A strain of Bacillus brevis 9-1 and its application in microbial fertilizer

Abstract

The present invention provides a strain of Brevibacillus sp. 9-1 and an application thereof in a microbial fertilizer, belonging to the field of microbial technology. The Brevibacillus sp. is isolated from soil, has a deposit number of CCTCC NO: M 20242933, a deposit date of December 30, 2024, and is deposited in the China Center for Type Culture Collection. The 16S rDNA sequence is shown in SEQ ID NO: 1. The Brevibacillus sp. 9-1 has the functional activities of solubilizing phosphate and potassium, fixing nitrogen, and producing ACC. The microbial fertilizer is obtained by expanding the culture of the Brevibacillus sp. 9-1. Compared with chemical fertilizers, the microbial fertilizer used in the present invention is more environmentally friendly, can not only improve soil fertility, but also promote the balance and diversity of soil microbial communities, and help maintain the health and stability of the soil ecosystem.

Description

Technical Field

[0001] The invention relates to a strain of Bacillus brevis 9-1 and application thereof in microbial fertilizer, belonging to the technical field of microorganisms. Background Art

[0002] In recent years, my country's grassland ecosystems have faced severe degradation due to climate change and human activities such as overgrazing, manifesting as declining soil fertility, reduced vegetation cover, and a sharp drop in productivity. While traditional fertilization methods (such as chemical fertilizers) can improve grassland productivity in the short term, long-term use can easily lead to soil compaction, imbalanced microbial communities, and even exacerbate ecological degradation. Therefore, developing sustainable soil improvement strategies is an urgent issue.

[0003] Microbial fertilizers are considered an important alternative to chemical fertilizers due to their environmental friendliness and ability to promote soil health. Currently, the most studied functional microorganisms include Bacillus spp., Pseudomonas aeruginosa, and Trichoderma spp. These strains can promote plant growth through nitrogen fixation, phosphorus solubilization, and secretion of plant hormones. However, existing microbial fertilizers still have the following problems:

[0004] Single bacterial strain function: Most commercial bacterial fertilizers only target a specific function (such as phosphorus solubilization or nitrogen fixation), making it difficult to comprehensively improve soil nutrient status;

[0005] High application cost: Some microbial fertilizers need to be mixed with organic carriers (such as straw and sawdust), which increases production and transportation costs and limits large-scale promotion;

[0006] Insufficient environmental adaptability: The survival rate of some strains is low in drought and poor soil, which affects the actual application effect. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a strain of Bacillus brevis 9-1 and its application in microbial fertilizer, as follows:

[0008] A Brevibacillus sp. 9-1 was isolated from soil, with a deposit number of CCTCCNO: M 20242933 and a deposit date of December 30, 2024, at the China Center for Type Culture Collection (address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province). The 16S rDNA sequence of Brevibacillus 9-1 is shown in SEQ ID NO: 1. The strain is classified as Brevibacillus sp.

[0009] Moreover, the Brevibacillus 9-1 is a Gram-positive bacterium, facultative anaerobic, and can grow under aerobic and anaerobic conditions.

[0010] Moreover, the Brevibacillus sp. 9-1 has the functional activities of solubilizing phosphate, solubilizing potassium, fixing nitrogen and producing 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase).

[0011] Furthermore, the preparation method of Brevibacillus sp. 9-1 is as follows:

[0012] a) Weigh 5 g of soil sample from the grazing ban site, place it in a 100 mL Erlenmeyer flask, add 50 mL of sterile distilled water, mix thoroughly, and obtain 10 -1 Concentration of soil suspension;

[0013] b) Pipette 1 mL of the above soil suspension into a 10 mL sterile centrifuge tube, add 9 mL of sterile distilled water, mix thoroughly, and obtain 10 mL of the soil suspension. -2 Repeat the above steps to dilute the solution to a final concentration of 10 -6 ;

[0014] c) Draw 10 -3 -10 -6 100 μL of the concentrated soil suspension was spread on R2A solid culture medium and cultured at 25°C for 7 days. After separation and purification, Bacillus brevis 9-1 was obtained.

[0015] In another aspect, the present invention provides a microbial fertilizer comprising the aforementioned Bacillus brevis.

