Novel bacillus and application thereof

Abstract

The invention discloses a novel bacillus, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No. 1.62901, and the preservation date of which is September 20, 2024. Gene identification shows that the strain belongs to a new microbial species in taxonomy. Researches show that the strain has nitrogen fixation capacity, can grow in a Mengjinna inorganic phosphorus culture medium, exerts phosphorus dissolving capacity, converts insoluble phosphorus elements which are difficult to absorb and utilize by organisms into dissolved phosphate ions which can be efficiently absorbed and utilized by plants, and provides a large number of nutrient substances for crop production; in addition, the strain has good endurance capacity to low-temperature and high-salt environments, and can play an environment restoration function in the low-temperature environments or saline-alkali areas. Therefore, the novel bacillus disclosed by the invention has better application potential and prospect in the aspects of agricultural production (microbial phosphate solubilization) and environmental restoration.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial phosphate solubilization technology and microbial fertilizer technology, and in particular relates to a novel Bacillus and application thereof. Background Art

[0002] Phosphorus plays an important role in plant growth and plays an indispensable role in plant growth, metabolism and stress resistance. First, phosphorus is an important component of adenosine triphosphate (ATP), which is responsible for the storage and transfer of energy in plant cells, supporting anabolism and growth. Secondly, phosphorus participates in the process of photosynthesis, helping plants convert light energy into chemical energy and promoting plant growth and development. Phosphorus also has a positive effect on the flowering and fruiting of plants, and can improve the quality of flowers and the yield of fruits. At the same time, phosphorus helps the development of the root system, promotes root growth and branching, and thus enhances the plant's ability to absorb water and nutrients. Phosphorus interacts with other nutrients (such as nitrogen and potassium), promotes the plant's absorption of these elements, and improves the overall nutrient utilization efficiency. Finally, phosphorus helps enhance the plant's resistance to adversity (such as drought, pests and diseases) and improves the plant's survival ability.

[0003] As an important component of the environment, microorganisms play an important role in ecosystem regulation. Among them, phosphate-solubilizing bacteria are a type of microorganism that can dissolve insoluble phosphorus compounds in the soil. By producing organic acids, enzymes and other metabolites, they convert insoluble phosphorus in the soil (such as calcium phosphate in phosphate rock) into a form that can be absorbed by plants, thereby increasing the effective phosphorus content of the soil. The activity of phosphate-solubilizing bacteria can not only increase the availability of phosphorus, but also improve the soil structure, increase the microbial activity of the soil, and thus enhance the overall fertility of the soil. Phosphate-solubilizing bacteria coexist with plant roots, which can promote the absorption of other nutrients by plants and improve the overall nutrient utilization efficiency. Therefore, the use of phosphate-solubilizing bacteria for biological phosphate solubilization is an environmentally friendly method that reduces dependence on chemical fertilizers and contributes to sustainable agricultural development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a novel Bacillus with good phosphate solubilization ability and strong environmental tolerance and application thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The novel Bacillus sp. has a deposit number of CGMCC No. 1.62901 and a deposit date of September 20, 2024.

[0007] The 16S rDNA of the strain has the base sequence of SEQ ID NO.1 in the sequence listing.

[0008] A microbial preparation containing the novel Bacillus sp.

[0009] Application of the novel Bacillus or microbial preparation in microbial phosphate solubilization.

[0010] Application of the novel Bacillus or microbial preparation in environmental remediation.

[0011] The environment is a low-temperature site.

[0012] The low temperature is 15-30℃.

[0013] The environment is a high salinity site.

[0014] High salinity means that the mass concentration of sodium chloride reaches 2-6%.

[0015] The inventor isolated and screened a novel Bacillus sp. at a mining site in Guangxi, with a deposit number of CGMCC No. 1.62901 and a deposit date of September 20, 2024. Genetic identification showed that the strain belongs to a new species of microorganism in taxonomy. Studies have shown that the strain has the ability to fix nitrogen, can grow in Montgina inorganic phosphate culture medium, and exerts the ability to solubilize phosphorus, converting insoluble phosphorus elements that are difficult to be absorbed and utilized by organisms into dissolved phosphate ions that can be efficiently absorbed and utilized by plants, providing a large amount of nutrients for crop production; in addition, the strain has good tolerance to low temperature and high salt environments, and can play an environmental remediation role in low temperature environments or saline-alkali areas. Accordingly, the novel Bacillus sp. of the present invention has better application potential and prospects in agricultural production (microbial phosphate solubilization) and environmental remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the growth of the novel Bacillus sp. LXY-4 of the present invention on the Montkina inorganic phosphate medium.

[0017] Figure 2 This is the phylogenetic tree of the novel Bacillus LXY-4 of the present invention based on 16S rDNA.

[0018] Figure 3 is the OD of the novel Bacillus LXY-4 of the present invention in LB medium with different concentrations of sodium chloride 600 Change graph.

