Bacillus velezensis with relatively strong stress resistance, bacterial powder, biocontrol inoculant and preparation and application of bacillus velezensis

Abstract

The invention belongs to the field of microorganisms, and particularly relates to bacillus velezensis with high stress resistance, bacterial powder, a biocontrol microbial inoculum and preparation and application thereof. The bacillus velezensis is bacillus velezensis Shannan.BV80-12 and is preserved in the China Center for Type Culture Collection, and the preservation number of the bacillus velezensis is CCTCC (China Center for Type Culture Collection) NO: M 2024386. The bacillus velezensis Shannan.BV80-12 provided by the invention has relatively strong tolerance to drought, and has the capability of producing indoleacetic acid (IAA). Meanwhile, the bacterial strain also has a broad-spectrum antibacterial ability, and has a relatively strong inhibition effect on fusarium graminearum, alternaria alternata, magnaporthe oryzae, bipolaris maydis, exserohilum turcicum, sclerotinia sclerotiorum, physalospora piricola, alternaria solani and citrus brown rot pathogen.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a Velezius bacillus with strong stress resistance, bacterial powder, a biocontrol agent and preparation and application thereof. Background Art

[0002] In the field of agricultural planting, although chemical pesticides and fertilizers can quickly take effect and improve efficiency, their long-term use poses a serious threat to the ecological environment and food safety. In contrast, microbial agents are highly regarded for their high efficacy, long-lasting effect, low risk of drug resistance and environmental friendliness. When selecting strains for biological control, those microorganisms that adapt to special extreme environments show unique advantages. Under long-term adverse pressure, these microorganisms can evolve a variety of survival mechanisms, such as producing plant growth hormones: indoleacetic acid (IAA), antioxidant defense substances, and inhibiting plant pathogens, thereby promoting the absorption of nutrients by plants, reducing the impact of environmental pressure, and enhancing the ability of plants to resist adversity.

[0003] The Chinese patent application with publication number CN118726189A discloses Bacillus Velezii A13 and its application in promoting tobacco growth and preventing leaf spot disease. The bacterial agent prepared by the Bacillus Velezii A13 can inhibit the growth of tobacco brown spot pathogens and has a preventive and therapeutic effect on tobacco brown spot disease in tobacco detached leaf inoculation. However, the Bacillus Velezii A13 bacterial agent provided by the patent has a low inhibition rate on tobacco brown spot pathogens and has a poor preventive and therapeutic effect on tobacco brown spot disease in tobacco detached leaf inoculation.

[0004] The Chinese patent application with publication number CN118726189A discloses Bacillus velez and its application. The Bacillus velez provided in the patent can effectively inhibit the growth of plant pathogens, and its fermentation liquid has preventive effects on rape sclerotinia fungi, wheat fusarium fungi, tomato gray mold fungi, pepper phytophthora, and rice seedling fungi. However, the broad-spectrum antibacterial rate of the Bacillus velez provided in the patent is poor. Summary of the invention

[0005] The problem existing in the prior art is that the prior art lacks Bacillus velezine which can effectively inhibit the growth of multiple pathogenic fungi and has a broad-spectrum antibacterial ability.

[0006] In view of the above problems existing in the prior art, the present invention provides a Bacillus Velezii with strong stress resistance, bacterial powder, biocontrol agent and preparation and application thereof.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a Bacillus velezensis, wherein the Bacillus velezensis is Bacillus velezensis Shannan.BV80-12, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO:M 2024386.

[0009] Preferably, the 16srRNA gene sequence of Bacillus Velezii is shown as SEQ ID No.13.

[0010] Preferably, the Bacillus Velezii carries a gene for maintaining bacterial genetic diversity, wherein the gene for maintaining bacterial genetic diversity comprises the sequence shown in SEQ ID NO: 1, and / or comprises the sequence shown in SEQ ID NO: 2, and / or comprises the sequence shown in SEQ ID NO: 4, and / or comprises the sequence shown in SEQ ID NO: 5.

[0011] Preferably, the Bacillus Velez subtilis carries a gene for environmental adaptability, wherein the gene for environmental adaptability comprises a sequence as shown in SEQ ID NO:3.

[0012] Preferably, the amino acid sequence encoded by the sequence shown in SEQ ID NO: 1 is the amino acid sequence shown in SEQ ID NO: 6,

[0013] and / or the amino acid sequence encoded by the sequence shown in SEQ ID NO:2 is the amino acid sequence shown in SEQ ID NO:7,

[0014] and / or the amino acid sequence encoded by the sequence shown in SEQ ID NO:4 is the amino acid sequence shown in SEQ ID NO:9,

[0015] And / or the amino acid sequence encoded by the sequence shown in SEQ ID NO:5 is the amino acid sequence shown in SEQ ID NO:10.

[0016] Preferably, the amino acid sequence encoded by the sequence shown in SEQ ID NO:3 is the amino acid sequence shown in SEQ ID NO:8.

[0017] Preferably, the Velez subtilis has drought tolerance, wherein the drought tolerance refers to: the viable cell count is 1-2×10 8 CFU / mL of the bacterial liquid of Bacillus Velez is inoculated in an LB liquid medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 40 g / L at an inoculum amount of 1-3 volume %, and the Bacillus Velez is inoculated at an OD 600nm The value is greater than or equal to 0.48 and less than 2.40.

[0018] Preferably, the Velez subtilis has the ability to produce indoleacetic acid, wherein the ability to produce indoleacetic acid refers to: the viable cell count is 1-2×10 8 CFU / mL of Bacillus Velez is inoculated into a LB liquid medium with a pH of 9.0 at an inoculum rate of 1-3% by volume, wherein the amount of indoleacetic acid per L of the LB liquid medium is 25-30 mg, preferably 27-28 mg;

[0019] And / or the ability to produce indoleacetic acid refers to: the number of viable bacteria is 1-2×10 8 The bacterial liquid of Bacillus velez with a CFU / mL is inoculated into an LB+ liquid culture medium with a pH of 9.0 at an inoculation rate of 1-3 volume %, and the amount of indoleacetic acid in each L of LB+ liquid culture medium is 50-55 mg, preferably 50-51 mg, wherein the LB+ liquid culture medium refers to an LB liquid culture medium containing 0.50 g of tryptophan per L.

[0020] Preferably, the Velez subtilis has the ability to inhibit pathogens, wherein the ability to inhibit pathogens refers to: the number of viable bacteria is 1-2×10 8 The bacterial solution of Bacillus Velezii with a CFU / mL has an inhibition rate of more than 90% against pathogenic bacteria.

[0021] Preferably, the pathogen is a pathogenic fungus;

[0022] More preferably, the pathogenic fungi include one or more substances selected from the group consisting of wheat fusarium head blight, tobacco brown spot pathogen, rice blast pathogen, corn leaf spot pathogen, corn leaf spot pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen and citrus brown rot pathogen;

[0023] Most preferably, the pathogenic fungi are wheat fusarium rust, tobacco brown spot pathogen, rice blast pathogen, corn leaf blight pathogen, corn leaf blight pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen and citrus brown rot pathogen.

[0024] Preferably, the inhibition rate of the Bacillus Velez against wheat fusarium is greater than or equal to 99%, and / or the inhibition rate of tobacco brown spot pathogen is greater than or equal to 97%, and / or the inhibition rate of rice blast pathogen is greater than or equal to 95%, and / or the inhibition rate of corn leaf blight pathogen is greater than or equal to 92%, and / or the inhibition rate of corn leaf blight pathogen is greater than or equal to 91%, and / or the inhibition rate of rapeseed sclerotinia pathogen is greater than or equal to 89%, and / or the inhibition rate of apple ring rot pathogen is greater than or equal to 90%, and / or the inhibition rate of tomato early blight pathogen is greater than or equal to 88%, and / or the inhibition rate of citrus brown rot pathogen is greater than or equal to 88%.

[0025] In a second aspect, the present invention provides the use of the Bacillus Velezii in the preparation of bacterial powder or biocontrol agent.

[0026] In a third aspect, the present invention provides a bacterial powder containing the Bacillus Velezii.

[0027] Preferably, the viable bacteria count of the bacterial powder is 1.1-6.4×10 10 CFU / g.

[0028] Preferably, the bacterial powder is prepared by a method comprising the following steps: mixing diatomaceous earth and fermentation broth of Bacillus velez in a ratio of 15-25 g:1 L, drying and crushing to obtain bacterial powder.

[0029] In a fourth aspect, the present invention provides a method for preparing the bacterial powder, comprising the following steps: mixing diatomaceous earth and fermentation broth of Bacillus velez in a ratio of 15-25 g:1 L, drying and crushing to obtain bacterial powder.

[0030] Preferably, the method comprises the following steps:

[0031] (1) inoculating the activated Bacillus Velez subtilis into a liquid culture medium for fermentation to obtain a fermentation liquid of the Bacillus Velez subtilis,

[0032] (2) Mix diatomaceous earth and fermentation liquid of Bacillus velez in a ratio of 15-25 g:1 L, dry and grind to obtain bacterial powder.

