A composite microbial inoculum capable of improving the nitrogen fixation ability of plants and its application

Abstract

The present invention discloses a composite microbial agent capable of improving the nitrogen fixation ability of plants and its application, belonging to the field of agricultural production. The composite microbial agent is composed of Brevibacillus sp. GD296 and Bacillus velezensis DPT-03, and the ratio of the effective viable bacteria count of the two is 1.2:1. Also disclosed is a composite microbial fertilizer, which 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 flour, 1-3 parts of alkaline soil conditioner, and the above-mentioned composite microbial agent, and the total effective viable bacteria count of the composite microbial agent in the composite microbial fertilizer is ≥ 350 million / g. Through experiments, it is found that the composite microbial agent is a combination of nitrogen-fixing bacteria and non-nitrogen-fixing bacteria, and the two have a promoting effect and can improve the nitrogen fixation effect; and using the composite microbial fertilizer prepared with the above-mentioned composite microbial 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 particularly to a composite microbial inoculum capable of improving the nitrogen fixation ability of plants and its application. Background Art

[0002] The countryside is the main base for food production. However, due to the extensive use of chemical fertilizers and pesticides, the ecological environment has been damaged, directly affecting the yield of crops, resulting in an increasingly serious phenomenon of high input and low output in planting. Therefore, it is quite common for rural cultivated land to be left uncultivated in recent years. The commonly used chemical fertilizers for planting crops include nitrogen fertilizers, phosphorus fertilizers, potassium fertilizers or their compound fertilizers. Excessive use of nitrogen fertilizers will produce a large amount of nitrogen oxides, and the warming effect of nitrogen oxides is much higher than that of carbon dioxide, increasing the formation of greenhouse gases. And nitrogen fertilizers applied to the soil are mainly lost through three ways: leaching, runoff and gaseous nitrogen escape. A part of nitrogen fertilizers will flow into rivers, lakes and reservoirs with rainwater, causing eutrophication of water bodies, while only a small part of nitrogen remains in the soil. Beneficial microorganisms in the soil can promote the absorption and utilization of crops through nitrogen fixation, improve the ecological conditions of the rhizosphere environment of the soil, thereby reducing the cost of crop production and enhancing the utilization rate of nitrogen fertilizers.

[0003] Alkaline soil refers to soil with a soil pH value greater than 7, generally including calcareous soil, saline soil and alkaline soil. Improving soil structure and ecological environment conditions through microorganisms has attracted much attention at present. However, for most existing microbial composite inoculants or composite microbial fertilizers, their reproductive ability drops significantly under alkaline stress when applied to the soil, thereby reducing the fertilizer efficiency. Summary of the Invention

[0004] The purpose of the present invention is to provide a composite microbial inoculum capable of improving the nitrogen fixation ability of plants and its application to solve the problems existing in the above-mentioned prior art. Through the combination of nitrogen-fixing bacteria and non-nitrogen-fixing bacteria, namely the compounding of Brevibacillus sp. and Bacillus velezensis, the two have a promoting effect and can improve the nitrogen fixation effect. And the composite microbial inoculum constructed by the two kinds of bacteria is prepared into a composite microbial fertilizer, with corn flour as a water-retaining agent and the fermentation products of orange peel and distiller's grains as an alkaline conditioner, which can significantly improve the fertilizer efficiency of the composite microbial inoculum in alkaline soil and promote plant growth.

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

[0006] The present invention provides a composite microbial inoculum, which includes Brevibacillus sp. and Bacillus velezensis.

[0007] Preferably, the Brevibacillus azotofixans is Brevibacillus azotofixans GD296 with the preservation number of CGMCC No. 2745; the Bacillus velezensis is Bacillus velezensis DPT-03 with the preservation number of CGMCC NO. 20317.

[0008] Preferably, the ratio of the viable bacteria count of Brevibacillus azotofixans GD296 to that of Bacillus velezensis DPT-03 is 1.2:1.

[0009] The present invention also provides a compound bacterial fertilizer for improving nitrogen fixation ability, which 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 flour, 1-3 parts of alkaline soil conditioner, and the compound bacterial agent, and the total viable bacteria count of the compound bacterial agent in the compound bacterial fertilizer is ≥ 350 million / g.

[0010] Preferably, it 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, and the total viable 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 distiller's grains fermentation 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, fermenting in a sealed manner at 30 °C for 30 h to obtain a fermentation broth; freeze-drying the fermentation broth 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 20 g: 9 g: 100 mL, and the sugar water is a 20% aqueous solution of granulated sugar.

