Bacillus velezensis and application thereof in prevention and treatment of soybean diseases

Abstract

The application relates to the field of microorganisms, and specifically discloses a bacillus velezensis and application thereof in prevention and treatment of soybean diseases. The bacillus velezensis is bacillus velezensis CX-BL2, which is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M20262262, and is preserved on January 28, 2026. The bacillus velezensis CX-BL2 can inhibit the growth of sclerotinia sclerotiorum, rhizoctonia solani, gloeosporium abietinum, fusarium solani, fusarium oxysporum, sclerotium rolfsii, fusarium graminearum and botrytis cinerea, effectively prevents and treats crop sclerotinia disease and root rot, can reduce the number of pesticide applications, saves labor, and provides a new means for safe, green and environment-friendly biological control of plant diseases.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and more specifically, to a Bacillus belyes and its application in the control of soybean diseases. Background Technology

[0002] Soybeans, as an important oilseed crop in my country, are highly susceptible to various pathogens during their growth, directly impacting their yield and quality. Among these, sclerotinia sclerotiorum rot is one of the most destructive diseases affecting soybeans. Sclerotinia sclerotiorum can damage all above-ground parts of the soybean plant, leading to symptoms such as seedling blight, leaf rot, stem rot, and pod rot, which can reduce soybean yield by up to 60% in severe cases. Besides sclerotinia sclerotiorum rot, soybean root rot is also a significant disease affecting soybean yield, causing 25-50% loss. The pathogens causing soybean root rot are complex, mainly including *Pythium*, *Fusarium*, *Phytophthora*, *Rhizoctonia*, *Macrophomina phaseolina*, and *Sclerotium rolfsii*, among others.

[0003] In field production, soybean diseases such as sclerotinia stem rot and root rot often occur simultaneously, and current production mainly relies on chemical pesticides for control. However, applying chemical agents separately to different pathogens not only increases the workload and production costs, but also easily leads to problems such as pathogen resistance, excessive pesticide residues, and environmental pollution. Therefore, developing efficient, safe, and green biological control methods that can simultaneously inhibit multiple soybean pathogens is of great significance for ensuring the healthy and sustainable development of my country's soybean industry.

[0004] Bacillus velezensis, a beneficial microorganism with good biocontrol and lithium intercalation properties, has received widespread attention in the field of plant disease and pest control in recent years. Studies have shown that this bacterium can produce a variety of antibacterial active substances, exhibiting significant antagonistic effects against various plant pathogenic fungi and bacteria. However, systematic research and application of Bacillus velezensis in soybean disease control, especially in the simultaneous control of soybean sclerotinia rot caused by Sclerotinia sclerotiorum and soybean root rot caused by multiple pathogens such as Pythium and Fusarium, remain relatively limited. Therefore, screening for Bacillus velezensis strains with broad-spectrum antagonistic effects against major soybean pathogens and developing their application technologies in soybean disease and pest control has significant practical implications and broad application prospects. Summary of the Invention

[0005] In order to prevent and control diseases such as sclerotinia stem rot and root rot in soybeans, this application provides a Bacillus belyssus and its application in the prevention and control of soybean diseases.

[0006] In a first aspect, this application provides a Bacillus belyssus bacillus, employing the following technical solution: A type of Bacillus velezensis, specifically Bacillus velezensis CX-BL2, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20262262 and deposit date of January 28, 2026.

[0007] By adopting the above technical solution, Bacillus belyss CX-BL can inhibit pathogenic fungi and / or pathogenic bacteria, mainly Pythium, Fusarium, Phytophthora, Rhizoctonia, Macropomina phaseolina, and Sclerotium rolfsii.

[0008] Secondly, this application provides a microbial inoculant, which adopts the following technical solution: A microbial agent comprising Bacillus belye CX-BL2.

[0009] Preferably, the effective viable count of Bacillus cyclophosphamide CX-BL2 in the microbial agent is (1-1.5) × 10⁻⁶. 10 FU / g.

[0010] Preferably, the microbial agent is an aqueous suspension, granules, or powder.

[0011] By employing the above-mentioned technical solution, *Bacillus belye* CX-BL2 can be cultured, and with the addition of adjuvants and appropriate methods, microbial agents can be obtained. For example, the culture can be concentrated into a high-concentration liquid semi-finished product using vacuum film concentration, which can then be used to further prepare liquid formulations. Alternatively, solid semi-finished products can be obtained using methods such as ventilation drying or spray drying. The resulting solid semi-finished product can be pulverized and used to prepare the target formulation. The prepared microbial agents can be used for soil surface treatment or foliar spraying before crop seedling disease onset, effectively controlling diseases.