[0033] Preferably, the fermentation temperature is 37° C.-42° C., and / or the initial pH value of the fermentation is 6.0-7.0, and / or the fermentation speed is 200-250 r / min, and / or the fermentation time is 20-36 h.

[0034] Preferably, the feed rate during drying is 600-610 mL / h, and / or the air volume during spray drying is 80-85 m 3 / h, and / or the outlet temperature of spray drying is 52-55°C.

[0035] In a fifth aspect, the present invention provides a biocontrol agent containing the bacterial powder.

[0036] Preferably, the number of viable bacteria of Bacillus Velezii contained in the biocontrol agent is greater than or equal to 1.1×10 10 CFU / g, preferably, the number of viable bacteria is 1.1-6.4×10 10 CFU / g.

[0037] Preferably, in parts by weight, the biocontrol agent comprises 70-90 parts of the bacterial powder, 10-20 parts of sodium dodecylbenzene sulfonate, 2-10 parts of dipotassium hydrogen phosphate and 0.1-1 part of ascorbic acid.

[0038] More preferably, the bacterial powder is 77.7-87.9 parts, and / or sodium dodecylbenzene sulfonate is 10-15 parts, and / or dipotassium hydrogen phosphate is 2-7 parts, and / or ascorbic acid is 0.1-0.5 parts.

[0039] Preferably, the biocontrol agent is prepared by a method comprising the following steps: mixing the bacterial powder, sodium dodecylbenzene sulfonate, dipotassium hydrogen phosphate and ascorbic acid to obtain the biocontrol agent.

[0040] Preferably, the mixed solution containing the biocontrol agent at a concentration of 0.01-0.05 g / mL has the effect of promoting plant growth under drought stress. Preferably, the plant is a corn seedling. More preferably, the mixed solution is a mixture of water and the biocontrol agent.

[0041] Preferably, the mixed solution containing the biocontrol agent at a concentration of 1-5 g / L has the function of preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by pathogens, more preferably, the plant diseases caused by pathogens include wheat fusarium rust and / or tobacco brown spot disease, more preferably, the mixed solution is a mixture of water and the biocontrol agent.

[0042] In a sixth aspect, the present invention provides the use of the Bacillus Velezii, or the bacterial powder, or the bacterial powder prepared by the preparation method, or the biological bacterial agent in the preparation of fertilizers or pesticides.

[0043] Preferably, the fertilizer is a fertilizer that helps promote plant growth under drought stress.

[0044] Preferably, the pesticide is a pesticide for preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by pathogenic bacteria. Preferably, the plant diseases caused by pathogenic bacteria include wheat fusarium head blight and / or tobacco brown spot disease.

[0045] In a seventh aspect, the present invention provides a fertilizer containing the Bacillus Velezii, or the bacterial powder or the bacterial powder prepared by the preparation method, or the biological agent. Preferably, the fertilizer is a fertilizer that helps promote plant growth under drought stress.

[0046] In an eighth aspect, the present invention provides a pesticide containing the Bacillus Velezii, or the bacterial powder or the bacterial powder prepared by the preparation method, or the biological microbial agent. Preferably, the pesticide is a pesticide for preventing and treating plant diseases. Preferably, the plant diseases are plant diseases caused by pathogenic bacteria. More preferably, the plant diseases caused by pathogenic bacteria include wheat head blight and / or tobacco brown spot disease. Beneficial effects of the present invention:

[0047] (1) The Bacillus velezensis Shannan.BV80-12 provided by the present invention has a strong tolerance to drought.

[0048] (2) The Bacillus velezensis Shannan.BV80-12 provided by the present invention has the ability to produce indoleacetic acid (IAA).

[0049] (4) The Bacillus Velez Shannan.BV80-12 provided by the present invention has a broad-spectrum antibacterial ability, specifically the ability to broadly inhibit pathogenic fungi. The pathogenic fungi are specifically wheat fusarium rust, tobacco brown spot pathogen, rice blast pathogen, corn leaf blight pathogen, corn leaf blight pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen and citrus brown rot pathogen. Bacillus Velez Shannan.BV80-12 has a strong inhibitory effect on these 9 pathogenic fungi, and the inhibition rate is all above 90%.

[0050] (5) The Bacillus Velezii Shannan.BV80-12 biocontrol agent provided by the present invention can promote the growth of corn seedlings under drought.

[0051] (6) The Bacillus Velezii Shannan.BV80-12 biocontrol agent provided by the present invention can effectively prevent and control wheat fusarium head blight and tobacco brown spot disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the colony morphology of Bacillus Velezii Shannan.BV80-12.

[0053] Figure 2 This is a microscopic observation picture of Bacillus Velez Shannan.BV80-12.

[0054] Strain deposit information

[0055] The Bacillus velezensis Shannan.BV80-12 provided by the present invention was deposited in the China Center for Type Culture Collection on March 4, 2024, with a deposit number of CCTCCNO: M 2024386, a deposit address of Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. DETAILED DESCRIPTION

[0056] In order to better understand the above technical solution, the technical solution of the present invention is clearly and completely explained in conjunction with the specific implementation method below. It should be noted that the content in the specific implementation method is only a specific implementation and explanation of the technical solution of the present invention, and should not be understood as limiting the scope of protection of the present invention.

[0057] The present invention provides a strain of Bacillus velez Shannan.BV80-12, which contains 5 unique genes related to maintaining bacterial genetic diversity and adaptability, specifically 2 transposase genes, 1 organic hydroperoxide resistance protein gene, 1 chromosome partitioning protein gene and 1 DNA polymerase III subunit δ gene, wherein:

[0058] The transposase gene is part of the transposable element. Transposable elements are DNA sequences in the bacterial genome that can move by themselves. These sequences can move within or between bacterial genomes, causing changes in gene loci. The product of the transposase gene: transposase, can recognize the boundaries of these mobile elements and catalyze their movement from one location to another. The movement of transposable elements plays an important role in the evolution of bacterial genomes, which may cause gene mutations, deletions, and inversions, thereby helping bacteria resist complex environmental changes.

[0059] The organic hydroperoxide resistance protein gene belongs to the superfamily of antioxidant enzymes and plays an important role in the peroxidation reaction of bacteria when resisting stress.

[0060] Chromosome distribution protein gene and DNA polymerase III subunit δ gene can ensure the stable transmission of genetic material, maintain the genetic diversity and adaptability of bacteria, and can also affect the physiological functions of bacteria, such as cell cycle and spore formation, thus playing an important role in the survival and evolution of bacteria in adversity.

[0061] In some specific embodiments, the present invention provides a Bacillus velezensis, wherein the Bacillus velezensis is Bacillus velezensis Shannan.BV80-12, with a deposit number of CCTCC NO:M2024386, wherein the Bacillus velezensis has drought tolerance,

[0062] The drought tolerance means that in an LB liquid culture medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 40 g / L, the OD600nm value of the Bacillus Velez is greater than or equal to 0.48 and less than 2.40.

[0063] The drought tolerance refers to: the number of viable bacteria is 1-2×10 8 CFU / mL of the bacterial liquid of Bacillus Velez is inoculated in an LB liquid medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 40 g / L at an inoculum amount of 1-3 volume %, and the Bacillus Velez is inoculated at an OD 600nm The value of is greater than or equal to 0.46 and less than 2.40.

[0064] Preferably, the drought tolerance refers to: the number of viable bacteria is 1-2×10 8 CFU / mL of the bacterial liquid of Bacillus Velez is inoculated in an LB liquid medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 30 g / L at an inoculum amount of 1-3 volume %, and the Bacillus Velez is inoculated at an OD 600nm The value is greater than or equal to 1.07 and less than 2.40.

[0065] Preferably, the drought tolerance refers to: the number of viable bacteria is 1-2×10 8 CFU / mL of the bacterial liquid of Bacillus Velez is inoculated in an LB liquid medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 20 g / L at an inoculum amount of 1-3 volume %, and the Bacillus Velez is inoculated at an OD 600nm The value of is greater than or equal to 1.53 and less than 2.40.

[0066] Preferably, the drought tolerance refers to: the number of viable bacteria is 1-2×10 8 CFU / mL of the bacterial liquid of Bacillus Velez is inoculated in an LB liquid medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 10 g / L at an inoculum amount of 1-3 volume %, and the Bacillus Velez is inoculated at an OD 600nm The value is greater than or equal to 1.80 and less than 2.40.

[0067] Preferably, the drought tolerance refers to: the number of viable bacteria is 1-2×10 8 CFU / mL of the bacterial liquid of Bacillus Velez is inoculated in an LB liquid medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 5 g / L at an inoculum amount of 1-3 volume %, and the Bacillus Velez is inoculated at an OD 600nm The value is greater than or equal to 2.00 and less than 2.40.

[0068] Preferably, the drought tolerance means that in an LB liquid culture medium with a PEG-6000 concentration of greater than or equal to 5 g / L and less than or equal to 10 g / L, the OD600nm value of the Bacillus Velez is greater than or equal to 1.80 and less than or equal to 2.02.

