Oil suspending agent containing pleuromutilin and preparation method thereof

Abstract

The invention belongs to the field of pesticide preparations, and particularly relates to an oil suspending agent containing pleuromutilin and a preparation method of the oil suspending agent. According to the invention, by optimizing the formula, the obtained oil suspending agent can effectively prevent and treat bacterial diseases, the dosage form of the oil suspending agent is more beneficial to application to spraying method pesticide application, and the embodiment verifies that the prevention and treatment effect on the bacterial diseases can reach 90% or above under the condition that the concentration of pleuromutilin is 200PPM. In addition, according to a traditional method, an active compound is obtained through fermentation, and then the active compound is mixed with a corresponding solvent and an auxiliary agent to be processed into a dosage form, so that the cost is high, and great pollution is brought. The method provided by the invention not only meets the control requirements on bacterial diseases, but also saves the energy and power cost from the aspect of process, reduces the use of solvents, is more beneficial to environmental protection, and thus has great economic benefits, environmental benefits and ecological benefits.

Description

Technical Field

[0001] The invention belongs to the field of pesticide preparations, and particularly relates to an oil suspension containing pleuromutilin and a preparation method thereof. Background Art

[0002] Plant bacterial diseases mainly refer to diseases caused by bacteria infecting plants. They are the second largest category of diseases that infect plants, second only to fungal diseases. Plant bacterial diseases can enter through natural openings (stomata, lenticels, water holes, etc.) and wounds, and spread through running water, rain, insects, etc., especially when plants are damaged by insect pests, pruning, typhoons and other extreme weather, which is more conducive to the spread of bacterial diseases. In areas with high temperature and high humidity, bacterial diseases have shown a high incidence in recent years, and have gradually shifted from cash crops to field crops, seriously affecting the quality and yield of crops.

[0003] What’s worse is that there are relatively few products for preventing and controlling bacterial diseases, mainly copper preparations. However, copper preparations have been used for a long time, and firstly, they have resistance, and secondly, they are unsafe and easy to cause pesticide damage. Thirdly, the large-scale use of copper preparations will also lead to a large occurrence of mite damage. Therefore, it is urgent to develop a bacterial disease prevention and control agent with outstanding prevention effect and good safety.

[0004] At the same time, in recent years, due to the obvious trend of land intensification and the increase in labor costs, the mechanization trend of pesticide application has become more and more obvious, and aircraft spraying has become the mainstream trend of pesticide application, especially in the field of large fields, which is almost all sprayed by drones. However, drone spraying has strict requirements on pesticide formulations, and the development of formulations that prevent drifting, promote pesticide sedimentation, and resist evaporation is a practical need for aerial pesticide application.

[0005] Although CN117281117A proposes a pesticide suspension of pleuromutilin for preventing and controlling plant bacterial diseases, it is an aqueous solution and is prone to drifting and evaporation during aerial spraying, and it is difficult to ensure that the effective ingredients are sufficiently deposited on the crop surface, so it cannot meet the actual needs of aerial spraying.

[0006] Under the above background, it is necessary to develop a bacterial disease control agent that can be used for aerial spraying. Summary of the invention

[0007] The first object of the present invention is to provide an oil suspension, which uses vegetable oil as an oil phase carrier and comprises the following components in parts by weight: 5 to 30 parts of pleuromutilin, 10 to 15 parts of methyl oleate, 4 to 6 parts of vegetable oil polyoxyethylene ether, 2 to 4 parts of alkyl sulfonate, 3.0 to 4.5 parts of organic bentonite, 1 to 2 parts of white carbon black, and 2.0 to 2.5 parts of a thickener; wherein the pleuromutilin is provided in the form of a pleuromutilin mother liquor, and the pleuromutilin mother liquor is an ethyl acetate extract of pleuromutilin with a concentration of 30 to 45 wt%.

[0008] The second object of the present invention is to provide a method for preparing the oil suspension, which comprises: separating a filter residue from a fermentation broth containing pleuromutilin; extracting the filter residue with methanol having a concentration of 90 vol% or more to obtain an extract; extracting the extract with ethyl acetate having a concentration of 90 vol% or more to obtain an ethyl acetate extract; concentrating the ethyl acetate extract to obtain a pleuromutilin mother liquor; and mixing the pleuromutilin mother liquor with other components to obtain the oil suspension.

[0009] The third object of the present invention is to provide a method for preventing and controlling bacterial diseases, which comprises: allowing the oil suspension to act on pathogens or their living environment; wherein the pathogens include one or more of rice bacterial leaf streak pathogen, citrus canker pathogen, kiwi canker pathogen, Korla pear branch blight pathogen, cabbage soft rot pathogen, peach bacterial leaf streak pathogen, watermelon bacterial fruit rot pathogen, cucumber bacterial leaf streak pathogen, pitaya canker pathogen, and mango bacterial black spot pathogen.

[0010] Furthermore, the present invention also provides a method for preventing and controlling plant bacterial diseases, comprising: applying the oil suspension to plants; wherein the pathogens of the bacterial diseases include one or more of rice bacterial leaf streak pathogen, citrus canker pathogen, kiwi canker pathogen, Korla pear branch blight pathogen, cabbage soft rot pathogen, peach bacterial leaf streak pathogen, watermelon bacterial fruit rot pathogen, cucumber bacterial leaf streak pathogen, pitaya canker pathogen, and mango bacterial black spot pathogen.

[0011] The oil suspension of the present invention can effectively prevent and control bacterial diseases, and the drug formulation is more conducive to application in spraying. It is verified by the examples that the control effect of bacterial diseases can reach more than 90% at a concentration of 200 PPM of pleuromutilin.