[0012] Preferably, the microbial agent is obtained through the following steps: The Bacillus belye CX-BL2 was fermented to obtain the fermentation product; The fermentation product and wettable powder are mixed in a mass ratio of 1:1-3 to prepare the microbial inoculant.

[0013] Preferably, when preparing the microbial agent, at least one of the following is added: adjuvant, surfactant, carrier, dispersant, wetting agent, penetrant, spreading agent, adhesive, defoamer, foaming agent, thickener, stabilizer and film-forming agent.

[0014] Preferably, the additives include at least one of carboxymethyl cellulose, polyethylene glycol or gelatin, gum arabic, and xanthan gum; The surfactant includes at least one of alcohol sulfonates, alkyl aryl sulfonates, lignin sulfonates, polyoxyethylene glycol ethers, polyoxyethylene alkyl aryl ethers, and polyoxyethylene sorbitol monoalkyl esters. The carrier is selected from at least one of sawdust, talc, bentonite, clay, kaolin, diatomaceous earth, white carbon black, vermiculite, quicklime, silica sand, industrial glucose, water, isopropanol, and glycerol.

[0015] Preferably, the fermentation culture includes: activating and culturing Bacillus belye CX-BL2 to obtain a seed culture; The seed culture was inoculated onto a culture medium and cultured on a large scale to obtain the fermentation product.

[0016] Preferably, the solvent of the culture medium used for activation culture is water, and the culture medium formula is: sucrose 0.4-0.6%, corn starch 1.5-1.8%, soybean meal 1.2-1.5%, and calcium carbonate 0.2-0.5%.

[0017] Preferably, the culture medium used for the expanded culture has the following formulation: 3-5 g / L beef extract, 3-5 g / L soybean peptone, 18-20 g / L glucose, and pH 7.

[0018] Thirdly, this application provides the application of Bacillus vesiculosus or microbial agents in the prevention and control of soybean diseases.

[0019] Fourthly, this application provides a microbial fertilizer, which adopts the following technical solution: A microbial fertilizer, made using Bacillus belye or a microbial inoculant, comprises the following steps: Protein-containing waste and sugar-containing waste are mixed, water is added and they are ground into a slurry to obtain a fermentation substrate; Microbial agents or Bacillus cyclohexane CX-BL2 cultured after fermentation were activated and inoculated into the fermentation substrate. The pH was adjusted to 5-7, fermented for 24-120 h, and then concentrated to obtain the concentrated liquid. The concentrated liquid was mixed evenly with sodium butylnaphthalene sulfonate, calcium lignosulfonate and kaolin to prepare microbial fertilizer.

[0020] By adopting the above technical solution Preferably, the mass ratio of the concentrate, sodium butylnaphthalene sulfonate, calcium lignosulfonate, and kaolin is 1:0.5:0.5:8.

[0021] Preferably, the protein-containing by-product is selected from at least one of dried bean curd residue, oil pressing residue, tofu residue, soy sauce residue, and distiller's grains; The sugar-containing by-products include at least one of dried tangerine peel residue, tea residue, bagasse, and furfural residue.

[0022] Preferably, the inoculum amount of the microbial agent or the fermented Bacillus vesiculosus is 0.1-10% of the fermentation substrate mass.

[0023] Preferably, the mass ratio of fermentation substrate to water is 1:0.2-1.5; and the fermentation temperature is 30-45℃.

[0024] In summary, this application has the following beneficial effects: The Bacillus cyclohexenes CX-BL2 strain screened in this application has a highly effective antagonistic effect against Sclerotinia sclerotiorum and can effectively control root rot. It can inhibit plant pathogens and prepare products for inhibiting plant pathogens and preventing diseases caused by plant pathogens. Detailed Implementation

[0025] The present application will be further described in detail below with reference to the embodiments. Example

[0026] In the following examples, the formulations of LB solid culture medium are as follows: per 1L volume, it includes 10g tryptone, 5g yeast extract, 10g NaCl, and 15g agar; the formulations of LB liquid culture medium are as follows: per 1L volume, it includes 10g tryptone, 5g yeast extract, and 10g NaCl; PDA solid culture medium: per 1L volume, it includes 200g potato, 20g glucose, and 15g agar, at natural pH; PDA liquid culture medium: per 1L volume, it includes 200g potato and 20g glucose, at natural pH; NA (Nutrient Agar) culture medium: per 1L of deionized water, it contains 10g tryptone, 3g beef meal, 5g sodium chloride, and 13g agar, stirred evenly, adjusted to pH=7.0±0.2 with NaOH, and sterilized at 121℃ for 20min.