[0069] Preferably, the drought tolerance means that in an LB liquid culture medium with a PEG-6000 concentration of greater than or equal to 10 g / L and less than or equal to 20 g / L, the OD600nm value of the Bacillus Velez is greater than or equal to 1.53 and less than or equal to 1.86.

[0070] Preferably, the drought tolerance means that in an LB liquid culture medium with a PEG-6000 concentration of greater than or equal to 20 g / L and less than or equal to 30 g / L, the OD600nm value of the Bacillus Velez is greater than or equal to 1.07 and less than or equal to 1.57.

[0071] Preferably, the drought tolerance means that in an LB liquid culture medium with a PEG-6000 concentration of greater than or equal to 30 g / L and less than or equal to 40 g / L, the OD600nm value of the Bacillus Velez is greater than or equal to 0.47 and less than or equal to 1.09.

[0072] In some specific embodiments, the Velez subtilis has the ability to inhibit pathogens, wherein the ability to inhibit pathogens refers to: the number of viable bacteria is 1-2×10 8 The bacterial liquid of Bacillus Velez with a CFU / mL has an inhibition rate of more than 90% for pathogens, wherein the pathogens are pathogenic fungi, and the pathogens are wheat fusarium, tobacco brown spot pathogen, rice blast pathogen, corn leaf blight pathogen, corn leaf blight pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen and citrus brown rot pathogen.

[0073] Preferably, in some specific embodiments, the inhibition rate of the Bacillus Velez against wheat fusarium is greater than or equal to 99%, and / or the inhibition rate of tobacco brown spot pathogen is greater than or equal to 97%, and / or the inhibition rate of rice blast pathogen is greater than or equal to 95%, and / or the inhibition rate of corn leaf blight pathogen is greater than or equal to 92%, and / or the inhibition rate of corn leaf blight pathogen is greater than or equal to 91%, and / or the inhibition rate of rapeseed sclerotinia pathogen is greater than or equal to 89%, and / or the inhibition rate of apple ring rot pathogen is greater than or equal to 90%, and / or the inhibition rate of tomato early blight pathogen is greater than or equal to 88%, and / or the inhibition rate of citrus brown rot pathogen is greater than or equal to 88%.

[0074] More preferably, in some specific embodiments, the inhibition rate of the Velez Bacillus against wheat fusarium is 99.7-99.9%, and / or the inhibition rate of tobacco brown spot pathogen is 97-99%, and / or the inhibition rate of rice blast pathogen is 95-97%, and / or the inhibition rate of corn leaf blight pathogen is 93-95%, and / or the inhibition rate of corn leaf blight pathogen is 92-94%, and / or the inhibition rate of rapeseed sclerotinia pathogen is 90-92%, and / or the inhibition rate of apple ring rot pathogen is 90-92%, and / or the inhibition rate of tomato early blight pathogen is 90-92%, and / or the inhibition rate of citrus brown rot pathogen is 89-91%.

[0075] In some specific embodiments, the present invention provides a biocontrol agent containing 1.1-6.4×10 10CFU / g. Preferably, in some specific embodiments, the viable count of Bacillus velezensis in the biocontrol agent can be 1.1×10 10 CFU / g, 1.2×10 10 CFU / g, 1.3×10 10 CFU / g, 1.4×10 10 CFU / g, 1.5×10 10 CFU / g, 1.6×10 10 CFU / g, 1.7×10 10 CFU / g, 1.8×10 10 CFU / g, 1.9×10 10 CFU / g, 2.0×10 10 CFU / g, 2.1×10 10 CFU / g, 2.2×10 10 CFU / g, 2.3×10 10 CFU / g, 2.4×10 10 CFU / g, 2.5×10 10 CFU / g, 2.6×10 10 CFU / g, 2.7×10 10 CFU / g, 2.8×10 10 CFU / g, 2.9×10 10 CFU / g, 3.0×10 10 CFU / g, 3.1×10 10 CFU / g, 3.2×10 10 CFU / g, 3.3×10 10 3CFU / g, 3.4×10 10 CFU / g, 3.5×10 10 CFU / g, 3.6×10 10 CFU / g, 3.7×10 10 CFU / g, 3.8×10 10 CFU / g, 3.9×10 10 CFU / g, 4.0×10 10 CFU / g, 4.1×10 10 CFU / g, 4.2×10 10 CFU / g, 4.3×10 10 CFU / g, 4.4×10 10 CFU / g, 4.5×10 10 CFU / g, 4.6×10 10 CFU / g, 4.7×10 10 CFU / g, 4.8×10 10 CFU / g, 4.9×1010 CFU / g, 5.0×10 10 CFU / g, 5.1×10 10 CFU / g, 5.2×10 10 CFU / g, 5.3×10 10 CFU / g, 5.4×10 10 CFU / g, 5.5×10 10 CFU / g, 5.6×10 10 CFU / g, 5.7×10 10 CFU / g, 5.8×10 10 CFU / g, 5.9×10 10 CFU / g, 6.0×10 10 CFU / g, 6.1×10 10 CFU / g, 6.2×10 10 CFU / g, 6.3×10 10 CFU / g or 6.4×10 10 CFU / g, or the viable bacteria count of Bacillus Velezii in the biocontrol agent within a numerical range consisting of any two of the above specific values ​​as endpoints.

[0076] In some specific embodiments, the present invention provides a biocontrol agent, which comprises, by weight, 70-90 parts of Bacillus Velezii powder, 10-20 parts of sodium dodecylbenzene sulfonate, 2-10 parts of dipotassium hydrogen phosphate and 0.1-1 parts of ascorbic acid.

[0077] Preferably, in some specific embodiments, the content of the powder of Bacillus velez may be 70.0 parts by weight, 70.1 parts by weight, 70.2 parts by weight, 70.3 parts by weight, 70.4 parts by weight, 70.5 parts by weight, 70.6 parts by weight, 70.7 parts by weight, 70.8 parts by weight, 70.9 parts by weight, 71.0 parts by weight, 71.1 parts by weight, 71.2 parts by weight, 71.3 parts by weight, 71.4 parts by weight, 71.5 parts by weight, 71.6 parts by weight, 71.7 parts by weight, 71.8 parts by weight, 71.9 parts by weight, 72.0 parts by weight, 72.1 parts by weight, 72.2 parts by weight, 72.3 parts by weight, 72.4 parts by weight, 72.5 parts by weight, 72.6 parts by weight, 72.7 parts by weight. parts by weight, 72.8 parts by weight, 72.9 parts by weight, 73.0 parts by weight, 73.1 parts by weight, 73.2 parts by weight, 73.3 parts by weight, 73.4 parts by weight, 73.5 parts by weight, 73.6 parts by weight, 73.7 parts by weight, 73.8 parts by weight, 73.9 parts by weight, 74.0 parts by weight, 74.1 parts by weight, 74.2 parts by weight, 74.3 parts by weight, 74.4 parts by weight, 74.5 parts by weight, 74.6 parts by weight, 74.7 parts by weight, 74.8 parts by weight, 74.9 parts by weight, 75.0 parts by weight, 75.1 parts by weight, 75.2 parts by weight, 75.3 parts by weight, 75.4 parts by weight, 75.5 parts by weight, 75.6 parts by weight, 75.7 parts by weight, 75.8 parts by weight parts by weight, 75.9 parts by weight, 76.0 parts by weight, 76.1 parts by weight, 76.2 parts by weight, 76.3 parts by weight, 76.4 parts by weight, 76.5 parts by weight, 76.6 parts by weight, 76.7 parts by weight, 76.8 parts by weight, 76.9 parts by weight, 77.0 parts by weight, 77.1 parts by weight, 77.2 parts by weight, 77.3 parts by weight, 77.4 parts by weight, 77.5 parts by weight, 77.6 parts by weight, 77.7 parts by weight, 77.8 parts by weight, 77.9 parts by weight, 78.0 parts by weight, 78.1 parts by weight, 78.2 parts by weight, 78.3 parts by weight, 78.4 parts by weight, 78.5 parts by weight, 78.6 parts by weight, 78.7 parts by weight, 78.8 parts by weight, 78.9 parts by weight, 79.0 parts by weight, 79.1 parts by weight, 79.2 parts by weight, 79.3 parts by weight, 79.4 parts by weight, 79.5 parts by weight, 79.6 parts by weight, 79.7 parts by weight, 79.8 parts by weight, 79.9 parts by weight, 80.0 parts by weight, 80.1 parts by weight, 80.2 parts by weight, 80.3 parts by weight, 80.4 parts by weight, 80.5 parts by weight, 80.6 parts by weight, 80.7 parts by weight, 80.8 parts by weight, 80.9 parts by weight, 81.0 parts by weight, 81.1 parts by weight, 81.2 parts by weight, 81.3 parts by weight, 81.4 parts by weight, 81.5 parts by weight, 81.6 parts by weight, 81.7 parts by weight, 81.8 parts by weight, 81.9 parts by weight, 82.0 parts by weight, 82.1 part by weight, 82.2 parts by weight, 82.3 parts by weight, 82.4 parts by weight, 82.5 parts by weight, 82.6 parts by weight, 82.7 parts by weight, 82.8 parts by weight, 82.9 parts by weight, 83.0 parts by weight, 83.1 parts by weight, 83.2 parts by weight, 83.3 parts by weight, 83.4 parts by weight, 83.5 parts by weight, 83.6 parts by weight, 83.7 parts by weight, 83.8 parts by weight, 83.9 parts by weight, 84.0 parts by weight, 84.1 parts by weight, 84.2 parts by weight, 84.3 parts by weight, 84.4 parts by weight, 84.5 parts by weight, 84.6 parts by weight, 84.7 parts by weight, 84.8 parts by weight, 84.9 parts by weight, 85.0 parts by weight, 85.1 parts by weight, 85.2 parts by weight, 85.3 parts by weight, 85.4 parts by weight, 85.5 parts by weight, 85.6 parts by weight, 85.7 parts by weight, 85.8 parts by weight, 85.9 parts by weight, 86.0 parts by weight, 86.1 parts by weight, 86.2 parts by weight, 86.3 parts by weight parts by weight, 86.4 parts by weight, 86.5 parts by weight, 86.6 parts by weight, 86.7 parts by weight, 86.8 parts by weight, 86.9 parts by weight, 87.0 parts by weight, 87.1 parts by weight, 87.2 parts by weight, 87.3 parts by weight, 87.4 parts by weight, 87.5 parts by weight, 87.6 parts by weight, 87.7 parts by weight, 87.8 parts by weight, 87.9 parts by weight, 88.0 parts by weight, 88.1 parts by weight, 88.2 parts by weight, 88.3 parts by weight, 88.4 parts by weight , 88.5 parts by weight, 88.6 parts by weight, 88.7 parts by weight, 88.8 parts by weight, 88.9 parts by weight, 89.0 parts by weight, 89.1 parts by weight, 89.2 parts by weight, 89.3 parts by weight, 89.4 parts by weight, 89.5 parts by weight, 89.6 parts by weight, 89.7 parts by weight, 89.8 parts by weight, 89.9 parts by weight, 90.0 parts by weight, or the content of the powder of Bacillus velezensis within a numerical range consisting of any two of the above specific numerical values ​​as endpoints.

