A method for improving the pollination rate and pod-setting rate of alfalfa

Abstract

The present invention provides a method for improving the pollination rate and pod-setting rate of alfalfa, belonging to the field of biotechnology. The present invention provides a strain of salt-tolerant Brevibacillus brevis ( Brevibacillus halotolerans ) EB3, with the preservation number of CGMCC No. 33617. Further provided are a bacterial agent composition composed of this bacterium and marine yeast, a composition composed of the bacterial agent composition and pollen nutrient solution, and a method for improving the pollination rate and pod-setting rate of alfalfa by using this composition, effectively solving the problems of low seed setting rate, small flower abortion, serious flower dropping, etc. of alfalfa, significantly increasing the pollen quantity and pollen germination rate of alfalfa, reducing the small flower dropping rate, thereby achieving the effect of significantly increasing the seed yield of alfalfa, and the method is simple to operate, with stable effects and low cost.

Description

Technical Field

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

[0002] Alfalfa is a general term for plants of the genus Medicago ( Medicago L.), annual or perennial herbaceous plants, which are characterized by high yield, rich in protein, good palatability, strong adaptability, etc. China is one of the main distribution areas of Medicago plants, and the common ones are alfalfa ( Medicago sativa L.) and yellow alfalfa ( Medicago falcata L.), which are excellent forages for the establishment of artificial grasslands and the improvement of natural grasslands in China, and play an important role in the development of China's animal husbandry. However, alfalfa is a typical cross-pollinated plant with a very low self-pollination and seed setting rate. Moreover, alfalfa flowers have a unique opening mechanism, and their pollination is a complex process, often relying on external mechanical forces and insects collecting nectar to bounce open the tightly wrapped keel petals for pollination. Therefore, due to its self-incompatibility and special keel petal structure, the natural pollination rate is very low, the phenomenon of flower dropping is serious, and it is difficult to propagate by seeds.

[0003] Improving the pollination rate and pod setting rate during the flowering period of alfalfa 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 regulation technology, artificial pollination technology, and breeding technology. However, insect pollination technology usually uses insects such as alfalfa leafcutting bees for alfalfa pollination. The activities and reproduction of insects are affected by climate, the pollination rate is unstable, and feeding and management require professional technology and equipment, making large-scale application difficult. Environmental regulation technology requires a large amount of facility construction and energy consumption, and it is difficult to precisely regulate. If the temperature, humidity, light, wind speed, etc. are adjusted improperly, it is easy to cause negative effects instead. Artificial pollination requires a large amount of professional equipment and labor costs, and the efficiency is extremely low. Breeding technology is not yet mature, and there are problems such as a long time cycle, high technical requirements, and high R & D costs. Therefore, it is urgent to study a simple, efficient method that can stably improve the pollination rate and pod setting rate of alfalfa to solve problems such as low seed setting rate and short supply of seed production of alfalfa. Summary of the Invention

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

[0005] The purpose of the present invention is also 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 small flower abscission rate, and increase the pod setting rate and seed yield of alfalfa.

[0006] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides a halotolerant Brevibacillus brevis ( Brevibacillus halotolerans ) EB3, with the preservation number of CGMCC No. 33617.

[0008] The present invention also provides a bacterial agent composition, comprising a marine yeast suspension and a bacterial suspension of the above-mentioned halotolerant Brevibacillus brevis EB3.

[0009] The present invention also provides the application of the above-mentioned halotolerant Brevibacillus brevis EB3 or the above-mentioned bacterial agent composition in alfalfa planting.

[0010] The present 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.

[0011] Preferably, the pollen nutrient solution comprises: 8 - 12 g / L of sucrose, 40 - 60 mg / L of boric acid, 60 - 90 mg / L of calcium chloride, 30 - 50 mg / L of magnesium sulfate, 650 - 840 μg / L of naphthaleneacetic acid, 520 - 865 μg / L of gibberellin, and 1000 - 1200 μg / L of 2,4-D.

