High-efficiency synergistic compound microbial inoculant, preparation method and application thereof

Abstract

The application discloses a high-efficiency synergic compound microbial inoculant, which comprises Bacillus subtilis and Trichoderma harzianum, wherein the effective viable count of the Bacillus subtilis is greater than or equal to 1.5*10 10 CFU / g, the effective viable count of the Trichoderma harzianum is greater than or equal to 1.2*10 10 CFU / g, the ratio of the effective viable count of the Bacillus subtilis to that of the Trichoderma harzianum is 1:1.2 to 1.5:1, and the total effective viable count of the high-efficiency synergic compound microbial inoculant is greater than or equal to 3.0*10 10 CFU / g. The application further discloses a preparation method of the high-efficiency synergic compound microbial inoculant, which comprises microbial liquid culture, spore suspension preparation, mixing and compounding and low-temperature drying, wherein the moisture content of the obtained microbial inoculant is less than or equal to 8%, the total effective viable count is greater than or equal to 3.0*10 10 CFU / g, and the survival rate of the viable bacteria is greater than or equal to 92% after being stored at 25 DEG C for 12 months. The application is suitable for green agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a highly efficient synergistic compound microbial agent, its preparation method, and its application. Background Technology

[0002] Plant diseases are a significant factor restricting agricultural production and affecting the yield and quality of agricultural products. Traditional chemical control methods easily lead to problems such as increased pathogen resistance, pesticide residues, and environmental pollution. Biological control technologies centered on beneficial microorganisms have attracted much attention due to their environmental friendliness and low likelihood of developing resistance.

[0003] Bacillus subtilis ( Bacillus subtilis ) and Trichoderma harzianum ( Trichoderma harzianum These are two widely used biocontrol agents. Bacillus subtilis mainly inhibits pathogens by producing antimicrobial substances, competing for ecological niches, and inducing systemic resistance in plants; Trichoderma harzianum, on the other hand, possesses multiple biocontrol mechanisms, including hyperparasitism, competition, and the production of cell wall-degrading enzymes. However, in practical applications, single-agent biocontrol often suffers from problems such as a narrow spectrum of control, poor stability, and significant environmental influences on field efficacy. Combining strains with different mechanisms of action can theoretically achieve synergistic effects and broaden the spectrum of control. However, simple physical mixing often leads to poor results or even failure due to antagonism between strains, differences in culture conditions, or incompatibility of formulations. Existing commercially available Bacillus subtilis + Trichoderma harzianum compound agents are mostly blindly mixed in a 1:2 or 2:1 ratio, resulting in obvious antagonistic effects between strains. After 6 months of storage at 25°C, the survival rate of live bacteria is less than 60%. The design of the synergistic adjuvant system of existing agents is also relatively simple, mostly using only mannitol as a cell protectant, lacking a comprehensive design of compound protection and functional enhancement. After 6 months of storage, the number of live bacteria decreases by more than 50%, and the colonization rate of bacteria on crop surfaces and in soil is low after field application.

[0004] Furthermore, the application of existing microbial agents is mostly at a rudimentary stage, often using fixed dilution ratios for spraying or root drenching. There is a lack of refined application plans for different disease types, disease stages, and crop growth stages. Existing application plans for soil-borne diseases are all fixed dilution ratios for root drenching, without adjusting the dosage and concentration according to the crop growth stage, resulting in a control efficacy of only 60% to 70%. For foliar diseases, they are mostly applied by single foliar spraying, with a disease recurrence rate as high as 30% or more. This means that the agents cannot act on the target in the most suitable time and in the most effective way, resulting in resource waste and unstable control effects.

[0005] Therefore, this invention addresses the core pain points in the research and application of existing agricultural microbial agents, specifically solving four major technical problems: (1) solving the problems of inter-species antagonism, narrow control spectrum, and low field control efficacy caused by the unreasonable ratio of Bacillus subtilis and Trichoderma harzianum in existing compound microbial agents; (2) solving the problems of simple design of existing microbial agent adjuvant systems, poor microbial protection effect, insufficient storage stability, and low field colonization rate; (3) solving the problems of large-scale microbial inactivation and difficulty in achieving commercial application standards due to high-temperature treatment in existing microbial agent drying processes; (4) solving the problems of extensive field application methods, lack of differentiated grading schemes, and poor adaptability to different disease types, crop growth stages, and disease severity in existing microbial agents. Summary of the Invention

[0006] The object of the present invention is to overcome at least the aforementioned defects and to provide advantages that will be described later.

[0007] Another objective of this invention is to provide a highly efficient synergistic compound microbial agent with significant synergistic effects, good stability, and a precise application scheme, as well as its preparation method and application.

[0008] To achieve these objectives and other advantages of the present invention, a highly efficient synergistic compound microbial agent is provided, comprising Bacillus subtilis and Trichoderma harzianum; based on the total weight of the compound microbial agent, the effective viable count of Bacillus subtilis is ≥1.5 × 10⁻⁶. 10 CFU / g, effective viable count of Trichoderma harzianum ≥1.2×10⁻⁶ 10 CFU / g; the ratio of effective viable bacteria count of Bacillus subtilis to Trichoderma harzianum is 1:1.2 to 1.5:1, and the total effective viable bacteria count of the highly efficient synergistic compound microbial agent is ≥3.0×10⁻⁶. 10 CFU / g.

[0009] In the above scheme, within the ratio range of this invention, through precise control of the fermentation process, the effective viable counts of both biocontrol bacteria are higher than the lower limit threshold. Under the extreme condition of a ratio of 1.5:1, Bacillus subtilis reaches the lower limit of 1.8 × 10⁻⁶. 10 CFU / g and Trichoderma harzianum levels reached the lower limit of 1.2 × 10⁻⁶. 10 CFU / g, the sum of the two consistently reached 3.0 × 10⁻⁶. 10 CFU / g; Under the limiting conditions of a ratio of 1:1.2, Bacillus subtilis reached the lower limit of 1.8 × 10⁻⁶ CFU / g. 10 At CFU / g, the viable count of Trichoderma harzianum can reach 2.16 × 10⁻⁶. 10 CFU / g, the sum of the two is 3.96×10 10 CFU / g; both meet the requirements for commercialization.

[0010] Preferably, the high-efficiency synergistic compound microbial agent also includes a synergistic adjuvant system; the synergistic adjuvant system, based on the total weight of the compound microbial agent, consists of 5%~8% trehalose, 3%~5% mannitol, 2%~3% xanthan gum, 1%~2% chitosan oligosaccharide, 0.5%~1% matrine, and the balance being diatomaceous earth; wherein, the xanthan gum has a viscosity ≥1200 mPa•s in a 1% aqueous solution at 25℃; and the chitosan oligosaccharide has a molecular weight ≤3000 Da and a degree of deacetylation ≥90%.

[0011] Preferably, the diatomaceous earth has a particle size of 500-800 mesh and a specific surface area of ​​15-25 m². 2 / g, porosity 70%~80%.

[0012] Preferably, the compound microbial agent is a wettable powder with a suspension rate ≥80%, a pH value of 6.0~7.5, and a finished product particle size of 200~400 mesh.

[0013] Methods for preparing highly efficient synergistic compound microbial agents include: Bacillus subtilis was inoculated onto LB medium and cultured aerobicly at 30–32°C and 180–200 r / min for 18–24 h to obtain a viable count ≥10⁻⁶. 10 Bacillus subtilis culture at CFU / mL.

[0014] Trichoderma harzianum was inoculated onto PDA medium and cultured at 25–28°C in the dark for 48–72 h. Conidia were collected and prepared to a concentration ≥10. 9 CFU / mL Trichoderma harzianum spore suspension.

[0015] Bacillus subtilis bacterial suspension and Trichoderma harzianum spore suspension were mixed, and trehalose, mannitol and xanthan gum were added. The mixture was stirred at 25-30℃ and 120 r / min for 30-60 min. Then chitosan oligosaccharide, matrine and diatomaceous earth carrier were added and mixed evenly to obtain the mixture.

[0016] The mixture was spray-dried at a feed rate of 10 mL / min, an atomization pressure of 0.3 MPa, an inlet air temperature of 100–110°C, an outlet air temperature of 45–50°C, a material temperature ≤60°C during drying, and a drying time of 15–20 min until the moisture content was ≤8% and the total number of viable bacteria was ≥3.0 × 10⁻⁶. 10 With a CFU / g concentration, the product particle size after drying is 200~400 mesh, thus obtaining a highly efficient synergistic compound microbial agent.

