A polydopamine-loaded spinosad nanopesticide formulation, its preparation method and application

Abstract

This invention belongs to the field of biopesticide technology, specifically relating to a polydopamine-loaded spinosad nanopesticide formulation, its preparation method, and its application. The preparation method includes the following steps: (1) adding polydopamine nanoparticles to a spinosad solution and stirring, then drying to obtain polydopamine-loaded spinosad nanoparticles; dispersing the spinosad-loaded polydopamine nanoparticles in PBS solution to obtain a polydopamine-loaded spinosad nanoparticle dispersion; (2) mixing the polydopamine-loaded spinosad nanoparticle dispersion obtained in step (1) with a Harpin 403 protein dispersion, and centrifuging to obtain the polydopamine-loaded spinosad nanopesticide formulation. This nanopesticide formulation exhibits durable and stable performance and excellent control effect against rice leaf roller.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically relating to a polydopamine-loaded spinosad nanopesticide formulation, its preparation method, and its application. Background Technology

[0002] Spinosad, also known as spinosad, is a novel natural insecticide with highly effective contact and feeding toxicity. Studies have found that spinosad is effective not only against Lepidoptera, Diptera, and Thysanoptera pests, but also against certain leaf-feeding pests in the Coleoptera and Orthoptera orders. Due to its combination of the safety of biopesticides and the rapid action of chemical pesticides, spinosad is widely used in the field of biopesticide technology. To date, spinosad has been applied to more than 250 crops in 73 countries. However, with the widespread use of spinosad, pest resistance has gradually increased, leading to a decline in control efficacy. Furthermore, spinosad has poor photostability, rapidly decomposing under sunlight, resulting in poor product persistence. To address the persistence issue, researchers have chemically modified spinosad to produce ethyl spinosad. Simultaneously, combining it with other pesticide components, such as abamectin, can also delay resistance development and improve control efficacy. However, this method is relatively expensive and often fails to achieve ideal results.

[0003] With the development of science and technology, nanomaterials are increasingly being applied in various fields. Research has found that using nanomaterial carriers can not only protect pesticide active ingredients from harsh environmental conditions and significantly improve their chemical stability, but also enhance their dispersibility and wettability, reducing the risk of organic solvents entering the environment. Polydopamine, as a novel pesticide carrier, exhibits high application potential due to its unique biodegradability and excellent adhesion. Multifunctional modification of dopamine has become a key strategy for addressing many challenges in pesticide use.

[0004] Harpin protein can activate the plant's innate immune system and enhance its resistance to pests, while spinosad directly targets pests to exert its insecticidal effect. Based on this background, this invention provides a novel Harpin protein-modified polydopamine nanopesticide, aiming to improve the control efficacy of traditional pesticides. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for preparing a polydopamine-loaded spinosad nanopesticide formulation, which has a simple preparation process.

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

[0007] A method for preparing a polydopamine-loaded spinosad nanopesticide formulation includes the following steps:

[0008] (1) Add polydopamine nanoparticles to spinosad solution and stir. After drying, polydopamine-loaded spinosad nanoparticles are obtained. Disperse polydopamine-loaded spinosad nanoparticles in PBS solution to obtain polydopamine-loaded spinosad nanoparticle dispersion.

[0009] (2) The polydopamine-loaded spinosad nanoparticle dispersion and Harpin403 protein dispersion obtained in step (1) are mixed and centrifuged to obtain the polydopamine-loaded spinosad nanopesticide formulation.

[0010] Further, the nucleotide sequence of the Harpin403 protein described in step (2) is shown in SEQ ID NO:1.

