Bacillus subtilis and application thereof

Abstract

The invention discloses bacillus subtilis YK-1, which is characterized in that the bacillus subtilis is preserved in China General Microbiological Culture Collection Center on July 21, 2023, the preservation number is CGMCC No.27965, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing. The invention also discloses an application of the compound in a bio-fertilizer. The bacillus subtilis YK-1 disclosed by the invention not only has efficient phosphate solubilizing capacity, but also has relatively strong effects of resisting plant diseases, promoting crop growth and increasing crop yield.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a Bacillus subtilis strain and application thereof. Background Art

[0003] Compared with traditional soil improvement technology, microbial soil improvement technology is efficient and safe. While improving the soil, it can also improve the disease resistance and quality of crops. Therefore, screening excellent strains and developing them into functional biofertilizers can effectively improve the soil, improve soil quality, and thus improve the quality and yield of crops. However, due to the lack of efficient strains, the efficacy of functional microbial fertilizers is generally low, which has become a key problem that needs to be solved in the current process of improving and fertilizing the soil in my country using microbial technology. Therefore, screening and obtaining efficient strains and allowing them to reproduce and function in the soil is the core of solving the above problems.

[0004] Bacillus is a kind of microorganism with spore-forming characteristics that is widely used, highly safe, highly resistant to stress, and environmentally friendly. There are many types of Bacillus, and many strains of Bacillus have strong inhibitory and killing effects on a variety of plant pathogens and pests. Bacillus can produce a variety of active substances, such as alveolar bacillusin and iturin antimicrobial peptides, and are therefore favored by biological control workers. In addition, Bacillus produces plant hormones for plants to use, and provides plants with necessary nutrients through nitrogen fixation, phosphorus solubilization, potassium solubilization, etc.

[0005] After the functional biological fertilizer is applied, the balance of soil microbial communities is regulated to effectively improve the soil microecological environment and increase the activity of soil enzymes, thereby promoting the transformation of soil organic matter and generating nutrients such as small molecular organic carbon that can be absorbed and utilized by crops. At the same time, the various antibacterial active substances produced by beneficial microorganisms can inhibit the reproduction of pathogens, enhance the ability of crops to resist various diseases, reduce the occurrence of soil-borne diseases, and promote the healthy growth of crops. If active strains with multiple excellent functions are added to fertilizers and developed into active bacterial fertilizers with multiple functions at the same time, crops can improve quality and disease resistance, and at the same time improve soil and increase soil fertility. Therefore, breeding Bacillus strains with multiple functions, good application effects and stability is still a research focus in this field. Summary of the invention

[0006] The invention aims to provide a Bacillus subtilis strain and its application in biological fertilizer.

[0007] The present invention provides a strain of Bacillus subtilis YK-1, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 21, 2023, with a deposit number of CGMCC No. 27965, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0008] The Bacillus subtilis YK-1 is Gram-positive, has rod-shaped cells, 0.8-1.2×2.0-4.0 μm, grows well on agar culture medium plates, has white, opaque colonies, and has a rough, wrinkled surface. It can form oval spores and is an aerobic bacterium.

[0009] The invention also provides application of the Bacillus subtilis YK-1 in biological fertilizer.

[0010] The beneficial effects of the present invention are:

[0011] 1. The Bacillus subtilis strain provided by the present invention is screened from the forest soil of Northeast China. It has a significant phosphate-solubilizing effect, can antagonize plant diseases, resist premature aging of crops, and effectively improve crop yield and quality.

[0012] 2. After the Bacillus subtilis YK-1 of the present invention is made into a biological fertilizer and applied to the soil, it can improve soil fertility, resist plant diseases, and increase crop yield and quality. The Bacillus subtilis YK-1 of the present invention can be further made into a microbial agent / biological organic fertilizer, which is widely used in the field of agricultural production, has a significant effect of improving soil and increasing production and quality, and has a very broad application prospect. DETAILED DESCRIPTION

[0013] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0014] The equipment and reagents used in the embodiments of the present invention can be selected from any commercially available one. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional replacement selections based on the technical ideas of the present invention and according to existing technologies, and are not limited to the specific records of the embodiments of the present invention.