[0078] Preferably, in some specific embodiments, the content of sodium dodecylbenzene sulfonate can be 10.0 parts by weight, 10.1 parts by weight, 10.2 parts by weight, 10.3 parts by weight, 10.4 parts by weight, 10.5 parts by weight, 10.6 parts by weight, 10.7 parts by weight, 10.8 parts by weight, 10.9 parts by weight, 11.0 parts by weight, 11.1 parts by weight, 11.2 parts by weight, 11.3 parts by weight, 11.4 parts by weight, 11.5 parts by weight, 11.6 parts by weight, 11.7 parts by weight, 11.8 parts by weight, 11.9 parts by weight, 12.0 parts by weight, 12.1 parts by weight, 12.2 parts by weight, 12.3 parts by weight. parts by weight, 12.4 parts by weight, 12.5 parts by weight, 12.6 parts by weight, 12.7 parts by weight, 12.8 parts by weight, 12.9 parts by weight, 13.0 parts by weight, 13.1 parts by weight, 13.2 parts by weight, 13.3 parts by weight, 13.4 parts by weight, 13.5 parts by weight, 13.6 parts by weight, 13.7 parts by weight, 13.8 parts by weight, 13.9 parts by weight, 14.0 parts by weight, 14.1 parts by weight, 14.2 parts by weight, 14.3 parts by weight, 14.4 parts by weight, 14.5 parts by weight, 14.6 parts by weight, 14.7 parts by weight, 14.8 parts by weight, 14.9 parts by weight, 15.0 parts by weight, 15.1 parts by weight, 15.2 parts by weight, 15.3 parts by weight, 15.4 parts by weight, 15.5 parts by weight, 15.6 parts by weight, 15.7 parts by weight, 15.8 parts by weight, 15.9 parts by weight, 16.0 parts by weight, 16.1 parts by weight, 16.2 parts by weight, 16.3 parts by weight, 16.4 parts by weight, 16.5 parts by weight, 16.6 parts by weight, 16.7 parts by weight, 16.8 parts by weight, 16.9 parts by weight, 17.0 parts by weight, 17.1 parts by weight, 17.2 parts by weight, 17.3 parts by weight, 17.4 parts by weight, 17.5 parts by weight, 17.6 parts by weight, 17.7 parts by weight, 17.8 parts by weight, 17.9 parts by weight .8 parts by weight, 17.9 parts by weight, 18.0 parts by weight, 18.1 parts by weight, 18.2 parts by weight, 18.3 parts by weight, 18.4 parts by weight, 18.5 parts by weight, 18.6 parts by weight, 18.7 parts by weight, 18.8 parts by weight, 18.9 parts by weight, 19.0 parts by weight, 19.1 parts by weight, 19.2 parts by weight, 19.3 parts by weight, 19.4 parts by weight, 19.5 parts by weight, 19.6 parts by weight, 19.7 parts by weight, 19.8 parts by weight, 19.9 parts by weight, 20.0 parts by weight, or the content of sodium dodecylbenzene sulfonate within the numerical range consisting of any two of the above specific numerical values ​​as endpoints.

[0079] Preferably, in some specific embodiments, the content of dipotassium hydrogen phosphate can be 2.0 parts by weight, 2.1 parts by weight, 2.2 parts by weight, 2.3 parts by weight, 2.4 parts by weight, 2.5 parts by weight, 2.6 parts by weight, 2.7 parts by weight, 2.8 parts by weight, 2.9 parts by weight, 3.0 parts by weight, 3.1 parts by weight, 3.2 parts by weight, 3.3 parts by weight, 3.4 parts by weight, 3.5 parts by weight, 3.6 parts by weight, 3.7 parts by weight, 3.8 parts by weight. , 3.9 parts by weight, 4.0 parts by weight, 4.1 parts by weight, 4.2 parts by weight, 4.3 parts by weight, 4.4 parts by weight, 4.5 parts by weight, 4.6 parts by weight, 4.7 parts by weight, 4.8 parts by weight, 4.9 parts by weight, 5.0 parts by weight, 5.1 parts by weight, 5.2 parts by weight, 5.3 parts by weight, 5.4 parts by weight, 5.5 parts by weight, 5.6 parts by weight, 5.7 parts by weight, 5.8 parts by weight, 5.9 parts by weight, 6.0 parts by weight, 6. .1 parts by weight, 6.2 parts by weight, 6.3 parts by weight, 6.4 parts by weight, 6.5 parts by weight, 6.6 parts by weight, 6.7 parts by weight, 6.8 parts by weight, 6.9 parts by weight, 7.0 parts by weight, 7.1 parts by weight, 7.2 parts by weight, 7.3 parts by weight, 7.4 parts by weight, 7.5 parts by weight, 7.6 parts by weight, 7.7 parts by weight, 7.8 parts by weight, 7.9 parts by weight, 8.0 parts by weight, 8.1 parts by weight, 8.2 parts by weight, 8.3 parts by weight The content of dipotassium hydrogen phosphate is 0.1 parts by weight, 0.2 parts by weight, 0.3 parts by weight, 0.4 parts by weight, 0.5 parts by weight, 0.6 parts by weight, 0.7 parts by weight, 0.8 parts by weight, 0.9 parts by weight, 0.9 parts by weight, 10.0 parts by weight, or within the numerical range consisting of any two of the above specific numerical values ​​as endpoints.

