Bacillus velezensis and application thereof

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Bacillus belyssiensis* and its applications. The *Bacillus belyssiensis* strain is named *Bacillus belyssiensis* BB-1, and is classified as *Bacillus belyssiensis* (…). Bacillus velezensis It was deposited on June 11, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.39202. It can inhibit pathogens such as Fusarium graminearum and produce amylase and protease, and can be used to prevent and control plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus belye and its applications. Background Technology

[0002] Bacillus belye is a new species of Bacillus, a novel microbial pesticide that promotes crop growth and shows good efficacy in disease resistance. Current research indicates that Bacillus belye is being used to treat agricultural diseases: Bacillus belye Ag75 inhibits Rhizoctonia solani (…). Rhizoctonia solani The growth of *Bacillus belyceae* was improved, and yield increased by 17.80% in maize field trials. In field trials, application of *Bacillus belyceae* LJ02 to control cotton Verticillium wilt achieved a control effect of 61.10% and significantly promoted cotton growth. *Bacillus belyceae* E2 also showed resistance to *Aspergillus ochraceus* (…). Aspergillus westerdijkiae fc-1 The growth of ) and the OTA it produces Ochratoxin A Ochratoxin A has a significant inhibitory effect and can be used to control fungal diseases caused by ochratoxin in agricultural products. Therefore, it is necessary to develop Bacillus belyssus.

[0003] Existing Bacillus belycei culture medium formulations often use high-value raw materials such as glucose, peptone, and yeast extract to achieve high bacterial counts, resulting in high raw material costs. Furthermore, existing incremental culture equipment for Bacillus belycei primarily uses small-capacity laboratory fermenters, which are unsuitable for industrial-scale production demands. Moreover, large-scale microbial production, especially using ton-scale fermentation equipment, suffers from complex fermentation processes and poor process stability. Therefore, the main challenges are how to stably and effectively increase microbial counts while reducing raw material costs. Summary of the Invention

[0004] The first aspect of the present invention is to provide a strain of Bacillus belyssus.

[0005] A second aspect of the present invention is to provide a culture.

[0006] A third aspect of the present invention is to provide a microbial agent.

[0007] A fourth aspect of the present invention is to provide a composition.

[0008] The fifth aspect of this invention aims to provide the application of Bacillus berberis of the first aspect, the culture of the second aspect, the bacterial agent of the third aspect, or the composition of the fourth aspect.

[0009] The sixth aspect of the present invention is to provide a Bacillus belesiensis culture medium.

[0010] The seventh aspect of the present invention is to provide the application of the Bacillus berberis culture medium of the sixth aspect of the present invention.

[0011] The object of the eighth aspect of the present invention is to provide a method.

[0012] The ninth aspect of this invention aims to provide a Bacillus vesiculosus agent.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a strain of Bacillus belyssus, named Bacillus belyssus BB-1, and classified as Bacillus belyssus (… Bacillus velezensis It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 11, 2026, with accession number CGMCC NO.39202.

[0014] A second aspect of the present invention provides a culture obtained by culturing Bacillus belye of the first aspect of the present invention.

[0015] In some embodiments, the culture is obtained by inoculating *Bacillus belyssus* of the first aspect of the present invention into a *Bacillus belyssus* culture medium and culturing it.

[0016] In some embodiments, the Bacillus belye culture medium comprises the following components: molasses, corn steep liquor, calcium salts, and magnesium salts.

[0017] In some embodiments, the calcium salt includes at least one of calcium carbonate, calcium sulfate, calcium chloride, calcium phosphate, and calcium citrate; further comprising calcium chloride.

[0018] In some embodiments, the magnesium salt includes at least one of magnesium chloride and magnesium sulfate; further comprising magnesium chloride.

[0019] In some embodiments, the molasses content in the culture medium is 0.05-20% by mass, for example, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 4.5%, 5%, 5.5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or values ​​between these; further, 0.1-10%; even further, 1-6%; and still further, 4-6%.

