Bacillus subtilis and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to bacillus subtilis and application thereof. The bacillus subtilis comprises a nucleotide sequence of 16S rDNA (ribosomal deoxyribonucleic acid) as shown in SEQ ID: 1. According to the bacillus subtilis provided by the embodiment of the invention, the utilization of nutrients and water by plants in water culture can be improved, the chlorophyll content of plant leaves is increased, and green algae breeding is inhibited, so that the water culture efficiency is improved, and the water culture difficulty and cost are reduced.

Description

Technical Field

[0001] This application relates to the field of microbiology, specifically to a Bacillus subtilis strain and its applications. Background Technology

[0002] Bacillus subtilis belongs to the genus Bacillus and is a group of Gram-positive bacteria with strong resistance, wide distribution, and broad-spectrum bactericidal activity. Many natural strains of Bacillus provide different highly bioactive genes, playing an important role in both production and in the study of genomes and physical maps. They have wide applications in agriculture, industry, and medicine (such as in biofertilizers, biopesticides, enzyme production, anti-infection, and anti-tumor applications).

[0003] Hydroponics, as a novel soilless cultivation method for plants, boasts advantages such as low cost, controllable environment, and resource conservation, gaining widespread acceptance from the industry and consumers. Currently, hydroponics is applied in various fields, such as scientific research using hydroponic technology to study plant roots and rhizosphere microorganisms; and agricultural production using hydroponics for seedling cultivation, fruit tree cultivation, or the cultivation of certain endangered rare species. Important factors affecting the growth of hydroponic plants include the nutrient solution and other functional components added to the water, such as microbial agents. For example, frequent or excessive use of nutrient solution can easily lead to eutrophication, causing a large proliferation of green algae that compete with plants for nutrients and oxygen; insufficient nutrient solution will reduce plant growth and may even cause plants to wither. Therefore, it is necessary to strictly control the conditions for adding hydroponic nutrient solution and monitor water quality.

[0004] Therefore, there is an urgent need to develop new plant rhizosphere growth-promoting bacteria agents to improve the utilization of nutrients and water by plants in hydroponics and inhibit the reproduction of green algae, thereby improving hydroponic efficiency and reducing the difficulty and cost of hydroponics. Summary of the Invention

[0005] The present invention solves at least one of the problems of the related art in the following aspects.

[0006] The first aspect of this application provides a Bacillus subtilis strain containing a nucleotide sequence of 16S rDNA as shown in SEQ ID: 1.

[0007] In some embodiments, the Bacillus subtilis is Bacillus subtilis BJIB-BF-2024-105 with the preservation number CGMCC No. 31786.

[0008] A second aspect of this application provides a microbial inoculant comprising Bacillus subtilis as described in any of the first aspects of the embodiment, wherein the content of Bacillus subtilis is greater than or equal to 1 × 10⁻⁶ based on the total volume of the microbial inoculant. 8 cfu / mL.

[0009] In some embodiments, the content of Bacillus subtilis is 5 × 10⁻⁶ based on the total volume of the microbial agent. 8 cfu / mL.

[0010] In some embodiments, the solvent for the microbial agent is water.

[0011] In some embodiments, the microbial inoculant may further comprise agronomically acceptable adjuvants and / or nutrients that promote the growth of hydroponic plants.

[0012] The third aspect of this application provides the application of Bacillus subtilis as described in any of the embodiments of the first aspect or the microbial agent as described in any of the embodiments of the second aspect in hydroponic plants.

[0013] In some embodiments, the application includes increasing the chlorophyll content in hydroponic plants and / or inhibiting algal growth.

[0014] In some embodiments, the hydroponic plant is a pothos.

[0015] In some embodiments, the algae are green algae.

[0016] The embodiments of this application achieve the following beneficial effects:

[0017] This application proposes for the first time Bacillus subtilis BJIB-BF-2024-105, which can increase the chlorophyll content in hydroponic plants and inhibit the reproduction of green algae, thereby significantly improving hydroponic efficiency and reducing the difficulty and cost of hydroponics.

