Special complex microbial inoculant for alfalfa in arid region as well as preparation method and application of special complex microbial inoculant

Abstract

The invention discloses a special complex microbial inoculant for alfalfa in an arid region and a preparation method of the special complex microbial inoculant. The complex microbial inoculant is a bacterial strain culture solution of bacillus mycoides Gnyt1 and a bacterial strain culture solution of sinorhizobium meliloti GAU-0093. The compound microbial inoculant can effectively promote the plant height and root length of alfalfa, improve the overground and underground biomass and chlorophyll content of alfalfa, effectively adapt to a drought stress environment, relieve drought stress damage to plants, and effectively promote the growth of alfalfa and the overground biomass at the same time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a special composite microbial agent for alfalfa in arid areas, a preparation method and an application thereof. Background Art

[0002] Global warming has led to an increase in the frequency of drought stress. Drought stress is a major global problem limiting agricultural productivity and is considered one of the most severe abiotic stresses faced during the plant growth and development process. It can reduce plant yields, especially in arid and semi-arid regions. Alfalfa (Medicago sativa L.) is a perennial high-quality forage with rich nutrition and high feeding value. It has well-developed underground roots, thick rhizomes, strong tillering ability, and lush branches and leaves, and has natural advantages in improving the ecological environment and maintaining soil and water. In arid and semi-arid regions, the yield of crops mainly depends on the amount of water. Therefore, drought stress will seriously affect the growth, development and yield of alfalfa, restricting the formation of the industrial chain.

[0003] Microbial resources are regarded as one of the national strategic security biological resources and have great potential for industrial application. Rhizobium is a rod-shaped Gram-negative bacterium, which symbiotically forms root nodules with leguminous plants to convert molecular nitrogen in the air into plant-available nitrogen. In addition, rhizobium strains distributed under stress conditions all show good adaptability to the environment. Plant growth promoting rhizobacteria (PGPR) refers to a group of beneficial microorganisms that can colonize in large numbers in the rhizosphere of plants and promote plant growth. However, there is no report on the use of plant growth promoting rhizobacteria and rhizobia to alleviate plant drought stress and improve arid soil. Summary of the Invention

[0004] The present invention provides a special composite microbial agent for alfalfa in arid areas, a preparation method and an application thereof. By adding the composite microbial agent, namely Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093, the composite microbial agent can improve the rhizosphere microecological environment, improve the soil nitrogen utilization efficiency, reduce the damage caused by drought stress to plants, and promote the growth of alfalfa and nodulation of roots.

[0005] The technical solution provided by the present invention is as follows:

[0006] In the first aspect of the present invention, a special compound bacterial agent for alfalfa in arid regions is provided. The compound bacterial agent is a strain culture solution of Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093.

[0007] Furthermore, the growth-promoting bacterium Bacillus mycoides Gnyt1 was deposited at the China Center for Type Culture Collection on April 10, 2017, with the deposit number CCTCC NO: M2017177; the Sinorhizobium meliloti GAU-0093 was deposited at the China Center for Type Culture Collection on March 20, 2025, with the deposit number CCTCC NO: M2025541.

[0008] Furthermore, the compound bacterial agent is obtained by compounding the culture solutions of the growth-promoting bacterium Bacillus mycoides Gnyt1 and the Sinorhizobium meliloti GAU-0093 in the same volume. The viable bacteria concentrations in the culture solutions of the growth-promoting bacterium Bacillus mycoides Gnyt1 and the Sinorhizobium meliloti GAU-0093 are both greater than or equal to ≥10 9 cfu / mL.

[0009] In the second aspect of the present invention, a preparation method of a special compound bacterial agent for alfalfa in arid regions is provided, which is used to prepare the special compound bacterial agent for alfalfa in arid regions as described above, and includes the following steps:

[0010] S1. Screen a drought-tolerant growth-promoting bacterium Bacillus mycoides Gnyt1 with the abilities of dissolving phosphorus, fixing nitrogen, and secreting plant hormones, and a rhizobium Sinorhizobium meliloti GAU-0093 with strong nodulation and nitrogen fixation abilities;

[0011] S2. Liquid-culture the Bacillus mycoides Gnyt1 and the Sinorhizobium meliloti GAU-0093 to obtain single-strain culture solutions;

[0012] S3. After compounding the different single-strain culture solutions in a ratio of 1:1, obtain the microbial compound bacterial agent.

[0013] Further, in step S1, the antagonism between the selected Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093 was determined by the plate confrontation method, and it was found that there was no antagonistic effect between the strains.