[0011] The sequence of SEQ ID NO:1 is as follows:

[0012] ATGTCCCTGAACACCTCCGGTCTGGGCGCCTCCACCATGCAAATCTCTATCGGTG

[0013] GCGCAGGCGGTAACAACGGCCTGCTGGGTACCTCTCGTCAGAACGCCGGTCTGGGTG

[0014] GCAACTCCGCCCTGGGCCTGGGCGGTGGTAATCAGAACGACACCGTAAACCAGCTG

[0015] GCCGGTCTGCTGACTGGCATGATGATGATGATGTCTATGATGGGCGGTGGTGGCCTGA

[0016] TGGGTGGTGGTCTGGGTGGTGGCCTGGGTAACGGCCTGGGCGGCTCTGGCGGTCTGG

[0017] GTGAAGGCCTGTCTAACGCCCTGAACGACATGCTGGGTGGTTCCCTGAACACTCTGG

[0018] GCTCTAAGGGCGGCAACAACACCACTTCTACTACCAACTCCCCGCTGGATCAAGCGC

[0019] TGGGTATTAACTCTACCAGCCAGAACGACGATTCCACTTCCGGTGCCGACTCTACCTC

[0020] TGATAGCAGCGACCCAATGCAGCAGCTGCTGAAGATGTTCAGCGAGATCATGCAGAG

[0021] CCTGTTCGGTGACGGTCAGGATGGCACTCAGGGTTCTTCTTCCGGTGGTAAACAGCC

[0022] GACCGAGGGTGAACAGAACGCATACAAGAAAGGCGTGACTGATGCTCTGTCCGGTC

[0023] TGATGGGTAACGGCCTGAGCCAGCTGCTGGGTAACGGTGGTCTGGGCGGTGGCCAG

[0024] GGTGGCAACGCAGGTACCGGTCTGGACGGTTCCTCTCTGGGTGGTAAAGGCCTGCAG

[0025] AACCTGTCTGGCCCGGTGGACTATCAGCAGCTGGGCAACGCAGTAGGTACCGGTATC

[0026] GGTATGAAAGCGGGCATCCAGGCCCTGAACGATATTGGTACCCACAGCGACTCCTCC

[0027] ACCCGCTCTTTCGTGAACAAGGGTGATCGCGCGATGGCTAAAGAAATCGGCCAGTTC

[0028] ATGGATCAGTATCCGGAAGTGTTCGGTAAGCCTCAGTATCAGAAGGGTCCGGGCCAG

[0029] GAAGTAAAAACTGATGATAAAAGCTGGGCTAAGGCTCTGAGCAAACCTGATGATGAC

[0030] GGCATGACCCCGGCATCTATGGAGCAATTTAACAAGGCGAAAGGCATGATCAAATCC

[0031] GCTATGGCAGGCGACACTGGCAACGGTAACCTGCAGGCTCGTGGTGCAGGTGGTTCT

[0032] TCTCTGGGTATCGACGCCATGATGGCCGGTGACGCAATCAACAACATGGCGCTGGGC

[0033] AAACTGGGTGCAGCG

[0034] Furthermore, the amino acid sequence encoded by the Harpin403 protein in step (2) is shown in SEQ ID NO:2.

[0035] The sequence of SEQ ID NO:2 is as follows:

[0036] MSLNTSGLGASTMQISIGGAGGNNGLLGTSRQNAGLGGNSALGLGGGNQNDTVN

[0037] QLAGLLTGMMMSMMGGGGLMGGGLGGGLGNGLGGSGGLGGEGLSNALNDMLGG

[0038] SLNTLGSKGGNNTTSTTNSPLDQALGINSTSQNDDSTSGADSTSDSSDPMQQLLKMFSEI

[0039] MQSLFGDGQDGTQGSSSGGKQPTEGEQNAYKKGVTDALSGLMGNGLSQLLGNGGLGG

[0040] GQGGNAGTLGLDGSSLGGKGLQNLSGPVDYQQLGNAVGTGIGMKAGIQALNDIGTHSDS

[0041] STRSFVNKGDRAMAKEIGQFMDQYPEVFGKPQYQKGPGQEVKTDDKSWAKALSKPDD

[0042] DGMTPASMEQFNKAKGMIKSAMAGDTGNGNLQARGAGGSSLGIDAMMAGDAINNMA

[0043] LGKLGAA

[0044] Furthermore, the dispersion of polydopamine-loaded spinosad nanoparticles described in step (2), Harpin 403

[0045] The volume ratio of the protein dispersion is 1:1-1.5; the concentrations of the polydopamine-loaded spinosad nanoparticle dispersion and the Harpin403 protein dispersion are both 1-1.5 mg / mL.

