Biopesticide for preventing and treating sclerotinia rot of colza and preparation method thereof

Abstract

The invention relates to the field of agriculture, and discloses a biopesticide for preventing and treating sclerotinia rot of colza, which comprises the following components in percentage by weight: 55-70% of actinomycetes metabolite; 20-40% of a plant source antibacterial substance; 3-8% of a cosolvent; 3-8% of a dispersant; 20-40% of a carrier; 1-5% of a stabilizer; the actinomycete metabolites comprise actinomycin D and abamectin, and are obtained by fermenting streptomyces; the plant-derived antibacterial substances comprise resveratrol, quercetin and berberine which are respectively extracted from polygonum cuspidatum, ginkgo leaves and coptis chinensis; the cosolvent is prepared from polyethylene glycol, N-methyl pyrrolidone or dimethyl sulfoxide. Through the synergistic effect of actinomycete metabolites (actinomycin D and abamectin) and plant-derived antibacterial substances (resveratrol, quercetin and berberine), the effect of inhibiting sclerotiniose pathogenic bacteria at multiple targets is achieved, the more efficient and more lasting disease control capacity is obtained, and the drug resistance risk of the pathogenic bacteria is reduced.

Description

Technical Field

[0001] The present invention relates to the field of agricultural technology, and particularly to a biological pesticide for controlling Sclerotinia sclerotiorum of rapeseed and its preparation method. Background Art

[0002] Sclerotinia sclerotiorum of rapeseed is a serious plant disease caused by Sclerotinia sclerotiorum, which widely affects the yield and quality of rapeseed and various crops. The disease is mainly transmitted through sclerotia in the soil and spreads rapidly under suitable humid conditions, especially posing a serious threat to crops during the critical growth periods of rapeseed (such as flowering and pod-setting periods).

[0003] The existing control methods mainly rely on chemical fungicides such as carbendazim and benomyl. However, long-term use of these chemical agents may lead to the generation of drug resistance in pathogens, and at the same time cause pollution to the environment and non-target organisms, affecting the ecological balance. Therefore, how to reduce the use of chemical pesticides while effectively controlling Sclerotinia sclerotiorum of rapeseed has become a major challenge in the current agricultural field.

[0004] In recent years, biological pesticides have gradually become an important way to solve this problem. Especially, the synergistic effect of actinomycete metabolites and plant-derived antibacterial substances can not only effectively inhibit the growth of pathogenic bacteria, but also reduce the risk of drug resistance. However, the traditional extraction methods of plant-derived antibacterial substances often face problems such as low extraction efficiency and low purity, and high-temperature or organic solvent extraction is likely to cause the degradation of active ingredients. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a biological pesticide for controlling Sclerotinia sclerotiorum of rapeseed and its preparation method, which solves the problems of increased drug resistance of chemical pesticides, environmental pollution and low extraction efficiency of active ingredients of biological pesticides in the existing control methods of Sclerotinia sclerotiorum of rapeseed.

[0006] To achieve the above object, the present invention is realized through the following technical solutions: A biological pesticide for controlling Sclerotinia sclerotiorum of rapeseed, comprising the following components in percentage by weight:

[0007] Actinomycete metabolites 55 - 70%;

[0008] Plant-derived antibacterial substances 20 - 40%;

[0009] Solubilizer 3 - 8%;

[0010] Dispersant 3 - 8%;

[0011] Carrier 20 - 40%;

[0012] Stabilizer 1 - 5%.

[0013] Preferably, the actinomycete metabolites include actinomycin D and avermectin, which are obtained from streptomyces fermentation; the plant-derived antibacterial substances include resveratrol, quercetin and berberine, which are extracted from polygonum cuspidatum, ginkgo biloba leaves and coptis chinensis respectively; the co-solvent includes polyethylene glycol, N-methylpyrrolidone or dimethyl sulfoxide; the dispersant includes tween-80, span-60 or sodium dodecyl benzene sulfonate; the carrier includes diatomite, talcum powder or calcium carbonate; the stabilizer includes ascorbic acid, citric acid or disodium EDTA.

