Application method of leguminous green manure combined with growth promoting bacteria for emission reduction and efficiency increase

Abstract

The application discloses an application method of leguminous green manure combined with growth-promoting bacteria for emission reduction and efficiency increase, and relates to the technical field of crop planting. The method comprises the following steps: sowing black bean seeds in soil inoculated with bacillus velezensis, and culturing black bean plants to the full flowering stage, and then crushing and turning over the black bean plants as green manure. A special bacillus velezensis is separated, which has the effects of bacteriostasis, nitrogen fixation, phosphorus dissolution, potassium solution, biofilm formation and auxin production, and has excellent biological characteristics such as stress resistance, rapid growth and easy separation, and the advantages of easy cultivation, safety, harmlessness and no pollution. Meanwhile, the bacillus velezensis can ferment to produce rich metabolic products, has good antibacterial activity and stress resistance. The bacillus velezensis is particularly suitable for being combined with black beans for green manure cultivation, and can maximize the effects of soil fertilization and gas emission reduction of green manure.

Description

Technical Field

[0001] This invention relates to the field of crop cultivation technology, and in particular to a method for applying legume green manure in combination with growth-promoting bacteria to reduce emissions and enhance efficiency. Background Technology

[0002] In recent years, my country has proposed a series of policies to accelerate the comprehensive green transformation of agriculture and protect and improve arable land quality. People's demands for the safety and quality of agricultural products are also constantly increasing. In addition to increasing yields, agricultural production should also focus on shifting towards high quality, diversification, and safety. Long-term overuse of chemical fertilizers and excessive reliance on pesticides can lead to the degradation of arable land quality, affecting the quality and safety of agricultural products. Green manure, a quintessential element of traditional Chinese agriculture, is a clean organic fertilizer source that can not only improve soil fertility, improve soil environmental quality, prevent soil erosion, and improve the ecological environment, but also increase crop yield and quality. It is an important measure for reducing chemical fertilizer use, implementing green control of pests, diseases, and weeds, and strengthening arable land quality. However, current green manure application models in my country's dryland areas still face practical problems such as unclear comprehensive effects and unstable benefits, hindering the sustainable development of the green manure industry.

[0003] To fully realize the ecological benefits of green manure, rational planting is essential. Combining green manure with enhancement products can address the current problems of immature summer green manure cultivation and utilization models and unclear comprehensive benefits in my country. This can provide a scientific basis and theoretical reference for increasing the contribution of green manure crops to the development of dryland agriculture in my country, rationally utilizing green manure resources, and promoting the development of the green manure industry system. However, research on the selection of different green manure + enhancement product combinations is still very incomplete. This invention aims to develop a green manure cultivation and utilization model using rye bean green manure + Bacillus belyi, in order to maximize the soil fertility and gas emission reduction effects of green manure. Summary of the Invention

[0004] The purpose of this invention is to provide a method for combining leguminous green manure with growth-promoting bacteria to reduce emissions and enhance efficiency, thereby solving the problems existing in the prior art. This method, by combining Bacillus berberis with rye soybeans for green manure cultivation, maximizes the soil fertility improvement and gas emission reduction effects of the green manure.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a plant growth-promoting Bacillus velezensis strain, which was deposited on June 6, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20241160.

[0007] The present invention also provides the application of the above-mentioned Bacillus belye in the preparation of microbial agents that promote plant growth.

[0008] The present invention also provides a microbial inoculant for promoting plant growth, wherein the active ingredient includes the aforementioned Bacillus belye.

[0009] The present invention also provides a method for preparing the above-mentioned microbial agent, comprising the following steps: inoculating the above-mentioned Bacillus belye into a fermentation medium for fermentation treatment to obtain a fermentation broth, then separating the bacterial cells, and then performing freeze-drying treatment to obtain the microbial agent.

[0010] Furthermore, the fermentation medium is beef extract peptone medium.

[0011] Furthermore, the fermentation process is carried out at a temperature of 27°C for 2-3 days.

[0012] This invention also provides a method for applying legume green manure in combination with growth-promoting bacteria to reduce emissions and enhance efficiency, including sowing black rye seeds in soil inoculated with the above-mentioned Bacillus bellii, cultivating black rye plants to the full flowering period, and then using the black rye plants as green manure by crushing and turning them over.

[0013] Furthermore, the inoculation of Bacillus belesis is achieved by strip-seeding the aforementioned microbial inoculant into the soil.

[0014] Furthermore, the inoculation amount of the microbial agent is 2.5% of the sown amount of rye bean seeds.

[0015] Furthermore, the folding depth of the folding process is 10-15cm.

[0016] The present invention discloses the following technical effects:

[0017] This invention has isolated a special strain of Bacillus belye, which has antibacterial, nitrogen-fixing, phosphorus-solubilizing, potassium-solubilizing, biofilm-forming, and auxin-producing effects. It also has excellent biological characteristics such as stress resistance, rapid growth, and easy isolation. Furthermore, it is easy to culture, safe, harmless, and pollution-free. At the same time, it can ferment to produce abundant metabolites, exhibiting good antibacterial activity and stress resistance.

