A synthetic microbial community for breaking dormancy of alfalfa seeds and promoting seedling growth and its application

Abstract

The present invention relates to the field of alfalfa cultivation, and particularly to a synthetic microbial community that breaks the dormancy of alfalfa seeds and promotes the growth of seedlings and its application. The present invention provides a strain of Sinorhizobium meliloti NFB5 with the preservation number of CGMCC NO. 33618, which can break the dormancy of alfalfa seeds and promote the growth of alfalfa seedlings and roots. The present invention combines Sinorhizobium meliloti NFB5 with one or two of the screened salt-tolerant Brevibacillus brevis EB3 and Bacillus sp. PS06 to obtain a synthetic microbial community. By soaking alfalfa seeds in the bacterial liquid containing the synthetic microbial community, the number of hard seeds can be significantly reduced, and the seed germination rate and germination potential can be significantly increased, indicating that the synthetic microbial community can better break the dormancy of alfalfa seeds, and there is no loss to the seeds and the seed quality will not be reduced. In addition, the synthetic microbial community also has a significant growth-promoting effect, which can significantly increase the seedling plant height, root length, stem weight and root weight.

Description

Technical Field

[0001] The present invention relates to the field of alfalfa cultivation, and particularly to a synthetic bacterial community for breaking alfalfa seed dormancy and promoting seedling growth and its application. Background Art

[0002] Seed dormancy is a biological characteristic formed by species during the long process of evolution and development to resist harsh external environments. It is an important part of plant adaptability, which can prevent plant seeds from germinating under unsuitable conditions and ensure the survival and reproduction of species in harsh environments. Leguminous plant seeds generally have physical dormancy, also known as hard seeds. Hard seediness is considered a necessary condition for the long-term survival of wild species. Many forage grass seeds with strong stress resistance generally have hard or impermeable seed coats as a mechanism to maintain the long-term viability of seeds. However, the hard seed characteristics of seeds affect seed germination and seedling establishment in the field, resulting in uneven emergence, seriously affecting the rapid establishment of forage grasses, and becoming an important issue that cannot be ignored in the artificial cultivation of leguminous forage grasses.

[0003] Alfalfa is one of the important forage grasses, with strong cold tolerance and drought tolerance. It can be promoted and planted in cold, arid and relatively barren areas, and has become a very valuable forage grass for the establishment of artificial grasslands and the improvement of degraded grasslands in China, playing a key role in the healthy development of animal husbandry. However, its seed dormancy characteristics hinder the utilization of alfalfa germplasm and variety improvement. At present, in actual production, seed dormancy can be relieved by methods such as mechanical abrasion, acid-base corrosion, and high-temperature soaking. Although these methods can break seed dormancy to a certain extent, they seriously affect seed quality, resulting in an increase in damaged seeds and abnormal seedlings, greatly increasing the seed consumption. Moreover, due to the decline in seed quality, it is difficult to form seedlings, seriously affecting the economic benefits of the forage grass planting industry. Summary of the Invention

[0004] To solve the above problems, the present invention provides a synthetic bacterial community for breaking alfalfa seed dormancy and promoting seedling growth and its application. The Sinorhizobium meliloti NFB5 and its synthetic bacterial community provided by the present invention can break alfalfa seed dormancy and promote the growth of alfalfa seedlings and roots.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Sinorhizobium meliloti ( Sinorhizobium meliloti ) NFB5, characterized in that the preservation number is CGMCC NO. 33618.

[0007] The present invention provides a synthetic bacterial community, which is composed of the Sinorhizobium meliloti NFB5 described in the above technical solution and other microorganisms; the other microorganisms include Bacillus ( Bacillus pratensis), PS06 and / or Bacillus brevis halotolerans ( Brevibacillus halotolerans ), EB3; the preservation number of the Bacillus brevis halotolerans EB3 is CGMCC NO. 33617.

[0008] The present invention provides a synthetic bacterial agent, and the synthetic bacterial agent includes the synthetic bacterial community described in the above technical solution; the OD of the synthetic bacterial community in the synthetic bacterial agent 600 value ≥ 0.2.

[0009] Preferably, the OD of the Bacillus brevis halotolerans EB3 and Bacillus sp. PS06 600 is the same; the OD of the Bacillus brevis halotolerans EB3 and Sinorhizobium meliloti NFB5 600 is the same; the OD of the Bacillus sp. PS06 and Sinorhizobium meliloti NFB5 600 is the same.

