A soil-borne disease prevention and treatment composite microbial agent based on metabolic orientation and rational compounding, a preparation method and a use method

Abstract

The present application relates to the field of microbial agents, to solve the problem of poor prevention and control of overall and stable effect of existing microbial agents, and provides a soil-borne disease prevention and control composite microbial agent based on metabolic orientation and rational compounding, comprising: bacillus velezensis and streptomyces; the bacillus velezensis is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 36321; the live bacterial number ratio of the bacillus velezensis and the streptomyces is (4-6): 1. The present application optimizes the carbon and nitrogen source ratio, glucose: soybean meal = 3: 1, applies NaCl stress, and directionally regulates the metabolic pathway of bacillus velezensis, so that the iturin yield is increased by 50% compared with conventional fermentation, breaking the bottleneck of insufficient active substance yield of single strain; the bacillus velezensis and the streptomyces are compounded to construct a synergistic mechanism of membrane breaking, killing and immune activation, and the function is complementary and the drug resistance risk is greatly reduced, and the synergistic effect of the two makes the prevention and control effect of pepper bacterial wilt and cucumber fusarium wilt not less than 80%.

Description

Technical Field

[0001] This invention relates to the field of compound microbial agents, and more specifically, to a compound microbial agent for the prevention and control of soil-borne diseases based on metabolic guidance and rational compounding, its preparation method, and its application method. Background Technology

[0002] Soil-borne diseases are highly destructive types of diseases in agricultural production. Among them, bacterial wilt of peppers and wilt of cucumbers are caused by *Ralstonia solanacearum* and *Fusarium oxysporum*, respectively. They are characterized by wide transmission routes, rapid onset, and difficulty in control, and are particularly severe in continuously cropped fields, often leading to yield reductions of 30%-50%, or even total crop failure. Currently, the control of soil-borne diseases still relies mainly on chemical control and crop rotation. However, the long-term use of chemical fungicides can cause soil pollution, excessive pesticide residues in agricultural products, and increased drug resistance in pathogens. Crop rotation, on the other hand, is limited by land resources and difficult to promote in large-scale planting.

[0003] Biocontrol agents have become an important direction for the control of soil-borne diseases due to their environmental friendliness and strong sustainability. Most existing biocontrol agents are based on a single bacterial strain, such as *Bacillus amyloliquefaciens* and *Bacillus subtilis*, whose control mechanism mainly relies on a single antibacterial effect, such as secreting antimicrobial substances or competing for nutrient sites. However, these single-strain agents have significant technical bottlenecks: First, their single control mechanism is insufficient to cope with the complex defense systems of pathogens, such as biofilm barriers, resulting in limited efficacy; second, long-term use of a single strain can easily lead to drug resistance in pathogens, shortening the lifespan of the agent; and third, the colonization stability of the strain in the soil is insufficient, greatly affected by the soil microbial community and physicochemical properties, leading to significant fluctuations in actual application effects.

[0004] Existing patented technologies mostly focus on the screening of single strains or simple compounding, lacking in-depth design of strain metabolic regulation and synergistic mechanisms. For example, some patents enhance the antibacterial activity of single strains by optimizing culture media, but fail to address the issues of drug resistance and single control mechanisms; some compound bacterial agents are merely simple mixtures of strains, lacking clear functional complementarity design, and even exhibiting antagonistic effects between strains, resulting in control efficacy falling short of expectations; furthermore, existing technologies do not employ dual-target design against pathogen biofilm defense and plant immune activation, resulting in an insufficiently comprehensive control strategy.

[0005] Therefore, developing a compound microbial agent that enhances the yield of active substances through metabolic regulation, achieves functional complementarity through rational compounding, and possesses multiple mechanisms such as membrane breaking, bactericidal and immune activation has become the key to breaking through the limitations of existing biocontrol technologies and achieving efficient and sustainable control of soil-borne diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a compound microbial agent for the prevention and control of soil-borne diseases based on metabolism guidance and rational compounding, its preparation method and application method, which solves the problems of poor comprehensiveness and stability of existing microbial agents in terms of prevention and control, and significantly improves the yield of functional active substances.