[0018] The application of Bacillus subtilis BJIB-BF-2024-105 proposed in this application is of great significance in promoting the growth of hydroponic plants. It significantly reduces the high requirements for nutrient solution addition in hydroponics, improves the utilization of nutrients and water by plants, inhibits the reproduction of green algae, reduces the difficulty of hydroponics, and promotes the large-scale promotion and application of hydroponics.

[0019] The microbial agent containing Bacillus subtilis provided in this application has a simple composition, and its preparation process and application method are easy to operate. After use, it can produce a highly efficient algae-inhibiting effect (inhibition efficiency up to 77%) and promote the chlorophyll content of hydroponic plant leaves. Furthermore, the microbial agent has a clear composition, is non-toxic and harmless, and can effectively reduce the amount of chemical disinfectants used in hydroponic production, simplify the cleaning of hydroponic facilities, reduce site and labor costs, and improve economic and environmental benefits. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The colony morphology of Bacillus subtilis BJIB-BF-2024-105.

[0022] Figure 2 Cluster analysis diagram of Bacillus subtilis BJIB-BF-2024-105.

[0023] Figure 3 The SPAD (Soil and Plant Analyzer Development) test results for Example 2 of this application are shown. The vertical axis represents the relative chlorophyll content reading, reflecting the degree of greenness of the plant.

[0024] Figure 4 The results of the microbial agent provided in Example 3 of this application inhibiting green algae. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0026] This application is based on the inventor's following understanding:

[0027] Hydroponics, as a novel soilless cultivation method for plants, boasts advantages such as low cost, controllable environment, and resource conservation, gaining widespread acceptance from the industry and consumers. Currently, hydroponics is applied in various fields, such as scientific research using hydroponic technology to study plant roots and rhizosphere microorganisms; and agricultural production using hydroponics for seedling cultivation, fruit tree cultivation, or the cultivation of certain endangered rare species. Important factors affecting the growth of hydroponic plants include the nutrient solution and other functional components added to the water, such as microbial agents. For example, frequent or excessive use of nutrient solution can easily lead to eutrophication, causing a large proliferation of green algae that compete with plants for nutrients and oxygen; insufficient nutrient solution will reduce plant growth and may even cause plants to wither. Therefore, it is necessary to strictly control the conditions for adding hydroponic nutrient solution and monitor water quality.

[0028] To further reduce the difficulty and improve the efficiency of hydroponics, the inventors of this application provide a microbial agent containing Bacillus subtilis. Its components are simple, and its preparation and application methods are easy to operate. After use, it can produce a highly effective algae-inhibiting effect (inhibition efficiency up to 77%) and promote the chlorophyll content of hydroponic plant leaves. Furthermore, the agent's components are clearly defined, non-toxic, and harmless. In hydroponic production, it can effectively reduce the amount of chemical disinfectants used, simplify the cleaning of hydroponic facilities, reduce site and labor costs, and improve economic and environmental benefits.

[0029] The first aspect of this application provides a Bacillus subtilis strain containing a nucleotide sequence of 16S rDNA as shown in SEQ ID: 1.

[0030] TGGCTCCCTGATAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAA

[0031] GACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGG

[0032] TTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGC

[0033] TAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTG

[0034] ATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGG

[0035] AATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTT

[0036] TTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTA

[0037] CCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT

[0038] ACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTC

[0039] TTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGA

[0040] ACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGA

[0041] TGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAG

[0042] CGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT

[0043] GAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCAC

[0044] TCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGC

[0045] ACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCT

[0046] TGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGT

[0047] GGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC

[0048] GCACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTG

[0049] ACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCT

[0050] ACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAAT

[0051] CCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGA

[0052] ATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACCCCGCCCGTC (SEQ ID NO: 1)

[0053] In some embodiments, the Bacillus subtilis is Bacillus subtilis BJIB-BF-2024-105 with the preservation number CGMCC No. 31786.

[0054] The Bacillus subtilis BJIB-BF-2024-105 used in this application embodiment was deposited on August 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 31786.

