Complex microbial inoculant capable of improving nitrogen fixation capacity of plants and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant capable of improving the nitrogen fixation capacity of plants and application of the complex microbial inoculant, and belongs to the field of agricultural production. The compound bacterial agent is prepared from brevibacillus sp. GD296 and bacillus velezensis DPT-03, and the ratio of the effective viable count of the brevibacillus sp. GD296 to the effective viable count of the bacillus velezensis DPT-03 is 1.2 to 1. The invention also discloses a compound bacterial fertilizer which comprises the following components in parts by weight: 30-35 parts of soybean meal, 45-50 parts of rice hull powder, 8-10 parts of corn flour, 1-3 parts of an alkaline soil conditioner and the compound bacterial agent, and the total effective viable count of the compound bacterial agent in the compound bacterial fertilizer is greater than or equal to 3.5 billion / g. Experiments show that the composite microbial agent is formed by combining nitrogen-fixing bacteria and non-nitrogen-fixing bacteria, and the nitrogen-fixing bacteria and the non-nitrogen-fixing bacteria have promoting effects and can improve the nitrogen-fixing effect; the compound bacterial fertilizer prepared from the compound bacterial agent can also significantly improve the growth of plants in alkaline soil.

Description

Technical Field

[0001] The present invention relates to the field of agricultural production, and in particular to a composite bacterial agent capable of improving the nitrogen fixation ability of plants and applications thereof. Background Art

[0002] Rural areas are the primary base for grain production. However, the extensive use of chemical fertilizers and pesticides has led to ecological damage, directly impacting crop yields and causing a growing problem of high input and low yield. Consequently, abandoned farmland in rural areas has become common in recent years. Commonly used fertilizers for crop cultivation include nitrogen, phosphorus, and potassium fertilizers, or their combination. Excessive use of nitrogen fertilizers produces large amounts of nitrogen oxides, which have a much greater warming effect than carbon dioxide, contributing to greenhouse gas formation. Furthermore, nitrogen fertilizer applied to the soil is primarily lost through leaching, runoff, and gaseous nitrogen escape. A portion of nitrogen fertilizer is carried by rainwater into rivers, lakes, and reservoirs, contributing to eutrophication, while only a small fraction remains in the soil. Beneficial microorganisms in the soil can enhance nitrogen absorption and utilization by crops through nitrogen fixation, improving the ecological conditions of the soil rhizosphere, thereby reducing crop production costs and increasing nitrogen fertilizer utilization.

[0003] Alkaline soil refers to soil with a pH greater than 7, generally including calcareous soil, saline soil, and alkaline soil. Improving soil structure and ecological conditions through microorganisms is currently receiving considerable attention. However, most existing microbial compound agents or fertilizers experience a significant decline in their reproductive capacity when applied to soil under alkaline conditions, which in turn reduces their fertilizer effectiveness. Summary of the Invention

[0004] The object of the present invention is to provide a composite bacterial agent capable of improving the nitrogen-fixing ability of plants and its application, so as to solve the problems existing in the above-mentioned prior art. By combining nitrogen-fixing bacteria and non-nitrogen-fixing bacteria, that is, compounding Brevibacillus azotobacter and Bacillus velezinoff, both of which have a promoting effect, the nitrogen-fixing effect can be improved. The composite bacterial agent constructed by the two bacteria is prepared into a composite bacterial fertilizer, with corn flour as a water-retaining agent and orange peel and wine lees fermentation products as alkaline conditioners, which can significantly improve the fertilizer effect of the composite bacterial agent in alkaline soil and promote plant growth.

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

[0006] The invention provides a composite bacterial agent, which comprises Brevibacillus sp. and Bacillus velezensis.

[0007] Preferably, the Brevibacillus azotobacter is Brevibacillus azotobacter GD296, with a deposit number of CGMCC No. 2745; and the Bacillus velez is Bacillus velez DPT-03, with a deposit number of CGMCC No. 20317.

[0008] Preferably, the ratio of the effective viable counts of the Brevibacillus azotobacter GD296 and the Bacillus velezensis DPT-03 is 1.2:1.

[0009] The present invention also provides a composite bacterial fertilizer for improving nitrogen fixation ability, comprising the following components in parts by weight: 30-35 parts of soybean meal powder, 45-50 parts of rice husk powder, 8-10 parts of corn flour, 1-3 parts of an alkaline soil conditioner, and the composite bacterial agent, wherein the total effective viable bacterial count of the composite bacterial agent in the composite bacterial fertilizer is ≥350 million / g.

