Bacillus and application thereof in restoration and improvement of saline alkali soil and / or promotion of plant growth

Abstract

The invention provides a bacillus and application thereof in saline alkali soil remediation and improvement and / or plant growth promotion, the bacillus is bacillus (Bacillus sp.) B8961 and is preserved in the China Center for Type Culture Collection on November 11, 2024, the preservation date is November 11, 2024, and the preservation number is CCTCC NO: M 20242511. Compared with the prior art, the novel bacillus B8961 provided by the invention has remarkable advantages in the aspects of saline-alkali soil remediation and crop growth promotion. The strain not only can effectively improve the saline-alkaline property of soil and reduce the salinity and pH value of the soil, but also can remarkably improve the fertility of the soil and create a good soil environment for the growth and development of crops.

Description

Technical Field

[0001] The invention belongs to the technical field of bacillus, and in particular relates to a bacillus and application thereof in saline-alkali soil restoration and improvement and / or plant growth promotion. Background Art

[0002] Soil salinization is a global ecological problem, leading to land degradation, reduced agricultural productivity and even food security issues. A large amount of salt ions gather in saline-alkali soil, causing salt to gradually accumulate on the soil surface, affecting the normal growth of plants; under these conditions, saline-alkali soil becomes compacted, and the porosity, air permeability and water permeability of the soil become increasingly poor, affecting the growth and metabolism of functional microorganisms in the soil and the activity of soil enzymes, and reducing the conversion rate of soil organic matter. Soil salinization will destroy soil structure, reduce soil fertility, and affect the absorption and metabolic functions of plants. The total area of ​​arable land is decreasing, and the quality of arable land is declining, which has seriously affected the development of agriculture and animal husbandry and the construction of ecological civilization.

[0003] According to data released by UNESCO and FAO, the total area of ​​saline-alkali land in the world is 954.38 million hectares, which is equivalent to the existing arable land area.

[0004] At present, Bacillus, as an important microbial agent resource, has shown broad application prospects in soil management and agricultural production. Bacillus has strong environmental adaptability and stress resistance, can survive in extreme environments such as saline-alkali and drought, and can significantly improve the salt tolerance and stress resistance of plants by secreting plant growth regulators (such as indoleacetic acid, gibberellins, etc.) and stress resistance substances (such as organic acids, extracellular polysaccharides, etc.). Bacillus can also decompose organic matter in the soil, release fast-acting nutrients, and improve soil structure and fertility. In addition, Bacillus can also inhibit the growth of pathogens and reduce the number of harmful microorganisms in the soil, thereby improving the structure of soil microbial communities and promoting the balance of soil ecosystems. In recent years, the application research on single strains in saline-alkali land improvement has gradually increased, but strains that can adapt to extreme saline-alkali environments and have significant growth-promoting functions are still relatively scarce.

[0005] However, consumers have limited knowledge of the effects and safety of microbial agents. The production process of microbial agents is complex and the production cost is relatively high, which leads to a higher price than traditional fertilizers, restricting their market competitiveness. At the same time, the total amount of publicly accessible resources in China's agricultural microbial resource professional strain bank is about 1.75×104 strains, which is only 17.8% of the world's largest agricultural microbial resource bank.

[0006] Although there are related microbial agents in China, most of the existing microbial agents on the market are mainly for conventional farmland, and customized microbial agents specifically for saline-alkali land are relatively scarce. Moreover, the effect of these traditional microbial agents in saline-alkali land is not ideal. The main reason is the lack of salt-tolerant growth-promoting strains and the poor adaptability of traditional formulas in saline-alkali environments, which has become a bottleneck restricting their effectiveness. Summary of the invention

[0007] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a strain of Bacillus and its application in saline-alkali soil remediation and improvement and / or plant growth promotion. The strain has excellent salt tolerance (salt tolerance range can reach 10% NaCl), can survive and reproduce in a high-salt environment, and secrete nutrients that promote plant growth. The strain can be used to improve the quality of saline-alkali soil and increase the salt tolerance and yield of crops.

[0008] Technical solution: To achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a Bacillus sp., which is Bacillus sp. B8961, deposited in the China Center for Type Culture Collection on November 11, 2024, and with a deposit number of CCTCCNO: M 20242511.

[0010] The Bacillus B8961 has a 16S rDNA sequence as shown in SEQ ID NO.1.

[0011] In a second aspect, the present invention provides a composition comprising the Bacillus B8961.

