Method for improving pollination rate and podding rate of alfalfa

Abstract

The invention provides a method for improving pollination rate and podding rate of alfalfa, and belongs to the technical field of biology. The invention provides a strain of salt-tolerant Bacillus brevis EB3, the preservation number of which is CGMCC No.33617, and further provides a microbial inoculum composition composed of the strain and marine yeast, a composition composed of the microbial inoculum composition and a pollen nutrient solution, and a method for improving the pollination rate and the pod bearing rate of alfalfa by using the composition. The method effectively solves the problems of low setting rate of alfalfa, abortion of florets, serious flower drop and the like, can significantly improve the number of alfalfa pollen and the germination rate of the pollen, and reduces the drop rate of the florets, so that the effect of significantly improving the seed yield of the alfalfa is achieved, and the method is simple to operate, stable in effect and low in cost.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving the pollination rate and pod setting rate of alfalfa. Background Art

[0002] Alfalfa is a plant of the genus Medicago ( Medicago L.) is a general term for annual or perennial herbaceous plants with the characteristics of high yield, rich protein, good palatability and strong adaptability. China is one of the main distribution areas of alfalfa plants. Common ones include purple alfalfa ( Medicago sativa L.) and Medicago truncatula ( Medicago falcate L.) is an excellent forage grass for artificial grassland construction and natural grassland improvement in my country, and plays an important role in the development of my country's animal husbandry. However, alfalfa is a typical cross-pollinating plant, and its self-pollination fruiting rate is very low. In addition, alfalfa flowers have a unique opening mechanism, and its pollination is a complex process, often relying on external mechanical forces and insects to open the tightly packed keel petals for pollination. Therefore, due to its self-incompatibility and special keel petal structure, the natural pollination rate is very low, the flower drop phenomenon is serious, and seed reproduction is difficult.

[0003] Improving the pollination rate and pod-setting rate of alfalfa during flowering is the key to increasing alfalfa yield. At present, feasible methods for improving the pollination rate and pod-setting rate of alfalfa include insect pollination technology, environmental control technology, artificial pollination technology and breeding technology. However, insect pollination technology is usually used for alfalfa pollination by breeding insects such as alfalfa leaf-cutting bees. The activities and reproduction of insects are affected by climate, the pollination rate is unstable, and breeding and management require professional technology and equipment, which makes large-scale application difficult. Environmental control technology requires a lot of investment in facility construction and energy consumption, and it is difficult to accurately control. If the temperature, humidity, light, wind speed, etc. are not properly adjusted, it is easy to cause negative effects. Artificial pollination requires a lot of professional equipment and manpower costs, and the efficiency is extremely low. Breeding technology is not yet mature, and there are problems such as long time cycle, high technical requirements, and high R&D costs. Therefore, it is urgent to study a simple, efficient, and stable method for improving the pollination rate and pod-setting rate of alfalfa, so as to solve the problems of low alfalfa fruit setting rate and insufficient supply of seed yield. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the object of the present invention is to provide a salt-tolerant Brevibacillus ( Brevibacillus halotolerans )EB3, with the deposit number of CGMCC No.33617, can be used to improve the pollination rate, pod setting rate and seed yield of alfalfa during flowering period.

[0005] The present invention also aims to provide a bacterial agent composition, composition and method for improving the pollination rate and / or pod setting rate of alfalfa, which can improve the pollen germination rate, reduce the floret shedding rate, and improve the pod setting rate and seed yield of alfalfa.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a salt-tolerant Brevibacillus ( Brevibacillus halotolerans )EB3, the deposit number is CGMCC No.33617.

[0007] The present invention also provides a bacterial agent composition, comprising a marine yeast suspension and a bacterial suspension of the above-mentioned salt-tolerant Brevibacillus EB3.

[0008] The present invention also provides application of the salt-tolerant Brevibacillus EB3 or the bacterial agent composition in alfalfa planting.