[0007] The embodiments of the present invention are achieved through the following technical solutions:

[0008] Preservation instructions:

[0009] Bacterial strain name: Bacillus belesii;

[0010] Latin name: Bacillus velezensis;

[0011] Strain number: BSA-05;

[0012] Classification and nomenclature: Bacillus velezensis;

[0013] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0014] The abbreviation for the depository institution is CGMCC.

[0015] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0016] Deposit date: October 23, 2025;

[0017] Registered with the China Museum Collection Center: CGMCC NO. 36321.

[0018] A compound microbial agent for the prevention and control of soil-borne diseases based on metabolism guidance and rational compounding includes: Bacillus bellis and Streptomyces;

[0019] The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321;

[0020] The viable count ratio of Bacillus belyssus and Streptomyces is (4-6):1.

[0021] Preferably, the viable bacterial concentrations of both Bacillus belyi and Streptomyces are not less than 1×10⁻⁶. 9 CFU / g.

[0022] Preferably, the soil-borne disease control compound microbial agent further includes: kaolin; the kaolin accounts for 4wt%-6wt% of the high-stress-resistant compound microbial agent.

[0023] A method for preparing the aforementioned soil-borne disease control compound microbial agent includes the following steps:

[0024] S100. Inoculate Bacillus belye into the induction medium and culture it at 25-35℃ and 150-250rpm for 60-80h with vibration to obtain Bacillus belye fermentation broth.

[0025] The induction medium comprises: 25-35 g / L glucose, 8-12 g / L soybean meal, 15-25 g / L NaCl, and 1.5-2.5 g / L KH2PO4;

[0026] Streptomyces was inoculated into the culture medium and cultured at 25-30℃ and 160-200 rpm for 90-100 h with shaking to obtain Streptomyces bacterial suspension;

[0027] S200, mixed with Bacillus cereus fermentation broth and Streptomyces bacterial broth, is then added to kaolin and spray-dried to obtain a soil-borne disease control compound microbial agent.

[0028] Preferably, the inlet air temperature of the spray drying process is 110-120℃, and the outlet air temperature is 50-60℃.

[0029] Preferably, the culture medium for inoculating Streptomyces includes: 18-24 g / L soluble starch, 0.8-1.2 g / L KNO3, 0.4-0.6 g / L KH2PO4, 0.4-0.6 g / L MgSO4·7H2O, 0.4-0.7 g / L NaCl, and 0.01-0.015 g / L FeSO4·7H2O.

[0030] Preferably, the yield of isochoric acid in the Bacillus bellis fermentation broth is ≥2.5 g / L.

[0031] A method of applying the aforementioned compound microbial agent for controlling soil-borne diseases includes: root irrigation and / or hole application.

[0032] Preferably, the root drenching method includes: performing the first root drenching 7-10 days after planting, and performing the second root drenching 8-12 days later; during root drenching, the soil-borne disease control compound microbial agent is diluted with water to a concentration of 4×10⁻⁶. 9 -6×10 9 CFU / mL, the amount of water used for root irrigation per plant is 150-200 mL.

[0033] Preferably, the method of applying the fungicide in the holes includes: mixing 5-10g of the fungicide with the soil in each hole before planting the plants.

[0034] The present invention has at least the following beneficial effects:

[0035] This invention optimizes the carbon-nitrogen source ratio (glucose:soybean meal = 3:1) and applies NaCl stress to directionally regulate the metabolic pathway of *Bacillus berreatus*, increasing the yield of iturin by 50% compared to conventional fermentation. This enhances the ability to destroy pathogen biofilms and overcomes the bottleneck of insufficient active substance production by single strains. The invention utilizes a complex microbial design: scientifically combining *Bacillus berreatus* and *Streptomyces* to construct a triple synergistic mechanism of biofilm disruption, killing, and immune stimulation. *Bacillus berreatus*'s lipopeptides destroy pathogen biofilms and activate plant immunity, while *Streptomyces*'s antibiotics and hydrolytic enzymes directly kill pathogens. This complementary function significantly reduces the risk of drug resistance. The synergistic effect of both results in a control efficacy of no less than 80% against bacterial wilt in peppers and wilt in cucumbers. This invention features a multi-target control system, targeting the pathogen's defense barriers: biofilms and bacterial activity, while simultaneously activating the plant's own immunity, forming a dual protection of pathogen inhibition and enhanced plant immunity. This more comprehensive control strategy is suitable for complex disease-causing environments such as continuous cropping fields. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a colony culture diagram of Bacillus belyssus. Detailed Implementation