[0012] In addition, the traditional method is to first obtain the original drug through fermentation, and then mix the original drug with the corresponding solvent and auxiliary agent to form a dosage form, which is not only costly but also causes great pollution. The method of the present invention not only meets the demand for prevention and control of bacterial diseases, but also saves energy and power costs from a process perspective, reduces the use of solvents, and is more environmentally friendly, so it has great economic benefits, environmental benefits and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 These are the in vivo experimental results of the control effects of the four groups of samples in the examples of the present invention on rice bacterial leaf streak disease. DETAILED DESCRIPTION

[0015] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0016] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0017] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0018] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0019] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0020] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0021] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0022] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0023] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0024] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0025] In the present invention, "Oil Suspension Concentrate (OSC)" refers to a form of pesticide formulation in which the active ingredients of the pesticide are ground into fine particles and dispersed in a non-aqueous oil phase to form a stable suspension.

[0026] In the present invention, when "pleuromutilin" is mentioned alone, it means pure pleuromutilin. For example, when it is mentioned that 5 to 30 parts of pleuromutilin or 5 to 30 wt% of pleuromutilin is contained, it refers to the content of pure pleuromutilin, and the weight of other substances in the pleuromutilin mother solution except pleuromutilin is not included in the calculation basis of the weight part and wt% of the oil suspension. Those skilled in the art can easily confirm the required amount of pleuromutilin mother solution to be added in combination with the concentration / content of pleuromutilin in the pleuromutilin mother solution.

[0027] The invention first provides an oil suspension, which uses vegetable oil as an oil phase carrier and comprises the following components in parts by weight: 5 to 30 parts of pleuromutilin, 10 to 15 parts of methyl oleate, 4 to 6 parts of vegetable oil polyoxyethylene ether, 2 to 4 parts of alkyl sulfonate, 3.0 to 4.5 parts of organic bentonite, 1 to 2 parts of white carbon black, and 2.0 to 2.5 parts of a thickener; wherein the pleuromutilin is provided in the form of a pleuromutilin mother liquor, and the pleuromutilin mother liquor is an ethyl acetate extract of pleuromutilin with a concentration of 30 to 45 wt%.

[0028] The present invention finds that the above system can significantly improve the dispersibility, wettability and stability of pleuromutilin in the oil phase carrier, thereby ensuring the efficacy while making the drug dosage form more suitable for spray application.

[0029] In the present invention, the vegetable oil polyoxyethylene ether is a nonionic surfactant formed by polymerizing a certain number of ethylene oxide (EO) units on the alcohol group of the vegetable oil. Its molecular structure is usually expressed as RO(CH2CH2O)nH, where R is a vegetable oil group and n is the number of EO units. In some specific embodiments, R is selected from soybean oil group, rapeseed oil group, cottonseed oil group, sunflower seed oil group, palm oil group, linseed oil group, olive oil group. In some specific embodiments, n is selected from any natural number between 1 and 20, more preferably any natural number between 1 and 15, and further preferably any natural number between 1 and 10. The selection of the vegetable oil polyoxyethylene ether in the present invention is not particularly limited, and the vegetable oil polyoxyethylene ether obtained from commercial sources based on common sense by those skilled in the art can achieve the technical effects mentioned in the present invention.

[0030] In the present invention, the general formula of the alkyl sulfonate is RSO3Me, wherein R represents an alkyl chain and Me represents a metal ion. It is an anionic surfactant, that is, it can dissociate in an aqueous solution to generate anions with surface activity. In some specific embodiments, the alkyl sulfonate is selected from one or more of linear alkyl sulfonates (general formula R-SO3Me, wherein R is a linear alkyl chain, usually containing 10-18 carbon atoms), branched alkyl sulfonates (general formula R-SO3Me, wherein R is a branched alkyl chain, usually containing 10-18 carbon atoms), α-olefin sulfonates (general formula R-SO3Me-CH2CH2NH2, wherein R is an alkyl chain) and taurine derivatives (general formula R-SO3Me-CH2CH2NH2, wherein R is an alkyl chain). As an example, the alkyl sulfonate can be C12~C18 linear alkyl sulfonates, isotridecyl sulfonates, isopentadecyl sulfonates, C12~C16α-olefin sulfonates, and C12~C16 taurine derivatives. In some specific embodiments, the metal ion (Me) in the alkyl sulfonate is selected from one or more of sodium ion (Na+), potassium ion (K+), ammonium ion (NH4+), magnesium ion (Mg2+) and calcium ion (Ca2+). The present invention does not specifically limit the selection of the alkyl sulfonate, and the alkyl sulfonates obtained from commercial sources based on common sense by those skilled in the art can achieve the technical effects mentioned in the present invention.

[0031] In some embodiments, the thickener is a nonionic polyurethane associative thickener. The present invention further finds that the above thickener can further improve the stability of the product.

[0032] In specific implementation, those skilled in the art can combine common sense and obtain the nonionic polyurethane associative thickener through commercial channels, and all of them can obtain the technical effects mentioned in the present invention. As an example, the nonionic polyurethane associative thickener includes Dow Chemical's Foamaster series (such as Foamaster VL-60, Foamaster VL-70, etc.), Emery Oleochemicals' FoamStar series (such as FoamStar A-40, FoamStar A-88, etc.), Dow Chemical's FoamKill series (such as FoamKill AT-50, FoamKill AT-100, etc.), DowChemical's FoamDis series (such as FoamDis A-10, FoamDis A-20, etc.), Dow Chemical's FoamTrol series (such as FoamTrol A-100, FoamTrol A-200, etc.), etc.