[0017] In the above scheme, when the drying temperature exceeds 60℃, the spore germination rate of Bacillus subtilis decreases by more than 70%, and the conidia germination rate of Trichoderma harzianum decreases by more than 60%. Therefore, controlling the heating temperature to ≤60℃ is the key to ensuring the activity of the bacteria. Spray drying with an inlet air temperature of 100~110℃ and an outlet air temperature of 45~50℃ ensures drying efficiency while avoiding direct contact between the material and high temperature.

[0018] Application of highly efficient synergistic compound microbial agents in the prevention and control of fungal and / or bacterial diseases.

[0019] Methods for controlling plant fungal and / or bacterial diseases using highly effective synergistic compound microbial agents include: applying the highly effective synergistic compound microbial agent to target crops such as strawberries, cucumbers, tomatoes, peppers, watermelons, citrus, and cotton; plant diseases including fungal and bacterial diseases; fungal diseases including root rot (…). Fusarium solani ), wilt disease ( Fusarium oxysporum ),powdery mildew( Sphaerotheca fuliginea ), gray mold ( Botrytis gray ) and downy mildew ( Pseudoperonospora cubensis Bacterial diseases include soft rot ( Erwinia carrot-eating Bacterial wilt ( Ralstonia solanacearum ), peptic ulcer ( Clavibacter michiganensis Bacterial leaf spot ( Xanthomonas campestris ).

[0020] Preferably, the application method when the disease occurs is as follows: when the disease incidence rate is ≤5%, the highly efficient synergistic compound microbial agent is diluted 500-700 times with water and sprayed on the diseased parts, leaves, and stems of the plant, with a spraying amount of 45-60 L per acre; when the disease incidence rate is >5%, the plant is first treated with a biopesticide for the corresponding disease or a chemical pesticide with low toxicity to biocontrol bacteria. After 7 days of drug treatment, the highly efficient synergistic compound microbial agent is diluted 600-800 times with water and sprayed on the diseased parts, leaves, and stems of the plant, with a spraying amount of 35-50 L per acre.

[0021] Preferably, the application method for soil-borne diseases is as follows: root irrigation is performed 1-3 days after transplanting, and full-field irrigation is performed 13-16 days after planting. Root irrigation is implemented according to the plant's growth stage, specifically: when the plant height is ≤10cm and the number of true leaves is ≤4, dilute the highly efficient synergistic compound microbial agent 700-800 times with water and irrigate 200-300 mL per plant; when the plant height is 10-30cm and the number of true leaves is 4-8, dilute the highly efficient synergistic compound microbial agent 600-700 times with water and irrigate 300-400 mL per plant; when the plant height is ≥30cm or the budding / fruit setting rate is ≥50%, dilute the highly efficient synergistic compound microbial agent 500-600 times with water and irrigate 400-500 mL per plant. mL; Irrigation treatment: Dilute the high-efficiency synergistic compound bacterial agent with clean water 900~1000 times, and irrigate according to the amount of 2000 L of diluted solution per acre.

[0022] In the above scheme, root irrigation treatment specifically refers to targeted application of pesticides to the roots of plants, directly acting on the rhizosphere of the crop; watering treatment is defined as uniform application of pesticides to the entire soil layer of the crop planting area. The root irrigation parameters for soil-borne diseases are general standards. For crops with well-developed root systems, such as strawberries, the dilution ratio can be appropriately reduced and the amount of pesticide applied to each plant can be increased according to the actual growth conditions to improve the rhizosphere colonization effect.

[0023] Preferably, the active preventative application method is to apply it at key growth stages of the crop, specifically including: 1-3 days before sowing, dilute the high-efficiency synergistic compound microbial agent with water at a ratio of 800-1000 times and irrigate with 2000-3000 L of diluted solution per acre; within 3 days of transplanting, dilute the high-efficiency synergistic compound microbial agent with water at a ratio of 500-800 times and irrigate the roots with 200-500 mL per plant; 7±1 days before flowering, dilute the high-efficiency synergistic compound microbial agent with water at a ratio of 800-1000 times and spray it on both the upper and lower surfaces of the leaves at a rate of 30-45 L per acre; simultaneously, dilute the high-efficiency synergistic compound microbial agent with water at a ratio of 500-600 times and irrigate the roots with 300-500 mL per plant.

[0024] All field applications of this invention should be carried out in the early morning (before sunrise to 9:00 AM) or late afternoon (after 5:00 PM to sunset) under rainless conditions and wind speeds ≤3 m / s. Avoid application during periods of high temperature (>32℃), strong sunlight (light intensity >50000 Lux), or when rainfall is expected within 4 hours. Use clean fresh water with a pH of 6.5-7.5 (tap water should be left to stand for at least 24 hours to remove chlorine, or well water or river water should be used). Do not mix with strong acids, strong alkalis, copper-based pesticides, or antibiotics. When sequential application with chemical pesticides is required, prioritize pesticides with low toxicity or no inhibitory effect on Bacillus subtilis and Trichoderma harzianum, including but not limited to: biopesticides (kasugamycin, jinggangmycin, polyoxin, kasugamycin), and fungicides other than benzimidazoles (azoxystrobin, pyraclostrobin, cyazofamid, etc.). Do not use continuously within 7 days with copper-based pesticides, streptomycin, tetracycline, or other highly inhibitory pesticides. The safe interval between chemical pesticides and microbial agents should be no less than 7 days. If chemical pesticides are used first, the microbial agent can only be applied after an interval of 7 days or more; if microbial agents are applied first, the chemical pesticide can only be applied after an interval of 5 days or more.

[0025] Advantages of this invention: Firstly, this invention fundamentally avoids interspecies antagonism by limiting the effective viable cell ratio of Bacillus subtilis to Trichoderma harzianum to 1:1.2 to 1.5:1, coupled with a lower limit threshold for single-cell viable cell count. This achieves complementary advantages and synergistic effects between the two biocontrol mechanisms. Simultaneously, fermentation process control ensures that the total viable cell count consistently reaches ≥3.0 × 10⁻⁶. 10 The high commercial standard of CFU / g enhances the control spectrum compared to single-agent bacterial agents.

[0026] Secondly, this invention utilizes a synergistic adjuvant system composed of trehalose, mannitol, xanthan gum, chitosan oligosaccharide, matrine, and diatomaceous earth with specific parameters to achieve synergistic complementarity in bacterial cell protection, field colonization, antibacterial and disease prevention, and plant immune activation, thus solving the problems of poor storage stability and low field colonization rate of existing bacterial agents.

[0027] Furthermore, this invention, through a precisely defined low-temperature spray drying process, strictly controls the material heating temperature to ≤60℃, while clearly defining the drying time and drying endpoint. This solves the technical bottleneck of large-scale cell inactivation caused by high-temperature drying, ensuring minimal loss of cell activity during the preparation process. The process is highly repeatable and suitable for large-scale production.

[0028] Furthermore, this invention provides a refined application scheme for different disease severity, disease types, and crop growth stages, including graded spraying, graded root irrigation for soil-borne diseases, and active prevention throughout the entire growth period. This solves the problems of extensive application of existing microbial agents and unstable efficacy, improves the utilization rate of microbial agents, significantly reduces the application of chemical pesticides, and has the comprehensive effects of disease prevention, yield increase, and soil improvement. Detailed Implementation

[0029] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0030] All raw materials used in all embodiments of this invention are commercially available conventional agricultural / biological grade reagents. The Bacillus subtilis and Trichoderma harzianum used were purchased from the China General Microbiological Culture Collection Center (accession numbers CGMCC1.1729 and CGMCC3.10154, respectively). All embodiments follow the core technical solution of this invention and are reproducible. General rules for application of this invention: (1) Application time: All field applications should be carried out in the early morning (before sunrise to 9 am) or evening (after 5 pm to sunset) under rainless conditions and wind speed ≤3 m / s. Avoid application during periods of high temperature (>32℃), strong light (light intensity >50000 Lux) or when rainfall is expected within 4 hours. (2) Dilution water: Clean fresh water with pH 6.5~7.5 should be used (tap water should be left to stand for more than 24 hours to remove chlorine or well water or river water should be used). It should not be mixed with strong acid, strong alkaline substances, copper preparations, or antibiotic pesticides. (3) Combined use of chemical pesticides: When it is necessary to apply chemical pesticides in sequence, pesticide varieties with low toxicity or no inhibition against Bacillus subtilis and Trichoderma harzianum should be given priority, including but not limited to: biological pesticides (kasugamycin, jinggangmycin, polyoxin, kasugamycin), fungicides other than benzimidazole (azoxystrobin, pyraclostrobin, cyproconazole, etc.). Do not use this fungicide consecutively within 7 days with highly inhibitory pesticides such as copper-based pesticides, streptomycin, and tetracycline. The safe interval between chemical pesticides and fungicides should be no less than 7 days. If a chemical pesticide is used first, this fungicide should be applied only after an interval of at least 7 days; if a fungicide is applied first, a chemical pesticide should be applied only after an interval of at least 5 days.