[0027] Example 1: Isolation and identification of Bacillus belyssus CX-BL2: (1) Sample collection: Rhizosphere soil from healthy plants was collected from Jiaxiang, Jining, Shandong, an area with a high incidence of soybean root rot. The soil was mixed evenly and ensured that there were no clumps, and used as soil samples.

[0028] (2) Strain isolation: Weigh 10g of soil sample and add it to an Erlenmeyer flask containing 90mL of sterile water. Place the Erlenmeyer flask in a constant temperature shaker at 150r / min and 28℃ for 30min to obtain a sample suspension. Use the gradient dilution method to isolate antagonistic bacteria. Take the samples at dilution factor 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Six gradient dilutions were prepared, with 100 μL of each dilution evenly spread onto LB agar plates. Each gradient was repeated three times. The plates were incubated at 28°C for 48 hours. Single colonies of bacteria with different morphologies were picked using a sterile inoculation loop, transferred to new LB agar plates, and then incubated at 28°C for 48 hours. If only one type of colony morphology was observed on the LB agar plates after incubation, the isolation and purification were considered complete. The purified LB agar plates were then stored at 4°C for later use.

[0029] (3) Strain screening: 3.1 Activate the isolated and purified strains, inoculate them into LB liquid medium, and place the inoculated LB liquid medium in a constant temperature shaker at 150 r / min and 28℃ for 24 h; after the culture, the OD600 of the strains in the LB liquid medium reaches more than 1.2, and the test bacterial solution is obtained; 3.2. *Sclerotinia sclerotiorum*, *Bacillus sclerotiorum*, *Rhizoctonia solani*, and *Bacillus bengalus* were activated on PDA solid medium plates to obtain activated pathogen plates. A sample was taken from the activated pathogen plate using a perforator to obtain pathogen blocks. These pathogen blocks were placed in the center of a blank PDA solid medium plate. Two 6mm sterile filter paper discs were placed parallel to each other 20mm from the center of the PDA solid medium plate. 10μL of the activated test bacterial solution was added to one filter paper disc as the test sample, and 10μL of sterile PDA liquid medium was added to the other filter paper disc as the control sample. The PDA solid medium plates were incubated at 28℃ for 72h. The antagonistic effect was observed, and the size of the inhibition zone was measured. The length of the inhibition zone (cm) was the distance between *Bacillus bengalus* CX-BL2 and the fungus, as detailed in Table 1.

[0030] Table 1. Inhibition band length of Bacillus belyss CX-BL2 against different pathogens

[0031] As shown in Table 1, a strain was finally obtained that showed significant inhibitory effects on Sclerotinia sclerotiorum, Sclerotinia sclerotiorum, Rhizoctonia solani, and Coccidioidomyces beanus. The strain was named Bacillus belyi CX-BL2. Bacillus belyi CX-BL2 was cultured in LB liquid medium and then stored in glycerol at -80℃.

[0032] (4) Strain identification 4.1 Morphological Characteristics: *Bacillus belyeceae* CX-BL2 was cultured on LB solid medium. Colony morphology was observed after 36 hours of culture, following the methods described in *Common Bacterial Systematic Identification Manual* (Dong Xiuzhu et al., 2001) and *Berge's Manual of Bacterial Identification* (Buchanan et al., 1984). Microscopic morphology was observed using 2.5% glutaraldehyde for sample fixation, followed by dehydration with 20%-100% ethanol seven times, 10 minutes each time. Scanning electron microscopy was performed after dehydration.

[0033] Bacillus vesiculosus CX-BL2 is a short rod-shaped, pale yellow bacterium with regular, nearly round, slightly convex edges, a moist surface, and is opaque. It is a Gram-positive bacterium.

[0034] 4.2 Physiological and biochemical characteristics: The physiological and biochemical characteristics of Bacillus bereaves CX-BL2 were identified using micro-bacterial biochemical identification tubes. All identification tubes were purchased from Qingdao Haibo Biotechnology Co., Ltd. The specific operating methods were in accordance with the instruction manual and the relevant methods in Berger's Manual of Bacterial Identification (8th Edition) were used for testing. See Table 2 for details.

[0035] Table 2 Physiological and chemical characteristics of Bacillus belyss CX-BL2

[0036] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0037] In summary, based on the above analysis of morphological characteristics, physiological and biochemical properties, and molecular biological identification, *Bacillus velezensis* CX-BL2 was identified as *Bacillus velezensis*. It has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M2026262.

