Brevibacillus brevis 9-1 and application thereof in microbial fertilizer

Abstract

The invention provides brevibacillus brevis 9-1 and application thereof in bacterial manure, and belongs to the technical field of microorganisms, the brevibacillus brevis 9-1 is separated from soil, the preservation number of the brevibacillus brevis 9-1 is CCTCC NO: M 20242933, the preservation date is December 30, 2024, the brevibacillus brevis 9-1 is preserved in the China Center for Type Culture Collection, and a 16S rDNA sequence is shown as SEQ ID NO: 1; the brevibacillus sp. 9-1 has the functional activity of solubilizing phosphorus, solubilizing potassium, fixing nitrogen and producing ACC (Acetobacter Cellulose), and the brevibacillus sp. 9-1 has the functional activity of solubilizing potassium, fixing nitrogen and producing ACC; the microbial fertilizer is obtained by enlarged culture of brevibacillus brevis 9-1, and compared with a chemical fertilizer, the microbial fertilizer used in the invention is more environment-friendly, not only can improve soil fertility, but also can promote balance and diversity of soil microbial communities, and is helpful for maintaining health and stability of a soil ecosystem.

Description

Technical Field

[0001] The present invention relates to a Brevibacillus sp. 9-1 and its application in microbial fertilizer, belonging to the field of microbial technology. Background Art

[0002] In recent years, affected by climate change and human activities such as overgrazing, the grassland ecosystem in China has faced serious degradation problems, manifested as a decline in soil fertility, a decrease in vegetation coverage, and a sharp reduction in productivity. Although traditional fertilization methods (such as chemical fertilizer application) can improve grassland productivity in the short term, long-term use is likely to cause soil compaction, microbial community imbalance, and even exacerbate the deterioration of the ecological environment. Therefore, developing sustainable soil improvement strategies has become an urgent problem to be solved.

[0003] Microbial fertilizers are regarded as an important alternative to chemical fertilizers due to their environmental friendliness and the ability to promote soil health. Currently, the functional microorganisms that have been studied more include Bacillus spp., Pseudomonas spp., and Trichoderma spp., etc. These strains can promote plant growth by nitrogen fixation, phosphorus solubilization, secreting plant hormones, etc. However, the existing microbial fertilizers still have the following problems: Single function of strains: Most commercial microbial fertilizers only target a specific function (such as phosphorus solubilization or nitrogen fixation), and it is difficult to comprehensively improve the soil nutrient status; Higher application cost: Some microbial fertilizers need to be mixed with organic carriers (such as straw, wood chips), which increases the production and transportation costs and limits large-scale promotion; Insufficient environmental adaptability: The survival rate of some strains is low in arid and barren soils, affecting the actual application effect. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a Brevibacillus sp. 9-1 and its application in microbial fertilizer, specifically as follows: A Brevibacillus sp. 9-1, isolated from soil, with the preservation number of CCTCC NO: M 20242933, the preservation date being: December 30, 2024, and the preservation unit being: China Center for Type Culture Collection (Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province); the 16S rDNA sequence of Brevibacillus sp. 9-1 is as shown in SEQ ID NO:1; and it is classified and named as Brevibacillus sp.

[0005] Moreover, the Brevibacillus sp. 9-1 is a Gram-positive bacterium, facultatively anaerobic, and can grow under aerobic and anaerobic conditions.

[0006] Moreover, the Brevibacillus sp. 9-1 has the functional activities of phosphorus solubilization, potassium solubilization, nitrogen fixation, and production of 1-aminocyclopropane-1-carboxylic acid (ACC).

[0007] Moreover, the preparation method of Brevibacillus sp. 9-1 is as follows: Weigh 5 g of the soil sample from the grazing-excluded plot, put it into a 100 mL Erlenmeyer flask, add 50 mL of sterile distilled water, and mix well to obtain a soil suspension with a concentration of 10 -1 ; Pipette 1 mL of the above soil suspension into a 10 mL sterile centrifuge tube, add 9 mL of sterile distilled water, and mix well to obtain a soil suspension with a concentration of 10 -2 . Repeat the above steps to dilute the solution to a final concentration of 10 -6 ; c) Pipette 100 μL of the soil suspension with a concentration of 10 -3 -10 -6 onto the R2A solid medium, and purify and culture it at 25 °C for 7 days. After separation and purification, Brevibacillus 9-1 is obtained.

[0008] On the other hand, the present invention provides a microbial fertilizer containing the above-mentioned Brevibacillus.

[0009] Moreover, the preparation method of the microbial fertilizer is as follows: The Brevibacillus sp. 9-1 strain is propagated and cultured in a 500 mL Erlenmeyer flask using a liquid R2A medium, and then centrifuged to obtain a bacterial sludge. The bacterial sludge is diluted with sterile water to 1×10 8 CFU / mL to prepare the microbial fertilizer, and the Brevibacillus microbial fertilizer is obtained.

