Bacillus megaterium with extreme salt tolerance and application thereof

Abstract

The invention provides a strain of bacillus megaterium with extreme salt tolerance and application thereof, the bacillus megaterium is bacillus megaterium B761, and is preserved in the China Center for Type Culture Collection, the preservation date is January 8, 2025, and the preservation number is CCTCC NO: M 2025058. The bacillus megaterium B761 has an excellent capability in the aspect of salt stress resistance, has an extreme salt tolerance characteristic, can survive in an environment with the salt concentration of 10%, and shows a remarkable plant growth promoting effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of Bacillus, and particularly relates to a Bacillus megaterium with extreme salt tolerance and its application. Background Art

[0002] Soil salinization is a global ecological problem, leading to land degradation, decreased agricultural productivity, and even threatening food security. A large amount of salt ions accumulate in saline-alkali soil, causing salts to gradually accumulate on the soil surface and affecting the normal growth of plants. Under the action of salts, saline-alkali soil shows hardening phenomenon, resulting in poor soil porosity, air permeability and water permeability, which in turn affects the growth and metabolism of functional microorganisms in the soil and the activity of soil enzymes, and reduces the conversion rate of soil organic matter. Soil salinization not only destroys the soil structure and reduces soil fertility, but also affects the absorption and metabolism functions of plants, severely restricting agricultural production.

[0003] With the reduction of the total cultivated land area and the decline of cultivated land quality, the development of agriculture and animal husbandry and the construction of ecological civilization are facing severe challenges. According to the data of UNESCO and FAO, the total area of saline-alkali land in the world is about 954.38 million hectares, and its reasonable development and utilization is of great significance for ensuring food security.

[0004] At present, the treatment technologies for saline-alkali land mainly include physical method, chemical method and biological method. Among them, compound microbial inoculants, as the main means of biological method, have gradually become a research hotspot for saline-alkali land treatment due to their advantages such as low cost, simple operation, small pollution and good treatment effect. Compound microbial inoculants improve the soil structure and promote plant growth through the action of microorganisms, and have significant eco-friendly characteristics. With the rise of ecological agriculture, the market demand for compound microbial inoculants has been increasing year by year. However, the popularization of compound microbial inoculants still faces many challenges. First, consumers have limited understanding of their effects and safety; second, the production process is complex and the cost is relatively high, resulting in their prices being higher than those of traditional fertilizers and limited market competitiveness.

[0005] Although there are already a variety of compound microbial inoculants, they mainly target conventional cultivated land, and there are few customized products specifically for saline-alkali land. The traditional compound microbial inoculants have poor effects in saline-alkali land, mainly because they lack salt-tolerant and growth-promoting patented strains, and the traditional formula has poor adaptability in saline-alkali environment. Summary of the Invention

[0006] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a Bacillus megaterium with extreme salt tolerance and its application. This strain has excellent ability in resisting salt stress. This strain has the characteristic of extreme salt tolerance, can survive in an environment with a salt concentration of up to 10%, and shows a significant plant growth promotion effect.

[0007] Technical solution: To achieve the above-mentioned invention objectives, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a strain of Bacillus megaterium, which is Bacillus megaterium B761, deposited in the China Center for Type Culture Collection on January 8, 2025, with the deposit number CCTCC NO: M 2025058.

[0009] The Bacillus megaterium B761 has a 16S rDNA sequence as shown in SEQ ID NO.1.

[0010] The present invention collects saline-alkali soil from saline-alkali areas in Xinjiang, China, and screens out Bacillus megaterium B761, which has excellent tolerance to extreme saline-alkali environments and has a growth-promoting function.

[0011] The Bacillus megaterium B761 strain of the present invention has good nitrogen fixation, potassium solubilization, and IAA secretion capabilities, can promote the cyclic transformation of soil nutrients, and has excellent growth-promoting effects. The Bacillus megaterium B761 strain provided by the present invention has excellent salt tolerance, and the salt tolerance range is about 10%.