[0080] Preferably, in some specific embodiments, the content of ascorbic acid can be 0.1 parts by weight, 0.11 parts by weight, 0.12 parts by weight, 0.13 parts by weight, 0.14 parts by weight, 0.15 parts by weight, 0.16 parts by weight, 0.17 parts by weight, 0.18 parts by weight, 0.19 parts by weight, 0.2 parts by weight, 0.21 parts by weight, 0.22 parts by weight, 0.23 parts by weight, 0.24 parts by weight, 0.25 parts by weight, 0.26 parts by weight, 0.27 parts by weight, 0.28 parts by weight, 0.29 parts by weight, 0.3 parts by weight, 0.31 parts by weight, 0.32 parts by weight, 0.33 parts by weight, 0.34 parts by weight, 0.36 parts by weight, 0.37 parts by weight, 0.38 parts by weight, 0.39 parts by weight, 0.40 parts by weight, 0.41 parts by weight, 0.42 parts by weight, 0.43 parts by weight, 0.44 parts by weight, 0.45 parts by weight, 0.46 parts by weight, 0.47 parts by weight, 0.48 parts by weight, 0.49 parts by weight, 0.50 parts by weight, 0.51 parts by weight, 0.52 parts by weight, 0.53 parts by weight, 0.54 parts by weight, 0.55 parts by weight, 0.56 parts by weight, 0.57 parts by weight, 0.58 parts by weight, 0.59 parts by weight, 0.60 parts by weight, 0.61 parts by weight, 0.62 parts by weight, 0.63 parts by weight, 0.64 parts by weight, 0.65 parts by weight, 0. , 0.46 parts by weight, 0.47 parts by weight, 0.48 parts by weight, 0.49 parts by weight, 0.5 parts by weight, 0.51 parts by weight, 0.52 parts by weight, 0.53 parts by weight, 0.54 parts by weight, 0.55 parts by weight, 0.56 parts by weight, 0.57 parts by weight, 0.58 parts by weight, 0.59 parts by weight, 0.6 parts by weight, 0.61 parts by weight, 0.62 parts by weight, 0.63 parts by weight, 0.64 parts by weight, 0.65 parts by weight, 0.66 parts by weight, 0.67 parts by weight, 0.68 parts by weight, 0.69 parts by weight, 0.7 parts by weight, 0.71 parts by weight, 0.72 parts by weight, 0.73 parts by weight, 0.74 parts by weight, 0.75 parts by weight, 0.76 parts by weight, 0.77 parts by weight, 0.78 parts by weight, 0.79 parts by weight, 0.80 parts by weight The content of ascorbic acid in the range of 0.90 weight part, 0.96 weight part, 0.97 weight part, 0.98 weight part, 0.99 weight part, 1.0 weight part, or 1.0 weight part, or 0.81 weight part, 0.82 weight part, 0.83 weight part, 0.84 weight part, 0.85 weight part, 0.86 weight part, 0.87 weight part, 0.88 weight part, 0.89 weight part, 0.9 weight part, 0.91 weight part, 0.92 weight part, 0.93 weight part, 0.94 weight part, 0.95 weight part, 0.96 weight part, 0.97 weight part, 0.98 weight part, 0.99 weight part, 1.0 weight part, or ...

[0081] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0082] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercially available products. The sources of the experimental reagents / instruments used in the present invention are shown in Table 1.

[0083] Table 1 Experimental reagents / instrument information

[0084]

[0085]

[0086] The preparation method of the culture medium used in the examples is as follows:

[0087] 1. LB liquid culture medium with a PEG-6000 concentration of 5 g / L: Mix 10 g of NaCl, 10 g of yeast extract powder, 10 g of peptone, 50 g of PEG-6000 and water and make up to 1 L, and sterilize at 121°C for 20 min to obtain an LB liquid culture medium with a PEG-6000 concentration of 5 g / L.

[0088] 2. LB liquid culture medium with a PEG-6000 concentration of 10 g / L: Mix 10 g of NaCl, 10 g of yeast extract powder, 10 g of peptone, 100 g of PEG-6000 and water and make up to 1 L, and sterilize at 121°C for 20 min to obtain an LB liquid culture medium with a PEG-6000 concentration of 10 g / L.

[0089] 3. LB liquid culture medium with a PEG-6000 concentration of 20 g / L: Mix 10 g of NaCl, 10 g of yeast extract powder, 10 g of peptone, 200 g of PEG-6000 and water and make up to 1 L, and sterilize at 121°C for 20 min to obtain an LB liquid culture medium with a PEG-6000 concentration of 20 g / L.

[0090] 4. LB liquid culture medium with a PEG-6000 concentration of 30 g / L: Mix 10 g of NaCl, 10 g of yeast extract powder, 10 g of peptone, 300 g of PEG-6000 and water and make up to 1 L, and sterilize at 121°C for 20 min to obtain an LB liquid culture medium with a PEG-6000 concentration of 30 g / L.

[0091] 5. LB liquid culture medium with a PEG-6000 concentration of 40 g / L: Mix 10 g of NaCl, 10 g of yeast extract powder, 10 g of peptone, 400 g of PEG-6000 and water and make up to 1 L, and sterilize at 121°C for 20 min to obtain an LB liquid culture medium with a PEG-6000 concentration of 40 g / L.

[0092] 6. LB liquid culture medium: Mix 10 g of NaCl, 10 g of yeast extract powder, 10 g of peptone and water and dilute to 1 L, then adjust the pH to 9.0 and sterilize at 121°C for 20 min to obtain an LB liquid culture medium with a pH of 9.0.

[0093] 7. LB+ liquid culture medium: Mix 10 g of NaCl, 10 g of yeast extract powder, 10 g of peptone, 0.5 g of tryptophan and water and make up to 1 L, then adjust the pH value to 9.0 and sterilize at 121°C for 20 min to obtain LB+ liquid culture medium with a pH of 9.0.

[0094] 8. PDA solid culture medium: Boil 200 g of peeled potatoes in boiling water for 20 min, collect the filtrate by filtering with gauze, mix the filtrate, 20 g of glucose, 15 g of agar and distilled water, and make up to 1 L with distilled water.

[0095] Example 1 Isolation, purification and identification of strains

[0096] (1) Isolation of strains: 25 g of the digestive tract contents of Tibetan pigs from Nadong District, Shannan City, Tibet, was mixed with 225 mL of sterile physiological saline to obtain a sample solution, which was then diluted in a gradient manner. 10 -3 , 10 -4 , 10 -5 , 10 -6 The diluted solution of the concentration was spread on an LB solid culture medium plate and cultured at 37°C for 48 hours. A single colony with a milky white, wrinkled, slightly convex and viscous morphology was picked for purification.

[0097] (2) Purification of strains: Streak the single colony obtained in step (1) on an LB solid culture medium plate, culture at 37°C for 48 hours, then pick a single colony and streak it on an LB solid culture medium plate, and purify for three generations until the morphology of the bacteria is consistent and there is no contamination by other bacteria, and then the purification is completed. Pick a single colony on the three-generation plate and culture it in LB liquid culture medium at 37°C for 24 hours to obtain a bacterial solution. After mixing the purified bacterial solution with 50% glycerol in equal proportions, store it at -80°C.

[0098] (2) Identification of strains: Streak the purified bacterial solution on an LB solid medium plate, incubate at 37°C for 48 hours, and observe the colony morphology of the strain. At the same time, pick a single colony and incubate it in LB liquid medium at 37°C for 24 hours. Then, aspirate 10 μL onto a glass slide and perform Gram staining. Magnify 100 times under an optical microscope to observe the microscopic morphology of the strain.

[0099] The colony morphology of this strain is: milky white, wrinkled, slightly convex, sticky in texture, forming cysts and capsule mucus, such as Figure 1 shown.

[0100] The microscopic morphology of the strain is: after Gram staining, the bacteria appear purple, and the bacteria are in the form of rods, that is, the bacteria are Gram-positive rods, such as Figure 2 shown.

[0101] The genome of the strain was extracted, and the 16S rDNA sequencing and strain identification were performed using a high-fidelity enzyme. The primer information, PCR amplification reaction system and conditions used are shown below.

[0102] Primer information

[0103]

[0104] PCR amplification reaction system and conditions

[0105]

[0106]

[0107] After the PCR product was tested to be qualified, it was sent to a sequencing company for sequencing, and the 16srRNA gene sequence was obtained as shown in SEQ ID NO:13.

[0108]

[0109]

[0110] Combined with morphological analysis and molecular identification, the bacteria was confirmed to be Bacillus Velez subtilis and named Bacillus Velez subtilis Shannan.BV80-12. It was deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO:M2024386. Figure 1 This is the colony morphology of Bacillus Velezii Shannan.BV80-12. Figure 2 This is a microscopic observation picture of Bacillus Velez Shannan.BV80-12.

[0111] Example 2 Whole genome sequencing analysis of strains

[0112] The Bacillus Velez subsp. Shannan.BV80-12 obtained in Example 1 was entrusted to Shenzhen BGI Co., Ltd. for whole genome sequencing and comparative genomics analysis. The results showed that the strain contained 5 unique genes related to maintaining bacterial genetic diversity and environmental adaptability (i.e., 2 bacterial transposase genes, 1 organic hydroperoxide resistance protein gene, 1 chromosome distribution protein gene, and 1 DNA polymerase III subunit δ gene), among which 2 bacterial transposase genes, 1 chromosome distribution protein gene, and 1 DNA polymerase III subunit δ gene have a direct role in maintaining bacterial genetic diversity, and 1 organic hydroperoxide resistance protein gene has a direct role in environmental adaptability. And these 5 genes all play an important role in bacterial stress resistance, helping Bacillus Velez subsp. Shannan.BV80-12 to resist complex environmental changes, improve the stress resistance of Bacillus Velez subsp. Shannan.BV80-12, and enable it to better exert its beneficial properties. The gene IDs corresponding to the above 5 genes, and the amino acid sequences corresponding to the above 5 genes, are as follows:

[0113] (1) Bacterial transposase gene: 1-2GL000284 (SEQ ID NO: 1), with the ID number in KEGG being K07483, and the amino acid sequence encoded by the gene being SEQ ID NO: 6.