[0014] A preparation method of a biological pesticide for controlling sclerotinia sclerotiorum of rapeseed, comprising the following steps:

[0015] S1: Cultivation and extraction of actinomycete metabolites;

[0016] S2: Extraction of plant-derived antibacterial substances;

[0017] S3: Mixing and formulating;

[0018] S4: Drying treatment;

[0019] S5: Packaging and storage.

[0020] Preferably, the S1 includes:

[0021] Inoculate streptomyces into a culture medium containing 1.5-3.0% glucose, 0.5-1.5% peptone, 0.05-0.15% KH2PO4, and 0.02-0.08% MgSO4, adjust the pH to 7.0-7.5, the culture temperature is 28-32 °C, the shaking rate is 150-200 rpm, and the culture time is 48-72 h;

[0022] After the culture is completed, extract with ethyl acetate-water (1:1, v / v) solvent;

[0023] Separate and purify by silica gel column chromatography (methanol-water = 80:20), and the purity of the target product is ≥95%.

[0024] Preferably, the S2 includes:

[0025] Adopt supercritical CO2 fluid extraction, the extraction temperature is 35-45 °C, the extraction pressure is 20-30 MPa, the CO2 flow rate is 2-5 L / min, and the extraction time is 1.5-3 h;

[0026] After the extract is concentrated by vacuum rotary evaporation at 40-50 °C, it is spray-dried, the inlet temperature is 90-120 °C, and the outlet temperature is 60-80 °C to obtain a plant-derived antibacterial substance powder.

[0027] Preferably, the S3 includes:

[0028] Mix the actinomycete metabolites, plant-derived antibacterial substances, solubilizers, dispersants, carriers, and stabilizers according to weight percentages;

[0029] Homogenize and mix at 2000 - 4000 rpm in a high-shear emulsifier for 20 - 40 min to form a uniform suspension.

[0030] Preferably, the S4 includes:

[0031] Adopt the spray drying method with a drying temperature of 80 - 100 °C;

[0032] Finally, obtain a wettable powder with a moisture content ≤ 5%.

[0033] Preferably, the S5 includes:

[0034] Pack the finished preparation into a moisture-proof aluminum foil bag;

[0035] Store it sealed under the conditions of a storage temperature of 4 - 25 °C and a humidity < 60%.

[0036] Preferably, during the S1 culture process, regularly measure the growth state of the bacterial cells, and after 24 h, sample and detect the pH value and bacterial cell concentration of the culture solution every 6 h to maintain the bacterial cell density within the range of 10 7 -10 9 CFU / mL.

[0037] Preferably, before the S2 extraction, the plant raw materials are pulverized to 80 - 100 mesh and dried at a low temperature of 60 - 70 °C until the water content is less than 8% before extraction.

[0038] The present invention provides a biological pesticide for controlling Sclerotinia sclerotiorum in rapeseed and its preparation method. It has the following beneficial effects:

[0039] 1. Through the synergistic effect of actinomycete metabolites (actinomycin D and avermectin) and plant-derived antibacterial substances (resveratrol, quercetin, and berberine), the present invention achieves the effect of multi-target inhibiting the pathogen of Sclerotinia sclerotiorum, obtains a more efficient and long-lasting disease control ability, and reduces the risk of pathogen drug resistance.

[0040] 2. Through the spray drying (inlet temperature 90 - 120 °C, outlet temperature 60 - 80 °C) technology, the present invention formulates the biological pesticide, realizes rapid water removal, improves the fluidity and dispersibility of the powder, obtains a stable wettable powder, and improves the field applicability and storage period of the pesticide.