[0014] Further, in step S2, the method for preparing the single-strain culture solution is as follows: Single colonies of Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093 were picked and inoculated into 250 mL Erlenmeyer flasks containing 150 mL of LB and YMA liquid media, respectively, and cultured on a shaker at 28 °C and 180 r / min for 48 - 72 h, so that the viable cell concentration of the liquid inoculum was greater than or equal to ≥10 9 cfu / mL.

[0015] The third aspect of the present invention provides an application of the above-mentioned special composite inoculant for alfalfa in arid areas in the growth of vegetation and soil improvement in the arid regions of northwest China.

[0016] Further, the application method of the composite inoculant is seed dressing, drip irrigation or direct spraying, and the application amount is 5.0 kg - 6.0 kg per mu.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The growth-promoting bacteria in the composite inoculant provided by the present invention have the growth-promoting abilities of nitrogen fixation, phosphorus solubilization, and secretion of plant hormones. At the same time, the selected Sinorhizobium meliloti has strong specificity. While promoting the growth of alfalfa, it can improve the nodulation and nitrogen fixation ability of plants. It is safe to use, has good ecological adaptability, and can improve the soil structure, increase the nitrogen utilization efficiency of the soil, improve soil fertility, promote plant growth, and alleviate the damage caused by drought stress to plants. Specific Embodiments

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the embodiments described below are some, but not all, of the embodiments of the present application.

[0020] Example 1

[0021] This embodiment provides a special compound microbial inoculum for alfalfa in arid areas. The compound microbial inoculum is a strain culture solution of Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093. Among them, the growth-promoting bacterium Bacillus mycoides Gnyt1 was deposited in the China Center for Type Culture Collection in 2017, and the deposit number is CCTCC NO: M2017177; the Sinorhizobium meliloti GAU-0093 was deposited in the China Center for Type Culture Collection in 2024, and the deposit number is CCTCC NO: M2025541.

[0022] The preparation method of the above compound microbial inoculum includes the following steps:

[0023] Step (1): Screen a drought-tolerant growth-promoting bacterium Bacillus mycoides Gnyt1 with the ability to dissolve phosphorus, fix nitrogen, and secrete plant hormones, inoculate it on an LB solid medium by the streaking method, and place it in an incubator at 28°C for 2 days. At the same time, screen a rhizobium Sinorhizobium meliloti GAU-0093 with strong nodulation and nitrogen fixation ability, inoculate it on a YMA solid medium by the streaking method, and place it in an incubator at 28°C for 3 days.

[0024] Step (2): Prepare a single-strain culture solution. Select single colonies and inoculate them into 250 mL Erlenmeyer flasks containing 150 mL of LB and YMA liquid media respectively, and culture them on a shaker at 28°C and 180 r / min for 48 h, so that the viable cell concentration of the liquid microbial inoculum is greater than 10 9 cfu / mL.

[0025] Step (3): Prepare a microbial compound inoculant. After mixing the single-strain culture solutions of Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093 in a volume ratio of 1:1, the microbial compound inoculum is obtained.

[0026] It should be noted that in this invention, the plate confrontation method is used to prove that there is no antagonistic effect between Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093, and they can be mixed as a microbial compound inoculum.

[0027] This compound microbial agent combines the advantages of plant growth-promoting rhizobacteria and alfalfa rhizobia, and simultaneously has the functions of nitrogen fixation, phosphorus solubilization, and secretion of plant hormones, promoting plant growth and root development, improving the nodulation and nitrogen fixation ability of plants, having good ecological adaptability, and at the same time improving the soil structure. It can be directly sprayed for application, and the microbial agent is safe to use.

[0028] The usage method of the compound microbial agent provided by the present invention is as follows: When used for seed dressing, the microbial compound microbial agent and seeds are fully mixed and air-dried before sowing; when used for drip irrigation, the microbial agent can be applied along with the drip irrigation tape when applied to the soil equipped with drip irrigation equipment; when used for spraying, the prepared microbial compound microbial agent is filled into a sprayer or watering can and evenly sprayed onto the alfalfa cultivation land. The dosage is 5.0 kg - 6.0 kg per mu.

[0029] Example 2

[0030] In this example, the first experiment was carried out on the above-prepared compound microbial agent, and the specific experiment is as follows:

[0031] Select alfalfa seeds with plump and uniform size. After disinfecting with 2% sodium hypochlorite for 2 minutes, wash them 4 - 5 times with sterile water, germinate them at 23°C, and plant them in a hydroponic box. Each box retains 18 seedlings with consistent growth and uniform distribution. After the seedlings grow for one week, inoculate the bacterial suspension. A total of four inoculation treatments are set, no inoculation (B1), single application of growth-promoting bacteria (B2), single application of rhizobia (B3), and mixed bacterial system (B4). After the seedlings are inoculated and grow for 7 days, a water potential condition of -0.4 MPa is set by using the method of simulating drought stress with PEG-6000. After the treatment, replace the nitrogen-free Hoagland's nutrient solution containing PEG-6000 with different water potentials every 3 days to maintain the solution concentration, and continuously stress-treat for 15 days. Measure the fresh weight, malondialdehyde (MDA) content, hydrogen peroxide (H2O2) content, peroxidase (POD), and superoxide dismutase (SOD) of the seedlings. During this period, water 30 mL of distilled water every two days and water 30 mL of full-strength Hoagland nutrient solution every seven days, and adjust appropriately according to the dryness and humidity. The specific experimental results are shown in Table 1:

[0032] Table 1: Usage effects of the special compound microbial agent for alfalfa in arid areas

[0033] Inoculation treatment Fresh weight (g) MDA (μmol / g FW) <![CDATA[H2O2 (nmol / gFW)]]> POD (U / g FW) SOD (U / g FW) B1 2.09±0.01 77.20±1.78 19.27±1.48 50.67±3.22 37.43±1.42 B2 2.32±0.09 58.24±2.24 17.32±0.54 144.26±6.12 14.35±0.79 B3 2.66±0.11 47.68±0.28 14.84±0.75 161.33±4.50 20.48±1.11 B4 3.68±0.15 38.72±0.34 12.79±0.81 166.35±2.33 154.82±4.01

[0034] As can be seen from Table 1, when applying the special compound microbial inoculant for alfalfa in arid areas prepared in Example 1 of the present invention, under the drought stress environment of -0.8 MPa, after inoculating the microbial inoculant, the fresh weight of alfalfa increased, with a 76.08% increase compared to the treatment without applying the microbial inoculant. Meanwhile, the MDA content and H2O2 content decreased significantly, with a 49.84% and 33.63% decrease respectively compared to the treatment without applying the microbial inoculant. The POD activity and SOD activity of the plants increased by 228.30% and 313.63% respectively compared to the treatment without inoculating the microbial inoculant. From the results, it can be seen that the microbial compound microbial inoculant provided by the present invention can effectively promote the fresh weight of alfalfa, alleviate the osmotic stress and ion toxicity of plants. Under drought stress, inoculating the microbial compound microbial inoculant reduced the malondialdehyde content and hydrogen peroxide content, and alleviated the oxidative damage caused to plants under drought stress by regulating the antioxidant enzymes and antioxidant system, thereby reducing the damage caused by drought stress to plants.

[0035] Example 3

[0036] In this example, a second experiment was conducted on the above-prepared compound microbial inoculant. The specific experiment is as follows:

[0037] The test site was set in Shuiyuan Town, Yongchang County, Jinchang City, Gansu Province (102°30′E, 38°12′W, altitude 1487 m). The test area belongs to the mid-temperate continental arid climate, with dry and windy weather all year round, sufficient sunlight, an average annual rainfall of 235 mm, an average annual temperature of 7.7 °C, and an annual evaporation of 2000.6 mm.

[0038] The test adopted a randomized block design in the field, with a total of 4 treatments, each treatment repeated 3 times. The sowing method of alfalfa was drill sowing, the plot area was 3 m × 5 m, the seed rate was 22.5 kg·hm -2 , the ridge width was 30 cm, the row spacing was 25 cm, and daily watering was carried out in the form of subsurface drip irrigation, with an annual irrigation volume of 6750 m 3 / hm 2 . The microbial inoculant formula had 4 levels: single application of rhizobia (B1), single application of growth-promoting bacteria (B2), mixed bacterial strains (B3), and non-inoculation (B4). The microbial inoculant was used for seed dressing, with a dosage of 0.15 L / kg. Sowing was carried out on April 15, 2024, with a sowing depth of 1 - 2 cm, and conventional field management.

[0039] The specific experimental treatments and results are shown in Table 2:

[0040] Table 2: Application effects of the special compound microbial inoculant for alfalfa in arid areas

[0041]

[0042] As can be seen from the above table, when applying the special compound microbial agent for alfalfa in arid areas of Example 1 of the present invention in an arid environment, after inoculating the compound microbial agent, the plant height, stem diameter, chlorophyll content, and above-ground biomass of the first cut of alfalfa are effectively increased by 8.67%, 8.33%, 18.08%, and 74.41% respectively; after inoculating the compound microbial agent, the plant height, stem diameter, chlorophyll content, and above-ground biomass of the second cut of alfalfa are effectively increased by 35.11%, 33.66%, 8.93%, and 71.91% respectively; after inoculating the compound microbial agent, the plant height, stem diameter, chlorophyll content, and above-ground biomass of the third cut of alfalfa are effectively increased by 29.88%, 61.97%, 28.44%, and 34.71% respectively. From the results, it can be known that the microbial compound microbial agent of the present invention can effectively promote the plant height and root length of alfalfa, increase the above-ground and underground biomass and chlorophyll content of alfalfa. The microbial agent can effectively adapt to the drought stress environment, relieve the drought stress damage suffered by plants, and at the same time effectively promote the growth and above-ground biomass of alfalfa.