[0010] Preferably, the compound fertilizer comprises the following components in parts by weight: 35 parts of soybean meal powder, 48 parts of rice husk powder, 8.5 parts of corn flour, 1.5 parts of alkaline soil conditioner, and the compound bacterial agent, wherein the total effective live bacteria count of the compound bacterial agent in the compound bacterial fertilizer is ≥350 million / g.

[0011] Preferably, the alkaline soil conditioner is orange peel and wine lees baking powder.

[0012] Preferably, the preparation method of the orange peel and distiller's grains fermentation powder comprises the following steps: mixing orange peel powder, distiller's grains powder and sugar water, and fermenting in a closed manner at 30° C. for 30 hours to obtain a fermentation liquid; and freeze-drying the fermentation liquid to prepare the orange peel and distiller's grains fermentation powder.

[0013] Preferably, the ratio of the orange peel powder, distiller's grains powder and sugar water is 20g:9g:100mL, and the sugar water is a white sugar aqueous solution with a concentration of 20%.

[0014] The present invention also provides application of the composite bacterial agent or the composite bacterial fertilizer in improving the nitrogen fixation ability of plants.

[0015] The present invention also provides application of the composite bacterial agent or the composite bacterial fertilizer in improving the alkali resistance of plants.

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

[0017] The composite bacterial agent of the present invention is compounded by Brevibacillus azotobacter GD296 and Bacillus velezensis DPT-03, which is a combination of nitrogen-fixing bacteria and non-nitrogen-fixing bacteria. The compound of the two can significantly improve the nitrogen-fixing effect of the nitrogen-fixing bacteria, thereby reducing the use of chemical nitrogen fertilizers and helping to reduce environmental pollution.

[0018] The composite bacterial fertilizer of the present invention can regulate the pH value of alkaline soil, thereby promoting plant growth and increasing plant yield. Furthermore, the raw materials for the composite bacterial fertilizer are readily available, the preparation method is simple and easy to operate, no environmentally polluting gases are generated, and the fertilizer can also promote the reuse of orange peels and distiller's grains, turning waste into treasure. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] The strains involved in the following examples are: Brevibacillus sp. GD296, which was deposited with the China General Microbiology Center (CGMCC) on November 17, 2008, with a deposit number of CGMCC No. 2745. Bacillus velezensis DPT-03, deposited with the China General Microbiology Center (CGMCC) on July 8, 2020, with a deposit number of CGMCC No. 20317. The above strains are available to the public from the above institutions.

[0025] Soybean meal meal, rice husk meal, corn meal, orange peel meal, and distiller's grains meal are all commercially available.

[0026] Example 1

[0027] A composite bacterial agent is prepared by mixing Brevibacillus azotobacter GD296 and Bacillus velezensis DPT-03. After mixing the two strains according to the ratio shown in Table 1 below, the nitrogenase activity of the strains in the composite bacterial system is determined by the acetylene reduction method (reference "Screening of High-Efficiency Nitrogen-Fixing Bacillus and Its Biological Characteristics", Sun Jianguang et al.).

[0028] Table 1

[0029]

[0030] As shown in the table above, Bacillus velezensis DPT-03 alone did not fix nitrogen. However, compared to Brevibacillus azotobacter GD296 alone, a combination of the two bacteria significantly improved nitrogen fixation. However, a ratio of Brevibacillus azotobacter GD296 to Bacillus velezensis DPT-03 of 1.2:1 significantly promoted nitrogen fixation. This optimal ratio served as the basis for subsequent experiments.

[0031] Example 2

[0032] A compound bacterial fertilizer for improving nitrogen fixation capacity comprises the following components, in parts by weight: 30 parts soybean meal powder, 45 parts rice husk powder, 8 parts corn flour, 1 part alkaline soil conditioner, and a compound bacterial agent, wherein the total effective viable bacterial count of the compound bacterial agent in the compound bacterial fertilizer is ≥350 million / g; the compound bacterial agent is obtained by mixing powder of Brevibacillus azotobacter GD296 and powder of Bacillus velezensis DPT-03 at a ratio of 1.2:1. The total effective viable bacterial count of the compound bacterial agent in the compound bacterial fertilizer is ≥350 million / g.