[0046] Furthermore, the dispersant used in the Harpin403 protein dispersion in step (2) is a PBS solution.

[0047] Furthermore, the mixing time in step (2) is 12-14 hours.

[0048] Further, in step (1), the mass ratio of polydopamine nanoparticles to spinosad is 1:(0.2-0.5), and the stirring time is 12-14h.

[0049] Further, the preparation process of the polydopamine nanoparticles in step (1) is as follows: dopamine hydrochloride is added to a mixed solution of Tris-HCl buffer and isopropanol, and the mixture is stirred at room temperature in the dark for 24-28 hours. After the reaction is completed, the reaction solution is purified to obtain polydopamine nanoparticles. The concentration of dopamine hydrochloride in the mixed solution is 0.5-0.8 mg / mL, and the volume ratio of Tris-HCl buffer to isopropanol is 1:(0.2-0.4).

[0050] The second objective of this invention is to provide a polydopamine-loaded spinosad nanopesticide formulation, which has long-lasting and stable performance and excellent control effect on rice leaf roller.

[0051] To achieve the above objectives, the present invention adopts the following technical solution:

[0052] A polydopamine-loaded spinosad nanopesticide formulation was prepared using the method described above.

[0053] The third objective of this invention is to provide an application of a polydopamine-loaded spinosad nanopesticide formulation, which has broad application prospects.

[0054] To achieve the above objectives, the present invention adopts the following technical solution:

[0055] The above-mentioned polydopamine-loaded spinosad nanopesticide formulations are used for pest control.

[0056] Compared with the prior art, the beneficial effects of this invention are as follows:

[0057] (1) In this invention, a dispersion of polydopamine-loaded spinosad nanoparticles is mixed with a Harpin403 protein dispersion. The amino group of Harpin protein is effectively combined with the catechol group of dopamine to form highly efficient Harpin protein-modified polydopamine nanoparticles. This can improve the encapsulation rate and drug loading of polydopamine-loaded spinosad, while enhancing the adhesion of the formulation to plants and promoting the absorption and utilization of spinosad by plants, thereby achieving the purpose of efficient pest control.

[0058] (2) The polydopamine-loaded spinosad nanopesticide formulation prepared by this invention has good stability, and the decomposition rate of spinosad is less than 3% after 20 days of light exposure. In addition, the polydopamine-loaded spinosad nanopesticide formulation prepared by this invention has excellent control effect on rice leaf folder, with a control effect of over 88.72% on rice leaf folder 7 days after application and over 93.45% on rice leaf folder 14 days after application. Attached Figure Description

[0059] Figure 1 This is a diagram showing the PCR verification results of the engineered E. coli BL21 / Harpin403 strain obtained in Example 1;

[0060] Figure 2 This is a graph showing the SDS-PAGE detection results of Example 1;

[0061] Figure 3 This is a microscopic morphology diagram of the polydopamine-loaded spinosad nanopesticide formulation obtained in Example 2;

[0062] Figure 4 This is a microscopic morphology diagram of the polydopamine-loaded spinosad nanopesticide formulation obtained in Example 3;

[0063] Figure 5 This is a microscopic morphology diagram of the polydopamine-loaded spinosad nanopesticide formulation obtained in Example 4.

[0064] Figure 6 This is a microscopic morphology diagram of the polydopamine-loaded spinosad nanopesticide formulation obtained in Comparative Example 1. Detailed Implementation

[0065] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments shall be performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0066] Example 1

[0067] Expression and purification of Harpin403 protein

[0068] The Harpin403 nucleotide sequence was synthesized by Genewiz, and then integrated into the pET28a plasmid to obtain the recombinant plasmid. The nucleotide sequence of the Harpin403 protein is shown in SEQ ID NO:1; the recombinant system is shown in Table 1. The recombinant plasmid was transformed into E. coli DH5α, and positive clones were screened and sent for sequencing. For clones with correct sequencing, the plasmid was extracted after amplification culture and transformed into E. coli BL21(DE3). Positive clones were identified by PCR. The positive clones were the successfully constructed E. coli BL21 / Harpin403 engineered bacteria. The PCR verification results are shown in Table 1. Figure 1 As shown.