[0038] To make the objectives, methods, and advantages of the embodiments of the present invention clearer, the methods in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0039] Example 1: A compound microbial agent for the prevention and control of soil-borne diseases based on metabolic guidance and rational compounding includes: Bacillus belye and Streptomyces;

[0040] The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321;

[0041] The ratio of viable Bacillus berleis to Streptomyces is 4:1.

[0042] The viable bacterial concentrations of both Bacillus belyi and Streptomyces were not less than 1×10⁻⁶. 9 CFU / g.

[0043] Example 2: A compound microbial agent for the prevention and control of soil-borne diseases based on metabolic guidance and rational compounding includes: Bacillus bellis and Streptomyces;

[0044] The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321;

[0045] The ratio of viable Bacillus berleis to Streptomyces is 6:1.

[0046] The viable bacterial concentrations of both Bacillus belyi and Streptomyces were not less than 1×10⁻⁶. 9 CFU / g.

[0047] Example 3: A compound microbial agent for the prevention and control of soil-borne diseases based on metabolic guidance and rational compounding includes: Bacillus belye and Streptomyces;

[0048] The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321;

[0049] The ratio of viable Bacillus berleis to Streptomyces is 5:1.

[0050] The viable bacterial concentrations of both Bacillus belyi and Streptomyces were not less than 1×10⁻⁶. 9 CFU / g.

[0051] Example 4: A compound microbial agent for the prevention and control of soil-borne diseases based on metabolic guidance and rational compounding includes: Bacillus vesiculosus, Streptomyces and kaolin;

[0052] The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321;

[0053] The ratio of viable Bacillus berleis to Streptomyces is 5:1.

[0054] The viable bacterial concentrations of both Bacillus belyi and Streptomyces were not less than 1×10⁻⁶. 9 CFU / g.

[0055] The kaolin accounts for 4 wt% of the high-stress-resistant compound microbial agent.

[0056] Example 5: A compound microbial agent for the prevention and control of soil-borne diseases based on metabolic guidance and rational compounding includes: Bacillus vesiculosus, Streptomyces and kaolin;

[0057] The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321;

[0058] The ratio of viable Bacillus berleis to Streptomyces is 5:1.

[0059] The viable bacterial concentrations of both Bacillus belyi and Streptomyces were not less than 1×10⁻⁶. 9 CFU / g.

[0060] The kaolin accounts for 6 wt% of the high-stress-resistant compound microbial agent.

[0061] Example 6: A compound microbial agent for the prevention and control of soil-borne diseases based on metabolic guidance and rational compounding includes: Bacillus vesiculosus, Streptomyces and kaolin;

[0062] The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321;

[0063] The ratio of viable Bacillus berleis to Streptomyces is 5:1.

[0064] The viable bacterial concentrations of both Bacillus belyi and Streptomyces were not less than 1×10⁻⁶. 9 CFU / g.

[0065] The kaolin accounts for 5 wt% of the high-stress-resistant compound microbial agent.

[0066] Example 7: A method for preparing the aforementioned compound microbial agent for controlling soil-borne diseases, comprising the following steps:

[0067] S100. Inoculate Bacillus belye into the induction medium and culture at 25℃ and 150 rpm for 60 h with shaking. By adjusting the carbon-nitrogen ratio and inducing salt stress, the yield of iturin was ≥2.5 g / L, and the fermentation broth of Bacillus belye was obtained.