[0055] A second aspect of this application provides a microbial inoculant comprising Bacillus subtilis as described in any of the first aspects of the embodiment, wherein the content of Bacillus subtilis is greater than or equal to 1 × 10⁻⁶ based on the total volume of the microbial inoculant. 8 cfu / mL (e.g., 1.5 × 10⁻⁶) 8 cfu / mL, 2×10 8 cfu / mL, 3×108 cfu / mL, 4×10 8 cfu / mL, 5×10 8 cfu / mL, 6×10 8 cfu / mL, 7×10 8 cfu / mL, 8×10 8 cfu / mL, 9×10 8 cfu / mL, 1×10 9 cfu / mL, 2×10 9 cfu / mL, 3×10 9 cfu / mL, 4×10 9 cfu / mL, 5×10 9 cfu / mL, 6×10 9 cfu / mL, 7×10 9 cfu / mL, 8×10 9 cfu / mL, 9×10 9 cfu / mL, 1×10 10 (cfu / mL, etc.).

[0056] In some embodiments, the content of Bacillus subtilis is 5 × 10⁻⁶ based on the total volume of the microbial agent. 8 cfu / mL.

[0057] In some embodiments, the solvent for the microbial agent is water.

[0058] In some embodiments, the microbial inoculant may further comprise agronomically acceptable adjuvants and / or nutrients that promote the growth of hydroponic plants.

[0059] In some embodiments, the microbial agent further includes excipients, wherein the excipients may be organic matter and / or inorganic matter.

[0060] In the embodiments of this application, the organic matter can be any microbial agent or nutrient other than Bacillus subtilis BJIB-BF-2024-105 (accession number CGMCC No. 31786) that can promote the growth of hydroponic plants or inhibit the growth of competing aquatic microorganisms or plants. In the embodiments of this application, the organic matter can also be organic fertilizer required for the growth of hydroponic plants. It is understood that the organic matter in the embodiments of this application, alone or in combination, is sufficient as long as it can promote the growth of hydroponic plants and inhibit the reproduction of green algae; this application does not impose any limitations on this.

[0061] In the embodiments of this application, the inorganic matter can be a chemical component that can be used in agriculture, such as an agriculturally acceptable carrier, excipient, diluent, adjuvant, medium, excipient, carrier or combination thereof, and / or an inorganic fertilizer that does not affect the concentration of microbial activity in the microbial agent. This application does not limit this.

[0062] The third aspect of this application provides the application of Bacillus subtilis as described in any of the embodiments of the first aspect or the microbial agent as described in any of the embodiments of the second aspect in hydroponic plants.

[0063] In some embodiments, the application includes increasing the chlorophyll content in hydroponic plants and / or inhibiting algal growth.

[0064] In some embodiments, the hydroponic plant is a pothos.

[0065] In some embodiments, the algae are green algae.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0067] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0068] Example 1

[0069] The inventors screened a strain of Bacillus subtilis BJIB-BF-2024-105 through a large number of random mutation experiments. This strain can increase the chlorophyll content of hydroponic plant leaves and inhibit algae growth. The identification of the strain is as follows.

[0070] Colony morphology

[0071] The morphological characteristics of the strains were determined according to the standard methods described in the "Handbook of Systematic Identification of Common Bacteria". Figure 1 As shown.

[0072] Based on observation, this strain appears as smooth, white, opaque, circular protrusions with a diameter of 2-6 mm on MRS solid medium, with irregular edges.

[0073] 16S rDNA sequence amplification and analysis

[0074] Genomic DNA was extracted using a bacterial genomic DNA extraction kit and used as a template for PCR reactions.

[0075] The 16S rDNA sequence of Bacillus subtilis BJIB-BF-2024-105 was amplified using the universal bacterial primers 27F (5'-3'):AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 2) and 1492R (5'-3'):CGGTTACCTTGTTACGACTT (SEQ ID NO: 3).