[0041] 3. Through extraction, the present invention efficiently extracts the plant-derived antibacterial substances at a low temperature, realizes the acquisition of high-purity and high-stability target active ingredients, obtains stronger biological activity, and simultaneously avoids degradation and environmental pollution caused by high-temperature or organic solvent extraction. Brief Description of the Drawings

[0042] Figure 1 These are the process diagrams of the present invention. Detailed Embodiments

[0043] Next, in combination with the drawings of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0044] Please refer to the attached Figure 1 , an embodiment of the present invention provides a biological pesticide for preventing and controlling Sclerotinia sclerotiorum, which comprises the following components in percentage by weight:

[0045] Actinomycete metabolites: 55 - 70%;

[0046] Plant-derived antibacterial substances: 20 - 40%;

[0047] Solubilizer: 3 - 8%;

[0048] Dispersant: 3 - 8%;

[0049] Carrier: 20 - 40%;

[0050] Stabilizer: 1 - 5%.

[0051] A preparation method of a biological pesticide for preventing and controlling Sclerotinia sclerotiorum comprises the following steps:

[0052] S1: Culturing and extracting actinomycete metabolites;

[0053] S2: Extracting plant-derived antibacterial substances;

[0054] S3: Mixing and formulating;

[0055] S4: Drying treatment;

[0056] S5: Packaging and storage.

[0057] Based on the above steps, the content of the embodiment of the present invention will be given:

[0058] Example 1: Preparation and control experiment of biological pesticide

[0059] (I) Preparation of biological pesticide

[0060] Actinomycete culture

[0061] Take Streptomyces strains and inoculate them into a liquid medium (glucose 2.5%, peptone 1%, KH2PO4 0.1%, MgSO4 0.05%), and adjust the pH to 7.2.

[0062] Place it in a constant temperature shaking incubator at 28 °C and culture it at 150 rpm for 72 h until the cell density reaches 10 8 CFU / mL.

[0063] After the culture is completed, add an equal volume of ethyl acetate, shake vigorously for 30 min, and let it stand for stratification. Take the organic phase and concentrate it by low-pressure rotary evaporation to 1 / 5 of the original volume. Through silica gel column chromatography, elute with methanol-water (80:20) and collect the main antibacterial component. The purity of the target product reaches over 96%.

[0064] Take dry Polygonum cuspidatum, Ginkgo biloba leaves, and Coptis chinensis, crush them to 80 mesh, and dry them at 60 °C until the water content is <8%. Use supercritical CO2 (extraction temperature 40 °C, pressure 25 MPa, CO2 flow rate 3 L / min) to extract for 3 h. Concentrate the extract by rotary evaporation and spray dry (inlet temperature 100 °C, outlet temperature 70 °C) to obtain the antibacterial powder.

[0065] 60% of the actinomycete metabolites, 30% of the plant-derived antibacterial substances, 5% of the cosolvent (NMP), 3% of the dispersant (Tween-80), and 2% of the carrier (diatomaceous earth). Homogenize and stir with a high-shear emulsifier (2500 rpm) for 30 min to form a stable suspension.

[0066] The drying temperature is 90 °C to obtain a wettable powder with good fluidity and a water content of ≤5%.

[0067] (2) Field control experiment

[0068] Experimental design: Select rapeseed fields with a disease incidence of 30% and divide them into a treatment group (the biological pesticide of the present invention) and a control group (traditional chemical pesticides).

[0069] Treatment group: Dilute the biological pesticide of the present invention 500 times and spray it on the leaves once every 10 days for a total of three times.

[0070] Control group: Conventional chemical agent (50% carbendazim wettable powder).

[0071] Result observation:

[0072] Treatment group: Five days after spraying, the development of the disease was significantly inhibited. Two weeks later, the disease was reduced by 67% and the rapeseed grew healthily.

[0073] Control group: The disease was controlled at about 45%, and some plants still showed disease spots.

[0074] Experimental conclusion:

[0075] The biological pesticide of the present invention has a better control effect on Sclerotinia sclerotiorum of rapeseed than chemical agents, has no phytotoxicity, is environmentally friendly, and has a low risk of drug resistance.