[0068] The induction medium consists of: 25 g / L glucose, 8 g / L soybean meal, 15 g / L NaCl, and 1.5 g / L KH2PO4; pH 7.0.

[0069] Streptomyces was inoculated into a culture medium and cultured at 25°C and 160 rpm for 90 h with shaking to obtain a Streptomyces culture solution. The culture medium used for inoculation of Streptomyces consisted of: 18 g / L soluble starch, 0.8 g / L KNO3, 0.4 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 0.4 g / L NaCl, 0.01 g / L FeSO4·7H2O, and pH 7.2.

[0070] S200, mixed with Bacillus cereus fermentation broth and Streptomyces bacterial broth, is then added to kaolin and spray-dried to obtain a soil-borne disease control compound microbial agent.

[0071] The inlet air temperature of the spray drying process is 110°C, and the outlet air temperature is 50°C.

[0072] Example 8: A method for preparing the aforementioned soil-borne disease control compound microbial agent, comprising the following steps:

[0073] S100. Inoculate Bacillus belye into the induction medium and culture at 35℃ and 250rpm for 80h with shaking. By adjusting the carbon-nitrogen ratio and inducing salt stress, the yield of iturin is ≥2.5g / L, and the fermentation broth of Bacillus belye is obtained.

[0074] The induction medium comprises: 35 g / L glucose, 12 g / L soybean meal, 25 g / L NaCl, 2.5 g / L KH2PO4; pH 7.5.

[0075] Streptomyces was inoculated into a culture medium and cultured at 30℃ and 200 rpm for 100 h with shaking to obtain a Streptomyces inoculum. The culture medium used for inoculation of Streptomyces consisted of: 24 g / L soluble starch, 1.2 g / L KNO3, 0.6 g / L KH2PO4, 0.6 g / L MgSO4·7H2O, 0.7 g / L NaCl, 0.015 g / L FeSO4·7H2O, pH 7.4.

[0076] S200, mixed with Bacillus cereus fermentation broth and Streptomyces bacterial broth, is then added to kaolin and spray-dried to obtain a soil-borne disease control compound microbial agent.

[0077] The inlet air temperature of the spray drying process is 120°C, and the outlet air temperature is 60°C.

[0078] Example 9: A method for preparing the aforementioned soil-borne disease control compound microbial agent, comprising the following steps:

[0079] S100. Inoculate Bacillus belye into the induction medium and culture at 30℃ and 200rpm for 72h with shaking. By adjusting the carbon-nitrogen ratio and inducing salt stress, the yield of iturin was ≥2.5g / L, and the fermentation broth of Bacillus belye was obtained.

[0080] The induction medium comprises: 30 g / L glucose, 10 g / L soybean meal, 20 g / L NaCl, 2 g / L KH2PO4; pH 7.0.

[0081] Streptomyces was inoculated into a culture medium and cultured at 28°C and 180 rpm for 96 h with shaking to obtain a Streptomyces culture solution. The culture medium used for inoculation of Streptomyces consisted of: 20 g / L soluble starch, 1 g / L KNO3, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.01 g / L FeSO4·7H2O, and pH 7.3.

[0082] S200, mixed with Bacillus cereus fermentation broth and Streptomyces bacterial broth, is then added to kaolin and spray-dried to obtain a soil-borne disease control compound microbial agent.

[0083] The inlet air temperature of the spray drying process is 120°C, and the outlet air temperature is 50°C.

[0084] Example 10: A method of using a compound microbial agent for the prevention and control of soil-borne diseases, including: root irrigation and / or hole application.

[0085] The root drenching application method includes: the first root drenching is carried out 7-10 days after the plants are transplanted, and the second root drenching is carried out 8-12 days later; during root drenching, the soil-borne disease control compound microbial agent is diluted with water to a concentration of 4×10⁻⁶. 9 -6×10 9 CFU / mL, the amount of water used for root irrigation per plant is 150-200 mL.

[0086] The method of applying the fungicide in the holes includes: mixing 5-10g of the fungicide with the soil in each hole before planting the plants.