[0069] The above-mentioned E. coli BL21 / Harpin403 engineered bacteria were inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured in a constant temperature shaker at 37℃ and 200 rpm for 10 h to obtain seed culture. The seed culture was then inoculated into LB medium (containing 50 μg / mL kanamycin) at a 5% inoculation rate and cultured in a constant temperature shaker at 37℃ and 200 rpm until OD... 600 To a final concentration of 0.8, add 1 mM IPTG and induce at low temperature for 4 hours. After induction culture, obtain the bacterial culture and store at -80℃ for later use.

[0070] Thaw the bacterial culture stored at -80℃ on ice, resuspend the bacteria in lysis buffer at 5 mL / g, add lysozyme to a final concentration of 1 mg / mL, place on ice for 30 min, centrifuge the culture at 8000 rpm for 10 min to collect the bacterial cells, sonicate to break up the cell walls, centrifuge again at 8000 rpm for 30 min, obtain the supernatant, and store on ice for later use.

[0071] After filtering the supernatant through a 0.45 μm filter membrane, 50% Ni-NTA packing material was added at a ratio of 1 mL of 50% Ni-NTA packing material per 4 mL of supernatant. The mixture was incubated at 4 °C for 60 min by rotation. The column was then packed, washed twice with 1 mL of wash buffer each time, and then washed four times with 1 mL of elution buffer each time. The eluent was collected. SDS-PAGE analysis showed that the target protein Harpin403 was clearly visible in lane 1, with a molecular weight of approximately 45 kDa. The results are shown in the figure below. Figure 2 As shown.

[0072] Table 1 Reorganization System

[0073] reagents volume Enzyme digestion products 4μL T4 DNA ligase 1μL 10×T4 DNA ligase buffer 1μL Deionized water Add to 10μL

[0074] Example 2

[0075] (1) According to the concentration of dopamine hydrochloride in the mixed solution being 0.7 mg / mL, dopamine hydrochloride was added to a mixed solution of 0.1 mol / L Tris-HCl buffer and isopropanol. The volume ratio of Tris-HCl buffer to isopropanol in the mixed solution was 1:0.4. The mixture was stirred at room temperature in the dark for 24 h. After the reaction was completed, the reaction solution was placed in a centrifuge tube and centrifuged at 15000 rpm for 15 min to obtain the precipitate. The precipitate was washed three times with deionized water to obtain polydopamine nanoparticles. The polydopamine nanoparticles were added to a spinosad solution at a mass ratio of 1:0.5. The pH of the reaction solution was maintained at 8.5 using Tris-HCl buffer, and the mixture was stirred at room temperature for 12 hours. After purification by dialysis and freeze-drying under vacuum, polydopamine-loaded spinosad nanoparticles were obtained. The spinosad-loaded polydopamine nanoparticles were then dispersed in PBS solution (pH=7.4) to obtain a polydopamine-loaded spinosad nanoparticle dispersion with a concentration of 1 mg / mL.

[0076] (2) The polydopamine nanoparticle dispersion loaded with spinosad obtained in step (1) and 1 mg / mL Harpin403 protein dispersion (dispersant is PBS, pH=7.4) were mixed at room temperature for 12 h at a volume ratio of 1:1. After centrifugation at 15000 rpm for 3 min, the nanopesticide formulation loaded with polydopamine (Harpin-SP-PDA-NPs) was obtained after washing.

[0077] Example 3

[0078] (1) According to the concentration of dopamine hydrochloride in the mixed solution being 0.8 mg / mL, dopamine hydrochloride was added to a mixed solution of 0.1 mol / L Tris-HCl buffer and isopropanol. The volume ratio of Tris-HCl buffer to isopropanol in the mixed solution was 1:0.3. The mixture was stirred at room temperature in the dark for 26 h. After the reaction was completed, the reaction solution was placed in a centrifuge tube and centrifuged at 15000 rpm for 15 min to obtain the precipitate. The precipitate was washed three times with deionized water to obtain polydopamine nanoparticles. The polydopamine nanoparticles were added to a spinosad solution at a mass ratio of 1:0.2. The pH of the reaction solution was maintained at 8.5 using Tris-HCl buffer, and the mixture was stirred at room temperature for 14 hours. After purification by dialysis and freeze-drying under vacuum, polydopamine-loaded spinosad nanoparticles were obtained. The polydopamine-loaded spinosad nanoparticles were then dispersed in PBS solution (pH=7.4) to obtain a dispersion of polydopamine-loaded spinosad nanoparticles with a concentration of 1.2 mg / mL.