[0033] In some embodiments, the oil suspension further comprises the following components by weight: 2 to 5 parts of antifreeze. Preferably, the antifreeze is ethylene glycol. Adding antifreeze helps to ensure the fluidity and stability of the oil suspension under low temperature conditions. Compared with other antifreeze agents such as glycerol, the stability and fluidity of ethylene glycol in the system of the present invention are significantly better.

[0034] In the present invention, the vegetable oil includes but is not limited to one or more of soybean oil, rapeseed oil, cottonseed oil, corn oil, sunflower seed oil, palm oil, linseed oil and olive oil. The above vegetable oils all have good wettability, dispersibility, stability and compatibility, can effectively disperse and stabilize pleuromutilin, and improve the use effect of the oil suspension.

[0035] In some embodiments, the vegetable oil is soybean oil. In the system of the present invention, the use of soybean oil has a better dispersion and stabilization effect on pleuromutilin, and is more conducive to improving the use effect of the oil suspension.

[0036] In some embodiments, the oil suspension contains no or very little (usually <0.5%) water.

[0037] In some embodiments, the oil suspension uses vegetable oil as the oil phase carrier and comprises the following components in parts by weight: 5 to 30 parts of pleuromutilin, 10 to 15 parts of methyl oleate, 4 to 6 parts of vegetable oil polyoxyethylene ether, 2 to 4 parts of alkyl sulfonate, 3.0 to 4.5 parts of organic bentonite, 1 to 2 parts of white carbon black, 2.0 to 2.5 parts of non-ionic polyurethane associative thickener, and 2 to 5 parts of ethylene glycol.

[0038] In some embodiments, the oil suspension comprises the following components: 5-30wt% pleuromutilin, 10-15wt% methyl oleate, 4-6wt% vegetable oil polyoxyethylene ether, 2-4wt% alkyl sulfonate, 3.0-4.5wt% organic bentonite, 1-2wt% white carbon black, 2.0-2.5wt% nonionic polyurethane associative thickener, and 2-5wt% ethylene glycol, with the balance being soybean oil.

[0039] In some specific embodiments, the oil suspension comprises the following components: 5wt% pleuromutilin, 10wt% methyl oleate, 4wt% vegetable oil polyoxyethylene ether, 2wt% alkyl sulfonate, 3.0wt% organic bentonite, 1wt% white carbon black, 2.0wt% nonionic polyurethane associative thickener, and 2wt% ethylene glycol, with the balance being soybean oil.

[0040] In some specific embodiments, the oil suspension comprises the following components: 30 wt% pleuromutilin, 15 wt% methyl oleate, 6 wt% vegetable oil polyoxyethylene ether, 4 wt% alkyl sulfonate, 4.5 wt% organic bentonite, 2 wt% white carbon black, 2.5 wt% nonionic polyurethane associative thickener, and 5 wt% ethylene glycol, with the balance being soybean oil.

[0041] In some specific embodiments, the oil suspension comprises the following components: 15wt% pleuromutilin, 12wt% methyl oleate, 4.5wt% vegetable oil polyoxyethylene ether, 2.5wt% alkyl sulfonate, 3.5wt% organic bentonite, 1.2wt% white carbon black, 2.2wt% nonionic polyurethane associative thickener, and 3wt% ethylene glycol, with the balance being soybean oil.

[0042] In some specific embodiments, the oil suspension comprises the following components: 25 wt% pleuromutilin, 14 wt% methyl oleate, 5.5 wt% vegetable oil polyoxyethylene ether, 3.5 wt% alkyl sulfonate, 4.0 wt% organic bentonite, 1.8 wt% white carbon black, 2.4 wt% nonionic polyurethane associative thickener, and 4 wt% ethylene glycol, with the balance being soybean oil.

[0043] In some specific embodiments, the oil suspension comprises the following components: 20 wt% pleuromutilin, 15 wt% methyl oleate, 4.5 wt% vegetable oil polyoxyethylene ether, 3 wt% alkyl sulfonate, 3.0 wt% organic bentonite, 1.5 wt% white carbon black, 2.0 wt% nonionic polyurethane associative thickener, and 2 wt% ethylene glycol, with the balance being soybean oil.

[0044] In some specific embodiments, the pleuromutilin mother liquor is an ethyl acetate extract of pleuromutilin having a concentration of 30wt%, 33wt%, 35wt%, 38wt%, 40wt%, 42wt%, 45wt%, or any value within the range of 30-45wt%.

[0045] In some embodiments, the particle size D90 of the oil suspension is less than or equal to 3 μm. The present invention further found that within the above particle size range, the product has less oil separation, a higher product suspension rate, and better product stability. In a specific embodiment, considering the production cost, it is more preferred that the particle size D90 of the oil suspension is 2 to 3 μm.

[0046] Those skilled in the art can combine the above-mentioned solutions and select any numerical value within the mentioned numerical range to obtain more specific embodiments of the oil suspension concentrate of the present invention.

[0047] The present invention also provides a method for preparing the oil suspension, which comprises: separating a filter residue from a fermentation broth containing pleuromutilin; extracting the filter residue with methanol having a concentration of 90 vol% or more to obtain an extract; extracting the extract with ethyl acetate having a concentration of 90 vol% or more to obtain an ethyl acetate extract; concentrating the ethyl acetate extract to obtain a pleuromutilin mother liquor; and mixing the pleuromutilin mother liquor with other components to obtain the oil suspension.

[0048] The present invention finds that by directly mixing the pleuromutilin mother liquor obtained by the above method with other components, an oil suspension with good dispersibility, wettability and stability can be obtained, and this can omit the steps of further concentrating, crystallizing, centrifuging and drying the mother liquor to obtain a pure product in the traditional preparation method, which is more conducive to controlling production costs and improving production efficiency.

