High permeability and antibacterial and insect-resistant nano-pesticide and preparation method thereof

Abstract

The application discloses high-permeability nano pesticide with antibacterial and anti-insect effects and a preparation method thereof, and relates to the technical field of pesticides. Step 1: chitosan, carboxymethyl chitosan, pectin and a crosslinking agent are respectively dissolved in acetic acid aqueous solution, deionized water, deionized water and deionized water to obtain a chitosan solution, a carboxymethyl chitosan aqueous solution, a pectin aqueous solution and a crosslinking agent aqueous solution; Step 2: at 35-45 DEG C, emulsifiers and an insecticide ethanol solution are sequentially added to the chitosan solution; at 25-35 DEG C, the crosslinking agent aqueous solution, the carboxymethyl chitosan solution and the pectin solution are sequentially added to the chitosan solution and stirred, and the pH value is adjusted to 6-7 to obtain the nano pesticide; the pectin is apple pectin. The nano pesticide prepared in the application has high permeability, bacteriostasis and anti-scouring properties when being sprayed on pine trees and other thick-bark hardwoods, and can efficiently prevent and control boring pests.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, specifically to highly penetrating nanopesticides with antibacterial and insecticidal effects and their preparation methods. Background Technology

[0002] Wood-boring insects are a group of pests that cause extremely serious damage in agricultural and forestry production. Representative species include longhorn beetles, jewel beetles, wood-boring moths, and clearwing moths. The larvae of these insects feed primarily inside the bark and trunk of plants, exhibiting characteristics such as concealed living habits, deep feeding sites, and long-lasting damage. They easily damage the tree's vascular tissues, weaken its growth, and in severe cases, lead to plant death, causing significant economic losses to fruit trees, forest trees, and garden plants. Furthermore, plant branches and trunks damaged by insects are highly susceptible to infection by pathogens, leading to fungal or bacterial diseases such as branch rot, canker, and twig dieback, further exacerbating plant mortality.

[0003] Currently, the control of trunk-boring pests mainly relies on chemical control methods, including surface application, surface spraying, and injection through drilling. However, methods such as injection through drilling or surface application are labor-intensive, labor-intensive, and inefficient, making them difficult to promote and apply in large-scale orchards or forest areas, and they can easily cause mechanical damage to the trees. Surface spraying, on the other hand, is simple to operate, highly efficient, and suitable for large-scale application. Existing technologies have developed the use of nano-pesticides in conjunction with surface spraying; however, most current research on nano-pesticides for conventional foliar spraying has the following drawbacks when used for trunk spraying: First, the pesticide penetration is insufficient. Since trunk-boring pests often lurk inside the bark or xylem, conventional insecticides cannot effectively penetrate the bark after spraying, resulting in poor insecticidal and antibacterial effects and ineffective control. Second, the pesticide has poor environmental stability and poor adhesion and spreadability, making it easily washed away by rainwater.

[0004] In summary, solving the above problems and preparing nano-pesticides with high penetration and antibacterial and insecticidal effects is of great significance for improving the control of stem borers. Summary of the Invention

[0005] The purpose of this invention is to provide a nano-pesticide with high penetration and antibacterial and insecticidal effects, and a method for preparing the same, in order to solve the problems mentioned in the background art.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for preparing highly penetrating nanopesticides with antibacterial and insecticidal effects includes the following steps: Step 1: Dissolve chitosan, carboxymethyl chitosan, pectin and crosslinking agent in acetic acid aqueous solution, deionized water, deionized water and deionized water respectively to obtain chitosan solution, carboxymethyl chitosan aqueous solution, pectin aqueous solution and crosslinking agent aqueous solution; Step 2: At 35~45℃, emulsifier and insecticide ethanol solution are added to chitosan solution in sequence; at 25~35℃, cross-linking agent aqueous solution, carboxymethyl chitosan solution and pectin solution are added in sequence and stirred, and the pH is adjusted to 6~7 to obtain nano-pesticide; The insecticide is one or more of abamectin, emamectin benzoate, thiamethoxam, and imidacloprid.