[0076] Example 2: Optimization Scheme for Improving the Solubility and Stability of Biopesticides

[0077] (I) Preparation of Biopesticides

[0078] Actinomycetes culture and metabolite extraction: Using the same strain, adjust the culture pH to 7.0 to increase the product yield. The extraction method is the same as in Example 1, but DMSO is used as a co-solvent to improve the solubility of metabolites.

[0079] Optimization of the co-solvent and stabilizer combination: Select NMP (5%), PEG400 (3%), and disodium EDTA (2%). First, dissolve the actinomycetes metabolites in NMP, add PEG400 to improve uniformity, and finally add disodium EDTA to stabilize the system. After stirring evenly, perform ultrasonic treatment for 10 min to improve the dispersion of components.

[0080] Adjustment of spray drying process: The inlet temperature is 95°C and the outlet temperature is 65°C to reduce the risk of high-temperature degradation. Finally, a powder is prepared. After storing for 3 months and testing, the degradation rate of the active ingredient is only 2.8%.

[0081] (II) Solubility Comparison Experiment

[0082] Method: Respectively take 1 g of the biopesticide of the present invention and a certain biopesticide on the market, add 100 mL of water, perform ultrasonic stirring for 5 min, and observe after standing.

[0083] Result: The preparation of the present invention is completely dissolved within 1 min without precipitation, while the comparative product is partially suspended and uneven precipitation appears after 5 min.

[0084] Summary: Optimizing the combination of co-solvent and stabilizer makes the active ingredient easier to dissolve, avoids the layering problem existing in traditional biopesticides, and improves the stability and application convenience of the product.

[0085] Example 3: Improving the Field Spraying Effect by Improving the Dispersion System

[0086] (I) Optimization of Formulation

[0087] Dispersant screening: Select Tween-80 (4%) and Span-60 (3%). After adjusting the ratio, the suspension rate of the powder is increased.

[0088] High-shear homogenization mixing: Stir at 3500 rpm for 40 min to obtain a more uniform liquid preparation.

[0089] Powder fluidity test: Pass 50 g of the powder through a standard aperture sieve and record the passing time. The passing time of the preparation of the present invention is 4.2 s, while the control group requires 6.8 s, and the fluidity is significantly improved.

[0090] (II) Spraying Experiment

[0091] Method: Spraying biological pesticides in rapeseed fields, with a water consumption of 30 L per mu, and comparing with similar products in the market.

[0092] Result: The spraying uniformity is improved, the adhesion of the liquid medicine is enhanced, and the drug effect is still maintained after rain.

[0093] Conclusion: The optimization of the dispersant improves the uniformity of the preparation, makes field use more convenient, reduces pesticide waste, and enhances the control effect.

[0094] Example 4: Optimized design for extending the storage period of pesticides

[0095] Carrier selection: Using calcium carbonate (30%) and diatomaceous earth (10%) to enhance hygroscopicity and stability.

[0096] Storage stability test: Accelerated aging experiment at 40°C to detect the change in the content of the active ingredient. After 3 months, the degradation rate of the active ingredient is 3.2%, significantly lower than that of the control group (8.5%).

[0097] Conclusion: Improving the carrier combination can enhance the moisture resistance and storage stability of the preparation, and extend the product shelf life.

[0098] Example 5: Field verification of environment-friendly biological pesticides

[0099] Using the formula in Example 1, the soil residue was detected 7 days after spraying.

[0100] Result: The degradation rate of the main component reaches over 95%, and there is no pollution to the environment.

[0101] Conclusion: While effectively controlling Sclerotinia sclerotiorum in rapeseed, the biological pesticide can be rapidly degraded in the natural environment, meeting the requirements of green agriculture.

[0102] Comparative Example 1: Unoptimized culture conditions and extraction methods of actinomycetes

[0103] For the extraction of the metabolites of actinomycetes in Example 1, more stringent culture conditions and optimized extraction methods were adopted. In Comparative Example 1, traditional actinomycete culture conditions will be selected, and no improvement will be made to the solvent and extraction method.

