A nano pesticide preparation and its application

Abstract

The invention discloses a nano pesticide preparation and its application, and belongs to the technical field of pesticides. The nano pesticide preparation is prepared by the following method: adding azadirachtin and fenpropicyclopyralidone to ethanol, and then adding polylactic acid-glycolic acid copolymer, stirring evenly, and obtaining an organic phase; dissolving Tween 20 in water to obtain an aqueous phase; under ice-water bath conditions, the organic phase is added dropwise to the aqueous phase, and stirred at high speed for 30-45min to obtain an emulsion, and ethanol is removed by rotary evaporation, washed, and freeze-dried to obtain a nano pesticide preparation. The present invention effectively combines azadirachtin and fenpropicyclopyralidone through nano-ization, significantly improves the biological activity and utilization rate of azadirachtin and fenpropicyclopyralidone, reduces the dosage of pesticides, and improves the control effect of soybean sclerotinia.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticides, and in particular to a nano pesticide preparation and application thereof. Background Art

[0002] Pesticides play an important role in preventing and controlling crop diseases, insects, and weeds, ensuring the normal growth of crops and increasing yields. At present, the market is still dominated by chemical pesticides, but the long-term use of chemical pesticides will inevitably put pressure on the environment, destroy the natural balance, and pose a serious threat to human health and life. In addition, some pests and diseases have developed resistance to chemical pesticides after long-term use, which not only fails to achieve the purpose of prevention and control, but even makes some pests and diseases more rampant.

[0003] Plant fungicides include plants with antibacterial ability or antibacterial active ingredients obtained from plants to make pesticides. Compared with chemical pesticides, plant-based pesticides have the characteristics of easy degradation, safety to non-target organisms, not easy to produce resistance to pests, special mode of action, multiple development channels, and multiple types. Psoralea corylifolia is an annual herb. The seeds of Psoralea corylifolia are rich in effective active ingredients such as isopsoralea chalcone, psoralea methyl and bakuchiol. Fenpyraclostrobin is the pesticide name of isopsoralea chalcone. It is an effective antibacterial active ingredient extracted from the seeds of Psoralea corylifolia, a leguminous plant. It is a new type of plant-based fungicide newly created and developed in my country. It has the characteristics of broad-spectrum bactericidal, immune induction, and growth promotion. It meets the requirements of green prevention and control, and is green, environmentally friendly, and residue-free. Neem was used to treat digestive tract parasitic diseases in ancient times. Neem trees are widely used in traditional agriculture in India, Southeast Asia, Africa, and other places. Its leaves, seeds, bark, and fruits are often used as natural pesticides to prevent and control plant diseases and insect pests. The active ingredients in neem mainly include azadirachtin, azadirachtic acid and azadirachtin. These compounds have multiple biological activities, especially azadirachtin, which performs outstandingly in insecticidal and antibacterial aspects.

[0004] In the prior art, although azadirachtin and fenpropicyclopentane each exhibit significant biological activity, such as broad-spectrum bactericidal, disease inhibition and insect repellent, there is currently no pesticide product that uses azadirachtin and fenpropicyclopentane in combination. In addition, most botanical pesticides are prone to photodegradation, non-biological oxidation or loss through volatilization. Compared with chemical pesticides, their poor stability and easy decomposition have restricted the application of botanical pesticides in production. As one of the "key technologies of the 21st century", nanotechnology has been widely used in various disciplines. With the continuous development of nanotechnology, the research and development of nanoformulations has also attracted more and more attention from scholars at home and abroad. The nano drug delivery system developed based on nanocarriers can effectively control the drug release rate, change the route of administration, reduce the toxic and side effects of drugs, and thus improve the utilization of drugs. It is of great significance in both medical and agricultural aspects. Therefore, combining nanotechnology and botanical pesticides is of great significance for improving the stability of botanical pesticides, enhancing their biological activity and extending the duration of effectiveness. Summary of the invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide a nanopesticide preparation and application thereof.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a nanopesticide preparation prepared by the following method:

[0008] (1) Adding azadirachtin and phenylpropenil to ethanol, and then adding polylactic acid-co-glycolic acid (PLGA), stirring evenly to obtain an organic phase;

[0009] (2) Dissolve Tween 20 in water to obtain an aqueous phase;

[0010] (3) In an ice-water bath, the organic phase is added dropwise to the aqueous phase, and stirred at high speed for 30-45 min to obtain an emulsion. The ethanol is removed by rotary evaporation, and the mixture is washed and freeze-dried to obtain a nanopesticide preparation.

[0011] Preferably, in step (1), the ratio of the added amounts of azadirachtin, phenylpropenamide and ethanol is (1-10) mg: 1 mg: (15-25) mL.

[0012] Preferably, in step (1), the ratio of the added amount of polylactic acid-co-glycolic acid (PLGA) to ethanol is (5-27.5) mg: (15-25) mL.

[0013] Preferably, in step (2), the mass concentration of Tween 20 in the aqueous phase is 0.2%-1%.

[0014] Preferably, in step (3), the volume ratio of the organic phase to the aqueous phase is 1:(3-10).

[0015] Preferably, in step (3), the dropping speed is 1-2 mL / min and the stirring speed is 1000-1500 rpm.

[0016] The second aspect of the present invention provides the use of the above-mentioned nanopesticide preparation in the preparation of a sclerotinia sclerotiorum inhibitor.

[0017] The third aspect of the present invention provides the use of the above-mentioned nano pesticide formulation in the preparation of a product for preventing and treating soybean sclerotinia disease, wherein the soybean sclerotinia disease is caused by Sclerotinia sclerotiorum.

[0018] Beneficial effects of the present invention:

[0019] 1. The present invention effectively combines azadirachtin and fenpropicyclopentane through nano-crystallization, significantly improving the biological activity and utilization rate of azadirachtin and fenpropicyclopentane, reducing the amount of pesticides used, and improving the control effect on soybean sclerotinia disease.

[0020] 2. The present invention compounds azadirachtin and fenpropicyclopyralid. Both azadirachtin and fenpropicyclopyralid are natural pesticides of plant origin. Azadirachtin mainly inhibits fungi by interfering with fungal cell membranes, enzyme activity, and reproduction processes, while fenpropicyclopyralid works by inhibiting the electron transport chain, destroying cell membranes, and inhibiting nucleic acid synthesis. After compounding, the two have different action pathways and can act together on multiple metabolic links of fungi, significantly improving the fungicidal effect on fungi.

[0021] 3. The present invention uses nano-sizing and the high specific surface area of ​​nanoparticles can make the active ingredients more evenly distributed in the target area, reduce drug loss and residue, use polylactic acid-glycolic acid copolymer (PLGA) as a carrier, have good biodegradability, will not cause persistent chemical pollution in the soil, and meet the requirements of green environmental protection of modern agriculture. At the same time, the nanopesticide coated with polylactic acid-glycolic acid copolymer (PLGA) can slowly release the active ingredients, thereby increasing the duration of the preparation, reducing the need for frequent application of pesticides, and reducing farmers' labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Photograph of the nanopesticide preparation prepared in Example 2 at an electron microscope resolution of 400 nanometers;

[0023] Figure 2 : Photograph of the nanopesticide preparation prepared in Example 2 at an electron microscope resolution of 4 μm. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0025] As described in the background technology, botanical pesticides are easier to degrade than chemical pesticides, safe for non-target organisms, less likely to develop resistance in pests, have a special mode of action, have multiple development pathways, and are of many types. However, botanical pesticides are easily photodegraded, non-biologically oxidized, or lost through volatilization. Compared with chemical pesticides, their poor stability and easy decomposition have restricted their application in production.