[0010] Moreover, the culture temperature for the expanded culture in the R2A medium is 28 ± 0.5 °C, and the pH value is 7.2 ± 0.2.

[0011] Moreover, the microbial fertilizer is used for seed coating, promoting plant growth, or improving soil.

[0012] Moreover, the plant is alfalfa or crested wheatgrass.

[0013] Moreover, the improvement of the soil includes significantly increasing the contents of organic carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen, and / or available phosphorus in the soil by applying the fertilizer.

[0014] Beneficial effects: Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The Bacillus brevis 9-1 provided by the present invention has multiple functional activities of phosphorus solubilization, potassium solubilization, nitrogen fixation, and ACC deaminase production, and can comprehensively improve the soil nutrient status, increase soil fertility, and thus effectively promote plant growth.

[0015] 2. The Bacillus brevis 9-1 provided by the present invention can grow under both aerobic and anaerobic conditions, and can survive and function in different types of soil environments, including arid and barren soils, thereby improving the actual application effect of microbial fertilizers.

[0016] 3. Compared with chemical fertilizers, the microbial fertilizer used in the present invention is more environmentally friendly. It can not only increase soil fertility, but also promote the balance and diversity of soil microbial communities, and contribute to maintaining the health and stability of the soil ecosystem.

[0017] 4. The Bacillus brevis 9-1 and its microbial fertilizer provided by the present invention can be obtained from the soil, with simple and extensive sources. There is no need to combine with other fertilizers, and it is convenient to use with remarkable effects. Description of the Drawings

[0018] Figure 1 Colonies formed by soil suspension on R2A medium; Figure 2 Colonies formed by the Bacillus brevis 9-1 of the present invention on R2A medium; Figure 3 Appearance of a transparent circle when the Bacillus brevis 9-1 is in the phosphorus-solubilizing medium; Figure 4 Effect of the microbial fertilizer of Bacillus brevis on the physical and chemical properties of soil after planting alfalfa; Figure 5 Effect of the microbial fertilizer of Bacillus brevis on the physical and chemical properties of soil after planting Agropyron cristatum; Figure 6 Morphologies of 10 strains (No. 1-1, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, 8-1, 9-1) in Example 1; Figure 7 Picture of alfalfa seeds placed in a culture dish in Example 5; Figure 8 Picture of germinated alfalfa in Example 5; Figure 9 Picture of the potted alfalfa experiment in Example 5. Detailed Embodiments

[0019] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be described. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of 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.

[0020] The short Bacillus was isolated and cultured by the Grassland Research Office of the Chinese Academy of Agricultural Sciences, and the preparation of microbial fertilizer of short Bacillus and field experiments were carried out; subsequent genome sequencing and phylogenetic tree construction were carried out by Shanghai Personalbio.

[0021] Example 1 1. Isolation and purification of soil microorganisms Weigh 5 g of soil samples from the grazing-ban plot, add 50 mL of sterile distilled water, and mix well to prepare a 10 -1 soil suspension; obtain dilutions from 10 -2 to 10 -6 by gradient dilution method (successively pipette 1 mL of the suspension into 9 mL of sterile water); take 10 -3 -10 -6 dilution of 100 μL and coat it on the R2A solid medium. After culturing at 25 °C, 10 strains (numbered 1-1, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, 8-1, 9-1) were isolated and purified, and their morphologies are as Figure 6 shown.

[0022] 2. Screening of functional strains The following functional assays were performed on the above 10 strains: Phosphate-solubilizing ability: Inoculate on the Mengjinna medium and culture at 28 °C for 3-4 days. Those with a clear zone around the colony are positive (+), indicating that they have phosphate-solubilizing ability, and those otherwise are negative (-), indicating that they do not have phosphate-solubilizing ability.

[0023] Potassium-solubilizing ability: Inoculate on the potassium-solubilizing medium. Those with a clear zone are positive, indicating that they have potassium-solubilizing ability.

[0024] Nitrogen-fixing ability: Inoculate on the Ashby medium. Those that can grow are positive, indicating that they have nitrogen-fixing ability.

[0025] ACC deaminase-producing ability: Culture in DF and ADF liquid media respectively. Those with a significantly higher absorbance in the ADF group than in the DF group are positive, indicating that they have ACC deaminase-producing ability.

[0026] 3. Screening results As shown in Table 1, only strain 9-1 has the abilities of nitrogen fixation (+), phosphorus solubilization (+), potassium solubilization (+) and ACC deaminase production (+) simultaneously. Therefore, strain 9-1 was selected as the candidate strain for the subsequent preparation of bacterial fertilizer.