[0012] Preferably, in the above-mentioned bacterial agent composition, the viable count of the bacterial suspension of halotolerant Brevibacillus brevis EB3 ≥ 1×10 8 cfu / mL; the viable count of the marine yeast suspension is 1×10 11 ~9×10 11 cells / L.

[0013] The present invention also provides the application of the above-mentioned composition for improving the pollination rate and / or pod setting rate of alfalfa in alfalfa planting.

[0014] The present invention also provides a method for improving the pollination rate and / or pod setting rate of alfalfa, spraying the above-mentioned composition for improving the pollination rate and / or pod setting rate of alfalfa on the alfalfa leaf surface, and the spraying steps are as follows: spraying the bacterial suspension of halotolerant Brevibacillus brevis EB3 at the budding stage of alfalfa; spraying the pollen nutrient solution at the early flowering stage of alfalfa; spraying the marine yeast suspension at the full flowering stage of alfalfa.

[0015] Preferably, the spraying amount of the pollen nutrient solution is 50 - 70 L / hm 2 .

[0016] Preferably, the spraying amount of the bacterial suspension of halotolerant Brevibacillus brevis EB3 is 45 - 55 L / mu; the spraying amount of the marine yeast suspension is 45 - 55 L / mu.

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

[0018] The present invention has obtained a strain of salt-tolerant Brevibacillus ( Brevibacillus halotolerans ) EB3 for the first time, and based on this strain combined with marine yeast, a microbial 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, effectively solving the problems of low seed setting rate of alfalfa, abortion of small flowers, serious flower dropping, etc., can significantly increase the pollen quantity and pollen germination rate of alfalfa, reduce the abscission rate of small flowers, 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 effects and low cost.

[0019] Biological deposit description

[0020] Salt-tolerant Brevibacillus ( Brevibacillus halotolerans ) EB3, classified and named as Salt-tolerant Brevibacillus ( Brevibacillus halotolerans ), depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms, address of depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 33617, deposit date: February 24, 2025. Description of the drawings

[0021] Figure 1 : Colony morphology of salt-tolerant Brevibacillus EB3. Detailed implementation manners

[0022] The present invention provides a strain of salt-tolerant Brevibacillus ( Brevibacillus halotolerans ) EB3, and the deposit number of the salt-tolerant Brevibacillus EB3 is CGMCC No. 33617. The salt-tolerant Brevibacillus EB3 of the present invention is isolated and purified from the fresh vegetative roots of Stipa breviflora, and it is proved by identification that it belongs to Brevibacillus halotolerans , and it is deposited in the General Microbiology Center of China Committee for Culture Collection of Microorganisms on February 24, 2025, and the address of the depositary institution is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] The present invention also provides a microbial agent composition comprising marine yeast and the above-mentioned salt-tolerant Brevibacillus EB3, and the microbial agent composition includes a marine yeast suspension and a bacterial suspension of the above-mentioned salt-tolerant Brevibacillus EB3. The marine yeast is a marine yeast strain obtained by conventional purchase.

[0024] The present invention also provides the application of the above-mentioned microbial agent composition in alfalfa planting. The bacterial suspension of salt-tolerant Brevibacillus EB3 and the marine yeast suspension are respectively sprayed on the alfalfa leaves, which can improve the visiting rate of pollinating insects, and further improve the pollination rate during the alfalfa flowering period, the pod setting rate of alfalfa and the seed yield of alfalfa. The alfalfa includes but is not limited to Medicago falcata and Medicago sativa.

[0025] 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 above-mentioned bacterial agent composition. The alfalfa includes, but is not limited to, Medicago falcata and Medicago sativa.

[0026] 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 naphthylacetic 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 naphthylacetic 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 naphthylacetic acid, gibberellin and 2,4-D, adjusting the pH value to 5 - 6 with hydrochloric acid or sodium hydroxide, and then making up the volume to 1 L. The pH value of the finally obtained pollen nutrient solution of the present invention is preferably 5.5.