[0009] The invention also provides a composition for improving the pollination rate and / or pod-setting rate of alfalfa, comprising a pollen nutrient solution and the above-mentioned bacterial agent composition.

[0010] Preferably, the pollen nutrient solution comprises: 8-12 g / L sucrose, 40-60 mg / L boric acid, 60-90 mg / L calcium chloride, 30-50 mg / L magnesium sulfate, 650-840 μg / L naphthaleneacetic acid, 520-865 μg / L gibberellin and 1000-1200 μg / L 2,4-D.

[0011] Preferably, in the bacterial agent composition, the number of viable bacteria of the salt-tolerant Brevibacillus EB3 bacterial suspension is ≥ 1×10 8 cfu / mL; the number of viable bacteria in the marine yeast suspension is 1×10 11 ~9×10 11 cells / L.

[0012] The present invention also provides application of the composition for improving the pollination rate and / or pod setting rate of alfalfa in alfalfa planting.

[0013] The present invention also provides a method for improving the pollination rate and / or pod setting rate of alfalfa, which comprises spraying the above-mentioned composition for improving the pollination rate and / or pod setting rate of alfalfa on the leaf surface of alfalfa, and the spraying steps are as follows: spraying a salt-tolerant Brevibacillus EB3 bacterial suspension at the budding stage of alfalfa; spraying a pollen nutrient solution at the initial flowering stage of alfalfa; and spraying a marine yeast suspension at the full flowering stage of alfalfa.

[0014] Preferably, the spraying amount of the pollen nutrient solution is 50-70 L / hm 2 .

[0015] Preferably, the spraying amount of the salt-tolerant Bacillus brevis EB3 bacterial suspension is 45-55 L / mu; the spraying amount of the marine yeast suspension is 45-55 L / mu.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The present invention obtains a salt-tolerant Brevibacillus for the first time. Brevibacillus halotolerans ) EB3, and based on the combination of the strain and marine yeast, a bacterial agent composition is obtained; the present invention further provides a composition and method for improving the pollination rate and / or pod setting rate of alfalfa, which effectively solves the problems of low fruit setting rate, floret abortion, and severe flower drop of alfalfa, and can significantly increase the number of alfalfa pollen and pollen germination rate, and reduce the floret drop rate, thereby achieving the effect of significantly improving the pollination rate and pod setting rate of alfalfa, and increasing the seed yield of alfalfa. The method of the present invention is simple to operate, has stable effect, and is low in cost.

[0017] Biological Deposit Description Halophilic Bacillus Brevibacillus ( Brevibacillus halotolerans EB3, classified as Brevibacillus halodurans ( Brevibacillus halotolerans ), depository: General Microbiology Center of China Culture Collection Administration, depository address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No.33617, deposit date February 24, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :The colony morphology of halodurable Brevibacillus EB3. DETAILED DESCRIPTION

[0019] The present invention provides a salt-tolerant Brevibacillus ( Brevibacillus halotolerans ) EB3, the salt-tolerant Brevibacillus EB3 is deposited as CGMCC No.33617. The salt-tolerant Brevibacillus EB3 of the present invention is isolated and purified from the fresh nutrient roots of Stipa breviflora, and is proved to belong to Brevibacillus halotolerans , and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on February 24, 2025. The address of the deposit unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] The present invention also provides a bacterial composition comprising marine yeast and the above-mentioned salt-tolerant Brevibacillus EB3, wherein the bacterial composition comprises a marine yeast suspension and a bacterial suspension of the above-mentioned salt-tolerant Brevibacillus EB3. The marine yeast is a conventionally purchased marine yeast strain.

[0021] The present invention also provides the use of the above-mentioned bacterial agent composition in alfalfa planting, wherein the salt-tolerant Brevibacillus EB3 bacterial suspension and the marine yeast suspension are sprayed on the alfalfa leaf surface respectively, which can increase the pollinating insect flower visiting rate, thereby increasing the alfalfa flowering pollination rate, alfalfa pod setting rate and alfalfa seed yield. The alfalfa includes but is not limited to yellow alfalfa and purple alfalfa.

