A bactericidal composition for preventing and treating asparagus stem blight and its preparation process

Abstract

The present invention relates to the technical field of plant disease prevention and control, and in particular to a bactericidal composition for preventing and controlling asparagus stem blight and a preparation process thereof. The preparation process comprises the following steps: preparing azoxystrobin; screening degradation bacteria; preparing a degradation slow-release bacterial agent; and preparing a bactericidal composition. The present invention degrades azoxystrobin by preparing a degradation slow-release bacterial agent, and the screened strains can accurately degrade azoxystrobin, and the strains are coated with a dopamine solution, and the dopamine solution can adhere to the surface of the azoxystrobin dominant degradation strains, and dopamine has good biocompatibility and will not pollute plants. Subsequently, after azoxystrobin takes effect to kill the asparagus stem blight pathogen, the cumulative release amount of the azoxystrobin dominant degradation strains coated with dopamine reaches a certain level, and azoxystrobin begins to be degraded in large quantities, thereby preventing azoxystrobin from polluting the environment.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, and in particular to a bactericidal composition for preventing and controlling asparagus stem blight and a preparation process thereof. Background Art

[0002] Asparagus stem blight, also known as asparagus black spot, is a fungal disease that affects the growth of asparagus plants, mainly caused by fungi such as Alternaria. This disease causes black or dark brown spots to appear on the asparagus stems, and in severe cases, it can cause the stems to die, affecting yield and quality.

[0003] Azoxystrobin is a broad-spectrum and highly efficient fungicide used in agricultural production, and it belongs to the methoxyacrylate fungicide class. This type of fungicide exerts its biological activity by inhibiting the respiration of fungi, and is particularly effective in preventing and controlling fungal diseases on a variety of crops, including asparagus stem blight. Therefore, azoxystrobin is often used to prepare a fungicide composition for preventing and controlling asparagus stem blight. However, the large-scale use of azoxystrobin is likely to leave residues in the soil and water, which has adverse effects on beneficial organisms such as pollinating insects, and there are also requirements for the residual amount of pesticides such as azoxystrobin in the crop products on the market. The prior art often achieves the purpose of reducing the concentration of azoxystrobin by compounding azoxystrobin with other chemical substances, thereby reducing the residual amount of azoxystrobin in the environment. Although this reduces the residual amount of azoxystrobin, the ability to prevent and control stem blight is also weakened, and even if azoxystrobin is compounded, the natural environment alone cannot degrade azoxystrobin in large quantities, resulting in the common fungicide composition for preventing and controlling asparagus stem blight causing environmental pollution. Therefore, the present invention provides a bactericidal composition for preventing and treating asparagus stem blight and a preparation process thereof, which are used to solve the above problems. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a fungicide composition for preventing and treating asparagus stem blight and a preparation process thereof.

[0005] A preparation process of a bactericidal composition for preventing and treating asparagus stem blight comprises the following steps:

[0006] S1: Preparation of azoxystrobin

[0007] DMF, intermediate (E)-2-[2-(6-chloropyridin-4-yloxy)phenyl]-3-methoxyacrylate, potassium carbonate, cuprous iodide and salicylonitrile were added to a three-necked flask, and the mixture was heated and stirred to react to obtain a reaction solution, and the reaction solution was filtered to obtain a filtrate, and the filtrate was distilled under reduced pressure to obtain azoxystrobin crude product, and the azoxystrobin crude product was added to methanol, and the mixture was heated and stirred to dissolve the azoxystrobin crude product, and the mixture was cooled until brown crystals were obtained, and the brown crystals were filtered out, washed, and vacuum dried to obtain azoxystrobin;

[0008] S2: Screening of degrading bacteria

[0009] The microbial environmental samples collected from the natural environment were used as the initial inoculum for enrichment culture of degradation microorganisms. 100 mL of MSM culture medium was placed in a 250 mL Erlenmeyer flask for sterilization. After cooling, the standard working stock solution of azoxystrobin was added under aseptic conditions to make the final mass concentration of azoxystrobin 50 mg / L. At the same time, 10 g of the microbial environmental sample was added. After culturing on a shaker for 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a 10% inoculum. After culturing under the same conditions for 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a 10% inoculum. The amount of the inoculum was transferred to an MSM medium containing 200 mg / L of azoxystrobin, and the culture was continued for 7 days, and then the inoculum was transferred to an MSM medium containing 400 mg / L of azoxystrobin at a 10% inoculum amount, and the culture was continued for 7 days to obtain an MSM medium fermentation liquid, and the MSM medium fermentation liquid was diluted by a gradient dilution multiple of 10 times, and 0.2 mL of the diluted MSM medium fermentation liquid after 4 dilutions was evenly spread on an LB solid plate containing 400 mg / L of azoxystrobin for culture, and the azoxystrobin dominant degrading bacteria were isolated from the LB solid plate;