[0010] The present invention provides a preparation method of the synthetic bacterial agent described in the above technical solution, including the following steps:

[0011] Culturing the strains in the synthetic bacterial community separately to obtain the bacterial suspension of each strain; the culture medium used for the culture includes LB medium;

[0012] Mixing the bacterial suspensions of each strain to obtain the synthetic bacterial agent.

[0013] Preferably, the conditions for the culture include: the temperature is 28 - 32 °C, and the pH is 6.8 - 7.2.

[0014] The present invention provides the application of the Sinorhizobium meliloti NFB5 described in the above technical solution, or the synthetic bacterial community described in the above technical solution, or the synthetic bacterial agent described in the above technical solution, or the synthetic bacterial agent prepared by the preparation method described in the above technical solution in breaking the dormancy of alfalfa seeds and / or promoting the growth of alfalfa seedlings.

[0015] Preferably, the breaking of the dormancy of alfalfa seeds includes: increasing the germination rate and / or germination potential of alfalfa seeds; the promoting of the growth of alfalfa seedlings includes: increasing one or more of the plant height, root length, stem weight, and root weight of alfalfa seedlings.

[0016] The present invention provides a method for breaking the dormancy of alfalfa seeds, including the following steps:

[0017] Soaking the disinfected alfalfa seeds in the microbial bacterial liquid, and subjecting the soaked alfalfa seeds to germination treatment; the microbial bacterial liquid includes the Sinorhizobium meliloti NFB5 described in the above technical solution, or the synthetic bacterial community described in the above technical solution, or the synthetic bacterial agent described in the above technical solution, or the synthetic bacterial agent prepared by the preparation method described in the above technical solution.

[0018] The present invention provides a method for promoting the growth of alfalfa seedlings, comprising: applying a microbial bacterial solution to the soil planted with alfalfa seedlings; the microbial bacterial solution comprises Sinorhizobium meliloti NFB5 described in the above technical solution, or the synthetic microbial community described in the above technical solution, or the synthetic bactericide described in the above technical solution, or the synthetic bactericide prepared by the preparation method described in the above technical solution.

[0019] Beneficial effects:

[0020] The present invention provides a strain of Sinorhizobium meliloti NFB5, with the preservation number of CGMCC NO.33618. The Sinorhizobium meliloti NFB5 provided by the present invention is a strain isolated from the roots of Stipa breviflora, which can break the dormancy of alfalfa seeds and promote the growth of alfalfa seedlings and roots.

[0021] Furthermore, the present invention combines Sinorhizobium meliloti NFB5 with one or two of the screened salt-tolerant Brevibacillus sp. EB3 and Bacillus sp. PS06 to obtain a synthetic microbial community. By soaking alfalfa seeds in the bacterial solution containing the synthetic microbial community, it can significantly ( P <0.05) reduce the number of hard seeds, and significantly ( P <0.05) improve the seed germination rate and germination potential, indicating that the synthetic microbial community can better break the dormancy of alfalfa seeds, and does not cause damage to the seeds, does not reduce the seed quality, and the effect is better than that of a single strain. In addition, the synthetic microbial community also has a significant growth-promoting effect, which can significantly ( P <0.05) increase the seedling plant height, root length, stem weight and root weight, and the growth-promoting effect is better than that of a single strain. Description of the drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0023] Figure 1 It is the colony morphology diagram of EB3 strain and NFB5 strain;

[0024] Figure 2 It is the alfalfa seedling diagram treated with different test bacterial solutions in the test example;

[0025] Figure 3 It is the physical diagram of Medicago falcata plants treated with different test bacterial solutions in the test example.

[0026] Biological preservation description

[0027] Sinorhizobium meliloti NFB5, classified and named as Sinorhizobium meliloti, deposited on February 24, 2025 at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 33618.

[0028] Brevibacillus salitolerans EB3, taxonomically named Brevibacillus halotolerans , deposited on February 24, 2025 at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 33617. Detailed implementation manners

[0029] The present invention provides a strain of Sinorhizobium meliloti NFB5, with the deposit number being CGMCC NO. 33618.

[0030] The Sinorhizobium meliloti NFB5 provided by the present invention is a strain isolated from the rhizosphere soil of Medicago falcata. The colony morphology is rod-shaped, oval, slightly yellow, smooth surface, aerobic, egg-white-like, and can break the dormancy of Medicago sativa seeds and promote the growth of Medicago sativa seedlings and roots.