[0012] The colonies of the Bacillus megaterium B761 strain of the present invention are circular on the solid LB medium plate, with a rough and opaque surface, produce spores, have no capsule, are positive for catalase, and the cells are straight rods, 0.6 - 0.7 μm × 2.0 - 3.0 μm. Bacillus megaterium B761 is Gram-positive, aerobic, and belongs to chemoheterotrophic microorganisms. The present invention uses the 16S rRNA gene sequence of Bacillus megaterium B761 to compare with the Standard database and 16S ribosomal RNA sequencedatabases in NCBI, preliminarily determines the taxonomic information of the strain, constructs a phylogenetic tree of strain B761, and combines the colony morphological characteristics of strain B761 for identification, indicating that strain B761 is a Bacillus megaterium strain.

[0013] In the second aspect, the present invention provides a composition, which contains the above-mentioned Bacillus megaterium B761.

[0014] As a specific implementation, the composition is a bacterial suspension, culture solution or bacterial cells containing Bacillus megaterium B761; in the composition, the effective viable count of Bacillus megaterium B761 is ≥ 4.2×10 9 CFU / g, further preferably ≥ 5×10 9 CFU / g, and the spore rate is ≥ 96%, more preferably ≥ 98%.

[0015] As a further embodiment, the method for preparing the bacterial suspension, the culture solution or the bacterial cells comprises the following steps:

[0016] Inoculate the Bacillus megaterium B761 in a culture medium for culturing to obtain a culture solution, centrifuge the culture solution and then resuspend it to obtain a bacterial suspension; centrifuge the culture solution to obtain bacterial cells;

[0017] Preferably, the temperature for culturing is 28°C to 37°C, more preferably 35°C to 37°C; the culturing time is 1 to 2 days, more preferably 2 days. The OD of the culture solution 600 is 0.7 to 1.0, further preferably 0.8 to 0.9; the culture medium of the present invention preferably comprises LB medium, and the LB medium preferably uses distilled water as a solvent and comprises the following components: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 - 20 g of agar and 1 L of distilled water, with natural pH.

[0018] The viable count of the bacterial suspension is preferably ≥4.2×10 9 CFU / g, further preferably ≥5×10 9 CFU / g.

[0019] After obtaining the culture solution, the present invention preferably centrifuges the culture solution to obtain bacterial cells, and the bacterial cells are preferably resuspended with sterile water to obtain a Bacillus megaterium B761 bacterial suspension. The above-mentioned centrifugation speed should be ≤6 pm, and there are no special limitations on other centrifugation parameters, and conventional methods can be used.

[0020] In a third aspect, the present invention provides the application of the above-mentioned Bacillus megaterium B761, or the above-mentioned composition in the remediation and improvement of saline-alkali soil and / or the promotion of plant growth under salt stress.

[0021] As a specific embodiment, in the saline-alkali soil or under salt stress, the mass concentration of sodium chloride is 9 - 11%.

[0022] As a specific embodiment, the plant is cotton.

[0023] In a fourth aspect, the present invention provides a method for remediating and improving saline-alkali soil, which comprises applying the above-mentioned Bacillus megaterium B761, or applying the above-mentioned composition in the saline-alkali soil.

[0024] In a fifth aspect, the present invention provides a method for promoting plant growth under salt stress, which comprises applying the above-mentioned Bacillus megaterium B761, or applying the above-mentioned composition in a plant growth medium or on a plant body.

[0025] The application method preferably comprises root irrigation and / or foliar spraying.

[0026] More preferably, the application method includes irrigating the roots with the B761 bacterial suspension; the effective viable count of the B761 bacterial suspension is preferably ≥4.2×10 9 CFU / g, the application amount per plant per time is preferably 50 - 100 mL, more preferably 100 mL; the number of application times per single growth cycle of each plant is preferably ≥2 times, more preferably 2 - 3 times.

[0027] Beneficial effects: Compared with the prior art, the present invention provides a new strain of Bacillus megaterium B761, which has the characteristics of extreme salt tolerance, can improve the salt tolerance of plants, can also degrade organic phosphorus compounds in the soil, and has a plant growth promoting effect at the same time. The results of the examples show that after applying the bacterial suspension containing Bacillus megaterium B761 in saline-alkali soil, the salt content and pH value of the saline-alkali soil are both significantly reduced, and the soil pH drops from 8.5 before application to about 7.6. In addition, after applying the bacterial suspension of Bacillus megaterium, the emergence rate of cotton is significantly increased, and at the same time, the plant height, root length, fresh weight and dry weight of cotton plants are all significantly increased. The above results indicate that the application of Bacillus megaterium B761 can effectively improve the soil nutrient status and increase the yield and quality of crops such as cotton. Description of the Drawings