[0114] (2) Bacterial transposase gene: 1-2GL000285 (SEQ ID NO: 2), with the ID number in KEGG being K07497. The amino acid sequence encoded by the gene is shown in SEQ ID NO: 7.

[0115] (3) Organic hydroperoxide resistance protein gene: 1-2GL000490 (SEQ ID NO: 3), with the ID number in KEGG being K23775, and the amino acid sequence encoded by the gene being SEQ ID NO: 8.

[0116] (4) Chromosome-distributed protein gene: 1-2GL001197 (SEQ ID NO: 4), with the ID number in KEGG being K03496, and the amino acid sequence encoded by the gene being SEQ ID NO: 9.

[0117] (5) DNA polymerase III subunit δ gene: 1-2GL002519 (SEQ ID NO: 5), with the ID number in KEGG being K02340. The amino acid sequence encoded by the gene is shown in SEQ ID NO: 10.

[0118] Example 3 Determination of drought tolerance of Bacillus velez Shannan.BV80-12

[0119] The Bacillus velez Shannan.BV80-12 and Bacillus velez CICC 24099 bacterial solutions described in Example 1 after activation for two generations in LB liquid medium were adjusted to OD 600nm 0.8 (the number of viable bacteria is 2×10 8 CFU / mL), and inoculated into LB liquid culture medium with PEG-6000 concentration of 0 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L or 40 g / L at a 2% volume inoculation amount (the higher the PEG-6000 concentration in the LB liquid culture medium with different PEG-6000 concentrations, the stronger the drought degree). After culturing at 37°C and 160 r / min for 24 h, the OD was measured 600nm , with OD 600nm The size of represents the growth and reproduction of the strain under different drought environments. Before the measurement, the UV-Visible light photometer was zeroed with LB liquid culture medium with the corresponding PEG-6000 concentration. The results are shown in Table 2 (three parallel experiments were set). 600nm <0.5 indicates that the strain is highly sensitive to drought environment; 0.5≤OD 600nm ≤1 indicates strains sensitive to drought environment; OD 600nm ≥1 indicates strains that are tolerant to drought environments.

[0120] Table 2. Growth and reproduction of strains under different drought environments

[0121]

[0122] As shown in Table 2, the results showed that in a drought environment with a PEG-6000 concentration of 30 g / L, Bacillus Velez Shannan.BV80-12 had tolerance, and in a drought environment with a PEG-6000 concentration of 40 g / L, it had no tolerance, and the tolerance effect of Bacillus Velez Shannan.BV80-12 was significantly better than that of Bacillus Velez CICC 24099.

[0123] Example 4 Determination of the ability of Bacillus velez Shannan.BV80-12 to produce indoleacetic acid (IAA)

[0124] The Bacillus velezensis Shannan.BV80-12 bacterial solution (viable count 2×10 8 CFU / mL) and Bacillus Velezii CICC 24099 culture (viable count 2×10 8CFU / mL), were inoculated in their respective LB medium with a pH of 9.0 and LB+ medium with a pH of 9.0 at an inoculum size of 2%, and cultured in flasks at 30°C and 150 r / min for 7 days to obtain fermentation broth, the fermentation broth was centrifuged at 4°C and 12000 r / min for 5 min to obtain supernatant, and the IAA content in the supernatant was determined by Salkowkin colorimetry. The results are shown in Table 3 (three parallel experiments were set up).

[0125] The specific determination method of the Salkowkin colorimetric method is as follows: take 2 mL of supernatant, add an equal amount of Salkowski reagent and place it in a dark place at room temperature, and after 30 minutes, use a UV-visible spectrophotometer to determine its absorbance at a wavelength of 530 nm. Calculate the IAA content in the fermentation broth per unit volume by comparing with the standard relationship curve, which is drawn by gradient dilution of the IAA analysis standard.

[0126] Table 3 Determination results of indoleacetic acid (IAA) production capacity

[0127]

[0128] As shown in Table 3, the results show that in LB medium, the IAA yield of Bacillus velez Shannan.BV80-12 is 27.03 mg / L, and the IAA yield in LB+ medium is 50.12 mg / L, which is about 2.61 times the IAA yield of Bacillus velez CICC 24099. The Bacillus velez Shannan.BV80-12 provided by the present invention can efficiently secrete IAA, and the IAA yield is better than that of Bacillus velez CICC 24099.

[0129] Example 5 Determination of the broad-spectrum antibacterial ability of Bacillus velez Shannan.BV80-12

[0130] The pathogenic fungus mycelium was inoculated in the center of the PDA solid culture plate. The bacterial solution of Bacillus velezensis Shannan.BV80-12 (viable count 2×10 8 CFU / mL) or Bacillus Velezii CICC 24099 culture (viable count 2×10 8 CFU / mL) was used as the treatment group; at the same time, a PDA solid culture plate inoculated with only pathogenic fungal hyphae was used as the control group. After culturing at 28°C in the dark for 5 days, the diameters of the pathogenic fungi hyphae were counted and the inhibition rate of the pathogenic fungi hyphae growth was calculated based on the diameters of the pathogenic fungi hyphae. The results are shown in Table 4 (three parallel experiments were set up).

[0131] Among them, the pathogenic fungi are wheat fusarium rust, tobacco brown spot pathogen, rice blast pathogen, corn leaf blight pathogen, corn leaf blight pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen or citrus brown rot pathogen.

[0132] The calculation formula of the pathogenic fungus hyphae growth inhibition rate is: pathogenic fungus hyphae growth inhibition rate = (pathogenic fungus hyphae diameter of the control group - pathogenic fungus hyphae diameter of the treatment group) / pathogenic fungus hyphae diameter of the control group × 100%.

[0133] Table 4. Inhibition rate of strains on mycelial growth of pathogenic fungi

[0134]

[0135] As shown in Table 4, the results show that Bacillus Velez Shannan.BV80-12 has a strong inhibitory effect on 9 pathogenic fungi, including wheat fusarium head blight, tobacco red spot pathogen, rice blast pathogen, corn leaf spot pathogen, corn leaf spot pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen and citrus brown rot pathogen, with inhibition rates higher than 90% and significantly better than Bacillus Velez CICC 24099. Among them, Bacillus Velez Shannan.BV80-12 has the best inhibitory effect on wheat fusarium head blight, with an inhibition rate of 99.8%, which is 3.02 times that of Bacillus Velez CICC 24099. The inhibition rate on tobacco red spot pathogen is 98.6%, which is 2.45 times that of Bacillus Velez CICC 24099.

[0136] Example 6 Preparation of Bacillus Velezii Shannan.BV80-12 Biocontrol Agent

[0137] (1) Activating Bacillus velez Shannan.BV80-12: The Bacillus velez Shannan.BV80-12 stored in the glycerol tube was inoculated into 10 mL of LB liquid culture medium at an inoculum size of 2%, and cultured at 37° C. for 24 h to obtain a primary activated bacterial solution; then, the primary activated bacterial solution was inoculated into 500 mL of LB liquid culture medium at an inoculum size of 2%, and cultured at 37° C. for 24 h to obtain a secondary activated bacterial solution;

[0138] (2) Preparation of Bacillus velez Shannan.BV80-12 fermentation broth: The secondary activated bacterial broth was inoculated into 2 L of LB liquid medium at a 10% inoculum amount, and high-density culture and fermentation were carried out for 20 h at a fermentation temperature of 37°C, an initial pH value of 7.0, and a rotation speed of 250 r / min to obtain Bacillus velez Shannan.BV80-12 fermentation broth (viable cell count of 1.1×10 10 CFU / mL).

[0139] (3) Preparation of Bacillus velez Shannan.BV80-12 powder: diatomaceous earth: Bacillus velez Shannan.BV80-12 fermentation liquid was mixed in a ratio of 20 g:1 L, spray-dried and pulverized to obtain Bacillus velez Shannan.BV80-12. The number of viable bacteria of Bacillus velez Shannan.BV80-12 in the powder was 6.0×10 10 CFU / mL. Spray drying is carried out by using a spray dryer, and the spray drying parameters are feed rate 600mL / h, air volume 80m 3 / h, outlet temperature 52℃.

[0140] (4) Preparation of biocontrol agent: Bacillus velez Shannan.BV80-12 powder: sodium dodecylbenzene sulfonate: dipotassium hydrogen phosphate: ascorbic acid = 87.9 g: 10 g: 2 g: 0.1 g were mixed evenly to obtain Bacillus velez Shannan.BV80-12 biocontrol agent, wherein the number of viable bacteria of Bacillus velez Shannan.BV80-12 in the biocontrol agent was 4.2×10 10 CFU / g.

[0141] Example 7 Effect of Bacillus velez Shannan.BV80-12 biocontrol agent on the growth of corn seedlings under drought stress

[0142] (1) Corn seeds with full grains and uniform size are first treated with 75% alcohol for 10 minutes and then washed twice with sterile water. Then, they are treated with 10% sodium hypochlorite for 10 minutes and then washed 5 times with sterile water to obtain sterile corn seeds.

