A composite microbial inoculant for saline-alkali soil improvement, its preparation method and application

Abstract

The present invention relates to a compound microbial inoculum, which comprises Marinobacter gudaonensis HMF12 with the preservation number of CGMCC No. 30478, Bacillus velezensis HM-3 and Bacillus subtilis HM-1. The compound microbial inoculum of the present invention can effectively improve the soil structure of saline-alkali land, reduce the soil pH and promote the growth of crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of agriculture, and particularly relates to a composite microbial inoculant for saline-alkali soil improvement, its preparation method and application. Background Art

[0002] As an important land resource, the improvement and utilization of saline-alkali land have always been the focus and hotspot in the field of agricultural research, and are also the basis for the development of cultivated land reserve resources and the potential guarantee for food production increase. The pH of saline-alkali land is generally high, the content of saline-alkali components is high, and the activity of soil microorganisms is poor. Therefore, the yield of crops in saline-alkali land is generally not high. At present, mainly three methods of physical, chemical and agricultural are adopted for the restoration and treatment of saline-alkali land, and each of these methods has its own deficiencies.

[0003] The biological improvement in the methods for improving saline-alkali land has the characteristics of high desalination rate, persistence and stability. By applying microbial inoculants to saline-alkali soil, the microorganisms in the inoculants can reproduce in the soil, thereby improving the soil nutrient status, increasing soil enzyme activity, increasing microbial diversity, promoting soil desalination, inhibiting salt return, reducing the toxicity of high salt concentration and improving plant growth, and at the same time repairing degraded saline soil. It can be seen that microbial inoculants will surely play an increasingly important role in the green development of agriculture, and are a sustainable method for developing and repairing saline-alkali land and improving the productivity of agricultural ecosystems. Therefore, in view of the current situation of soil salinization, it is of great significance to research and develop microbial composite inoculants for saline-alkali land improvement. Summary of the Invention

[0004] The purpose of the present invention is to provide a composite microbial inoculant that can effectively improve the soil structure of saline-alkali land, reduce the soil pH and promote crop growth.

[0005] The present invention adopts the following technical scheme:

[0006] A composite microbial inoculant, which comprises Marinobacter gudaonensis HMF12, Bacillus velezensis HM-3 and Bacillus subtilis HM-1.

[0007] Further, the preservation number of Marinobacter gudaonensis HMF12 is CGMCC No. 30478, and it was preserved in the China General Microbiological Culture Collection Center on April 30, 2024, with the address being Beijing, China.

[0008] Further, the preservation number of Bacillus velezensis HM-3 is CGMCC No. 23739, and it was preserved in the China General Microbiological Culture Collection Center on November 8, 2021, with the address being Beijing, China.

[0009] Furthermore, the preservation number of the Bacillus subtilis HM-1 is CGMCC No. 21752, and it was preserved at the China General Microbiological Culture Collection Center on January 28, 2021, with the address being Beijing, China.

[0010] In the compound microbial inoculum, the viable count ratio of the Marinobacter gudaonensis HMF12, Bacillus velezensis HM-3, and Bacillus subtilis HM-1 is 20 - 40∶25 - 45∶25 - 45.

[0011] Furthermore, the total viable count of the compound microbial inoculum is not less than 5×10 8 cfu / g.

[0012] Furthermore, the compound microbial inoculum further includes granular organic fertilizer, binder, and anti-caking agent.

[0013] A preparation method of the above compound microbial inoculum includes the following steps:

[0014] (1) Prepare the Marinobacter gudaonensis HMF12 bacterial powder, Bacillus velezensis HM-3 bacterial powder, and Bacillus subtilis HM-1 bacterial powder respectively;

[0015] (2) Mix the Marinobacter gudaonensis HMF12 bacterial powder, Bacillus velezensis HM-3 bacterial powder, and Bacillus subtilis HM-1 bacterial powder to obtain a bacterial powder mixture, and then mix the bacterial powder mixture evenly with granular organic fertilizer, binder, and anti-caking agent.