[0020] In some embodiments, the corn steep liquor in the culture medium is 0.05-10% by mass, for example, 0.05%, 0.1%, 0.5%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 7%, 8%, 9%, 10%, or values ​​between these; further, 0.1-5%; even further, 1-5%; and still further, 2.5-3.5%.

[0021] In some embodiments, the calcium salt content in the culture medium is 0.01-0.2% by mass, for example, it may be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, or values ​​between these; further, 0.01-0.1%; even further, 0.04-0.08%; and still further, 0.05-0.07%.

[0022] In some embodiments, the magnesium salt content in the culture medium is 0.05-1% by mass, for example, it may be 0.05%, 0.1%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or values ​​between these; further, 0.1-1%; even further, 0.2-0.6%; and still further, 0.3-0.5%.

[0023] In some embodiments, the Bacillus berberis culture medium also includes water.

[0024] In some implementations, the water content is a surplus.

[0025] A third aspect of the present invention provides a microbial agent comprising: Bacillus berleis of the first aspect of the present invention or a culture of the second aspect.

[0026] A fourth aspect of the present invention provides a composition comprising: *Bacillus belye* of the first aspect, a culture of the second aspect, or a bacterial agent of the third aspect; and Additional surfactants and / or excipients.

[0027] In some embodiments, the adjuvant activator includes one or more biocontrol agents, one or more chemical agents, one or more fertilizers, one or more herbicides, one or more growth promoters, or any combination thereof.

[0028] In some embodiments, the excipients are selected from agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film-forming agents, spontaneous accelerators, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, or any combination thereof.

[0029] A fifth aspect of the present invention provides the use of Bacillus berberis of the first aspect, the culture of the second aspect, the inoculant of the third aspect, or the composition of the fourth aspect in the prevention and control of plant diseases.

[0030] In some implementations, the plant disease is caused by a pathogen.

[0031] In some implementations, the pathogen includes Fusarium graminearum.

[0032] A sixth aspect of the present invention provides a Bacillus belye culture medium comprising the following components: molasses, corn steep liquor, calcium salts and magnesium salts.

[0033] In some embodiments, the calcium salt includes at least one of calcium carbonate, calcium sulfate, calcium chloride, calcium phosphate, and calcium citrate; further comprising calcium chloride.

[0034] In some embodiments, the magnesium salt includes at least one of magnesium chloride and magnesium sulfate; further comprising magnesium chloride.

[0035] In some embodiments, the molasses content in the culture medium is 0.05-20% by mass, for example, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 4.5%, 5%, 5.5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or values ​​between these; further, 0.1-10%; even further, 1-6%; and still further, 4-6%.

[0036] In some embodiments, the corn steep liquor in the culture medium is 0.05-10% by mass, for example, 0.05%, 0.1%, 0.5%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 7%, 8%, 9%, 10%, or values ​​between these; further, 0.1-5%; even further, 1-5%; and still further, 2.5-3.5%.

[0037] In some embodiments, the calcium salt content in the culture medium is 0.01-0.2% by mass, for example, it may be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, or values ​​between these; further, 0.01-0.1%; even further, 0.04-0.08%; and still further, 0.05-0.07%.

[0038] In some embodiments, the magnesium salt content in the culture medium is 0.05-1% by mass, for example, it may be 0.05%, 0.1%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or values ​​between these; further, 0.1-1%; even further, 0.2-0.6%; and still further, 0.3-0.5%.

[0039] In some embodiments, the Bacillus berberis culture medium also includes water.

[0040] In some implementations, the water content is a surplus.

[0041] A seventh aspect of the invention provides the use of the Bacillus belye culture medium of the sixth aspect of the invention in any one of (a1)-a5): a1) Cultivate Bacillus belye; a2) Increase the number of Bacillus belyssus cells; a3) Improve the sporulation rate of Bacillus belyss; a4) Preparation of Bacillus belesi inoculum; a5) Prepare a product, which is used in any one of a1)-a4).