[0022] Based on the bacterial agent composition, the present invention further provides a composition for improving the pollination rate and / or pod setting rate of alfalfa, comprising a pollen nutrient solution and the bacterial agent composition. The alfalfa includes but is not limited to yellow alfalfa and purple alfalfa.

[0023] In the present invention, the pollen nutrient solution comprises: 8-12 g / L sucrose, 40-60 mg / L boric acid, 60-90 mg / L calcium chloride, 30-50 mg / L magnesium sulfate, 650-840 μg / L naphthaleneacetic acid, 520-865 μg / L gibberellin and 1000-1200 μg / L 2,4-D. Preferably, it comprises 10 g / L sucrose, 50 mg / L boric acid, 80 mg / L calcium chloride, 40 mg / L magnesium sulfate, 750 μg / L naphthaleneacetic acid, 700 μg / L gibberellin and 1100 μg / L 2,4-D. The preparation method of the pollen nutrient solution of the present invention comprises the following steps: dissolving sucrose, boric acid, calcium chloride and magnesium sulfate in water, adding naphthaleneacetic acid, gibberellin and 2,4-D, adjusting the pH value to 5-6 with hydrochloric acid or sodium hydroxide and then settling to 1 L. The pH value of the pollen nutrient solution finally obtained by the present invention is preferably 5.5.

[0024] In the bacterial composition of the present invention, the number of viable bacteria of the salt-tolerant Brevibacillus EB3 bacterial suspension is ≥ 1×10 8 cfu / mL; the number of viable bacteria in the marine yeast suspension is 1×10 11 ~9×10 11 cells / L, preferably 2×10 11 ~8×10 11 As an optional embodiment, the preparation of the bacterial suspension of the present invention comprises the following steps: preparing 100 mL of LB liquid culture medium, adding salt-tolerant Brevibacillus EB3 after sterilization and cooling, and shaking and culturing at 28°C and 150 r / min for 36 hours, and the OD of the bacterial liquid is 600 When the value was 0.2, centrifuge at 5000r / min for 10min, discard the supernatant, and wash the bacteria adhering to the bottom of the centrifuge tube with sterile water to obtain the OD 600 The effective viable count of the salt-tolerant Bacillus brevis EB3 obtained by the bacterial suspension with a value of 0.2 was 1×10 8 As an optional embodiment, the method for preparing the cell suspension of marine yeast of the present invention comprises the following steps: adding a monoclonal strain of marine yeast to a sterile aqueous solution containing 300-400 g / L of sucrose, culturing at 24-26°C for 40-55 h, and obtaining a viable cell count of 1×10 11 ~9×10 11 cells / L marine yeast suspension. The culture temperature is preferably 25°C, and the culture time is preferably 48h.

[0025] The present invention also provides a method for improving the pollination rate and / or pod setting rate of alfalfa, comprising the following steps: spraying a salt-tolerant Bacillus brevis EB3 bacterial suspension during the budding period of alfalfa; spraying a pollen nutrient solution during the initial flowering period of alfalfa; and spraying a cell suspension of the above-mentioned marine yeast during the full flowering period of alfalfa. The present invention promotes the reproductive growth of alfalfa by first spraying a salt-tolerant Bacillus brevis EB3 bacterial suspension on the leaves of alfalfa plants in the budding period; then spraying a pollen nutrient solution on the leaves of alfalfa plants in the initial flowering period to stimulate pollen germination and improve the pollen germination rate; and spraying a cell suspension of marine yeast during the full flowering period to increase the flower visiting rate of pollinating insects, thereby increasing the pollination rate and pod setting rate of alfalfa during flowering, effectively solving the problems of low fruit setting rate, floret abortion, and severe flower drop of alfalfa. The alfalfa includes but is not limited to yellow alfalfa and purple alfalfa.