[0087] For the control of bacterial wilt in peppers: apply the inoculant by root drenching. Perform the first root drenching 7 days after transplanting, diluting the inoculant to 5×10⁻⁶. 8 CFU / mL, 150mL per plant for root irrigation, repeat the second root irrigation after 10 days, and apply twice consecutively;

[0088] For the prevention and control of cucumber wilt: a combination of hole application and root irrigation is adopted: apply the fungicide to the hole before transplanting, and irrigate the roots once 15 days after transplanting. The control effect reaches more than 85%, and the number of soil pathogens in continuously cropped fields is reduced by 80%.

[0089] Experimental Example: A method for preparing the aforementioned compound microbial agent for controlling soil-borne diseases, comprising the following steps:

[0090] S100. *Bacillus belye* was inoculated into induction medium and cultured at 30℃ and 200 rpm for 72 h with shaking. Fermentation broth of *Bacillus belye* was obtained through carbon-nitrogen ratio adjustment and salt stress induction; the yield of iturobrine was measured to be 2.8 g / L.

[0091] The induction medium comprises: 30 g / L glucose, 10 g / L soybean meal, 20 g / L NaCl, 2 g / L KH2PO4; pH 7.0.

[0092] Streptomyces was inoculated into a culture medium and cultured at 28℃ and 180 rpm with shaking for 96 h to obtain a Streptomyces bacterial suspension. The culture medium used for inoculation of Streptomyces consisted of: 20 g / L soluble starch, 1 g / L KNO3, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl, and 0.01 g / L FeSO4·7H2O, with a pH of 7.3. The viable cell concentration was measured to be 1.3 × 10⁻⁶. 9 CFU / g.

[0093] S200, at a live bacteria ratio of 3:1, mix Bacillus berberis fermentation broth and Streptomyces bacterial broth, then add kaolin, and after spray drying, obtain a soil-borne disease control compound microbial agent. The kaolin accounts for 5 wt% of the high-stress-resistant compound microbial agent.

[0094] The inlet air temperature of the spray drying process is 120°C, and the outlet air temperature is 50°C.

[0095] Product testing: The product is in the form of a wettable powder with a moisture content of 6.5%, a suspension rate of 78%, and a wetting time of 60 seconds. After being placed at a pH of 5.0-8.0 and a high temperature of 45℃ for 72 hours, the survival rate of the strain is still not less than 80%.

[0096] When applied continuously for 2-3 seasons in fields with continuous cropping, pathogens did not develop resistance, and soil microbial diversity increased by 15%-20%, resulting in improved soil fertility.

[0097] Trial of control of bacterial wilt in peppers

[0098] Experimental materials: The crop used was horn pepper, with seedlings having 4-5 true leaves. Soil samples were taken from a continuously cropped field with a high incidence of bacterial wilt in peppers; the concentration of Ralstonia solanacearum in the soil was 1.2 × 10⁻⁶. 5 CFU / g soil, pH 6.8-7.2.

[0099] The Streptomyces was purchased from Shanghai Yushao Biotechnology Co., Ltd., and the product name is Streptomyces liani.

[0100] Comparative Example 1: The difference from the experimental example is that it does not contain Streptomyces.

[0101] Comparative Example 2: The difference from the experimental example is that the induction culture medium does not contain sodium chloride.

[0102] Comparative Example 3: The difference from the experimental example is that Bacillus belysin was purchased from Shanghai Xuanke Biotechnology Co., Ltd., product name: Bacillus belysin.

[0103] Blank example: clear water.

[0104] Application method:

[0105] The experimental case, comparative example 1, and blank case were all applied by root drenching. The inoculum was diluted to 5 × 10⁻⁶. 9 CFU / mL, apply 150mL to each plant twice, once on the 7th and 17th day after transplanting.

[0106] The incidence of bacterial wilt and yield of peppers were investigated 60 days after transplanting. The results are shown in Table 1 below.