[0038] Example 2: A microbial inoculant, in powder form, prepared as follows: (1) Activation culture: Bacillus CX-BL2 preserved in glycerol at -80℃ was inoculated onto NA solid medium and activated in a constant temperature incubator at 30℃ for 24h. Then, a single colony was selected and inoculated into 100mL of water-based medium and activated in a constant temperature incubator at 30℃ and 180rpm for 24h to obtain seed culture. The water-based medium formula was: 0.6% sucrose, 1.8% corn starch, 1.2% soybean meal and 0.5% calcium carbonate.

[0039] (2) Expanded culture: The concentration was 1×10 7 The seed culture at a concentration of CFU / mL was inoculated onto the expansion culture medium at an inoculum size of 5%. The culture was incubated for 72 h at 30℃, 300 rpm, 0.05 MPa, and an aeration rate of 0.4 (V / V·min) to obtain the fermentation product. The effective bacterial concentration of the fermentation product was 1.5 × 10⁻⁶. 10 The culture medium, with a concentration of CFU / mL, consisted of 5g beef extract, 3g / L soybean peptone, and 20g / L glucose, with a pH of 7, and was prepared by autoclaving at 120°C for 0.5h.

[0040] (3) The fermentation product and wettable powder (talc) are mixed at a mass ratio of 1:3 to obtain a blend. 2% of the blend mass of wetting agent (sodium dodecylbenzenesulfonate) and 2% of dispersant (sodium methylene bisnaphthalenesulfonate) are added, mixed evenly, and spray-dried to obtain a powdered microbial agent. During spray drying, the inlet air temperature is 180℃, the outlet air temperature is 65℃, and the feed rate is 3000ml / h.

[0041] Example 3: A microbial inoculant, an aqueous suspension, prepared as follows: (1) Activation culture: Bacillus CX-BL2 preserved in glycerol at -80℃ was inoculated onto NA solid medium and activated in a constant temperature incubator at 30℃ for 24h. Then, a single colony was selected and inoculated into 100mL of water-based medium and activated in a constant temperature incubator at 30℃ and 180rpm for 24h to obtain seed culture. The water-based medium formula was: 0.6% sucrose, 1.8% corn starch, 1.2% soybean meal and 0.5% calcium carbonate.

[0042] (2) Expanded culture: The concentration was 1×10 7 The seed culture at a concentration of CFU / mL was inoculated onto the expansion culture medium at an inoculum size of 5%. The culture was incubated for 72 h at 30℃, 300 rpm, 0.05 MPa, and an aeration rate of 0.4 (V / V·min) to obtain the fermentation product. The effective bacterial concentration of the fermentation product was 1.5 × 10⁻⁶. 10The culture medium, with a concentration of CFU / mL, consisted of 5g beef extract, 3g / L soybean peptone, and 20g / L glucose, with a pH of 7, and was prepared by autoclaving at 120°C for 0.5h.

[0043] (3) The fermentation product and wettable powder (talc) are mixed at a mass ratio of 1:3 to obtain a blend. 0.05% xanthan gum, 0.05% coconut oil and 0.1% wetting agent (sodium dodecyl sulfate) of the blend are added and mixed evenly to obtain a water suspension microbial agent.

[0044] Example 4: A microbial inoculant, in granule form, prepared as follows: (1) Activation culture: Bacillus CX-BL2 preserved in glycerol at -80℃ was inoculated onto NA solid medium and activated in a constant temperature incubator at 30℃ for 24h. Then, a single colony was selected and inoculated into 100mL of water-based medium and activated in a constant temperature incubator at 30℃ and 180rpm for 24h to obtain seed culture. The water-based medium formula was: 0.6% sucrose, 1.8% corn starch, 1.2% soybean meal and 0.5% calcium carbonate.

[0045] (2) Expanded culture: The concentration was 1×10 7 The seed culture at a concentration of CFU / mL was inoculated onto the expansion culture medium at an inoculum size of 5%. The culture was incubated for 72 h at 30℃, 300 rpm, 0.05 MPa, and an aeration rate of 0.4 (V / V·min) to obtain the fermentation product. The effective bacterial concentration of the fermentation product was 1.5 × 10⁻⁶. 10 The culture medium, with a concentration of CFU / mL, consisted of 5g beef extract, 3g / L soybean peptone, and 20g / L glucose, with a pH of 7, and was prepared by autoclaving at 120°C for 0.5h.

