp. HMF09 and an application thereof. Background Art

[0002] Saline-alkali soil is a general term for saline soil and alkaline soil. It is a kind of obstructive soil widely distributed all over the world. According to the severity of salinization, it can be usually divided into mild, moderate and severe saline-alkali soil. Due to the poor properties of saline-alkali soil, it is not conducive to the normal growth of crops, and a large amount of salinized soil cannot be effectively utilized. How to utilize this part of saline-alkali soil is an urgent problem to be solved.

[0003] The methods of improving saline-alkali land can be divided into water conservancy, agriculture, biology and chemical improvement measures. Flooding and salt washing is the most common treatment method for improving saline-alkali soil, but it requires a lot of water and is not conducive to implementation in arid and water-scarce areas. Agricultural measures often use deep plowing, organic fertilizer application, straw return to the field, etc. These measures can increase the content of soil organic matter. Chemical measures often use improvers such as phosphoric acid, gypsum, and wood vinegar to improve soil quality, but the use of chemical improvers will cause secondary pollution to the soil. Biological measures are to reduce the accumulation of salt on the soil surface by increasing vegetation coverage and improving the regional microclimate environment, such as using salt-alkali tolerant crops, and secondly, use microbial methods for improvement.

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

[0005] The purpose of the present invention is to provide a composite microbial agent which can effectively reduce soil pH, reduce soil salinity, improve crop emergence rate and salt-alkali resistance, and increase crop yield.

[0006] The present invention adopts the following technical solution: A composite microbial agent comprises halophilic bacillus HMF09 and bacillus subtilis.

[0007] Further, the halophilic Bacillus ( Halobacillus sp. ) The deposit number of HMF09 is CGMCC No.30475, and it is deposited in the General Microbiology Center of China Culture Collection Administration. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 30, 2024.

[0008] Furthermore, the ratio of the viable bacteria count of the halophilic Bacillus HMF09 to that of the Bacillus subtilis is 1-10:2.

[0009] Furthermore, the total number of viable bacteria in the composite microbial agent is not less than 3×10 10 cfu / g.

[0010] A method for preparing the composite microbial agent comprises the following steps: (a) Activation of HMF09 strain; (b) Preparation of HMF09 seed solution; (c) preparing HMF09 fermentation broth; (d) The HMF09 fermentation liquid and soluble starch are uniformly mixed in a mass ratio of 5 to 15:1, and sprayed in a freeze dryer to obtain the halophilic Bacillus HMF09 bacterial powder, the effective viable bacterial count of which is not less than 3.0×10 10 cfu / g; (e) The prepared halophilic Bacillus HMF09 powder and the Bacillus subtilis powder are mixed evenly according to a certain proportion.

[0011] In the preparation method, LB liquid culture medium is used in steps (a) to (c), the pH value is 7.2 to 7.4, and the culture is shaken at 60 rpm and 35° C.

[0012] In the preparation method, the effective live bacteria count of the Bacillus subtilis powder is not less than 110 billion / g.

[0013] An application of the above composite microbial agent in improving soil properties in saline-alkali land.

[0014] An application of the composite microbial agent in promoting the growth of crops.

[0015] The method of using the above-mentioned composite microbial agent is to mix with seeds, or spray the agent on the soil surface before planting, and then turn the agent into the soil through tillage.

[0016] The beneficial effect of the present invention is that the composite microbial agent of the present invention is compounded by halophilic Bacillus HMF09 and Bacillus subtilis, and the two can not only grow synergistically with each other, but also cooperate with each other to achieve the effects of lowering soil pH, reducing soil salinity, improving crop emergence rate and salt-alkali resistance, and increasing crop yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the colony morphology of Halobacillus sp. HMF09 cultured on solid LB medium for 24 hours.

[0018] Figure 2 This is the morphology of Halobacillus sp. HMF09 under an optical microscope after Gram staining.

[0019] Figure 3This is the 16S rDNA phylogenetic tree of Halobacillus sp. HMF09.