[0076] PCR reaction system: 10 μL of 2×PhantaMasterMix, 0.5 μL of DNA template, 0.5 μL of universal primer 27F, 0.5 μL of universal primer 1492R, and 8.5 μL of sterile distilled water.

[0077] PCR reaction procedure: 95℃ for 3 min, 95℃ for 15 s denaturation, 55℃ for 15 s annealing, 72℃ for 50 s extension, denaturation-annealing-extension for 35 cycles, 72℃ for 10 min, and finally cooling and maintaining the product at 16℃.

[0078] The PCR products were excised and purified before being resequencing.

[0079]

[0080] The sequencing results were compared with the BLASTn sequence on the NCBI website. The strain with the highest homology to the 16S rDNA sequence (Query_2661175) of the tested strain Bacillus subtilis BJIB-BF-2024-105 was Bacillus subtilis (sequence number MT437043.1), with a similarity of 99.92%.

[0081] Cluster analysis

[0082] Based on the sequence alignment results, suitable strain 16S rDNA sequences were selected, and a phylogenetic tree was constructed using the Neighbor-Joining method. The results are as follows: Figure 2 As shown.

[0083] strain preservation

[0084] Bacillus subtilis strain BJIB-BF-2024-105 was deposited at the China General Microbiological Culture Collection Center (CGMCC); deposit registration number: CGMCC No. 31786; deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; postcode: 100101; deposit date: August 30, 2024.

[0085] Example 2

[0086] Bacillus subtilis BJIB-BF-2024-105 (CGMCC No. 31786) was cultured in liquid form for 20 hours at 30℃ and 200 rpm. After incubation, it was diluted with nutrient solution and mixed thoroughly to prepare a liquid microbial inoculum with a cell count of 1×10⁻⁶. 8 cfu / mL.

[0087] Treatment group: Liquid microbial inoculant was prepared at a ratio of 10... 3 The initial concentration of cfu / mL was mixed and applied to the nutrient solution tank of the hydroponic facility. The inoculant was added once on the first day of the pothos cultivation, and then once every other day for a total of 3 times.

[0088] Control group: Deionized water was applied in the same volume and frequency as the treatment group to serve as the blank control group.

[0089] Green algae inhibition detection

[0090] On the 8th day of cultivation, the culture medium near the roots of the pothos was collected to count the growth of green algae and calculate the inhibition efficiency of green algae. The above-ground fresh weight and underground fresh weight of the pothos were also measured to calculate the growth rate.

[0091] Inhibition efficiency (%) = (Green algae count in control group on day 8 - Green algae count in experimental group on day 8) / Green algae count in control group on day 8) × 100%

[0092] Growth rate (%) = (Measurement data on day 8 - Measurement data on day 1) / Measurement data on day 1 × 100%

[0093] Compared to the control group that was not treated with liquid microbial agents, the growth of green algae in the root culture medium of the pothos in the treatment group was significantly inhibited, and the growth rates of both the above-ground and underground fresh weight of the pothos in the treatment group were higher than those in the control group.

[0094] Chlorophyll content test of pothos leaves

[0095] On day 14 of cultivation, SPAD levels in the leaves of the treatment and control groups were measured. The results are as follows: Figure 3 As shown.

[0096] Example 3

[0097] This embodiment uses the liquid microbial inoculant prepared in Example 1, which contains 1×10 8 Bacillus subtilis BJIB-BF-2024-105 (CFU / mL, accession number CGMCC No. 31786)

[0098] Four groups of pothos plants with the same growth were transplanted into hydroponic trays, namely control group 1, control group 2, experimental group 1, and experimental group 2.

[0099] Control Group 1 (control addition): Sterile water was added directly to the plant liquid culture medium in the same volume as the liquid microbial agent added in Experiment Group 1. The treatment was carried out once on the first day after transplanting, and then once every other day for a total of three treatments. On the 8th day of cultivation, the culture medium near the roots was taken to count the growth of green algae and measure the above-ground and underground fresh weight of the pothos.