[0026] The alfalfa budding period of the present invention is set as follows: when the growth point of the alfalfa plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, it indicates that the alfalfa begins to enter the budding period. The spraying of the salt-tolerant Brevibacillus EB3 bacterial suspension of the present invention is preferably foliar spraying, and the spraying amount is 45-55 L / mu, preferably 50 L / mu.

[0027] The initial flowering period of alfalfa in the present invention is set as: when the number of open florets on 75-85% of the inflorescences accounts for 15-25% of the total number of florets. The spraying of the pollen nutrient solution in the present invention is preferably foliar spraying, and the spraying amount is 50-70 L / hm 2 , preferably 60 L / hm 2 .

[0028] The alfalfa flowering period for spraying the marine yeast cell suspension of the present invention is 6 to 8 days after spraying the pollen nutrient solution, preferably 7 days after spraying the pollen nutrient solution, and the spraying amount of the marine yeast cell suspension is 45 to 55 L / mu, preferably 50 L / mu.

[0029] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] In a specific embodiment of the present invention, the marine yeast was purchased from Inner Mongolia Shengkang Biotechnology Co., Ltd.

[0031] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Example 1 Halophilic Bacillus Brevibacillus ( Brevibacillus halotolerans ) Isolation and identification of EB3: Take fresh nutrient roots of Stipa brevis, rinse them repeatedly with sterile water for 3-4 times, soak them in 75% alcohol for 1 min, rinse them with sterile water for 3-4 times, disinfect them with 1% sodium hypochlorite solution for 30 seconds, rinse them with sterile water for 3-4 times, and dry them with sterile filter paper. Select 0.1g of the sterilized roots, cut them into small pieces of about 1cm in length with sterilized scissors under sterile conditions, place them in a sterilized mortar, add 1mL of sterile water, and grind the roots thoroughly into a homogenate. Take 200µL of the ground homogenate and spread it on NB culture medium, invert and culture at 30℃; 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.

[0034] The total bacterial DNA was extracted, and the 16S rDNA universal primers were selected for PCR amplification. The DNA was then sent to Shanghai Meiji Biotechnology for sequencing. By sequence comparison, the strain EB3 was identified as Halophilic Brevibacillus ( Brevibacillus halotolerans ).

[0035] The EB3 strain was inoculated into LB solid medium and cultured at 30°C and pH 7 for 52 h. The colony morphology was observed. Figure 1 The results showed that the colony morphology of the EB3 strain was rod-shaped, oval, slightly yellow, opaque, with a rough surface and irregular edges.

[0036] Example 2 A bacterial agent composition comprises a salt-tolerant brevis bacillus EB3 bacterial suspension and a marine yeast suspension.

[0037] Halophilic Bacillus brevis EB3 bacterial suspension: prepare 100 mL LB liquid medium, sterilize and cool, add halophilic Bacillus brevis EB3, shake and culture at 28°C, 150 r / min for 36 h, and the bacterial suspension OD 600 When the value was 0.2, centrifuge at 5000r / min for 10min, discard the supernatant, and wash the bacteria adhering to the bottom of the centrifuge tube with sterile water to obtain the OD 600 The effective viable count of the salt-tolerant Brevibacillus EB3 obtained by the bacterial suspension with a value of 0.2 was 2×10 8 cfu / mL.

[0038] Marine yeast suspension: add marine yeast monoclonal strain to a sterile aqueous solution containing 386 g / L sucrose and culture at 25 °C for 40 h to obtain a viable cell count of 2.6 × 10 11 cells / L of marine yeast suspension.

[0039] Example 3 A bacterial agent composition comprises a salt-tolerant brevis bacillus EB3 bacterial suspension and a marine yeast suspension.