[0020] Figure 4 These are the results of a synergistic test between strain HMF09 and Bacillus subtilis. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the examples and drawings. The protection scope of the present invention is not limited to the examples, and any changes made by those skilled in the art within the scope defined by the claims also belong to the protection scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0022] Example 1 Screening and Identification of Halobacillus sp. HMF09 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 District, Xinjiang Uygur Autonomous Region, China.

[0023] (1) Morphological characteristics The colonies are round and orange-yellow in color, with smooth and neat edges and no protrusions. Figure 1 As shown. The bacteria are rod-shaped, have spores, and are Gram-positive. Figure 2 shown.

[0024] (2) Physiological and biochemical characteristics The physiological and biochemical characteristics of strain HMF09 were identified, and the results are shown in Table 1.

[0025] Table 1 Physiological and biochemical characteristics of strain HMF09 .

[0026] (3) Molecular biological characteristics The genomic DNA of strain HMF09 was extracted and used as a template to perform PCR amplification using bacterial 16S rDNA universal primers. The amplified product was recovered and sequenced to obtain a DNA sequence containing 1500 bp (as shown in SEQ ID No. 1). The sequencing results were entered into the GeneBank database for BLAST comparison analysis. The 16S rDNA sequence was compared with the NCBI database, and it was found that the consistency between HMF09 and Halobacillus halophilus reached 99%. The results of constructing the phylogenetic tree are shown in the following figure. Figure 3 According to the strain morphology, sequencing analysis and physiological and biochemical test results, HMF09 was identified as Halobacillus sp. Halobacillus sp. ).

[0027] The halophilic Bacillus HMF09 was deposited in the China General Microbiological Culture Collection Center on April 30, 2024, with the address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No.30475.

[0028] Example 2 Synergy test between Halobacillus sp. HMF09 and Bacillus subtilis.

[0029] LB solid culture medium formula: 5g yeast extract, 10g peptone, 10g sodium chloride, 18g agar, placed in a 1000mL beaker, add 900mL distilled water and heat to dissolve, adjust the pH to 7.2-7.4, make up to 1L with distilled water, sterilize at 121℃ for 30 minutes, and set aside.

[0030] The strains to be tested include: Halobacillus sp. HMF09 and Bacillus subtilis. Among them, Bacillus subtilis powder (purchased from Tianjin Kunhe Biotechnology Co., Ltd., with an effective viable count of 110 billion / g) was used.

[0031] The Bacillus subtilis strain was separated and purified from the Bacillus subtilis powder (microbial agent) using LB medium, and stored at -4°C for later use. The Halobacillus halophilus HMF09 was activated using LB medium.

[0032] Use an inoculation loop to pick up a Bacillus subtilis colony and place it in 1 mL of sterile water. Use a sterile pipette to blow and mix repeatedly to prepare a bacterial suspension, then add it to the LB medium that has not solidified but is not hot. After shaking, pour the plate immediately, place the bacterium cake of Bacillus halophilus HMF09 in the center of the cooled and solidified plate, mark it, and repeat 3 times. Place it in a 37 ℃ constant temperature incubator and culture it for 3 days. The observation results are as follows: Bacillus subtilis and Bacillus halophilus HMF09 grow together, and no inhibition zone is produced, indicating that Bacillus subtilis and Bacillus halophilus HMF09 do not inhibit each other. Results are shown in Figure 4 .

[0033] Example 3 Preparation of Halobacillus sp. HMF09 powder (1) LB liquid medium: Place 5 g of yeast extract, 10 g of peptone, and 10 g of sodium chloride in a 1000 mL beaker, add 900 mL of distilled water and heat to dissolve, adjust the pH to 7.2-7.4, make up to 1 L with distilled water, sterilize at 121°C for 30 min, and set aside.

[0034] (2) Activation of strains: Pick a colony of Halobacillus subtilis HMF09, inoculate it into a test tube containing 10 mL of LB liquid culture medium, and culture it at a constant temperature of 160 rpm and 35°C for 24 h for activation.