[0042] In some embodiments, the product is a reagent or a kit.

[0043] In some embodiments, the *Bacillus belyssus* is the *Bacillus belyssus* of the first aspect of the present invention.

[0044] An eighth aspect of the present invention provides a method comprising the step of using a Bacillus belye culture medium according to a sixth aspect of the present invention; The method is used for any one of a1)-a4): a1) Cultivate Bacillus belye; a2) Increase the number of Bacillus belyssus cells; a3) Improve the sporulation rate of Bacillus belyss; a4) Prepare Bacillus belysin inoculum.

[0045] In some embodiments, the method includes the following steps: inoculating Bacillus belye seed culture into the Bacillus belye culture medium of the sixth aspect of the present invention, and fermenting.

[0046] In some embodiments, the inoculation amount of the Bacillus belye seed culture is 1-15% (v / v); more commonly, it is 9-11%.

[0047] In some embodiments, the fermentation temperature is 25-40°C; more specifically, it is 35-39°C.

[0048] In some embodiments, the fermentation time is 35-40 hours.

[0049] In some embodiments, the dissolved oxygen during the fermentation process is 1-50%.

[0050] In some embodiments, dissolved oxygen during the fermentation process is controlled by stirring and aeration.

[0051] In some embodiments, the stirring speed is 30-600 rpm (e.g., 30-70 rpm or 200-600 rpm).

[0052] In some embodiments, the ventilation rate is 1-6000 L / min (e.g., 1-3 L / min or 3000-6000 L / min).

[0053] In some embodiments, the method for preparing the Bacillus belye seed culture includes the following steps: inoculating activated Bacillus belye into a seed culture medium for a first culture.

[0054] In some embodiments, the temperature of the first culture is 25-40°C; more specifically, it is 35-39°C.

[0055] In some embodiments, the first culture is carried out when the culture reaches OD. 600 2.5 or higher (preferably cultured to OD) 600 (2.5).

[0056] In some embodiments, the method for preparing the Bacillus belye seed culture further includes the following steps: inoculating the product of the first culture into a seed culture medium for a second culture, and then inoculating the product of the second culture into a seed culture medium for a third culture.

[0057] In some embodiments, the inoculum amount of the product from the first culture is 0.1-10%; more specifically, 0.9-1.1%.

[0058] In some embodiments, the temperature of the second culture is 25-40°C; more specifically, it is 35-39°C.

[0059] In some embodiments, the second culture is carried out until the OD (Oxygen Demand) is reached. 600 2.5 or higher (preferably cultured to OD) 600 (2.5).

[0060] In some embodiments, the inoculum of the product from the second culture is 0.1-10%; more specifically, 2.9-3.1%.

[0061] In some embodiments, the temperature of the third culture is 25-40°C; more specifically, it is 35-39°C.

[0062] In some embodiments, the third culture is carried out until the OD (Oxygen Demand) is reached. 600 2.5 or higher (preferably cultured to OD) 600 (2.5).

[0063] In some embodiments, the seed culture medium includes peptone, yeast extract, and sodium chloride.

[0064] In some embodiments, the method for preparing the activated Bacillus belye includes the following steps: inoculating Bacillus belye into an activation culture medium and performing a fourth culture.

[0065] In some embodiments, the temperature of the fourth culture is 25-40°C; more specifically, it is 35-39°C.

[0066] In some embodiments, the fourth culture time is 12-20 hours; more specifically, 15-17 hours.

[0067] In some embodiments, the activation culture medium includes peptone, yeast extract, sodium chloride, and agar powder.

[0068] In some embodiments, the *Bacillus belyssus* is the *Bacillus belyssus* of the first aspect of the present invention.

[0069] A ninth aspect of the present invention provides a Bacillus bellis inoculant comprising Bacillus bellis and a Bacillus bellis culture medium according to the sixth aspect of the present invention.

[0070] In some embodiments, the *Bacillus belyssus* is the *Bacillus belyssus* of the first aspect of the present invention.