[0016] In the preparation method, the effective viable count of the Marinobacter gudaonensis HMF12 bacterial powder is not less than 3×10 10 cfu / g, the effective viable count of the Bacillus velezensis HM-3 bacterial powder is not less than 2×10 10 cfu / g, and the effective viable count in the Bacillus subtilis HM-1 bacterial powder is not less than 2.5×10 10 cfu / g.

[0017] In the preparation method, the mass ratio of the bacterial powder mixture, granular organic fertilizer, binder, and anti-caking agent is 5 - 20∶1000∶1 - 10∶5 - 15.

[0018] An application of the above compound microbial inoculum in improving saline-alkali soil.

[0019] An application of the above compound microbial inoculum in promoting the growth of crops planted in saline-alkali soil.

[0020] The beneficial effects of the present invention are as follows: The microbial complex bactericide of the present invention, whose main components are Marinobacter gudaonensis HMF12, Bacillus velezensis HM-3, and Bacillus subtilis HM-1, can effectively regulate the soil flora structure, reduce the soil pH, promote the growth of wheat roots, promote tillering, improve the drought and saline-alkali resistance of crops, alleviate the impact of salt stress on plants, and increase crop yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the colony morphology of Marinobacter gudaonensis HMF12.

[0022] Figure 2 It is the cell morphology of Marinobacter gudaonensis HMF12.

[0023] Figure 3 It is the 16S rDNA phylogenetic tree of Marinobacter gudaonensis HMF12.

[0024] Figure 4 It is the co-culture result of Marinobacter gudaonensis HMF12, Bacillus velezensis HM-3, and Bacillus subtilis HM-1.

[0025] Figure 5 It is the growth situation of Bacillus subtilis HM-1 in Gibson modified medium.

[0026] Figure 6 It is the growth situation of Bacillus velezensis HM-3 in Gibson modified medium.

[0027] Figure 7 It is the growth situation of Marinobacter gudaonensis HMF12 in Gibson modified medium. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention will be further described below in conjunction with the embodiments and the drawings. The protection scope of the present invention is not limited to the embodiments, 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 embodiments are all conventional methods unless otherwise specified. The reagents used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0029] Example 1 Characteristics of Marinobacter gudaonensis HMF12

[0030] Marinobacter gudaonensis ( Oceanobacillus jeddahense ) HMF12 was screened and isolated from the rhizosphere soil of wheat in saline-alkali land in Huanghua City, and was deposited in the China General Microbiological Culture Collection Center on April 30, 2024. The address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No. 30478.

[0031] (1)Morphological characteristics

[0032] The colonies are round, with neat edges and milky white, as Figure 1 shown. Gram staining is positive, as Figure 2 shown. The phylogenetic tree of Marinobacter jeddahensis HMF12 constructed based on 16S rDNA is as Figure 3 shown.

[0033] (2)Physiological and biochemical characteristics

[0034] Identification experiments were carried out on the physiological and biochemical characteristics of strain HMF12, and the results are shown in Table 1.

[0035] Table 1 Physiological and biochemical characteristics of strain HMF12

[0036] .

[0037] Example 2 Biocompatibility among Marinobacter jeddahensis HMF12, Bacillus velezensis HM-3, and Bacillus subtilis HM-1.

[0038] Bacillus velezensis ( Bacillus velezensis ), HM-3, is deposited in the China General Microbiological Culture Collection Center, located in Beijing, China, with the deposit number CGMCC No. 23739 and the deposit date of November 08, 2021.

[0039] Bacillus subtilis ( Bacillus subtilis ), HM-1, is deposited in the China General Microbiological Culture Collection Center, located in Beijing, China, with the deposit number CGMCC No. 21752 and the deposit date of January 28, 2021.

[0040] Use an inoculation loop to pick up the colonies of Marinobacter jeddahensis HMF12 and place them in 1 mL of sterile water. Use a sterile pipette to repeatedly blow and wash to mix evenly to prepare a bacterial suspension, and then add it to the LB medium that is not solidified but not too hot to the touch. After shaking well, immediately pour the plate. Place the bacterial cakes of Bacillus velezensis strain HM-3 and Bacillus subtilis strain HM-1 evenly on the cooled and solidified plate, mark them well, and repeat 3 times. Place them in an incubator at 37 °C for 3 days. Observe the results: Bacillus velezensis HM-3 and Bacillus subtilis HM-1 grow together with Marinobacter jeddahensis HMF12, and no inhibition zone is produced, indicating that Bacillus velezensis HM-3 and Bacillus subtilis HM-1 do not inhibit each other with Marinobacter jeddahensis HMF12. The results are shown in Figure 4 .

