Pesticide composition containing chlorfenapyr and polypeptide and application thereof

Abstract

The invention belongs to the technical field of pesticides, and provides a pesticide composition containing chlorfenapyr and polypeptide, and the pesticide composition contains chlorfenapyr and enzymolysis enteromorpha protein. The hydrolyzed enteromorpha protein is added into the composition, so that the oxidative damage of chlorfenapyr to leaves is effectively reduced, and the adverse effect of chlorfenapyr on plant health and production performance is overcome.

Description

Technical Field

[0001] This application pertains to the field of pesticides, and specifically, this application provides pesticide compositions comprising chlorfenapyr and polypeptides and their applications. Background Technology

[0002] Chlorfenapyr is a novel pyrrole insecticide belonging to the pyrethroid derivative class, possessing unique contact and stomach poison mechanisms of action. Its target is the insect mitochondrial respiratory chain complex III (cytochrome bc1 complex), inhibiting cellular energy synthesis by blocking the electron transport chain, leading to pest death. Compared to traditional chemical pesticides, chlorfenapyr is characterized by high efficiency, low residue, and broad-spectrum insecticidal activity. It exhibits significant control efficacy against Lepidoptera, Coleoptera, and Hemiptera pests (such as diamondback moth, beet armyworm, aphids, and spider mites), and has low toxicity to mammals. It is widely used in integrated pest management for crops such as rice, cotton, vegetables, and fruit trees.

[0003] However, chlorfenapyr still has limitations in practical applications and carries the risk of phytotoxicity: high doses or sensitive crops (such as many rice varieties) are prone to phytotoxicity symptoms such as leaf yellowing, curling, and even growth inhibition, affecting crop yield and quality. At present, measures to alleviate chlorfenapyr phytotoxicity mainly include rotating the use of the pesticide, adding adjuvants or safeners, but these methods have problems such as high cost, short effective period, or poor environmental compatibility.

[0004] Ulva prolifera is a common seaweed species along the eastern coast of my country. Its outbreaks not only affect the coastal ecology and tourism industry, but the disposal of the large quantities of harvested Ulva prolifera is also a challenge. Its uses and value in fields such as feed and fertilizer are limited, while the disposal costs are high. Developing efficient and low-toxicity plant-derived or biological detoxifying agents (such as protein / peptide preparations) from Ulva prolifera has become a promising research direction in the field of green plant protection. Summary of the Invention

[0005] This application attempts to utilize inexpensive raw materials, such as seaweed protein or its enzymatic hydrolysate, in combination with fipronil to reduce its damage to plants.

[0006] On the one hand, this application provides a pesticide composition comprising chlorfenapyr and a polypeptide, wherein the pesticide composition comprises chlorfenapyr and enzymatically hydrolyzed seaweed protein.

[0007] Furthermore, the amount of enzymatically hydrolyzed Ulva protein in the pesticide composition is 4-6% of the mass of chlorfenapyr, preferably 5%.

[0008] Furthermore, the pesticide composition is a suspension concentrate.

[0009] Furthermore, the pesticide composition is a solid formulation.

[0010] Furthermore, the method for preparing the enzymatically hydrolyzed seaweed protein includes extracting seaweed protein by alkaline extraction and acid precipitation, and hydrolyzing seaweed protein using bromelain or alcalase.

[0011] Furthermore, the method for preparing the enzymatically hydrolyzed seaweed protein is as follows:

[0012] (A) After washing the seaweed, cut it into small pieces and place the seaweed pieces in distilled water with a wet weight of 4-6 times the seaweed pieces.

[0013] (B) Freeze at -20℃ for 10-12 hours, then thaw at room temperature for 4-5 hours. Repeat twice.

[0014] (C) Centrifuge the mixture after the second thawing in step (B) to obtain the supernatant, adjust the pH of the supernatant to 8.5 and then stir to extract for 1-2 hours;

[0015] (D) Centrifuge the mixture obtained in step (C) to collect the supernatant, adjust the pH of the supernatant to 4.0, let it stand at room temperature for 1-2 hours, centrifuge to collect the precipitate and freeze dry it to obtain the seaweed protein.

[0016] (E) Prepare a 4-8% w / v aqueous solution of Ulva prolifera protein containing 900-1100 U / mL of bromelain or 900-1100 U / mL of Alcalase; adjust the pH of the solution to 6.0-8.0 and enzymatically hydrolyze at 50-60℃ for 3-8 hours; inactivate the enzyme in a boiling water bath; concentrate and freeze dry to obtain enzymatically hydrolyzed Ulva prolifera protein.

[0017] Further, in step (E), the seaweed protein is prepared as a 4-6% w / v aqueous solution containing 900-1000 U / mL of bromelain; the pH of the solution is adjusted to 6.0, and enzymatic hydrolysis is carried out at 50-60℃ for 6-7 hours; the enzyme is inactivated by boiling water bath; and the solution is concentrated and freeze-dried to obtain enzymatically hydrolyzed seaweed protein.

[0018] On the other hand, this application provides the application of the above-mentioned pesticide composition in the control of rice pests.