[0071] In some embodiments, the Bacillus berberis agent is prepared by the method described in the eighth aspect of the present invention.

[0072] The beneficial effects of this invention are: This invention provides a strain of Bacillus belyssus, named Bacillus belyssus BB-1, and classified as Bacillus belyssus (… Bacillus velezensis It was deposited on June 11, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.39202. It can inhibit pathogens such as Fusarium graminearum and produce amylase and protease, and can be used to prevent and control plant diseases.

[0073] This invention provides a Bacillus belye culture medium, which includes the following components: molasses, corn steep liquor, calcium salts and magnesium salts; by using inexpensive molasses and corn steep liquor to replace more expensive raw materials such as glucose and yeast powder, the cost is reduced.

[0074] Furthermore, by optimizing the culture medium formula through experiments, the problem of low cell density during the fermentation of Bacillus belysus was effectively solved, while the sporulation rate was improved.

[0075] The culture medium has a simple formula and uses economical raw materials, making it suitable for large-scale production in ton-level fermenters and able to meet the industry's demand for high output. Attached Figure Description

[0076] Figure 1 A microscopic (1000X) photograph of Bacillus belyss BB-1 is shown.

[0077] Figure 2 The image shows an LB plate photograph of the colony morphology of Bacillus belyss BB-1.

[0078] Figure 3 The ability of Bacillus belyss BB-1 to produce proteases was demonstrated.

[0079] Figure 4 The ability of Bacillus belyss BB-1 to produce amylase was demonstrated. Detailed Implementation

[0080] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0081] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0082] The present invention will be further described in detail below through specific embodiments.

[0083] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0084] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0085] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. For reference, please refer to "Molecular Cloning: A Laboratory Manual (3rd Edition)" (Science Press), "Microbiology Experiments (4th Edition)" (Higher Education Press), and the manufacturer's instructions for the corresponding instruments and reagents.

[0086] The peptone and yeast powder used in the experiment were purchased from Angel Yeast Co., Ltd., sodium chloride and magnesium chloride were purchased from Wuxi Yasheng Chemical Co., Ltd., agar powder was purchased from Sinopharm Chemical Reagent Co., Ltd., and molasses and corn steep liquor were from Anhui Fengyuan Biotechnology Co., Ltd.

[0087] The culture media involved in the following examples are as follows: Protease identification medium: 10 g / L skim milk powder, 15 g / L agar powder; Amylase identification medium: soluble starch 20 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L; Counting medium (LB medium): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L; Activation medium (LB medium): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L; Seed culture medium (LB medium): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0088] Example 1: Isolation, Purification and Identification of Strains 1. Isolation, purification, and screening of biocontrol bacteria Rhizosphere soil samples were collected from corn plantations in Lienchan Town, Guzhen County, Anhui Province. The samples were placed in sterile bags and brought back to the laboratory for bacterial isolation.

[0089] Biocontrol bacteria were isolated using a dilution-coating method. 10g of rhizosphere soil sample was weighed and diluted with sterile water to prepare a solution of 10... -4 10-5 10 -6 Soil suspensions of different gradients were taken in batches of 100 μL and evenly spread onto LB plates. Each gradient was replicated three times. The plates were incubated in a constant temperature incubator at 28 °C. After 24 h, bacterial colonies of different morphologies were picked and streaked. The obtained single colonies were streaked onto LB solid slant medium and stored in a refrigerator at 4 °C.

[0090] The biocontrol bacteria were screened using the plate confrontation method. Single colonies were picked and inoculated into LB liquid medium and cultured at 32°C and 180 rpm for 20 hours with shaking to prepare a fermentation broth. Fusarium graminearum (…) was then placed in a clean bench… Fusarium graminearum The bacteria (purchased from the Institute of Microbiology, Chinese Academy of Sciences, catalog number CGMCC 3.3488) were inoculated onto PDA plates and activated for 3–5 days. A 5 mm diameter *Fusarium graminearum* bacterial dish was placed in the center of the PDA plate, and 2 μL of bacterial fermentation broth was inoculated around the dish in a cross pattern. Uninoculated fermentation broth served as the control (CK). Each treatment was replicated in triplicate. After incubation at 28°C for 3–7 days, the diameter of the pathogen in both the control and treatment groups was measured, and the inhibition rate was calculated.