[0015] The culture medium formula involved in the present invention is as follows:

[0016] LB solid culture medium: 10 g peptone, 5 g yeast powder, 10 g NaCl, 15 g agar, distilled water to 1 L, pH adjusted to 7.0, sterilized at 121°C for 15 min, poured into a culture dish when cooled to 55-70°C, and placed at room temperature for 6-8 hours before use.

[0017] Insoluble inorganic phosphorus liquid culture medium: glucose 10 g, (NH)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·H2O 0.03 g, Ca3(PO4) 25.0 g, distilled water 1000 mL, pH 7.0-7.5, sterilize at 115 ° C for 30 min, and set aside after aseptic packaging.

[0018] Insoluble inorganic phosphorus solid culture medium: Add 2% agar powder to the above-mentioned insoluble inorganic phosphorus liquid culture medium, sterilize at 115°C for 30 minutes, pour into a culture dish when cooled to 55-70°C, and place at room temperature for 6-8 hours before use.

[0019] The present invention is further described below in conjunction with specific embodiments.

[0020] Example 1 Preliminary screening of Bacillus phosphate solubilizing in soil

[0021] 1. Isolation and purification of soil microorganisms:

[0022] (1) Soil samples: collected from forest soil in Northeast China.

[0023] (2) Separation and purification:

[0024] Weigh 5g of soil sample and dissolve it in a glass beaded flask containing 100ml of sterile water. Shake it on a shaker for 30min to make a soil suspension. Use a sterile pipette to take 10ml of soil suspension and add it to the liquid culture medium. Place it in a constant temperature incubator at 37℃ and culture it for 48h. Heat the culture medium in an 80℃ water bath for 10 minutes to kill the bacteria and enrich the spores.

[0025] 1 ml of the culture solution was transferred into a sterile test tube containing 9 ml of sterile water and diluted to 1:10 by gradient dilution. -3 -10 -6 of soil gradient dilution solution.

[0026] Use a pipette to transfer 0.1 mL of each diluted solution and spread it evenly on the LB solid plate; culture it in a constant temperature incubator at 32°C for 48 h, then take it out and pick a single colony to continue separation and purification until the colonies grown on each plate have the same morphology and color, which is a single strain.

[0027] Using the above method, the applicant screened out a total of 219 bacterial strains.

[0028] Example 2 Bacillus phosphate-solubilizing rescreening

[0029] The 219 bacterial strains obtained from the initial screening of Example 1 were inoculated onto a poorly soluble inorganic phosphorus solid culture medium, and cultured at 30°C for 3 days. The sizes of the transparent zones around the colonies were observed, and the three bacterial strains with the largest transparent zones were screened and named YK-1, YK-2 and YK-3, respectively.

[0030] The three strains with the largest transparent zones were inoculated into 50 mL of insoluble inorganic phosphorus liquid culture medium, respectively, and cultured at 30°C and 200 rpm for 6 days. At the same time, a liquid phosphate-dissolving culture medium without any bacteria was used as a control group, and the number of live bacteria in the culture medium was detected.

[0031] 2.1 Drawing of phosphorus standard curve

[0032] Pipette 0.0, 0.2, 0.4, 0.8, 1.6, 2.0, 3.2, 4.0 mL of 5 mg / L phosphorus standard solution into test tubes, then add 2 mL of molybdenum antimony anti-colorimetric agent to each test tube, distilled water to 20 mL, shake well and let stand for 20 minutes, and measure the absorbance value at a wavelength of 700 nm. At this time, the phosphorus concentration in each tube is divided into: 0.00, 0.05, 0.10, 0.20, 0.40, 0.50, 0.80, 1.00 mg / mL. Draw a phosphorus standard curve with phosphorus concentration as the horizontal axis and absorbance as the vertical axis.