[0031] In this invention, the suspension rate of the microbial agent was determined according to GB / T19136-2003 "Determination of Suspension Rate of Pesticide Wettable Powders"; the pH value was determined according to GB / T1601-2001 "Determination of pH Value of Pesticides"; and the particle size of the finished product was determined according to GB / T 21782.2-2008 "Determination of Powder Particle Size Distribution by Laser Diffraction". All tests were performed in triplicate, and the data are expressed as mean ± standard deviation.

[0032] Example 1 This highly efficient synergistic compound microbial agent consists of Bacillus subtilis (purchased from the China General Microbiological Culture Collection Center, CGMCC 1.1729), Trichoderma harzianum (purchased from the China General Microbiological Culture Collection Center, CGMCC 3.10154), and a synergistic adjuvant system. Based on the total weight of the compound microbial agent, the effective viable count of Bacillus subtilis is 1.7 × 10⁻⁶. 10The effective viable count of *Trichoderma harzianum* was 2.1 × 10⁻⁶ CFU / g. 10 The effective viable count ratio of Bacillus subtilis to Trichoderma harzianum is 1:1.2, and the total effective viable count of the highly efficient synergistic compound microbial agent is 3.8 × 10⁻⁶. 10 CFU / g.

[0033] The synergistic adjuvant system, based on the total weight of the compound bacterial agent, consists of 6% trehalose, 4% mannitol, 2.5% xanthan gum, 1.5% chitosan oligosaccharide, 0.8% matrine, and the balance diatomaceous earth. Specifically, the xanthan gum has a viscosity of 1500 mPa·s in a 1% aqueous solution at 25°C; the chitosan oligosaccharide has a molecular weight of 2000 Da and a degree of deacetylation of 92%; and the diatomaceous earth has a particle size of 600 mesh and a specific surface area of ​​20 m². 2 / g, porosity 75%.

[0034] The product obtained in this embodiment has a particle size of 320 mesh, a suspension rate of 87.5±0.6%, and a pH value of 6.6±0.1, which meets the quality index requirements of this invention.

[0035] Example 2 This highly efficient synergistic compound microbial agent consists of Bacillus subtilis (purchased from the China General Microbiological Culture Collection Center, CGMCC 1.1729), Trichoderma harzianum (purchased from the China General Microbiological Culture Collection Center, CGMCC 3.10154), and a synergistic adjuvant system. Based on the total weight of the compound microbial agent, the effective viable count of Bacillus subtilis is 1.9 × 10⁻⁶. 10 The effective viable count of *Trichoderma harzianum* was 1.5 × 10⁻⁶ CFU / g. 10 The ratio of CFU / g to the effective viable count of the two agents is 1.3:1, and the total effective viable count of the compound microbial agent is 3.4 × 10⁻⁶. 10 CFU / g.

[0036] The synergistic adjuvant system, based on the total weight of the compound bacterial agent, consists of 5% trehalose, 3% mannitol, 2% xanthan gum, 1% chitosan oligosaccharide, 0.5% matrine, and the balance diatomaceous earth. Specifically, the xanthan gum has a viscosity of 1300 mPa·s in a 1% aqueous solution at 25°C; the chitosan oligosaccharide has a molecular weight of 2500 Da and a degree of deacetylation of 90%; and the diatomaceous earth has a particle size of 500 mesh and a specific surface area of ​​15 m². 2 / g, porosity 70%.

[0037] The product obtained in this embodiment has a particle size of 260 mesh, a suspension rate of 85.8±0.7%, and a pH value of 6.4±0.1, which meets the quality index requirements of this invention.

[0038] Example 3 This highly efficient synergistic compound microbial agent consists of Bacillus subtilis (purchased from the China General Microbiological Culture Collection Center, No. CGMCC 1.1729), Trichoderma harzianum (purchased from the China General Microbiological Culture Collection Center, No. CGMCC 3.10154), and a synergistic adjuvant system. Based on the total weight of the compound microbial agent, the effective viable count of Bacillus subtilis is 2.2 × 10⁻⁶. 10 The effective viable count of *Trichoderma harzianum* was 1.5 × 10⁻⁶ CFU / g. 10 The ratio of CFU / g to the effective viable count of the two agents is 1.5:1, and the total effective viable count of the compound microbial agent is 3.7 × 10⁻⁶. 10 CFU / g.

[0039] The synergistic adjuvant system, based on the total weight of the compound bacterial agent, consists of 8% trehalose, 5% mannitol, 3% xanthan gum, 2% chitosan oligosaccharide, 1% matrine, and the balance diatomaceous earth. Specifically, the xanthan gum has a viscosity of 1800 mPa·s in a 1% aqueous solution at 25°C; the chitosan oligosaccharide has a molecular weight of 1500 Da and a degree of deacetylation of 95%; and the diatomaceous earth has a particle size of 800 mesh and a specific surface area of ​​25 m². 2 / g, porosity 80%.

[0040] The product obtained in this embodiment has a particle size of 380 mesh, a suspension rate of 86.2±0.5%, and a pH value of 7.0±0.1, which meets the quality index requirements of this invention.

[0041] Example 4 A method for preparing a highly efficient synergistic compound microbial agent includes the following steps: Step 1: Bacillus subtilis (purchased from China General Microbiological Culture Collection Center, catalog number CGMCC 1.1729) was inoculated into LB liquid medium and cultured at 32℃ and 190 r / min for 20 hours with shaking, yielding a viable count of 1.2 × 10⁻⁶. 10 Bacillus subtilis culture at CFU / mL.

[0042] Step 2: Inoculate *Trichoderma harzianum* (purchased from the China General Microbiological Culture Collection Center, catalog number CGMCC3.10154) onto PDA agar plates and incubate at 26°C in the dark for 60 hours. Wash away conidia with sterile water containing 0.05% Tween-80, filter through gauze, and prepare a viable count concentration of 1.5 × 10⁻⁶. 9 CFU / mL Trichoderma harzianum spore suspension.

[0043] Step 3: Mix the above-mentioned Bacillus subtilis bacterial suspension and Trichoderma harzianum spore suspension at an effective viable cell ratio of 1:1.2. Add 6% trehalose, 4% mannitol, and 2.5% xanthan gum (1% aqueous solution, viscosity ≥1200 mPa•s at 25℃) to the mixed bacterial suspension. Stir continuously at 28℃ and 120 r / min for 45 minutes to ensure the protective agent is fully dissolved and in uniform contact with the bacterial cells. While continuously stirring, add 1.5% chitosan oligosaccharide (molecular weight ≤3000 Da, degree of deacetylation ≥90%), 0.8% matrine, and sufficient 600-mesh diatomaceous earth carrier to the above mixture, and stir until a uniform paste-like mixture is formed.

[0044] Step 4: Spray dry the mixture. Set the spray drying parameters as follows: feed rate 10 mL / min, atomization pressure 0.3 MPa, inlet air temperature 105℃, outlet air temperature 48℃, and material residence time in the drying tower approximately 2 seconds, thus ensuring that the temperature of the material particles does not exceed 60℃ throughout the drying process. Stop drying when the material moisture content reaches 6.2%. During spray drying, due to the absorption of a large amount of latent heat by water evaporation, the actual temperature of the material particles is much lower than the inlet air temperature; simultaneously, trehalose and mannitol form a protective film on the surface of the bacteria, further reducing heat conduction damage to the bacteria.

[0045] Testing showed that the total number of viable bacteria in the product obtained in this embodiment reached 3.8 × 10⁻⁶. 10 The product has a CFU / g, a particle size of 300 mesh, a suspension rate of 88.2±0.6%, and a pH value of 6.7±0.1, indicating that the drying conditions can effectively protect the bacterial activity and the effective viable count of the product meets the requirements.

