Bacillus subtilis and application thereof

Abstract

The invention discloses bacillus subtilis and application thereof, and relates to the technical field of microorganisms, the bacillus subtilis S1-17L provided by the invention is preserved in the China General Microbiological Culture Collection Center, the strain preservation number is GGMCC NO: 31323, the preservation date is July 16, 2024, and the 16S rDNA sequence of the bacillus subtilis S1-17L is as shown in SEQ ID NO.1; the strain can secrete a plurality of hydrolases including protease, phosphatase and cellulase, has relatively high capacity of dissolving insoluble phosphorus in soil and a plurality of biological functions, is a growth-promoting and environment-friendly strain, and lays a foundation for research and development of efficient and green microbial fertilizers.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Bacillus subtilis and application thereof. Background Art

[0002] In recent years, the excessive use of fertilizers has led to serious ecological problems, such as eutrophication of water bodies, soil fertility decline, soil compaction and reduced quality of agricultural products. With the introduction of the new concept of green development, people are increasingly concerned about the pollution of water bodies and soil caused by the application of fertilizers and chemical pesticides, and limiting their use has become a consensus of the international community. Since plants absorb nutrients required for growth and reproduction from the soil, their growth may be affected by soil activity or toxic metal elements. Bioremediation has the advantages of being economical, environmentally friendly and not easy to destroy the ecological balance, and has advantages in repairing soil with low concentrations of pesticide residues. The interaction between microorganisms and roots significantly increases the absorption of essential compounds and prevents the accumulation of toxic compounds. In addition, some herbicides are not easily hydrolyzed under non-alkaline conditions and can only be decomposed by microorganisms. Microbial fertilizer is a safe, efficient and environmentally friendly microbial living product that can effectively increase the content of nitrogen, phosphorus, potassium and other elements in the soil, and can also produce key substances such as hydrolases and auxins (such as IAA) in the rhizosphere of plants, thereby activating related signal pathways and significantly promoting plant growth.

[0003] Microbial fertilizers are microbial preparations made by mixing microbial fertilizers with carriers after large-scale cultivation. They are called microbial fertilizers or microbial agents. Compared with ordinary biological fertilizers, microbial fertilizers can act on various trees or crops. At the same time, they are not strict with soil requirements, can balance soil pH, improve soil physical and chemical properties, and promote healthy plant growth. In addition, microbial fertilizers can greatly increase the number and activity of plant rhizosphere growth-promoting bacteria in the soil, enrich organic matter in the soil, enhance plant resistance, and reduce pathogen invasion. More importantly, microbial fertilizers play a positive role in protecting farmland environment and promoting straw composting. Due to its unique social, economic and ecological effects, microbial fertilizers are becoming increasingly popular among the public. They not only do not pollute the environment, but also have low production costs, and can meet the needs of improving the natural environment, maintaining ecological balance, and safe production of agricultural and forestry products. Therefore, microbial fertilizers have huge development and application potential.

[0004] In summary, screening out a microbial strain that can simultaneously dissolve multiple insoluble phosphorus in the soil, promote rapid plant growth and is harmless to the environment and humans is of great significance to various related biological protection fields such as plant protection and biological fertilizer. Summary of the invention

[0005] In view of this, the present invention separates and purifies a Bacillus subtilis S1-17L from soil with specific properties. The strain is currently deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, with a strain deposit number of GGMCC NO: 31323 and a deposit date of July 16, 2024. The strain can secrete a variety of hydrolases, can dissolve a variety of insoluble phosphorus in the soil and is harmless to the environment and humans. It can provide a solid foundation for subsequent plant protection, biological fertilizers and other related biological protection fields. That is, one of the purposes of the present invention is to propose a Bacillus subtilis S1-17L.

[0006] Furthermore, the 16S rDNA sequence of the Bacillus subtilis S1-17L is shown in SEQ ID NO.1.

[0007] The second object of the present invention is to provide a bacterial agent, which contains the above-mentioned Bacillus subtilis S1-17L.

[0008] The third object of the present invention is to provide the use of the above-mentioned Bacillus subtilis S1-17L or bacterial agent in decomposing the poorly soluble organic macromolecular phosphorus in the soil into small-molecule inorganic phosphorus.