[0019] Figure 4 This is a graph showing the change in the amount of solubilized phosphorus of the novel Bacillus sp. LXY-4 of the present invention in Montgena inorganic phosphorus culture medium containing different concentrations of sodium chloride.

[0020] Figure 5 is the OD of the novel Bacillus LXY-4 in LB medium at different temperatures 600 Change graph.

[0021] Figure 6This is a graph showing the change in the amount of solubilized phosphorus of the novel Bacillus LXY-4 of the present invention in Montkina inorganic phosphorus culture medium at different temperatures.

[0022] Preservation Information

[0023] Neobacillus sp. LXY-4, deposit number CGMCC No. 1.62901, deposit date: September 20, 2024, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, deposited by: General Microbiology Center, China Culture Collection Administration.

[0024] Storage conditions: Glycerol tube -80℃ storage

[0025] Colony morphology: Yellow colonies with a yellow halo around them, with a rough, raised, and moist surface DETAILED DESCRIPTION

[0026] Example 1 Isolation and identification of strains

[0027] The test site was located in a mining area in Guangxi. A representative soil area was selected to avoid contamination and interference. Topsoil samples were collected using a sterilized shovel or sampler. The samples were placed in sterile plastic bags or containers and labeled. The collected samples were then shipped to a laboratory at low temperatures for microbial isolation and screening.

[0028] Montgena's inorganic phosphate medium is used for the isolation and screening of phosphate-solubilizing bacteria. Its insoluble phosphorus is tricalcium phosphate. Its specific composition is as follows: 10.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, and 5.0g tricalcium phosphate per 1L of distilled water. After thorough shaking, adjust the pH to 7 with NaOH or HCl, sterilize at 121°C and autoclave for 20 minutes, and cool until ready for use. For the solid medium, add 15g agar powder, adjust the pH to 7, and autoclave at 121°C for 20 minutes. Pour the solid medium into a sterile Petri dish while still hot and cool to obtain a solid medium plate.

[0029] LB medium is used for strain expansion. Its composition is: 5g yeast powder, 10g sodium chloride, and 10g tryptone per 1L of distilled water. After thorough shaking, adjust the pH to 7 with NaOH or HCl. Sterilize at 121°C and autoclave for 20 minutes. Cool and set aside.

[0030] Take 5g of soil sample and add it to 45mL of sterile saline or buffer. Shake thoroughly for 10 minutes to fully suspend the soil particles. Let the soil mixture stand for 10 minutes to precipitate the solid particles. The supernatant is the bacterial suspension used for screening. Take 100μL of bacterial suspension and add it to 900μL of sterile saline and mix thoroughly to form 10 -1 diluent. Take 100μL10 -1 The dilution was added to 900 μL of sterile saline to form 10 -2 dilution, and so on, up to 10 -6 Dilution. Take 200 μL of 10 -4 , 10 -5 and 10 -6 Add the gradient dilutions to the Asbestos nitrogen-free medium and spread evenly on the plate using a sterile applicator. Place the plate upside down in a 30°C incubator for 48 hours and observe the growth of the colonies.

[0031] Once a single colony has clearly grown on the plate, select a representative, well-growing colony and pick it with a sterile inoculating loop. Streak the plate onto fresh Aspergillus medium to ensure a single colony is isolated. Place the plate upside down in a 30°C incubator and incubate for 48 hours. Observe the growth of the colony and repeat the streaking process until a purified strain is obtained.

[0032] The genomic DNA was extracted from the purified strain using a DNA extraction kit and PCR amplification was performed. The PCR product was subjected to agarose gel electrophoresis to confirm the amplification effect. The 16S rRNA of the bacterium was amplified by PCR and sequenced. The sequence (SEQ ID NO.1) was Blast-matched to the NCBI database. The comparison results showed that ( Figure 2 ), with the highest homology of 97.75% with Bacillus marasmi strain Marseille-P3556, the strain was named Neobacillus sp. LXY-4, referred to as Neobacillus sp. LXY-4 or LXY-4 strain.

[0033] Under normal circumstances, a similarity of less than 98% can be considered a new species of microorganism. The similarity between the LXY-4 strain and its closest strain is 97.75%, so it is considered a potential new species of microorganism in genetic identification.

[0034] Example 2 Phosphate-dissolving ability test of bacterial strains

[0035] The LXY-4 strain was inoculated into liquid LB medium and cultured at 30°C and 180 rpm for 24 h.600 = 1. Pipette 10 μL of seed solution and inoculate onto solid inorganic phosphate culture medium, place in an incubator at 30°C and culture upside down for 3 days, and measure the colony diameter and phosphate-solubilizing ring diameter.