[0027] In the bacterial agent composition of the present invention, the viable count of the Bacillus pumilus EB3 suspension ≥ 1×10 8 cfu / mL; the viable count of the marine yeast suspension is 1×10 11 ~9×10 11 cells / L, preferably 2×10 11 ~8×10 11 cells / L. As an alternative embodiment, the preparation of the bacterial suspension of the present invention comprises the following steps: preparing 100 mL of LB liquid medium, adding Bacillus pumilus EB3 after sterilization and cooling, shaking and culturing at 28 °C and 150 r / min for 36 h. When the OD 600 value is 0.2, centrifuging at 5000 r / min for 10 min, discarding the supernatant, and washing the bacteria adhered to the bottom of the centrifuge tube with sterile water to obtain a bacterial suspension with an OD 600 value of 0.2. The effective viable count of the obtained Bacillus pumilus EB3 is above 1×10 8 cfu / mL. As an alternative embodiment, the preparation method of the cell suspension of the 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 sucrose, culturing at 24 - 26 °C for 40 - 55 h to obtain a marine yeast suspension with a viable count of 1×10 11 ~9×10 11 cells / L. The culture temperature is preferably 25 °C, and the culture time is preferably 48 h.

[0028] 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 bacterial suspension of Bacillus brevis EB3 with salt tolerance during the budding stage of alfalfa; spraying a pollen nutrient solution during the early flowering stage of alfalfa; spraying a cell suspension of the above-mentioned marine yeast during the full flowering stage of alfalfa. By first spraying the bacterial suspension of Bacillus brevis EB3 with salt tolerance on the leaf surface of alfalfa plants at the budding stage, the reproductive growth of alfalfa is promoted; then spraying the pollen nutrient solution on the leaf surface of alfalfa plants at the early flowering stage to stimulate pollen germination and increase the pollen germination rate; and spraying the cell suspension of marine yeast during the full flowering stage to increase the flower-visiting rate of pollinating insects, thereby improving the pollination rate and pod-setting rate during the flowering stage of alfalfa, effectively solving the problems of low seed-setting rate, small flower abortion, and serious flower dropping of alfalfa. The alfalfa includes but is not limited to Medicago falcata L. and Medicago sativa L.

[0029] The budding stage of alfalfa in the present invention is defined as follows: 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, it indicates that alfalfa begins to enter the budding stage. The spraying of the bacterial suspension of Bacillus brevis EB3 with salt tolerance in the present invention is preferably by leaf spraying, and the spraying amount is 45 - 55 L / mu, preferably 50 L / mu.

[0030] The early flowering stage of alfalfa in the present invention is defined as follows: when the number of small flowers opened on 75 - 85% of the inflorescences accounts for 15 - 25% of the total number of small flowers. The spraying of the pollen nutrient solution in the present invention is preferably by leaf spraying, and the spraying amount is 50 - 70 L / hm 2 , preferably 60 L / hm 2 .

[0031] The full flowering stage of alfalfa for spraying the cell suspension of marine yeast in the present invention is 6 - 8 days after spraying the pollen nutrient solution, preferably 7 days after spraying the pollen nutrient solution, and the spraying amount of the cell suspension of marine yeast is 45 - 55 L / mu, preferably 50 L / mu.

[0032] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0033] In the specific embodiments of the present invention, the marine yeast is purchased from Inner Mongolia Shengkang Biotechnology Co., Ltd.

[0034] In the following embodiments, unless otherwise specified, all are conventional methods.

[0035] The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0036] Example 1

[0037] Halophilic Bacillus Brevibacillus ( Brevibacillus halotolerans ) Isolation and identification of EB3:

[0038] 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.

[0039] 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, it was determined that strain EB3 was a salt-tolerant Brevibacillus ( Brevibacillus halotolerans ).