[0046] Example 5 A method for preparing a highly efficient synergistic compound microbial agent includes the following steps: Step 1: Inoculate Bacillus subtilis (purchased from China General Microbiological Culture Collection Center, catalog number CGMCC 1.1729) into LB liquid medium and culture at 30℃ and 200 r / min for 24 hours with shaking to obtain a viable count of 1.5 × 10⁻⁶. 10 Bacillus subtilis culture at CFU / mL.

[0047] Step 2: Inoculate *Trichoderma harzianum* (purchased from the China General Microbiological Culture Collection Center, catalog number CGMCC3.10154) onto PDA agar plates and incubate at 28°C in the dark for 72 hours. Wash away conidia with sterile water containing 0.05% Tween-80, filter through gauze, and prepare a viable count concentration of 1.2 × 10⁻⁶. 9 CFU / mL Trichoderma harzianum spore suspension.

[0048] Step 3: Mix the above Bacillus subtilis bacterial suspension and Trichoderma harzianum spore suspension at an effective viable cell ratio of 1.5:1. Add 8% trehalose, 5% mannitol, and 3% xanthan gum (1% aqueous solution, viscosity ≥1200 mPa•s at 25℃) to the mixed bacterial suspension. Stir continuously for 60 minutes at 30℃ and 120 r / min to ensure the protective agent is fully dissolved and in uniform contact with the bacterial cells. While continuously stirring, add 2% chitosan oligosaccharide (molecular weight ≤3000 Da, degree of deacetylation ≥90%), 1% matrine, and sufficient 800-mesh diatomaceous earth carrier to the above mixture, and stir until a uniform paste-like mixture is formed.

[0049] Step 4: Spray dry the mixture. Set the spray drying parameters as follows: feed rate 10 mL / min, atomization pressure 0.3 MPa, inlet air temperature 110℃, outlet air temperature 50℃, and the residence time of the material in the drying tower is approximately 2 seconds, thus ensuring that the temperature of the material particles does not exceed 60℃ throughout the drying process. Stop drying when the moisture content of the material reaches 5.8%. During spray drying, due to the absorption of a large amount of latent heat by water evaporation, the actual temperature of the material particles is much lower than the inlet air temperature; at the same time, trehalose and mannitol form a protective film on the surface of the bacteria, further reducing the damage to the bacteria caused by heat conduction.

[0050] Testing showed that the total number of viable bacteria in the product obtained in this embodiment reached 3.7 × 10⁻⁶. 10 The product has a CFU / g, a particle size of 350 mesh, a suspension rate of 86.5±0.5%, and a pH value of 6.9±0.1, indicating that the drying conditions can effectively protect the bacterial activity and the effective viable count of the product meets the requirements.

[0051] Example 6 A method for preparing a highly efficient synergistic compound microbial agent includes the following steps: Step 1: Inoculate Bacillus subtilis (purchased from China General Microbiological Culture Collection Center, catalog number CGMCC 1.1729) into LB liquid medium and culture at 31℃ and 180 r / min with shaking for 18 hours to obtain a viable count of 1.0 × 10⁻⁶. 10 Bacillus subtilis culture at CFU / mL.

[0052] Step 2: Inoculate *Trichoderma harzianum* (purchased from the China General Microbiological Culture Collection Center, catalog number CGMCC3.10154) onto PDA agar plates and incubate at 25°C in the dark for 48 hours. Wash away conidia with sterile water containing 0.05% Tween-80, filter through gauze, and prepare a viable count concentration of 1.0 × 10⁻⁶. 9 CFU / mL Trichoderma harzianum spore suspension.

[0053] Step 3: Mix the above-mentioned Bacillus subtilis bacterial suspension and Trichoderma harzianum spore suspension at an effective viable cell ratio of 1:1.3. Add 7% trehalose, 3.5% mannitol, and 2.2% xanthan gum (1% aqueous solution, viscosity ≥1200 mPa•s at 25℃) to the mixed bacterial suspension. Stir continuously for 30 minutes at 25℃ and 120 r / min to ensure the protective agent is fully dissolved and in uniform contact with the bacterial cells. While continuously stirring, add 1.2% chitosan oligosaccharide (molecular weight ≤3000 Da, degree of deacetylation ≥90%), 0.6% matrine, and sufficient 600-mesh diatomaceous earth carrier to the above mixture, and stir until a uniform paste-like mixture is formed.

[0054] Step 4: Spray dry the mixture. Set the spray drying parameters as follows: feed rate 10 mL / min, atomization pressure 0.3 MPa, inlet air temperature 100℃, outlet air temperature 45℃, and material residence time in the drying tower approximately 2 seconds to ensure that the temperature of the material particles does not exceed 60℃ throughout the drying process. Stop drying when the material moisture content reaches 7.5%. During spray drying, due to the absorption of a large amount of latent heat by water evaporation, the actual temperature of the material particles is much lower than the inlet air temperature; simultaneously, trehalose and mannitol form a protective film on the surface of the bacteria, further reducing heat conduction damage to the bacteria.

[0055] Testing showed that the total number of viable bacteria in the product obtained in this embodiment reached 3.2 × 10⁻⁶. 10 The product has a CFU / g, a particle size of 280 mesh, a suspension rate of 85.3±0.7%, and a pH value of 6.5±0.1, indicating that the drying conditions can effectively protect the bacterial activity and the effective viable count of the product meets the requirements.

[0056] Example 7 The application methods for preventing and controlling strawberry root rot (soil-borne disease) and powdery mildew (foliar disease) include the following steps: Step 1: On the second day after transplanting the strawberry seedlings, apply root irrigation treatment: for strawberry plants with a height ≤10 cm and ≤4 true leaves, take the compound microbial agent prepared in Example 4, dilute it with water 750 times, and apply 250 mL to the roots of each plant.

[0057] Step 2: On the 15th day after strawberry planting, carry out full-field irrigation treatment: Dilute the compound bacterial agent with water 950 times, and irrigate at a rate of 2000L of diluted solution per acre.

[0058] Step 3: Seven days before the strawberry flowering period, use a combination of foliar spraying and root irrigation: Dilute the compound microbial agent with water 900 times and spray it evenly on both sides of the strawberry leaves, with a spraying amount of 35L per acre; at the same time, dilute the compound microbial agent with water 550 times and irrigate the roots of each plant with 350mL.

[0059] Step 4: When sporadic powdery mildew lesions are first discovered in the field (and the diseased leaf rate is ≤3%), foliar spraying treatment should be carried out immediately: dilute the compound bacterial agent 600 times and spray it on the front and back of the diseased leaves and the leaves of the surrounding plants. The spraying amount is 50L per acre. After an interval of 8 days, spray a second time in the same way.

[0060] Example 8 The application method for preventing and controlling cucumber soft rot (bacterial disease) and downy mildew (fungal foliar disease) is to immediately adopt a combination of whole-field irrigation and foliar spraying at the early stage of disease occurrence (soft rot disease incidence ≤2%, downy mildew disease leaf incidence ≤3%).

[0061] Step 1: Take the compound microbial agent prepared in Example 4, dilute it 900 times with water, and irrigate the entire field with 2500L of diluted solution per acre; at the same time, take the compound microbial agent, dilute it 550 times with water, and spray it on both sides of the cucumber leaves and the diseased areas, with a spraying amount of 50L per acre.

[0062] Step 2: Six days after the first foliar spray, dilute the compound microbial agent 550 times and spray it for the second time, with a spraying amount of 55L per acre.

[0063] Example 9 Application methods for controlling tomato bacterial wilt (a bacterial disease) and leaf mold (a fungal foliar disease) involve tiered spraying based on the severity of the disease, specifically including: When the disease incidence rate in the field is ≤5%, the compound microbial agent prepared in Example 5 is diluted 600 times with water and sprayed on the diseased parts, leaves and stems of the tomato plants at a rate of 50L per acre.

[0064] When the disease incidence rate in the field is >5%, first use kasugamycin to treat bacterial wilt and pyraclostrobin to treat leaf mold. After 7 days of drug treatment, take compound bacterial agent, dilute it 700 times with water, and spray it on the diseased parts, leaves and stems of tomato plants. The spraying amount is 40L per acre.