[0012] As a specific embodiment, the composition is a bacterial suspension, culture solution or bacterial cells containing Bacillus B8961; in the composition, the effective viable count of Bacillus B8961 is ≥4.8×10 9 CFU / g, more preferably ≥5.1×10 9 CFU / g, more preferably ≥1×10 10 CFU / g; spore rate ≥96%, more preferably ≥98%.

[0013] As a further embodiment, the method for preparing the bacterial suspension, culture solution or bacterial cells comprises the following steps:

[0014] The Bacillus B8961 is inoculated into a culture medium for cultivation to obtain a culture solution, the culture solution is centrifuged and then resuspended to obtain a bacterial suspension; the culture solution is centrifuged to obtain bacterial cells;

[0015] Preferably, the culture temperature is preferably 25 to 37°C, more preferably 28 to 30°C; the culture time is preferably 1 to 3 days, more preferably 2 days; the OD of the culture solution is preferably 600 The medium preferably comprises LB medium, which preferably uses ultrapure water as solvent and comprises the following components: 5 g yeast extract, 10 g trypsin, 10 g sodium chloride and 1 L distilled water, and 20 g agar is added to each liter of solid LB medium.

[0016] After obtaining the culture solution, the present invention preferably centrifuges the culture solution to obtain bacterial cells, and the bacterial cells are preferably resuspended in sterile water to obtain a Bacillus B8961 bacterial suspension.

[0017] In a third aspect, the present invention provides the use of the Bacillus B8961 or the composition in the remediation and improvement of saline-alkali soil and / or the promotion of plant growth in a saline-alkali environment.

[0018] As a specific implementation scheme, the mass concentration of sodium chloride in the saline-alkali soil or saline-alkali environment is 9-11%.

[0019] As a specific embodiment, the plant is corn.

[0020] In a fourth aspect, the present invention provides a method for repairing and improving saline-alkali soil, comprising applying the Bacillus B8961 or the composition in the saline-alkali soil.

[0021] In a fifth aspect, the present invention provides a method for promoting plant growth in a saline-alkali environment, comprising applying the Bacillus B8961 in a plant growth medium or on a plant body, or applying the composition.

[0022] The application method preferably includes root irrigation and / or foliar spraying.

[0023] When irrigating the roots, the steps include: inoculating the Bacillus B8961 into the soil at the roots of the plants. Preferably, B8961 bacterial suspension is used for root irrigation, and the effective viable bacteria count of the B8961 bacterial suspension is preferably ≥4.8×10 9 CFU / g, the application amount per plant each time is preferably 50-100 mL, more preferably 100 mL; the number of applications per plant in a single growth cycle is preferably ≥2 times, more preferably 2-3 times.

[0024] Beneficial effects: Compared with the prior art, the present invention provides a new strain of Bacillus B8961, which shows significant advantages in saline-alkali soil remediation and crop growth promotion. This strain can not only effectively improve the salinity of the soil, reduce soil salinity and pH, but also significantly improve soil fertility, creating a good soil environment for the growth and development of crops. The results of the implementation case showed that after the application of extreme Bacillus B8961, the salinity and pH value of the saline-alkali soil decreased significantly, and the soil pH dropped from 8.8 before application to 7.7. At the same time, the application of the bacteria increased the key soil fertility indicators such as alkaline nitrogen, available phosphorus, available potassium and organic matter in saline-alkali soil by 20.4%, 19.2%, 32.1% and 31.6%, respectively. In addition, the application of the bacteria significantly increased the germination rate of corn (34.3%), and increased the plant height and root length of corn by 35.3% and 44.6%, respectively. The above results show that the application of Bacillus B8961 can effectively improve soil nutrient conditions and provide excellent nutritional support for the growth of crops such as corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The colony morphology of Bacillus subtilis B8961 strain in LB medium plate with 10% NaCl mass concentration;

[0026] Figure 2 This is the effect of temperature on the growth of Bacillus B8961 strain;

[0027] Figure 3 This is a graph showing the effect of the initial pH value of the culture medium on the growth of Bacillus B8961 strain;

[0028] Figure 4 This is a graph showing the effect of different salt concentrations on the growth of Bacillus sp. B8961 strain;

[0029] Figure 5 is the phylogenetic tree of Bacillus strain B8961;

[0030] Figure 6 It is the soil fertility improvement rate of different elements. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0032] Example 1 Isolation and purification of halotolerant Bacillus

[0033] 1. Take 10 g of saline-alkali soil collected from the saline-alkali land area of ​​Xinjiang and add it to a conical flask containing glass beads and 100 mL of deionized water, then place it in a shaker at 30°C and 200 rpm for 12 h to obtain a soil dilution solution.