[0040] 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.

[0041] Example 2

[0042] A bacterial agent composition comprises a salt-tolerant brevis bacillus EB3 bacterial suspension and a marine yeast suspension.

[0043] 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.

[0044] 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 11Marine yeast suspension of cells / L.

[0045] Example 3

[0046] A bacterial agent composition: including the suspension of Bacillus brevis EB3 with salt tolerance and the marine yeast suspension.

[0047] Suspension of Bacillus brevis EB3 with salt tolerance: Prepare 100 mL of LB liquid medium. After sterilization and cooling, add Bacillus brevis EB3 with salt tolerance. Shake and culture at 28 °C and 150 r / min for 36 h. When the OD 600 value is 0.2, centrifuge at 5000 r / min for 10 min, discard the supernatant, and wash the bacteria adhered to the bottom of the centrifuge tube with sterile water to obtain a bacterial suspension with an OD 600 value of 0.2. The effective viable count of Bacillus brevis EB3 obtained is 4.6×10 8 cfu / mL.

[0048] Marine yeast suspension: Add the marine yeast monoclonal strain to the sterile aqueous solution containing 345 g / L of sucrose. Culture at 25 °C for 55 h to obtain a marine yeast suspension with a viable count of 6.5×10 11 cells / L.

[0049] Example 4

[0050] A composition for improving the pollination rate and / or pod-setting rate of alfalfa: pollen nutrient solution and the bacterial agent composition of Example 3.

[0051] Pollen nutrient solution: 8 g / L of sucrose, 40 mg / L of boric acid, 60 mg / L of calcium chloride, 30 mg / L of magnesium sulfate, 650 μg / L of naphthaleneacetic acid, 520 μg / L of gibberellin, and 1000 μg / L of 2,4-D. Preparation method: Dissolve sucrose, boric acid, calcium chloride, and magnesium sulfate in water. After adjusting the pH value to 5 with hydrochloric acid / sodium hydroxide, add naphthaleneacetic acid, gibberellin, and 2,4-D, and make up the volume to 1 L.

[0052] Example 5

[0053] A composition for improving the pollination rate and / or pod-setting rate of alfalfa: pollen nutrient solution and the bacterial agent composition of Example 3.

[0054] Pollen nutrient solution: 12 g / L of sucrose, 60 mg / L of boric acid, 90 mg / L of calcium chloride, 50 mg / L of magnesium sulfate, 840 μg / L of naphthaleneacetic acid, 865 μg / L of gibberellin, and 1200 μg / L of 2,4-D. Preparation method: Dissolve sucrose, boric acid, calcium chloride, and magnesium sulfate in water. After adjusting the pH value to 5.5 with hydrochloric acid / sodium hydroxide, add naphthaleneacetic acid, gibberellin, and 2,4-D, and make up the volume to 1 L.

[0055] Example 6

[0056] A composition for increasing the pollination rate and / or pod-setting rate of alfalfa: pollen nutrient solution and the bacterial agent composition of Example 3.

[0057] Pollen nutrient solution: 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. Preparation method: Dissolve sucrose, boric acid, calcium chloride, and magnesium sulfate in water, adjust the pH value to 6 with hydrochloric acid / sodium hydroxide, then add naphthaleneacetic acid, gibberellin, and 2,4-D, and make up the volume to 1 L.

[0058] Example 7

[0059] A method for increasing the pollination rate and / or pod-setting rate of alfalfa:

[0060] Use the composition described in Example 4, spray the bacterial suspension of Bacillus brevis EB3 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 enters the budding stage), and the spraying amount on the leaf surface is 50 L per mu. At the early flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounts for 20% of all small flower numbers), spray the pollen nutrient solution on the leaf surface, and the spraying amount is 50 L / hm 2 ; At the full flowering stage of alfalfa (6 days after spraying the pollen nutrient solution), spray the cell suspension of marine yeast on the leaf surface, and the spraying amount is 45 L per mu.