[0091] Inhibition rate = (Diameter of control pathogen - Diameter of treatment pathogen) / (Diameter of control pathogen - Diameter of fungal cake) × 100%; Among the obtained strains, one strain was screened that had the strongest inhibitory effect on Fusarium graminearum, with an inhibition rate of 70.05%.

[0092] 2. Identification of biocontrol strains (1) Plate and microscopic observation The selected strains were streaked on LB solid medium for 24 hours. The colony morphology, size, edge, surface, raised shape, transparency and color were observed. Gram staining was performed, and the colony morphology was photographed and recorded.

[0093] like Figure 1 , 2 As shown, the bacteria grow well on LB medium, with milky white colonies, producing extracellular secretions. Picks of the bacteria are quite viscous, Gram-positive, and appear as rod-shaped cells under a microscope, with spores produced.

[0094] (2) Molecular biological identification Genomic DNA was extracted from the biocontrol strain. Using a set of PCR primers for bacterial 16S rDNA, the genomic DNA was amplified by PCR and sequenced. The 16S rDNA sequence is shown in SEQ ID NO: 1. The strain was identified as *Bacillus belyssae*. Bacillus velezensisIt was named Bacillus belyss BB-1, and its classification name was Bacillus belyss ( Bacillus velezensis The gene was deposited on June 11, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.39202, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0095] Example 2: Determination of the protease production capacity of Bacillus belyssus BB-1 Bacillus belye BB-1 strain was inoculated onto activation medium and cultured at 37°C for 16 h. It was then inoculated onto seed medium and cultured at 37°C until OD (Organic Depth) was reached. 600 When the concentration is 2.5, seed culture is obtained. 10 μL of seed culture is inoculated onto protease identification medium containing circular filter paper discs (5 mm in diameter) and incubated at 37°C for 24 h. The size of the transparent zone around the filter paper discs is then observed.

[0096] The results are as follows Figure 3 As shown, the presence of a clear zone around the colony of *Bacillus belye* BB-1 indicates that this bacterium produces proteases and has the ability to degrade proteins. This ability is an important characteristic of biocontrol bacteria in inhibiting plant pathogens and is a crucial indicator of biocontrol properties.

[0097] Example 3: Determination of the amylase-producing ability of Bacillus belyssus BB-1 Bacillus belye BB-1 strain was inoculated onto activation medium and cultured at 37°C for 16 h. It was then inoculated onto seed medium and cultured at 37°C until OD (Organic Depth) was reached. 600 When the concentration is 2.5, seed culture is obtained. 10 μL of seed culture is inoculated onto amylase identification medium containing circular filter paper discs (5 mm in diameter), and incubated at 37°C for 24 h. Color development is performed with 0.1% dilute iodine solution, and the size of the transparent zone around the filter paper disc is observed.

[0098] The results are as follows Figure 4 As shown, the presence of a clear zone around the colony of *Bacillus belye* BB-1 indicates that this bacterium produces amylase and has the ability to degrade starch. This ability is an important characteristic of biocontrol bacteria in inhibiting plant pathogens and is a crucial indicator of biological control performance.

[0099] Example 4: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 5%, corn steep liquor 3%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0100] Example 5: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 0.1%, corn steep liquor 3%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0101] Example 6: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 0.5%, corn steep liquor 3%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0102] Example 7: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 1%, corn steep liquor 3%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0103] Example 8: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 10%, corn steep liquor 3%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0104] Example 9: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 5%, corn steep liquor 0.1%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0105] Example 10: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 5%, corn steep liquor 0.5%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0106] Example 11: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 5%, corn steep liquor 1%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0107] Example 12: A Bacillus belye BB-1 culture medium A Bacillus belye BB-1 culture medium, comprising the following components by weight percentage: molasses 5%, corn steep liquor 5%, calcium chloride 0.06%, magnesium chloride 0.4%, and water balance.