[0100] Control group 2 (root soaking control): The roots of each plant were soaked in 50 mL of sterile water. The treatment was carried out once on the first day after transplanting, and then once every other day for a total of three times. On the 8th day of cultivation, the culture medium near the roots was taken to count the growth of green algae and measure the above-ground and underground fresh weight of the pothos.

[0101] Experimental Group 1 (with added microbial agent): at 10 3The initial concentration of liquid microbial agent was added to the nutrient solution at a concentration of cfu / mL. The microbial agent was added directly on the first day after transplanting, and then added once every other day for a total of three times. On the 8th day of cultivation, the culture solution near the roots was taken to count the growth of green algae and to measure the above-ground and underground fresh weight of the pothos.

[0102] Experimental Group 2 (root soaking with microbial agent): The roots of each plant were soaked in 50 mL of liquid microbial agent. The root soaking treatment was performed once on the first day after transplanting, and then once every other day for a total of 3 times. On the 8th day of cultivation, the culture solution near the roots was taken to count the growth of green algae and measure the above-ground fresh weight and underground fresh weight of the pothos.

[0103] Inhibition efficiency (%) = (Green algae count in control group on day 8 - Green algae count in experimental group on day 8) / Green algae count in control group on day 8 × 100%

[0104] according to Figure 4 As can be seen, compared with the control group that was not treated with liquid microbial agent, the growth of green algae in the root culture medium of the pothos treated with liquid microbial agent was significantly inhibited. The inhibition efficiency of green algae in experimental group 1 (with added microbial agent) was as high as 77%, and the inhibition efficiency of green algae in experimental group 2 (with root soaking in microbial agent) was close to 20%.

[0105] Calculation of the growth rate of above-ground and below-ground fresh weight of pothos:

[0106] Growth rate (%) = (Measurement data on day 8 - Measurement data on day 1) / Measurement data on day 1 × 100%

[0107] In addition, the growth rates of above-ground and below-ground fresh weight in Experiment 1 and Experiment 2 were significantly higher than those in Control Group 1 and Control Group 2.

[0108] In summary, the microbial agent containing Bacillus subtilis provided in this application has a simple composition, and its preparation process and application method are easy to operate. It improves the utilization of nutrients and water by plants and inhibits the reproduction of green algae (the inhibition efficiency can reach 77%), reduces the difficulty of hydroponics, and promotes the large-scale promotion and application of hydroponics. The agent has a clear composition, is non-toxic and harmless, and can effectively reduce the amount of chemical disinfectants used in hydroponic production, simplify the difficulty of cleaning hydroponic facilities, reduce site and labor costs, and improve economic and environmental benefits.

[0109] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0110] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A Bacillus subtilis strain, characterized in that, Nucleotide sequence containing 16S rDNA as shown in SEQ ID:

1.

2. The Bacillus subtilis according to claim 1, characterized in that, The Bacillus subtilis is BJIB-BF-2024-105 with the preservation number CGMCC No. 31786.

3. A microbial inoculant, characterized in that, Contains Bacillus subtilis as described in claim 1 or 2, wherein the content of Bacillus subtilis is greater than or equal to 1 × 10⁻⁶ based on the total volume of the microbial agent. 8 cfu / mL.

4. The microbial agent according to claim 3, characterized in that, Based on the total volume of the microbial agent, the content of Bacillus subtilis is 5 × 10⁻⁶. 8 cfu / mL.

5. The microbial agent according to claim 3 or 4, characterized in that, The solvent for the microbial agent is water.

6. The microbial inoculant according to any one of claims 3 to 5, characterized in that, It also contains agronomically acceptable additives and / or nutrients that promote the growth of hydroponic plants.

7. The application of Bacillus subtilis as described in claim 1 or 2, or the microbial agent as described in any one of claims 3 to 6, in hydroponic plants.

8. The application according to claim 7, characterized in that, The applications include increasing chlorophyll content in hydroponic plants and / or inhibiting algal growth.

9. The application according to claim 7 or 8, characterized in that, The hydroponic plant is a pothos.

10. The application according to any one of claims 7 to 9, characterized in that, The algae mentioned are green algae.

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