[0016] The preparation method of the microbial fertilizer is as follows: the Brevibacillus sp. 9-1 strain is propagated in a 500 mL triangular flask using liquid R2A culture medium, and then centrifuged to obtain bacterial sludge, which is diluted with sterile water to 1×10 8 CFU / mL, and made into bacterial fertilizer to obtain Brevibacillus microbial fertilizer.

[0017] Furthermore, 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.

[0018] Moreover, microbial fertilizers are used for seed coating, to promote plant growth or to improve soil.

[0019] Furthermore, the plant is alfalfa or wheatgrass.

[0020] Moreover, 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.

[0021] Beneficial effects:

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The Bacillus brevis 9-1 provided by the present invention has multiple functional activities such as phosphate solubilization, potassium solubilization, nitrogen fixation and ACC deaminase production, which can comprehensively improve soil nutrient conditions and enhance soil fertility, thereby effectively promoting plant growth.

[0024] 2. The Brevibacillus 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 practical application effect of microbial fertilizers.

[0025] 3. Compared with chemical fertilizers, the microbial fertilizer used in the present invention is more environmentally friendly. It can not only improve soil fertility, but also promote the balance and diversity of soil microbial communities, and help maintain the health and stability of the soil ecosystem.

[0026] 4. The Bacillus brevis 9-1 and the microbial fertilizer provided by the present invention can be obtained from the soil, the material is simple and extensive, and there is no need to use it in combination with other fertilizers. It is easy to use and has significant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the colony formed by soil suspension on R2A medium;

[0028] Figure 2 The colonies formed by Brevibacillus 9-1 of the present invention on R2A medium;

[0029] Figure 3 The clear zone appears when Bacillus brevis 9-1 is in phosphate-solubilized medium;

[0030] Figure 4 The study is about the effect of Bacillus brevis microbial fertilizer on the physical and chemical properties of soil after planting alfalfa.

[0031] Figure 5 The study is about the effect of Bacillus brevis microbial fertilizer on the physical and chemical properties of soil after planting wheatgrass.

[0032] Figure 6 The morphologies of the 10 strains (numbered 1-1, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, 8-1, and 9-1) in Example 1 are shown;

[0033] Figure 7This is a picture of alfalfa seeds placed in a culture dish in Example 5;

[0034] Figure 8 This is a picture of alfalfa after germination in Example 5;

[0035] Figure 9 This is a picture of the alfalfa pot experiment in Example 5. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be described below in conjunction with the embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] The present invention isolated and cultured Bacillus brevis by the Grassland Research Laboratory of the Chinese Academy of Agricultural Sciences, and carried out preparation of Bacillus brevis microbial fertilizer and field experiments; Shanghai Paisono performed subsequent genome sequencing and evolutionary tree construction.

[0038] Example 1

[0039] 1. Isolation and purification of soil microorganisms

[0040] Weigh 5 g of soil sample from the grazing ban site, add 50 mL of sterile distilled water, mix well and prepare 10 -1 Soil suspension; 10 mL suspension was obtained by gradient dilution (1 mL suspension was added to 9 mL sterile water). -2 to 10 -6 dilution; take 10 -3 -10 -6 100 μL of the dilution was applied to R2A solid medium and cultured at 25°C. Ten strains (numbered 1-1, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, 8-1, and 9-1) were isolated and purified. Figure 6 shown.

[0041] 2. Screening of functional strains

[0042] The following functional assays were performed on the above 10 strains:

[0043] Phosphate solubilization ability: Inoculate in Montgina medium and culture at 28℃ for 3-4 days. If a transparent area appears around the colony, it is positive (+), indicating that it has the ability to solubilize phosphate. Otherwise, it is negative (-), indicating that it does not have the ability to solubilize phosphate.

[0044] Potassium-solubilizing ability: When inoculated into potassium-solubilizing culture medium, the appearance of transparent area is positive, proving that it has potassium-solubilizing ability.

[0045] Nitrogen fixation ability: When inoculated into Ashby medium, those that can grow are positive, proving that they have the ability to fix nitrogen.

[0046] Ability to produce ACC deaminase: The cells were cultured in DF and ADF liquid culture media respectively. Those with significantly higher absorbance in the ADF group than in the DF group were considered positive, indicating that they had the ability to produce ACC deaminase.