[0049] In a specific embodiment, the concentration of methanol used in the leaching can be 90 vol%, 95 vol%, 100 vol%, or any value between 90 and 100 vol%.

[0050] In a specific embodiment, the concentration of ethyl acetate used in the extraction can be 90 vol%, 95 vol%, 100 vol%, or any value between 90 and 100 vol%.

[0051] In some embodiments, the content of pleuromutilin in the pleuromutilin mother solution is 30-45 wt %.

[0052] In some specific embodiments, the content of pleuromutilin in the pleuromutilin mother solution may be 30wt%, 33wt%, 35wt%, 38wt%, 40wt%, 42wt%, 45wt%, or any value within the range of 30-45wt%.

[0053] In some embodiments, the leaching specifically comprises: soaking the filter residue having a water content of 15-20wt% in methanol having a concentration of 90vol% or more for 6-10 hours, and after the feed and liquid are separated, taking the supernatant as the leaching liquid.

[0054] In some embodiments, the waste residue produced during the leaching is dried, and the methanol therein is recovered by condensation for subsequent leaching.

[0055] In some embodiments, the methanol phase separated in the extraction is fractionated, and the methanol therein is recovered for subsequent leaching.

[0056] In some embodiments, the gas generated during the concentration is collected, and the ethyl acetate therein is recovered by condensation for subsequent extraction.

[0057] In the present invention, the process of separating the filter residue from the fermentation broth containing pleuromutilin is not particularly limited, and the specific operation can be determined by those skilled in the art in combination with common sense. In some specific embodiments, the fermentation broth of pleuromutilin is obtained after bacterial culture, primary seed fermentation, secondary fermentation and tertiary fermentation. In some specific embodiments, the step of separating the filter residue from the fermentation broth includes filter pressing and flash drying.

[0058] In some embodiments, the method for preparing the oil suspension further comprises the step of shearing and / or grinding the mixture of the pleuromutilin mother solution and other components, which is more conducive to component mixing and particle size control.

[0059] In some specific embodiments, the preparation method of the oil suspension further includes: mixing and dissolving the raw materials except the thickener, then adding the thickener, mixing evenly under the action of a shearing machine, then pumping the mixed slurry into a sander for grinding, then taking samples and measuring the particle size using a particle size distribution instrument, and when D90 is less than or equal to 3 μm, stopping stirring and filtering the oil suspension.

[0060] The third object of the present invention is to provide a method for preventing and controlling bacterial diseases, which comprises: allowing the oil suspension to act on pathogens or their living environment; wherein the pathogens include one or more of rice bacterial leaf streak pathogen, citrus canker pathogen, kiwi canker pathogen, Korla pear branch blight pathogen, cabbage soft rot pathogen, peach bacterial leaf streak pathogen, watermelon bacterial fruit rot pathogen, cucumber bacterial leaf streak pathogen, pitaya canker pathogen, and mango bacterial black spot pathogen.

[0061] Furthermore, the present invention also provides a method for preventing and controlling plant bacterial diseases, comprising: applying the oil suspension to plants; wherein the pathogens of the bacterial diseases include one or more of rice bacterial leaf streak pathogen, citrus canker pathogen, kiwi canker pathogen, Korla pear branch blight pathogen, cabbage soft rot pathogen, peach bacterial leaf streak pathogen, watermelon bacterial fruit rot pathogen, cucumber bacterial leaf streak pathogen, pitaya canker pathogen, and mango bacterial black spot pathogen.

[0062] In some embodiments, the oil suspension is applied by spraying.

[0063] In the present invention, the spray method refers to a technique of using a spray device to disperse the oil suspension into tiny particles (droplets) and spraying them uniformly on crops, soil or other target areas. In some specific embodiments, the spray device is selected from one or more of a manual sprayer, a backpack sprayer, a tractor-mounted sprayer, a self-propelled sprayer, an unmanned aerial vehicle sprayer, and an airplane equipped with a spray system.

[0064] In some preferred embodiments, the spraying method is aerial spraying.

[0065] In the present invention, the aerial pesticide application refers to the technology of using an aircraft as a spraying platform, dispersing pesticides into tiny particles (droplets) through the spraying equipment on the aircraft, and spraying them evenly to the target area. The aircraft can be a drone or a traditional manned aircraft (such as a light fixed-wing aircraft or a helicopter).

[0066] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0067] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0068] In the following examples, in order to facilitate comparison of the effects, the plant oil polyoxyethylene ethers were purchased from Jiangsu Qingyu Chemical Technology Co., Ltd., with the product model SP-OF3418, in which the number of EO units was 8-10; the alkyl sulfonates were purchased from Jiangsu Qingyu Chemical Technology Co., Ltd., with the product model SP-OF3462; the nonionic polyurethane associative thickeners were purchased from Jiangsu Qingyu Chemical Technology Co., Ltd., with the product model SP-OF3472B. It should be understood that the description here does not constitute a limitation to the present invention, and other applicable products obtained by those skilled in the art through commercial channels can also obtain the technical effects mentioned in the present invention.

[0069] In the following examples, the pleuromutilin mother solution was prepared by the following method:

[0070] 1. Bacteria culture

[0071] After the prepared culture medium is sterilized at 121℃ for 30min, it is inoculated into a 500mL eggplant bottle under sterile conditions and cultured for 10 days. After passing the test, it is inoculated into a seed bottle and cultured at 25℃ for 2 days at 200 rpm. After passing the test, it is inoculated into a 1000mL fermentation bottle and cultured at 25℃ for 3 days. After passing the test, it is transferred to a first-level seed tank. Among them, the formula of the culture medium is: 3 grams of peptone, 3 grams of beef extract, 3 grams of yeast extract, 15 grams of agar, 5 grams of sodium chloride, and 1000 milliliters of distilled water.