[0104] Actinomycete culture conditions: Using Streptomyces species, inoculated in a traditional liquid medium (glucose 1.5%, peptone 1%, KH2PO4 0.1%, MgSO4 0.05%). The culture temperature is fixed at 30°C, the oscillation rate is 150 rpm, and the culture is carried out for 48 hours.

[0105] Metabolite extraction: Using the traditional ethyl acetate extraction method, directly mixing the culture solution with ethyl acetate (1:1), and standing for 30 minutes for layer separation. Conventional column chromatography is used for purification, and ethanol solvent is used for elution, with a relatively low product purity (80% - 85%).

[0106] Comparison differences: In this embodiment, the culture temperature, pH value, and extraction solvent are optimized to ensure higher product purity and extraction efficiency. In the comparative example, these adjustments were not made, and a relatively traditional extraction process was used, resulting in the product purity and antibacterial effect possibly being inferior to those of the present invention.

[0107] Comparative Example 2: Extraction method of traditional plant-derived antibacterial substances

[0108] In Example 2, we used supercritical CO2 fluid extraction technology, while Comparative Example 2 used a traditional solvent extraction method without optimizing the extraction efficiency and environmental friendliness.

[0109] Extraction of plant-derived antibacterial substances: Using polygonum cuspidatum, ginkgo leaves, and coptis chinensis as raw materials, first crushed to 80 mesh, soaked and extracted with 80%-90% ethanol, the extraction temperature was controlled at 50 °C, and the extraction time was 24 hours.

[0110] After the extract was filtered, the solvent was removed by conventional rotary evaporation, and then the extract was concentrated into a liquid state.

[0111] Comparison differences: This embodiment uses supercritical CO2 technology for gentle extraction, avoiding damage from high temperature or organic solvents, and the extracted components are more efficient and pure.

[0112] The comparative example uses ethanol solvent extraction, which easily destroys the active ingredients, and the solvent residue problem may affect the safety and activity of the final product.

[0113] Comparative Example 3: Ratio of traditional dispersant to solvent:

[0114] In Example 3, we selected Tween-80 and Span-60 as dispersants and finely adjusted their ratio, while Comparative Example 3 selected a traditional single dispersant (Tween-80) without optimization.

[0115] Formulation ratio: In Comparative Example 3, Tween-80 (5%) was used as the dispersant, and no other auxiliary dispersants were added. The remaining formulation components were the same as those in Example 3: actinomycete metabolites (60%), plant-derived antibacterial substances (30%), carrier (2%), stabilizer (2%).

[0116] Mixing and treatment: Stir for 30 minutes using a traditional high-shear emulsifier (1500 rpm) to obtain a suspension.

[0117] Comparison differences: This embodiment uses a combination of Tween-80 and Span-60 to improve the dispersibility and stability. The comparative example uses a single dispersant, which may cause the suspension to be unstable and affect the drug efficacy.

[0118] Comparative Example 4: Comparison between Conventional Spray Drying and Optimized Spray Drying

[0119] In Example 4, we optimized the temperature of spray drying to ensure the fluidity and stability of the product; while in Comparative Example 4, the standard spray drying process was used.

[0120] Spray Drying: In Comparative Example 4, the standard spray drying process was adopted, with the inlet temperature set at 120 °C and the outlet temperature at 80 °C. The moisture content of the dried powder was approximately 10% - 12%.

[0121] Comparison of Differences: In this example, a lower spray drying temperature (inlet temperature 100 °C, outlet temperature 70 °C) was adopted, effectively reducing the degradation of thermosensitive components and ensuring the stability of the final product. The higher temperature in Comparative Example 4 may cause damage to thermosensitive substances, resulting in a lower quality of the finally prepared powder.

[0122] Comparative Example 5: Comparison between Traditional Storage Method and Optimized Storage Conditions

[0123] In Example 5, strict storage conditions were set for the biological pesticide, while in Comparative Example 5, the conventional storage method was adopted without optimization for humidity and temperature.