[0010] S3: Preparation of degradation slow-release bacterial agent

[0011] First, a calcium chloride solution, a sodium carbonate solution and a dopamine solution are prepared, and then a myclobutanil-degrading bacterial strain is added to the calcium chloride solution, and stirred evenly with a magnetic stirrer to obtain a mixed solution, and then sodium polytetrastyrene sulfonate is added to the mixed solution, and after stirring evenly, the solution is added dropwise to the sodium carbonate solution, and the stirring is continued after the dropwise addition is completed to obtain a milky white solution, the milky white solution is allowed to stand, and a supernatant and a precipitate are collected respectively, and a dopamine solution is added to the precipitate, and after the precipitate is completely suspended, a dark brown precipitate is collected, the dark brown precipitate is washed with ultrapure water, and the washed dark brown precipitate is dried into powder to obtain a degradation slow-release bacterial agent;

[0012] S4: Preparation of bactericidal composition

[0013] Add ethyl acetate to a crystallizer, add myclobutanil and a degradation slow-release bacterial agent to dissolve, stir, filter the solid using a Buchner funnel preheated at 50°C to obtain a mixed saturated solution at 50°C, quickly pour the 50°C mixed saturated solution into a 4°C ethyl acetate precooled anti-solvent, stir until crystals precipitate, filter, and dry the crystals at room temperature to obtain a bactericidal composition.

[0014] Furthermore, step S1 prepares azoxystrobin, comprising the following steps:

[0015] S1.1: Add 11 parts by mass of DMF, 1.6 parts by mass of the intermediate (E)-2-[2-(6-chloropyridin-4-yloxy)phenyl]-3-methoxyacrylate, 1 part by mass of potassium carbonate, 0.05 parts by mass of cuprous iodide and 0.7 parts by mass of salicylonitrile to a three-necked flask, heat to 120-140° C., stir and react for 4.5-5 hours to obtain a reaction solution;

[0016] S1.2: Filter the reaction solution to obtain a filtrate, and distill the filtrate under reduced pressure at 65-70°C to obtain a crude product of azoxystrobin. Add the crude product of azoxystrobin to methanol, heat to 50-60°C and stir to dissolve the crude product of azoxystrobin, cool until brown crystals are obtained, filter out the brown crystals, wash the brown crystals with petroleum ether 2-3 times, and dry in vacuo to obtain azoxystrobin.

[0017] Furthermore, step S2 of screening degradation bacteria comprises the following steps:

[0018] S2.1: The microbial environmental samples collected from the natural environment were used as the initial inoculum for enrichment culture of degradation microorganisms. 100 mL of MSM culture medium was placed in a 250 mL conical flask for sterilization. After cooling, the standard working stock solution of azoxystrobin was added under aseptic conditions to make the final mass concentration of azoxystrobin 50 mg / L. At the same time, 10 g of the microbial environmental sample was added. After culturing on a shaker for 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a rate of 10%. After culturing under the same conditions for 7 days, the inoculum was transferred to MSM culture medium containing 200 mg / L azoxystrobin at a rate of 10%, and the culture was continued for 7 days. Then, the inoculum was transferred to MSM culture medium containing 400 mg / L azoxystrobin at a rate of 10%, and the culture was continued for 7 days to obtain MSM culture medium fermentation liquid;

[0019] S2.2: The MSM culture medium fermentation liquid was diluted 10 times in a gradient dilution, and 0.2 mL of the diluted MSM culture medium fermentation liquid after 4 dilutions was evenly applied to an LB solid plate containing 400 mg / L of azoxystrobin. After culturing at 30° C. for 2 days, the azoxystrobin dominant degrading bacteria were isolated from the LB solid plate.

[0020] Furthermore, in step S1.2, the vacuum drying condition is 60-70° C. for 5-6 hours.

[0021] Furthermore, the shaking culture conditions in step S2.1 are 30° C. and 200 r / min.

[0022] Furthermore, step S3 of preparing a degradation sustained-release bacterial agent comprises the following steps:

[0023] S3.1: First, prepare 0.33 mol / L calcium chloride solution and sodium carbonate solution, and 1 mg / mL dopamine solution, then add the dominant azoxystrobin-degrading bacteria to the calcium chloride solution, and stir evenly with a magnetic stirrer to obtain a mixed solution;

[0024] S3.2: Add 0.3 parts by mass of sodium polytetrastyrene sulfonate to the mixed solution, stir evenly, and then dropwise add to 100-120 parts by volume of sodium carbonate solution. After the dropwise addition is complete, continue stirring for 1-2 minutes to obtain a milky white solution;

[0025] S3.3: After the milky white solution is allowed to stand for 30-35 minutes, the supernatant and the precipitate are collected separately, 200-220 parts by volume of the dopamine solution are added to the precipitate, and after the precipitate is completely suspended, the mixture is stirred at a speed of 300-350 r / min for 20-24 hours, and the dark brown precipitate is collected;

[0026] S3.4: Wash the dark brown precipitate three times with ultrapure water of pH=8.5, dry the washed dark brown precipitate into powder in an oven at 45-50℃ to obtain a degradation slow-release bacterial agent.

[0027] Furthermore, in step S3.1, the solid-to-liquid ratio of the azoxystrobin dominant degrading bacteria to the calcium chloride solution is 1:100 g / L.