[0072] 2. Primary seed fermentation

[0073] The prepared culture medium was put into the first-level seed tank, sterilized by steam at 121°C, maintained at pressure for 30 minutes, cooled by 25°C using circulating water, and transferred to qualified 1000mL fermentation bottle seeds under sterile conditions, and cultured at 25°C for 2 days with ventilation. The formula of the culture medium is: 2wt% corn starch, 2wt% glucose, 2wt% soybean cake powder, 1wt% ammonium sulfate, 0.5wt% calcium carbonate, and 92.5wt% pure water.

[0074] 3. Secondary fermentation

[0075] The prepared culture medium is put into the secondary seed tank, sterilized at 121℃ by steam, maintained at pressure for 30 minutes, cooled by 25℃ by circulating water, transferred to the qualified primary seed liquid under sterile conditions, and cultured at 25℃ for 2 days with ventilation. The formula of the culture medium is: 2wt% corn starch, 2wt% glucose, 2wt% soybean cake powder, 1wt% ammonium sulfate, 0.5wt% calcium carbonate, and 92.5wt% pure water.

[0076] 4. Tertiary fermentation

[0077] The qualified raw materials were slurried according to the following formula: 2.5wt% glucose, 2wt% soybean cake powder, 1wt% ammonium sulfate, 2wt% corn steep liquor, 92.5wt% pure water, and then put into the fermentation tank, sterilized by steam at 121℃, maintained at pressure for 30min, cooled by 25℃ by circulating water, transferred to the qualified secondary seed liquid under sterile conditions, and cultured at 25℃ for 11 days with ventilation. The qualified titer (reaching 10000IU / mL) entered the filter press process.

[0078] 5. Filter Press

[0079] The qualified fermentation liquid is sent to the ceramic membrane for filtration, the filtrate is sent to the sewage treatment station for centralized treatment, and the filter residue is dried.

[0080] 6. Flash drying

[0081] The filter residue is broken into small particles by high-speed rotating blades. Hot air enters from the bottom of the dryer, forming a high-speed rotating upward airflow, and the dried and qualified materials are carried out by the airflow.

[0082] 7. Extraction and filtration

[0083] The filtered residue containing 20 wt% water was soaked in 95 vol% methanol for 8 hours. After the feed and liquid were separated, the clear liquid was sent to the extraction process. The turbid liquid containing the bacterial residue was filtered again. The waste residue was dried and condensed to recover the methanol for further treatment.

[0084] 8. Extraction and concentration to obtain pleuromutilin mother solution

[0085] The leached liquid is extracted with ethyl acetate, separated with a centrifuge, and the ethyl acetate extract is concentrated to obtain a pleuromutilin mother liquor with a content of 35 wt%, and the extracted and separated ethyl acetate enters a distillation tower for recovery.

[0086] Example 1 Confirmation of the formula of oil suspension

[0087] 1. Choice of surfactant

[0088] Taking 20wt% pleuromutilin (calculated as pure product content) oil suspension as an example, in order to screen the optimal dosage of adjuvants for vegetable oil polyoxyethylene ether, alkyl sulfonate, organic bentonite, etc., three contents of 1.5wt%, 3wt%, and 4.5wt% were set for screening, wherein the dispersibility results were obtained by referring to GB / T32775-2016 "Method for Determining Dispersibility of Pesticides", the wettability results were obtained by referring to GB / T3441-2001 "Method for Determining Wettability of Pesticides", and the stability results were obtained by referring to GB / T1603-2023 "Method for Determining Stability of Pesticide Emulsions". The specific results are shown in Table 1.

[0089] Table 1

[0090]

[0091] As shown in the results of Table 1, at different contents of the three surfactants, the stability of pleuromutilin after hot storage and cold storage all reached above medium. And under the premise of selecting the best in dispersibility, wettability and stability, the surfactant formula was finally determined to be: vegetable oil polyoxyethylene ether (4.5wt%), alkyl sulfonate (3.0wt%), organic bentonite (3.0wt%), which has good stability, high suspension rate, and more outstanding dispersibility and wettability.

[0092] 2. Selection of Thickener Dosage

[0093] The above formula is prone to sedimentation and oil separation under long-term storage. In view of this situation, this experimental example increases the sedimentation stability by adding a thickener to improve the stability and suspension rate of the oil suspension, and observes the stability of the product through hot storage and cold storage. The results are shown in Table 2. Among them, the suspension rate results are obtained by referring to GB / T14825-2023 "Method for Determination of Pesticide Suspension Rate", and the dispersibility results are obtained by referring to GB / T32775-2016 "Method for Determination of Pesticide Dispersibility". The oil separation rate is mainly obtained by naked eye observation. Since the oil suspension agent belongs to a thermally unstable system, if the formula system is unstable, the suspended particles will settle, resulting in the separation of oil clear liquid from the upper layer. For those with severe oil separation, the oil separation rate is evaluated as poor; if no oil separation is observed, the oil separation rate is evaluated as excellent; the intermediate situation is evaluated as medium oil separation rate.

[0094] Table 2

[0095]

[0096]

[0097] Finally, it was determined that the optimal addition amount of nonionic polyurethane associative thickener was 2-2.5wt%.