[0026] Based on this, the present invention combines the advantages of botanical pesticides and nanotechnology, and provides a nanopesticide preparation, which not only significantly improves the biological activity and utilization rate of azadirachtin and phenylpropenil, but also effectively solves the problems of poor stability and easy degradation of botanical pesticides. Through nanotechnology, the efficacy of the preparation is prolonged, the stability is enhanced, and the drug residue and environmental pollution are reduced, which meets the needs of green, environmentally friendly and sustainable development of modern agriculture. The application of the present invention in preventing and controlling soybean sclerotinia disease has also proved its significant effect and potential, and has broad application prospects.

[0027] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0028] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0029] The present invention uses Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ) strain number is CGMCC 3.7083, which was purchased from the General Microbiology Center of China Culture Collection Administration.

[0030] The polylactic acid-glycolic acid copolymer (PLGA) used in the present invention was purchased from Shandong Xinfu Industrial Co., Ltd., and the product model is B6029-1.

[0031] The azadirachtin used in the present invention was purchased from Hubei Wei's Chemical Reagent Co., Ltd., CAS No.: 58812-37-6, and the active ingredient content: 95%.

[0032] The fenpropicyclopentyl used in the present invention was purchased from Shenyang Tongxiang Biological Pesticide Co., Ltd., CAS registration number: 20784-50-3, and the mass fraction of fenpropicyclopentyl was 1.5%.

[0033] Example 1: Preparation of nanopesticide preparation:

[0034] (1) Weigh 1 mg of azadirachtin and 1 mg of fenpropimorph, add them to 20 mL of ethanol, then add 5 mg of polylactic acid-co-glycolic acid (PLGA), and stir at room temperature until the solution is uniform to obtain an organic phase;

[0035] (2) Dissolve 0.5 g of Tween 20 in 100 mL of deionized water to obtain an aqueous phase;

[0036] (3) In an ice-water bath, the organic phase was added dropwise to the aqueous phase at a rate of 1.5 mL / min and stirred at 1200 rpm for 40 minutes to form an emulsion. The emulsion was then transferred to a rotary evaporator and ethanol was slowly evaporated at 30°C by rotary evaporation until the organic solvent was completely evaporated. As the ethanol evaporated, PLGA gradually encapsulated azadirachtin and phenylbutaclostrobin to form nanopesticide particles.

[0037] (4) The emulsion was ultracentrifuged using an ultracentrifuge (13,000 rpm, 20 min, 4°C) to separate the nanoparticles. The nanoparticles were washed twice with 100 mL of phosphate buffered saline (PBS, pH 7.4) to remove the unencapsulated active ingredients and residual surfactants. The separated nanoparticles were freeze-dried to obtain a nanopesticide preparation.

[0038] Example 2: Preparation of nanopesticide preparation:

[0039] (1) Weigh 5 mg of azadirachtin and 1 mg of fenpropimorph, add them to 20 mL of ethanol, then add 15 mg of polylactic acid-co-glycolic acid (PLGA), and stir at room temperature until the solution is uniform to obtain an organic phase;

[0040] (2) Dissolve 0.5 g of Tween 20 in 100 mL of deionized water to obtain an aqueous phase;

[0041] (3) In an ice-water bath, the organic phase was added dropwise to the aqueous phase at a rate of 1.5 mL / min and stirred at 1200 rpm for 40 minutes to form an emulsion. The emulsion was then transferred to a rotary evaporator and ethanol was slowly evaporated at 30°C by rotary evaporation until the organic solvent was completely evaporated. As the ethanol evaporated, PLGA gradually encapsulated azadirachtin and phenylbutaclostrobin to form nanopesticide particles.

[0042] (4) The emulsion was ultracentrifuged using an ultracentrifuge (13,000 rpm, 20 min, 4°C) to separate the nanoparticles. The nanoparticles were washed twice with 100 mL of phosphate buffered saline (PBS, pH 7.4) to remove the unencapsulated active ingredients and residual surfactants. The separated nanoparticles were freeze-dried to obtain a nanopesticide preparation.