[0009] Specifically, the poorly soluble organic macromolecular phosphorus includes lecithin and calcium phytate.

[0010] The present invention also aims to provide the use of the above-mentioned Bacillus subtilis S1-17L or bacterial agent in the preparation of bacterial fertilizer for promoting plant growth.

[0011] The present invention also aims to provide the use of the above-mentioned Bacillus subtilis S1-17L or bacterial agent in treating soil containing insoluble phosphorus.

[0012] The present invention also aims to provide the use of the above-mentioned Bacillus subtilis S1-17L or bacterial agent in the production of hydrolases.

[0013] Furthermore, the hydrolase comprises at least one of a protease, a phosphatase and a cellulase.

[0014] The present invention also aims to provide the use of the above-mentioned Bacillus subtilis S1-17L or bacterial agent in the production of indoleacetic acid.

[0015] Compared with the prior art, the present invention has the following advantages: (1) The strain provided by the present invention can dissolve a variety of insoluble phosphorus in the soil and is harmless to the environment and humans. It reduces agricultural phosphorus pollution to a certain extent, expands the range of phosphorus sources for plants, and promotes plants' absorption of soil phosphorus. It is a strain that is both growth-promoting and environmentally friendly, laying a foundation for the research and development of efficient and green microbial fertilizers.

[0016] (2) The strain provided by the screen of the present invention can produce a large amount of auxin IAA and a variety of hydrolases, which can promote the rapid growth of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the morphological characteristic of strain S1-17L provided in the embodiment of the present invention.

[0018] Figure 2 It is a phylogenetic tree of strain S1-17L constructed based on 16S rRNA gene sequence provided in an embodiment of the present invention.

[0019] Figure 3 The following is a summary of the ecological characteristics of the Burkholderia paludis strain S1-17L provided in the embodiments of the present invention. a: protease; b: phosphatase; c: cellulase. DETAILED DESCRIPTION

[0020] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] The materials of the embodiments of the present invention are as follows: 1. Soil The pecan forest in Linglong Street, Lin'an District, Hangzhou (30°18′N, 119°65′E) was selected as the sampling site. Five standard pecan trees with a spacing greater than 10m, uniform growth and good health were randomly selected as sampling objects in the sampling site. After removing the dead branches and leaves and other surface coverings within a radius of 2m from the base of the trees with a shovel, carefully dig to the end of the root to collect fine root samples, collect soil samples at a depth of 0−30cm, and collect rhizosphere soil by shaking the roots. Mix them evenly, put one portion in a sterile sealed bag and store at 4℃, and the other portion is air-dried for later use. The specific soil physical and chemical properties are as follows: pH 5.21, ω (organic matter) 55.87 g·kg −1,ω (alkaline hydrolyzed nitrogen) 132.8mg·kg −1 ,ω(available phosphorus)8.65 mg·kg −1 ,ω (fast-acting potassium) 147.0 mg·kg −1 .

[0023] 2. Culture medium LB medium (g / L): 10.0% tryptone, 5.0% yeast extract, 10.0% sodium chloride, distilled water to 1 L, pH 7.2–7.4.

[0024] NA medium (g / L): beef extract 3.0, peptone 10.0, NaCl 5.0, agar 20.0, distilled water to 1 L, pH 7.0–7.5.

[0025] NBRIP medium (g / L): glucose 10.0, Ca3(PO4)2 5.0, MgCl2 5.0, KCL 0.2, MgSO4·7H2O 0.25, (NH4)2SO4 0.1, agar powder 20.0, pH 7.0.

[0026] King medium (g / L): peptone 20.0, K2HPO4 1.5, MgSO4·7H2O 1.5, tryptophan 0.1, glycerol 15.0 mL, distilled water to 1 L, pH 7.2.

[0027] Protease detection medium (g / L): skim milk powder 15.0, agar powder 20.0, distilled water to 1 L.