[0065] Example 10 The application method for controlling watermelon wilt (a soil-borne fungal disease) and vine blight (a foliar fungal disease) includes the following steps: Step 1: Two days before watermelon sowing, dilute the compound microbial agent prepared in Example 6 with water 900 times, and irrigate the entire field with 2500L of diluted solution per acre. Step 2: On the first day after transplanting watermelon seedlings, apply root irrigation treatment: When the watermelon plants are 10-30cm tall and have 4-8 true leaves, dilute the compound bacterial agent 650 times with water and apply 350mL to the roots of each plant. Step 3: Six days before the watermelon flowering period, use a combination of foliar spraying and root irrigation: Dilute the compound microbial agent 950 times with water and spray it evenly on both sides of the watermelon leaves, with a spraying amount of 40L per acre; at the same time, dilute the compound microbial agent 550 times with water and irrigate the roots of each plant with 400mL.

[0066] The high-efficiency synergistic compound microbial agent of the present invention is vacuum-packed in aluminum foil composite bags, with a net content of 1 kg or 5 kg per bag, and the outer packaging is a cardboard box. Under sealed, light-proof, and dry conditions, it is stored in a cool place below 25℃ (relative humidity ≤60%), with a shelf life of 12 months (live bacteria survival rate ≥92%); when stored in a cold storage at 4℃, the shelf life can be extended to 24 months (live bacteria survival rate ≥95%). (1) Short-term storage at room temperature: suitable for planting bases or agricultural material dealers to use in the current season, no refrigeration equipment is required, it is recommended to use within 7 days after opening the bag, and the unused part should be sealed and placed in a cool place. (2) Long-term cold chain storage: suitable for production enterprises or large distributors to stock up across seasons, it is recommended to store in a cold storage at 4℃, which can guarantee the product activity within two years. (3) Transportation conditions: the product activity is not affected by short-distance transportation (≤7 days) within the range of -5℃ to 40℃, but direct sunlight and rain should be avoided.

[0067] Experiment and Analysis All experiments in this section used the highly efficient synergistic compound microbial agent prepared in Example 4 as the core test material. The test strains were Bacillus subtilis CGMCC 1.1729 and Trichoderma harzianum CGMCC 3.10154, both purchased from the China General Microbiological Culture Collection Center (CGMCC).

[0068] In all the following experiments, the formula for calculating the "control effect" is: Control effect (%) = [(Disease index of blank control group - Disease index of treatment group) / Disease index of blank control group] × 100%; The disease index is calculated according to the grading standard of the corresponding disease in the "Guidelines for Field Efficacy Tests of Pesticides" issued by the Ministry of Agriculture and Rural Affairs; All experimental data were compared using Duncan's new multiple range method, with p < 0.05 indicating significant difference and p < 0.01 indicating extremely significant difference. Each treatment group was set up with 3 biological replicates, and the data are expressed as "mean ± standard deviation".

[0069] Experiment 1: Verification of the antagonistic effect, fermentation performance, and synergistic effect of the tested strains. 1. Experimental Materials and Instruments Tested strains: Bacillus subtilis CGMCC 1.1729 and Trichoderma harzianum CGMCC 3.10154.

[0070] Culture media: LB liquid medium, PDA solid medium, PDA semi-solid medium, LB+PDA mixed medium (1:1, v / v).

[0071] Test pathogen: Phytophthora capsici ( Phytophthora capsicum ), cucumber downy mildew ( Pseudoperonospora cubensis ).

[0072] Instruments: constant temperature shaking incubator, biochemical incubator, clean bench, hemocytometer, ultraviolet spectrophotometer.

[0073] 2. Test Plan (1) Verification of antagonistic effect of strains (plate confrontation method): PDA medium was poured into plates, and after solidification, a 5 mm diameter Trichoderma harzianum CGMCC 3.10154 mycelial cake was inoculated in the center of the plate; Bacillus subtilis CGMCC1.1729 bacterial suspension (1×10⁻⁶) was inoculated at the four corners 25 mm away from the center mycelial cake. 8 CFU / mL), 5 µL was inoculated at each point; a blank control plate was set up with only Trichoderma harzianum inoculated, and the plates were incubated at 28℃ in the dark for 72 h. The growth of the two strains was observed, the width of the inhibition zone was measured, and the presence of antagonistic effects was determined.

[0074] (2) Validation of the fermentation performance of the strain: A. A single colony of Bacillus subtilis CGMCC 1.1729 was inoculated into LB liquid medium and cultured aerobically at 32°C and 190 r / min according to the process parameters of Example 4 of this invention. Samples were taken at 12h, 18h, 20h, and 24h of culture, and the viable count was determined using the serial dilution plating method to verify whether ≥10⁻⁶ cells / cells could be reached within 18-24h. 10 The index is CFU / mL.

[0075] B. Inoculate Trichoderma harzianum CGMCC 3.10154 onto PDA plates and incubate at 26°C in the dark. Samples are taken at 48h, 60h, and 72h. Conidia are washed away with sterile water, and spore concentration is counted using a hemocytometer to verify whether ≥10⁻⁶ spores can be reached within 48–72h. 9 The index is CFU / mL.

[0076] (3) Verification of the synergistic effect of strains: A. *Bacillus subtilis* CGMCC 1.1729 and *Trichoderma harzianum* CGMCC 3.10154 were mixed at a viable viable count ratio of 1:1.2 and inoculated onto LB+PDA mixed medium (1:1, v / v). *Bacillus subtilis* CGMCC 1.1729 and *Trichoderma harzianum* CGMCC 3.10154 were used as controls. The mixture was incubated at 28℃ and 120 r / min with shaking. Samples were taken every 4 h, and the viable counts of both strains in each treatment were counted using the serial dilution plate method to plot co-culture growth curves.

[0077] B. The combination of the present invention (Bacillus subtilis CGMCC 1.1729 + Trichoderma harzianum CGMCC 3.10154, ratio 1:1.2) and single Bacillus subtilis CGMCC 1.1729 and single Trichoderma harzianum CGMCC 3.10154 were used to conduct plate confrontation inhibition tests on Phytophthora capsici and Pseudoperonospora cubensis, respectively. The diameter of the inhibition zone was measured and the synergy coefficient was calculated (synergy coefficient = diameter of the combined inhibition zone / average diameter of the inhibition zones of the two single bacteria).

[0078] 3. Testing Methods Antagonistic effect determination: If there is no inhibition zone and the two strains can grow normally across each other, it is determined that there is no antagonism; if there is a clear inhibition zone, it is determined that there is antagonism.

[0079] Viable bacterial count / spore concentration determination: The serial dilution plate method was used, with 3 replicates for each sample. The bacteria were incubated at 37℃ for 24 h (Bacillus subtilis) and at 28℃ for 48 h (Trichoderma harzianum) before counting.

[0080] 4. Test Results Table 1: Results of the antagonistic effect test between the two strains Table 2: Changes in viable cell count during Bacillus subtilis fermentation process Table 3: Changes in spore concentration during Trichoderma harzianum culture Table 4: Comparison of antibacterial activity between the combination of the present invention and a single bacterial agent The co-culture growth curve results showed that during the 0-48h culture period, the viable counts of both strains continuously increased without mutual inhibition; at 48h, the viable count of Bacillus subtilis was 1.35 × 10⁻⁶. 8CFU / mL, viable count of Trichoderma harzianum was 9.8 × 10⁻⁶. 7 CFU / mL, consistent with the growth trend during monoculture.

[0081] 5. Data Analysis (1) The results of confrontation culture showed that there was no inhibition zone when Bacillus subtilis CGMCC 1.1729 and Trichoderma harzianum CGMCC 3.10154 were co-cultured. Both strains could grow normally without any antagonistic effect, which fully met the 1:1.2~1.5:1 ratio requirement of this invention and could achieve stable compounding.

[0082] (2) The fermentation performance test results showed that the viable count of Bacillus subtilis CGMCC 1.1729 could stably reach 1.21 × 10⁻⁶ cells after 20 h of culture. 10 CFU / mL, fully meeting the requirement of ≥10 in this invention. 10 Requirements for preparing bacterial suspensions with CFU / mL; *Trichoderma harzianum* CGMCC 3.10154 achieves a spore concentration of 1.52 × 10⁻⁶ after 60 hours of cultivation. 9 CFU / mL, meeting the requirement of ≥10 in this invention. 9 The preparation requirements for spore suspensions at CFU / mL are met, and the fermentation / sporogenesis performance of the two strains is adapted to the preparation process of this invention.