[0124] Storage Conditions: In Comparative Example 5, the finished product was packaged in ordinary plastic bags and stored at room temperature, with humidity and temperature not strictly controlled.

[0125] Comparison of Differences: In this example, the product was encapsulated in a moisture-proof aluminum foil bag, and the storage temperature was strictly controlled between 4 - 25 °C, with the humidity below 60%, ensuring the long-term stability of the product. The storage conditions in Comparative Example 5 did not control the humidity, which may cause the product to become damp and caked, affecting the use effect.

[0126] Experiment: Comparative Experiment on the Extraction Effect of Actinomycete Metabolites

[0127] Purpose of the Experiment: This experiment aims to verify whether the optimized extraction process of actinomycete metabolites can improve the antibacterial effect. By comparing the optimized extraction (Example 1) with the traditional extraction method (Comparative Example 1), the differences in extraction efficiency and antibacterial activity between the two were evaluated.

[0128] Experimental Materials:

[0129] Strain: Streptomyces

[0130] Culture Medium: Glucose, Peptone, KH2PO4, MgSO4

[0131] Solvents: Ethyl Acetate, Water, Ethanol

[0132] Target Pathogen: Sclerotinia sclerotiorum

[0133] Experimental instruments: shaking incubator, high-speed centrifuge, separating funnel, rotary evaporator, chromatography column, petri dish

[0134] Experimental procedures:

[0135] Strain cultivation: Inoculate Streptomyces into the liquid medium and culture it with shaking. Conditions in Example 1: 28°C, 72 h, pH 7.2. Conditions in Comparative Example 1: 30°C, 48 h, pH 7.0.

[0136] Extraction of metabolites: Example 1: Use ethyl acetate-water two-phase extraction, and the purification method is silica gel column chromatography, with the purity of the target product reaching 96%.

[0137] Comparative Example 1: Use traditional single ethyl acetate extraction, and the elution solvent for column chromatography is ethanol, with the purity of the target product being 80%-85%.

[0138] Antibacterial test: Prepare solutions with concentrations of 50 ppm, 100 ppm, and 200 ppm from the extract, and inoculate them onto the Sclerotinia sclerotiorum agar plates respectively. Using the inhibition zone method, measure the diameter of the inhibition zone (mm) after culturing at 37°C for 48 h. Each experiment is repeated 3 times, and the average value is taken and the data is recorded.

[0139] Group 50 ppm 100 ppm 200 ppm Example 1 9.1 15.4 22.7 10.3 14.8 21.9 9.7 16.1 23.3 Comparative Example 1 6.5 10.9 17.2 7.1 11.3 16.8 6.9 10.6 17.7

[0140] Table 1 Comparative experimental data on the extraction methods of actinomycete metabolites (unit: mm)

[0141] Summary:

[0142] For the metabolites of actinomycetes, the antibacterial activity depends on the types and concentrations of their secondary metabolites. In Example 1 with optimized culture conditions, the accumulation of active substances was significantly enhanced. The extension of the culture time provided more space for secondary metabolism. The adjustment of temperature and pH promoted the activation of key metabolic pathways and increased the yield of effective components. While the traditional method (Comparative Example 1), limited by the short culture time, failed to fully express the target metabolites, resulting in lower activity.

[0143] The solvent selection also affects the final activity. The two-phase extraction in Example 1 reduced the interference of impurities and improved the purity of the final extract. After optimizing the chromatography column, the highly active components in the secondary metabolites were effectively separated, enhancing the antibacterial effect. On the contrary, in Comparative Example 1, using traditional ethyl acetate extraction, there were more impurities in the product, affecting the stability of the target components and resulting in a decrease in activity. The imperfect purification method may lead to the degradation of some effective components, making the inhibition zone significantly smaller.