[0033] 2.2 Determination of available phosphorus content in culture medium

[0034] Under sterile conditions, 5 mL of the culture medium of the above three strains of bacteria were taken respectively, and centrifuged at 8000 rpm for 5 minutes. The supernatant was taken and diluted to an appropriate concentration. 0.5 ml of the dilution was pipetted into a test tube, and 5 ml of distilled water, 2 drops of 2,4-dinitrophenol indicator, and 2 ml of molybdenum antimony anticolorimetric agent were added. The volume was then made up to 20 ml with distilled water, and the mixture was shaken and allowed to stand for 20 minutes. The colorimetry was performed at a wavelength of 700 nm, and the absorbance value was substituted into the standard curve to calculate the available phosphorus content in the supernatant. The specific results are shown in Table 1.

[0035] Table 1 Phosphate solubilization effect of different strains

[0036] sample Viable bacteria count (CFU / mL) Available phosphorus content (mg / L) Blank control group 0 14 YK-1 <![CDATA[2.2×10 8 ]]> 129 YK-2 <![CDATA[9.3×10 7 ]]> 105 YK-3 <![CDATA[8.7×10 7 ]]> 79

[0037] From the data in Table 1, it can be seen that the three strains screened by the applicant from the soil samples all have strong phosphate solubilization capabilities and can decompose the insoluble phosphorus (Ca3(PO4)2) in the culture medium into soluble, fast-acting phosphorus with significant effects. Among them, the YK-1 strain has the highest phosphate solubilization efficiency, with a fast-acting phosphorus content of up to 129 mg / L.

[0038] Example 3 Identification of strains

[0039] (1) Colony morphology and physiological and biochemical test results of YK-1 strain:

[0040] The colony of the YK-1 strain is white, opaque, has wrinkles on the surface, and forms spores. Gram staining is positive, the suitable growth temperature range is 27-35°C, and the suitable growth pH range is 5.5-8.0. Physiological and biochemical results show that the YK-1 strain can grow in a 5% sodium chloride medium, the VP test result is positive, the methyl red test is negative, the starch hydrolysis, gelatin liquefaction, nitrate reduction test results are positive, D-mannitol can be fermented to produce acid, but propionate cannot be utilized.

[0041] (2) Molecular biological identification of YK-1 strain:

[0042] The YK-1 strain obtained by the above screening was identified by molecular biological methods, its 16s rDNA sequence was measured, and blast comparison was performed in the GenBank nucleic acid database.

[0043] Its 16s rDNA sequence is:

[0044] GCAGTGCGGCGTGCCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGC

[0045] TCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGT

[0046] AAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGA

[0047] ACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGA

[0048] CCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGAT

[0049] GCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGC

[0050] CCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAA

[0051] GTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGC

[0052] TCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACG

[0053] GTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA

[0054] CGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGC

[0055] GGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATT

[0056] GGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTA

[0057] GCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTC

[0058] TCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGA

[0059] TTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGG

[0060] GGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG

[0061] AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAA

[0062] GCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGT

[0063] CTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGA

[0064] GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT

[0065] AAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGG

[0066] GCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0067] TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACA

[0068] GAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTT

[0069] CTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTA

[0070] GTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACAC

[0071] ACCGCCCGTCACAACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTAGGAGCCAGCCGCCGAAGGTGGACCCCA.

[0072] Based on the colony morphology, physiological and biochemical characteristics and 16s rDNA comparison results of the YK-1 strain, it was determined that the YK-1 strain was Bacillus subtilis and named Bacillus subtilis YK-1.

[0073] The applicant (Shenyang Yikan Environmental Biotechnology Development Co., Ltd., address: No. 31, Xihejiu North Street, Shenyang Economic and Technological Development Zone, Shenyang Province) deposited Bacillus subtilis YK-1 at the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on July 21, 2023, with the deposit number CGMCC No. 27965, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0074] Example 4 Phosphate solubilization and yield-increasing test of Bacillus YK-1 in field cultivation of Chinese cabbage

[0075] 1. Test location: Liaozhong District, Shenyang City, Liaoning Province.