[0083] (3) The synergistic effect verification results showed that the co-culture growth curve indicated that the viable counts of both strains continuously increased during the 0-48h culture period, with no mutual inhibition. At 48h, the viable counts of Bacillus subtilis and Trichoderma harzianum reached 1.35 × 10⁻⁶. 8 CFU / mL and 9.8×10 7 The CFU / mL ratio was consistent with the growth trend observed during single-culture, indicating that the two bacteria can coexist and proliferate normally under this ratio. The comparative results of antibacterial activity showed that the inhibition zone diameter of the combination of the present invention against both pathogens was significantly larger than that of the single bacterial agent (p<0.05), with a synergistic coefficient of 1.35, greater than 1, demonstrating a positive synergistic effect. This proves that the two bacteria have a significant synergistic effect under the 1:1.2 ratio specified in this invention, rather than a simple additive effect.

[0084] 6. Conclusion The selected Bacillus subtilis CGMCC 1.1729 and Trichoderma harzianum CGMCC 3.10154 showed no antagonistic effect, and their fermentation and sporulation performance fully met the process requirements of this invention. Under the specified ratio of this invention, the antibacterial activity and co-culture growth performance of the two strains were significantly better than those of a single agent, with a synergistic coefficient of 1.35, proving that this ratio can achieve positive synergistic effects. Both strains can be used as the core production strains of the compound agent of this invention, exhibiting excellent compatibility.

[0085] Experiment 2: Validation Experiment of Low-Temperature Drying Process for Compound Microbial Agent 1. Experimental materials and instruments: Test material: The mixture prepared according to steps 1 to 3 of Example 4 (two strains in a ratio of 1:1.2, including a complete set of synergistic adjuvants).

[0086] Instruments: small spray dryer, constant temperature forced air drying oven, ultra-clean workbench.

[0087] Testing indicators: moisture content of materials, total number of viable bacteria in compound microbial agent, and bacterial survival rate.

[0088] 2. Test Plan Two treatment groups were set up, with three replicates in each group: Treatment I (low-temperature drying process group of this invention): strictly following the parameters of Example 4, the spray drying feed rate was 10 mL / min, the atomization pressure was 0.3 MPa, the inlet air temperature was 105℃, the outlet air temperature was 48℃, and the drying time was 18 min, controlling the material temperature ≤60℃ during the drying process. Treatment II (conventional high-temperature drying control group): using industry-standard high-temperature spray drying parameters, the inlet air temperature was 140℃, the outlet air temperature was 70℃, and the remaining feed rate and atomization pressure were the same as Treatment I. Simultaneously, a drying time gradient group was set up: 15 min, 18 min, 20 min, and 25 min, with the remaining parameters consistent with Treatment I, to verify the optimal drying time.

[0089] 3. Testing Methods Moisture content determination: determined by drying method in GB 20287-2006 "Agricultural Microbial Agents".

[0090] Determination of total viable count: The viable counts of Bacillus subtilis and Trichoderma harzianum were counted separately using the serial dilution plate method, and the total viable count was calculated.

[0091] Calculation of bacterial survival rate: Survival rate (%) = (Total number of viable bacteria after drying / Total number of viable bacteria in the mixture before drying) × 100%.

[0092] 4. The test results are shown in Tables 5 and 6.

[0093] Table 5: Effects of different drying processes on the core indicators of microbial agents Table 6: Effects of different drying times on microbial inoculant properties 5. Data Analysis (1) The total number of viable bacteria in the low-temperature drying process group of the present invention reaches 3.81 × 10⁻⁶. 10The CFU / g cell survival rate was as high as 94.2%, significantly higher than that of the high-temperature control group (p<0.01); the high-temperature drying process resulted in more than 70% cell inactivation, failing to meet the ≥3.0×10⁻⁶ CFU / g requirement of this invention. 10 The core indicator of CFU / g proves that the drying process parameters defined in this invention are the key to ensuring the high activity of the microbial agent.

[0094] (2) The drying time gradient test showed that when the drying time was 15~20 min, the moisture content of the inoculant was ≤8%, and the total number of effective viable bacteria was ≥3.0×10. 10 The optimal drying time was 18 min, during which both the viable cell count and moisture content reached their optimal levels. When the drying time exceeded 20 min, the bacteria were exposed to heat for too long, resulting in a significant decrease in the viable cell count (p<0.05).

[0095] 6. Conclusion The spray drying process parameters specified in this invention can ensure drying efficiency while maximizing the preservation of bacterial activity. The final product fully meets the requirements of this invention: moisture content ≤8% and total effective viable bacteria count ≥3.0 × 10⁻⁶. 10 The core indicator, CFU / g, shows that the process is stable and repeatable, making it suitable for large-scale production.

[0096] Experiment 3: Storage Stability Verification Test of Compound Microbial Agent 1. Experimental materials and instruments: Test materials: the high-efficiency synergistic compound microbial agent prepared in Example 4, and the commercially available conventional Bacillus subtilis + Trichoderma harzianum compound microbial agent (control group, effective viable count 2.0 × 10⁻⁶). 10 CFU / g).

[0097] Instruments: Biochemical incubator, 4℃ refrigerator, ultra-clean workbench.

[0098] Storage conditions: Store at 25℃ in a dark, sealed container; store at 4℃ in a refrigerated, sealed container; or store at 37℃ using accelerated storage (simulating long-term storage at room temperature).

[0099] 2. Test Plan (1) Dispense the test bacterial agent into aluminum foil vacuum bags, 100g per bag, and store them at 25℃, 4℃ and 37℃ respectively.

[0100] (2) Samples were taken at 0 months, 3 months, 6 months, 12 months and 24 months of storage, the total number of viable bacteria was determined and the viable bacteria survival rate was calculated.

[0101] (3) Survival rate calculation formula: viable bacteria survival rate (%) = (total number of viable bacteria after storage / initial total number of viable bacteria) × 100%, with 3 replicates per group.

[0102] 3. Testing Methods Determination of total viable count: The viable counts of Bacillus subtilis and Trichoderma harzianum were counted separately using the serial dilution plate method, and the total viable count and survival rate were calculated.

[0103] 4. The test results are shown in Tables 7 and 8.

[0104] Table 7: Changes in viable bacterial survival rate under storage conditions at 25℃ Table 8: Comparison of viable bacterial survival rates under different storage conditions over 12 months 5. Data Analysis (1) After being stored at room temperature (25°C) for 12 months, the viable bacteria survival rate of the bacterial agent of the present invention is still as high as 92.7%, and the total number of effective viable bacteria remains at 3.53 × 10⁻⁶. 10 The CFU / g is significantly higher than the ≥3.0×10⁻⁶ of this invention. 10 The CFU / g standard was significantly better than that of the commercially available control agent (p<0.01); the survival rate of the commercially available control agent was less than 60% after 6 months of storage.

[0105] (2) Under refrigeration at 4℃, the survival rate of the bacterial agent of the present invention can still reach more than 95% after 24 months of storage, and the survival rate is ≥85% after 90 days of accelerated storage at 37℃. According to the Arrhenius equation, the shelf life of the bacterial agent of the present invention can be stably reached 12 months at room temperature at 25℃ and 24 months at refrigeration at 4℃, which fully meets the requirements of commercial production and distribution.

[0106] 6. Conclusion The highly efficient synergistic compound microbial agent prepared in Example 4, through precise strain ratio and synergistic protection of the synergistic adjuvant system, exhibits storage stability far superior to similar commercially available products. After 12 months of storage at room temperature (25°C), the survival rate of live bacteria is ≥92%, fully meeting the technical requirements of this invention and possessing excellent commercial application value.

[0107] Experiment 4: Field Disease Control Efficacy Verification Experiment of Compound Microbial Agent 1. Experimental materials and locations Test bacterial agent: The highly efficient synergistic compound bacterial agent prepared in Example 4.

[0108] The tested crops were strawberry (variety "Hongyan") and cucumber (variety "Guiyan No. 2").

[0109] Target diseases: Strawberry root rot (soil-borne fungal disease), strawberry powdery mildew (foliar fungal disease); cucumber downy mildew (foliar fungal disease), cucumber soft rot (bacterial disease).

[0110] Experimental locations: Strawberry planting base in the suburbs of Nanning City, Guangxi Zhuang Autonomous Region, and greenhouse cucumber planting base in Guilin City.