[0107] Table 1

[0108]

[0109] The results showed that the compound microbial agent of the present invention achieved a control efficacy of 85.3% against bacterial wilt of pepper, which was significantly higher than that of single-strain preparations; the yield increased by 40.2%, and the number of soil pathogens in continuously cropped fields decreased by 80%, verifying the synergistic effect of metabolism-directed fermentation and rational compounding.

[0110] The data from Comparative Example 2 and the experimental example show that the effect of the compound bacterial agent was significantly improved after sodium chloride stress was used to target and regulate the metabolic pathway of Bacillus belyssus.

[0111] As can be seen from the comparison of data between Comparative Example 3 and the experimental examples, the bacterial agent prepared using the specific Bacillus berleis of this invention is far more effective than the conventional Bacillus berleis.

[0112] Cucumber Fusarium Wilt Control Experiment

[0113] Experimental materials: Cucumber variety "Bomei 8", seedlings with 3-4 true leaves. Soil was taken from a continuously cropped field with a high incidence of cucumber wilt disease, and the concentration of Fusarium in the soil was 9×10⁻⁶. 4 CFU / g soil, pH 6.0-6.5.

[0114] Control group 1: The difference from the experimental case is that it does not contain Bacillus belye.

[0115] Control group 2: Water.

[0116] Application method: In the experimental case, control group 1, and control group 2, a combination of hole application and root drenching was used. 5g of the inoculant was applied to each hole before planting, and the roots were drenched once 15 days after planting, diluted to 5×10⁻⁶ g. 8 CFU / mL, 200mL per plant.

[0117] The incidence of cucumber wilt and the number of soil pathogens were investigated 90 days after transplanting. The results are shown in Table 2 below.

[0118] Table 2

[0119]

[0120] The results showed that the compound microbial agent of the present invention achieved a control efficacy of 85.7% against cucumber wilt, reduced the number of soil pathogens by 80%, and increased soil microbial diversity by 18.3%, which was significantly better than the single Streptomyces preparation, demonstrating the synergistic advantages of the compound strain.

[0121] Stability and drug resistance test of compound microbial agents

[0122] 1. Test metrics

[0123] Storage stability: The compound microbial agent was stored at 25℃ and 4℃ for 18 months, and the survival rate of the strains and the retention rate of ituronic acid were tested regularly.

[0124] Environmental adaptability: The compound bacterial agent was placed in pH 5.0, pH 8.0 and 45℃ high temperature environments for 72 hours respectively, and the survival rate of the strains was tested.

[0125] Drug resistance test: Compound microbial agent was applied to continuously cropped fields for three seasons, and the changes in the sensitivity of pathogens to the agent were detected.

[0126] 2. Test Results

[0127] (1) Storage stability: After 18 months of storage at 25℃, the survival rate of Bacillus belyi (CGMCC No. 36321) and Streptomyces (CGMCC 4.1056) was 82%, and the retention rate of iturobacillus was 85%; after 18 months of storage at 4℃, the survival rate of the strains was 90%, and the retention rate of iturobacillus was 92%.

[0128] (2) Environmental adaptability: The survival rate of the strain was 83% under pH 5.0 conditions, 81% under pH 8.0 conditions, and 80% under high temperature of 45℃, all of which were better than conventional single strain preparations (the survival rate of strains under the same conditions is ≤65%).

[0129] (3) Resistance test: After three consecutive seasons of application, the sensitivity of pathogens to compound microbial agents did not decrease significantly, and no drug-resistant strains of pathogens appeared in the soil, which verified the effectiveness of "rational compounding" in reducing the risk of drug resistance.

[0130] Test results show that the compound microbial agent of the present invention has excellent stability and environmental adaptability, and there is no risk of drug resistance with long-term application, making it suitable for the needs of large-scale and sustainable agricultural production.

[0131] Example 11: Screening culture of Bacillus belye

[0132] Take 10g of farmland soil sample and add it to a 90mL Erlenmeyer flask containing sterile water. Shake to mix and let stand. Take the middle layer bacterial suspension and immediately heat-treat it in an 80℃ water bath for 15min to enrich Bacillus. After cooling, perform a 10-fold serial dilution of the bacterial suspension. Spread each dilution onto LB agar plates. The LB agar formulation is: 10g / L tryptone, 5g / L yeast extract, 10g / L NaCl, 15-20g / L agar, pH 7.0-7.2. Incubate the spread plates upside down in a 30-37℃ incubator for 24-48 hours.