[0061] Example 8

[0062] A method for increasing the pollination rate and / or pod-setting rate of alfalfa:

[0063] Use the composition described in Example 5, spray the bacterial suspension of Bacillus brevis EB3 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 enters the budding stage), and the spraying amount on the leaf surface is 50 L per mu. At the early flowering stage of alfalfa (when the number of small flowers opened on 80% of the inflorescences accounts for 20% of all small flower numbers), spray the pollen nutrient solution on the leaf surface, and the spraying amount is 70 L / hm 2 ; At the full flowering stage of alfalfa (6 days after spraying the pollen nutrient solution), spray the cell suspension of marine yeast on the leaf surface, and the spraying amount is 55 L per mu.

[0064] Example 9

[0065] A method for increasing the pollination rate and / or pod-setting rate of alfalfa:

[0066] Using the composition described in Example 6, spray the bacterial suspension of Brevibacillus halotolerans EB3 on alfalfa at the budding stage (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, it indicates that alfalfa enters the budding stage), and the spraying amount on the leaf surface is 50 L per mu. At the early flowering stage of alfalfa (when the number of small flowers open on 80% of the inflorescences accounts for 20% of all small flower numbers), spray the pollen nutrient solution on the leaf surface, and the spraying amount is 60 L / hm 2 ; At the full flowering stage of alfalfa (6 days after spraying the pollen nutrient solution), spray the cell suspension of marine yeast on the leaf surface, and the spraying amount is 50 L per mu.

[0067] Test Example 1

[0068] 1. Test variety: Medicago falcata

[0069] 2. Experimental site: Baiyinxile Ranch, Xilingol League, Inner Mongolia

[0070] 3. Experimental grouping:

[0071] Treatment group 1: Use the composition described in Example 6; 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, it indicates that alfalfa enters the budding stage, on June 1, 2024), spray the bacterial suspension of Brevibacillus halotolerans EB3 on the leaf surface at 50 L per mu; at the early flowering stage of alfalfa (when the number of small flowers open on 80% of the inflorescences accounts for 20% of all small flower numbers, on June 20, 2024), spray the pollen nutrient solution on the leaf surface, and the spraying amount is 60 L / hm 2 ; At the full flowering stage of alfalfa (7 days after spraying the pollen nutrient solution), spray the cell suspension of marine yeast on the leaf surface, and the spraying amount is 50 L per mu.

[0072] Treatment group 2: Use 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, it indicates that alfalfa enters the budding stage, on June 1, 2024), spray the bacterial suspension of Brevibacillus halotolerans EB3 on the leaf surface at 50 L per mu; at the early flowering stage of alfalfa (when the number of small flowers open on 80% of the inflorescences accounts for 20% of all small flower numbers, on June 20, 2024), spray the pollen nutrient solution on the leaf surface, and the spraying amount is 60 L / hm 2 ; At the full flowering stage of alfalfa (7 days after spraying the pollen nutrient solution), spray the cell suspension of marine yeast on the leaf surface, and the spraying amount is 50 L per mu.

[0073] Treatment group 3: The composition described in Example 4 was used; 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 to the naked eye appears, indicating that alfalfa enters the budding stage, June 1, 2024), a bacterial suspension of Brevibacillus salitolerans EB3 was sprayed on the leaf surface at a rate of 50 L per mu; at the early flowering stage of alfalfa (when the number of small flowers open on 80% of the inflorescences accounts for 20% of the total number of small flowers, June 20, 2024), a pollen nutrient solution was sprayed on the leaf surface, and the spraying amount was 60 L / hm 2 ; at the full flowering stage of alfalfa (7 days after spraying the pollen nutrient solution), a cell suspension of marine yeast was sprayed on the leaf surface, and the spraying amount was 50 L per mu.