[0031] The present invention provides a synthetic microbial community, which is composed of the Sinorhizobium meliloti NFB5 described in the above technical solution and other microorganisms; the other microorganisms include Bacillus PS06 and / or Brevibacillus salitolerans EB3; the deposit number of Brevibacillus salitolerans EB3 is CGMCC NO. 33617. The Bacillus PS06 of the present invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the deposit number being CGMCC 1.17355 T , and is disclosed in the thesis [Shi Jiajia. Identification of a new strain of Bacillus PS06 isolated from the rhizosphere soil of Stipa breviflora and its growth promotion effect [D]. Inner Mongolia: Inner Mongolia University, 2021.].

[0032] As an implementation manner, the other microorganisms include Bacillus PS06 and Sinorhizobium meliloti NFB5.

[0033] The Brevibacillus salitolerans EB3 provided by the present invention is a strain isolated from the roots of Stipa breviflora. The colony morphology is rod-shaped, oval, slightly yellowish, opaque, rough surface, aerobic, irregular edges, and can break the dormancy of Medicago sativa seeds and promote the growth of Medicago sativa seedlings and roots.

[0034] The present invention combines the Sinorhizobium meliloti NFB5 with one or two of the screened Brevibacillus salitolerans EB3 and Bacillus PS06 to obtain a synthetic microbial community, and this synthetic microbial community can significantly (P <0.05) reduce the number of hard seeds, significantly ( P <0.05) improve the seed germination rate and germination potential, indicating that the synthetic microbial community can better break the dormancy of alfalfa seeds, and there is no loss to the seeds, the seed quality is not reduced, and the effect is better than that of a single strain. In addition, the synthetic microbial community also has a significant growth-promoting effect, which can significantly ( P <0.05) increase the seedling plant height, root length, stem weight and root weight, and the growth-promoting effect is better than that of a single strain.

[0035] Based on the above advantages, the present invention provides a synthetic microbial agent, and the synthetic microbial agent includes the synthetic microbial community described in the above technical solution; the OD of the synthetic microbial community in the synthetic microbial agent 600 value ≥ 0.2. As an implementation manner, the OD of the synthetic microbial community in the synthetic microbial agent 600 value is 0.2.

[0036] As an implementation manner, the OD of the halotolerant Brevibacillus sp. EB3 and Bacillus sp. PS06 600 values are the same; the OD of the halotolerant Brevibacillus sp. EB3 and Sinorhizobium meliloti NFB5 600 values are the same; the OD of the Bacillus sp. PS06 and Sinorhizobium meliloti NFB5 600 values are the same.

[0037] The present invention provides a preparation method of the synthetic microbial agent described in the above technical solution, including the following steps:

[0038] Cultivate the strains in the synthetic microbial community respectively to obtain the bacterial suspensions of each strain; the culture medium used for the cultivation includes LB medium;

[0039] Mix the bacterial suspensions of each strain to obtain the synthetic microbial agent.

[0040] As an implementation manner, the conditions for the cultivation include: the temperature is 28 - 32 °C, and the pH is 6.8 - 7.2.

[0041] As an implementation manner, the OD of each bacterial suspension 600 value ≥ 0.2; as another implementation manner, the OD of each bacterial suspension 600 value is 0.2. As an implementation manner, mix the bacterial suspensions of each strain in equal volume to obtain the synthetic microbial agent.

[0042] Based on the above advantages, the present invention provides the application of the synthetic microbial agent prepared by the Sinorhizobium meliloti NFB5 described in the above technical solution, or the synthetic microbial community described in the above technical solution, or the synthetic microbial agent described in the above technical solution, or the preparation method described in the above technical solution in breaking the dormancy of alfalfa seeds and / or promoting the growth of alfalfa seedlings.

[0043] As an embodiment, breaking the dormancy of alfalfa seeds includes: increasing the germination rate and / or germination potential of alfalfa seeds; promoting the growth of alfalfa seedlings includes: increasing one or more of the plant height, root length, stem weight and root weight of alfalfa seedlings.

[0044] Based on the above advantages, the present invention provides a method for breaking alfalfa seed dormancy, comprising the following steps:

[0045] The sterilized alfalfa seeds are soaked in a microbial solution, and the soaked alfalfa seeds are germinated; the microbial solution includes the alfalfa Sinorhizobium NFB5 described in the above technical solution, or the synthetic bacterial community described in the above technical solution, or the synthetic bacterial agent described in the above technical solution, or the synthetic bacterial agent prepared by the preparation method described in the above technical solution.