[0034] 2. Take 10 mL of the soil dilution obtained in step 1, add it to a flask containing 100 mL of sterile salt-free LB medium, add NaCl, and make the final mass concentration of NaCl in the LB medium reach 10%. Place the flask in a shaker at 30°C and 200 rpm for 24 hours to obtain the first round of culture solution. Then take 10 mL of the first round of culture solution, repeat the above culture process, and perform 5 rounds of culture in total to finally obtain the culture solution obtained from the 5th round of culture.

[0035] 3. Take 1 mL of the final culture solution and -1 , 10 -2 ……10 -7 The concentration was serially diluted, and then 10 -7 The dilutions of different concentrations were spread on LB medium plates with different NaCl concentrations, the NaCl concentrations in the LB medium plates were 0%, 5%, 10%, 15%, and 20%, and then inverted at 30°C until colonies appeared on the plates. A single colony was streaked onto an LB medium plate containing 15% NaCl, inverted at 30°C, and the operation was repeated 3 to 5 times until a pure culture strain was obtained.

[0036] A single colony was obtained by pure culture and named B8961. The strain B8961 was streaked and isolated on a LB medium plate containing 10% NaCl. The colony morphology formed by culturing at 30°C for about 24 hours is shown in Figure 1. The colony is a slightly yellow colony visible to the naked eye.

[0037] The colony morphology of strain B8961 on the solid LB medium plate is round, flat and moist, slightly yellow, and the inside of the colony is mucous. Strain B8961 belongs to Gram-positive bacteria. The solid LB medium consists of: 10g of trypsin, 5g of yeast extract, 10g of sodium chloride, 15g of agar, and 1L of ultrapure water.

[0038] Strain B8961 was deposited in the China Center for Type Culture Collection (CCTCC) under a patented preservation procedure. The specific address is Wuhan University, Wuchang District, Wuhan City, Hubei Province. The preservation number is CCTCC NO: M20242511, and the preservation date is November 11, 2024.

[0039] Example 2 Study on the growth characteristics of Bacillus B8961

[0040] 1. Pick a single colony of strain B8961 from the LB medium plate obtained in Example 1 and inoculate it into a test tube containing liquid LB medium, and culture it at 30°C and 200 rpm until OD 600 It is about 0.8, which is used as seed liquid.

[0041] 2. The strain B8961 seed solution prepared in step 1 was inoculated into LB medium respectively, and cultured at 10°C, 20°C, 25°C, 30°C, 40°C, and 50°C for 24 hours, and the OD of the obtained culture solution was measured. 600 The results showed that strain B8961 grew well in the range of 20-40℃, and the OD 600 When the temperature rises to 50℃, the growth of strain B8961 is significantly inhibited, but it can still maintain a certain growth capacity. The OD of the culture fluid obtained after 24h of culture is 600 Can rise to 1.667 (see Figure 2 ).

[0042] 3. The strain B8961 seed solution prepared in step 1 was inoculated into LB medium with initial pH values ​​of 4, 5, 6, 7, 8, 9, and 10, respectively. After culturing at 30°C and 200 rpm for 36 h, the OD of the resulting culture solution was measured. 600 The results showed that strain B8961 could grow in the pH range of 5 to 10, and grew best in the pH range of 5 to 9. The OD of the culture solution obtained after 24 hours of cultivation was 600 When the pH value rises to 10, strain B8961 can still maintain a certain growth ability. The OD of the culture fluid obtained after 24 hours of cultivation is 600 Can rise to 1.746 (see Figure 3 ).

[0043] 4. The strain B8961 seed solution prepared in step 1 was inoculated into LB medium with NaCl concentrations of 0%, 5%, 10%, 15%, and 20%, respectively. After culturing at 30°C and 200 rpm for 48 h, the OD of the resulting culture solution was measured. 600 The results showed that strain B8961 had excellent salt tolerance (see Figure 4 ). The strain B8961 grew well in LB medium with a NaCl concentration of 1% to 10%. In LB medium with a NaCl concentration of 10%, the OD 600 Although strain B8961 could not grow within 24 hours in an environment with a NaCl concentration of 15% or more, after 3 days of cultivation, flocculent bacteria were observed in LB medium with a NaCl concentration of 15%. The OD of the culture fluid obtained after 7 days of cultivation was 600 It can rise to 0.354. This shows that strain B8961 can not only tolerate an environment as high as 15% NaCl, but also maintain a certain growth ability under this condition.