[0047] 3. Filter results

[0048] As shown in Table 1, only strain 9-1 had the ability to fix nitrogen (+), solubilize phosphorus (+), solubilize potassium (+), and produce ACC deaminase (+), so strain 9-1 was selected as the candidate strain for subsequent bacterial fertilizer preparation.

[0049] The functions of the remaining strains are as follows:

[0050] 1-1, 2-1: only dissolve phosphorus and potassium;

[0051] 3-1: only produces ACC deaminase;

[0052] 4-1: Nitrogen fixation and phosphorus / potassium solubilization ability;

[0053] 5-1: Nitrogen fixation and potassium release ability

[0054] 5-2: Nitrogen fixation and phosphorus solubilization ability;

[0055] 6-1: Phosphate solubilization and production of ACC deaminase.

[0056] 7-1: Nitrogen fixation and potassium solubility ability;

[0057] 8-1: Nitrogen fixation and phosphorus solubilization capabilities.

[0058] Table 1 Functional assay results of 10 strains

[0059]

[0060] Example 2

[0061] The method of Example 1 was used to prepare Bacillus brevis 9-1. Bacillus brevis 9-1 was cultured on R2A medium at a constant temperature of 28°C. The colonies formed by the soil suspension on the R2A medium were as follows: Figure 1 As shown, the colonies formed by Bacillus brevis 9-1 on R2A medium are as follows Figure 2 shown.

[0062] Whole genome sequencing was performed by Beijing Housheng Botai Biotechnology Co., Ltd., and the 16S rRNA gene sequence of Bacillus brevis 9-1 was compared with the GenBank database for homology analysis. It was found that Bacillus brevis 9-1 had 100% similarity with Bacillus brevis Brevis and belonged to the genus Brevis. The specific 16S rDNA sequence is shown in SEQ ID NO: 1.

[0063] Example 3

[0064] The function of Bacillus brevis 9-1 was identified again. The Bacillus brevis 9-1 prepared in Example 2 was cultured at 28°C on R2A medium. The purified Bacillus brevis 9-1 was inoculated into Montina medium and cultured at 28±0.5°C in complete darkness for 3-4 days. The strain was able to grow on the medium and had a transparent area near the strain, indicating that it had the ability to solubilize phosphate. Figure 3 shown.

[0065] Inoculate Bacillus brevis 9-1 into potassium-solubilizing medium and culture at 28±0.5℃ in complete darkness for 3-4 days. If it can grow on the potassium-solubilizing medium and a transparent area is formed near the strain, it is identified as having potassium-solubilizing ability.

[0066] The purified cultured Bacillus brevis 9-1 was inoculated into Ashby medium and cultured at 28±0.5°C in complete darkness for 3-4 days. If it was able to grow on the medium, it was identified as having nitrogen fixation ability.

[0067] The purified cultured Bacillus brevis 9-1 (three groups: 9-1-1, 9-1-2, and 9-1-3) was inoculated into liquid DF medium (containing a conventional nitrogen source, and the strain can grow without relying on ACC deaminase) and liquid ADF medium (containing ACC as the only nitrogen source, and the strain must rely on ACC deaminase to decompose ACC to obtain nitrogen for growth); the culture was shaken on a shaker for 3 days, and the absorbance of each culture solution was measured using a microplate reader. The results are shown in Table 2.

[0068] Table 2 Absorbance measurement results

[0069]

[0070] As shown in Table 2, the growth rate of Bacillus brevis 9-1 strain in ADF medium was better than that in DF medium (OD 540 higher), it proves that it can utilize ACC as a nitrogen source, that is, it has ACC deaminase activity.

[0071] Example 4

[0072] Bacillus brevis 9-1 was transferred to liquid R2A culture medium and cultured on a shaker, and the culture medium components (drugs) used for the shaking culture were all of analytical grade. The culture medium components were then removed by centrifugation to obtain Bacillus brevis bacterial sludge. The Bacillus brevis microbial sludge was dissolved in sterile distilled water to prepare a Bacillus brevis microbial liquid. Sterilized alfalfa seeds were soaked in the Bacillus brevis microbial liquid to coat the alfalfa seeds.