[0035] (3) Preparation of seed solution: Take 5 mL of the activated bacterial solution and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid culture medium. Incubate the culture at 160 rpm and 35°C for 24 h to obtain the seed solution.

[0036] (4) Preparation of fermentation broth: 180 mL of the prepared seed solution was inoculated into a 6 L small fermentation tank containing 3.6 L of LB liquid medium, and cultured at 160 rpm and 35 °C for 48 h to obtain the fermentation broth of Halobacillus sp. HMF09. The effective viable count was 3.31 × 10 9 cfu / mL.

[0037] (5) Preparation of bacterial agent: The fermentation liquid and soluble starch were mixed evenly at a mass ratio of 10:1, and sprayed in a freeze dryer to obtain the powder of Halobacillus sp. HMF09. The effective viable bacterial count was 3.15×10 10 cfu / g.

[0038] Example 4 Preparation of composite microbial agent The bacillus halophilus HMF09 powder prepared in Example 3 and the bacillus subtilis agent (purchased from Tianjin Kunhe Biotechnology Co., Ltd., with an effective viable count of 110 billion / g) were mixed at a viable count ratio of 1:2, 5:2, and 5:1 to obtain composite microbial agent 1, composite microbial agent 2, and composite microbial agent 3. After testing, the total viable count of the prepared composite microbial agent 1 was 60.19 billion / g, the total viable count of the composite microbial agent 2 was 39.57 billion / g, and the total viable count of the composite microbial agent 3 was 35.75 billion / g.

[0039] Examples 5-7 Potted plant seed dressing test with three composite microbial agents under sodium chloride salt stress 1. Test materials Wheat variety: Cangmai 6002.

[0040] The physical and chemical indicators of the original soil are: organic matter 8.76 g / kg, alkaline nitrogen 30.96 mg / kg, available phosphorus 15.98 mg / kg, available potassium 34.28 mg / kg, salt 0.22 g / kg, and pH 6.58.

[0041] Flower pot: 10cm diameter.

[0042] 2. Test methods (1) Preparation of test soil: Local soil was selected as the test object. The soil had a low organic matter content and was easy to harden after watering. It was watered once with 1.2% NaCl and 1% Na2CO3 solution and then dried to constant weight. The soil was sieved through a 2mm sieve to serve as the test soil. The soil pH was 7.82 using the potentiometric method, and the soil soluble salt content was 6.33 g / kg dried soil using the oven drying method.

[0043] (2) Pretreatment: Transfer the prepared saline-alkali soil into a 10 cm diameter flower pot, 350 g per pot; select wheat seeds with full grains and uniform size, soak them in 50°C warm water for 30 minutes for disinfection, and set aside.

[0044] (3) Experimental design: The experiment set up 6 treatments, each with 8 pots, and 10 wheat grains were 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, and treatment 4 was not treated with microbial agent 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 seed. After sowing, all wheat treatments were transferred to a light incubator for continued cultivation.

[0045] (4) Detection indicators: After 30 days of cultivation, the growth indicators of wheat seedlings (plant height, longest root length, and number of roots) in the blank control group, the single bacterial agent treatment group, and the composite microbial treatment group under saline-alkali stress were tested. Four soil samples were randomly collected from each treatment, mixed, and 200 g was taken out by the quartering method for drying. The pH value of the dried soil and the soluble salt content of the soil were tested.

[0046] 3. Results and analysis From the results in Table 2, it can be seen that the composite microbial agents 1 to 3 prepared by Example 4 can significantly reduce the soluble salt content in the soil, reduce the soil pH, and alleviate the inhibition of saline-alkali stress on wheat growth. The effects of the composite microbial agents 1 to 3 are significantly better than those of the single microbial agents Bacillus subtilis and Bacillus halophilus HMF09. The experiment shows that HMF09 and Bacillus subtilis have a synergistic effect.

[0047] Table 2 Statistical results of survey indicators for each treatment .