[0111] Control agents: Local conventional chemical control agents (50% carbendazim wettable powder, 25% pyraclostrobin suspension, 47% kasugamycin-copper oxychloride wettable powder).

[0112] 2. Test Plan A fully randomized block design was used, with each treatment having 3 replicates and each cell having an area of ​​30 m². 2 Protective zones are set up between communities to prevent cross-contamination.

[0113] (1). Strawberry field trial grouping Group T1 (treatment group of the present invention): applied according to the scheme of Example 7.

[0114] Group T2 (Conventional Chemical Control Group): Following the local growers' conventional approach, drench the roots with 50% carbendazim at 800 times dilution at the initial stage of root rot, and spray with 25% pyraclostrobin at 1500 times dilution at the initial stage of powdery mildew.

[0115] CK group (clean water blank control group): The same amount of clean water was sprayed / irrigated at the same time, and the rest of the agricultural management was completely the same.

[0116] (2). Cucumber field experiment grouping Group T1 (treatment group of the present invention): applied according to the scheme of Example 8.

[0117] Group T2 (Conventional Chemical Control Group): Following the local growers' conventional treatment plan, alternately spray and drench the roots with 47% Chunlei·Wangtong 800 times solution and 68% Jingjiashuang·Mangzinc 600 times solution every 5-7 days.

[0118] CK group (clean water blank control group): The same amount of clean water was sprayed / irrigated at the same time, and the rest of the agricultural management was exactly the same.

[0119] 3. Testing Methods During the peak harvest period (strawberries) and 14 days after the second pesticide application (cucumbers), a five-point sampling method was used for investigation. 100 plants were randomly sampled in each plot, and the number of diseased plants and leaves were recorded. The disease index and control effect were calculated according to the "Guidelines for Field Efficacy Trials of Pesticides". The disease incidence rate was calculated as follows: Disease incidence rate (%) = (Number of diseased plants / Total number of plants surveyed) × 100%.

[0120] 4. The test results are shown in Tables 9 to 11.

[0121] Table 9: Comparison of Strawberry Disease Control Effects Table 10: Comparison of the effectiveness of cucumber disease control Table 11: Comparison of control efficacy of application programs for different disease severity levels (verification of pepper blight) 5. Data Analysis (1) In the strawberry field trial, the treatment group of the present invention achieved a control effect of 86.8% on strawberry root rot and 87.2% on powdery mildew, which was significantly better than the conventional control schemes of farmers. In the cucumber field trial, the treatment group of the present invention achieved a control effect of 89.9% on cucumber bacterial soft rot and 85.7% on fungal downy mildew, which was also significantly better than the conventional chemical control group (p<0.05). This proves that the compound microbial agent of the present invention can simultaneously and effectively control fungal and bacterial diseases, with a broad control spectrum and stable control effect.

[0122] (2) The results of the graded application scheme verification show that the overall average control efficacy of the graded application strategy of the present invention is 88.9%, which is significantly better than the uniform application scheme (p<0.05). Especially in severely diseased areas with a disease rate of >5%, the scheme of first chemical suppression and then biological consolidation solves the problem of poor control efficacy of fungal agents under high pathogen base, and the control efficacy is improved by 18.7%.

[0123] 6. Conclusion The highly efficient synergistic compound microbial agent prepared in Example 4, combined with the refined application scheme of the present invention, has excellent control effects on fungal and bacterial diseases of strawberries and cucumbers, meeting the requirements of green agricultural production.

[0124] Experiment 5: Verification Experiment on the Effects of Compound Microbial Agents on Crop Yield Increase, Quality Improvement, and Soil Improvement 1. Experimental Materials and Methods Test materials: Fruit and root zone soil samples from each treatment group of the strawberry and cucumber field experiments in Experiment 4.

[0125] Test indicators: Individual harvesting and yield calculation for each plot, including average yield per acre and yield increase rate; soluble solids content of strawberry fruit (handheld refractometer method), vitamin C content of cucumber (2,6-dichlorophenolindophenol titration method); soil bulk density (ring cutter method), and total soil microbial biomass (phospholipid fatty acid PLFA method).

[0126] 2. The test results are shown in Tables 12 to 14.

[0127] Table 12: Comparison of Strawberry Yield Increase and Quality Improvement Effects Table 13: Comparison of Cucumber Yield Increase and Quality Improvement Effects Table 14: Comparison of Soil Physicochemical and Biological Indicators (Before and After the Experiment) 3. Data Analysis (1) The average yield of strawberries in the treatment group of this invention increased by 17.3% and cucumbers by 15.3% compared with the blank control group. The soluble solids and vitamin C content of the fruit were significantly higher than those of the chemical control group and the blank control group (p<0.05), which proves that the compound microbial agent of this invention can not only control diseases, but also significantly improve crop yield and commercial quality.

[0128] (2) After the experiment, the soil bulk density of the treatment group of the present invention decreased by 8.5%, the total soil microbial biomass increased by 36.2%, and the abundance of beneficial microorganisms increased by 35.8%, which was significantly better than the chemical control group and the blank control group (p<0.01), proving that the compound microbial agent of the present invention can effectively improve the soil physical structure, optimize the soil micro-ecology, and alleviate the continuous cropping obstacles.

[0129] 4. Conclusion The highly efficient synergistic compound microbial agent prepared in Example 4 can significantly improve crop yield and quality while effectively controlling diseases. It also improves soil physicochemical properties and optimizes rhizosphere microecology, achieving a comprehensive effect of disease prevention, yield increase and soil improvement, and has excellent ecological benefits and economic value.

[0130] Experiment 6: Environmental Safety Verification Test of Compound Microbial Agent 1. Experimental Materials and Methods Test bacterial agent: The highly efficient synergistic compound bacterial agent prepared in Example 4. Test organisms: zebrafish, silkworm, bee, and earthworm (all of which are test organisms for environmental risk assessment).

[0131] Experimental methods: Acute toxicity tests were conducted in accordance with the standard methods of the "Guidelines for Environmental Risk Assessment of Pesticide Registration" to detect the toxicity of the fungicide to non-target organisms, and at the same time, the residues in the soil and plants after application were detected.

[0132] 2. The test results are shown in Tables 15 and 16.

[0133] Table 15: Acute toxicity results of microbial agents to non-target organisms Table 16: Results of the Experiment on the Influence of Beneficial Microorganisms on Soil 3. Data Analysis (1) The results of the acute toxicity test showed that the compound microbial agent of the present invention was practically non-toxic to zebrafish, silkworms and earthworms, and low toxic to bees, which met the safety requirements of the "Guidelines for Environmental Risk Assessment of Pesticide Registration".

[0134] (2) Soil microbial test results showed that after 30 days of application of the microbial agent of the present invention, the number of beneficial microorganisms such as soil rhizobia and actinomycetes increased significantly without inhibition, proving that the microbial agent is friendly to the soil micro-ecological environment.

[0135] (3) The residue test results showed that no harmful residues were detected in the soil, plants and fruits 30 days after the application of this microbial agent, which meets the safety standards for green food production.

[0136] 4. Conclusion The highly efficient synergistic compound microbial agent prepared in Example 4 is non-toxic to non-target organisms, has no inhibitory effect on beneficial soil microorganisms, leaves no environmental residues, and has excellent environmental safety, meeting the production safety requirements of green agriculture and organic agriculture.

[0137] Experiment 7: Toxicological Safety Evaluation of the Strains 1. Experimental materials: Bacillus subtilis CGMCC 1.1729, Trichoderma harzianum CGMCC 3.10154.

[0138] 2. Test Methods Safety classification and identification were conducted according to NY / T 1109 "General Technical Guidelines for Biosafety of Microbial Fertilizers", and a third party was commissioned to conduct acute oral toxicity, acute dermal toxicity, skin irritation, and sensitization tests. Antibiotic resistance testing was conducted according to EUCAST standards, detecting the minimum inhibitory concentration (MIC) of two strains against eight clinically used antibiotics, including tetracycline, erythromycin, streptomycin, and chloramphenicol, and also detecting whether they carried acquired resistance genes.

[0139] 3. Experimental Results Bacillus subtilis CGMCC 1.1729 belongs to the first-level strains exempt from toxicology testing according to NY / T 1109; the toxicology test of Trichoderma harzianum CGMCC 3.10154 showed that the acute oral LD50 was >5000 mg / kg and the acute dermal LD50 was >5000 mg / kg, with no skin irritation or sensitization, meeting the safety requirements.