[0044] In summary, strain B8961 exhibits excellent environmental adaptability, can grow well in a wide range of temperatures (20-40°C), pH values ​​(5-10) and salt concentrations (1%-10% NaCl), and can still maintain a certain growth activity under extreme conditions (such as 50°C high temperature, pH 10 strong alkali, 15% NaCl high salt). In addition, strain B8961 also has the ability to fix nitrogen and dissolve potassium, and can secrete proteases and convert tryptophan into indoleacetic acid, thereby effectively promoting the recycling and transformation of soil nutrients and providing favorable conditions for plant growth. These characteristics indicate that strain B8961 has broad application prospects in saline-alkali land improvement and crop growth promotion.

[0045] Example 3 Classification and Identification of Bacillus B8961

[0046] 1. Sequence determination and analysis of 16S rDNA of Bacillus sp. B8961 strain:

[0047] 1) Extraction of PCR template DNA:

[0048] The purified strain B8961 was inoculated into LB medium containing 10% NaCl and cultured at 30° C. on a shaking platform for 24 h. The bacteria were collected and the total genomic DNA was extracted using a DNA extraction kit.

[0049] 2) PCR amplification

[0050] Using universal primers:

[0051] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3',

[0052] 1492R: 5'-TACGACTTAACCCCAATCGC-3';

[0053] The total volume of the PCR reaction system was 25uL, the DNA of strain B8961 was 10ng, the upstream and downstream primers were 2uL each, dDTP (10mM) was 2uL, Taq enzyme was 0.5uL, 10×Buffer was 5uL, and ddH2O was added to the total system to 25uL. The PCR amplification conditions were 95℃, 3min; 95℃, 30s; 65℃, 30s; 72℃, 90s; 35 cycles; 72℃, 10min; 4℃, 5min.

[0054] 3) 16S rRNA sequence determination

[0055] The PCR amplification product was detected by electrophoresis and purified, and then sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The specific sequence is shown in SEQ ID NO.1 provided in the attached. The obtained sequences were spliced, compared and analyzed on the NCBI website, and a phylogenetic tree was constructed. The phylogenetic tree of the bacteria is shown in Figure 5 As shown, comparison analysis revealed that strain B8961 was most closely related to Bacillus sp. ITP21 and Bacillus endophyticus strain OBT-KSU309, and the similarity of 16S rDNA sequence with both of them was as high as 100% (query cover 100%).

[0056] The 16s rRNA gene sequences of the 12 strains most closely related to strain B8961 were downloaded from the above database, and a phylogenetic tree was established using the Neighbor-Joining method using MEGA 11 software commonly used in the art.

[0057] Based on the 16S rRNA gene sequence similarity, phylogenetic tree analysis and colony morphology, strain B8961 was preliminarily identified as Bacillus sp., and this strain was closely related to Bacillus endophyticus.

[0058] The 16S rRNA nucleotide sequence of Bacillus B8961 is shown in SEQ ID NO.1.

[0059] Example 4 Effect of strain B8961 on pH value of saline-alkali soil

[0060] B8961 bacterial suspension (1×10 8 CFU / mL) was evenly mixed into the soil, 10mL of bacterial solution was added per kilogram of soil, and appropriate humidity (60%-70% of field water holding capacity) and temperature (usually 25°C) were maintained, and cultured for 90 days. After the culture was completed, a 0-20cm cultivated layer saline-alkali soil sample was taken and naturally air-dried, and then passed through a 2mm sieve. 10.00g of soil sample was accurately weighed and added with ultrapure water at a soil-water ratio of 1:2.5. The test solution was obtained by constant temperature shaking at 25°C for 30 minutes, centrifugation at 5000rpm for 10 minutes, and filtration with a 0.45μm filter membrane. Using a pre-calibrated pH meter, the electrode was immersed 3cm below the surface of the test solution. Each sample was measured in parallel 3 times and the average was taken, and the comparison results of pH 8.81±0.02 in the control group and pH 7.70±0.02 in the treatment group were finally obtained.

[0061] Table 1 Effects of different treatments on pH value of saline-alkali land

[0062]

[0063] The experimental results showed that the pH value of saline-alkali soil treated with B8961 bacterial suspension decreased significantly compared with the control treatment. The soil pH value was 8.8 before application, and it dropped to 7.7 90 days after application, a decrease of 12.5%. This shows that B8961 bacterial agent can effectively improve the pH of saline-alkali soil, reduce soil salinity, and provide a more suitable soil environment for crop growth.