[0043] Applying the compound microbial agent provided by the present invention can effectively promote the growth of alfalfa, fix nitrogen in the air, provide nitrogen nutrition for alfalfa, and at the same time can convert insoluble phosphorus, potassium and other nutrients in the soil into forms that can be absorbed and utilized by alfalfa, increase the availability of soil nutrients, and promote the growth and development of alfalfa. After the bacteria reproduce and grow, they produce metabolites such as antibiotics and hormones, which can enhance the stress resistance of alfalfa and improve its adaptability to adverse environments such as drought. When the beneficial microorganisms in the compound microbial agent colonize in the soil, they can compete with pathogenic bacteria for nutrients and living space, inhibit the growth and reproduction of pathogenic bacteria, and thus reduce the occurrence of pests and diseases. For the arid soil environment, applying the compound microbial agent can promote the decomposition and transformation of organic matter in the soil, increase the content of soil organic matter, improve the soil structure, enhance the air permeability and water permeability of the soil, and create a good soil microecological environment for the growth of alfalfa.

[0044] The above is only the optimal specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions within the technical scope disclosed in this application should be covered by the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claimed rights.

Claims

1. A special compound microbial agent for alfalfa in arid regions, characterized in that: The compound microbial agent is a strain culture solution of Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093.

2. The special compound microbial agent for alfalfa in arid regions according to claim 1, characterized in that: The growth-promoting bacterium Bacillus mycoides Gnyt1 was deposited in the China Center for Type Culture Collection in 2017, and the deposit number is CCTCC NO: M2017177; the Sinorhizobium meliloti GAU-0093 was deposited in the China Center for Type Culture Collection in 2024, and the deposit number is CCTCC NO: M2025541.

3. The special compound microbial agent for alfalfa in arid regions according to claim 1, characterized in that: The composite microbial agent is obtained by mixing the culture solution of the growth-promoting bacterium Bacillus mycoides Gnyt1 and the culture solution of Sinorhizobium meliloti GAU-0093 in the same volume. The viable cell concentrations in the culture solution of the growth-promoting bacterium Bacillus mycoides Gnyt1 and the culture solution of Sinorhizobium meliloti GAU-0093 are both greater than or equal to 10 9 cfu / mL.

4. A preparation method of a special composite microbial inoculum for alfalfa in arid areas, which is used to prepare the special composite microbial inoculum for alfalfa in arid areas as described in any one of claims 1-3, characterized in that, It includes the following steps: S1. Screen a drought-tolerant growth-promoting bacterium Bacillus mycoides Gnyt1 with the ability to dissolve phosphorus, fix nitrogen, and secrete plant hormones, and a rhizobium Sinorhizobium meliloti GAU-0093 with strong nodulation and nitrogen fixation ability; S2. Liquid-culture the Bacillus mycoides Gnyt1 and Sinorhizobium meliloti GAU-0093 to obtain single-strain culture solutions; S3. After compounding different single-strain culture solutions in a ratio of 1:1, obtain the microbial compound microbial agent.

5. The preparation method of the special compound microbial agent for alfalfa in arid regions according to claim 4, characterized in that: In step S1, the plate confrontation method is used to screen the Bacillus mycoides Gnyt1 and the Sinorhizobium meliloti GAU-0093 to determine that there is no antagonistic effect between the strains.

6. The preparation method of the special compound microbial agent for alfalfa in arid regions according to claim 4, characterized in that: In step S2, the specific method for preparing the single-strain culture solution is as follows: Pick single colonies of the Bacillus mycoides Gnyt1 and the Sinorhizobium meliloti GAU-0093 and inoculate them into 250 mL Erlenmeyer flasks containing 150 mL of LB and YMA liquid media respectively. Incubate them on a shaker at 28 °C and 180 r / min for 48 - 72 h, so that the viable cell concentration of the liquid inoculum is greater than or equal to ≥10 9 cfu / mL.

7. Application of the special compound microbial agent for alfalfa in arid regions according to any one of claims 1-3 in the vegetation growth and soil improvement in the arid northwest region.

8. Use of the special compound microbial inoculum for alfalfa in arid regions according to claim 7, characterized in that, The usage method of the compound microbial agent is seed dressing, drip irrigation or direct spraying, and the dosage is 5.0 kg - 6.0 kg per mu.

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