[0108] Example 13: Effect of molasses on the cell mass and sporulation rate of Bacillus belyss BB-1 Bacillus belye BB-1 strain was inoculated onto activation medium and cultured at 37°C for 16 h. It was then inoculated onto seed medium and cultured at 37°C until OD (Organic Depth) was reached. 600 When the pH was 2.5, seed culture was obtained and inoculated at 10% into a 5L fermenter containing 3L of the culture medium from Examples 4, 5, 6, 7, or 8. The initial pH was 6.8, the aeration rate was 1-3 L / min, and the stirring speed was 200-600 rpm. Dissolved oxygen was controlled at 30% by controlling stirring and aeration. The total fermentation time was 35 hours (fermentation at 37°C). 2 mL of fermentation broth from each of the above culture systems was serially diluted with sterile water, and 0.1 mL of the diluted solution was spread onto counting medium. 2 mL of the same fermentation broth from each culture system was then incubated in an 80°C water bath for 20 minutes, followed by serial dilution with sterile water, and 0.1 mL of the diluted solution was spread onto counting medium. After incubation at 28°C for 24 hours, plates with colony counts between 30 and 300 CFU and no spreading colony growth were selected, with 3 replicates per group, and the colony count and sporulation rate were calculated.

[0109] Sporulation rate = Number of colonies produced by coating with heated bacterial solution ÷ Number of colonies produced by coating with unheated bacterial solution × 100%.

[0110] The results are shown in Table 1: The number of cells increased significantly with increasing molasses concentration, but the sporulation rate decreased significantly when the molasses concentration was greater than 5%. Furthermore, increasing the molasses concentration did not significantly increase the number of cells in the fermentation broth, but increased costs, which was not economically viable. Therefore, choosing 5% molasses as the carbon source for Bacillus belyssus BB-1 fermentation is more reasonable.

[0111] Table 1. Effects of molasses on cell mass and sporulation rate of Bacillus belyss BB-1

[0112] Note: The bacterial count is the number of colonies produced by coating the unheated bacterial solution.

[0113] Example 13: Effect of corn steep liquor on the bacterial count and sporulation rate of Bacillus belyssus BB-1 Bacillus belye BB-1 strain was inoculated onto activation medium and cultured at 37°C for 16 h. It was then inoculated onto seed medium and cultured at 37°C until OD (Organic Depth) was reached. 600When the pH was 2.5, seed culture was obtained and inoculated at 10% into a 5L fermenter containing 3L of the culture medium from Examples 4, 9, 10, 11, or 12. The initial pH was 6.8, the aeration rate was 1-3 L / min, and the stirring speed was 200-600 rpm. Dissolved oxygen was controlled at 30% by controlling stirring and aeration. The total fermentation time was 35 hours (fermentation at 37°C). 2 mL of fermentation broth from each of the above culture systems was serially diluted with sterile water, and 0.1 mL of the diluted solution was spread onto counting medium. 2 mL of the same fermentation broth from each culture system was then incubated in an 80°C water bath for 20 minutes, followed by serial dilution with sterile water, and 0.1 mL of the diluted solution was spread onto counting medium. After incubation at 28°C for 24 hours, plates with colony counts between 30 and 300 CFU and no spreading colony growth were selected, with 3 replicates per group, and the colony count and sporulation rate were calculated.

[0114] Sporulation rate = Number of colonies produced by coating with heated bacterial solution ÷ Number of colonies produced by coating with unheated bacterial solution × 100%.