[0007] Ideally, the chitosan solution contains 0.1-0.5% chitosan, and the acetic acid aqueous solution contains 0.5-1.5 wt%. In the aqueous solution of carboxymethyl chitosan, the concentration of carboxymethyl chitosan is 0.5-1%; The concentration of pectin in the pectin aqueous solution is 0.5-1%.

[0008] More preferably, the crosslinking agent aqueous solution has a mass concentration of 0.5-1.5%; the crosslinking agent is either sodium tripolyphosphate or glutaraldehyde.

[0009] More optimally, the emulsifier is composed of Span 60 and Tween 80 in a mass ratio of (1:1) to (1:5).

[0010] Ideally, the pectin is apple pectin.

[0011] In a further embodiment, the pectin is thermally modified apple pectin, which is prepared by dissolving apple pectin in deionized water, stirring at 50-60°C to dissolve, adjusting the pH to 4-5; treating with high-pressure steam at 120-130°C for 20-30 minutes, cooling, adding ethanol to precipitate, washing, and drying to obtain thermally modified apple pectin.

[0012] More optimally, the pectin is phenolic acid and heat-modified apple pectin, and its preparation method is as follows: heat-modified apple pectin is added to deionized water, p-hydroxybenzoic acid-DMF solution is added; lipase is added, and the mixture is reacted at 60~65℃ for 24 hours. After cooling, ethanol is added to precipitate the mixture, and the mixture is washed and dried to obtain phenolic acid and heat-modified apple pectin.

[0013] In a more optimized manner, the mass ratio of the phenolic acid and the thermally modified apple pectin raw materials, the thermally modified apple pectin, the p-hydroxybenzoic acid and the lipase is 10:(1~1.5):1.

[0014] The nanopesticide prepared by the aforementioned method for preparing a penetrating nanopesticide with antibacterial and insecticidal effects is used in the prevention and control of plant diseases and pests caused by stem borers.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) Compared with the injection method in the prior art, the nano-pesticide prepared in this application can exert its effect by spraying with drones, which saves time and effort.

[0016] (2) This application uses chitosan, carboxymethyl chitosan and pectin to form a composite polysaccharide nanocarrier, loads insecticides to form nano pesticides, improves the comprehensive performance of pesticides, and makes the nano pesticides highly permeable, antibacterial and erosion resistant when sprayed on thick-barked trees such as pine and camphor trees, and can effectively prevent and control trunk borers and twig blight.

[0017] Among them, chitosan and carboxymethyl chitosan provide stable positive charge, which can generate electrostatic attraction with the negatively charged bark surface, enhancing the adhesion and directional migration of pesticide on the bark. Compared with the introduction of chitosan alone, the introduction of carboxymethyl chitosan can improve film formation and increase the encapsulation and controlled release capabilities of insecticides. The pectin used is apple pectin, which can respond to the pectinase secreted by trunk borers, realizing the targeted degradation and precise release of nanocarriers at the insect infection sites, significantly improving the utilization efficiency of pesticides.

[0018] Meanwhile, the further proposed solution uses heat-modified apple pectin, which has a looser structure and lower molecular weight compared to conventional apple pectin, thus reducing penetration resistance; it is supplemented with a mild cross-linking agent to regulate swelling, prevent rapid loss of efficacy, prolong the duration of effect, and better meet the long-term control needs of forest trees.

[0019] Meanwhile, a further formulation utilizes phenolic acids and thermally modified apple pectin. Based on thermal modification, an appropriate amount of p-hydroxybenzoic acid is introduced, increasing the phenolic hydroxyl functional groups and significantly enhancing the hydrogen bonds and hydrophobic interactions between the pectin and the hydroxyl groups and suberin on the bark surface. This significantly improves the adhesion of the pesticide solution to the rough, hydrophobic bark surface and its resistance to rain erosion. Furthermore, p-hydroxybenzoic acid itself possesses excellent antibacterial activity and can synergistically inhibit pathogens such as *Pinus sylvestris* twig blight pathogen.