[0074] Treatment group 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 to the naked eye appears, indicating that alfalfa enters the budding stage, June 1, 2024), sterile deionized water was sprayed on the leaf surface at a rate of 50 L per mu. At the early flowering stage of alfalfa (when the number of small flowers open on 80% of the inflorescences accounts for 20% of the total number of small flowers, June 20, 2024), sterile deionized water was sprayed on the leaf surface, and the spraying amount was 60 L / hm 2 ; at the full flowering stage of alfalfa (7 days after spraying sterile deionized water), sterile deionized water was sprayed on the leaf surface, and the spraying amount was 50 L per mu.

[0075] Treatment group 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 to the naked eye appears, indicating that alfalfa enters the budding stage, June 1, 2024), sterile deionized water was sprayed on the leaf surface at a rate of 50 L per mu; at the early flowering stage of alfalfa (when the number of small flowers open on 80% of the inflorescences accounts 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 leaf surface, and the spraying amount was 60 L / hm 2 ; at the full flowering stage 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 leaf surface, and the spraying amount was 50 L per mu.

[0076] Treatment group 6: 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 to the naked eye appears, indicating that alfalfa enters the budding stage, June 1, 2024), the bacterial suspension of Brevibacillus salitolerans EB3 in the composition described in Example 6 was sprayed on the leaf surface at a rate of 50 L per mu; at the early flowering stage of alfalfa (when the number of small flowers open on 80% of the inflorescences accounts for 20% of the total number of small flowers, June 20, 2024), sterile deionized water was sprayed on the leaf surface, and the spraying amount was 60L / hm 2During 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.

[0077] 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.

[0078] Other field managements were the same among the groups.

[0079] 4. Result measurement and statistics

[0080] (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.

[0081] The number of pollen grains per floret (grains / flower) = the total number of pollen grains on the slide × 200.

[0082] (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.

[0083] Pollen germination rate (%) = number of germinated pollen / total number of pollen grains on the slide × 100%.

[0084] (3)Determination of small flower abscission rate: On July 8, 2024, after entering the full-bloom stage, 1 m at each end of each row in each planting area was reserved as a protected area. Three 1-m-long sample segments were randomly selected from the middle area, and the number of main branches at the base of the plants near the ground surface was counted. Thirty healthy reproductive branches were randomly selected from each area to measure the number of inflorescences on each reproductive branch and the number of small flowers on each inflorescence;

[0085] Small flower abscission rate (%) = (Average number of small flowers on each inflorescence - Average number of pods on each inflorescence) / Average number of small flowers on each inflorescence × 100%

[0086] (4)Determination of pod-setting rate: On July 23, 2024, after entering the pod-setting stage, thirty healthy reproductive branches were randomly selected from each planting area to measure the number of pod-bearing inflorescences on each reproductive branch, the number of pods on each inflorescence, and the number of seeds in each pod and other yield components;

[0087] Pod-setting rate (%) = Average number of pods on each inflorescence / Average number of small flowers on each inflorescence × 100%

[0088] (5)Determination of seed yield: On August 20, 2024, at the seed maturity stage, when 3 / 4 of the pods turned dark brown, 1 m of protected rows were left on both sides of each plot. Three 1 m × 1 m quadrats were randomly selected from the middle area. After cutting the above-ground part, it was sun-dried and threshed, and then weighed. After calculating the average value, the seed yield per unit area was converted.

[0089] The measurement results of steps (1) to (5) are shown in Table 1.

[0090] Table 1 Effects of different treatments on alfalfa pollen quantity, pollen germination rate, pod-setting rate and seed yield

[0091]

[0092] The results show that the quantity of alfalfa pollen, pollen germination rate and pod-setting rate are high after being treated by the method described in the present invention, which can significantly reduce the small flower abscission rate and increase the yield of alfalfa seeds.

[0093] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

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

2. Use of Brevibacillus halotolerans EB3 according to claim 1 in alfalfa cultivation.

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