[0043] Example 3: Preparation of nanopesticide preparation:

[0044] (1) Weigh 10 mg of azadirachtin and 1 mg of fenpropimorph, add them to 20 mL of ethanol, then add 27.5 mg of polylactic acid-co-glycolic acid (PLGA), and stir at room temperature until the solution is uniform to obtain an organic phase;

[0045] (2) Dissolve 0.5 g of Tween 20 in 100 mL of deionized water to obtain an aqueous phase;

[0046] (3) In an ice-water bath, the organic phase was added dropwise to the aqueous phase at a rate of 1.5 mL / min and stirred at 1200 rpm for 40 minutes to form an emulsion. The emulsion was then transferred to a rotary evaporator and ethanol was slowly evaporated at 30°C by rotary evaporation until the organic solvent was completely evaporated. As the ethanol evaporated, PLGA gradually encapsulated azadirachtin and phenylbutaclostrobin to form nanopesticide particles.

[0047] (4) The emulsion was ultracentrifuged using an ultracentrifuge (13,000 rpm, 20 min, 4°C) to separate the nanoparticles. The nanoparticles were washed twice with 100 mL of phosphate buffered saline (PBS, pH 7.4) to remove the unencapsulated active ingredients and residual surfactants. The separated nanoparticles were freeze-dried to obtain a nanopesticide preparation.

[0048] Comparative Example 1:

[0049] (1) Weigh 6 mg of azadirachtin, add it to 20 mL of ethanol, then add 1 mg of polylactic acid-co-glycolic acid (PLGA), and stir at room temperature until the solution is uniform to obtain an organic phase;

[0050] (2) Dissolve 0.5 g of Tween 20 in 100 mL of deionized water to obtain an aqueous phase;

[0051] (3) In an ice-water bath, the organic phase was added dropwise to the aqueous phase at a rate of 1.5 mL / min and stirred at 1200 rpm for 40 minutes to form an emulsion. The emulsion was then transferred to a rotary evaporator and ethanol was slowly evaporated at 30°C by rotary evaporation until the organic solvent was completely evaporated. As the ethanol evaporated, PLGA gradually encapsulated azadirachtin and phenylbutaclostrobin to form nanopesticide particles.

[0052] (4) The emulsion was ultracentrifuged using an ultracentrifuge (13,000 rpm, 20 min, 4°C) to separate the nanoparticles. The nanoparticles were washed twice with 100 mL of phosphate buffered saline (PBS, pH 7.4) to remove the unencapsulated active ingredients and residual surfactants. The separated nanoparticles were freeze-dried to obtain a nanopesticide preparation.

[0053] Comparative Example 2:

[0054] (1) Weigh 6 mg of fenpropimorph, add it to 20 mL of ethanol, then add 1 mg of polylactic acid-co-glycolic acid (PLGA), and stir at room temperature until the solution is uniform to obtain an organic phase;

[0055] (2) Dissolve 0.5 g of Tween 20 in 100 mL of deionized water to obtain an aqueous phase;

[0056] (3) In an ice-water bath, the organic phase was added dropwise to the aqueous phase at a rate of 1.5 mL / min and stirred at 1200 rpm for 40 minutes to form an emulsion. The emulsion was then transferred to a rotary evaporator and ethanol was slowly evaporated at 30°C by rotary evaporation until the organic solvent was completely evaporated. As the ethanol evaporated, PLGA gradually encapsulated azadirachtin and phenylbutaclostrobin to form nanopesticide particles.

[0057] (4) The emulsion was ultracentrifuged using an ultracentrifuge (13,000 rpm, 20 min, 4°C) to separate the nanoparticles. The nanoparticles were washed twice with 100 mL of phosphate buffered saline (PBS, pH 7.4) to remove the unencapsulated active ingredients and residual surfactants. The separated nanoparticles were freeze-dried to obtain a nanopesticide preparation.