[0040] Halophilic Bacillus brevis EB3 bacterial suspension: prepare 100 mL LB liquid medium, sterilize and cool, add halophilic Bacillus brevis EB3, shake and culture at 28°C, 150 r / min for 36 h, and the bacterial suspension OD 600 When the value was 0.2, centrifuge at 5000r / min for 10min, discard the supernatant, and wash the bacteria adhering to the bottom of the centrifuge tube with sterile water to obtain the OD 600 The effective viable count of the salt-tolerant Brevibacillus EB3 obtained from the bacterial suspension with a value of 0.2 was 4.6×10 8 cfu / mL.

[0041] Marine yeast suspension: add marine yeast monoclonal strain to a sterile aqueous solution containing 345 g / L sucrose and culture at 25 °C for 55 h to obtain a viable cell count of 6.5 × 10 11 cells / L of marine yeast suspension.

[0042] Example 4 A composition for improving the pollination rate and / or pod setting rate of alfalfa: pollen nutrient solution and the bacterial agent composition of embodiment 3.

[0043] Pollen nutrient solution: 8g / L sucrose, 40mg / L boric acid, 60mg / L calcium chloride, 30mg / L magnesium sulfate, 650μg / L naphthaleneacetic acid, 520μg / L gibberellin and 1000μg / L 2,4-D. Preparation method: Dissolve sucrose, boric acid, calcium chloride and magnesium sulfate in water, adjust the pH value to 5 with hydrochloric acid / sodium hydroxide, add naphthaleneacetic acid, gibberellin and 2,4-D, and adjust the volume to 1L.

[0044] Example 5 A composition for improving the pollination rate and / or pod setting rate of alfalfa: pollen nutrient solution and the bacterial agent composition of embodiment 3.

[0045] Pollen nutrient solution: 12g / L sucrose, 60mg / L boric acid, 90mg / L calcium chloride, 50mg / L magnesium sulfate, 840μg / L naphthaleneacetic acid, 865μg / L gibberellin and 1200μg / L 2,4-D. Preparation method: Dissolve sucrose, boric acid, calcium chloride and magnesium sulfate in water, adjust the pH value to 5.5 with hydrochloric acid / sodium hydroxide, add naphthaleneacetic acid, gibberellin and 2,4-D, and adjust the volume to 1L.

[0046] Example 6 A composition for improving the pollination rate and / or pod setting rate of alfalfa: pollen nutrient solution and the bacterial agent composition of embodiment 3.

[0047] Pollen nutrient solution: 10g / L sucrose, 50mg / L boric acid, 80mg / L calcium chloride, 40mg / L magnesium sulfate, 750μg / L naphthaleneacetic acid, 700μg / L gibberellin and 1100μg / L 2,4-D. Preparation method: Dissolve sucrose, boric acid, calcium chloride and magnesium sulfate in water, adjust the pH value to 6 with hydrochloric acid / sodium hydroxide, add naphthaleneacetic acid, gibberellin and 2,4-D, and adjust the volume to 1L.

[0048] Example 7 A method for improving the pollination rate and / or pod setting rate of alfalfa: The composition described in Example 4 was used to spray the salt-tolerant Brevibacillus EB3 suspension at the budding stage of alfalfa (observe the top of the alfalfa plant. When the growth point of the plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, it indicates that the alfalfa has entered the budding stage). The amount of foliar spraying was 50 L / mu. At the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounts for 20% of the total number of small flowers), the pollen nutrient solution was sprayed on the leaves at a spraying rate of 50 L / hm 2 ; During the flowering period of alfalfa (6 days after spraying pollen nutrient solution), spray the cell suspension of marine yeast on the leaves at a spraying rate of 45L / mu.

[0049] Example 8 A method for improving the pollination rate and / or pod setting rate of alfalfa: The composition described in Example 5 was used to spray the salt-tolerant Brevibacillus EB3 suspension at the budding stage of alfalfa (observe the top of the alfalfa plant. When the growth point of the plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, it indicates that the alfalfa has entered the budding stage). The amount of foliar spraying was 50 L / mu. At the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounts for 20% of the total number of small flowers), the pollen nutrient solution was sprayed on the leaves at a spraying rate of 70L / hm 2 ; During the flowering period of alfalfa (6 days after spraying pollen nutrient solution), spray the cell suspension of marine yeast on the leaves at a spraying rate of 55L / mu.