[0073] Example 5

[0074] Soil was collected from the grazing ban area of ​​the Sharqin Experimental Station of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. A potted experiment was conducted in the greenhouse of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. The coated alfalfa seeds (experimental group) prepared in Example 4 and the uncoated alfalfa seeds (CK) were grown in sterile culture dishes. 50 seeds ( Figure 7 ), and calculate the germination rate. Figure 8 ), select seedlings with consistent growth and transplant them into potted plants for experiment ( Figure 9 ), ensuring that both seed groups were exposed to identical environmental conditions. Germination rates were recorded for both seed groups, and aboveground and belowground biomass, as well as plant height, were measured 60 days later. Details are shown in Table 3. Our results show that inoculation with bacterial fertilizer can increase germination rates, aboveground and belowground biomass, and plant height of alfalfa seeds.

[0075] Table 3 Comparison of coated and uncoated seeds

[0076]

[0077] Example 6

[0078] Bacillus brevis 9-1 was transferred to liquid R2A culture medium and cultured on a shaker. All the culture medium components (drugs) used for the shaking culture were of analytical grade. The culture medium components were then removed by centrifugation to obtain Bacillus brevis bacterial slurry. The Bacillus brevis microbial slurry was dissolved in sterile distilled water to prepare a Bacillus brevis microbial liquid. The sterilized wheatgrass seeds were soaked in the Bacillus brevis microbial liquid to coat the wheatgrass seeds.

[0079] Example 7

[0080] Soil was collected at the Experimental Station of the Agricultural and Pastoral Interlaced Area of ​​the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. A potted experiment was conducted indoors at the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. The coated and uncoated wheatgrass seeds prepared in Example 6 were respectively grown in culture dishes for seedling cultivation. 50 seeds were placed in each culture dish to calculate the germination rate. After seven days of germination, seedlings with consistent growth were selected for transplanting in a potted experiment. Ensure that the two groups of seeds are under the same environmental conditions. The germination rate of the two groups of seeds was recorded, and the aboveground biomass, underground biomass, and plant height were measured after 60 days, as shown in Table 4 below.

[0081] The results showed that inoculation with bacterial fertilizer could increase the germination rate, aboveground biomass, underground biomass and plant height of wheatgrass seeds.

[0082] Table 4 Comparison of coated and uncoated seeds

[0083]

[0084] Example 8

[0085] Soil was collected from the grazing ban area of ​​the Sharqin Experimental Station of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences, and a pot experiment was carried out in the greenhouse of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. The coated and uncoated alfalfa seeds prepared in Example 5 were respectively grown in culture dishes for seedling cultivation. Seven days after germination, seedlings with consistent growth were selected for transplanting for pot experiments. Ensure that the two groups of seeds are under the same environmental conditions. After 60 days, the total nitrogen, organic carbon, available phosphorus, ammonium nitrogen, and nitrate nitrogen contents 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, Figure 4 The results showed that the inoculation of bacterial fertilizer could increase the content of organic carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen and available phosphorus in the soil where alfalfa was planted.

[0086] Example 9

[0087] Soil was collected from the grazing ban area of ​​the Sharqin Experimental Station of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. A potted experiment was conducted in the greenhouse of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. The coated and uncoated wheatgrass seeds prepared in Example 6 were grown in petri dishes. After seven days of germination, seedlings with consistent growth were selected and transplanted for potted experiments. The two groups of seeds were ensured to be under the same environmental conditions. After 60 days, the soil organic carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen, and available phosphorus contents were measured (detection method is the same as Example 8), as follows: Figure 5 The results showed that the inoculation of bacterial fertilizer could increase the content of total nitrogen, organic carbon, available phosphorus, ammonium nitrogen and nitrate nitrogen in the soil of wheatgrass.

Claims

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

1.

2. A microbial fertilizer, characterized in that: The invention is obtained by expanding and culturing the Brevibacillus sp. 9-1 strain according to claim 1.

3. The application of the microbial fertilizer according to claim 2, characterized in that Used for seed coating.

4. The application of the microbial fertilizer according to claim 2, characterized in that Used to promote plant growth.

5. The use of the microbial fertilizer according to claim 4, characterized in that: The plant is alfalfa or wheatgrass.

6. The use of the microbial fertilizer according to claim 2, characterized in that: Used to improve soil.

7. The use of the microbial fertilizer according to claim 6, characterized in that: The soil improvement includes 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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