[0018] Black rye soybeans possess advantages such as strong adaptability and resistance to adverse conditions, a short growth cycle, and strong nitrogen-fixing ability. The *Bacillus belyi* isolated in this invention is particularly suitable for green manure cultivation in combination with black rye soybeans, maximizing the soil fertility and reducing gas emissions. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a single colony image of strain ANB1;

[0021] Figure 2 This is a phylogenetic tree diagram of strain ANB1. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1: Isolation, Identification and Preservation of Bacillus belyssus

[0028] 1. Separation

[0029] A strain of bacteria was isolated from soil in Inner Mongolia and named ANB1.

[0030] 2. Identification

[0031] The strain ANB1 was identified as belonging to Bacillus velezensis. The 16S rDNA sequence of strain ANB1 is shown in SEQ ID NO.1, and a single colony diagram is shown below. Figure 1 Phylogenetic tree Figure 2 .

[0032] SEQ ID NO.1:

[0033] GTGGCGTCTGCTATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCTTCGGGGGCAAAGGGCAGGGTGGGGATGGTTGCCCCAACTCCGGCCGGAAATT.

[0034] 3. Preservation of microbial strains

[0035] Bacillus belyeiasis ANB1 was deposited on June 6, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20241160.

[0036] Example 2

[0037] Preparation of Bacillus vesiculosus inoculum:

[0038] The Bacillus belyss ANB1 isolated in Example 1 was inoculated into a fermentation medium and cultured at 27°C for 2 days (up to 3 days) to obtain a fermentation broth. The fermentation broth was centrifuged to obtain a precipitate, which was then freeze-dried to obtain Bacillus belyss inoculum.

[0039] The fermentation medium used was beef extract peptone medium.

[0040] Example 3: Pot Experiment

[0041] The pot experiment was conducted in two groups: a control group and a black soybean + Bacillus vesicles group, which were planted according to the method shown in Table 1.

[0042] Table 1. Grouping and treatment methods for potted plant experiments

[0043]

[0044] Forty-five days after planting, the biomass dry weight, total plant nitrogen (N), total plant phosphorus (P), total plant potassium (K), and soil pH of black rye beans in the control group and the black rye bean + Bacillus belyssum group were measured. The results are shown in Table 2. The results showed that compared with the single black rye bean treatment, the biomass of the black rye bean + Bacillus belyssum treatment increased significantly by 44%.

[0045] Table 2. Effects of potted Bacillus belyi inoculation on rye soybean.

[0046] index control group Black rye soybean + Bacillus betel nut group Biomass dry weight (g / pot) 1.84 3.69 pH 8.26 8.24

[0047] Example 4 Field Trial

[0048] Field trials were conducted in Yongshou and Yangling respectively to verify the results, using the following methods:

[0049] Sowing: In early July 2023, the *Bacillus beryl* inoculum prepared in Example 2 was inoculated into the planting location of black rye beans using a row sowing method, followed by sowing of black rye beans. The inoculation amount of *Bacillus beryl* inoculum was 2.5% of the black rye bean seed weight, and the sowing rate of black rye beans was 8 kg / mu. A control group consisting of only black rye beans (i.e., without inoculation with *Bacillus beryl* inoculum) was used.

[0050] Green manure incorporation: When the black rye soybean plants reach their full flowering stage (about 60 days of growth), they can be directly incorporated as green manure using a local crushing and returning machine. The incorporation depth of the green manure crop should be 10-15cm, and the incorporation time should be 30-40 days before the sowing of the subsequent winter wheat crop.

[0051] The following indicators were measured for each group: plant biomass, nitrogen return to the field after green manure incorporation, soil pH, available phosphorus in the soil, N2O accumulation, CH4 accumulation, CO2 accumulation, global warming potential (GWP), and greenhouse gas emission intensity. The results are shown in Table 3. The results showed that in the Yongshou experiment, black rye soybean + Bacillus belysinensis inoculation significantly increased biomass by 11.5% and nitrogen return to the field by 13% compared to the control group (black rye soybean alone). In the Yangling field experiment, black rye soybean + Bacillus belysinensis inoculation significantly increased biomass by 11% and nitrogen return to the field by 30% compared to the control group (black rye soybean alone). The technique of inoculating with Bacillus belysinensis inoculation can significantly increase the biomass of black rye soybean. Compared to single black rye bean, inoculation with microbial agents can enhance the soil's ability to lower pH. In Yongshou, after turning in green manure, the soil available phosphorus from black rye bean + Bacillus beleilli increased significantly by 67.4% compared to single black rye bean. In Yangling, after turning in green manure, the soil available phosphorus from black rye bean + Bacillus beleilli increased by 8.4% compared to single black rye bean.

[0052] Table 3 Effects of field inoculation with Bacillus belyi on rye soybean

[0053]

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for applying leguminous green manure in combination with growth-promoting bacteria to reduce emissions and enhance efficiency, characterized in that, The process includes sowing black rye bean seeds in soil inoculated with Bacillus velezensis, cultivating black rye bean plants to full bloom, and then using the black rye bean plants as green manure by crushing and turning them over. The aforementioned Bacillus belyes was deposited on June 6, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20241160.

2. The application method according to claim 1, characterized in that, The inoculation of Bacillus belesis is achieved by strip-seeding microbial agents into the soil; The microbial agent is obtained by inoculating the Bacillus belye into a fermentation medium for fermentation treatment, obtaining a fermentation broth, separating the bacterial cells, and then freeze-drying them.

3. The application method according to claim 2, characterized in that, The inoculation amount of the microbial agent is 2.5% of the amount of rye bean seeds sown.

4. The application method according to claim 1, characterized in that, The folding depth of the folding process is 10-15cm.

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