Bacillus sp. L01 and application thereof

Abstract

The invention relates to the technical field of microbiology and biology, and provides a Bacillus sp. L01 and application thereof. The water barren bacillus L01 has super-strong stress resistance, can maintain metabolic activity under extreme composite stress of 9% NaCl and 30% PEG6000, has triple functions of phosphate solubilization, potassium solubilization and protease secretion, and can synchronously realize coordinated regulation of saline-alkali remediation, drought adaptation, phosphorus and potassium activation, organic matter conversion and the like by using a single strain. The method has the core advantages of being high in stress tolerance, high in function integration level and low in application cost, and a new bioremediation scheme is provided for degraded soil.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, and more specifically, to a strain of Bacillus subtilis L01 and its applications. Background Technology

[0002] The continued degradation of arable land quality leads to exposed land surfaces, exacerbating wind erosion and dust transport, and significantly worsening the regional atmospheric environment. In addition, the decline in vegetation cover further disrupts the water and heat balance and biogeochemical cycles, weakening the ecosystem's functions of water conservation, carbon sequestration, and biodiversity maintenance.

[0003] Traditional remediation methods are no longer sufficient to meet the restoration needs of degraded land. Microbial remediation technology is therefore considered the most promising alternative. For example, the salt-tolerant strains disclosed in CN202411598263.7 can significantly improve crop yield and quality; the *Halomonas* strains disclosed in CN202411394087.5 can survive in extreme salinity environments of 16% NaCl and pH 8.5. *Bacillus* spp., due to their spore production, strong resistance, and good compatibility with chemical pesticides, have become a core species in biocontrol research both domestically and internationally. Among them, strains such as *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus polymyxa*, *Bacillus cereus*, *Bacillus sonorensis*, *Bacillus megaterium*, and *Bacillus pumilus* have been reported most frequently. Taking Bacillus pumilus as an example, the strain BP-20231025 disclosed in CN202410727271.0 can grow in the range of 2%-8% NaCl (optimal salt concentration 4%-6%), but lacks systematically verified growth-promoting and disease-resistant functions. This indicates a significant gap in the comprehensive adaptability of existing strains under multiple adversities and their growth-promoting effects on plants. Therefore, screening biocontrol Bacillus strains with multiple functions including high salt tolerance, drought tolerance, tolerance to poor soil conditions, growth promotion, and disease resistance will provide a new biotechnological pathway for the ecological restoration and agricultural utilization of saline-alkali and desertified lands. However, to date, there have been no systematic studies or reports on the application of Bacillus inaquosorum in the improvement of saline-alkali and desertified lands and crop growth promotion, indicating that this direction has significant innovative potential and application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus subtilis L01 and its applications.

[0005] This invention innovatively utilizes Bacillus inaquosorum (CGMCC No. 35658) to achieve a breakthrough in salt and drought tolerance: this strain maintains metabolic activity under extreme combined stress of 9% NaCl and 30% PEG6000, and also has functions such as phosphorus solubilization, potassium solubilization and protease secretion, which can provide a new bioremediation solution for degraded soil.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a strain L01 isolated and cultured from the topsoil of the Yellow River region in Kenli District, Dongying City, Shandong Province, classified and named *Bacillus ininaquosorum*, which is now deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 35658, deposit date August 18, 2025.

[0007] Secondly, the present invention provides a microbial agent or bio-fertilizer prepared from the aforementioned Bacillus subtilis.

[0008] Thirdly, the present invention provides plant growth promoters, agricultural fertilizers, phosphorus activators or soil activators prepared from the aforementioned Bacillus aquaticus or its inoculum.

[0009] Fourthly, the present invention provides any of the following applications of the *Bacillus silenstracum* or its inoculum:

[0010] 1) Used for phosphorus solubilization;

[0011] 2) Used for potassium solubilization;

[0012] 3) Used for the production of amylase;

[0013] 4) Used for protease production;

[0014] 5) Used to promote plant growth or to prepare plant growth promoters;

[0015] 6) Used to improve soil nutrient content or prepare soil activators;

[0016] 7) Used for the improvement of salinized and desertified land.

[0017] Furthermore, the phosphorus mentioned in 1) is inorganic phosphorus, such as calcium phosphate.

[0018] Furthermore, the plant growth promotion method described in 5) includes: irrigating the plants with the bacterial solution of the *Bacillus silenstilae*.

[0019] Furthermore, the plant includes, but is not limited to, cotton.

[0020] Furthermore, the improvement of soil nutrient content mentioned in 6) includes increasing the content of absorbable phosphorus and potassium nutrients in the soil.