[0098] 3. Choice of antifreeze

[0099] In order to make the above oil suspension still maintain stability and fluidity under severe cold and low temperature conditions, it is usually necessary to add a certain amount of suitable antifreeze. When glycerol is used as the antifreeze in the formulation of the present invention, the system viscosity is too high and the fluidity is biased; while when ethylene glycol is used as the antifreeze, the viscosity of the oil suspension is moderate, and the stability and fluidity have obvious advantages over the formulation using glycerol. Therefore, in the oil suspension of the present invention, ethylene glycol is selected as the antifreeze.

[0100] 4. Effect of particle size on product stability after hot and cold storage

[0101] After determining the above-mentioned auxiliary agent, the raw materials except the thickener are mixed and stirred to dissolve, and then the thickener is added and mixed evenly under the action of a shearing machine, and then the mixed slurry is pumped into a sander for grinding, and a sample is taken to determine the particle size using a particle size distribution instrument to prepare truncated pleurotus ostreatus oil suspension concentrates of different particle sizes, which are numbered 1 to 5, respectively. According to the relevant national standards or industry standards for pesticides (GB / T14825-2023 "Method for Determination of Pesticide Suspension Rate", GB / T19136-2003 "Method for Determination of Pesticide Thermal Storage Stability", GB / T19137-2003 "Method for Determination of Pesticide Low Temperature Stability"), the main technical indicators of the truncated pleurotus ostreatus oil suspension concentrate of the present invention are measured, and the specific results are shown in Table 3.

[0102] Table 3

[0103] serial number Particle size (D90, μm) Oil separation Suspension rate (%) 1 1.0 none 98 2 3.0 none 95 3 5.0 Small amount 85 4 8.0 Small amount 80 5 10.0 Large 70

[0104] As can be seen from Table 3, for the pleurotus ostreatus oil suspension of the present invention, the smaller the particle size of the product particles, the less oil separation of the product, the higher the product suspension rate, and the better the product stability. Although the smaller the particle size, the better the product stability, it is directly proportional, but if it is too small, it is difficult to industrialize and the production cost is relatively high. Therefore, the particle size of 2-3 μm not only takes into account the economy, but also maximizes the stability of the product.

[0105] 5. Determination of the optimal formulation of pleuromutilin

[0106] Based on the above optimization process, taking 20 wt% pleuromutilin content as an example, the preferred formula of the oil suspension concentrate of the present invention is shown in Table 4 below.

[0107] Table 4

[0108] Components Content (wt%) Pleuromutilin 20％ Methyl oleate 15％ Vegetable oil polyoxyethylene ether 4.5％ White Carbon Black 1.5％ Alkyl sulfonate 3.0％ Organic bentonite 3.0％ Nonionic polyurethane associative thickener 2.0％ Ethylene glycol antifreeze 2.0％ Soybean Oil Make up margin

[0109] 6.20% pleurotus ostreatus oil suspension concentrate test results

[0110] According to the relevant national standards or industry standards for pesticides (GB / T19136-2003), the main properties of the pleuromutilin oil suspension were tested, as shown in Table 5. The results showed that under this formula, the pleuromutilin oil suspension was a light yellow oily liquid with excellent dispersibility and suspension rate, and had good stability at room temperature and after hot storage.

[0111] Table 5

[0112]

[0113]

[0114] Example 2 Pesticide efficacy in vitro test

[0115] In this example, the turbidity method was used to test the inhibitory effect of the oil suspension containing 20 wt % of pleuromutilin determined in Example 1 on pathogens such as rice bacterial leaf streak and citrus canker.

[0116] 1. Test materials: pipette tips (5mL, 1mL, 200μL), 15mL centrifuge tubes, secondary water, 2mL EP tubes, NB culture medium, all need to be sterilized.

[0117] 2. NB medium: 3 g beef peptone, 5 g peptone, 1 g yeast powder, 10 g glucose, 1 L secondary water. Dispense into test tubes (4 mL / tube), pH = 7.0-7.2.

[0118] 3. Operation steps:

[0119] (1) Weigh a certain amount of the preparation to be tested, and then dissolve it with a corresponding volume (200 μL) of DMSO. Prepare a 5% solution system, dilute it to the required concentration in proportion, and adjust the volume to 4 mL with 0.1% Tween solution.

[0120] (2) Use a pipette to draw 1 mL of the drug-Tween water mixture into a test tube containing 4 mL of culture medium.

[0121] (3) Use a pipette to aspirate 200 μL of the mixed solution (culture medium), measure its OD value using a 96-well plate, and record it.

[0122] (4) Add 50 μL of the bacterial solution of Pathogen of Rice Bacterial Leaf Streak Pathogen and Pathogen of Citrus Canker into each test tube.

[0123] (5) Place the plate in a shaker set at 28°C and 180 rpm.

[0124] (6) After 24h-36h, measure and record the OD value, and then calculate the inhibition rate. Generally, the OD value of CK is 0.6-0.8, which indicates that the bacteria are not contaminated and are growing normally.

[0125] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium.

[0126] Inhibition rate % = (corrected OD value of the control culture medium bacterial solution - corrected OD value of the culture medium containing bacteria) / corrected OD value of the control culture medium bacterial solution * 100.

[0127] 4. Activity data:

[0128] The inhibitory activity (inhibition rate %) of 20% zinc thiazole suspension (agent A), 3% zhongshengmycin wettable powder (agent B), 20% pleuromutilin oil suspension (agent C) and 2% kasugamycin aqueous solution (agent D) against the pathogens of rice bacterial leaf streak and citrus canker at drug concentrations of 800, 400, 200, and 100 ppm was tested, and the results are shown in Table 6. In Table 6, "A-800" means the group in which the inhibitory activity of agent A against the pathogens was tested at a concentration of 800 ppm, and the reference method of other agent groups is similar.