[0079] (2) The polydopamine-loaded spinosad nanoparticle dispersion obtained in step (1) and 1.2 mg / mL Harpin403 protein dispersion (dispersant is PBS, pH=7.4) were mixed at room temperature for 13 h at a volume ratio of 1:1.2. After centrifugation at 15000 rpm for 3 min, the polydopamine-loaded spinosad nanopesticide formulation (Harpin-SP-PDA-NPs) was obtained after washing.

[0080] Example 4

[0081] (1) The concentration of dopamine hydrochloride in the mixed solution was 0.5 mg / mL. Dopamine hydrochloride was added to a mixed solution of 0.1 mol / L Tris-HCl buffer and isopropanol. The volume ratio of Tris-HCl buffer to isopropanol in the mixed solution was 1:0.2. The mixture was stirred at room temperature in the dark for 28 h. After the reaction was completed, the reaction solution was placed in a centrifuge tube and centrifuged at 15000 rpm for 15 min to obtain the precipitate. The precipitate was washed three times with deionized water to obtain polydopamine nanoparticles. The polydopamine nanoparticles were added to a spinosad solution at a mass ratio of 1:0.4. The pH of the solution was maintained at 8.5 using Tris-HCl buffer. The mixture was stirred at room temperature for 13 hours. After dialysis purification and vacuum freeze-drying, polydopamine-loaded spinosad nanoparticles were obtained. The polydopamine-loaded spinosad nanoparticles were dispersed in PBS solution (pH=7.4) to obtain a polydopamine-loaded spinosad nanoparticle dispersion with a concentration of 1.5 mg / mL.

[0082] (2) The polydopamine-loaded spinosad nanoparticle dispersion obtained in step (1) and 1.5 mg / mL Harpin403 protein dispersion (dispersant is PBS, pH=7.4) were mixed at room temperature for 14 h at a volume ratio of 1:1.5. After centrifugation at 15000 rpm for 3 min, the polydopamine-loaded spinosad nanopesticide formulation (Harpin-SP-PDA-NPs) was obtained after washing.

[0083] Comparative Example 1

[0084] The difference between Comparative Example 1 and Example 2 is that step (2) is omitted, i.e., the Harpin403 protein is not used to modify polydopamine.

[0085] Experimental Example 1

[0086] The microstructure of the nano-pesticide formulations prepared in Examples 2-4 and Comparative Example 1 was observed using a transmission electron microscope (TEM, Tecnai G2 F30 S-TWIN, USA). The specific process is as follows:

[0087] The nano-pesticide formulations prepared in Examples 2-4 and Comparative Example 1 were ultrasonically dispersed in ethanol to obtain nano-pesticide formulation samples with a concentration of 100 μg / mL. 20 μL of each sample was dropped onto a copper grid, allowed to air dry, and then observed. The results are as follows: Figure 3-6 As shown.

[0088] Depend on Figure 3-6 It can be seen that the dry average particle size of the polydopamine-loaded spinosad nanopesticide formulations in Examples 2-4 is 203 nm. According to TEM results ( Figure 3-5 Compared to unmodified PDA-NPs (), Figure 6 The nanoparticles modified with Harpin403 protein exhibited a distinct thin film structure around their periphery, further demonstrating the successful modification of the Harpin403 protein.

[0089] Experiment Example 2

[0090] The polydopamine-loaded spinosad nanopesticide formulations prepared in Examples 2-4 and the nanopesticide formulation obtained in Comparative Example 1 were dispersed in water. The encapsulation efficiency and loading of spinosad were detected by high performance liquid chromatography. The results are shown in Table 2.