[0020] (3) The introduction of Tween 80 and Span 60 emulsifiers can further reduce the contact angle of the pesticide solution compared with a single emulsifier, so that the nano pesticides can spread, wet and penetrate quickly on the rough and hydrophobic pine bark surface; avoid droplet bouncing and loss, and further improve the adhesion and retention efficiency. Attached Figure Description

[0021] Figure 1 The images show the penetration effect of different nano-pesticides on pine tree segments after spraying. Figure 2 This is a scanning electron microscope image of the nano-pesticide in Example 1; Figure 3 This is a comparative diagram showing the inhibitory effects of nano-pesticides on the blight pathogen of Pinus sylvestris var. mongolica in Example 1. Figure 4 This is a diagram illustrating the effect of rainwater erosion resistance in Example 1. Figure 5 This is a diagram illustrating the effect of rainwater erosion resistance in Example 2. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] It should be noted in the following embodiments that there are no special restrictions on the purchasers of all raw materials involved in this invention. Exemplary examples include: chitosan with a degree of deacetylation of 90% and a viscosity of ≤500cps; and carboxymethyl chitosan with a degree of carboxylation of ≥80%.

[0024] Preparation of thermally modified apple pectin: Dissolve 2g of apple pectin in 200mL of deionized water, stir at 55℃ to dissolve, and adjust the pH to 4.2; place in a steam sterilizer, treat with high pressure steam at 125℃ for 30 minutes, cool, add ethanol for precipitation treatment, wash and dry the precipitated product multiple times to obtain thermally modified apple pectin.

[0025] Preparation method of phenolic acid and heat-modified apple pectin: 1g of heat-modified apple pectin was added to 200mL of deionized water, and 10g of 1.5wt% p-hydroxybenzoic acid-DMF solution was added; 100mg of lipase was added, and the mixture was reacted at 60℃ for 24 hours. After cooling, ethanol was added for precipitation treatment, and the precipitate was collected, washed and dried repeatedly to obtain phenolic acid and heat-modified apple pectin.

[0026] Example 1: A method for preparing a highly permeable nanopesticide with antibacterial and insecticidal effects, comprising the following steps: Step 1: Dissolve 0.3g of chitosan in 100mL of 1wt% acetic acid aqueous solution to obtain a 0.3% chitosan solution; dissolve 0.4g of carboxymethyl chitosan in 100mL of deionized water to obtain a 0.4% carboxymethyl chitosan aqueous solution; dissolve 0.75g of pectin in 75mL of deionized water to obtain a 1% pectin aqueous solution; add 1g of sodium tripolyphosphate to 100mL of deionized water to obtain a 1wt% sodium tripolyphosphate aqueous solution. Step 2: At 40℃, add 0.2g of Tween 80 and 0.1g of Span 60 as emulsifiers and 1g of 1wt% emamectin benzoate ethanol solution to the chitosan solution in sequence; at 30℃, add sodium tripolyphosphate aqueous solution, carboxymethyl chitosan solution and pectin solution in sequence and stir, adjust pH=6.8 to obtain nano-pesticide; In this embodiment, the pectin used is phenolic acid and thermally modified apple pectin.

[0027] Example 2: The implementation process is the same as in Example 1, except that in this example, heat-modified apple pectin is used.

[0028] Example 3: The implementation process is the same as in Example 1, except that apple pectin is used in this example.