[0021] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0022] The *Bacillus spp.* L01 of this invention possesses superior stress resistance and functionality:

[0023] (1) Strong salt tolerance: The strain can tolerate salt concentrations of 6%-9% NaCl (equivalent to about 1.7%-3.0% of total salt content), with the best growth at 6%-7% NaCl (about 1.7%-2.4% of total salt content), and the biomass accumulation rate reaches its peak at 6% NaCl; its tolerance to extreme salt stress is up to 9% NaCl (about 3.0% of total salt content), and it is fully adapted to the weakly alkaline environment (pH 7.5-8.5) of coastal saline-alkali land.

[0024] (2) Strong tolerance to barren environment: The strain can grow at glucose concentrations of 15-500 mg / L (equivalent to carbon source concentration of 6-200 mg / L), exceeding the international threshold for oligotrophic bacteria (Kuznetsov standard: ≤15 mg C / L), indicating that it can overcome the low carbon source limitation of saline soil and achieve colonization and metabolic activity in an environment with scarce organic matter (≤8 g / kg).

[0025] (3) Drought tolerance: The growth of the strain was not significantly affected when the concentration of PEG6000 was 10% and 20%, and the growth of the strain was slow but still possible when the concentration was 30%. The strain maintained its growth activity under simulated drought stress of 30% PEG6000 and could adapt to sandy soil with annual precipitation <200 mm and organic matter content <0.5%; it could improve soil microstructure and increase the survival rate of artificial vegetation.

[0026] (4) Fast-acting nutrient release function: The strain has the function of phosphorus and potassium solubilization, which accelerates the mineralization of plant-absorbable phosphorus and potassium nutrients and helps to improve barren soil.

[0027] (5) Disease resistance and growth promotion: The strain can secrete amylase and protease to accelerate the mineralization of organic matter into nutrients that can be absorbed by plants and promote plant growth.

[0028] (6) High temperature resistance: The strain can withstand high temperatures up to 95°C and can be used for high temperature granulation.

[0029] (7) Diverse functions and applicable to various environments: This strain has the functions of salt tolerance, drought tolerance, barrenness tolerance, high temperature tolerance, phosphorus solubilization, potassium solubilization, amylase production, and protease production, and can be applied to the improvement of degraded land in various scenarios. Attached Figure Description

[0030] Figure 1 This is the colony morphology of Bacillus silicifica L01 of the present invention.

[0031] Figure 2 The results of the nutrient deficiency stress experiment of strain L01 in the preferred embodiment of the present invention are shown.

[0032] Figure 3 The results of the high-temperature stress experiment of strain L01 at 95°C are shown in the preferred embodiment of the present invention.

[0033] Figure 4 The results of experiments on the production of amylase, protease, phosphorus solubilization, and potassium solubilization by strain L01 in the preferred embodiment of the present invention are shown. Detailed Implementation

[0034] Currently, some progress has been made in land improvement for salinization and semi-desertification, but several key issues remain unresolved: 1) Chemical improvement methods (such as gypsum application and water washing for desalination) can alleviate salt damage in the short term, but they are costly per acre and can trigger secondary salinization. 2) Most salt-tolerant bacterial agents cannot simultaneously possess the functions of tolerance to poor soil, drought, salt, and plant growth promotion. Degraded land often exhibits these characteristics, so most salt-tolerant bacterial agents are easily inactivated under multiple stresses, resulting in low survival rates in the field and an inability to synergistically resist the combined stresses of salinity and drought. 3) Conventional phosphorus-solubilizing bacterial agents require high carbon source content, and their phosphorus-solubilizing function is greatly reduced in poor soils, making them unable to adapt to the low-carbon source stress in saline-alkali / desert areas. 4) Existing drought-resistant bacterial agents generally lack mineral activation capacity and have low efficiency in releasing solid phosphorus and potassium nutrients from the soil.

[0035] To address the aforementioned problems, this invention provides a salt- and drought-tolerant Bacillus inauspicious water bacterium (Bacillus inauspicious water bacterium) L01, which can simultaneously achieve synergistic regulation of salt and alkali remediation, drought adaptation, phosphorus and potassium activation, and organic matter transformation using a single strain. It possesses core advantages such as strong stress resistance, high functional integration, and low application cost.

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0037] The culture medium used in the following examples:

[0038] Glucose medium: (NH4)2HPO4 1 g; KNO3 1 g; (NH4)2SO4 0.5 g; K2HPO4·3H2O 1.3 g; NaCl 5 g; MgSO4·7H2O 0.41 g; 1.5–2% agar added to solid medium.

[0039] LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl.

[0040] Drought-resistant culture medium: LB medium + different concentrations of PEG6000.