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

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

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

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

[0018] The compound bacterial fertilizer of the present invention can regulate the pH value of alkaline soil, thereby facilitating the growth of plants and increasing the plant yield. In addition, the raw materials of the compound bacterial fertilizer are easily available, the preparation method is simple and easy to operate, no environmentally polluting gases are generated, and it can also promote the reuse of orange peels and distiller's grains, turning waste into treasure. Detailed Embodiments

[0019] The 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, characteristics, and embodiments of the present invention.

[0020] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0022] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0023] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0024] The strains involved in the following examples: Brevibacillus sp. GD296, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 17, 2008, with the deposit number CGMCC No. 2745. Bacillus velezensis DPT-03, deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC), with the deposit number CGMCC NO. 20317 and the deposit date of July 8, 2020. The public can obtain the above strains from the above institutions.

[0025] Soybean meal powder, rice husk powder, corn flour, orange peel powder, and distiller's grains powder can all be obtained commercially.

[0026] Example 1

[0027] A compound microbial agent is composed of Brevibacillus azotofixans GD296 and Bacillus velezensis DPT-03. After mixing the two strains according to the ratio shown in Table 1 below, the acetylene reduction method is used to measure the nitrogenase activity of the strains in the compound microbial system (reference: "Screening of highly efficient nitrogen-fixing Bacillus and its biological characteristics", Sun Jianguang, etc.).

[0028] Table 1

[0029]

[0030] It can be seen from the above table that the single strain Bacillus velezensis DPT-03 has no nitrogen-fixing effect. Compared with the single strain Brevibacillus azotofixans GD296, the combination of the two strains is beneficial to improving nitrogen fixation. However, when the effective viable count ratio of Brevibacillus azotofixans GD296: Bacillus velezensis DPT-03 is 1.2:1, it has a significant promoting effect on nitrogen fixation. The optimal ratio is used as the basis for subsequent experiments.

[0031] Example 2

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

[0033] The preparation method is as follows:

[0034] (1) Mix soybean meal powder, rice husk powder, and corn flour in proportion, sterilize, and cool for later use;

[0035] (2) Mix orange peel powder, distiller's grains powder and sugar water in a ratio of 20 g: 9 g: 100 mL, mix evenly, and ferment in a sealed manner at 30 °C for 24 - 36 h to obtain a fermentation broth; then freeze-dry the fermentation broth to obtain an alkaline soil conditioner; the sugar water concentration is 20%, which is prepared by mixing granulated sugar and sterilized water in a mass-to-volume ratio of 20 g: 100 mL;

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

[0037] (4) Add the bacterial powders of Brevibacillus azotofixans GD296 and Bacillus velezensis DPT-03 to the material in step (3) according to the required effective viable bacteria count, and then dry at a low temperature of 45 °C to obtain the compound bio-fertilizer.

[0038] Example 3

[0039] A compound bio-fertilizer for improving nitrogen fixation ability, comprising 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 alkaline soil conditioner, and a compound bacterial agent, and the total effective viable bacteria count of the compound bacterial agent in the compound bio-fertilizer is ≥ 350 million / g; the compound bacterial agent is obtained by mixing the bacterial powders of Brevibacillus azotofixans GD296 and Bacillus velezensis DPT-03 in an effective viable bacteria count ratio of 1.2:1.

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

[0041] Example 4

[0042] A compound bio-fertilizer for improving nitrogen fixation ability, comprising 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 a compound bacterial agent, and the total effective viable bacteria count of the compound bacterial agent in the compound bio-fertilizer is ≥ 350 million / g; the compound bacterial agent is obtained by mixing the bacterial powders of Brevibacillus azotofixans GD296 and Bacillus velezensis DPT-03 in an effective viable bacteria count ratio of 1.2:1.

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

[0044] Example 5

[0045] A compound bio-fertilizer for improving nitrogen fixation ability, comprising 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 alkaline soil conditioner, and a compound bacterial agent, and the total effective viable bacteria count of the compound bacterial agent in the compound bio-fertilizer is ≥ 350 million / g; the compound bacterial agent is obtained by mixing the bacterial powders of Brevibacillus azotofixans GD296 and Bacillus velezensis DPT-03 in an effective viable bacteria count ratio of 1.2:1.

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

[0047] Comparative Example 1

[0048] The difference from Example 4 is that the compound bacterial fertilizer does not contain corn flour. Other method 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 only obtained by fermenting orange peel powder. Other method 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 only obtained by fermenting distiller's grains powder. Other method steps are the same.