[0028] Figure 1 is the colony morphology of Bacillus megaterium B761 strain on the LB medium plate with a NaCl mass concentration of 10%;

[0029] Figure 2 is the influence diagram of the salt concentration of the medium on the growth amount of Bacillus megaterium B761;

[0030] Figure 3 is the influence diagram of the initial pH value of the medium on the growth amount of Bacillus megaterium B761;

[0031] Figure 4 is the influence diagram of temperature on the growth amount of Bacillus megaterium B761;

[0032] Figure 5 is the phylogenetic tree of strain B761 and its closely related strains in the 16S ribosomal RNA sequencedatabases based on 16S rRNA gene similarity. Detailed Embodiments

[0033] The technical solutions of the present invention will be further described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1 Isolation and Purification of Salt-Tolerant Bacillus megaterium

[0035] 1. Take 10 g of saline-alkali soil collected from Xinjiang saline-alkali land and add it to a triangular flask containing glass beads and 100 mL of deionized water. Then place it in a shaker at 37 °C and 200 rpm for 12 h to obtain a soil dilution.

[0036] 2. Take 10 mL of the soil dilution obtained in step 1 and add it to a triangular flask containing 100 mL of sterile and salt-free LB medium. Add NaCl to make the final mass concentration of NaCl in the LB medium 10%. Then culture it in a shaker at 37 °C and 200 rpm for 24 h to obtain the first culture solution. Subsequently, take 10 mL of the first culture solution and repeat the above culture process. After repeating it 5 times in total, obtain the culture solution obtained from the last culture.

[0037] 3. Take 1 mL of the culture solution obtained from the last culture and sequentially perform gradient dilution according to the concentrations of 10 -1 、10 -2 ……10 -7 Then take the dilution solution with a concentration of 10 -7 and coat it on LB medium plates supplemented with different mass concentrations of NaCl. The mass concentration of NaCl in the LB medium plates is 0%, 5%, 10%, 15%, and 20%. Then incubate it upside down at 37 °C until colonies appear on the plate. Pick a single colony and streak it on an LB medium plate containing 10% NaCl by mass concentration, and incubate it upside down at 37 °C. Repeat the operation 3 - 5 times until pure culture is obtained.

[0038] A single strain of bacteria was obtained through pure culture and named B761. Streak and isolate the strain B761 on an LB medium plate with a NaCl mass concentration of 10%. The colony morphology formed after culturing at 37 °C for about 24 h is shown in Figure 1 , and the colony is a white colony visible to the naked eye.

[0039] The colony of strain B761 is circular on the solid LB medium plate, indicating rough and opaque, presenting white, with irregular edges and being flat. Strain B761 is Gram-positive, aerobic, and belongs to chemoheterotrophic microorganisms. The composition of the solid LB medium is: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, and 1 L of ultrapure water.

[0040] Strain B761 was deposited at the China Center for Type Culture Collection (CCTCC) on January 8, 2025, in accordance with the patent deposit procedure, with the deposit number CCTCC NO: M 2025058. The address of the depositary institution is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0041] Example 2 Study on the growth characteristics of Bacillus megaterium B761

[0042] 1. Pick a single colony of strain B761 from the LB medium plate obtained by culturing in Example 1 and inoculate it into a test tube containing liquid LB medium. Culture it at 37°C and 200 rpm until the OD 600 is about 0.8 to obtain a seed solution.

[0043] 2. Inoculate the seed solution of strain B761 in step 1 into LB media with NaCl concentrations of 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, and 15% respectively. After culturing at 200 rpm and 37°C for 24 h, measure the OD 600 of the obtained culture solution. Strain B761 has very excellent salt tolerance. As shown in Figure 2 and Table 1, it can be seen that strain B761 can grow well in LB media with 1% - 10% (mass concentration) NaCl; in the LB medium with 10% NaCl, its OD 600 can still rise to 1.58 within 24 h.