[0027] The functions of the remaining strains are as follows: 1-1, 2-1: Only phosphorus and potassium solubilization; 3-1: Only ACC deaminase production; 4-1: Nitrogen fixation, phosphorus and / or potassium solubilization ability; 5-1: Nitrogen fixation, potassium solubilization ability 5-2: Nitrogen fixation, phosphorus solubilization ability; 6-1: Phosphorus solubilization and ACC deaminase production.

[0028] 7-1: Nitrogen fixation, potassium solubilization ability; 8-1: Nitrogen fixation, phosphorus solubilization ability.

[0029] Table 1 Results of functional determination of 10 strains

[0030] Example 2 Bacillus brevis 9-1 was prepared by the method of Example 1. Bacillus brevis 9-1 was cultured at a constant temperature of 28 °C on R2A medium. The colonies formed by the soil suspension on R2A medium were as Figure 1 shown, and the colonies formed by Bacillus brevis 9-1 on R2A medium were as Figure 2 shown.

[0031] The whole genome sequencing was carried out by Beijing Housheng Botai Biotechnology Co., Ltd. The 16S rRNA gene sequence of Bacillus brevis 9-1 was subjected to homology comparison and analysis in the GenBank database. It was found that Bacillus brevis 9-1 had 100% similarity with Bacillus Brevis and belonged to the genus Bacillus. The specific 16S rDNA sequence was as shown in SEQ ID NO:1.

[0032] Example 3 The function of Bacillus brevis 9-1 was identified again. Bacillus brevis 9-1 prepared in Example 2 was cultured at a constant temperature of 28 °C on R2A medium; the purified Bacillus brevis 9-1 was inoculated into the Mengjinna medium and cultured for 3-4 days at 28 ± 0.5 °C in the dark. Its performance was that it could grow on the medium and there was a transparent area near the strain, identifying that it had the ability of phosphorus solubilization, as Figure 3 shown.

[0033] Inoculate Bacillus brevis 9-1 into a potassium-solubilizing medium and culture it at 28 ± 0.5 °C in total darkness for 3 - 4 days. Its performance is that it can grow on the potassium-solubilizing medium and there is a transparent area near the strain, indicating that it has the ability to solubilize potassium; Inoculate the purified Bacillus brevis 9-1 into an Ashby medium and culture it at 28 ± 0.5 °C in total darkness for 3 - 4 days. The performance is that it can grow on this medium, indicating that it has the ability to fix nitrogen.

[0034] Inoculate the purified Bacillus brevis 9-1 (three groups: 9-1-1, 9-1-2, 9-1-3) into a liquid DF medium (containing a conventional nitrogen source and the strain can grow without relying on ACC deaminase) and a liquid ADF medium (containing ACC as the sole nitrogen source and the strain needs to rely on ACC deaminase to decompose ACC to obtain nitrogen for growth); culture it on a shaker for 3 days, and use an enzyme-labeling instrument to measure the absorbance of each culture broth. The results are shown in Table 2.

[0035] Table 2 Results of absorbance measurement

[0036] As shown in Table 2, the growth of Bacillus brevis 9-1 strain in the ADF medium is better than that in the DF medium (OD 540 is higher), which proves that it can use ACC as a nitrogen source, that is, it has ACC deaminase activity.

[0037] Example 4 Transfer Bacillus brevis 9-1 into a liquid R2A medium and culture it on a shaker. All the components (drugs) of the culture medium used for shaking culture are of analytical purity. Then, centrifuge to remove the culture medium components to obtain a Bacillus brevis mud. Dissolve the Bacillus brevis microbial mud in sterile distilled water to prepare a Bacillus brevis microbial liquid, and soak the disinfected alfalfa seeds in the Bacillus brevis microbial liquid to coat the alfalfa seeds.

[0038] Example 5 Collect soil in the prohibited grazing area of the Sharqin Experimental Station of the Institute of Grassland Research, Chinese Academy of Agricultural Sciences, and conduct a pot experiment in the greenhouse of the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. Sow the alfalfa coated seeds (experimental group) and uncoated alfalfa seeds (CK) prepared in Example 4 in sterile petri dishes for seedling raising. Put 50 seeds in each petri dish ( Figure 7 ), and calculate the germination rate accordingly. Seven days after germination ( Figure 8 ), select the seedlings with consistent growth and transplant them for pot experiments ( Figure 9), ensuring that the two groups of seeds are under the same environmental conditions. Record the germination rates of the two groups of seeds, and measure the aboveground biomass, underground biomass, and plant height after 60 days, as shown in Table 3 below. Our results show that inoculation with the microbial fertilizer can increase the germination rate, aboveground biomass, underground biomass, and plant height of alfalfa seeds.