[0041] LB medium formulation: 5.0 g of yeast extract, 10.0 g of peptone, 10.0 g of NaCl, 1000 mL of water, pH 7.0. Sterilize at 121 °C for later use.

[0042] Bacillus velezensis HM-3, Bacillus subtilis HM-1, and Marinobacter gudaonensis HMF12 were respectively cultured on Gibson modified medium in a 37 °C constant temperature incubator for 3 days. The results are as Figures 5 - 7 shown. All three strains can grow on this medium, indicating that all three strains can grow and survive in a high-salt and high-alkali environment.

[0043] Gibson modified medium formulation: 5.0 g of casein, 3.0 g of sodium citrate, 10.0 g of yeast extract powder, 2.0 g of KCl, 5.0 g of peptone, 2.0 g of magnesium sulfate, 60 g of NaCl, 1000 mL of water, pH 8.5.

[0044] Example 3 Preparation of Marinobacter gudaonensis HMF12 bacterial powder

[0045] (1) LB liquid medium: 5 g of yeast extract, 10 g of peptone, 10 g of sodium chloride. Place them in a 1000 mL beaker, add 900 mL of distilled water, heat and dissolve, adjust the pH to 7.2 - 7.4, make up the volume to 1 L with distilled water, sterilize at 121 °C for 30 minutes, and reserve for later use.

[0046] (2) Activation of the strain: Pick up a loop of the colony of Marinobacter gudaonensis HMF12 and inoculate it into a 150 mL Erlenmeyer flask containing 50 mL of LB liquid medium. Incubate it at a constant temperature of 35 °C with shaking at 160 rpm for 24 h for activation.

[0047] (3) Preparation of the seed liquid: Take 5 mL of the activated bacterial liquid and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid medium. Incubate it at a constant temperature of 35 °C with shaking at 160 rpm for 24 h to obtain the seed liquid.

[0048] (4) Preparation of the fermentation broth: Take 180 mL of the prepared seed liquid and inoculate it into a 6 L small fermenter containing 3.5 L of LB liquid medium. Incubate it at a constant temperature of 35 °C with shaking at 160 rpm for 48 h to obtain the fermentation broth of Marinobacter gudaonensis HMF12, and the effective viable count is 3.31×10 9 cfu / mL.

[0049] (5) Preparation of the microbial agent: Mix the fermentation broth and diatomaceous earth evenly according to a mass ratio of 10:1, and spray it with a freeze dryer to obtain the powder of Marinobacter gudaonensis HMF12. After detection, the effective viable count is 3.15×10 10 cfu / g.

[0050] Example 4 Preparation of Bacillus velezensis HM-3 Bacterial Powder

[0051] Prepare a Bacillus velezensis HM-3 bacterial agent using diatomaceous earth as a carrier. The specific steps are as follows:

[0052] (1) Prepare LB liquid medium: 3 g of beef extract, 10 g of peptone, and 5 g of sodium chloride are placed in a 1000 mL beaker, dissolved by heating with 900 mL of distilled water, adjust the pH to 7.2 - 7.4, make up the volume to 1 L with distilled water, and sterilize at 121 °C for 30 minutes for standby.

[0053] (2) Activate the strain: Pick one loop of strain HM-3 colony and inoculate it into a 150 mL Erlenmeyer flask containing 50 mL of LB liquid medium, and culture it at a constant temperature of 37 °C with shaking at 160 rpm for 24 h for activation.

[0054] (3) Preparation of seed liquid: Take 4 mL of the activated bacterial liquid and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid medium, and culture it at a constant temperature of 37 °C with shaking at 160 rpm for 24 h to obtain the seed liquid.