[0076] 2. Test process and results

[0077] Three treatments were set up: ① blank control group: a control group without adding Bacillus fermentation broth; ② fermentation broth sterilization treatment group: Bacillus subtilis YK-1 fermentation broth fermented for 72 hours (viable count 2.1×10 8 CFU / mL), sterilized at 121℃ for 20min, and the sterilized fermented liquid was applied to the experimental plot with water at a dosage of 10L / mu on the day of planting pakchoi. ③ Fermented liquid treatment group: YK-1 fermented liquid fermented for 72h (viable bacteria count 2.1×10 8 CFU / mL), at a dosage of 10L / mu, it was applied to the test plot with water on the day of planting Chinese cabbage. Except for the different application methods and application amounts of fungicides in each treatment group, other farmland water, fertilizer and management measures were exactly the same. There were 6 test plots in each treatment, for a total of 18 test plots. The plots of each treatment were randomly distributed, each test area was a square (2m×2m), and each experimental area was kept at a distance of 0.5 meters. 50 days after sowing, all the Chinese cabbage was harvested, and the fresh weight and dry weight of the Chinese cabbage in each test area were tested separately, and the average fresh weight and average dry weight of the Chinese cabbage in each treatment group were calculated for comparison and analysis. At the same time, soil samples were collected from each treatment test area, and the content of available phosphorus in the soil samples was detected by the Olsen method.

[0078] Test results: Compared with the blank control group (treatment ①), the average fresh weight of Chinese cabbage in the Bacillus subtilis YK-1 treatment group (③ microbial agent treatment group) increased by 39.62% (Table 1), and the soil available phosphorus content increased by 21.4%. The results show that the live Bacillus subtilis YK-1 in the fermentation liquid has the effect of significantly increasing the yield of Chinese cabbage and the content of soil available phosphorus.

[0079] Table 2 Effects of different treatments on fresh weight of Chinese cabbage

[0080]

[0081] Example 5 Experiment on Phosphate Solubilization and Powdery Mildew Resistance of Bacillus subtilis YK-1 in Muskmelon Cultivation

[0082] 1. Test location: Xinmin City, Liaoning Province.

[0083] 2. Test process and results:

[0084] Three treatments were set up: ① blank control group: no Bacillus fermentation broth was added; ② fermentation broth sterilization treatment group: YK-1 fermentation broth fermented for 72 hours (viable count 2.0×10 8CFU / mL), sterilized at 121℃ for 20min, and the sterilized fermentation liquid was applied to the test plot three times with water on the 30th, 45th and 60th days of melon planting. ③ Fermentation liquid treatment group: YK-1 fermentation liquid (viable bacteria count 2.0×10 8 CFU / mL), at a dosage of 10L / mu, on the 30th, 45th and 60th days of melon planting, it was applied to the test plot three times with water. Except for the different application amounts of fungicides in each treatment group, other farmland water, fertilizer and management measures were exactly the same. Three test plots were set for each treatment, for a total of 9 test plots. The plots of each treatment were randomly distributed, each test area was a rectangle (10m×8m), and a distance of 0.5 meters was maintained between each experimental area. During the planting process, the melons were picked and the yield was recorded, the vitamin C and chlorophyll contents in the leaves of plants with different treatments in the middle and late stages of growth were determined, and the occurrence of powdery mildew was recorded. At the same time, soil samples were collected from the test areas of each treatment, and the content of available phosphorus in the soil samples was detected by the Olsen method.