[0044] 3. Inoculate the seed solution of strain B761 in step 1 into LB media with initial pH values of 4, 5, 6, 7, 8, 9, and 10 respectively. After culturing at 200 rpm and 37°C for 24 h, measure the OD 600 of the obtained culture solution. The results of the influence of the initial pH value on the growth of strain B761 are shown in Figure 3 and Table 1. Strain B761 grows in the pH range of 5 - 10, and grows well in the pH range of 5 - 9. The OD 600 of the obtained culture solution after culturing for 24 h rises above 2. When the pH rises to 10, strain B761 can still grow, and the OD 600 of the obtained culture solution after culturing for 24 h can rise to 1.65.

[0045] 4. Inoculate the seed solution of strain B761 in step 1 into LB media, and then place them in static culture at temperatures of 10°C, 20°C, 30°C, 40°C, and 50°C respectively. After culturing for 24 h, measure the OD 600 of the obtained culture solution. The influence of temperature on the growth of B761 is shown in Figure 4 and Table 1. Strain B761 grows well at temperatures of 20 - 40°C, and the OD 600 rises above 2.20 within 24 h. When the temperature rises to 50°C, the growth trend of strain B761 is significantly inhibited, but it can still grow well, and the OD 600 of the obtained culture solution after culturing for 24 h rises to 1.31.

[0046] Table 1 Growth characteristics of strain B761

[0047]

[0048]

[0049] Example 3 Identification of Bacillus megaterium Strain B761

[0050] The identification of Bacillus megaterium was carried out by combining molecular biology methods with the analysis of colony morphological characteristics.

[0051] 1. Extraction of PCR template DNA:

[0052] The purified strain B761 was inoculated into LB medium containing 10% NaCl and cultured in a shaker at 37 °C for 24 h. The cells were collected, and the total genomic DNA was extracted using a DNA extraction kit.

[0053] 2. PCR amplification.

[0054] Using the bacterial universal primers 27F (SEQ ID NO.2): 5'-AGAGTTTGATCCTGGCTCAG-3’ and 1492R (SEQ ID NO.3): 5’-TACGACTTAACCCCAATCGC-3’ as primers, the 16S rRNA gene of strain B761 was amplified by PCR. PCR amplification system (25 μL): 10 ng of DNA of strain B761 obtained in step 2, 2 μL of each upstream and downstream primer, 2 μL of dDTP (10 mM), 0.5 μL of Taq enzyme, 5 μL of 10×PCR buffer, and ddH2O was added to 25 μL. PCR amplification conditions: 95 °C, 5 min; 95 °C, 30 s; 65 °C, 30 s; 72 °C, 1.5 min, 35 cycles; 72 °C, 10 min; 4 °C, 5 min.

[0055] 3. 16S rRNA sequence determination

[0056] The PCR amplification products were detected by electrophoresis, purified, and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The specific sequence is shown in SEQ ID NO.1 provided in the appendix. The obtained sequences were spliced, compared and analyzed on the NCBI website, and a phylogenetic tree was constructed. The phylogenetic tree of this bacterium is as Figure 5 shown. Through comparative analysis, it was found that strain B761 has the closest genetic relationship with Bacillus megaterium, and the similarity of the 16S rDNA sequence with the Bacillus megaterium sequence is as high as 99.86% (query cover 100%).

[0057] Download the 16S rRNA gene sequences of 12 strains that are closest to the strain in the above database, and use the common MEGA 11 software in the field to establish a phylogenetic tree by the Neighbor-Joining method.

[0058] Based on the 16S rRNA gene sequence similarity, phylogenetic tree analysis, and colony morphology, strain B761 was preliminarily identified as Bacillus sp., and this strain was closely related to Bacillus megaterium.

[0059] The 16S rRNA nucleotide sequence of Bacillus megaterium B761 is shown in SEQ ID NO.1.

[0060] Example 4 Effect of Strain B761 on Cotton Growth

[0061] 1. Select seeds of the Xinluzao cotton variety. Disinfect the cotton seeds with 75% ethanol for 1 minute, and rinse them with sterile water 3 - 5 times. Finally, soak the seeds in sterile water for 3 - 5 hours to allow them to fully absorb water. Prepare PVC seedling pots filled with saline - alkali soil from Xinjiang, with drainage holes at the bottom. The pH value of the saline - alkali soil from Xinjiang selected this time was 8.5, the available nitrogen content was 45 mg / kg, the available phosphorus content was 12 mg / kg, the organic matter content was 10 g / kg, the available potassium content was 200 mg / kg, and the salt content was 0.6 g / kg. In this group of experiments, an inoculation group and a control group were set up. In the inoculation group, 10 mL of B761 bacterial suspension was inoculated into the soil, and the effective viable bacteria count of the bacterial suspension was 5×10 9 CFU / g, and the bacterial suspension was replenished once a week; the control group added an equal amount of sterile water, and each group had 5 replicates. Sow the cotton seeds into the seedling pots, with 5 seeds sown in each pot. The experiment was carried out in a greenhouse with a light cycle of 12 hours of light / 12 hours of darkness, and water was regularly added to maintain the soil humidity.