[0046] (3) The fermentation product and wettable powder (talc) are mixed at a mass ratio of 1:3 to obtain a blend. 5% light calcium carbonate, 2% wetting agent (sodium dodecylbenzenesulfonate), 2% dispersant (sodium methylene bisnaphthalenesulfonate), 1% industrial glucose and 15% sawdust are added to the blend. The mixture is then pulverized, and an appropriate amount of water is added for extrusion granulation. The mixture is then dried in an oven at 50°C for 2 hours to obtain granular microbial inoculant.

[0047] Example 5: A microbial fertilizer, prepared by the following method: Protein-containing by-products (soy sauce residue) and sugar-containing by-products were mixed in a 1:1 mass ratio to obtain the fermentation substrate. The main components of the soy sauce residue were as follows: moisture 7.2%, salt 2.7%, fat 8.9%, SDF 1.8%, crude fiber 28.4%, crude protein 26.6%, and ash 23.19%. The main components of the tea residue were as follows: moisture 5.43%, theanine 5.22 g / kg, tea polyphenols 9.32%, and crude protein 17.56%. The powdered microbial agent prepared in Example 2 was diluted with sterile water to prepare 10... 6 -10 10 Take 0.1 mL of the solution and spread it evenly on a NA plate. Incubate at 28°C for 48 h. Then inoculate it into the fermentation substrate, adjust the pH to 7, ferment for 24 h, and concentrate it to 1 / 3 of the original volume to obtain the concentrated solution. 10g of concentrated liquid was mixed evenly with 5g of sodium butylnaphthalene sulfonate, 5g of calcium lignosulfonate and 80g of kaolin to prepare microbial fertilizer.

[0048] Example 6: A microbial fertilizer, prepared by the following method: Protein-containing by-products (soy sauce residue) and sugar-containing by-products were mixed in a 1:1 mass ratio to obtain the fermentation substrate. The main components of the soy sauce residue were as follows: moisture 7.2%, salt 2.7%, fat 8.9%, SDF 1.8%, crude fiber 28.4%, crude protein 26.6%, and ash 23.19%. The main components of the tea residue were as follows: moisture 5.43%, theanine 5.22 g / kg, tea polyphenols 9.32%, and crude protein 17.56%. The aqueous suspension of microbial agent prepared in Example 3 was diluted with sterile water to prepare 10... 6 -10 10 Take 0.1 mL of the solution and spread it evenly on a NA plate. Incubate at 28°C for 48 h. Then inoculate it into the fermentation substrate, adjust the pH to 7, ferment for 24 h, and concentrate it to 1 / 3 of the original volume to obtain the concentrated solution. 10g of concentrated liquid was mixed evenly with 5g of sodium butylnaphthalene sulfonate, 5g of calcium lignosulfonate and 80g of kaolin to prepare microbial fertilizer.

[0049] Example 7: A microbial fertilizer, prepared by the following method: Protein-containing by-products (soy sauce residue) and sugar-containing by-products were mixed in a 1:1 mass ratio to obtain the fermentation substrate. The main components of the soy sauce residue were as follows: moisture 7.2%, salt 2.7%, fat 8.9%, SDF 1.8%, crude fiber 28.4%, crude protein 26.6%, and ash 23.19%. The main components of the tea residue were as follows: moisture 5.43%, theanine 5.22 g / kg, tea polyphenols 9.32%, and crude protein 17.56%. The granular microbial agent prepared in Example 4 was diluted with sterile water to prepare 10... 6 -10 10 Take 0.1 mL of the solution and spread it evenly on a NA plate. Incubate at 28°C for 48 h. Then inoculate it into the fermentation substrate, adjust the pH to 7, ferment for 24 h, and concentrate it to 1 / 3 of the original volume to obtain the concentrated solution. 10g of concentrated liquid was mixed evenly with 5g of sodium butylnaphthalene sulfonate, 5g of calcium lignosulfonate and 80g of kaolin to prepare microbial fertilizer.

[0050] Example 8: A microbial fertilizer, prepared by the following method: (1) Activation culture: Bacillus CX-BL2 preserved in glycerol at -80℃ was inoculated onto NA solid medium and activated in a constant temperature incubator at 30℃ for 24h. Then, a single colony was selected and inoculated into 100mL of water-based medium and activated in a constant temperature incubator at 30℃ and 180rpm for 24h to obtain seed culture. The water-based medium formula was: 0.6% sucrose, 1.8% corn starch, 1.2% soybean meal and 0.5% calcium carbonate.