[0036] 1 mL of seed liquid was inoculated into 100 mL of liquid inorganic phosphorus culture medium at an inoculum rate of 1%, and the culture was carried out in a shaking incubator at 30°C and 180 r / min for 3 days. The water-soluble phosphorus content in the culture medium was determined by the molybdenum antimony colorimetric method.

[0037] The results showed that after three days of culture, a clear phosphorus-dissolving ring appeared around the colony, indicating that the bacterium has the ability to dissolve insoluble tricalcium phosphate into soluble phosphorus. This indicates that the bacterium can provide the phosphorus required for plant growth and can be absorbed and utilized. At the same time, the dissolved phosphorus in the supernatant of the liquid culture medium was tested (Table 1). After three days of culture, the phosphorus content in the supernatant reached 195.7 mg / L, and the pH of the culture medium reached 4.2, indicating that the bacterium functions by secreting organic acids to dissolve phosphorus.

[0038] Table 1 Determination of phosphate solubilization ability of strains

[0039]

[0040] Example 3 Adaptability of strains to high salinity conditions

[0041] Prepare liquid LB medium and inorganic phosphate medium, divide into 50mL conical flasks, add different concentrations of sodium chloride to simulate high salinity conditions, set up 2%, 4%, 6%, 8%, 10% for a total of 5 gradient experiments. 600 = 1 seed solution was inoculated into the above LB medium and inorganic phosphorus medium at a 1% inoculum amount, and the OD of the bacterial suspension was measured after culturing at 30 ° C and 160 r / min for 24 h. 600 Value and dissolved phosphorus amount.

[0042] result( Figure 3 ) It can be seen that the increase in salt concentration inhibited the growth of the bacteria. Under the condition of 2% OD 600 =3.72, OD under 4% conditions 600 =3.44, the growth of the strain did not change significantly, indicating that the bacteria can fully tolerate 4% salt concentration. Under 6% conditions, OD 600 =2.49, its growth was slightly inhibited, but it could still reach a high OD value, indicating that the bacteria had good tolerance to 6% salt concentration. Under 8% and 10% conditions, the bacteria had difficulty growing normally.

[0043] The water-soluble phosphorus content in the culture medium was determined using the molybdenum antimony colorimetric method. At a 2% salt concentration, the soluble phosphorus content was 163.3 mg / L, and at a 4% salt concentration, it was 158.5 mg / L. At a 6% salt concentration, the solubility was 141.1 mg / L. This suggests that salt concentrations of 6% and below do not significantly inhibit its phosphorus-solubilizing function. Combined with its growth pattern, this suggests that the bacterium can function normally at a 6% salt concentration, making it highly effective in environmental remediation of high-salt sites. Even at an 8% salt concentration, it still retained a certain amount of phosphorus-solubilizing capacity, with the solubility in the culture system reaching 65.3 mg / L.

[0044] In summary, the bacteria has excellent adaptability to a high salt concentration of 6% and has great application prospects in the ecological restoration of saline sites.

[0045] Example 4 Adaptability of strains in low temperature environments

[0046] Prepare liquid LB medium and inorganic phosphate medium, divide into 50 mL conical flasks, set up 30℃, 25℃, 20℃, 15℃, 10℃ for a total of 5 gradient experiments. 600 = 1 seed solution was inoculated into the above LB medium and inorganic phosphorus medium at a 1% inoculum amount, and the OD of the bacterial suspension was measured after shaking culture at different temperatures and 160r / min for 24h. 600 Value and dissolved phosphorus amount.

[0047] The results show that at 25°C, the OD600 was 4.34, and the solubility of phosphorus was 150.9 mg / L. While lowering the temperature inhibited the growth and function of the strain, 25°C had no effect on the strain. At 20°C, the OD600 was 4.22, and the solubility of phosphorus was 131.1 mg / L. At 15°C, the OD600 was 3.42, and the solubility of phosphorus was 95.3 mg / L. The strain also exhibited excellent tolerance to temperatures of 15°C. Growth and function were significantly inhibited at 10°C.

Claims

1. A novel Bacillus strain, whose deposit number is CGMCC No. 1.62901 and the deposit date is September 20, 2024.

2. The novel Bacillus according to claim 1, characterized in that The 16S rDNA of the strain has the base sequence of SEQ ID NO.1 in the sequence listing.

3. A microbial preparation, characterized in that Contains the novel Bacillus according to claim 1.

4. Use of the novel Bacillus according to claim 1 or the microbial preparation according to claim 3 in microbial phosphate solubilization.

5. Use of the novel Bacillus according to claim 1 or the microbial preparation according to claim 3 in environmental remediation.

6. The use according to claim 5, characterized in that: The environment is a low-temperature site.

7. The use according to claim 6, characterized in that: The low temperature is 15-30°C.

8. The use according to claim 5, characterized in that: The environment is a high-salinity site.

9. The use according to claim 8, characterized in that: The high salinity means that the mass concentration of sodium chloride reaches 2-6%.

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