[0115] The results are shown in Table 2. While the bacterial count increased significantly with increasing corn steep liquor concentration, the sporulation rate decreased significantly when the corn steep liquor concentration exceeded 3%. Furthermore, increasing the corn steep liquor concentration did not significantly increase the bacterial count in the fermentation broth, but increased costs, which was not economically viable. Therefore, selecting 3% corn steep liquor as the nitrogen source for Bacillus belyss BB-1 fermentation is more reasonable.

[0116] Table 2. Effects of corn steep liquor on the cell count and sporulation rate of Bacillus belyssus BB-1

[0117] Note: The bacterial count is the number of colonies produced by coating the unheated bacterial solution.

[0118] Example 14: Investigation of cell mass and sporulation rate of Bacillus belyss BB-1 in ton-scale fermentation Bacillus belye BB-1 strain was inoculated onto activation medium and cultured at 37°C for 16 h. It was then inoculated onto seed medium and cultured at 37°C until OD (Organic Depth) was reached. 600 When the OD value is 2.5, the seed culture is obtained and inoculated into a primary seed tank containing seed culture medium at a 1% inoculation rate. The culture is then incubated at 37°C until the OD value reaches 2.5. 600 When the OD value is 2.5, inoculate 3% into a secondary seed tank containing seed culture medium and culture at 37°C until the OD value reaches 2.5. 600 When the concentration reaches 2.5, inoculate 10% of the culture medium from Example 4 (30 ml). 3 In the fermentation broth, the initial pH was 6.8, and the aeration rate was 180–360 m³ / h. 3The stirring speed was 30-70 rpm, and the dissolved oxygen was controlled at 30% by controlling the stirring and aeration rate. The total fermentation time was 35 hours (fermentation at 37℃). 2 mL of the above culture broth was serially diluted with sterile water, and 0.1 mL of the diluted solution was spread onto counting medium. Another 2 mL of the same culture broth was incubated in an 80℃ water bath for 20 minutes, followed by another serial dilution with sterile water, and 0.1 mL of the diluted solution was spread onto counting medium. The culture was incubated at 28℃ for 24 hours. Plates with colony counts between 30 and 300 CFU and no spreading colony growth were selected, with three replicates per group. The colony count and sporulation rate were calculated.

[0119] Sporulation rate = Number of colonies produced by coating with heated bacterial solution ÷ Number of colonies produced by coating with unheated bacterial solution × 100%.

[0120] This example demonstrates a comparative scale-up experiment of Bacillus belyss BB-1 using a ton-scale fermentation unit. The final cell count in the fermentation broth (the cell count is the number of colonies produced by coating the unheated bacterial solution) reached 2.2 × 10⁻⁶. 10 The concentration of cfu / mL and the sporulation rate reached 97%, compared to the 1×10⁻⁶ colony count obtained by existing ton-scale fermentation. 9 Compared to previous versions, the cfu / mL ratio shows a significant improvement. Furthermore, the use of inexpensive molasses and corn steep liquor instead of more expensive raw materials such as glucose and yeast powder reduces costs. Therefore, this invention demonstrates that the Bacillus BB-1 fermentation broth provided is suitable for ton-scale fermentation equipment and can meet the production demands of industry.

[0121] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

[0122] SEQ ID NO: 1

Claims

1. A strain of Bacillus belyss, named Bacillus belyss BB-1, is classified as Bacillus belyss ( Bacillus velezensis It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 11, 2026, with accession number CGMCC NO.39202.