[0046] Based on the above advantages, the present invention provides a method for promoting the growth of alfalfa seedlings, comprising: applying a microbial solution to soil in which alfalfa seedlings are planted; the microbial solution comprises the alfalfa Sinorhizobium NFB5 described in the above technical scheme or the synthetic bacterial community described in the above technical scheme or the synthetic bacterial agent described in the above technical scheme or the synthetic bacterial agent prepared by the preparation method described in the above technical scheme.

[0047] To further illustrate the present invention, a synthetic bacterial community for breaking alfalfa seed dormancy and promoting seedling growth and its application provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] (1) Isolation and identification of bacterial strains

[0050] EB3: Take fresh nutrient roots of Stipa brevis, rinse them repeatedly with sterile water for 3-4 times, soak them in 75% alcohol by volume for 1 min, rinse them with sterile water for 3-4 times, disinfect them with 1% sodium hypochlorite solution by mass for 30 s, rinse them with sterile water for 3-4 times, and dry them with sterile filter paper. Select 0.1 g of the sterilized roots, cut them into small pieces of about 1 cm in length with sterile scissors under sterile conditions, place them in a sterile mortar, add 1 mL of sterile water, and grind the roots thoroughly into a homogenate. Take 200 µL of the ground homogenate and spread it on NB medium, invert and culture at 30°C; according to the morphology of the colonies, pick colonies of different morphologies under sterile conditions, inoculate them on LB purification medium, and pass them three times to obtain pure cultured bacteria, which are recorded as EB3.

[0051] NFB5: Take 1 g of rhizosphere soil of yellow alfalfa and dilute it to 10 with 10 mL of sterile water. -1 , 10-2 and 10 -3 and 10 -4 Suspensions with dilution factors of -2 and -3 were separately spread on NFM nitrogen-fixing medium and cultured in an inverted position at 30 °C. According to the colony morphology, colonies with different morphologies were picked under sterile conditions and inoculated onto LB purification medium for three passages to obtain pure-cultured bacteria, denoted as NFB5.

[0052] Total bacterial DNA was extracted, and universal primers for 16S rDNA were selected for PCR amplification, which was then sent to Shanghai Majorbio for sequencing. Through sequence alignment, strain EB3 was preliminarily identified as belonging to the genus Brevibacillus, with a similarity of 99.02% to the 16S rRNA gene of the known type strain of the genus Brevibacillus. Strain NFB5 was identified as belonging to the genus Sinorhizobium meliloti, with a similarity of 99.85% to the 16S rRNA gene of the known type strain of the genus Sinorhizobium meliloti.

[0053] (2)Physiological and biochemical identification of EB3 and NFB5

[0054] Strains EB3 and NFB5 were separately inoculated onto LB solid medium and cultured at a temperature of 28 - 32 °C and a pH of 7.0 for 72 h, and the colony morphology was observed. The results are shown in Figure 1 . The results showed that the colony morphology of strain EB3 was rod-shaped, oval, slightly yellowish, opaque, with a rough surface and irregular edges; the colony morphology of strain NFB5 was rod-shaped, oval, slightly yellow, with a smooth surface and an egg-white shape.

[0055] In summary, strain EB3 was named Brevibacillus halotolerans EB3 and deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC NO. 33617; strain NFB5 was named Sinorhizobium meliloti NFB5 and deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC NO. 33618.

[0056] Example 2

[0057] 1. Preparation of bacterial suspensions of different strains: 100 mL of LB liquid medium was prepared, and after sterilization and cooling, Bacillus sp. PS06, Brevibacillus halotolerans EB3 and Sinorhizobium meliloti NFB5 screened in Example 1 were separately added. The mixture was shaken and cultured at 28 °C and 150 r / min for 36 h. The growth of bacteria was measured with the uninoculated medium as a negative control. The OD 600 value of the bacterial solution was adjusted to 0.2, and it was centrifuged at 5000 r / min for 10 min. The supernatant was discarded, and the bacteria adhering to the bottom of the centrifuge tube were washed with sterile water to obtain three bacterial suspensions with an OD 600 value of 0.2 (denoted as PS06, EB3 and NFB5 respectively).

[0058] 2. Bacterial suspension of synthetic microbiota 1 (PS06 + EB3 + NFB5): Equal volumes of the 3 bacterial suspensions prepared in step 1 were mixed, and sterile water was added to adjust the OD 600 value of the mixed bacterial solution to 0.2, obtaining the bacterial suspension of synthetic microbiota 1.