[0129] Table 6

[0130]

[0131]

[0132] From the results, it can be seen that the in vitro activity of 20% pleuromutilin oil suspension (agent C) against rice bacterial leaf streak pathogen and citrus canker pathogen at concentrations of 100ppm, 200ppm, 400ppm and 800ppm is generally better than that of 2% kasugamycin aqueous solution, 20% zinc thiazole suspension and 3% sinomenine wettable powder.

[0133] Example 3: Pesticide efficacy test on rice bacterial leaf streak in living potted plants

[0134] This example uses the leaf clipping method to test the protective and therapeutic effects of the oil suspension containing 20 wt % of pleuromutilin in the preferred formulation determined in Example 1 against rice bacterial leaf streak at a concentration of 200 μg / mL in living pot plants.

[0135] 1. Protective activity:

[0136] The 20% pleuromutilin oil suspension of the present invention is selected and dissolved with 200 μL of DMSO, and a 0.1% Tween-80 solution is used to prepare a medicament with a concentration of 200 μg / mL, and the medicament is evenly sprayed on rice leaves until liquid drops fall. After 24 hours, the leaves are cut and inoculated with scissors containing rice bacterial leaf streak pathogens, and the bacterial liquid is at the tip of the rice leaf 1 / 3. At the same time, a clear water control without the medicament is set. Each treatment is 10-15 leaves, and repeated 3 times. The length of the lesions on the rice leaves is measured 14 days after the application of the medicament, and the control effect is calculated according to the length of the lesions.

[0137] The specific calculation formula is: preventive effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group * 100.

[0138] The results are shown in Table 7 and Figure 1 .

[0139] Table 7

[0140]

[0141] From Table 7 and Figure 1 It can be seen that at a concentration of 200 μg / mL, the protective activities of the 20% pleuromutilin oil suspension, 20% zinc thiazole suspension, 3% zhongshengmycin wettable powder, and 2% kasugamycin aqueous solution against rice bacterial leaf streak disease are 92.6%, 78.4%, 45.6%, and 82.1%, respectively. That is, the 20% pleuromutilin oil suspension has the highest in vivo protective activity, followed by the 20% zinc thiazole suspension and the 2% kasugamycin aqueous solution, and the 3% zhongshengmycin wettable powder has the worst protective activity.

[0142] 2. Therapeutic activity:

[0143] Use scissors dipped in the bacterial solution of rice bacterial leaf streak to cut off the 1 / 3 cm portion of the tip of the rice leaf. After 24 hours, select the 20% pleurotus ostreatus oil suspension of the present invention and add DMSO to dissolve it, then use 0.1% Tween-80 solution to prepare a medicament with a concentration of 200 μg / mL, and evenly spray it on the rice leaves until liquid drops fall. At the same time, set a clear water control without the medicament. Treat 10-15 leaves per rice plant. Measure the length of the lesions on the rice leaves 14 days after the application of the medicament, and calculate the control effect according to the length of the lesions.

[0144] The specific calculation formula is: preventive effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group * 100.

[0145] The results are shown in Table 8.

[0146] Table 8

[0147]

[0148]

[0149] It can be seen from Table 8 that at a concentration of 200 μg / mL, the therapeutic activities of the 20% pleuromutilin oil suspension, 20% zinc thiazole suspension, 3% zhongshengmycin wettable powder, and 2% kasugamycin aqueous solution on rice bacterial leaf streak disease are 95.6%, 88.4%, 55.8%, and 82.5%, respectively. That is, the in vivo therapeutic activity of the 20% pleuromutilin oil suspension is the highest, followed by the 20% zinc thiazole suspension and the 2% kasugamycin aqueous solution, and the in vivo therapeutic activity of the 3% zhongshengmycin wettable powder is the worst.

[0150] Example 4 Pesticide efficacy test on living potted plants with citrus canker

[0151] At a concentration of 200 μg / mL, the protective and therapeutic effects of the oil suspension containing 20 wt % of pleuromutilin determined in Example 1 on citrus canker in living pot plants were tested by needle pricking method.

[0152] 1. Protective activity

[0153] Use an analytical balance to accurately weigh 18.0 mg of 20% pleuromutilin dispersible oil suspension, add 200 μL of DMSO to dissolve, and use 0.1% Tween-80 solution to prepare a medicament with a concentration of 200 μg / mL. Spray it evenly on citrus leaves until liquid drops. After 24 hours, use a disposable syringe containing citrus canker bacteria for inoculation. Each leaf is pierced with 18 small holes, and a new filter paper soaked with Xac suspension (OD595=0.6) is attached to the above wound surface for 24 hours. The filter paper containing the medicine is discarded, and a clear water control without the medicine is set. Each treatment is 10-15 leaves, and repeated 3 times. The incidence of citrus leaves is measured 21 days after the application of the medicine, and the control effect is calculated based on the disease index.

[0154] The specific calculation formula is: preventive effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group * 100.

[0155] The results are shown in Table 9.

[0156] Table 9

[0157]

[0158]

[0159] It can be seen from Table 9 that at a concentration of 200 μg / mL, the protective activities of the 20% pleuromutilin oil suspension, 20% zinc thiazole suspension, 3% zhongshengmycin wettable powder, and 2% kasugamycin aqueous solution against citrus canker are 89.6%, 78.4%, 38.2%, and 77.5%, respectively. That is, the 20% pleuromutilin oil suspension has the highest in vivo protective activity, followed by the 20% zinc thiazole suspension and the 2% kasugamycin aqueous solution, and the 3% zhongshengmycin wettable powder has the worst protective activity.