[0143] (2) Soak the sterile corn seeds obtained in step (1) in sterile water for 12 hours. Select sterile corn seeds of uniform size and showing white spots for sowing, with a sowing depth of about 1 cm, one hole per pot, and one seed per hole.

[0144] (3) Biocontrol agent group: 6 g of Bacillus velez Shannan.BV80-12 biocontrol agent was mixed with water and fixed to 300 mL, and sterilized corn seeds were irrigated once every 5 days. After the 20th day of irrigating, the sterilized corn seeds were subjected to drought treatment. The drought treatment method was: 30 mL of the biocontrol agent was used to irrigate the sterilized corn seeds once every 5 days. After the 15th day of drought treatment, corn seedlings were obtained, and then the plant height, stem diameter, dry weight of the aboveground part and dry weight of the underground part of the corn seedlings were measured, and the root-crown ratio (ratio of the dry weight of the underground part to the dry weight of the aboveground part) was calculated based on the dry weight of the aboveground part and the dry weight of the underground part. The results are shown in Table 5 (three parallel experiments were set up).

[0145] Blank control group: sterile corn seeds were irrigated with 300 mL of water.

[0146] Positive control group: 6 g of Bacillus Velezii inoculant (cwiRPn3U) was mixed with water and the volume was adjusted to 300 mL.

[0147] Table 5. Plant height, stem diameter, aboveground dry weight, underground dry weight, and root-to-shoot ratio of maize seedlings

[0148]

[0149] As shown in Table 5, the plant height increased by 13 cm and 15 cm compared with the positive control group and the blank control group, the stem diameter increased by 2.8 cm and 3.7 cm compared with the positive control group and the blank control group, and the root-crown ratio increased by 4.69% and 9.59% compared with the positive control group and the blank control group, respectively. This shows that the biocontrol agent group has a significant promoting effect on the plant height, stem diameter, aboveground dry weight, and underground dry weight of corn seedlings, and the biocontrol agent group has a better growth state and a stronger root system.

[0150] Example 8 Control effect of Bacillus velez Shannan.BV80-12 biocontrol agent on wheat scab in the field

[0151] The field control of wheat fusarium rust with the biocontrol agent Bacillus Velezii Shannan.BV80-12 was carried out in the experimental field of Jiangling County, Jingzhou City.

[0152] Positive control group: 50 mL of 8% metconazole suspension was mixed with water and the volume was fixed to 45 L to obtain a mixed solution, and the mixture was added per mu (one mu = 666.67 m 2 ) Spray 45L of the mixed solution and spray the field.

[0153] Blank control group: water.

[0154] Biocontrol agent group: 100g of Bacillus velez Shannan.BV80-12 biocontrol agent was mixed with water and the volume was fixed to 45L to obtain a mixed solution, and the mixture was added per mu (one mu = 666.67m 2 ) Spray 45L of the mixed solution and spray the field.

[0155] Each treatment was repeated three times, and the area of ​​the plot was 60m 2 All plots were arranged in random blocks. The first spraying was carried out at the early stage of wheat heading and flowering, and the second spraying was carried out 7 days later. At the milky stage of wheat, the diseased ear rate, disease index and control effect of wheat scab were calculated according to the standard NY-T1464.15-2007, and the results are shown in Table 6.

[0156] Table 6. Disease rate, disease index and control effect of wheat fusarium head blight

[0157] Diseased ear rate (%) Disease index Control effect (%) Blank control group 89.25±0.2 70.34±0.03 / Positive control group 60.24±0.1 19.26±0.01 72.62±0.1 Biocontrol agent group 61.77±0.1 20.23±0.01 71.24±0.1

[0158] As shown in Table 6, the results showed that the field control effect of the biocontrol agent group was significant, at 71.24%, close to the positive control group, at 72.62%.

[0159] Example 9: Effect of the biocontrol agent Bacillus Velezii Shannan.BV80-12 on indoor control of tobacco brown star disease

[0160] Biocontrol agent group: When the tobacco seedlings grow to 12 fully expanded true leaves (about 100 days), pick 2 to 3 tobacco leaves in the middle and lower parts (keep 3 fully expanded tobacco leaves in the upper part), mix 2g of Bacillus Velezii Shannan.BV80-12 biocontrol agent with water and dilute to 1L to obtain a mixed solution, spray the mixed solution on the tobacco leaves (based on no droplets dripping), dry at room temperature and repeat the spraying to complete the spraying. Then, inoculate tobacco brown star pathogen on the tobacco leaves after spraying, and set 3 replicates. The method of inoculating tobacco brown star pathogen is: use the tip of the gun to lightly poke the front veins of the tobacco leaves to cause a wound (without piercing the leaves), and use the hanging drop method to inoculate the tobacco brown star pathogen hyphae suspension at the punctured site (the number of viable bacteria is 1×10 6 / mL), 8 to 10 points were inoculated on each tobacco leaf, and 25 μL of tobacco brown spot mycelium suspension was inoculated on each point. The leaves were moisturized and cultured at a temperature of 28°C and a humidity of 70% for 7 days. The incidence was counted according to the grading standard for the incidence of tobacco brown spot disease on detached leaves in Table 7, and the disease index and control effect were calculated. The results are shown in Table 8.

[0161] The calculation formula of the disease index is:

[0162] The calculation formula of the control effect is: control effect = [(disease index of the blank control group - disease index of the biocontrol agent group) / disease index of the blank control group] × 100%.

[0163] Blank control group: The difference from the biocontrol agent group is that the spraying of Bacillus Velezii Shannan.BV80-12 biocontrol agent was replaced by spraying of clean water.

[0164] Positive control group: The difference from the biocontrol agent group is that the biocontrol agent Shannan.BV80-12 sprayed with Bacillus Velezii was replaced with a 40% dilution of Sclerotium Clean (the preparation method of the dilution is: 2 mL of 40% Sclerotium Clean and water are mixed and the volume is fixed to 1 L to obtain a 40% dilution of Sclerotium Clean).

[0165] Table 7. Grading criteria for the incidence of tobacco brown spot disease on detached leaves

[0166] grade Disease 0 No disease, no lesions 1 The leaves around the inoculated part turn slightly green 3 Pale yellow halo around the inoculated part of the leaf 5 <![CDATA[Yellow lesions, the center does not wither or withers slightly, and the lesion area is less than 1 cm 2 > 7 <![CDATA[Yellow lesion, central necrosis, lesion area 1 - 2 cm 2 > 9 <![CDATA[Yellow lesions, central necrosis, lesion area greater than 2 cm 2 >

[0167] Table 8. Incidence, disease index and control effect of tobacco brown spot disease

[0168] Incidence (%) Disease index Preventive effect (%) Blank control group 99±0.8 70.34±0.05 / Positive control group 80±0.3 22.29±0.01 68.31±0.03 Biocontrol agent group 79±0.2 15.13±0.01 78.50±0.02

[0169] As shown in Table 8, the results showed that the biocontrol agent group had a better control effect on tobacco brown spot disease, which was significantly higher than the control effect of the positive control group (chemical pesticide Myrrhotide) on tobacco brown spot disease.

[0170] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not limitations of the present invention. Any non-prominent substantial features and non-significant improvements made by those skilled in the art on this basis should fall within the scope of protection of the present invention.

[0171] Table 9. Sequence Listing

[0172]

[0173]

[0174]

[0175]

[0176]

Claims

1. A Bacillus Velez, characterized in that The Bacillus velezensis is Bacillus velezensis Shannan.BV80-12, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO:M 2024386.

2. The Bacillus Velezii according to claim 1, characterized in that The 16srRNA gene sequence of the Bacillus Velezii is shown as SEQ ID No.

13.

3. The Bacillus Velezii according to claim 1 or 2, characterized in that The Bacillus Velezii carries genes that maintain bacterial genetic diversity. Among them, the gene for maintaining bacterial genetic diversity comprises the sequence shown in SEQ ID NO: 1, and / or comprises the sequence shown in SEQ ID NO: 2, and / or comprises the sequence shown in SEQ ID NO: 4, and / or comprises the sequence shown in SEQ ID NO:

5.

4. The Bacillus Velezii according to any one of claims 1 to 3, characterized in that The Velez bacillus carries genes that are environmentally adaptable. Among them, the gene for environmental adaptability comprises the sequence shown in SEQ ID NO:

3.

5. The Bacillus Velezii according to claim 3, characterized in that The amino acid sequence encoded by the sequence shown in SEQ ID NO: 1 is the amino acid sequence shown in SEQ ID NO: 6, and / or the amino acid sequence encoded by the sequence shown in SEQ ID NO:2 is the amino acid sequence shown in SEQ ID NO:7, and / or the amino acid sequence encoded by the sequence shown in SEQ ID NO:4 is the amino acid sequence shown in SEQ ID NO:9, And / or the amino acid sequence encoded by the sequence shown in SEQ ID NO:5 is the amino acid sequence shown in SEQ ID NO:

10.