[0050] Example 9 A method for improving the pollination rate and / or pod setting rate of alfalfa: The composition described in Example 6 was used to spray the salt-tolerant Brevibacillus EB3 suspension at the budding stage of alfalfa (observe the top of the alfalfa plant. When the growth point of the plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, it indicates that the alfalfa has entered the budding stage). The amount of foliar spraying was 50 L / mu. At the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounts for 20% of the total number of small flowers), the pollen nutrient solution was sprayed on the leaves at a spraying rate of 60L / hm 2; During the flowering period of alfalfa (6 days after spraying pollen nutrient solution), spray the cell suspension of marine yeast on the leaves at a spraying rate of 50L / mu.

[0051] Test Example 1 1. Test variety: Yellow Medicago 2. Experimental location: Baiyinxile Pasture, Xilin Gol League, Inner Mongolia 3. Experimental groups: Treatment group 1: using the composition described in Example 6; in the budding stage of alfalfa (observing the top of the alfalfa plant, when the growth point of the plant begins to differentiate into small flower buds, and the first flower bud recognizable to the naked eye appears, indicating that the alfalfa has entered the budding stage, June 1, 2024), the salt-tolerant Brevibacillus EB3 suspension was sprayed on the leaves at 50 L / mu; in the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounted for 20% of the total number of small flowers, June 20, 2024), the pollen nutrient solution was sprayed on the leaves at a spraying rate of 60 L / hm 2 ; During the flowering period of alfalfa (7 days after spraying pollen nutrient solution), spray the cell suspension of marine yeast on the leaves at a spraying rate of 50 L / mu.

[0052] Treatment group 2: using the composition described in Example 5; at the budding stage of alfalfa (observing the top of the alfalfa plant, when the growth point of the plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, indicating that the alfalfa has entered the budding stage, June 1, 2024), spraying the salt-tolerant Brevibacillus EB3 suspension at 50 L / mu; at the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounts for 20% of the total number of small flowers, June 20, 2024), spraying the pollen nutrient solution on the leaves, the spraying amount is 60L / hm 2 ; During the flowering period of alfalfa (7 days after spraying pollen nutrient solution), spray the cell suspension of marine yeast on the leaves at a spraying rate of 50 L / mu.

[0053] Treatment group 3: using the composition described in Example 4; at the budding stage of alfalfa (observing the top of the alfalfa plant, when the growth point of the plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, indicating that the alfalfa has entered the budding stage, June 1, 2024), the salt-tolerant Brevibacillus EB3 suspension was sprayed on the leaves at 50 L / mu; at the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounted for 20% of the total number of small flowers, June 20, 2024), the pollen nutrient solution was sprayed on the leaves at a spraying rate of 60 L / hm 2 ; During the flowering period of alfalfa (7 days after spraying pollen nutrient solution), spray the cell suspension of marine yeast on the leaves at a spraying rate of 50 L / mu.

[0054] Treatment group 4: During the budding stage of alfalfa (observe the top of the alfalfa plant, when the growth point of the plant begins to differentiate into small buds, and the first bud recognizable to the naked eye appears, indicating that the alfalfa has entered the budding stage, June 1, 2024), spray sterile deionized water on the leaves at 50 L / mu. During the initial flowering stage of alfalfa (when the number of small flowers blooming on 80% of the inflorescences accounts for 20% of the total number of small flowers, June 20, 2024), spray sterile deionized water on the leaves at a spraying rate of 60 L / hm 2 ; During the flowering period of alfalfa (7 days after spraying sterile deionized water), spray sterile deionized water on the leaves at a spraying rate of 50 L / mu.