[0055] (4) Preparation of fermentation broth: Take 180 mL of the prepared seed liquid and inoculate it into a 6 L small fermenter containing 3.5 L of LB liquid medium, and culture it at a constant temperature of 37 °C with shaking at 160 rpm for 48 h to obtain the HM-3 fermentation broth, and the effective viable count is 2.26×10 9 cfu / mL.

[0056] (5) Mix the fermentation broth and diatomaceous earth evenly according to a mass ratio of 10:1, and spray it with a freeze dryer to obtain the Bacillus velezensis powder. After detection, the effective viable count is 4.12×10 10 cfu / g.

[0057] Example 5 Preparation of Bacillus subtilis HM-1 Bacterial Powder

[0058] According to the steps of Example 3, prepare the Bacillus subtilis HM-1 bacterial powder. After detection, the effective viable count is 2.6×10 10 cfu / g.

[0059] Example 6 Preparation of Compound Microbial Bacterial Agent

[0060] (1) Materials

[0061] Binder: Caramel color, purchased from the market, and dissolved with warm water at a weight ratio of 1:1 when in use;

[0062] Granular organic fertilizer: Purchased from the market, its content: organic matter 50.0%, N+P+K = 9.2%;

[0063] Anti-caking agent: Prepared from talcum powder and diatomaceous earth in a mass ratio of 1:1. Both the talcum powder and diatomaceous earth are purchased from the market.

[0064] (2) Preparation method

[0065] Take the HMF12 powder of Marinobacter gudaonensis prepared in Examples 3 to 5 (effective viable count is 3.15×10 10 cfu / g), the HM-3 powder of Bacillus velezensis (effective viable count is 4.12×10 10 cfu / g), and the HM-1 powder of Bacillus subtilis (effective viable count is 2.6×10 10 cfu / g). According to the requirement that the viable count ratio of Marinobacter gudaonensis HMF12, Bacillus velezensis HM-3 and Bacillus subtilis HM-1 is 20 - 40:25 - 45:25 - 45, take 5 g of Marinobacter gudaonensis HMF12 powder, 4 g of Bacillus velezensis HM-3 powder, and 8.0 g of Bacillus subtilis HM-1 powder to obtain 17 g of a mixture of powder.

[0066] Mix 17 g of the mixture of powder with 1 kg of granular organic fertilizer, stir for 5 minutes, then add 5 g of caramel color and stir for 5 minutes, and then add 10 g of anti-caking agent and stir for 5 - 8 minutes. The prepared product has non-sticky particles, does not form lumps when held in the hand, has a shiny black appearance, and no powder, thus obtaining the compound microbial inoculant. After testing, the total viable count of the prepared compound microbial inoculant is 5.1×10 8 cfu / g.

[0067] Example 7 Field test of compound microbial inoculant

[0068] (1) Planting location and time

[0069] The test is set up in the dry-alkali wheat test field in Huanghua. The saline-alkali land area is 23.7 mu, belonging to a severely saline-alkali plot. On October 17, 2023, wheat is sown in the saline-alkali land. The sowing rate is 15 kg / mu. Before sowing, 28 kg / mu of diammonium phosphate is applied along with deep soil turning operation. Wheat variety: Jiemai 19.

[0070] (2) Test treatment

[0071] The inoculant treatment is to apply the compound microbial inoculant prepared in Example 6 by ditch application along with the machine during wheat sowing; the control treatment is to use granular organic fertilizer without adding powder instead. Parallel tests are carried out on 2 test fields with different salinities respectively. The soil samples before planting are collected on October 12, 2023, and the soil samples after planting are collected on June 6, 2024. The average values are measured after mixing the samples taken at five points.

[0072] (3) Test results

[0073] a) Effects of applying the compound microbial inoculant on soil physical and chemical indexes

[0074] As can be seen from Table 2, after applying the compound microbial inoculant prepared in Example 6 of the present invention, the total amount of water-soluble salts in Test Field 1 decreased by 14.29%, and that in Test Field 2 decreased by 8%; the pH of Test Field 1 decreased from 8.32 to 7.76, and the pH of Test Field 2 decreased from 7.91 to 7.45. The results show that applying the compound microbial inoculant prepared by the present invention can effectively reduce the pH, reduce the content of water-soluble salts in the soil, improve the soil environment, and relieve the damage caused by salt stress to plants.