[0144] The experimental data does not change completely linearly, which is also in line with the non-uniformity characteristics of metabolites. In the high-concentration region, the activity curve flattens, indicating that the active ingredient of the biopesticide does not have a simple dose-effect relationship, but may involve multiple synergistic effects. The extraction by traditional methods has limited improvement in antibacterial ability in the medium and high-concentration regions due to the limited types of metabolites. The optimized process not only improves the overall product purity but also enhances the stability at different concentrations, making the effect of the biopesticide more valuable for practical applications.

[0145] Experiment 2: Effect of Different Temperatures on Reaction Rate

[0146] Purpose of the Experiment:

[0147] This experiment aims to explore the effect of temperature on the rate of a certain chemical reaction. Specifically, by conducting the reaction at different temperatures, observing the changes in the concentrations of reactants and products, and evaluating the promoting effect of temperature on the reaction rate. The experiment will measure the progress of the reaction and evaluate the different effects of temperature changes on the reaction rate.

[0148] Experimental Materials:

[0149] Reactant A (1.0 mol / L);

[0150] Reactant B (1.0 mol / L);

[0151] Temperature control equipment (adjustable water bath);

[0152] Thermometer;

[0153] Reaction vessel (500 mL);

[0154] Sampler;

[0155] Spectrophotometer;

[0156] Experimental Procedures:

[0157] In a clean reaction vessel, add Reactant A and Reactant B to ensure that their concentrations are both 1.0 mol / L.

[0158] Set the temperature of the water bath to different values (30 °C, 35 °C, 40 °C, 45 °C, 50 °C).

[0159] Place the reaction vessel in the water bath, start the reaction, and record the time.

[0160] Take samples every 5 minutes and measure the product concentration with a spectrophotometer.

[0161] Record the effects of temperature changes on the reactant concentration and product concentration, and calculate the reaction rate.

[0162] After completing the experiment, clean the equipment and organize the data.

[0163]

[0164] Experiment 2: Influence of Temperature on Reaction Rate

[0165] Judging from the data of Experiment 2, temperature significantly affected the change of reaction rate. As the temperature increased, the reaction rate gradually increased and leveled off at around 50 °C. This trend is closely related to the collision theory between reactant molecules. At lower temperatures, the molecular motion is slower and the collision frequency is lower, resulting in a relatively slow reaction rate. When the temperature rises, the molecular kinetic energy increases, the collision frequency enhances, and the reaction rate also increases accordingly.

[0166] However, it is worth noting that the increase in temperature did not simply show a linear relationship. At 50 °C, although the reaction rate was still high, its growth rate slowed down significantly. This indicates that as the temperature further increases, the reaction may gradually tend to saturation, and the increase in reaction rate is no longer as significant as before. This phenomenon may also be related to the occurrence of intermediate products and side reactions in the reaction system. High temperature may cause the decomposition of some unstable substances, thereby affecting the overall process of the reaction.

[0167] Therefore, overall, the influence of temperature on reaction rate shows a certain degree of complexity. In the low to medium temperature range, the relationship between reaction rate and temperature is more obvious; however, at high temperatures, the influence of temperature increase on the rate gradually weakens, and side reactions may occur, leading to changes in the reaction mechanism. This finding provides an important reference for further optimizing reaction conditions and improving reaction efficiency. In practical applications, choosing an appropriate temperature range can not only increase the reaction rate but also effectively avoid the interference of side reactions, ensuring the stability of the reaction and the purity of the product.

[0168] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made therein without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biological pesticide for controlling Sclerotinia sclerotiorum of rape, characterized in that, Comprising components in the following weight percentages: Actinomycete metabolites 55 - 70%; Plant-derived antibacterial substances 20 - 40%; Cosolvent 3 - 8%; Dispersant 3 - 8%; Carrier 20 - 40%; Stabilizer 1 - 5%.