[0055] Treatment group 5: During the budding stage of alfalfa (observe the top of the alfalfa plant, when the growth point of the plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, indicating that the alfalfa has entered the budding stage, June 1, 2024), spray sterile deionized water 50 L / mu on the leaves; during the initial flowering stage of alfalfa (when the number of small flowers blooming on 80% of the inflorescences accounts for 20% of the total number of small flowers, June 20, 2024), spray the pollen nutrient solution in the composition described in Example 6 on the leaves, and the spraying amount is 60 L / hm 2 During the flowering period of alfalfa (7 days after spraying the pollen nutrient solution), the cell suspension of marine yeast in the composition described in Example 6 was sprayed on the leaves at a spraying rate of 50 L / mu.

[0056] Treatment group 6: During the budding stage of alfalfa (observe the top of the alfalfa plant, when the growth point of the plant begins to differentiate into small flower buds and the first flower bud recognizable by the naked eye appears, indicating that the alfalfa has entered the budding stage, June 1, 2024), the salt-tolerant Brevibacillus EB3 suspension in the composition described in Example 6 was sprayed on the leaves at 50 L / mu; during the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounted for 20% of the total number of small flowers, June 20, 2024), sterile deionized water was sprayed on the leaves at a spraying rate of 60 L / hm 2 During the flowering period of alfalfa (7 days after spraying with sterile deionized water), the cell suspension of marine yeast in the composition described in Example 6 was sprayed on the leaves at a spraying rate of 50 L / mu.

[0057] Treatment group 7: During the budding stage of alfalfa (observe the top of the alfalfa plant, when the growth point of the plant begins to differentiate into small flower buds, and the first flower bud recognizable to the naked eye appears, indicating that the alfalfa has entered the budding stage, June 1, 2024), the salt-tolerant Brevibacillus EB3 suspension in the composition described in Example 6 was sprayed on the leaves at 50 L / mu; during the initial flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounted for 20% of the total number of small flowers, June 20, 2024), the pollen nutrient solution in the composition described in Example 6 was sprayed on the leaves at a spraying rate of 60 L / hm 2; During the flowering period of alfalfa (7 days after spraying pollen nutrient solution), spray sterile deionized water on the leaves at a spraying rate of 50 L / mu.

[0058] Other field managements were the same among the groups.

[0059] 4. Result measurement and statistics (1) Pollen number determination: On July 3, 2024, 7 days after spraying the bacterial solution / deionized water, 10 newly opened single flowers of the same size were randomly selected from different parts of different plants in each planting area, placed in a 15-mL centrifuge tube, added with 1 mL of 2.5% cellulase for 24 h, and then added with 9 mL of 2.5% sucrose solution. After thorough mixing, 5 μL of the solution was taken with a pipette and observed under an optical microscope. Five dilutions were taken for observation for each treatment.

[0060] The number of pollen grains per floret (grains / flower) = the total number of pollen grains on the slide × 200.

[0061] (2) Pollen germination rate determination: On July 3, 2024, 7 days after spraying the bacterial solution / deionized water, 10 single flowers of the same size that had just opened were randomly selected from different parts of different plants in each planting area, and their anthers were mixed and placed in a 1.5 mL centrifuge tube at 4°C for testing. Use a glass rod to drop a drop of sucrose boric acid culture medium (prepared with 10% sucrose, 0.015% boric acid and 1% agar) on a glass slide. After cooling, use a clean brush to pick up a small amount of pollen and evenly spread it on the surface of the culture medium. Place it in a 25°C incubator and incubate it for 1.5 hours. Observe the germination of pollen under a microscope. The pollen tube length exceeding the pollen grain diameter is used as the germination standard. The pollen germination rate is calculated, and each treatment is repeated 3 times.

[0062] Pollen germination rate (%) = number of germinated pollen / total number of pollen grains on the slide × 100%.