[0041] Salt-tolerant medium: LB medium + different concentrations of NaCl.

[0042] Inorganic phosphorus medium: MgSO4·7H2O 0.1 g; Ca3(PO4)2 3–5 g; sucrose 10 g; KCl 0.2 g; (NH4)2SO4 0.5 g; MnSO4 1 mL (0.004 g / L); yeast extract 0.5 g; FeSO4 (Fe-EDTA) 0.1 mL (0.002 g / L); NaCl 0.2 g; 1.5–2% agar added to the solid medium.

[0043] Potassium feldspar powder solid culture medium: (NH4)2SO4 0.5 g; MgSO4·7H2O 0.5 g; Na2HPO4 1.5 g; sucrose 2 g; potassium feldspar powder 5 g.

[0044] Example 1: Isolation and Identification of Bacillus subtilis

[0045] I. Isolation of Bacillus subtilis

[0046] Bacillus silenstracum was isolated and cultured from the topsoil along the Yellow River in Kenli District, Dongying City, using the following method:

[0047] (1) Take soil samples, disinfect them to kill microorganisms on the surface of the test materials, and spread them on LB solid medium;

[0048] (2) Pick a single colony and streak it repeatedly on LB solid medium for purification until a pure strain is obtained;

[0049] (3) Inoculate the pure strain into LB liquid medium and culture at 28℃ for 24h. Take 500μl of bacterial solution and add it to 500μl of 50% glycerol. Mix and store in a freezer at -80℃.

[0050] (4) The obtained pure strains were identified and preserved after whole-genome sequencing. One of the isolated and purified rhizosphere bacteria was numbered L01.

[0051] II. Characteristics of Bacillus subtilis

[0052] The *Bacillus siltatifolius* L01 of this invention has the following biological characteristics:

[0053] 1. Colony morphology characteristics

[0054] After culturing on LB medium for 24 hours, the colonies of *Bacillus aquaticus* are yellow, irregularly shaped, 2-10 mm in diameter, with uneven, flat, and dry edges.

[0055] 2. Genetic characteristics

[0056] Based on the whole genome sequencing results, strain L01 is a Bacillus subtilis species, and some gene sequences are shown in SEQ ID NO:1.

[0057] 3. Some physiological and biochemical characteristics of strain L01

[0058] It can grow well on LB medium, with an optimal growth temperature of 35℃ and an optimal growth pH of 7.2.

[0059] In addition, the strain was tested for tolerance to poor soil, salt, drought, high temperature, starch hydrolysis, protein decomposition, phosphorus solubility, and potassium solubility.

[0060] This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 18, 2025, with accession number CGMCC No. 35658.

[0061] Example 2 Functional testing of Bacillus subtilis

[0062] I. Tolerance to Oligometabolism: Different concentrations of glucose culture medium were prepared, and 2 mL of inoculum was used to inoculate each medium. The cultures were then incubated at 37 ℃ with shaking at 150 r / min. The viable cell count was performed using the plate count method. The results are shown in Table 1.

[0063] Table 1 Results of tolerance to malnutrition

[0064] Glucose concentration gradient (mg / L) Count of viable bacteria after 72 hours Proliferation rate 0 0.043 8.6% 15 0.60 120% 50 0.70 140% 100 42 8400% 500 53 10600% 5g / L 114 22800%

[0065] II. Drought Tolerance: The glucose concentration in the culture medium was adjusted to the optimal level. Different concentrations of PEG6000 were added to simulate mild, moderate, and severe drought, and a control group was set up. Each experiment was conducted in triplicate, and the absorbance value at 600 nm was measured. The results are shown in Table 2.

[0066] Table 2 Results of drought resistance experiment

[0067] PEG6000 concentration (%) <![CDATA[OD 600 (72h growth value) Proliferation rate 0 2.138 17816% 10 2.004 16700% 20 1.863 15525% 30 0.420 3500%

[0068] III. Temperature Tolerance: After culturing the strain in liquid LB medium for 48 h, the supernatant was collected by centrifugation, diluted, and placed in water baths at 95℃ and 100℃ for 30 min respectively. The mixture was then cooled under running water, diluted, and spread to determine its temperature tolerance. The results showed that the strain could tolerate a high temperature of 95℃. (See attached results). Figure 3 .

[0069] IV. Salt Tolerance: The glucose concentration in the culture medium was adjusted to the optimal level, and different concentrations of NaCl were added, with no NaCl added as a control. Each group of experiments was set up in triplicate, and the absorbance value at a wavelength of 600 nm was measured. The results are shown in Table 3.