[0051] (2) Expanded culture: The concentration was 1×10 7 The seed culture at a concentration of CFU / mL was inoculated onto the expansion culture medium at an inoculum size of 5%. The culture was incubated for 72 h at 30℃, 300 rpm, 0.05 MPa, and an aeration rate of 0.4 (V / V·min) to obtain the fermentation product. The effective bacterial concentration of the fermentation product was 1.5 × 10⁻⁶. 10 The culture medium, with a concentration of CFU / mL, consisted of 5g beef extract, 3g / L soybean peptone, and 20g / L glucose, with a pH of 7, and was prepared by autoclaving at 120°C for 0.5h.

[0052] (3) Protein-containing by-products (soy sauce residue) and sugar-containing by-products were mixed in a 1:1 mass ratio to obtain a fermentation substrate. The main components of the soy sauce residue were as follows: moisture 7.2%, salt 2.7%, fat 8.9%, SDF 1.8%, crude fiber 28.4%, crude protein 26.6%, and ash 23.19%. The main components of the tea residue were as follows: moisture 5.43%, theanine 5.22 g / kg, tea polyphenols 9.32%, and crude protein 17.56%. (4) Dilute the fermentation product obtained in step (2) with sterile water to prepare 10 6 -10 10 Take 0.1 mL of the solution and spread it evenly on a NA plate. Incubate at 28°C for 48 h. Then inoculate it into the fermentation substrate, adjust the pH to 7, ferment for 24 h, and concentrate it to 1 / 3 of the original volume to obtain the concentrated solution. (5) Mix 10g of concentrated liquid with 5g of sodium butylnaphthalene sulfonate, 5g of calcium lignosulfonate and 80g of kaolin to obtain microbial fertilizer.

[0053] Performance testing I. Inhibitory effect of Bacillus belyceta CX-BL2 on sclerotinia stem rot (1) The antibacterial effect of Bacillus belyi CX-BL2 on Sclerotinia sclerotiorum on isolated soybean leaves Take 3g of the microbial inoculant prepared in Examples 2-4, and dilute it in 30mL of sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL, yielding treatment solution 1, treatment solution 3, and treatment solution 4.

[0054] Each detached soybean leaf was treated with a microbial inoculant solution, approximately 10 μL per leaf. Water-treated leaves served as controls. Each treatment was repeated four times (four replicates refer to four soybean plants, with three detached true leaves selected from each soybean plant). After the leaves dried, a 0.6 cm diameter block of Sclerotinia sclerotiorum NJ13 mycelium was inoculated at the same location on the right side of the leaf aorta. A small amount of water was then sprayed onto the leaves. The soybean leaves were placed in a plastic box, sealed with plastic wrap, and cultured in a 22°C incubator with light / dark (12h / 12h) humidity control for 7 days. The size of the lesions was recorded, as shown in Table 4.

[0055] Table 4

[0056] The results showed that 7 days after inoculation, when Sclerotinia sclerotiorum was inoculated under water treatment conditions, the bacteria could normally infect soybean leaves, and the lesion diameter reached 12.31 mm. However, when soybean leaves were treated with a microbial agent containing Bacillus belyss CX-BL2 and then inoculated with Sclerotinia sclerotiorum, the infection of the bacteria on soybean leaves was significantly limited, and the lesion diameter was controlled below 9.14 mm.

[0057] (2) The antibacterial effect of Bacillus belyss CX-BL2 on Sclerotinia sclerotiorum in live soybean. Take 3g of the microbial inoculant prepared in Examples 2-4, and dilute it in 30mL of sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL, treatment solutions 1, 3, and 4 were obtained. Water was used as a control. The treatment solutions and water were sprayed onto the leaves of living soybean plants with similar growth. After the liquid dried, a 0.6 cm diameter mycelial block of Sclerotinia sclerotiorum NJ13 was inoculated at the same position on the right side of the main vein of the leaf. Two leaves were inoculated per plant, and four parallel replicates were performed. The plants were incubated at 20℃ with humidity. The size of the lesions was recorded after 7 days, as shown in Table 5.

[0058] Table 5. Control efficacy of Bacillus belye CX-BL2 against sclerotinia stem rot in live soybean leaves.

[0059] It is evident that microbial agents containing Bacillus belye CX-BL2 can prevent Sclerotinia sclerotiorum infection on living soybean plants.

[0060] (II) Inhibitory effect of Bacillus belyi CX-BL2 on Fusarium solani in live soybean. After growing Fusarium oxysporum on PDA plates for 5 days, 5 mycelial cakes were punched using a sterile puncher and placed in 1 LPD medium for 7 days of shaking incubation. The medium was then diluted to 1:10 with sterile water. 6 One spore / mL for use.