[0064] In the above experiment, the initial soil fertility was first determined, including alkaline nitrogen, available phosphorus, available potassium and organic matter. After the soil and bacterial solution were cultured, the soil fertility after bacterial solution treatment was determined and the soil fertility improvement rate was calculated. The results are as follows: Figure 6 As shown, the application of B8961 bacteria increased key soil fertility indicators such as alkaline nitrogen, available phosphorus, available potassium and organic matter in saline-alkali soil by 20.4%, 19.2%, 32.1% and 31.6%, respectively.

[0065] Example 5 Effect of strain B8961 on corn yield and quality in saline-alkali land in Xinjiang

[0066] Select corn seeds with no obvious damage on the surface and full grains, disinfect them with 1% sodium hypochlorite solution for 5 minutes, rinse them with sterile water three times, and dry them for later use. 8 CFU / mL) for 4 hours; the control group soaked the seeds in sterile distilled water for 4 hours and sowed directly after soaking.

[0067] A 2×3 hole pot was used. The soil soaked with an appropriate amount of 5% NaCl solution was used as the pot soil. Tap water that had been placed overnight was added and mixed. The soil moisture content was maintained at 20% and the salt content reached 0.75%. The experimental design was to treat 10 holes in each hole. The treated seeds were planted in the pot, 3 seeds per hole, with a sowing depth of about 1.5 cm. The seeds were covered with plastic film and placed in an artificial climate chamber for cultivation. The culture conditions were 20°C, no light for 8 hours; 22°C, 30μmol / m 2 / s light for 2h; 25℃, 100μmol / m 2 / s light for 12h; 30μmol / m 2 / s light for 2h. After the seeds germinate, remove the film and irrigate once every 5 days (about 10mL per hole). After 15 days of germination, measure the plant height, root length and plant weight of corn plants in the experimental group and the control group.

[0068] Table 2 Effects of different treatments on the growth of corn plants

[0069]

[0070] The experimental results showed that compared with the control treatment, the plant height and root length of corn seeds soaked in B8961 bacterial suspension increased significantly by 35.3% and 44.6% respectively after 15 days of growth, and the fresh weight and dry weight increased by 40% and 45.7% respectively, indicating that soaking in B8961 bacterial agent diluted solution has a significant promoting effect on corn.

[0071] The implementation modes of the present invention have been described in detail above in conjunction with specific embodiments, but the present invention is not limited to the above-mentioned implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A Bacillus strain, characterized in that: The bacillus is Bacillus sp. B8961, deposited in the China Center for Type Culture Collection, the preservation date is November 11, 2024, and the preservation number is CCTCC NO:M 20242511.

2. The Bacillus according to claim 1, characterized in that The Bacillus B8961 has a 16S rDNA sequence as shown in SEQ ID NO.

1.

3. A composition, characterized in that The composition comprises the Bacillus B8961 described in claim 1.

4. The composition according to claim 3, characterized in that The composition is a bacterial suspension, culture solution or bacterial cells containing Bacillus B8961; in the composition, the effective viable count of Bacillus B8961 is ≥4.8×10 9 CFU / g, spore rate ≥96%.

5. The composition according to claim 4, characterized in that The preparation method of the bacterial suspension, culture solution or bacterial cells comprises the following steps: The Bacillus B8961 is inoculated into a culture medium for cultivation to obtain a culture solution, the culture solution is centrifuged and then resuspended to obtain a bacterial suspension; the culture solution is centrifuged to obtain bacterial cells; Preferably, the culture temperature is 25°C to 37°C, and the culture time is 1 to 2 days.

6. Use of the Bacillus B8961 according to claim 1 or 2, or the composition according to any one of claims 3 to 5, in remediating and improving saline-alkali soil and / or promoting plant growth in a saline-alkali environment.

7. The use according to claim 6, characterized in that: In the saline-alkali soil or saline-alkali environment, the mass concentration of sodium chloride is 9-11%.

8. The use according to claim 6, characterized in that: The plant is corn.

9. A method for repairing and improving saline-alkali soil, characterized in that: The method comprises applying the Bacillus B8961 according to claim 1 or 2, or applying the composition according to any one of claims 3 to 5 in saline-alkali soil.

10. A method for promoting plant growth in a saline-alkali environment, characterized in that: The method comprises applying the Bacillus B8961 of claim 1 or 2 in a plant growth medium or on a plant body, or applying the composition of any one of claims 3-5.

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