[0028] Furthermore, step S4 prepares the bactericidal composition, comprising the following steps:

[0029] S4.1: Set the ice machine to a constant temperature of 50-55°C, connect the crystallizer, add ethyl acetate into the crystallizer, wait until the temperature reaches the set temperature, add azoxystrobin and a degradation slow-release bacterial agent to dissolve, stir at a rate of 150-230 r / min, stir for 10-12 hours, turn off the magnetic stirring, filter the solid using a Buchner funnel preheated at 50°C, and obtain a mixed saturated solution at 50°C;

[0030] S4.2: Pour the mixed saturated solution at 50°C rapidly into a pre-cooled anti-solvent of ethyl acetate at 4°C, stir until crystals precipitate, filter, and dry the crystals at room temperature to obtain a bactericidal composition.

[0031] Furthermore, in step S4.1, the mass ratio of azoxystrobin, the degradation slow-release bacterial agent and ethyl acetate is 27:13:100.

[0032] A bactericidal composition for preventing and treating asparagus stem blight is prepared by any one of the above-mentioned preparation processes of the bactericidal composition for preventing and treating asparagus stem blight.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects:

[0034] 1. The present invention degrades azoxystrobin by preparing a degradation slow-release bacterial agent. The screened bacterial strain can accurately degrade azoxystrobin. The bacterial strain is coated with a dopamine solution. The dopamine solution can adhere to the surface of the azoxystrobin-dominant degrading bacterial strain. In addition, dopamine has good biocompatibility and will not pollute plants. Subsequently, after azoxystrobin takes effect to kill the asparagus stem blight pathogen, the cumulative release amount of the azoxystrobin-dominant degrading bacterial strain coated with dopamine reaches a certain level, and azoxystrobin begins to be degraded in large quantities, thereby preventing azoxystrobin from polluting the environment.

[0035] 2. In the present invention, azoxystrobin is subjected to polycrystallization treatment when preparing the fungicidal composition, and azoxystrobin is compounded with a degradation sustained-release bacterial agent and ethyl acetate to prepare a solvate. After the degradation sustained-release bacterial agent is dissolved in ethyl acetate, since azoxystrobin and ethyl acetate contain similar polar groups, azoxystrobin is dissolved in large quantities in ethyl acetate according to the principle of like dissolves like, and adheres to the degradation sustained-release bacterial agent dispersed in ethyl acetate due to the intermolecular interaction force. Subsequently, crystallization and drying are performed to ensure the integrity of the dopamine coating layer on the surface of the degradation sustained-release bacterial agent. The obtained fungicidal composition has azoxystrobin as a main component and is used for sterilization. After the azoxystrobin adhered to the outer layer of the dopamine coating layer volatilizes, the dopamine coating layer volatilizes and disintegrates over time, and the azoxystrobin dominant degradation bacteria in the inner layer begin to degrade the residual azoxystrobin, thereby preventing excessive azoxystrobin residues from affecting plant growth and soil environment.

[0036] 3. The present invention prepares azoxystrobin and carries out specialized iterative cultivation of the prepared azoxystrobin, thereby culturing a natural strain capable of using azoxystrobin as a main food source in a high-concentration azoxystrobin solution, and preparing azoxystrobin-dominant degrading bacteria. The azoxystrobin-dominant degrading bacteria are selective for azoxystrobin, and can not only survive in a high-concentration azoxystrobin environment, but also use azoxystrobin as a food source for unidirectional selective decomposition of azoxystrobin, thereby ensuring that the degradation slow-release bacterial agent can degrade azoxystrobin and reduce azoxystrobin residues in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.

[0038] Figure 1 The present invention is a flowchart of a process for preparing a fungicide composition for preventing and treating asparagus stem blight used in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The following is a detailed description of a bactericidal composition for preventing and treating asparagus stem blight and its preparation process provided by the present invention in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternatives to implement some known technologies; and the accompanying drawings are only for more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0040] Embodiment 1:

[0041] A preparation process of a bactericidal composition for preventing and controlling asparagus stem blight, such as Figure 1 As shown, the following steps are included:

[0042] S1: Preparation of azoxystrobin

[0043] S1.1: In a three-necked flask, 11 parts by mass of DMF, 1.6 parts by mass of the intermediate (E)-2-[2-(6-chloropyridin-4-yloxy)phenyl]-3-methoxyacrylate, 1 part by mass of potassium carbonate, 0.05 parts by mass of cuprous iodide and 0.7 parts by mass of salicylonitrile were added, and the mixture was heated to 120° C. and stirred for reaction for 5 h to obtain a reaction solution;

[0044] S1.2: Filter the reaction solution to obtain a filtrate, and distill the filtrate under reduced pressure at 70°C to obtain a crude product of azoxystrobin. Add the crude product of azoxystrobin to methanol, heat to 60°C and stir to dissolve the crude product of azoxystrobin, and cool until brown crystals are obtained. Filter and remove the brown crystals, wash the brown crystals with petroleum ether 3 times, and vacuum dry them at 60°C for 5 hours to obtain azoxystrobin.

[0045] S2: Screening of degrading bacteria

[0046] S2.1: The microbial environmental samples collected from the natural environment were used as the initial inoculum for enrichment culture of degradation microorganisms. 100 mL of MSM culture medium was placed in a 250 mL conical flask for sterilization. After cooling, the standard working stock solution of azoxystrobin was added under aseptic conditions to make the final mass concentration of azoxystrobin 50 mg / L. At the same time, 10 g of the microbial environmental sample was added and placed in a shaking incubator for culture (the conditions for the shaking incubator were 30°C and 200 r / min). After 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a rate of 10%. After 7 days of culture under the same conditions, the inoculum was transferred to the MSM culture medium containing 200 mg / L azoxystrobin at a rate of 10% and cultured for another 7 days. Then, the inoculum was transferred to the MSM culture medium containing 400 mg / L azoxystrobin at a rate of 10% and cultured for another 7 days to obtain the MSM culture medium fermentation liquid.