[0019] Furthermore, the pest in question is an aphid.

[0020] Furthermore, the above-mentioned pesticide composition is sprayed in the application.

[0021] The formulation of pesticide compositions can be designed by those skilled in the art in accordance with existing technology. For example, chlorfenapyr and enzymatically hydrolyzed seaweed protein can be prepared in the same system in solid or mixed form, provided separately in the same package, or provided separately in separate packages and then applied together.

[0022] Those skilled in the art may use processing methods known in the art, such as pulverization and high-pressure homogenization, when preparing or using the composition.

[0023] The crops and pests applicable to the pesticide composition of this application are not limited to those shown in the examples. Chlorfenapyr can be used for any known pests and their corresponding effects. Pests include, but are not limited to, diamondback moth, beet armyworm, aphid, spider mite, etc., and crops include, but are not limited to, rice, corn, cotton, vegetables, fruit trees, etc.

[0024] This application uses inexpensive, even garbage-like, seaweed as raw material. After treatment with commonly used food-grade protease, only a small amount is needed to combine it with chlorfenapyr to effectively reduce the harm of chlorfenapyr. This provides an effective and practical approach for the wider use and better efficacy of this drug. Attached Figure Description

[0025] Figure 1 The SOD levels in rice leaves after treatment with different enzymatic hydrolysis times and formulations. Detailed Implementation

[0026] Main materials:

[0027] The 10% chlorfenapyr suspension was produced by the applicant (the active ingredient was pulverized at low temperature to an average particle size of <100μm and then prepared with deionized water).

[0028] The seaweed (Enteromorpha prolifera) was collected from the coastal area of ​​Rizhao, Shandong.

[0029] Bromelain, papain, and fig protease were purchased from Xiasheng Biotechnology. The protease is a Novozymes product.

[0030] The SOD, POD, and MDA detection kits use products from Shanghai Yanqi Biotechnology.

[0031] All other reagents were standard domestically produced.

[0032] Example 1: Preparation of Ulva prolifera protein and its enzymatic hydrolysate

[0033] Preparation of seaweed protein:

[0034] Using the alkaline extraction and acid precipitation method: After washing and chopping the seaweed, place the seaweed fragments in distilled water at 5 times their wet weight; freeze at -20℃ for 12 hours and thaw at room temperature for 4 hours, repeating twice; centrifuge the mixture after the second thawing to collect the supernatant, adjust the pH of the supernatant to 8.5 and stir to extract for 1 hour; centrifuge to collect the supernatant; adjust the pH of the supernatant to 4.0, let it stand at room temperature for 1 hour, centrifuge to collect the precipitate and freeze-dry.

[0035] Enzymatic hydrolysis of seaweed protein:

[0036] The *Ulva prolifera* protein was prepared as a 5% w / v aqueous solution containing 1000 U / mL of bromelain, papain, and figase. Adjust the pH of the solution to 6.0 (bromelain, papain, fig protease) or 8.0. Enzymatic hydrolysis was performed at 50°C for 4 hours; the enzyme was inactivated by boiling water bath; the hydrolysate was concentrated and freeze-dried to obtain the hydrolysate.

[0037] Example 2: Efficacy and Toxicity Tests

[0038] Experimental field:

[0039] The experimental rice paddy was located in Jiaozuo, Henan Province. The rice variety was Yu Nong Jing 6. The experimental plot area was 4m×5m. There were isolation strips with a width of more than 1m between the plots. Each group had 3 parallel replicates.

[0040] Experimental reagents:

[0041] Preliminary experiments in potted rice plants in the laboratory determined the following pesticide formulation: Add 5% (by weight of chlorfenapyr) of *Ulva prolifera* protein or its enzymatic hydrolysate to 10% chlorfenapyr suspension (control groups were established with no pesticide application and those with only peptide application). After a 500-fold dilution, apply at a slightly higher dose than normal, 30 mL chlorfenapyr suspension per acre. Use a backpack electric sprayer (2 MPa pressure) to apply once during the seedling stage and once during the expected peak of aphid nymphal infestation, approximately 7 days apart. (During spraying, ensure even coverage of both sides of the plant leaves; the atomization effect should be a fine mist. Apply on sunny mornings between 9-11 am, avoiding high-temperature periods (>35℃) or rainy days). Other water, fertilizer, and pesticide management were the same for all groups.

[0042] Pest control efficacy testing:

[0043] Insect population density was investigated on days 7 and 14 after pesticide application: 5 locations were randomly selected in each plot, and 6 crops were investigated at each location. The number of live insects was recorded, and the mortality rate was calculated relative to the control group that did not receive pesticide application.

[0044] Crop physiological index testing:

[0045] Rice leaves were collected from plants undergoing efficacy assessment on days 7 and 14 after pesticide application to determine antioxidant enzyme activity (SOD, POD) and malondialdehyde (MDA) content (spectrophotometric method). After maturity, the grains were manually threshed, and the number of effective panicles, seed setting rate, thousand-grain weight, and yield of each plot were recorded.