[0029] Example 4: The implementation process is the same as in Example 3, except that pectin is not used in this example, and the following steps are included: Step 1: Dissolve 0.6g of chitosan in 150mL of 1wt% acetic acid aqueous solution to obtain a chitosan solution; dissolve 0.9g of carboxymethyl chitosan in 150mL of deionized water to obtain a carboxymethyl chitosan aqueous solution; add 1g of sodium tripolyphosphate to 100mL of deionized water to obtain a 1wt% sodium tripolyphosphate aqueous solution. Step 2: At 40°C, add 0.2g of Tween 80 and 0.1g of Span 60 as emulsifiers and 1g of 1wt% emamectin benzoate ethanol solution to the chitosan solution in sequence; at 30°C, add sodium tripolyphosphate aqueous solution and carboxymethyl chitosan solution in sequence and stir, adjust pH=6.8 to obtain nano-pesticide.

[0030] Example 5: The implementation process is the same as in Example 3, except that in this example, only chitosan is used, without the addition of carboxymethyl chitosan; it includes the following steps: Step 1: Dissolve 0.7g of chitosan in 200mL of 1wt% acetic acid aqueous solution to obtain a chitosan solution; dissolve 0.75g of pectin in 75mL of deionized water to obtain a 1% pectin aqueous solution; add 1g of sodium tripolyphosphate to 100mL of deionized water to obtain a 1wt% sodium tripolyphosphate aqueous solution. Step 2: At 40℃, add 0.2g of Tween 80 and 0.1g of Span 60 as emulsifiers and 1g of 1wt% emamectin benzoate ethanol solution to the chitosan solution in sequence; at 30℃, add sodium tripolyphosphate aqueous solution and pectin solution in sequence and stir, adjust pH=6.8 to obtain nano-pesticide.

[0031] Example 6: The implementation process is the same as in Example 1, except that the proportion of phenolic acid in the phenolic acid and heat-modified apple pectin is too high, and the ratio of heat-modified apple pectin to p-hydroxybenzoic acid is 10:3. The preparation process is as follows: Preparation method of phenolic acid and heat-modified apple pectin: 1g of heat-modified apple pectin is added to 200mL of deionized water, 10g of 3wt% p-hydroxybenzoic acid-DMF solution is added, 100mg of lipase is added, and the mixture is reacted at 60℃ for 24 hours. After cooling, ethanol is added for precipitation treatment. The precipitate is collected, washed and dried multiple times to obtain phenolic acid and heat-modified apple pectin.

[0032] Performance Characterization 1: The permeability of the nano-pesticides prepared in each embodiment was characterized. Simulating drone application, the FITC-containing nano-pesticides were sprayed onto pine trees. Seven days later, samples were taken from the top 3, 5, 7, 9, and 11 cm of the tree trunk and photographed using a small animal live imaging system. The results are as follows: Figure 1 As shown, Figure 1 The corresponding fluorescence intensities are shown in Table 1 below: Table 1

[0033] Results and Discussion: In Example 1, the use of phenolic acid and thermally modified apple pectin resulted in the highest fluorescence intensity at all points, indicating the best intraplant penetration, translocation, and retention effects. In Example 2, only thermally modified apple pectin was used without phenolic acid functional modification, leading to decreased drug permeability. In Example 3, ordinary apple pectin was used, showing acceptable short-range penetration but poor long-range drug penetration. In Example 4, the absence of pectin resulted in a lack of plant-compatible carriers, leading to poor long-range penetration. In Example 5, the use of chitosan alone without carboxymethyl chitosan reduced the system's dispersion stability and resulted in poor mid- to long-range penetration. In Example 6, excessively high phenolic acid content easily caused pesticide flocculation and aggregation, severely reducing the permeability of nano-pesticides.

[0034] Performance Characterization 2: The nanopesticide prepared in Example 1 was tested using scanning electron microscopy; the results are as follows: Figure 2 As shown in the figure, the nano-pesticides prepared in this application are regular spherical or near-spherical in shape, with complete particle morphology and no obvious agglomeration, indicating the formation of a stable nanostructure.