[0048] Examples 8-10 Field Test of Composite Microbial Agents Overview of the test site: The test site is located in Huanghua City. It is a drought-alkali wheat field planted by the applicant. After harvesting the drought-alkali wheat, corn will be planted in the next crop. The soil texture of the plot is loam. The data of the soil sample taken before sowing are as follows: pH 7.62, alkaline nitrogen 114.1mg / kg, available phosphorus 30.18mg / kg, available potassium 86mg / kg, organic matter 9.97g / kg, salt content 2.93g / kg, which is moderate saline-alkali land.

[0049] Experimental design: The experiment set up 6 treatments, each treatment was 0.5 mu, repeated 3 times, and each treatment was randomly arranged. One day before planting, 250 g of each of the three composite microbial agents prepared in Example 4 were used per mu in treatments 1 to 3, 250 g of a single halophilic bacillus HMF09 agent was used per mu in treatment 4, 250 g of a single Bacillus subtilis agent was used per mu in treatment 5, and no composite microbial agent was sprayed in treatment 6, which was replaced by clean water, as a blank control (CK). Each treatment agent was diluted 150 times and evenly sprayed on the soil surface. After spraying, the diluted agent sprayed on the soil surface was immediately turned into the soil using a tillage machine.

[0050] Experimental site management: On June 10, 2023, the land was prepared, 40 kg of compound fertilizer (N: P2O5: K2O = 28: 6: 7) and 160 kg of organic fertilizer were used as base fertilizer per mu, and sowing was carried out the next day. Planting depth: 5-8 cm, planting density: 5000 plants / mu, plant spacing: 30 cm, row spacing: 33 cm, planting variety: Wei Ke 985. Other conventional production management measures were carried out.

[0051] Investigation method: 20 days after sowing, investigate the emergence and plant height of corn in each treatment plot; at maturity, measure the yield of each treatment plot.

[0052] Table 3 Statistics of corn germination rate, plant height and yield in each treatment .

[0053] The results in Table 3 show that the effects of composite microbial agents 1 to 3 are significantly better than those of single microbial agents Bacillus subtilis and Bacillus halobacterium HMF09, and HMF09 and Bacillus subtilis have a synergistic effect. Composite microbial agents 1 to 3 prepared using Example 4 can alleviate the inhibition of corn growth under saline-alkali stress, improve corn emergence rate, and increase corn yield.

[0054] The above is only the best specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions within the technical scope disclosed in the present application shall be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A composite microbial agent, characterized in that: These include Halobacillus Halobacillus sp. ) HMF09 and Bacillus subtilis.

2. The composite microbial agent according to claim 1, characterized in that: The deposit number of the halophilic Bacillus HMF09 is CGMCC No.30475.

3. The composite microbial agent according to claim 2, characterized in that: The ratio of the number of live bacteria of Halobacillus sp. HMF09 and Bacillus subtilis was 1-10:

2.

4. The composite microbial agent according to claim 3, characterized in that: The total viable count is not less than 3×10 10 cfu / g.

5. A method for preparing the composite microbial agent according to any one of claims 1 to 4, characterized in that: It includes the following steps: (a) Activation of HMF09 strain; (b) Preparation of HMF09 seed solution; (c) preparing HMF09 fermentation broth; (d) The HMF09 fermentation liquid and the soluble starch are uniformly mixed in a mass ratio of 5 to 15:1, and sprayed in a freeze dryer to obtain the halophilic Bacillus HMF09 bacterial powder, the effective viable bacterial count of which is not less than 3.0×10 10 cfu / g; (e) The prepared halophilic Bacillus HMF09 powder and the Bacillus subtilis powder are mixed evenly according to a certain proportion.

6. The preparation method according to claim 5, characterized in that: In the steps (a) to (c), LB liquid culture medium is used, the pH value is 7.2 to 7.4, and the culture is shaken at 60 rpm and 35° C.

7. Use of the composite microbial agent as claimed in any one of claims 1 to 4 in improving soil properties in saline-alkali land.

8. Use of the composite microbial agent according to any one of claims 1 to 4 in promoting the growth of crops.

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