[0091] Table 2

[0092] Group Encapsulation efficiency (%) Drug loading (%) Example 2 Nanopesticide Formulation 69.37±1.24 41.18±1.65 Example 3 Nanopesticide Formulation 68.95±1.17 41.06±1.72 Example 4 Nanopesticide Formulation 68.29±1.36 40.67±1.49 Comparative Example 1: Nanopesticide Formulation 58.33±1.85 36.25±2.03

[0093] The results in Table 2 show that the polydopamine-loaded spinosad nanopesticide formulation prepared in this invention has a high encapsulation rate and high pesticide loading.

[0094] Experimental Example 3

[0095] The polydopamine-loaded spinosad nanopesticide formulations obtained in Examples 2-4, the nanopesticide formulation obtained in Comparative Example 1, and the methanol solution of spinosad were placed in 25 mL quartz tubes, respectively. Two quartz tubes were set up for each group. One group was directly sealed, and the other group was sealed and wrapped with tin foil to protect it from light. Then, the three groups of quartz tubes were exposed to near-ultraviolet light at the same time. The experiment was repeated 3 times. Samples were taken at 0, 3, 10, and 20 days after treatment and stored in a -4℃ refrigerator. The absorbance A of spinosad was measured using a UV / Vis spectrophotometer. The decomposition rate of spinosad in each group was calculated according to the following formula: Decomposition rate = (1-Ai / A0)×100%, where Ai is the absorbance of spinosad after light exposure and A0 is the absorbance of spinosad before light exposure. The results are shown in Table 3.

[0096] Table 3

[0097]

[0098]

[0099] As shown in Table 3, after 20 days of light irradiation, the decomposition rate of the polydopamine-loaded spinosad nanopesticide formulation obtained in this invention is less than 3%. These results demonstrate that the polydopamine-loaded spinosad nanopesticide formulation obtained in this invention possesses excellent photostability.

[0100] Experiment Example 4

[0101] The polydopamine-loaded spinosad nanopesticide formulations obtained in Examples 2-4 and the nanopesticide formulation obtained in Comparative Example 1 were dispersed in water and sprayed onto fresh, clean cabbage leaves. After air drying, the leaves were soaked in 100 mL of deionized water. The mass of spinosad in the water was determined and analyzed using UV-Vis, and the residual rate (%) of spinosad was calculated according to the following formula:

[0102] Residual rate (%) = [1 - (mass of spinosad in water / mass of spinosad at the start of spraying)] × 100%

[0103] The experimental results are shown in Table 4. In this experiment, a spinosad ethanol solution was used as the control group.

[0104] Table 4

[0105] Grouping Residual rate of spinosad (%) Example 2: Nanopesticide Formulation Group 46.77 Example 3: Nanopesticide Formulation Group 46.58 Example 4: Nanopesticide Formulation Group 46.24 Comparative Example 1: Nanopesticide Formulation Group 39.51 spinosad ethanol solution group 32.85

[0106] Table 4 shows that after washing, the residual rate of spinosad ethanol solution on cabbage leaves was 32.85%, the residual rate of the nano-pesticide formulation in Comparative Example 1 was 39.51%, and the residual rate of the nano-pesticide formulations in Examples 2-4 was over 46.24%. Comparative analysis indicates that the polydopamine-loaded spinosad nano-pesticide formulation obtained in this invention has good adhesion properties to plants.

[0107] Experimental Example 5

[0108] A randomized block design was used with six treatments: the polydopamine-loaded spinosad nanopesticide formulation prepared in Examples 2-4, the nanopesticide formulation of Comparative Example 1, the 5% spinosad suspension (control group), and a water treatment (blank control group). Each treatment was configured with three replicates, for a total of 18 plots, each plot area being 15m². 2 .