[0035] Performance Characterization 3: The antibacterial activity of the nano-pesticide prepared in Example 1 was tested. A piece of actively growing fungus with a diameter of approximately 5 mm was placed in the center of each petri dish. Blank PDA medium was used as a control group. The prepared medium was placed upside down in an incubator and incubated at 28°C for 5 days. The diameter of the fungus was then measured. Using *Pinus sylvestris* var. *mongolica* as the target fungus, the mycelial growth rate method was used to determine the inhibitory activity of matrine and the nano-pesticide at different treatment times (24h, 48h, 72h, 96h). No pesticide treatment was used as a negative control. Five concentration gradients (1.0, 2.0, 3.0, 4.0, 5.0 mg / mL) were set for both matrine and the nano-pesticide, with three replicates for each group. The diameter of the inhibition zone (mm) at each concentration was recorded. The following section uses a concentration of 2.0 mg / mL as an example for direct comparison. The results are as follows. Figure 3 As shown in Table 2.

[0036] Table 2

[0037] Results and Discussion: Figure 3 As shown in Table 1, in the control group without any treatment, the mycelial growth rate of *F. truncatum* was the fastest. Within 24 hours, the mycelium had spread outwards from the inoculation point, with an average colony diameter of 12.5 mm; after 48 hours, distinct colonies formed, with a diameter expanding to 28.3 mm; after 72 hours, the colony diameter reached 45.6 mm, almost occupying most of the plate area; and after 96 hours, the mycelium covered the entire petri dish (90 mm in diameter), showing vigorous growth without any inhibitory effect. Matrine had a certain inhibitory effect on *F. truncatum*, but the effect was limited. At 24 hours, mycelial germination was delayed, with an inhibition zone diameter of 8.2 mm; after 48 hours, colony expansion was slow, with an inhibition zone diameter of 15.6 mm; after 72 hours, the colony diameter was approximately 32.1 mm, with sparse mycelium still visible at the edge; and after 96 hours, the colony diameter reached 54.7 mm, smaller than the control group, but the inhibition rate decreased to 39%, indicating that the late-stage persistence of matrine was insufficient. These results are consistent with EC... 50 The trend was consistent; the EC50 of matrine decreased from 3.30 mg / mL at 24 h to 1.58 mg / mL at 96 h, but the inhibition rate at 96 h was still significantly lower than that of nano-pesticides (P<0.05). The nano-pesticide treatment group showed a significant and long-lasting antibacterial effect. At 24 h, mycelia only germinated in small numbers around the inoculation point, with an inhibition zone diameter of only 3.5 mm, indicating strong inhibition of diffusion. At 48 h, colony expansion was extremely slow, with an inhibition zone diameter of 6.2 mm, much smaller than that of the matrine group at the same concentration. At 72 h, the colony diameter was only 9.8 mm, with mycelial growth essentially halted and clear edges without escape. At 96 h, the colony diameter remained below 11.3 mm, and the antibacterial effect remained stable with no significant recovery of growth. The EC50 of nano-pesticides... 50The values ​​were significantly lower than those of matrine at all time points: 1.18 mg / mL at 24 h and as low as 0.41 mg / mL at 96 h. The 95% confidence intervals did not overlap, indicating that the toxicity of nano-pesticides to twig blight pathogens was significantly better than that of matrine.

[0038] Performance Characterization 4: The nanopesticides prepared in Examples 1 and 2 were subjected to erosion resistance experiments. 5 mL of sample was sprayed onto the surface of pine needles and subjected to simulated rainwater erosion. Images were then captured using CLSM (Confocal Laser Surgery) imaging. The results are shown below. Figure 4 and Figure 5 As shown; Results and Discussion: Figure 4 It is evident that the nanoparticles exhibit excellent adhesion, maintaining high retention even after 7 minutes of rinsing, indicating that the nanopesticides in this application have high deposition efficiency on pine needles and effectively reduce pesticide loss due to rainfall. Further combining... Figure 5 It can be seen that the nano-pesticide prepared by single thermally modified apple pectin in Example 2 showed slight dispersion and detachment of particles as the rinsing time increased, and the residue rate showed a slow downward trend. In contrast, the nano-pesticide based on phenolic acid and thermally modified apple pectin in Example 1 had significant advantages. After long-term rinsing, the microstructure of the membrane was more intact, the pesticide residue was significantly higher than that in Example 2, and the retention stability was stronger.