[0070] Table 3 Results of Salt Tolerance Test

[0071] NaCl 0 5% 6% 7% 8% 9% <![CDATA[OD 600 Growth value 1.040 1.182 1.256 1.123 0.899 0.709 Proliferation rate 14857% 16885% 17942% 16042% 12842% 10128%

[0072] V. Phosphorus and Potassium Solubilization Ability: Inorganic phosphorus (calcium phosphate) medium and potassium feldspar powder solid medium were prepared separately. After inoculation, the medium was incubated at 28℃ for 7 days. The degradation of the clear zone and the growth of *Bacillus subtilis* on the medium were observed. Results are shown below. Figure 4 A liquid culture medium containing inorganic phosphorus and potassium feldspar powder was prepared, and 2 mL of bacterial suspension (bacterial concentration approximately 10⁸ CFU / mL) was inoculated. 2 mL of sterile water was used as a control. The culture was carried out at 37°C with shaking for 7 days. After centrifugation, the supernatant was collected, and the phosphate and potassium ion contents in the supernatant were determined using the molybdenum antimony colorimetric method and a flame spectrophotometer, respectively. The results showed that the net phosphorus solubility of the strain was 7.06 mg / L, and the net potassium solubility reached 65.32 mg / L.

[0073] Example 3: Experiment on the growth-promoting effect of Bacillus subtilis on cotton.

[0074] Soil samples were collected from the topsoil of the Yellow River region in Kenli District, Dongying City, Shandong Province, and then air-dried, ground, and sieved (20 mesh). The experiment was divided into two groups: a comparative group of "saline-alkali soil + normal watering" and an experimental group of "saline-alkali soil + normal watering + treatment with Bacillus subtilis in barren water".

[0075] Cotton (Lu Mian Yan 28) seeds were used as experimental material in a pot experiment. Before emergence, the seeds were cultured in the dark; after emergence, the lid and light source were removed for further cultivation. Treatment was performed after the first true leaf emerged. In this example, *Bacillus subtilis* (with a bacterial count of approximately 10) was used. 8The bacterial strain was infected by root irrigation with CFU / mL for 5 days, while the control group used sterile water instead of the bacterial solution. Three weeks later, morphological indicators (plant height, SPAD, leaf area, whole plant fresh weight, aboveground fresh weight, whole plant dry weight, aboveground dry weight) and soil physicochemical properties (pH, salinity) were measured. The results showed that this strain is drought and salt tolerant, grows stably under low carbon source and pH 5-9 conditions, and possesses phosphorus and potassium solubilizing and enzyme secretion functions. It has a significant growth-promoting effect on cotton and a certain degree of soil improvement. Specific results are shown in Tables 4 and 5.

[0076] Table 4. Growth-promoting effect of *Bacillus subtilis* on cotton.

[0077] Plant height (cm) SPAD <![CDATA[Leaf area (cm 2 )]]> Whole plant fresh weight (g) Fresh weight of above-ground parts (g) Whole plant dry weight (g) Dry weight of aboveground parts (g) Comparative Example 14.74 10.81 10.62 1.33 0.91 0.31 0.20 Example 24.35 21.13 20.71 2.94 2.19 0.59 0.41

[0078] Table 5. Soil improvement effect of Bacillus subtilis.

[0079] Physical and chemical properties of soil Comparative Example Experimental Example pH 8.32 7.93 Salt content (g / kg) 2.6 2.05

[0080] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Bacillus inaquosorum L01, accession number CGMCC No. 35658.

2. A microbial agent or bio-fertilizer prepared from the *Bacillus aquaticus* as described in claim 1.

3. Plant growth promoters, agricultural fertilizers, phosphorus activators, or soil activators prepared from the *Bacillus silens* or its inoculum as described in claim 1.

4. Any of the following applications of the *Bacillus subtilis* or its inoculum as described in claim 1: 1) Used for phosphorus solubilization; 2) Used for potassium solubilization; 3) Used for the production of amylase; 4) Used for protease production; 5) Used to promote plant growth or to prepare plant growth promoters; 6) Used to improve soil nutrient content or prepare soil activators; 7) Used for the improvement of salinized and desertified land.

5. The application according to claim 4, characterized in that, The phosphorus mentioned in 1) is inorganic phosphorus.

6. The application according to claim 4, characterized in that, The plant growth promotion method described in 5) includes: irrigating the plants with the bacterial solution of the Bacillus subtilis.

7. The application according to claim 6, characterized in that, The plant mentioned includes cotton.

8. The application according to claim 4, characterized in that, The improvement of soil nutrient content mentioned in 6) includes increasing the content of absorbable phosphorus and potassium nutrients in the soil.

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