6. The Bacillus Velezii according to claim 4, characterized in that The amino acid sequence encoded by the sequence shown in SEQ ID NO:3 is the amino acid sequence shown in SEQ ID NO:

8.

7. The Bacillus Velezii according to any one of claims 1 to 6, characterized in that The Bacillus Velezii has drought tolerance, The drought tolerance refers to: the number of viable bacteria is 1-2×10 8 CFU / mL of the bacterial liquid of Bacillus Velez is inoculated in an LB liquid medium with a PEG-6000 concentration of greater than 0 g / L and less than or equal to 40 g / L at an inoculum amount of 1-3 volume %, and the Bacillus Velez is inoculated at an OD 600nm The value is greater than or equal to 0.48 and less than 2.

40.

8. The Bacillus Velezii according to any one of claims 1 to 7, characterized in that The Velez bacillus has the ability to produce indoleacetic acid, The ability to produce indoleacetic acid refers to: the number of viable bacteria is 1-2×10 8 CFU / mL of Bacillus Velez is inoculated into a LB liquid medium with a pH of 9.0 at an inoculum rate of 1-3% by volume, wherein the amount of indoleacetic acid per L of the LB liquid medium is 25-30 mg, preferably 27-28 mg; And / or the ability to produce indoleacetic acid refers to: the number of viable bacteria is 1-2×10 8 The bacterial liquid of Bacillus velez with a CFU / mL is inoculated into an LB+ liquid culture medium with a pH of 9.0 at an inoculation rate of 1-3 volume %, and the amount of indoleacetic acid in each L of LB+ liquid culture medium is 50-55 mg, preferably 50-51 mg, wherein the LB+ liquid culture medium refers to an LB liquid culture medium containing 0.50 g of tryptophan per L.

9. The Bacillus Velezii according to any one of claims 1 to 8, characterized in that The Velez subtilis has the ability to inhibit pathogenic bacteria, wherein the ability to inhibit pathogenic bacteria refers to: the number of viable bacteria is 1-2×10 8 The bacterial solution of Bacillus Velezii with a CFU / mL has an inhibition rate of more than 90% against pathogenic bacteria.

10. The Bacillus Velezii according to any one of claims 1 to 9, characterized in that The pathogen is a pathogenic fungus; Preferably, the pathogenic fungi include one or more substances selected from the group consisting of wheat fusarium head blight, tobacco brown spot pathogen, rice blast pathogen, corn leaf spot pathogen, corn leaf spot pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen and citrus brown rot pathogen; More preferably, the pathogenic fungi are wheat fusarium rust, tobacco brown spot pathogen, rice blast pathogen, corn leaf blight pathogen, corn leaf blight pathogen, rapeseed sclerotinia pathogen, apple ring rot pathogen, tomato early blight pathogen and citrus brown rot pathogen.

11. The Bacillus Velezii according to claim 10, characterized in that The inhibition rate of the Bacillus Velez against wheat fusarium is greater than or equal to 99%, and / or the inhibition rate of tobacco brown spot pathogen is greater than or equal to 97%, and / or the inhibition rate of rice blast pathogen is greater than or equal to 95%, and / or the inhibition rate of corn leaf blight pathogen is greater than or equal to 92%, and / or the inhibition rate of corn leaf blight pathogen is greater than or equal to 91%, and / or the inhibition rate of rapeseed sclerotinia pathogen is greater than or equal to 89%, and / or the inhibition rate of apple ring rot pathogen is greater than or equal to 90%, and / or the inhibition rate of tomato early blight pathogen is greater than or equal to 88%, and / or the inhibition rate of citrus brown rot pathogen is greater than or equal to 88%.

12. Use of the Bacillus Velezii according to any one of claims 1 to 11 in the preparation of bacterial powder or biocontrol agent.

13. A bacterial powder, characterized in that: Contains the Bacillus Velezii according to any one of claims 1 to 11.

14. The bacterial powder according to claim 13, characterized in that: The number of viable bacteria is 1.1-6.4×10 10 CFU / g.

15. The bacterial powder according to claim 13 or 14, characterized in that: The bacterial powder is prepared by a method comprising the following steps: diatomaceous earth and fermentation liquid of Bacillus velezii are mixed in a ratio of 15-25 g:1 L, dried and crushed to obtain bacterial powder.

16. A method for preparing the bacterial powder according to any one of claims 13 to 15, characterized in that: The method comprises the following steps: mixing diatomaceous earth and fermentation liquid of Bacillus velez in a ratio of 15-25 g:1 L, drying and crushing to obtain bacterial powder.

17. The preparation method according to claim 16, characterized in that: The steps include: (1) inoculating the activated Bacillus Velez subtilis into a liquid culture medium for fermentation to obtain a fermentation liquid of the Bacillus Velez subtilis, (2) Mix diatomaceous earth and fermentation liquid of Bacillus velez in a ratio of 15-25 g:1 L, dry and grind to obtain bacterial powder.

18. The preparation method according to claim 17, characterized in that: The fermentation temperature is 37° C.-42° C., and / or the initial pH value of the fermentation is 6.0-7.0, and / or the fermentation speed is 200-250 r / min, and / or the fermentation time is 20-36 h.

19. The preparation method according to claim 17, characterized in that: The drying is performed by spray drying, wherein the feed rate of the spray drying is 600-610 mL / h, and / or the air flow rate of the spray drying is 80-85 m 3 / h, and / or the outlet temperature of spray drying is 52-55°C.

20. A biocontrol agent, characterized in that: Contains the bacterial powder according to any one of claims 13 to 15 or the bacterial powder prepared by the preparation method according to any one of claims 16 to 19.

21. The biocontrol agent according to claim 20, characterized in that The number of live bacteria of Bacillus Velezii contained in the biocontrol agent is greater than or equal to 1.1×10 10 CFU / g, preferably, the number of viable bacteria is 1.1-6.4×10 10 CFU / g.

22. The biocontrol agent according to claim 20 or 21, characterized in that: In parts by weight, the biocontrol agent comprises 70-90 parts of the bacterial powder, 10-20 parts of sodium dodecylbenzene sulfonate, 2-10 parts of dipotassium hydrogen phosphate and 0.1-1 part of ascorbic acid. Preferably, the bacterial powder is 77.7-87.9 parts, and / or sodium dodecylbenzene sulfonate is 10-15 parts, and / or dipotassium hydrogen phosphate is 2-7 parts, and / or ascorbic acid is 0.1-0.5 parts.

23. The biocontrol agent according to claim 22, characterized in that: The biocontrol agent is prepared by a method comprising the following steps: mixing the bacterial powder, sodium dodecylbenzene sulfonate, dipotassium hydrogen phosphate and ascorbic acid to obtain the biocontrol agent.

24. The biocontrol agent according to any one of claims 20 to 23, characterized in that The mixed solution containing the biocontrol agent at a concentration of 0.01-0.05 g / mL has the effect of promoting plant growth under drought stress. Preferably, the plant is a corn seedling. Preferably, the mixed solution is a mixture of water and the biocontrol agent.

25. The biocontrol agent according to any one of claims 20 to 24, characterized in that The mixed liquid containing the biocontrol agent at a concentration of 1-5g / L has the function of preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by pathogens. Preferably, the plant diseases caused by pathogens include wheat fusarium head blight and / or tobacco brown spot disease. Preferably, the mixed liquid is a mixture of water and the biocontrol agent.

26. Use of the Bacillus Velezii according to any one of claims 1 to 11, or the bacterial powder according to any one of claims 13 to 15, or the bacterial powder prepared by the preparation method according to any one of claims 16 to 19, or the biological bacterial agent according to any one of claims 20 to 25 in the preparation of fertilizers or pesticides, Preferably, the fertilizer is a fertilizer that helps promote plant growth under drought stress. Preferably, the pesticide is a pesticide for preventing and controlling plant diseases, wherein: The plant diseases are plant diseases caused by pathogenic bacteria. Preferably, the plant diseases caused by pathogenic bacteria include wheat head blight and / or tobacco brown spot disease.

27. A fertilizer, characterized in that: Containing the Bacillus Velezii according to any one of claims 1 to 11, or the bacterial powder according to any one of claims 13 to 15, or the bacterial powder prepared by the preparation method according to any one of claims 16 to 19, or the biological bacterial agent according to any one of claims 20 to 25, preferably, the fertilizer is a fertilizer that helps promote plant growth under drought stress.

28. A pesticide, characterized in that Containing the Bacillus Velez subtilis according to any one of claims 1 to 11, or the bacterial powder according to any one of claims 13 to 15, or the bacterial powder prepared by the preparation method according to any one of claims 16 to 19, or the biological bacterial agent according to any one of claims 20 to 25, preferably, the pesticide is a pesticide for preventing and controlling plant diseases, preferably, the plant disease is a plant disease caused by pathogenic bacteria, and more preferably, the plant disease caused by pathogenic bacteria includes wheat fusarium rust and / or tobacco brown spot disease.

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