[0059] 3. Bacterial suspension of synthetic microbiota 2 (PS06 + EB3): Equal volumes of the PS06 and EB3 bacterial suspensions prepared in step 1 were mixed, and sterile water was added to adjust the OD 600 value of the mixed bacterial solution to 0.2, obtaining the bacterial suspension of synthetic microbiota 2.

[0060] 4. Bacterial suspension of synthetic microbiota 3 (EB3 + NFB5): Equal volumes of the EB3 and NFB5 bacterial suspensions prepared in step 1 were mixed, and sterile water was added to adjust the OD 600 value of the mixed bacterial solution to 0.2, obtaining the bacterial suspension of synthetic microbiota 3.

[0061] 5. Bacterial suspension of synthetic microbiota 4 (PS06 + NFB5): Equal volumes of the PS06 and NFB5 bacterial suspensions prepared in step 1 were mixed, and sterile water was added to adjust the OD 600 value of the mixed bacterial solution to 0.2, obtaining the bacterial suspension of synthetic microbiota 4.

[0062] Comparative example 1

[0063] A synthetic supernatant was prepared as follows: The bacterial suspensions of the 3 strains prepared in Example 2 were centrifuged respectively, and the supernatants were taken and mixed in equal volumes to obtain the synthetic supernatant.

[0064] Test example

[0065] The bacterial suspensions of the 3 strains prepared in Example 2 were adjusted to an OD 600 value of 0.2 with sterile water respectively to obtain the test bacterial solutions of the 3 strains.

[0066] The bacterial suspensions of synthetic microbiota 1 - 4 prepared in Example 2, the test bacterial solutions of the 3 strains, sterile water, LB liquid medium, and the synthetic supernatant in Comparative example 1 were used as test reagents respectively to conduct germination experiments and pot experiments on alfalfa. Each kind of seed was subjected to 3 parallel repeated experiments with each test reagent. The steps are as follows:

[0067] 1. Selection of alfalfa seeds: Seeds of Medicago falcata and Medicago sativa with a hard seed rate of 57% were selected respectively.

[0068] 2. Seed disinfection: The alfalfa seeds were first soaked in sterile water for 5 min, then disinfected with 1% sodium hypochlorite for 30 s, and then washed 3 times with sterile water.

[0069] 3. Inoculation treatment: Add the sterilized alfalfa seeds into 5 mL of the test agent and shake at 30°C and 150 r / min for 6 hours.

[0070] 4. Germination experiment: After pouring out the suspension of synthetic bacteria, the seeds were placed on sterile filter paper, and after absorbing the water, they were placed in a culture dish covered with two layers of moist filter paper. 100 seeds were placed in each dish, and 3 replicates were placed in a light incubator. They were cultured at a constant temperature of 20°C, 8 hours of light, and 16 hours of darkness. The initial count began on the 4th day, and the number of germinated seedlings was counted daily. The last count day was the 10th day, and the number of normal seedlings, abnormal seedlings, hard seeds, dead seeds, germination rate, and germination potential were finally counted.

[0071] Germination rate = (number of seeds germinated within the specified germination days / total number of seeds tested) × 100%;

[0072] Germination potential = (total number of seeds germinated when the germination number reaches the peak / number of seeds tested) × 100%.

[0073] 5. Pot experiment: Select normal seedlings with consistent germination and plant them in pots (11 cm high × 12 cm in diameter) filled with 500 g sterilized soil. Plant 5 pots for each treatment, add 20 mL of the test agent diluted 3 times, and cultivate at a constant temperature of 25°C, 16 hours of light, 8 hours of darkness, and water regularly. After 60 days of cultivation, measure the plant height, root length, stem weight and root weight. Some results are shown in Figure 2 .

[0074] The preparation method of the sterilized soil is as follows: after removing roots, dead branches and gravel from the collected soil with a 2 mm sieve, vermiculite is added in a volume ratio of soil: vermiculite = 7:3, sterilized at 120° C. for 20 minutes, and sterilized again after an interval of 12 hours.

[0075] The results are shown in Tables 1~4.

[0076] Table 1 Effects of different tested agents on the germination rate of yellow alfalfa seeds

[0077]

[0078] Note: Different lowercase letters represent significant differences, and the same applies to the following tables.