[0039] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a highly permeable nano-pesticide with antibacterial and insecticidal effects, characterized in that: Includes the following steps: Step 1: Dissolve chitosan, carboxymethyl chitosan, pectin and crosslinking agent in acetic acid aqueous solution, deionized water, deionized water and deionized water respectively to obtain chitosan solution, carboxymethyl chitosan aqueous solution, pectin aqueous solution and crosslinking agent aqueous solution; Step 2: At 35~45℃, emulsifier and insecticide ethanol solution are added to chitosan solution in sequence; at 25~35℃, cross-linking agent aqueous solution, carboxymethyl chitosan solution and pectin solution are added in sequence and stirred, and the pH is adjusted to 6~7 to obtain nano-pesticide; The pectin is apple pectin.

2. The method for preparing the nano-pesticide with penetrating, antibacterial, and insecticidal effects according to claim 1, characterized in that: The pectin is a heat-modified apple pectin, which is prepared by dissolving apple pectin in deionized water, stirring at 50-60°C, adjusting the pH to 4-5, treating with high-pressure steam at 120-130°C for 20-30 minutes, cooling, adding ethanol to precipitate, washing, and drying to obtain the heat-modified apple pectin.

3. The method for preparing the nano-pesticide with penetrating, antibacterial, and insecticidal effects according to claim 2, characterized in that: The pectin is phenolic acid and heat-modified apple pectin, and its preparation method is as follows: heat-modified apple pectin is added to deionized water, p-hydroxybenzoic acid-DMF solution is added; lipase is added, and the mixture is reacted at 60~65℃ for 24 hours. After cooling, ethanol is added to precipitate the mixture, and the mixture is washed and dried to obtain phenolic acid and heat-modified apple pectin.

4. The method for preparing the nano-pesticide with penetrating, antibacterial, and insecticidal effects according to claim 3, characterized in that: In the raw materials of phenolic acid and thermally modified apple pectin, the mass ratio of thermally modified apple pectin, p-hydroxybenzoic acid, and lipase is 10:(1~1.5):

1.

5. The method for preparing the nano-pesticide with penetrating, antibacterial, and insecticidal effects according to claim 1, characterized in that: The insecticide is one or more of abamectin, emamectin benzoate, thiamethoxam, and imidacloprid; the concentration of the ethanol solution of the insecticide is 0.8~1.2wt%.

6. The method for preparing the nano-pesticide with penetrating, antibacterial, and insecticidal effects according to claim 1, characterized in that: The chitosan solution contains 0.1-0.5% chitosan; the acetic acid aqueous solution contains 0.5-1.5 wt% acetic acid. The concentration of carboxymethyl chitosan in the aqueous solution is 0.5-1%. The concentration of pectin in the pectin aqueous solution is 0.5-1%.

7. The method for preparing the nano-pesticide with penetrating, antibacterial, and insecticidal effects according to claim 1, characterized in that: The crosslinking agent aqueous solution has a mass concentration of 0.5-1.5%; the crosslinking agent is either sodium tripolyphosphate or glutaraldehyde.

8. The method for preparing the nano-pesticide with penetrating, antibacterial, and insecticidal effects according to claim 1, characterized in that: The emulsifier is composed of Span 60 and Tween 80 in a mass ratio of (1:1) to (1:5).

9. The nanopesticide prepared by the method for preparing the nanopesticide with penetrating, antibacterial, and insecticidal effects according to any one of claims 1 to 7, characterized in that: The application of the nano-pesticide in the prevention and control of plant diseases and pests caused by stem borers.

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