[0063] (3) Determination of floret shedding rate: On July 8, 2024, after the flowering period, 1m was left at both ends of each row in each planting area as a protection area, and three 1m long sample sections were randomly selected from the middle area to count the number of main branches near the base of the plant surface. Thirty healthy reproductive branches were randomly selected from each area to determine the number of inflorescences and florets per inflorescence on each reproductive branch; Floret shedding rate (%) = (average number of florets per inflorescence - average number of pods per inflorescence) / average number of florets per inflorescence × 100% (4) Pod-setting rate determination: On July 23, 2024, when the pod-setting period began, 30 healthy reproductive branches were randomly selected from each planting area to determine the yield components such as the number of pod-setting inflorescences, the number of pods per inflorescence, and the number of seeds per pod on each reproductive branch; Pod setting rate (%) = average number of pods per inflorescence / average number of florets per inflorescence × 100% (5) Seed yield determination: On August 20, 2024, when the seeds are mature and 3 / 4 of the pods turn dark brown, leave 1 m long protective rows on both sides of each plot. Randomly select three 1 m × 1 m sample plots in the middle area, cut the above-ground parts, dry them, thresh them, and weigh them. Calculate the average value and convert it into seed yield per unit area.

[0064] The measurement results of steps (1) to (5) are shown in Table 1.

[0065] Table 1 Effects of different treatments on alfalfa pollen number, pollen germination rate, pod setting rate and seed yield

[0066] The results showed that the alfalfa treated by the method of the present invention had high pollen quantity, pollen germination rate and pod setting rate, which could significantly reduce the floret shedding rate and increase the yield of alfalfa seeds.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A salt-tolerant Brevibacillus ( Brevibacillus halotolerans ) EB3, characterized in that The deposit number is CGMCC No.33617.

2. A bacterial agent composition, characterized in that: The invention comprises a marine yeast suspension and a bacterial suspension of the salt-tolerant Brevibacillus EB3 according to claim 1.

3. Use of the salt-tolerant Brevibacillus EB3 according to claim 1 or the bacterial agent composition according to claim 2 in alfalfa cultivation.

4. A composition for improving the pollination rate and / or pod setting rate of alfalfa, characterized in that: It comprises pollen nutrient solution and the bacterial agent composition according to claim 2.

5. The composition for improving the pollination rate and / or pod setting rate of alfalfa according to claim 4, characterized in that: The pollen nutrient solution comprises: 8-12 g / L sucrose, 40-60 mg / L boric acid, 60-90 mg / L calcium chloride, 30-50 mg / L magnesium sulfate, 650-840 μg / L naphthaleneacetic acid, 520-865 μg / L gibberellin and 1000-1200 μg / L 2,4-D.

6. The composition for improving the pollination rate and / or pod setting rate of alfalfa according to claim 4, characterized in that: In the bacterial agent composition, the number of viable bacteria of the salt-tolerant Brevibacillus EB3 bacterial suspension is ≥ 1×10 8 cfu / mL; the number of viable bacteria in the marine yeast suspension is 1×10 11 ~9×10 11 cells / L.

7. Use of the composition for improving the pollination rate and / or pod setting rate of alfalfa according to any one of claims 4 to 6 in alfalfa planting.

8. A method for increasing the pollination rate and / or pod setting rate of alfalfa, characterized in that: The composition for improving the pollination rate and / or pod setting rate of alfalfa according to any one of claims 4 to 6 is sprayed on the alfalfa leaves, and the spraying steps are: spraying a salt-tolerant Brevibacillus EB3 bacterial suspension at the alfalfa budding stage; spraying a pollen nutrient solution at the alfalfa initial flowering stage; and spraying a marine yeast suspension at the alfalfa full flowering stage.

9. The method according to claim 8, characterized in that The spraying rate of the pollen nutrient solution is 50-70 L / hm 2 .

10. The method according to claim 8, characterized in that The spraying amount of the salt-tolerant Bacillus brevis EB3 bacterial suspension is 45-55 L / mu; the spraying amount of the marine yeast suspension is 45-55 L / mu.

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