[0058] Comparative Example 3:

[0059] (1) Weigh 1 mg of azadirachtin and 5 mg of fenpropimorph, add them to 20 mL of ethanol, then add 15 mg of polylactic acid-co-glycolic acid (PLGA), and stir at room temperature until the solution is uniform to obtain an organic phase;

[0060] (2) Dissolve 0.5 g of Tween 20 in 100 mL of deionized water to obtain an aqueous phase;

[0061] (3) In an ice-water bath, the organic phase was added dropwise to the aqueous phase at a rate of 1.5 mL / min and stirred at 1200 rpm for 40 minutes to form an emulsion. The emulsion was then transferred to a rotary evaporator and ethanol was slowly evaporated at 30°C by rotary evaporation until the organic solvent was completely evaporated. As the ethanol evaporated, PLGA gradually encapsulated azadirachtin and phenylbutaclostrobin to form nanopesticide particles.

[0062] (4) The emulsion was ultracentrifuged using an ultracentrifuge (13,000 rpm, 20 min, 4°C) to separate the nanoparticles. The nanoparticles were washed twice with 100 mL of phosphate buffered saline (PBS, pH 7.4) to remove the unencapsulated active ingredients and residual surfactants. The separated nanoparticles were freeze-dried to obtain a nanopesticide preparation.

[0063] Comparative Example 4:

[0064] (1) Weigh 15 mg of azadirachtin and 1 mg of fenpropimorph, add them to 20 mL of ethanol, then add 40 mg of polylactic acid-co-glycolic acid (PLGA), and stir at room temperature until the solution is uniform to obtain an organic phase;

[0065] (2) Dissolve 0.5 g of Tween 20 in 100 mL of deionized water to obtain an aqueous phase;

[0066] (3) In an ice-water bath, the organic phase was added dropwise to the aqueous phase at a rate of 1.5 mL / min and stirred at 1200 rpm for 40 minutes to form an emulsion. The emulsion was then transferred to a rotary evaporator and ethanol was slowly evaporated at 30°C by rotary evaporation until the organic solvent was completely evaporated. As the ethanol evaporated, PLGA gradually encapsulated azadirachtin and phenylbutaclostrobin to form nanopesticide particles.

[0067] (4) The emulsion was ultracentrifuged using an ultracentrifuge (13,000 rpm, 20 min, 4°C) to separate the nanoparticles. The nanoparticles were washed twice with 100 mL of phosphate buffered saline (PBS, pH 7.4) to remove the unencapsulated active ingredients and residual surfactants. The separated nanoparticles were freeze-dried to obtain a nanopesticide preparation.

[0068] Comparative Example 5:

[0069] Azadirachtin and phenylpropenamide were mixed in a mass ratio of 5:1 as a pesticide preparation.

[0070] Test example: Indoor toxicity test of a mixture of azadirachtin and fenpropicyclopentane on Sclerotinia sclerotiorum:

[0071] 1. Test strain: Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ).

[0072] 2. Test methods:

[0073] 2.1 Refer to the "Guidelines for Indoor Bioassay of Pesticides NY / T 1156.2-2006" and use the growth rate method of drug-containing culture medium for determination.

[0074] The pesticide formulations prepared in Examples 1-3 and Comparative Examples 1-5 were formulated into mother liquors with an equal-ratio concentration gradient. After adding the test drug to the PDA medium at about 45°C and mixing well, it was poured into a petri dish with a diameter of 9 cm to make a drug-containing plate, and an equal amount of dimethyl sulfoxide was added as a control. A punch with an inner diameter of 5 mm was used to punch out a mycelial cake of Sclerotinia sclerotiorum ( Sclerotinia sclerotia cultured for 3 days and placed in the center of the plate, and then cultured in the dark in an incubator at 25±1°C. Each treatment was repeated 3 times. When the colony in the control group grew close to the edge of the petri dish, the colony diameter was measured by the cross method, and the inhibition rate was calculated.