[0061] Take 3g of the microbial inoculant prepared in Examples 2-4, and dilute it in 30mL of sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL, yielding treatment solution 1, treatment solution 3, and treatment solution 4.

[0062] At the 2-3 true leaf stage of soybeans, a small wound was made at the base of the stem using an inoculation needle, and 10 mL of the prepared Fusarium solani fermentation broth was poured in. Each replicate consisted of 15 seedlings, and the treatment was repeated 3 times. A control group was inoculated with 10 mL of sterile water. After inoculation, the plants were placed in a humidified glass room and cultured conventionally at 30℃ and 80%-90% humidity for 24-36 hours. Then, 10 mL of the treatment solution was poured into the base of the soybean stem, and the plants were placed in the glass room for another 7 days until the pathogen control showed complete disease development. The disease index and control efficacy were then investigated, and the disease grading standards were as follows (using a 0-4 scale). The disease control treatment was inoculated only with the Fusarium solani fermentation broth; the healthy control treatment was inoculated with an equal volume of water as the bacterial solution. The specific test results are shown in Table 6.

[0063] 0 = Healthy plant, no symptoms; 1 = Healthy above-ground parts of the plant, with visible brown symptoms only around the base of the stem; 2 = Obvious dark brown symptoms at the base of the stem, with yellowing leaves or curled or drooping leaves on the upper part of the plant; 3 = Severe yellowing of leaves or wilting of the above-ground parts of the plant, with severe rot at the base of the stem; 4 = The entire plant withers or dies.

[0064] Disease index = ∑(number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100.

[0065] Prevention and control effect (%) = [(disease index of disease control - disease index of each treatment) / disease index of disease control] × 100.

[0066] Table 6. Control efficacy of Bacillus belye CX-BL2 against Fusarium oxysporum f. sp. solanum rot.

[0067] It can be seen that 7 days after inoculation, the pathogen control showed full disease development, with soybean roots turning red, epidermis cracking, obvious browning of the main root and lateral roots, reddening spreading, and lateral roots falling off, with a disease index of 71.25%. The incidence of soybean root rot was significantly reduced after treatment with microbial agent containing Bacillus belyss CX-BL2, with a control efficacy of over 83%.

[0068] (III) Tests on the efficacy of microbial fertilizers against sclerotinia stem rot Experimental plots were selected in Anhui Province and randomly divided into subplots for the experiment. Water spraying served as the negative control group, 50% carbendazim as the positive control group, and microbial fertilizer spraying (prepared in Examples 5-8) served as the treatment group. The fertilizer was sprayed twice at different stages of soybean growth, namely the branching stage and the flowering stage.

[0069] The dosage of microbial fertilizer was 100g / 30L water / acre, and the dosage of prochloraz was 562.5ppm. The incidence of sclerotinia rot was recorded during the soybean maturity period, and soybean plant yields were measured.

[0070] The experimental field was divided into 5 plots, and each treatment was repeated 3 times. The spraying areas were randomly distributed. The disease incidence was measured in each plot using a five-point sampling method. 100 soybean plants were randomly selected from each point to count the disease incidence. 5 points were selected from each plot to measure the planting density (transplanted field). 5 points were randomly selected from the diagonal, and 10 soybean plants were selected from each point to measure the number of effective pods per plant, the number of grains per pod, and the weight of 1,000 grains.

[0071] The disease grading standards are as follows (using a 0-4 level rating scale): 0 = Healthy plant, no symptoms; 1 = Healthy above-ground parts of the plant, with visible brown symptoms only around the base of the stem; 2 = Obvious dark brown symptoms at the base of the stem, with yellowing leaves or curled or drooping leaves on the upper part of the plant; 3 = Severe yellowing of leaves or wilting of the plant above ground, with severe rot at the base of the stem; 4 = The entire plant withers or dies.

[0072] Incidence rate (%) = (number of infected plants / 1000) × 100.

[0073] Disease index = ∑(number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100.

[0074] Soybean conventional yield (kg / hm2) = (number of plants per hectare × number of effective siliques per plant × number of grains per silique × weight of 1000 grains × yield coefficient) / (1000 × 1000).

[0075] Table 7. Control effect of microbial fertilizer on sclerotinia stem rot

[0076] As can be seen from the data in Table 7, using microbial fertilizer containing Bacillus cyclohexane CX-BL2 for field control of soybeans can effectively reduce the incidence of sclerotinia rot and root rot, and improve yield.