[0047] S2.2: The MSM culture medium fermentation liquid was diluted 10 times in a gradient dilution, and 0.2 mL of the diluted MSM culture medium fermentation liquid after 4 dilutions was evenly applied to an LB solid plate containing 400 mg / L of azoxystrobin. After culturing at 30° C. for 2 days, the azoxystrobin dominant degrading bacteria were isolated from the LB solid plate.

[0048] S3: Preparation of degradation slow-release bacterial agent

[0049] S3.1: First, prepare 0.33 mol / L calcium chloride solution and sodium carbonate solution, and 1 mg / mL dopamine solution (pH=8.5). Then, add the dominant azoxystrobin-degrading bacteria to the calcium chloride solution, the solid-liquid ratio of the dominant azoxystrobin-degrading bacteria to the calcium chloride solution is 1:100 g / L, and stir evenly with a magnetic stirrer to obtain a mixed solution;

[0050] S3.2: Add 0.3 parts by mass of sodium polytetrastyrene sulfonate to the mixed solution, stir evenly, and then dropwise add to 100 parts by volume of the sodium carbonate solution. After the dropwise addition is complete, continue stirring for 1 minute to obtain a milky white solution;

[0051] S3.3: After the milky white solution was allowed to stand for 30 min, the supernatant and the precipitate were collected separately, 200 parts by volume of the dopamine solution were added to the precipitate, and after the precipitate was completely suspended, the mixture was stirred at a speed of 300 r / min for 24 h, and the dark brown precipitate was collected;

[0052] S3.4: Wash the dark brown precipitate three times with ultrapure water of pH=8.5, dry the washed dark brown precipitate into powder in an oven at 45°C to obtain a degradation slow-release bacterial agent.

[0053] S4: Preparation of bactericidal composition

[0054] S4.1: Set the constant temperature of the ice machine to 50°C, connect the crystallizer, add ethyl acetate into the crystallizer, wait until the temperature reaches the set temperature, add azoxystrobin and degradation sustained-release bacteria to dissolve, the mass ratio of azoxystrobin, degradation sustained-release bacteria to ethyl acetate is 27:13:100, the stirring rate is 150r / min, turn off the magnetic stirring after stirring for 12h, use a Buchner funnel preheated at 50°C to filter the solid, and obtain a mixed saturated solution at 50°C;

[0055] S4.2: Pour the mixed saturated solution at 50°C rapidly into a pre-cooled anti-solvent of ethyl acetate at 4°C, stir until crystals precipitate, filter, and dry the crystals at room temperature to obtain a bactericidal composition.

[0056] Embodiment 2:

[0057] A preparation process of a bactericidal composition for preventing and controlling asparagus stem blight, such as Figure 1As shown, the following steps are included:

[0058] S1: Preparation of azoxystrobin

[0059] S1.1: In a three-necked flask, 11 parts by weight of DMF, 1.6 parts by weight of the intermediate (E)-2-[2-(6-chloropyridin-4-yloxy)phenyl]-3-methoxyacrylate, 1 part by weight of potassium carbonate, 0.05 parts by weight of cuprous iodide and 0.7 parts by weight of salicylonitrile were added, and the mixture was heated to 140° C. and stirred for reaction for 4.5 hours to obtain a reaction solution;

[0060] S1.2: Filter the reaction solution to obtain a filtrate, and distill the filtrate under reduced pressure at 65°C to obtain a crude product of azoxystrobin. Add the crude product of azoxystrobin to methanol, heat to 50°C and stir to dissolve the crude product of azoxystrobin, and cool until brown crystals are obtained. Filter and remove the brown crystals, wash the brown crystals twice with petroleum ether, and vacuum dry them at 70°C for 6 hours to obtain azoxystrobin.

[0061] S2: Screening of degrading bacteria

[0062] S2.1: The microbial environmental samples collected from the natural environment were used as the initial inoculum for enrichment culture of degradation microorganisms. 100 mL of MSM culture medium was placed in a 250 mL conical flask for sterilization. After cooling, the standard working stock solution of azoxystrobin was added under aseptic conditions to make the final mass concentration of azoxystrobin 50 mg / L. At the same time, 10 g of the microbial environmental sample was added and placed in a shaking incubator for culture (the conditions for the shaking incubator were 30°C and 200 r / min). After 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a rate of 10%. After 7 days of culture under the same conditions, the inoculum was transferred to the MSM culture medium containing 200 mg / L azoxystrobin at a rate of 10% and cultured for another 7 days. Then, the inoculum was transferred to the MSM culture medium containing 400 mg / L azoxystrobin at a rate of 10% and cultured for another 7 days to obtain the MSM culture medium fermentation liquid.