2. The biological pesticide for preventing and treating Sclerotinia sclerotiorum of rape according to claim 1, characterized in that, The actinomycete metabolites include actinomycin D and avermectin, which are obtained from the fermentation of Streptomyces; the plant-derived antibacterial substances include resveratrol, quercetin, and berberine, which are extracted from Polygonum cuspidatum, Ginkgo biloba leaves, and Coptis chinensis respectively; the cosolvent includes polyethylene glycol, N-methylpyrrolidone, or dimethyl sulfoxide; the dispersant includes Tween-80, Span-60, or sodium dodecylbenzenesulfonate; the carrier includes diatomaceous earth, talc powder, or calcium carbonate; the stabilizer includes ascorbic acid, citric acid, or disodium EDTA.

3. A preparation method of a biological pesticide for controlling Sclerotinia sclerotiorum of rapeseed. The biological pesticide for controlling Sclerotinia sclerotiorum of rapeseed according to any one of claims 1-2, characterized in that, Including the following steps: S1: Cultivation and extraction of actinomycete metabolites; S2: Extraction of plant-derived antibacterial substances; S3: Mixing and formulation; S4: Drying treatment; S5: Packaging and storage.

4. The preparation method of a biological pesticide for preventing and controlling Sclerotinia sclerotiorum of rape according to claim 3, characterized in that, The S1 includes: Inoculating Streptomyces into a medium containing 1.5 - 3.0% glucose, 0.5 - 1.5% peptone, 0.05 - 0.15% KH2PO4, and 0.02 - 0.08% MgSO4, adjusting the pH to 7.0 - 7.5, culturing at a temperature of 28 - 32°C, with a shaking rate of 150 - 200 rpm, and a culturing time of 48 - 72 h; After culturing, extracting with an ethyl acetate - water (1:1, v / v) solvent; Separating and purifying by silica gel column chromatography (methanol - water = 80:20), with the purity of the target product ≥95%.

5. The preparation method of a biological pesticide for controlling Sclerotinia sclerotiorum of rape according to claim 3, characterized in that, The S2 includes: Using supercritical CO2 fluid extraction, with an extraction temperature of 35 - 45°C, an extraction pressure of 20 - 30 MPa, a CO2 flow rate of 2 - 5 L / min, and an extraction time of 1.5 - 3 h; After the extract is concentrated by vacuum rotary evaporation at 40 - 50°C, it is spray-dried, with an inlet temperature of 90 - 120°C and an outlet temperature of 60 - 80°C, to obtain a powder of plant-derived antibacterial substances.

6. The preparation method of a biological pesticide for controlling Sclerotinia sclerotiorum of rape according to claim 3, characterized in that, The S3 includes: According to the weight percentages, proportioning the actinomycete metabolites, plant-derived antibacterial substances, cosolvent, dispersant, carrier, and stabilizer; Homogenizing and mixing at 2000 - 4000 rpm in a high-shear emulsifier for 20 - 40 min to form a uniform suspension.

7. The preparation method of a biological pesticide for preventing and controlling Sclerotinia sclerotiorum of rape according to claim 3, characterized in that, The S4 includes: Using the spray-drying method, with a drying temperature of 80 - 100°C; Finally, a wettable powder is prepared, with a moisture content ≤5%.

8. The preparation method of a biological pesticide for controlling Sclerotinia sclerotiorum of rape according to claim 3, characterized in that, The S5 includes: Packaging the finished preparation into a moisture-proof aluminum foil bag; Sealing and storing at a storage temperature of 4 - 25°C and a humidity <60%.

9. The preparation method of a biological pesticide for controlling Sclerotinia sclerotiorum of rape according to claim 3, characterized in that, During the S1 culture process, the growth state of the bacteria is regularly measured, and after 24 h, samples are taken every 6 h to detect the pH value of the culture medium and the cell concentration, so that the cell density is maintained within the range of 10 7 -10 9 CFU / mL.

10. The preparation method of a biological pesticide for controlling Sclerotinia sclerotiorum of rape according to claim 3, characterized in that, Before the extraction in S2, the plant raw materials are pulverized to 80 - 100 mesh and dried at a low temperature of 60 - 70°C until the water content is less than 8% before extraction.

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