[0046] Table 1. Basic efficacy of various agents

[0047]

[0048]

[0049] Consistent with the results of the pot experiment, Table 1 shows that the use of Ulva protease alone had little effect on aphid control. When Ulva protease or its hydrolysate was used in combination with chlorfenapyr, the effects of the bromelain, fig protease and Alcalase hydrolysate groups seemed to be improved compared to the chlorfenapyr group, but not to a significant extent.

[0050] Table 2 Oxidative damage to leaves caused by various agents

[0051]

[0052]

[0053] The results in Table 2 show that, consistent with previous studies, chlorfenapyr does indeed cause some oxidative damage to leaves. Bromelain and Alcalase hydrolysates of Ulva protease can effectively alleviate this damage, while other proteins and hydrolysates have no significant effect. Savinasedan hydrolysate, on the contrary, exacerbates oxidative damage.

[0054] Table 3. Effects of various agents on yield and quality.

[0055]

[0056] The results in Table 3 show that the various pesticides had little effect on the number of effective panicles and the thousand-grain weight, but the bromelain and Alcalase hydrolysate of Ulva protease were significantly better in terms of seed setting rate and yield (the group that did not apply chlorfenapyr and other pesticides was not included in the statistics in Table 3 due to more severe pest infestation in the later stage).

[0057] In addition, the applicant further used the cage contact method to determine the toxicity of various agents to bees. Preliminary results showed that the bromelain, figase and alcalase hydrolysate groups of Ulva protease effectively reduced the mortality rate of contact bees (no dead bees were even observed with bromelain), which was a significant improvement compared to the approximately 7% mortality rate of contact bees with chlorfenapyr.

[0058] Example 3: Study on the degree of enzymatic hydrolysis

[0059] The effects of different degrees of enzymatic hydrolysis by bromelain and alcalase on SOD and MDA levels in potted rice plants were further investigated. The enzyme dosage and temperature in the hydrolysis system were fixed (1000 U / mL, 50℃), and the hydrolysis time was set to 2-8 hours. SOD levels in leaves were measured 7 days after application to the plants. The results are as follows: Figure 1As shown, the effect of Alcalase hydrolysate remained basically unchanged after 3 hours, and decreased slightly after 6 hours; while the effect of bromelain hydrolysate was improved after 6 hours compared with 4-5 hours. Therefore, the hydrolysis time of bromelain was adjusted to more than 6 hours.

Claims

1. A pesticide composition comprising chlorfenapyr and a polypeptide, characterized in that, The pesticide composition contains chlorfenapyr and enzymatically hydrolyzed seaweed protein.

2. The pesticide composition according to claim 1, wherein the amount of enzymatically hydrolyzed Ulva prolifera protein in the pesticide composition is 4-6% of the mass of chlorfenapyr.

3. The pesticide composition according to claim 1 or 2, wherein the pesticide composition is a suspension concentrate.

4. The pesticide composition according to claim 1 or 2, wherein the pesticide composition is a solid formulation.

5. The pesticide composition according to any one of claims 1-4, wherein the method for preparing the enzymatically hydrolyzed Ulva prolifera protein comprises extracting Ulva prolifera protein by alkaline extraction and acid precipitation, and hydrolyzing Ulva prolifera protein using bromelain or Alcalase.

6. The pesticide composition according to claim 5, wherein the method for preparing the enzymatically hydrolyzed Ulva prolifera protein is as follows: (A) After washing the seaweed, cut it into small pieces and place the seaweed pieces in distilled water with a wet weight of 4-6 times the seaweed pieces. (B) Freeze at -20℃ for 10-12 hours, then thaw at room temperature for 4-5 hours. Repeat twice. (C) Centrifuge the mixture after the second thawing in step (B) to obtain the supernatant, adjust the pH of the supernatant to 8.5 and then stir to extract for 1-2 hours; (D) Centrifuge the mixture obtained in step (C) to collect the supernatant, adjust the pH of the supernatant to 4.0, let it stand at room temperature for 1-2 hours, centrifuge to collect the precipitate and freeze dry it to obtain the seaweed protein. (E) Prepare a 4-8% w / v aqueous solution of the seaweed protein obtained in step (D), which contains 900-1100 U / mL of bromelain or 900-1100 U / mL of Alcalase; adjust the pH of the solution to 6.0-8.0, and enzymatically hydrolyze it at 50-60℃ for 3-8 hours; inactivate the enzyme in a boiling water bath; concentrate and freeze dry to obtain the enzymatically hydrolyzed seaweed protein.

7. The pesticide composition according to claim 6, wherein in step (E), the seaweed protein is prepared as a 4-6% w / v aqueous solution containing 900-1000 U / mL of bromelain; the pH of the solution is adjusted to 6.0, and enzymatically hydrolyzed at 50-60°C for 6-7 hours; the enzyme is inactivated by boiling water bath; and the solution is concentrated and freeze-dried to obtain the enzymatically hydrolyzed seaweed protein.

8. The application of the pesticide composition according to any one of claims 1-7 in the control of rice pests.

9. The application according to claim 8, wherein the pest is an aphid.

10. The application according to claim 8 or 9, wherein the pesticide composition according to any one of claims 1-7 is sprayed in the application.

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