[0075] Table 2 Soil test results of wheat fields in Huanghua saline-alkali land

[0076] 。

[0077] b) Effects of applying the compound microbial inoculant for saline-alkali land improvement on soil flora

[0078] As can be seen from Table 3, after applying the compound microbial inoculant prepared in Example 6 of the present invention, the total number of effective viable bacteria in the soils of Test Field 1 and Test Field 2 showed an increasing state, and the change of the control flora was not significant. Therefore, the compound microbial inoculant of the present invention can effectively regulate the soil flora structure.

[0079] Table 3 Flora test results of wheat fields in Huanghua saline-alkali land

[0080] 。

[0081] c) Effects of applying the compound microbial inoculant for saline-alkali land improvement on the growth of wheat in Huanghua saline-alkali land

[0082] As can be seen from Table 4, after applying the compound microbial inoculant prepared in Example 6 of the present invention, the plant height, stem diameter, number and length of fibrous roots, tiller number, ear length and number of ears per mu of wheat all exceeded the control, and the yield increased by about 30% compared with the control in the test field. From the results, it can be known that the compound microbial inoculant provided by the present invention can effectively promote the growth and yield of wheat in saline-alkali land.

[0083] Table 4 Growth test results of wheat fields in Huanghua saline-alkali land

[0084] 。

[0085] In summary, applying the compound microbial inoculant prepared by the present invention can effectively reduce the pH of saline-alkali soil, reduce the content of water-soluble salts in saline-alkali soil, relieve the damage caused by salt stress to plants, effectively regulate the soil flora structure, effectively promote the growth of wheat, and achieve high crop yields.

[0086] As described above, it is only the optimal specific implementation manner 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 should be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claimed rights.

Claims

1. A composite microbial agent, characterized in that: The invention comprises Bacillus jeddah HMF12, Bacillus velez HM-3 and Bacillus subtilis HM-1; the deposit number of the Bacillus jeddah HMF12 is CGMCC No.30478; the deposit number of the Bacillus velez HM-3 is CGMCC No.23739; the deposit number of the Bacillus subtilis HM-1 is CGMCC No.21752.

2. The composite microbial agent according to claim 1, characterized in that: The ratio of the number of live bacteria of the Jeddah Marine Bacillus HMF12, the Velez Bacillus HM-3 and the Bacillus subtilis HM-1 is 20-40:25-45:25-45.

3. The composite microbial agent according to claim 1, characterized in that: The total viable count is not less than 5×10 8 cfu / g.

4. The composite microbial agent according to any one of claims 1 to 3, characterized in that It also includes granular organic fertilizer, a binder and an anti-caking agent.

5. A method for preparing the composite microbial agent according to claim 4, characterized in that: It includes the following steps: (1) preparing bacterial powders of Bacillus maritimus HMF12, Bacillus velez HM-3 and Bacillus subtilis HM-1 respectively; (2) Mix the powder of Bacillus maritimus HMF12, the powder of Bacillus velez HM-3 and the powder of Bacillus subtilis HM-1 to obtain a powder mixture, and then mix the powder mixture with granular organic fertilizer, a binder and an anti-caking agent.

6. The preparation method according to claim 5, characterized in that: The effective viable bacteria count of the Jeddah Marine Bacillus HMF12 powder is not less than 3×10 10 cfu / g, the effective viable count of the Bacillus Velez HM-3 powder is not less than 2×10 10 cfu / g, the effective viable bacteria count in the Bacillus subtilis HM-1 powder is not less than 2.5×10 10 cfu / g.

7. The preparation method according to claim 5, characterized in that: The mass ratio of the bacterial powder mixture, the granular organic fertilizer, the binder and the anti-caking agent is 5-20:1000:1-10:5-15.

8. Use of the composite microbial agent as claimed in any one of claims 1 to 3 in improving saline-alkali soil.

9. Use of the composite microbial agent as claimed in any one of claims 1 to 3 in promoting the growth of crops planted in saline-alkali soil.

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