[0053] Comparative Example 4

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

[0055] Comparative Example 5

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

[0057] Application Example:

[0058] Corn seeds were sterilized with 75% alcohol for 1 min, then with 2.5% sodium hypochlorite for 5 min, and then washed 3 times with sterile water. The seeds were buried in a sterile quartz sand culture dish to a depth of 1 - 2 cm, and the quartz sand was thoroughly watered with sterile water. Under dark conditions, germination was induced at 30 °C for 3 days, and then transferred to a cultivation flower pot. Each pot was treated with 0.5 N nitric acid for sterilization for 24 h, and then filled with a mixed soil (at 2 / 3 of the flower pot, the mixed soil was a mixture of soil and vermiculite with a mass ratio of 10:3), and watered to a water content of 45%. 5 germinated seeds were planted in each pot, and 100 mL of a nitrogen-free corn nutrient solution (modified Hoagland nutrient solution, purchased from Qingdao Haibo Biotechnology Co., Ltd.) was poured every week. The compound bacterial fertilizers of Examples 2 - 5 and Comparative Examples 1 - 5 were applied as base fertilizers to each pot, and the pH of the mixed soil in each pot was adjusted to 8.0 before applying the base fertilizer. After one month, samples were collected by the breaking-soil method, and the plant height, root length, corn root biomass, and corn above-ground biomass were statistically analyzed. The group without applying the compound bacterial fertilizer was used as the blank control, and 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 sampling the soil 30 days after sowing, the pH values of the mixed soil in all flowerpots were measured to be between 7.2 and 7.6, which was close to neutral, indicating that the use of the compound bacterial fertilizer of the present invention could also significantly reduce the pH value of the soil.

[0064] The results of the pot experiment showed that the application of the compound bacterial fertilizers of Examples 2-5 could significantly promote the plant height and root growth of corn seedlings in alkaline soil, and also increase the biomass of roots and above-ground parts. Compared with the blank control group, the plant height of Comparative Examples 1-5 increased by 7.89%-16.67%, the root length increased by 1.89%-10.60%, the biomass of corn roots increased by 2.15%-9.18%, and the biomass of the above-ground parts of corn increased by 2.68%-8.64%, but there were no significant differences; the plant height of Examples 2-5 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 above-ground biomass increased by 21.18%-35.00%, all of which had significant differences. The use of alkaline conditioners could regulate the soil pH while providing rich nutrients. Corn flour, as a water-retaining agent and nutrient source, could maintain soil moisture, prevent nutrient loss, and adjust the microbial ecological balance. The compound bacterial agent could 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 components of the compound bacterial fertilizer, the fertilizer efficiency and alkali resistance of the bacterial fertilizer were significantly improved.

[0065] The above-described embodiments are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent is composed of Brevibacillus sp. and Bacillus velezensis; The Brevibacillus azotobacter is Brevibacillus azotobacter GD296, with a deposit number of CGMCC No. 2745; the Bacillus velez is Bacillus velez DPT-03, with a deposit number of CGMCC NO. 20317; The ratio of the effective viable counts of the Brevibacillus azotobacter GD296 and the Bacillus velezensis DPT-03 is 1.2:

1.

2. A composite bacterial fertilizer for improving nitrogen fixation ability, 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 flour, 1-3 parts of alkaline soil conditioner, and the composite bacterial agent according to claim 1, wherein the total effective live bacteria count of the composite bacterial agent in the composite bacterial fertilizer is ≥ 350 million / g; The alkaline soil conditioner is orange peel and distiller's lees fermentation powder; the preparation method of the orange peel and distiller's lees fermentation powder comprises the following steps: orange peel powder, distiller's lees powder and sugar water are mixed, and fermented in a closed manner at 30°C for 24-36 hours to obtain fermentation liquid; the fermentation liquid is freeze-dried to prepare orange peel and distiller's lees fermentation powder; the ratio of the orange peel powder, distiller's lees powder and sugar water is 20g:9g:100mL, and the sugar water is a white granulated sugar aqueous solution with a concentration of 20%.

3. The composite bacterial fertilizer according to claim 2, 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.

4. Use of the composite bacterial agent according to claim 1 or the composite bacterial fertilizer according to any one of claims 2 to 3 in improving the nitrogen fixation ability of plants.

5. Use of the composite bacterial fertilizer as described in any one of claims 2 to 3 in improving the alkaline resistance of plants.

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