[0039] Table 3 Comparison of coated seeds and uncoated seeds

[0040] Example 6 Transfer Bacillus brevis 9-1 into liquid R2A medium and shake culture it on a shaker. All the components (drugs) of the medium used for shake culture are of analytical purity. Then, centrifuge to remove the medium components to obtain Bacillus brevis bacterial sludge. Dissolve the Bacillus brevis microbial bacterial sludge in sterile distilled water to prepare a Bacillus brevis microbial bacterial solution, and soak the disinfected Agropyron cristatum seeds in the Bacillus brevis microbial bacterial solution to coat the Agropyron cristatum seeds.

[0041] Example 7 Collect soil at the agro-pastoral ecotone experimental station of the Institute of Grassland Research, Chinese Academy of Agricultural Sciences, and conduct a pot experiment indoors at the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. Sow the coated and uncoated Agropyron cristatum seeds prepared in Example 6 in petri dishes for seedling cultivation respectively. Put 50 seeds in each petri dish to calculate the germination rate. After seven days of germination, select seedlings with consistent growth vigor for transplanting to conduct a pot experiment. Ensure that the two groups of seeds are under the same environmental conditions. Record the germination rates of the two groups of seeds, and measure the aboveground biomass, underground biomass, and plant height after 60 days, as shown in Table 4 below.

[0042] The results show that inoculation with the microbial fertilizer can increase the germination rate, aboveground biomass, underground biomass, and plant height of Agropyron cristatum seeds.

[0043] Table 4 Comparison of coated seeds and uncoated seeds

[0044] Example 8 Soil was collected from the fenced area of the Sharqin Experimental Station of the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. A pot experiment was carried out in the greenhouse of the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. The coated and uncoated alfalfa seeds prepared in Example 5 were respectively used for seedling raising in petri dishes. Seven days after germination, seedlings with consistent growth were selected for transplanting to conduct the pot experiment. Ensure that the two groups of seeds are under the same environmental conditions. After 60 days, the contents of total nitrogen, organic carbon, available phosphorus, ammonium nitrogen, and nitrate nitrogen in the soil were measured. The total nitrogen in the soil was detected by the method specified in HJ 717—2014 "Kjeldahl Method", ammonium nitrogen and nitrate nitrogen were detected by the method specified in HJ634—2012 "Potassium Chloride Extraction-Spectrophotometry", available phosphorus was detected by the method specified in HJ 704-2014 "Sodium Bicarbonate Extraction-Molybdenum Antimony Anti-Spectrophotometry", and organic carbon was detected by HJ 615-2011 "Potassium Dichromate Oxidation-External Heating Method"; specifically as Figure 4 shown. The results showed that inoculation with the bacterial fertilizer could increase the contents of organic carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen, and available phosphorus in the soil where alfalfa was planted.

[0045] Example 9 Soil was collected from the fenced area of the Sharqin Experimental Station of the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. A pot experiment was carried out in the greenhouse of the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. The coated and uncoated crested wheatgrass seeds prepared in Example 6 were respectively used for seedling raising in petri dishes. Seven days after germination, seedlings with consistent growth were selected for transplanting to conduct the pot experiment. Ensure that the two groups of seeds are under the same environmental conditions. After 60 days, the contents of soil organic carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen, and available phosphorus were measured (the detection method was the same as that in Example 8), specifically as follows Figure 5 shown. The results showed that inoculation with the bacterial fertilizer could increase the contents of total nitrogen, organic carbon, available phosphorus, ammonium nitrogen, and nitrate nitrogen in the soil where crested wheatgrass was planted.

Claims

1. A Brevibacillus sp. 9-1, characterized in that: It was isolated from soil, its preservation number is CCTCC NO: M 20242933, the preservation date is December 30, 2024, and it is preserved in the China Center for Type Culture Collection. The 16S rDNA sequence is shown in SEQ ID NO:

1.

2. The Brevibacillus sp. 9-1 according to claim 1, characterized in that: The Brevibacillus sp. 9-1 is a Gram-positive bacterium that grows under both aerobic and anaerobic conditions.

3. The Brevibacillus sp. 9-1 according to claim 1, characterized in that: The Brevibacillus sp. 9-1 has the functional activities of solubilizing phosphate, solubilizing potassium, fixing nitrogen and producing 1-aminocyclopropane-1-carboxylic acid.

4. A microbial fertilizer, characterized in that: The invention is obtained by expanding and culturing the Brevibacillus sp. 9-1 strain according to any one of claims 1 to 3.

5. The use of the microbial fertilizer according to claim 4, characterized in that: Used for seed coating.

6. The use of the microbial fertilizer according to claim 4, characterized in that: Used to promote plant growth.

7. The use of the microbial fertilizer according to claim 6, characterized in that: The plant is alfalfa or wheatgrass.

8. The use of the microbial fertilizer according to claim 4, characterized in that: Used to improve soil.

9. The use of the microbial fertilizer according to claim 8, characterized in that: The soil improvement includes significantly increasing the content of organic carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen and / or available phosphorus in the soil by applying the bacterial fertilizer.

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