[0028] Phosphatase assay medium (g / L): glucose 10.0, CaCO3 5.0, (NH4)2SO4 0.5, NaCl 0.3, KCL0.3, MgSO4·7H2O 0.3, FeSO4.7H2O 0.03, distilled water to 1 L, pH 7.0–7.5. When pouring the plate, add 3–4 mL of egg yolk liquid (prepared with 1:1 egg yolk and 0.8% NaCl) for every 100 mL and shake well.

[0029] Cellulase detection medium (g / L): sodium carboxymethyl cellulose 2.0, Congo red 2.0, (NH4) SO4 2.0, NaCl 0.5, MgSO4·7H2O 0.5, K2HPO4 1.0, agar powder 20.0, distilled water to 1 L, pH 7.2.

[0030] Example 1 Isolation and purification of strain S1-17L Prepare three phosphate-solubilizing bacteria isolation culture plates with tricalcium phosphate, lecithin and calcium phytate as the sole phosphorus source, respectively, and set aside. Put 8 glass beads in a 200mL triangular flask and add 49.5mL of deionized water, then sterilize it in an autoclave at 121℃ for 0.5h. After cooling, weigh 0.5g of rhizosphere soil in an ultra-clean workbench and place it in the flask. Then place the triangular flask in a shaker and shake it at 180r / min for 1h. Let it stand for 15min, take the supernatant, and dilute it to 10 with sterile water by tenfold dilution method. −3 −10 −8 Gradient bacterial suspension, 0.1mL of bacterial suspension was applied to the phosphate-solubilizing bacteria screening medium, and 3 replicates were set for each gradient, and cultured in the dark in an incubator at (28±0.5)℃ for 3-7 days. Colonies with different morphologies and obvious transparent circles (D / d≥1, D: halo diameter; d: colony diameter) were selected for further streaking purification, and repeated several times until a pure strain with stable transparent circles was obtained. The purified strain suspension was then mixed with 40% glycerol at a ratio of 1:1 and stored in an ultra-low temperature refrigerator at -80℃.

[0031] The strain stored in the −80°C ultra-low temperature refrigerator was taken out, inoculated into LB solid medium by streaking method, and cultured in the dark in a bacterial incubator at (28±0.5)°C for 3−7 days. Prepare NA liquid medium, take 20 mL and dispense into 50 mL Erlenmeyer flasks, steam sterilize in a 121°C autoclave for 0.5 h, and cool for use. Take out the LB solid medium, pick a single colony strain and inoculate it into NA liquid medium, culture it in a constant temperature oscillator at (28±0.5)°C and 180 r / min for 3 days, then transfer the bacterial suspension to a sterile centrifuge tube in an ultra-clean workbench, centrifuge at 10000 r / min for 15 min, retain the bottom cells, wash with sterile saline, centrifuge again, and repeat 3 times; finally, adjust the concentration of the bacterial suspension to 1.0×10 3 , 1.0×10 6 CFU / mL, for future use.

[0032] Example 2 Identification of strain S1-17L After streaking in NA medium and culturing at 28°C for 24 hours. Figure 1 As shown, the colonies of the strain on NA medium are slightly yellow, with a smooth and translucent surface, and are opaque and non-flowable.

[0033] The strain was subjected to physiological and biochemical analysis, and the results are shown in Table 1. With reference to the Bergey Manual of Bacterial Identification and combined with morphological observation, the strain S1-17L was preliminarily identified as Bacillus subtilis.

[0034] Table 1 Physiological and biochemical characteristics of strain S1-17L Index Result Index Result Cell diameter>1.0m − Parasporal crystals − Spore round − Catalase + cyst enlargement − Anaerobic growth − VP determination + Produces D-glucose + Glucose gas production − Hydrolyzed casein + starch + gelatin + Utilizes: Citrate + Tyrosine Hydrolysis − Nitrate reduction to nitrite + Form: Dihydroxyacetone ND Requires NaCl and KC − Requires allantoin and urea salts − Growth pH: 6.8 Nutrient Broth + Growth NaCl: 2-7% + Growth in the presence of lysozyme d H+(O or CO autotrophic growth − Note: +: positive; −: negative; d: 11%~89% of strains are positive; ND: not determined A small amount of bacterial cells were scraped and DNA was extracted using the DNeasyⓇPowerSoilⓇDNAIsolationKit kit [Qiagen Enterprise Management (Shanghai) Co., Ltd.] according to the instructions. The 16SrRNA gene sequence was amplified using the bacterial primer pair 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO.2) and 1492R: 5′-CTACGGCTACCTTGTTACGA-3′ (SEQ ID NO.3). PCR amplification conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 1min30s, a total of 35 cycles; 72℃ final extension for 7min. After the reaction was completed, 3μL of PCR product was taken for 1% agarose gel electrophoresis to confirm the PCR amplification fragment. The sequencing service was entrusted to Shenzhen Micro-Science Group Co., Ltd. The base sequences obtained by sequencing were compared with BLAST in the National Center for Biotechnology Information (NCBI) for analysis of closely related species.