[0063] S2.2: The MSM culture medium fermentation liquid was diluted 10 times in a gradient dilution, and 0.2 mL of the diluted MSM culture medium fermentation liquid after 4 dilutions was evenly applied to an LB solid plate containing 400 mg / L of azoxystrobin. After culturing at 30° C. for 2 days, the azoxystrobin dominant degrading bacteria were isolated from the LB solid plate.

[0064] S3: Preparation of degradation slow-release bacterial agent

[0065] S3.1: First, prepare 0.33 mol / L calcium chloride solution and sodium carbonate solution, and 1 mg / mL dopamine solution (pH=8.5). Then, add the dominant azoxystrobin-degrading bacteria to the calcium chloride solution, the solid-liquid ratio of the dominant azoxystrobin-degrading bacteria to the calcium chloride solution is 1:100 g / L, and stir evenly with a magnetic stirrer to obtain a mixed solution;

[0066] S3.2: Add 0.3 parts by mass of sodium polytetrastyrene sulfonate to the mixed solution, stir evenly, and then dropwise add to 100 parts by volume of the sodium carbonate solution. After the dropwise addition is complete, continue stirring for 2 minutes to obtain a milky white solution;

[0067] S3.3: After the milky white solution was allowed to stand for 35 min, the supernatant and the precipitate were collected separately, 200 parts by volume of the dopamine solution were added to the precipitate, and after the precipitate was completely suspended, the mixture was stirred at a speed of 350 r / min for 20 h, and the dark brown precipitate was collected;

[0068] S3.4: Wash the dark brown precipitate three times with ultrapure water of pH=8.5, dry the washed dark brown precipitate into powder in an oven at 45°C to obtain a degradation slow-release bacterial agent.

[0069] S4: Preparation of bactericidal composition

[0070] S4.1: Set the constant temperature of the ice machine to 55°C, connect the crystallizer, add ethyl acetate into the crystallizer, wait until the temperature reaches the set temperature, add azoxystrobin and degradation slow-release bacteria to dissolve, the mass ratio of azoxystrobin, degradation slow-release bacteria to ethyl acetate is 27:13:100, the stirring rate is 230r / min, turn off the magnetic stirring after stirring for 10h, use a Buchner funnel preheated at 50°C to filter the solid, and obtain a mixed saturated solution at 50°C;

[0071] S4.2: Pour the mixed saturated solution at 50°C rapidly into a pre-cooled anti-solvent of ethyl acetate at 4°C, stir until crystals precipitate, filter, and dry the crystals at room temperature to obtain a bactericidal composition.

[0072] Embodiment 3:

[0073] A preparation process of a bactericidal composition for preventing and controlling asparagus stem blight, such as Figure 1 As shown, the following steps are included:

[0074] S1: Preparation of azoxystrobin

[0075] S1.1: In a three-necked flask, 11 parts by mass of DMF, 1.6 parts by mass of the intermediate (E)-2-[2-(6-chloropyridin-4-yloxy)phenyl]-3-methoxyacrylate, 1 part by mass of potassium carbonate, 0.05 parts by mass of cuprous iodide and 0.7 parts by mass of salicylonitrile were added, and the mixture was heated to 120° C. and stirred for reaction for 5 h to obtain a reaction solution;

[0076] S1.2: Filter the reaction solution to obtain a filtrate, and distill the filtrate under reduced pressure at 70°C to obtain a crude product of azoxystrobin. Add the crude product of azoxystrobin to methanol, heat to 60°C and stir to dissolve the crude product of azoxystrobin, and cool until brown crystals are obtained. Filter and remove the brown crystals, wash the brown crystals with petroleum ether 3 times, and vacuum dry them at 60°C for 5 hours to obtain azoxystrobin.

[0077] S2: Screening of degrading bacteria

[0078] S2.1: The microbial environmental samples collected from the natural environment were used as the initial inoculum for enrichment culture of degradation microorganisms. 100 mL of MSM culture medium was placed in a 250 mL conical flask for sterilization. After cooling, the standard working stock solution of azoxystrobin was added under aseptic conditions to make the final mass concentration of azoxystrobin 50 mg / L. At the same time, 10 g of the microbial environmental sample was added and placed in a shaking incubator for culture (the conditions for the shaking incubator were 30°C and 200 r / min). After 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a rate of 10%. After 7 days of culture under the same conditions, the inoculum was transferred to the MSM culture medium containing 200 mg / L azoxystrobin at a rate of 10% and cultured for another 7 days. Then, the inoculum was transferred to the MSM culture medium containing 400 mg / L azoxystrobin at a rate of 10% and cultured for another 7 days to obtain the MSM culture medium fermentation liquid.

[0079] S2.2: The MSM culture medium fermentation liquid was diluted 10 times in a gradient dilution, and 0.2 mL of the diluted MSM culture medium fermentation liquid after 4 dilutions was evenly applied to an LB solid plate containing 400 mg / L of azoxystrobin. After culturing at 30° C. for 2 days, the azoxystrobin dominant degrading bacteria were isolated from the LB solid plate.