[0085] Test results: The yield of muskmelon in the Bacillus subtilis YK-1 treatment group (3 fermentation liquid treatment group) increased by 8.5% compared with the blank control group (treatment 1), and increased by 6.7% compared with the sterilized fermentation liquid treatment group (treatment 2). In the late stage of muskmelon planting, the leaves of the blank control group (treatment 1) without the application of bacterial liquid were yellow and powdery disease was serious, while the leaves of the treatment group (treatment 3) with the application of active bacterial agent were green and no powdery disease occurred. In the late stage of muskmelon growth, the vitamin C content of the leaves of the treatment group 3 increased by 16.1% compared with the blank control group, the chlorophyll content increased by 22.3%, and the soil available phosphorus content increased by 30.7%. The results show that Bacillus subtilis YK-1 has the effect of increasing muskmelon yield, solubilizing phosphorus and resisting muskmelon diseases.

[0086] Example 6 Phosphate solubilization and yield increase test of Bacillus YK-1 in tomato cultivation

[0087] 1. Test location: Sujiatun District, Shenyang City, Liaoning Province.

[0088] 2. Test process

[0089] Three treatments were set up: ① blank control group: a control group without adding Bacillus fermentation broth; ② fermentation broth sterilization treatment group: YK-1 fermentation broth fermented for 72 hours (viable count 2.3×10 8 CFU / mL), sterilized at 121℃ for 20min, and then applied to the test plot three times with water at a dosage of 10L / mu on the 10th, 25th, and 35th days after tomato transplanting. ③ Fermentation liquid treatment group: YK-1 fermentation liquid fermented for 72h (viable bacteria count 2.3×10 8CFU / mL), at a dosage of 10L / mu, it was applied to the test plot three times with water on the 10th, 25th, and 35th days after tomato transplanting. ④ Fermentation liquid + small molecule carbon treatment group: YK-1 fermentation liquid fermented for 72 hours (viable bacteria count 2.3×10 8 CFU / mL), at a dosage of 10L / mu, on the 10th, 25th and 35th days after tomato transplanting, it was applied to the test plot three times with water. At the same time, small molecule carbon (vitamin C industrial fermentation waste liquor, organic matter content of 48.8%, citric acid content of 25.1%, acetic acid content of 4.3%) was applied twice on the 10th and 25th days after tomato transplanting, 10 kg of small molecule carbon was applied per mu each time. Except for the different application amounts of sterilization solution or small molecule carbon in each treatment group, other farmland water, fertilizer and management measures were completely consistent. There were 3 test plots for each treatment, for a total of 9 test plots. The plots of each treatment were randomly distributed, each test area was rectangular (4m×3m), and a distance of 0.5 meters was maintained between each experimental area. During the planting process, tomatoes were picked and the yield was recorded. At the same time, soil samples were collected from the test areas of each treatment, and the content of available phosphorus in the soil samples was detected by the Olsen method.

[0090] Test results: The tomato yield of the Bacillus subtilis YK-1 treatment group (treatment ③) increased by 12.4% compared with the blank control group (treatment ①) and increased by 10.0% compared with the sterilization group. However, the tomato yield of treatment ④ was significantly higher than that of treatment ③ (increased by 7.5%). In the late stage of tomato growth, the soil available phosphorus content of treatment ③ group increased by 29.1% compared with treatment ① and 28.6% compared with treatment ②. The results show that Bacillus subtilis YK-1 has the effect of significantly increasing tomato yield and increasing the available phosphorus content in the soil. When Bacillus subtilis YK-1 is applied together with small molecular carbon substances, it can more effectively increase crop yields.

[0091] The above results show that the Bacillus subtilis YK-1 screened by the present invention has a significant phosphate-solubilizing effect on the soil (increasing the available phosphorus content in the soil), and also has the effects of promoting crop growth, increasing crop yield and resisting plant diseases. It can be used as a strain raw material for producing microbial agents / bio-organic fertilizers and is widely used in the field of agricultural production.

[0092] The above-described embodiments are only preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several changes and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis YK-1, characterized in that: The Bacillus subtilis was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 21, 2023, with the deposit number: CGMCC No. 27965, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. Use of the Bacillus subtilis as claimed in claim 1 in biological fertilizer.

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