[0035] The 16S rRNA gene sequencing results were uploaded to NCBI, and the strain phylogeny analysis was obtained using the sequencing data and database information in MEGA7.0 software, such as Figure 2 As shown. The data were compared by BLAST in the NCBI database, and it was found that strain S1-17L had the closest evolutionary relationship with Bacillus subtilis, with a sequence homology of 99.48%. Based on the colony morphology observation and molecular biological identification results of the strain, strain S1-17L was identified as Bacillus subtilis. The NCBI accession number is: PP542101. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 16, 2024, with the deposit number GGMCCNO:31323. The base sequence of strain S1-17L is as follows: GTGATTCTTTTTCCGCTCTCAGCGTCAGTTACAGAC CAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGATCTAAAATTCTAT (SEQ.ID.NO.1).

[0036] Example 3 Analysis of phosphate-solubilizing ability of strain S1-17L NBRIP liquid culture medium (phosphorus mass concentration was set to 1 g P / L) containing different insoluble inorganic phosphorus (Ca3(PO4)2, AlPO4 and FePO4) and organic phosphorus (lecithin, sodium phytate) was prepared respectively, 20 mL was dispensed into 50 mL conical flasks, sterilized at 121℃ for 30 min, inoculated with the test strain suspension at 1% inoculation rate, cultured in a constant temperature shaking box at (28±0.5)℃ and 180 r / min for 3 days, centrifuged at 10000 r / min for 15 min, and the supernatant was retained. The soluble phosphorus content in the supernatant was determined by molybdenum blue colorimetry. The culture without bacteria was used as the control, and each treatment was repeated 3 times.

[0037] The mineralization ability of the strains for insoluble organic phosphorus varies with different phosphorus sources. After inoculation and cultivation in the culture medium with lecithin and calcium phytate as phosphorus sources, the net soluble phosphorus content in the solution (C 接种处理 With C 未接种处理 The difference between the values ​​of , the same below) are 25.09 and 174.84 mg / L respectively. At the same time, the strain also has the ability to dissolve insoluble inorganic phosphorus. When tricalcium phosphate, iron phosphate and aluminum phosphate are used as phosphorus sources, the net soluble phosphorus content in the culture solution is 232.14, 8.08 and 10.32 mg / L respectively. Compared with the phosphate-dissolving bacteria reported by previous researchers, this strain has a comprehensive solubility ability for insoluble phosphorus in the soil. It can convert insoluble phosphorus in the soil into a form that can be absorbed and utilized by plants by secreting protons, organic acids, hydrolases and other substances, thereby expanding the range of phosphorus sources for plants, promoting the absorption of soil phosphorus by plants, and then promoting plant growth, providing a good material basis for the production of microbial fertilizers.

[0038] Example 4 Determination of the ability of strain S1-17L to secrete indole-3-acetic acid (IAA) IAA is involved in the regulation and control of many physiological and biochemical processes and has a wide range of physiological effects. IAA can promote cell division, leaf enlargement, stem elongation, adventitious root formation, seed growth, fruit growth, fruit setting and apical dominance, etc., and plays an important role in promoting plant growth.