[0062] 2. Regularly observe the growth of cotton during the experiment. Record the emergence rate starting from the 7th day after sowing, and measure the plant height every 7 days. After the experiment ended, measure the fresh weight and dry weight of the whole cotton seedling and count the root length of the cotton seedling. At the same time, the five - point sampling method was used to regularly detect the pH of the rhizosphere soil of cotton seedlings during the experiment.

[0063] Table 2 Changes in Plant Height of Cotton Seedlings under Different Treatments

[0064]

[0065] Table 3 Growth Status of Cotton under Different Treatments after 45 Days of Growth

[0066]

[0067] Table 4 Changes in pH of Rhizosphere Soil of Cotton Seedlings

[0068]

[0069] Note: p ≤ 0.05

[0070] One week after sowing, the emergence rate of cotton in the inoculation group was 84%, while that in the control group was 64%. The plant height of cotton seedlings in the inoculation group (36.7 cm) was significantly higher than that in the control group (25.8 cm). The root length in the inoculation group was 40% higher than that in the control group, and the fresh weight and dry weight of the whole plant increased by 57.6% and 58.2% respectively. Through the analysis of the pH data of the rhizosphere soil of cotton seedlings, we found that the pH of the rhizosphere soil of cotton seedlings decreased significantly after the application of the bacterial suspension, while the change range of the pH of the soil far from the rhizosphere of cotton seedlings was small. To sum up, the soil inoculated with the B761 bacterial suspension can effectively promote the growth of cotton and improve the saline-alkali tolerance of cotton.

[0071] The embodiments of the present invention have been described in detail above in conjunction with specific embodiments. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art.

Claims

1. A Bacillus megaterium, characterized in that, The Bacillus megaterium is Bacillus megaterium B761, which is deposited in the China Center for Type Culture Collection. The deposit date is January 8, 2025, and the deposit number is CCTCC NO: M 2025058.

2. The Bacillus megaterium according to claim 1, wherein The Bacillus megaterium B761 has a 16S rDNA sequence as shown in SEQ ID NO.

1.

3. A composition, characterized in that, The composition contains the Bacillus megaterium B761 described in claim 1.

4. The composition according to claim 3, characterized in that, The composition is a bacterial suspension, a culture solution or bacterial cells containing Bacillus megaterium B761; in the composition, the effective viable count of Bacillus megaterium B761 is ≥4.2×10 9 CFU / g, and the spore rate is ≥96%.

5. The composition according to claim 4, wherein The preparation method of the bacterial suspension, culture solution or bacterial cells includes the following steps: Inoculate the Bacillus megaterium B761 in a medium for culture to obtain a culture solution, centrifuge the culture solution and resuspend it to obtain a bacterial suspension; centrifuge the culture solution to obtain bacterial cells; Preferably, the culture temperature is 28°C to 37°C, and the culture time is 1 to 2 days.

6. Use of the Bacillus megaterium B761 described in claim 1 or 2, or the composition described in any one of claims 3-5 in the remediation and improvement of saline-alkali soil and / or the promotion of plant growth under salt stress.

7. The application according to claim 6, wherein In the saline-alkali soil or under salt stress, the mass concentration of sodium chloride is 9-11%.

8. The application according to claim 6, characterized in that, The plant is cotton.

9. A method for repairing and improving saline-alkali soil, characterized in that, It includes applying the Bacillus megaterium B761 described in claim 1 or 2, or applying the composition described in any one of claims 3-5 in the saline-alkali soil.

10. A method for promoting plant growth under salt stress, characterized in that, It includes applying the Bacillus megaterium B761 described in claim 1 or 2, or applying the composition described in any one of claims 3-5 in the plant growth medium or on the plant body.

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