[0160] 2. Therapeutic activity

[0161] A disposable syringe containing citrus canker bacteria was used for inoculation. Each leaf was pierced with 18 small holes. A new filter paper soaked with Xac suspension (OD595=0.6) was attached to the wound surface 24 hours later. 18.0 mg of 20% pleuromutilin dispersible oil suspension was accurately weighed using an analytical balance, dissolved in DMSO, and then prepared into a 200 μg / mL agent with 0.1% Tween-80 solution. The agent was evenly sprayed on the citrus leaves until liquid dripped. A clear water control without the agent was set. 10-15 leaves were treated for each citrus plant. The disease condition of the citrus leaves was measured 21 days after the application of the agent, and the control effect was calculated based on the disease index.

[0162] The specific calculation formula is: preventive effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group * 100.

[0163] The results are shown in Table 10.

[0164] Table 10

[0165]

[0166] It can be seen from Table 10 that at a concentration of 200 μg / mL, the therapeutic activities of the 20% pleuromutilin oil suspension, 20% zinc thiazole suspension, 3% zhongshengmycin wettable powder, and 2% kasugamycin aqueous solution on rice bacterial leaf streak disease are 96.5%, 84.4%, 52.8%, and 88.5%, respectively. That is, the in vivo therapeutic activity of the 20% pleuromutilin oil suspension is the highest, followed by the 20% zinc thiazole suspension and the 2% kasugamycin aqueous solution, and the in vivo therapeutic activity of the 3% zhongshengmycin wettable powder is the worst.

[0167] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An oil suspension, which uses vegetable oil as an oil phase carrier and comprises the following components by weight: 5 to 30 parts of pleuromutilin, 10 to 15 parts of methyl oleate, 4 to 6 parts of vegetable oil polyoxyethylene ether, 2 to 4 parts of alkyl sulfonate, 3.0 to 4.5 parts of organic bentonite, 1 to 2 parts of white carbon black, and 2.0 to 2.5 parts of thickener; wherein, The pleuromutilin is provided in the form of a pleuromutilin mother solution, which is an ethyl acetate extract of pleuromutilin with a concentration of 30-45 wt%.

2. The oil suspension according to claim 1, wherein The thickener is a nonionic polyurethane associative thickener.

3. The oil suspension according to claim 1 or 2, wherein The oil suspension further comprises the following components by weight: 2 to 5 parts of antifreeze; preferably, the antifreeze is ethylene glycol.

4. The oil suspension concentrate according to any one of claims 1 to 3, wherein The vegetable oil is soybean oil.

5. The oil suspension concentrate according to any one of claims 1 to 4, wherein The oil suspension comprises the following components: 5-30wt% of pleuromutilin, 10-15wt% of methyl oleate, 4-6wt% of vegetable oil polyoxyethylene ether, 2-4wt% of alkyl sulfonate, 3.0-4.5wt% of organic bentonite, 1-2wt% of white carbon black, 2.0-2.5wt% of nonionic polyurethane associative thickener, and 2-5wt% of ethylene glycol, with the balance being soybean oil.

6. The oil suspension according to any one of claims 1 to 5, wherein The particle size D90 of the oil suspension is less than or equal to 3 μm.

7. A method for preparing an oil suspension according to any one of claims 1 to 6, comprising: separating a filter residue from a fermentation broth containing pleuromutilin; Using methanol with a concentration of 90 vol% or more to extract the filter residue to obtain an extract; Extracting the extract with ethyl acetate having a concentration of 90 vol% or more to obtain an ethyl acetate extract; Concentrating the ethyl acetate extract to obtain a pleuromutilin mother solution; The pleuromutilin mother solution is mixed with other components to obtain the oil suspension.

8. The method for preparing an oil suspension according to claim 7, wherein: In the pleuromutilin mother solution, the content of pleuromutilin is 30-45wt%.

9. The method for preparing an oil suspension according to claim 7 or 8, wherein: The extraction specifically comprises: The filter residue with a water content of 15-20wt% is soaked in methanol with a concentration of more than 90vol% for 6-10 hours, and after the material and liquid are separated, the supernatant is taken as the leaching liquid.

10. The method for preparing an oil suspension according to any one of claims 7 to 9, wherein: Drying the waste residue produced in the leaching process and condensing and recovering the methanol therein for subsequent leaching; and / or, fractionating the methanol phase separated in the extraction and recovering the methanol therein for subsequent leaching; And / or, the gas generated during the concentration is collected, and the ethyl acetate therein is recovered by condensation for subsequent extraction.

11. A method for preventing and controlling bacterial diseases, comprising: The oil suspension concentrate according to any one of claims 1 to 6 is allowed to act on pathogens or their living environment; wherein the pathogens include one or more of rice bacterial leaf streak pathogen, citrus canker pathogen, kiwi canker pathogen, Korla pear branch blight pathogen, cabbage soft rot pathogen, peach bacterial leaf streak pathogen, watermelon bacterial fruit rot pathogen, cucumber bacterial leaf streak pathogen, pitaya canker pathogen, and mango bacterial black spot pathogen.

12. A method for preventing and controlling plant bacterial diseases, comprising: Applying the oil suspension according to any one of claims 1 to 6 to plants; wherein the pathogens of bacterial diseases include one or more of rice bacterial leaf streak pathogen, citrus canker pathogen, kiwi canker pathogen, Korla fragrant pear branch blight pathogen, cabbage soft rot pathogen, peach bacterial leaf streak pathogen, watermelon bacterial fruit rot pathogen, cucumber bacterial leaf streak pathogen, pitaya canker pathogen, and mango bacterial black spot pathogen.

13. The method according to claim 11 or 12, wherein: The oil suspension is applied by spraying; preferably, the spraying is aerial spraying.

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