[0080] S3: Preparation of degradation slow-release bacterial agent

[0081] S3.1: First, prepare 0.33 mol / L calcium chloride solution and sodium carbonate solution, and 1 mg / mL dopamine solution (pH=8.5). Then, add the dominant azoxystrobin-degrading bacteria to the calcium chloride solution, the solid-liquid ratio of the dominant azoxystrobin-degrading bacteria to the calcium chloride solution is 1:100 g / L, and stir evenly with a magnetic stirrer to obtain a mixed solution;

[0082] S3.2: Add 0.3 parts by mass of sodium polytetrastyrene sulfonate to the mixed solution, stir evenly, and then dropwise add to 120 parts by volume of the sodium carbonate solution. After the dropwise addition is complete, continue stirring for 1 minute to obtain a milky white solution.

[0083] S3.3: After the milky white solution was allowed to stand for 30 min, the supernatant and the precipitate were collected separately, 220 parts by volume of the dopamine solution were added to the precipitate, and after the precipitate was completely suspended, the mixture was stirred at a speed of 300 r / min for 24 h, and the dark brown precipitate was collected;

[0084] S3.4: Wash the dark brown precipitate three times with ultrapure water of pH=8.5, dry the washed dark brown precipitate into powder in an oven at 45°C to obtain a degradation slow-release bacterial agent.

[0085] S4: Preparation of bactericidal composition

[0086] S4.1: Set the constant temperature of the ice machine to 50°C, connect the crystallizer, add ethyl acetate into the crystallizer, wait until the temperature reaches the set temperature, add azoxystrobin and degradation sustained-release bacteria to dissolve, the mass ratio of azoxystrobin, degradation sustained-release bacteria to ethyl acetate is 27:13:100, the stirring rate is 150r / min, turn off the magnetic stirring after stirring for 12h, use a Buchner funnel preheated at 50°C to filter the solid, and obtain a mixed saturated solution at 50°C;

[0087] S4.2: Pour the mixed saturated solution at 50°C rapidly into a pre-cooled anti-solvent of ethyl acetate at 4°C, stir until crystals precipitate, filter, and dry the crystals at room temperature to obtain a bactericidal composition.

[0088] Comparative Example 1:

[0089] Compared with Example 1, the difference of Comparative Example 1 is that step S3 is not performed, and the dominant azoxystrobin degrading bacteria are directly added in step S4 to prepare the bactericidal composition, specifically "S4.1: the ice machine is set to a constant temperature of 50°C, a crystallizer is connected, ethyl acetate is added to the crystallizer, and when the temperature rises to the set temperature, the dominant azoxystrobin degrading bacteria are added to dissolve, the solid-liquid ratio of the dominant azoxystrobin degrading bacteria to ethyl acetate is 13:100 g / L, the stirring rate is 150 r / min, and the magnetic stirring is turned off after stirring for 12 hours, and the solid is filtered using a Buchner funnel preheated at 50°C in advance to obtain a mixed saturated solution at 50°C;

[0090] S4.2: The mixed saturated solution at 50°C was quickly poured into a pre-cooled anti-solvent of ethyl acetate at 4°C, stirred until crystals precipitated, filtered, and the crystals were dried at room temperature to obtain a bactericidal composition. The prepared bactericidal composition was recorded as Comparative Example 1.

[0091] Comparative Example 2:

[0092] Compared with Example 1, the difference of Comparative Example 2 is that step S4 is not performed to prepare the solvate, but the degradation sustained-release bacterial agent is directly mixed with myclobutanil, specifically: "myclobutanil and the degradation sustained-release bacterial agent are mixed in a mass ratio of 27:13 to obtain a bactericidal composition" and the prepared bactericidal composition is recorded as Comparative Example 2.

[0093] Place the naturally collected soil samples in a cool and ventilated place to dry naturally, grind, and pass through a 2mm sieve. Prepare working mother solutions for Examples 1-3 and Comparative Examples 1-2 respectively, and soak them in diatomaceous earth to make them completely adsorbed. The soaked diatomaceous earth is placed in a fume hood to dry, and mixed into the soil to make the final concentration of the fungicide in the soil 50mg / kg. Take 500g of soil samples and culture them in a constant temperature and humidity incubator at 30℃, and the water holding capacity of the soil is maintained at 40%. Cultivate continuously for 10 days at 30℃ and avoid light, and take samples regularly, and use HPLC to determine the fungicide residue. Each treatment is repeated 3 times to obtain the treated soil samples. The treatment with an equal amount of clean water is used as the control group.

[0094] Extraction of the residual amount of azoxystrobin: Weigh 20g of the treated soil sample, place it in a 250mL conical flask, add 20mL of acetone, and ultrasonically extract for 20min. Add 20mL of ethyl acetate to the conical flask, shake and extract, filter the washing liquid through a small funnel with 20g of anhydrous sodium sulfate, and collect it in a 250mL round-bottom flask, repeat the extraction twice, and put the filtrate in the flask, evaporating it to dryness at 45℃, wash the round-bottom flask with acetone 3 times, transfer and make up to 10mL. Take 1mL and filter it through a 0.45μm microporous organic membrane injection filter and collect it in a sample injection bottle. Finally, the residual amount of azoxystrobin A1 was determined by high performance liquid chromatography, and the residual amount of azoxystrobin A0 measured in the control group was determined.

[0095] Degradation rate = (1-A1 / A0) * 100%

[0096] A1 is the residual concentration of azoxystrobin after treatment, and A0 is the residual concentration of azoxystrobin in the control group, as shown in Table 1.