[0140] Antibiotic resistance testing showed that the MIC values ​​of both strains for the eight antibiotics tested in this experiment were all below the resistance breakpoints specified by EUCAST, and no acquired antibiotic resistance genes were detected, indicating that there is no risk of transferable antibiotic resistance in either strain.

[0141] 4. Conclusion The strains used in this invention are non-pathogenic and pose no risk of antibiotic resistance, and fully comply with the safety requirements of GB 20287-2006 and NY / T1109.

[0142] Comprehensive test conclusions 1. The Bacillus subtilis CGMCC 1.1729 and Trichoderma harzianum CGMCC 3.10154 selected in this invention have no antagonistic effect and are safe and compliant. Under the ratio specified in this invention, the antibacterial activity and co-culture growth performance of the two bacteria combination are significantly better than those of the single bacteria agent, with a synergistic coefficient of 1.35, proving that this ratio can achieve positive synergistic effect and the fermentation performance is suitable for the process of this invention.

[0143] 2. The low-temperature drying process of this invention can maximize the preservation of bacterial activity, and the total number of effective viable bacteria in the final product is consistently ≥3.0×10⁻⁶. 10 CFU / g, with a live bacteria survival rate of ≥92% after 12 months of storage at room temperature (25℃), which is superior to similar products on the market.

[0144] 3. The compound microbial agent of this invention has excellent control effects on both fungal and bacterial diseases, and can significantly improve crop yield and quality, improve soil microecology, and achieve the three-in-one effect of disease prevention, yield increase and soil improvement.

[0145] 4. The compound microbial agent of this invention has excellent environmental safety, no pesticide residues, and is non-toxic to non-target organisms, meeting the requirements of green agricultural production and possessing broad prospects for commercial promotion.

[0146] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be readily implemented by those skilled in the art. Therefore, the present invention is not limited to the specific details without departing from the general concept defined by the claims and their equivalents.

Claims

1. A highly efficient synergistic compound microbial agent, characterized in that, Including Bacillus subtilis and Trichoderma harzianum; Based on the total weight of the compound microbial agent, the effective viable count of Bacillus subtilis is ≥1.5×10⁻⁶. 10 CFU / g, effective viable count of Trichoderma harzianum ≥1.2×10⁻⁶ 10 CFU / g; the ratio of effective viable bacteria count of Bacillus subtilis to Trichoderma harzianum is 1:1.2 to 1.5:1, and the total effective viable bacteria count of the highly efficient synergistic compound microbial agent is ≥3.0×10⁻⁶. 10 CFU / g.

2. The high-efficiency synergistic compound microbial agent as described in claim 1, characterized in that, It also includes synergistic adjuvant systems; The synergistic adjuvant system is based on the total weight of the compound microbial agent and consists of 5%~8% trehalose, 3%~5% mannitol, 2%~3% xanthan gum, 1%~2% chitosan oligosaccharide, 0.5%~1% matrine and the balance diatomaceous earth. The xanthan gum has a viscosity ≥1200 mPa·s in a 1% aqueous solution at 25°C; the chitosan oligosaccharide has a molecular weight ≤3000 Da and a degree of deacetylation ≥90%.

3. The high-efficiency synergistic compound microbial agent as described in claim 2, characterized in that, Diatomaceous earth has a particle size of 500-800 mesh and a specific surface area of ​​15-25 m². 2 / g, porosity 70%~80%.

4. The highly efficient synergistic compound microbial agent as described in any one of claims 1 to 3, characterized in that, The compound microbial agent is a wettable powder with a suspension rate of ≥80%, a pH value of 6.0~7.5, and a finished product particle size of 200~400 mesh.

5. A method for preparing a highly efficient synergistic compound microbial agent as described in any one of claims 1 to 4, characterized in that, include: Bacillus subtilis was inoculated onto LB medium and cultured aerobicly at 30–32°C and 180–200 r / min for 18–24 h to obtain a viable count ≥10⁻⁶. 10 CFU / mL Bacillus subtilis bacterial suspension; Trichoderma harzianum was inoculated onto PDA medium and cultured at 25–28°C in the dark for 48–72 h. Conidia were collected and prepared to a concentration ≥10. 9 CFU / mL Trichoderma harzianum spore suspension; Bacillus subtilis bacterial suspension and Trichoderma harzianum spore suspension were mixed, and trehalose, mannitol and xanthan gum were added. The mixture was stirred at 25-30℃ and 120 r / min for 30-60 min. Then chitosan oligosaccharide, matrine and diatomaceous earth carrier were added and mixed evenly to obtain the mixture. The mixture was spray-dried at a feed rate of 10 mL / min, an atomization pressure of 0.3 MPa, an inlet air temperature of 100–110℃, an outlet air temperature of 45–50℃, a material temperature ≤60℃ during drying, and a drying time of 15–20 min, until the moisture content was ≤8% and the total number of viable bacteria was ≥3.0 × 10⁻⁶. 10 With a CFU / g concentration, the product particle size after drying is 200~400 mesh, thus obtaining a highly efficient synergistic compound microbial agent.

6. The application of the highly efficient synergistic compound microbial agent as described in any one of claims 1 to 4 in the prevention and control of fungal and / or bacterial diseases.

7. A method for controlling plant fungal and / or bacterial diseases using a highly efficient synergistic compound microbial agent as described in any one of claims 1 to 4, characterized in that, Highly efficient synergistic compound microbial agents were applied to target crops, including strawberries, cucumbers, tomatoes, peppers, watermelons, citrus, and cotton. Plant diseases include fungal diseases and bacterial diseases; Fungal diseases include root rot, wilt, powdery mildew, gray mold, and downy mildew; Bacterial diseases include soft rot, bacterial wilt, canker, and bacterial leaf spot.

8. The method as described in claim 7, characterized in that, The application method for spraying when the disease occurs is as follows: When the disease incidence rate is ≤5%, dilute the aforementioned high-efficiency synergistic compound microbial agent with water at a ratio of 500 to 700 times and spray it on the diseased parts, leaves, and stems of the plants. The spraying amount is 45 to 60 L per acre. When the disease incidence rate is >5%, first treat the plants with biopesticides for the corresponding disease or chemical pesticides with low toxicity to biocontrol bacteria. Seven days after the treatment, dilute the highly efficient synergistic compound bacterial agent with water 600-800 times and spray it on the diseased parts, leaves and stems of the plants. The spraying amount is 35-50 L per acre.

9. The method as described in claim 7, characterized in that, The application method for soil-borne diseases is as follows: Root irrigation should be carried out 1-3 days after transplanting, and full field irrigation should be carried out 13-16 days after planting. The root irrigation treatment is implemented according to the plant's growth stage, and specifically includes: When the plant height is ≤10cm and the number of true leaves is ≤4, dilute the aforementioned high-efficiency synergistic compound bacterial agent with water 700-800 times and irrigate the roots of each plant with 200-300 mL. When the plant is 10-30cm tall and has 4-8 true leaves, dilute the highly efficient synergistic compound microbial agent with water 600-700 times and irrigate the roots of each plant with 300-400 mL. When the plant height is ≥30cm or the budding / fruit setting rate is ≥50%, take the highly efficient synergistic compound microbial agent, dilute it with water 500~600 times, and irrigate the roots of each plant with 400~500 mL. The irrigation treatment is as follows: Dilute the aforementioned high-efficiency synergistic compound microbial agent with clean water 900 to 1000 times, and irrigate at a rate of 2000 L of diluted solution per acre.

10. The method as described in claim 7, characterized in that, The application method for proactive prevention is to apply it at key stages of crop growth, specifically including: One to three days before sowing, dilute the high-efficiency synergistic compound microbial agent with water at a ratio of 800 to 1000 times, and irrigate with 2000 to 3000 L of diluted solution per acre. Within 3 days of transplanting, dilute the highly efficient synergistic compound microbial agent with water at a ratio of 500 to 800 times and apply it to the roots at a rate of 200 to 500 mL per plant. 7±1 days before flowering, dilute the high-efficiency synergistic compound microbial agent with water at a ratio of 800 to 1000 times and spray it on the front and back of the leaves at a rate of 30 to 45 L per acre. At the same time, dilute the high-efficiency synergistic compound microbial agent with water at a ratio of 500 to 600 times and drench the roots at a rate of 300 to 500 mL per plant.

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