[0133] Example 12: Classification and Identification of Bacillus belye

[0134] 1. Morphological characteristics of the strain

[0135] The colonies are milky white, round, 1.0-1.5 mm in diameter, with a smooth, slightly convex surface that is dry and deeply wrinkled, and irregular edges. Under a microscope, the bacteria are rod-shaped or large oval, often arranged in pairs, 0.6-0.8 μm wide and 1.5-3.0 μm long, with spores (elliptical), no capsule, peritrichous flagella, and are motile. They are Gram-negative, and the optimal growth pH is 7.0-7.5.

[0136] 2. Absorbance

[0137] At pH 8.4, the absorbance at a wavelength of 600 nm is 5.25.

[0138] 3.16S rRNA gene sequence

[0139]

[0140] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound microbial agent for the control of soil-borne diseases based on metabolic guidance and rational formulation, characterized in that, include: Bacillus bellis and Streptomyces; The Bacillus belyssus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.36321; The viable count ratio of Bacillus belyssus and Streptomyces is (4-6):

1.

2. The compound microbial agent for controlling soil-borne diseases according to claim 1, characterized in that, The viable bacterial concentrations of both Bacillus belyi and Streptomyces were not less than 1×10⁻⁶. 9 CFU / g.

3. The compound microbial agent for controlling soil-borne diseases according to claim 1 or 2, characterized in that, Also includes: Kaolin; the kaolin accounts for 4wt%-6wt% of the high-stress-resistant compound microbial agent.

4. A method for preparing a compound microbial agent for controlling soil-borne diseases as described in any one of claims 1-3, characterized in that, Includes the following steps: S100. Inoculate Bacillus belye into the induction medium and culture it at 25-35℃ and 150-250rpm for 60-80h with vibration to obtain Bacillus belye fermentation broth. The induction medium comprises: 25-35 g / L glucose, 8-12 g / L soybean meal, 15-25 g / L NaCl, and 1.5-2.5 g / L KH2PO4; Streptomyces was inoculated into the culture medium and cultured at 25-30℃ and 160-200 rpm for 90-100 h with shaking to obtain Streptomyces bacterial suspension; S200, mixed with Bacillus cereus fermentation broth and Streptomyces bacterial broth, is then added to kaolin and spray-dried to obtain a soil-borne disease control compound microbial agent.

5. The preparation method according to claim 4, characterized in that, The inlet air temperature of the spray drying process is 110-120℃, and the outlet air temperature is 50-60℃.

6. The preparation method according to claim 4, characterized in that, The culture medium for inoculating Streptomyces includes: 18-24 g / L soluble starch, 0.8-1.2 g / L KNO3, 0.4-0.6 g / L KH2PO4, 0.4-0.6 g / L MgSO4·7H2O, 0.4-0.7 g / L NaCl, and 0.01-0.015 g / L FeSO4·7H2O.

7. The preparation method according to claim 4, characterized in that, The yield of iturin in the Bacillus berberis fermentation broth is ≥2.5 g / L.

8. A method of using the compound microbial agent for controlling soil-borne diseases according to any one of claims 1-3, characterized in that, include: Root irrigation and / or hole application.

9. The method of use according to claim 8, characterized in that, The root drenching application method includes: the first root drenching is carried out 7-10 days after the plants are transplanted, and the second root drenching is carried out 8-12 days later; during root drenching, the soil-borne disease control compound microbial agent is diluted with water to a concentration of 4×10⁻⁶. 9 -6×10 9 CFU / mL, the amount of water used for root irrigation per plant is 150-200 mL.

10. The method of use according to claim 8, characterized in that, The method of applying the fungicide in the holes includes: mixing 5-10g of the fungicide with the soil in each hole before planting the plants.

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