2. A culture obtained by culturing Bacillus belye as described in claim 1.

3. The culture according to claim 2, characterized in that, The culture was obtained by the following method: inoculating the Bacillus berleis described in claim 1 into a Bacillus berleis culture medium and culturing it to obtain the culture. Preferably, the Bacillus belye culture medium comprises the following components: molasses, corn steep liquor, calcium salts, and magnesium salts; Preferably, the calcium salt includes at least one selected from calcium carbonate, calcium sulfate, calcium chloride, calcium phosphate, and calcium citrate; further comprising calcium chloride; Preferably, the magnesium salt comprises at least one of magnesium chloride and magnesium sulfate; further comprising magnesium chloride; Preferably, the molasses content in the culture medium is 0.05-20% by mass; more preferably 0.1-10%; even more preferably 1-6%; and still more preferably 4-6%. Preferably, the content of the corn steep liquor in the culture medium is 0.05-10% by mass; more preferably 0.1-5%; even more preferably 1-5%; and still more preferably 2.5-3.5%. Preferably, the calcium salt content in the culture medium is 0.01-0.2% by mass; more preferably 0.01-0.1%; even more preferably 0.04-0.08%; and still more preferably 0.05-0.07%. Preferably, the content of the magnesium salt in the culture medium is 0.05-1% by mass; further, 0.1-1%; even further, 0.2-0.6%; and still further, 0.3-0.5%.

4. A microbial agent comprising: The Bacillus belyssus of claim 1 or the culture of any one of claims 2-3.

5. A composition comprising: Bacillus belye as claimed in claim 1, the culture as claimed in any one of claims 2-3, or the bacterial agent as claimed in claim 4; and Additional surfactants and / or excipients; Preferably, the adjuvant activator includes one or more biocontrol agents, one or more chemical agents, one or more fertilizers, one or more herbicides, one or more growth promoters, or any combination thereof; Preferably, the excipients are selected from agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film-forming agents, spontaneous growth promoters, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, or any combination thereof.

6. The use of Bacillus belye as described in claim 1, the culture as described in any one of claims 2-3, the inoculant as described in claim 4, or the composition as described in claim 5 in the prevention and control of plant diseases; Preferably, the plant disease is caused by a pathogen; Preferably, the pathogen includes Fusarium graminearum.

7. A Bacillus belyss culture medium, which is the Bacillus belyss culture medium as described in claim 3.

8. The use of the Bacillus belyssus culture medium according to claim 7 in any one of (a1)-a5): a1) Cultivate Bacillus belye; a2) Increase the number of Bacillus belyssus cells; a3) Improve the sporulation rate of Bacillus belyss; a4) Preparation of Bacillus belesi inoculum; a5) Prepare a product, said product being used in any one of a1)-a4); Preferably, the Bacillus belesii is the Bacillus belesii as described in claim 1.

9. A method comprising the step of using the Bacillus belye culture medium of claim 7; The method is used for any one of a1)-a4): a1) Cultivate Bacillus belye; a2) Increase the number of Bacillus belyssus cells; a3) Improve the sporulation rate of Bacillus belyss; a4) Preparation of Bacillus belesi inoculum; Preferably, the method includes the following steps: inoculating the Bacillus belye seed culture into the Bacillus belye culture medium of claim 7, and fermenting; Preferably, the inoculum size of the Bacillus belyceae seed solution is 1-15%; more preferably, it is 9-11%. Preferably, the fermentation temperature is 25-40°C; more preferably, it is 35-39°C. Preferably, the fermentation time is 35-40 hours; Preferably, the dissolved oxygen level during the fermentation process is 1-50%; Preferably, the method for preparing the Bacillus belye seed culture includes the following steps: inoculating activated Bacillus belye into a seed culture medium for a first culture; Preferably, the method for preparing the Bacillus belye seed culture further includes the following steps: inoculating the product of the first culture into a seed culture medium for a second culture, and then inoculating the product of the second culture into a seed culture medium for a third culture; Preferably, the seed culture medium comprises peptone, yeast extract, and sodium chloride; Preferably, the method for preparing the activated Bacillus belye includes the following steps: inoculating Bacillus belye into an activation culture medium and performing a fourth culture; Preferably, the activation culture medium comprises peptone, yeast extract, sodium chloride, and agar powder; Preferably, the Bacillus belesii is the Bacillus belesii as described in claim 1.

10. A Bacillus bellis inoculum agent, comprising Bacillus bellis and the Bacillus bellis culture medium as described in claim 7; Preferably, the Bacillus belesii is the Bacillus belesii as described in claim 1.

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