[0039] Prepare King liquid culture medium, take 20mL and dispense it into 50mL Erlenmeyer flasks, sterilize the liquid in a 121℃ autoclave for 0.5h, inoculate the test strain, and culture it in a constant temperature oscillator at (28±0.5)℃ and 180r / min for 3d. Then transfer the bacterial suspension to a sterile centrifuge tube and centrifuge it at 10000r / min for 15min. The IAA concentration in the supernatant is determined by the Salkowski colorimetric method. In short, take equal volumes of supernatant and S2 colorimetric solution (1mL 0.5mol / LFeCl3 added to 50mL 35% HClO4) and mix them evenly. After standing at room temperature for 30min, observe the color change of the solution, and compare the color at a wavelength of 530nm. The IAA concentration in the culture medium is determined and calculated using the standard IAA calibration curve.

[0040] The experimental results show that strain S1-17L showed a dark pink color after reacting with Salkowski colorimetric solution. The IAA production was measured by spectrophotometry and the result was 44.73 mg / L. Cui Weiguo et al. screened out several strains of IAA-secreting bacteria from the potato rhizosphere, with secretion amounts ranging from 0.80 to 40.27 μg / mL. It can be seen that strain S1-17L is a potential strain for producing microbial fertilizers and has good application potential.

[0041] Example 5 Ability of strain S1-17L to secrete hydrolases Protease is an enzyme that can break the peptide bonds between protein amino acids and plays an important role in cell metabolism. Compared with plant and animal cellulases, microbial cellulases have the advantages of easy operation, fast growth, easy genetic material manipulation, good enzymatic stability under harsh conditions, simple production and low cost. In addition, phosphatase plays an important role in the phosphorus absorption process. Fertilizers or agents made from phosphate-solubilizing microorganisms are directly applied to the soil. Under the action of phosphatase, insoluble organic macromolecules of phosphorus are decomposed into small inorganic molecules of phosphorus. Plants can efficiently utilize phosphorus resources, which not only reduces the excessive input of phosphorus in agricultural production, but also dissolves the insoluble phosphorus in the soil for plants to absorb and utilize, reducing agricultural phosphorus pollution to a certain extent.

[0042] In this example, the solid plate method was used to qualitatively evaluate the ability of the strain to secrete hydrolases by observing the hydrolysis zone. Protease, phosphatase and cellulase solid culture medium detection plates were prepared respectively, and a single colony was picked and dotted in the center of the solid plate. Each treatment was repeated 3 times and cultured in a (28±0.5)℃ incubator in the dark for 3-7 days. If a transparent zone is produced around the strain, it means that it has the ability to produce the hydrolase, otherwise it is not.

[0043] The results are as follows Figure 3As shown, after the strain was inoculated into solid culture medium, corresponding transparent circles were generated around the strain, indicating that the strain had the ability to secrete protease, phosphatase and cellulase.

[0044] In summary, the strain screened by the present invention has a high ability to dissolve insoluble phosphorus in the soil and multiple biological functions. It is a strain that is both growth-promoting and environmentally friendly, laying a foundation for the research and development of efficient and green microbial fertilizers.

[0045] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0046] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Bacillus subtilis S1-17L, characterized in that: It is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the strain collection number is GGMCC NO:31323.

2. The Bacillus subtilis S1-17L according to claim 1, characterized in that The 16S rDNA sequence of the Bacillus subtilis S1-17L is shown in SEQ ID NO.

1.

3. A bacterial agent, characterized in that: Contains the Bacillus subtilis S1-17L according to claim 1.

4. Use of the Bacillus subtilis S1-17L according to claim 1 or 2 or the bacterial agent according to claim 3 in decomposing poorly soluble organic macromolecular phosphorus in soil into small molecular inorganic phosphorus.

5. Use of the Bacillus subtilis S1-17L according to claim 1 or 2 or the bacterial agent according to claim 3 in the preparation of bacterial fertilizer for promoting plant growth.

6. Use of the Bacillus subtilis S1-17L according to claim 1 or 2 or the bacterial agent according to claim 3 in treating soil containing insoluble phosphorus.

7. Use of the Bacillus subtilis S1-17L according to claim 1 or 2 or the bacterial agent according to claim 3 in producing hydrolases.

8. The use according to claim 7, characterized in that: The hydrolase comprises at least one of a protease, a phosphatase and a cellulase.

9. Use of the Bacillus subtilis S1-17L according to claim 1 or 2 or the bacterial agent according to claim 3 in the production of indoleacetic acid.

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