[0079] Table 2 Effects of different tested agents on the morphological characteristics of yellow-flowered alfalfa seedlings

[0080]

[0081] Table 3 Effects of different tested agents on the germination rate of alfalfa seeds

[0082]

[0083] Table 4 Effects of Different Test Reagents on the Morphological Characteristics of Alfalfa Seedlings

[0084]

[0085] The test results show that after treating the seeds of Medicago falcata and Medicago sativa with the synthetic microbial community constructed by the present invention, the number of hard seeds can be significantly reduced ( P <0.05), and the germination rate and germination potential of the seeds can be significantly increased ( P <0.05). Moreover, the synthetic microbial community 1 (PS06 + EB3 + NFB5) has the best effect, indicating that this synthetic microbial community can better break the dormancy of alfalfa seeds without causing damage to the seeds and without reducing the seed quality (Tables 1 and 3). In addition, this synthetic microbial community also has a significant growth-promoting effect, which can significantly increase the seedling plant height, root length, stem weight and root weight ( P <0.05) (Tables 2 and 4), and the synthetic microbial community 1 (PS06 + EB3 + NFB5) has the best effect ( Figure 3 ).

[0086] In summary, the strains and their synthetic microbial community provided by the present invention can break the dormancy of alfalfa seeds and promote the growth of alfalfa seedlings and roots.

[0087] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A synthetic microbial community, characterized in that, The synthetic bacterial community consists of Sinorhizobium meliloti ( Sinorhizobium meliloti ), NFB5, Bacillus sp. ( Bacillus pratensis ), PS06, and Brevibacillus salexigens ( Brevibacillus halotolerans ), EB3; The Rhizobium meliloti ( Sinorhizobium meliloti ) NFB5 has a deposit number of CGMCC NO.33618; the halotolerant Brevibacillus EB3 has a deposit number of CGMCC NO.33617; the Bacillus ( Bacillus pratensis ) PS06 was disclosed in the thesis: Shi Jiajia. Identification of a new strain of Bacillus PS06 isolated from the rhizosphere soil of Stipa breviflora and its growth-promoting effect [D]. Inner Mongolia: Inner Mongolia University, 2021.

2. A synthetic microbial agent, characterized in that, The synthetic microbial inoculum comprises the synthetic microbial community described in claim 1; the OD600 value of the synthetic microbial community in the synthetic microbial inoculum is ≥ 0.

2.

3. The synthetic microbial agent according to claim 2, wherein The OD600 values of the halotolerant Brevibacillus EB3 and Bacillus PS06 are the same; the OD600 values of the halotolerant Brevibacillus EB3 and Sinorhizobium meliloti NFB5 are the same; the OD600 values of Bacillus PS06 and Sinorhizobium meliloti NFB5 are the same.

4. The preparation method of the synthetic microbial agent according to claim 2 or 3, characterized in that, Comprising the following steps: Culturing the strains in the synthetic microbial community separately to obtain the bacterial suspensions of each strain; the culture medium used for the culture comprises LB medium. Mixing the bacterial suspensions of each strain to obtain the synthetic microbial inoculum.

5. The preparation method according to claim 4, characterized in that, The conditions for the culture include: the temperature is 28 - 32 °C, and the pH is 6.8 - 7.

2.

6. Application of the synthetic microbial community described in claim 1 or the synthetic microbial inoculum described in claim 2 or 3 or the synthetic microbial inoculum prepared by the preparation method described in claim 4 or 5 in breaking the dormancy of alfalfa seeds and / or promoting the growth of alfalfa seedlings.

7. The application according to claim 6, wherein The breaking of the dormancy of alfalfa seeds includes: increasing the germination rate and / or germination potential of alfalfa seeds; the promoting of the growth of alfalfa seedlings includes: increasing one or more of the plant height, root length, shoot weight, and root weight of alfalfa seedlings.

8. A method for breaking the dormancy of alfalfa seeds, characterized in that, Comprising the following steps: Soaking the disinfected alfalfa seeds in the microbial liquid, and subjecting the soaked alfalfa seeds to germination treatment; the microbial liquid comprises the synthetic microbial community described in claim 1 or the synthetic microbial inoculum described in claim 2 or 3 or the synthetic microbial inoculum prepared by the preparation method described in claim 4 or 5.

9. A method for promoting the growth of alfalfa seedlings, characterized in that, Including: Applying the microbial liquid to the soil planted with alfalfa seedlings; the microbial liquid comprises the synthetic microbial community described in claim 1 or the synthetic microbial inoculum described in claim 2 or 3 or the synthetic microbial inoculum prepared by the preparation method described in claim 4 or 5.

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