[0033] The preparation method is:

[0034] (1) Mix soybean meal powder, rice husk powder, and corn flour according to a certain proportion, sterilize, cool, and set aside;

[0035] (2) orange peel powder, distiller's grains powder and sugar water are mixed in a ratio of 20 g:9 g:100 mL, and the mixture is fermented in a sealed container at 30° C. for 24-36 hours to obtain a fermentation liquid; the fermentation liquid is then freeze-dried to obtain an alkaline soil conditioner; the sugar water concentration is 20%, and the mixture is prepared by mixing white sugar and sterilized water in a mass volume ratio of 20 g:100 mL;

[0036] (3) Mix the materials of steps (1) and (2) according to the proportions and set aside;

[0037] (4) Add the bacterial powder of Brevibacillus azotobacter GD296 and the bacterial powder of Bacillus velezensis DPT-03 to the material of step (3) according to the required effective viable bacteria count, and then dry at 45° C. to obtain the composite bacterial fertilizer.

[0038] Example 3

[0039] A compound bacterial fertilizer for improving nitrogen fixation capacity comprises the following components in parts by weight: 32 parts of soybean meal powder, 48 parts of rice husk powder, 9 parts of corn flour, 2 parts of an alkaline soil conditioner, and a compound bacterial agent, wherein the total effective viable bacterial count of the compound bacterial agent in the compound bacterial fertilizer is greater than or equal to 350 million / g; the compound bacterial agent is obtained by mixing bacterial powder of Brevibacillus azotobacter GD296 and bacterial powder of Bacillus velezensis DPT-03 at a ratio of effective viable bacterial count of 1.2:1.

[0040] The preparation method is the same as that of Example 2.

[0041] Example 4

[0042] A compound bacterial fertilizer for improving nitrogen fixation capacity comprises the following components in parts by weight: 35 parts of soybean meal powder, 48 parts of rice husk powder, 8.5 parts of corn flour, 1.5 parts of an alkaline soil conditioner, and a compound bacterial agent, wherein the total effective viable bacterial count of the compound bacterial agent in the compound bacterial fertilizer is greater than or equal to 350 million / g; the compound bacterial agent is obtained by mixing bacterial powder of Brevibacillus azotobacter GD296 and bacterial powder of Bacillus velezensis DPT-03 at a ratio of effective viable bacterial count of 1.2:1.

[0043] The preparation method is the same as that of Example 2.

[0044] Example 5

[0045] A compound bacterial fertilizer for improving nitrogen fixation capacity comprises the following components in parts by weight: 35 parts of soybean meal powder, 50 parts of rice husk powder, 10 parts of corn flour, 3 parts of an alkaline soil conditioner, and a compound bacterial agent, wherein the total effective viable bacterial count of the compound bacterial agent in the compound bacterial fertilizer is greater than or equal to 350 million / g; the compound bacterial agent is obtained by mixing bacterial powder of Brevibacillus azotobacter GD296 and bacterial powder of Bacillus velezensis DPT-03 at a ratio of 1.2:1 in terms of effective viable bacterial count.

[0046] The preparation method is the same as that of Example 2.

[0047] Comparative Example 1

[0048] The difference from Example 4 is that the compound bacterial fertilizer does not contain corn flour. The other steps are the same.

[0049] Comparative Example 2

[0050] The difference from Example 4 is that the alkaline conditioner in the compound bacterial fertilizer is obtained only by fermenting orange peel powder. The other steps are the same.

[0051] Comparative Example 3

[0052] The difference from Example 4 is that the alkaline conditioner in the compound bacterial fertilizer is obtained only by fermentation of vinasse powder. The other steps are the same.

[0053] Comparative Example 4

[0054] The difference from Example 3 is that the compound bacterial fertilizer does not contain Bacillus Velez subtilis DPT-03. The other steps are the same.

[0055] Comparative Example 5

[0056] The difference from Example 4 is that the compound bacterial fertilizer does not contain Brevibacillus azotobacter GD296. The other steps of the method are the same.

[0057] Application examples:

[0058] Corn seeds were sterilized with 75% alcohol for 1 minute, then with 2.5% sodium hypochlorite for 5 minutes, and then rinsed three times with sterile water. The seeds were then placed in sterile quartz sand trays at a depth of 1-2 cm. The sand was then thoroughly watered with sterile water. Germination was accelerated at 30°C in the dark for 3 days. The seeds were then transferred to pots and sterilized by soaking in 0.5N nitric acid for 24 hours. The pots were then filled with a soil mixture (soil to vermiculite in a 10:3 mass ratio) to fill the 2 / 3rd of the pot and watered to a moisture content of 45%. Five germinated seeds were planted in each pot, and 100 mL of nitrogen-free corn nutrient solution (modified Hoagland nutrient solution, purchased from Qingdao Haibo Biotechnology Co., Ltd.) was added every other week. The compound fertilizers of Examples 2-5 and Comparative Examples 1-5 were applied as base fertilizer to each pot. The pH of the mixed soil in each pot was adjusted to 8.0 before applying the base fertilizer. One month later, samples were collected by breaking the soil, and plant height, root length, root biomass, and aboveground biomass of the corn were calculated. A blank control without the compound fertilizer was used. The results are shown in Table 2.