[0097] Table 1

[0098]

[0099] Experiment on Sterilization of Asparagus Stem Blight

[0100] The soil of half-year-old potted asparagus seedlings was inoculated with an asparagus stem blight strain. After culturing for 1 day, 3 g of Examples 1-3 and Comparative Examples 1-2 were added to the soil, 30 plants in each group. The fungicide composition was evenly sprinkled on the soil and buried in the soil at a depth of 3 cm. The soil was cultured for 14 days, and the incidence of the asparagus seedlings was counted as shown in Table 2.

[0101] Table 2

[0102]

[0103] It can be seen that the incidence rate of Example 1 is 6.7%, and the degradation rate of azoxystrobin is 77.1%, the incidence rate of Example 2 is 10%, and the degradation rate of azoxystrobin is 75.2%, and the incidence rate of Example 3 is 6.7%, and the degradation rate of azoxystrobin is 75.6%. It can be seen that the examples can effectively degrade azoxystrobin and reduce the residue of azoxystrobin in the soil while reducing the incidence rate of stem blight.

[0104] The degradation rate of myclobutanil in Comparative Example 1 was 86.4%, and the incidence rate of asparagus was 83.3%. It can be seen that the myclobutanil-degrading dominant bacteria were not coated and directly mixed with myclobutanil. Although myclobutanil could have a good degradation effect on myclobutanil, myclobutanil could not prevent and control the stem blight pathogen, thereby increasing the incidence rate of asparagus and having almost no effect on preventing the occurrence of stem blight.

[0105] The degradation rate of myclobutanil in Comparative Example 2 was 50.3%, and the incidence rate of asparagus was 36.7%. It can be seen that without performing step S4, the degree of binding between the degradation sustained-release bacterial agent and myclobutanil decreased, the degradation rate of myclobutanil became lower, and myclobutanil was easily dispersed unevenly, which affected the killing of myclobutanil on stem blight pathogens and affected plant growth.

[0106] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A preparation process of a bactericidal composition for preventing and treating asparagus stem blight, characterized in that: The steps include: S1: Preparation of azoxystrobin DMF, intermediate (E)-2-[2-(6-chloropyridin-4-yloxy)phenyl]-3-methoxyacrylate, potassium carbonate, cuprous iodide and salicylonitrile were added to a three-necked flask, and the mixture was heated and stirred to react to obtain a reaction solution, and the reaction solution was filtered to obtain a filtrate, and the filtrate was distilled under reduced pressure to obtain azoxystrobin crude product, and the azoxystrobin crude product was added to methanol, and the mixture was heated and stirred to dissolve the azoxystrobin crude product, and the mixture was cooled until brown crystals were obtained, and the brown crystals were filtered out, washed, and vacuum dried to obtain azoxystrobin; S2: Screening of degrading bacteria The microbial environmental samples collected from the natural environment were used as the initial inoculum for enrichment culture of degradation microorganisms. 100 mL of MSM culture medium was placed in a 250 mL Erlenmeyer flask for sterilization. After cooling, the standard working stock solution of azoxystrobin was added under aseptic conditions to make the final mass concentration of azoxystrobin 50 mg / L. At the same time, 10 g of the microbial environmental sample was added. After culturing on a shaker for 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a 10% inoculum. After culturing under the same conditions for 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a 10% inoculum. The amount of the inoculum was transferred to an MSM medium containing 200 mg / L of azoxystrobin, and the culture was continued for 7 days, and then the inoculum was transferred to an MSM medium containing 400 mg / L of azoxystrobin at a 10% inoculum amount, and the culture was continued for 7 days to obtain an MSM medium fermentation liquid, and the MSM medium fermentation liquid was diluted by a gradient dilution multiple of 10 times, and 0.2 mL of the diluted MSM medium fermentation liquid after 4 dilutions was evenly spread on an LB solid plate containing 400 mg / L of azoxystrobin for culture, and the azoxystrobin dominant degrading bacteria were isolated from the LB solid plate; S3: Preparation of degradation slow-release bacterial agent First, a calcium chloride solution, a sodium carbonate solution and a dopamine solution are prepared, and then a myclobutanil-degrading bacterial strain is added to the calcium chloride solution, and stirred evenly with a magnetic stirrer to obtain a mixed solution, and then sodium polytetrastyrene sulfonate is added to the mixed solution, and after stirring evenly, the solution is added dropwise to the sodium carbonate solution, and the stirring is continued after the dropwise addition is completed to obtain a milky white solution, the milky white solution is allowed to stand, and a supernatant and a precipitate are collected respectively, and a dopamine solution is added to the precipitate, and after the precipitate is completely suspended, a dark brown precipitate is collected, the dark brown precipitate is washed with ultrapure water, and the washed dark brown precipitate is dried into powder to obtain a degradation slow-release bacterial agent; S4: Preparation of bactericidal composition Add ethyl acetate to a crystallizer, add myclobutanil and a degradation slow-release bacterial agent to dissolve, stir, filter the solid using a Buchner funnel preheated at 50°C to obtain a mixed saturated solution at 50°C, quickly pour the 50°C mixed saturated solution into a 4°C ethyl acetate precooled anti-solvent, stir until crystals precipitate, filter, and dry the crystals at room temperature to obtain a bactericidal composition.

2. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 1, characterized in that: Step S1 prepares azoxystrobin, comprising the following steps: S1.1: Add 11 parts by mass of DMF, 1.6 parts by mass of the intermediate (E)-2-[2-(6-chloropyridin-4-yloxy)phenyl]-3-methoxyacrylate, 1 part by mass of potassium carbonate, 0.05 parts by mass of cuprous iodide and 0.7 parts by mass of salicylonitrile to a three-necked flask, heat to 120-140° C., stir and react for 4.5-5 hours to obtain a reaction solution; S1.2: Filter the reaction solution to obtain a filtrate, and distill the filtrate under reduced pressure at 65-70°C to obtain a crude product of azoxystrobin. Add the crude product of azoxystrobin to methanol, heat to 50-60°C and stir to dissolve the crude product of azoxystrobin, cool until brown crystals are obtained, filter out the brown crystals, wash the brown crystals with petroleum ether 2-3 times, and dry in vacuo to obtain azoxystrobin.

3. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 2, characterized in that: Step S2 is to screen the degradation bacteria, comprising the following steps: S2.1: The microbial environmental samples collected from the natural environment were used as the initial inoculum for enrichment culture of degradation microorganisms. 100 mL of MSM culture medium was placed in a 250 mL conical flask for sterilization. After cooling, the standard working stock solution of azoxystrobin was added under aseptic conditions to make the final mass concentration of azoxystrobin 50 mg / L. At the same time, 10 g of the microbial environmental sample was added. After culturing on a shaker for 7 days, the inoculum was transferred to the second batch of MSM culture medium containing 100 mg / L azoxystrobin at a rate of 10%. After culturing under the same conditions for 7 days, the inoculum was transferred to MSM culture medium containing 200 mg / L azoxystrobin at a rate of 10%, and the culture was continued for 7 days. Then, the inoculum was transferred to MSM culture medium containing 400 mg / L azoxystrobin at a rate of 10%, and the culture was continued for 7 days to obtain MSM culture medium fermentation liquid; S2.2: The MSM culture medium fermentation liquid was diluted 10 times in a gradient dilution, and 0.2 mL of the diluted MSM culture medium fermentation liquid after 4 dilutions was evenly applied to an LB solid plate containing 400 mg / L of azoxystrobin. After culturing at 30° C. for 2 days, the azoxystrobin dominant degrading bacteria were isolated from the LB solid plate.

4. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 3, characterized in that: The vacuum drying condition in step S1.2 is 60-70°C for 5-6h.

5. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 4, characterized in that: The shaking culture conditions in step S2.1 are 30°C and 200 r / min.

6. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 5, characterized in that: Step S3 is preparing a degradation slow-release bacterial agent, comprising the following steps: S3.1: First, prepare 0.33 mol / L calcium chloride solution and sodium carbonate solution, and 1 mg / mL dopamine solution, then add the dominant azoxystrobin-degrading bacteria to the calcium chloride solution, and stir evenly with a magnetic stirrer to obtain a mixed solution; S3.2: Add 0.3 parts by mass of sodium polytetrastyrene sulfonate to the mixed solution, stir evenly, and then dropwise add to 100-120 parts by volume of sodium carbonate solution. After the dropwise addition is complete, continue stirring for 1-2 minutes to obtain a milky white solution; S3.3: After the milky white solution is allowed to stand for 30-35 minutes, the supernatant and the precipitate are collected separately, 200-220 parts by volume of the dopamine solution are added to the precipitate, and after the precipitate is completely suspended, the mixture is stirred at a speed of 300-350 r / min for 20-24 hours, and the dark brown precipitate is collected; S3.4: Wash the dark brown precipitate three times with ultrapure water of pH=8.5, dry the washed dark brown precipitate into powder in an oven at 45-50℃ to obtain a degradation slow-release bacterial agent.

7. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 6, characterized in that: In step S3.1, the solid-liquid ratio of the azoxystrobin dominant degrading bacteria to the calcium chloride solution is 1:100 g / L.

8. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 7, characterized in that: Step S4 prepares a bactericidal composition, comprising the following steps: S4.1: Set the ice machine to a constant temperature of 50-55°C, connect the crystallizer, add ethyl acetate into the crystallizer, wait until the temperature reaches the set temperature, add azoxystrobin and a degradation slow-release bacterial agent to dissolve, stir at a rate of 150-230 r / min, stir for 10-12 hours, turn off the magnetic stirring, filter the solid using a Buchner funnel preheated at 50°C, and obtain a mixed saturated solution at 50°C; S4.2: Pour the mixed saturated solution at 50°C rapidly into a pre-cooled anti-solvent of ethyl acetate at 4°C, stir until crystals precipitate, filter, and dry the crystals at room temperature to obtain a bactericidal composition.

9. The preparation process of a bactericidal composition for preventing and controlling asparagus stem blight according to claim 8, characterized in that: In step S4.1, the mass ratio of azoxystrobin, the degradation slow-release bacterial agent and ethyl acetate is 27:13:

100.

10. A fungicide composition for preventing and controlling asparagus stem blight, which is prepared by the preparation process of the fungicide composition for preventing and controlling asparagus stem blight according to any one of claims 1 to 9.

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