[0077] (iv) Tests on the efficacy of microbial fertilizers against root rot The efficacy of microbial fertilizer in controlling soybean root rot was verified using potted soybean seedlings (Dongdou 16). The seedlings were planted in sterile soil that had undergone high-pressure steam sterilization. The soil at the base of the soybean stem was removed, and micro-wounds were created by pricking the main root about 1 cm below the stem using a sterile inoculation needle 15 times. 1 mL of a 10% concentration was then inoculated. 5 A CFU / mL suspension of soybean root rot fungus spores was injected into the soil along the main root of soybeans for root rot inoculation.

[0078] Dilute the microbial fertilizer with sterile water to a viable bacteria count of 10. 7 CFU / mL was used as the treatment solution. Then, 1 mL of the treatment solution was inoculated into soybean potted seedlings in a suspension of root rot fungus spores. The control group was inoculated with 1 mL of sterile aqueous solution. After inoculation, all soybeans were placed at room temperature (25℃, photoperiod: 12h light: 12h darkness).

[0079] Forty days after treating soybean potted seedlings, the disease status of soybean roots was observed. Each soybean line was divided into five equal segments, and the severity of each segment was graded. The results were statistically analyzed, and the disease index and control effect were calculated. The severity grading standards for soybean root rot are as follows: Grade 0: Healthy; Grade I: Discoloration of the root cortex, cortex not detached; Grade II: Browning of the xylem, cortex detached; Grade III: Blackening of the xylem, cortex detached; Grade IV: Rotting and softening of the xylem, cortex detached.

[0080] Disease severity index = ∑(number of disease severity levels × representative level value) / (total number of levels × representative value of the highest severity level) × 100.

[0081] Prevention and control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0082] Table 8

[0083] Therefore, when using sterile water as the control group, soybean roots were severely affected, with the entire root cortex rotting and peeling off, and the xylem turning black and soft, with a disease index reaching 92.51. However, when soybeans were treated with microbial fertilizer containing Bacillus belye CX-BL2, the disease was mainly concentrated at the lower part of the root or the tip of the fibrous roots, with no disease in the middle and upper parts. The cortex peeled off slightly, and the xylem turned slightly brown, with the disease index controlled below 15.84, demonstrating a better control effect on root rot.

[0084] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A type of Bacillus belesii, characterized in that, The Bacillus velezensis mentioned is Bacillus velezensis CX-BL2, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20262262 and deposit date of January 28, 2026.

2. A microbial inoculant, characterized in that, Includes Bacillus cyclophosphamide CX-BL2 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The effective viable count of Bacillus cyclophosphamide CX-BL2 in the microbial agent is (1-1.5)×10⁻⁶. 10 FU / g.

4. The microbial agent according to claim 2, characterized in that, The microbial agent is an aqueous suspension, granules, or powder.

5. The microbial agent according to claim 2, characterized in that, The microbial inoculant is obtained through the following steps: The Bacillus belye CX-BL2 was fermented to obtain the fermentation product; The fermentation product and wettable powder are mixed in a mass ratio of 1:1-3 to prepare the microbial inoculant.

6. The microbial agent according to claim 5, characterized in that, The fermentation culture includes: activating and culturing Bacillus belye CX-BL2 to obtain a seed culture; The seed culture was inoculated onto a culture medium and cultured on a large scale to obtain the fermentation product.

7. The microbial agent according to claim 6, characterized in that, The medium used for activation culture is water as the solvent, and the medium formula is: sucrose 0.4-0.6%, corn starch 1.5-1.8%, soybean meal 1.2-1.5%, and calcium carbonate 0.2-0.5%.

8. The microbial agent according to claim 6, characterized in that, The culture medium used for the expanded culture was formulated as follows: 3-5 g / L beef extract, 3-5 g / L soybean peptone, 18-20 g / L glucose, with a pH of 7.

9. The application of Bacillus berberis as described in claim 1 or the microbial agent as described in any one of claims 2-8 in the prevention and control of soybean diseases.

10. A microbial fertilizer, characterized in that, Prepared using Bacillus belye as described in claim 1 or a microbial agent as described in any one of claims 2-8, comprising the following steps: Protein-containing waste and sugar-containing waste are mixed, water is added and they are ground into a slurry to obtain a fermentation substrate; Microbial agents or Bacillus cyclohexane CX-BL2 cultured after fermentation were activated and inoculated into the fermentation substrate. The pH was adjusted to 5-7, fermented for 24-120 h, and then concentrated to obtain the concentrated liquid. The concentrated liquid was mixed evenly with sodium butylnaphthalene sulfonate, calcium lignosulfonate and kaolin to prepare microbial fertilizer.

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