[0059] Table 2 Statistical results

[0060]

[0061]

[0062] Note: In the same column, different letters represent significant differences at the p=0.05 level.

[0063] After 30 days of soil sampling, the pH of the mixed soil in all the flower pots was measured to be between 7.2 and 7.6, which is close to neutral, indicating that the use of the composite bacterial fertilizer of the present invention can also significantly reduce the pH value of the soil.

[0064] The results of the potted plant experiments showed that the application of the composite fertilizers of Examples 2 to 5 significantly promoted the growth of corn seedlings in alkaline soil, including the height and root system, and increased the biomass of the root system and aboveground parts. Compared with the blank control group, the plant height of Comparative Examples 1 to 5 increased by 7.89%-16.67%, the root length increased by 1.89%-10.60%, the root biomass of corn increased by 2.15%-9.18%, and the aboveground biomass of corn increased by 2.68%-8.64%, but there were no significant differences. The plant height of Examples 2 to 5 increased by 55.19%-74.43%, the root length increased by 42.28%-56.93%, the root biomass increased by 20.43%-24.50%, and the aboveground biomass increased by 21.18%-35.00%, all of which were significantly different. The use of alkaline conditioners can regulate soil pH while providing rich nutrients. Corn flour, as a water-retaining agent and nutrient source, can maintain soil moisture, prevent nutrient loss, and adjust the microbial ecological balance. The compound bacterial agent can promote the microbial ecological balance while increasing the content of beneficial bacteria, thereby promoting the absorption and utilization of nitrogen sources and other nutrients. Through the synergistic effect of the various components of the compound bacterial fertilizer, the fertilizer efficiency and alkaline resistance of the fertilizer are significantly improved.

[0065] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A composite bacterial agent, It is characterized in that Including Brevibacillus sp. and Bacillus velezensis.

2. The composite bacterial agent according to claim 1, It is characterized in that The Brevibacillus azotobacter is Brevibacillus azotobacter GD296, with a preservation number of CGMCC No.2745; the Bacillus velez is Bacillus velez DPT-03, with a preservation number of CGMCC NO.20317.

3. The composite bacterial agent according to claim 2, It is characterized in that The ratio of the effective viable counts of the Brevibacillus azotobacter GD296 and the Bacillus velezensis DPT-03 is 1.2:

1.

4. A compound bacterial fertilizer that improves nitrogen fixation ability. It is characterized in that The invention comprises the following components in parts by weight: 30-35 parts of soybean meal powder, 45-50 parts of rice husk powder, 8-10 parts of corn powder, 1-3 parts of alkaline soil conditioner, and the composite bacterial agent according to any one of claims 1-3, wherein the total effective live bacteria count of the composite bacterial agent in the composite bacterial fertilizer is ≥350 million / g.

5. The composite bacterial fertilizer according to claim 4, It is characterized in that The invention comprises the following components in parts by weight: 35 parts of soybean meal powder, 48 parts of rice husk powder, 8.5 parts of corn powder, 1.5 parts of alkaline soil conditioner, and the composite bacterial agent, wherein the total effective live bacteria count of the composite bacterial agent in the composite bacterial fertilizer is ≥350 million / g.

6. The composite bacterial fertilizer according to claim 4, It is characterized in that The alkaline soil conditioner is orange peel and wine lees baking powder.

7. The composite bacterial fertilizer according to claim 6, It is characterized in that The preparation method of orange peel and distiller's lees fermentation powder comprises the following steps: mixing orange peel powder, distiller's lees powder and sugar water, and fermenting in a closed manner at 30° C. for 24-36 hours to obtain fermentation liquid; and freeze-drying the fermentation liquid to prepare orange peel and distiller's lees fermentation powder.

8. The composite bacterial fertilizer according to claim 7, It is characterized in that The ratio of the orange peel powder, distiller's grains powder and sugar water is 20g:9g:100mL, and the sugar water is a 20% white sugar aqueous solution.

9. Use of the composite bacterial agent according to any one of claims 1 to 3 or the composite bacterial fertilizer according to any one of claims 4 to 8 in improving the nitrogen fixation ability of plants.

10. Use of the composite bacterial agent according to any one of claims 1 to 3 or the composite bacterial fertilizer according to any one of claims 4 to 8 in improving the alkaline resistance of plants.

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