[0075] Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0076] Calculate the inhibition rate of the mycelial growth of Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), the pathogen of soybean sclerotinia, by different concentrations of the药剂, conduct data analysis and calculate the regression equation and EC 50 .

[0077] 2.2 Then calculate the co-toxicity coefficient (CTC) of the mixture according to the Sun Yunpei method. The co-toxicity coefficient is used to analyze the effect of the mixture. The calculation formula of the co-toxicity coefficient (CTC) is as follows:

[0078] Toxicity index (TI) = (EC50 of the standard drug / EC50 of the test drug) × 100;

[0079] Actual toxicity index of the mixture (ATI) = (EC50 of the standard drug / EC50 of the mixed drug) × 100;

[0080] Theoretical toxicity index of the mixture (TTI) = toxicity index of drug A × percentage content of drug A in the mixture + toxicity index of drug B × percentage content of drug B in the mixture;

[0081] Co-toxicity coefficient (CTC) = (ATI / TTI) × 100;

[0082] According to the classification standard of the co-toxicity coefficient (CTC), CTC ≤ 80 indicates antagonism, 80 < CTC < 120 indicates additive effect, and CTC ≥ 120 indicates synergistic effect.

[0083] Table 1 Indoor toxicity determination results of toosendanin and phenylpropenone on Sclerotinia sclerotiorum

[0084]

[0085] From the results in Table 1, it can be seen that the co-toxicity coefficient (CTC) of azadirachtin and fenpropicyclopyralid is greater than 120 when the ratio of the two is within the range of 1-10:1, and the synergistic effect is significant. It shows high activity against the pathogen of soybean sclerotinia, Sclerotinia sclerotiorum, among which 5:1 is the best ratio. In addition, in Comparative Example 5 and Example 2, the ratio of azadirachtin and fenpropicyclopyralid is 5:1, but the synergistic effect of Example 2 is stronger than that of Comparative Example 5, and EC 50 It is also lower, indicating that the synergistic effect between azadirachtin and phenoxyethanol is enhanced through nano-sizing, which significantly improves the biological activity and utilization rate of azadirachtin and phenoxyethanol, reduces the amount of pesticides used, and improves the control effect on soybean sclerotinia disease.

[0086] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A nanopesticide preparation, characterized in that: Prepared by the following method: (1) Adding azadirachtin and phenylpropenil to ethanol, and then adding polylactic acid-glycolic acid copolymer, stirring evenly to obtain an organic phase; (2) Dissolve Tween 20 in water to obtain an aqueous phase; (3) Add the organic phase dropwise into the aqueous phase under ice-water bath conditions, stir at high speed for 30-45 min to obtain an emulsion, remove ethanol by rotary evaporation, wash, and freeze-dry to obtain a nanopesticide preparation; The ratio of the added amounts of azadirachtin, phenylpropenamide and ethanol is (1-10) mg: 1 mg: (15-25) mL.

2. The nanopesticide preparation according to claim 1, characterized in that: In step (1), the ratio of the added amount of polylactic acid-glycolic acid copolymer and ethanol is (5-27.5) mg: (15-25) mL.

3. The nanopesticide preparation according to claim 1, characterized in that: In step (2), the mass concentration of Tween 20 in the aqueous phase is 0.2%-1%.

4. The nanopesticide preparation according to claim 1, characterized in that: In step (3), the volume ratio of the organic phase to the aqueous phase is 1:(3-10).

5. The nanopesticide preparation according to claim 1, characterized in that: In step (3), the dropping speed is 1-2 mL / min and the stirring speed is 1000-1500 rpm.

6. Use of the nanopesticide preparation according to any one of claims 1 to 5 in the preparation of a sclerotinia sclerotiorum inhibitor.

7. Use of the nanopesticide preparation according to any one of claims 1 to 5 in the preparation of a product for preventing and treating soybean sclerotinia rot, characterized in that: The soybean sclerotinia disease is caused by Sclerotinia sclerotiorum.

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