[0109] During the rice tillering stage, at the peak of the first and second instar larvae of the rice leaf roller, apply pesticide using a Gongnong-16 sprayer at a rate of 30L per acre. After the rice leaf roller infestation stabilizes, survey the number of rolled leaves twice, at 7 days and 14 days after application, for a total of two surveys. Samples were taken from 5 points in each plot, totaling 25 clumps. The number of rolled leaves was recorded, and the control effect was statistically analyzed. The formula for calculating the control effect is as follows:

[0110] Leaf curling rate (%) = Number of curled leaves surveyed / Total number of leaves surveyed × 100%;

[0111] Control efficacy (%) = (leaf curling rate of water group - leaf curling rate of pesticide formulation group after application) / leaf curling rate of water group × 100%; See Table 5 for results.

[0112] Table 5

[0113]

[0114] Table 5 shows that, compared with other spinosad emulsion groups, the polydopamine-loaded spinosad nanopesticide formulation prepared in this invention exhibits excellent control efficacy against rice leaf folder. The control efficacy at 7 days post-application was 88.72%–89.57%, and at 14 days post-application, it was 93.45%–94.18%, significantly higher than the comparative and control groups. These results also indicate that the polydopamine-loaded spinosad nanopesticide formulation modified with Harpin403 protein has better residual efficacy. In summary, the polydopamine-loaded spinosad nanopesticide formulation obtained in this invention demonstrates superior control efficacy and long-lasting stability against the target pests in practical applications, thus showing broad application prospects.

[0115] The above are merely preferred embodiments of the present invention and are not limited to the examples described above. Those skilled in the art will recognize that various modifications and variations can be made based on the principles of the present invention. Any modifications or improvements made should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a polydopamine-loaded spinosad nanopesticide formulation, characterized in that, Includes the following steps: (1) Add polydopamine nanoparticles to spinosad solution and stir. After drying, polydopamine-loaded spinosad nanoparticles are obtained. Disperse polydopamine-loaded spinosad nanoparticles in PBS solution to obtain polydopamine-loaded spinosad nanoparticle dispersion. (2) Mix the polydopamine-loaded spinosad nanoparticle dispersion and Harpin403 protein dispersion obtained in step (1), and centrifuge to obtain polydopamine-loaded spinosad nanopesticide formulation. The mass ratio of polydopamine nanoparticles to spinosad in step (1) is 1:(0.2-0.5).

2. The method for preparing the polydopamine-loaded spinosad nanopesticide formulation according to claim 1, characterized in that, The nucleotide sequence of the Harpin403 protein described in step (2) is shown in SEQ ID NO:

1.

3. The method for preparing the polydopamine-loaded spinosad nanopesticide formulation according to claim 1, characterized in that, The amino acid sequence encoded by the Harpin403 protein in step (2) is shown in SEQ ID NO:

2.

4. The method for preparing the polydopamine-loaded spinosad nanopesticide formulation according to claim 1, characterized in that, In step (2), the volume ratio of the polydopamine-loaded spinosad nanoparticle dispersion and the Harpin403 protein dispersion is 1:1-1.5; the concentrations of the polydopamine-loaded spinosad nanoparticle dispersion and the Harpin403 protein dispersion are both 1-1.5 mg / mL.

5. The method for preparing the polydopamine-loaded spinosad nanopesticide formulation according to claim 1, characterized in that, The dispersant used in the Harpin403 protein dispersion in step (2) is PBS solution.

6. The method for preparing the polydopamine-loaded spinosad nanopesticide formulation according to claim 1, characterized in that, The mixing time in step (2) is 12-14 hours.

7. The method for preparing the polydopamine-loaded spinosad nanopesticide formulation according to claim 1, characterized in that, The stirring time in step (1) is 12-14 hours.

8. The method for preparing the polydopamine-loaded spinosad nanopesticide formulation according to claim 1, characterized in that, The preparation process of polydopamine nanoparticles in step (1) is as follows: dopamine hydrochloride is added to a mixed solution of Tris-HCl buffer and isopropanol, and the mixture is stirred at room temperature in the dark for 24-28 hours. After the reaction is completed, the reaction solution is purified to obtain polydopamine nanoparticles. The concentration of dopamine hydrochloride in the mixed solution is 0.5-0.8 mg / mL, and the volume ratio of Tris-HCl buffer to isopropanol is 1:(0.2-0.4).

9. A nano-pesticide formulation of polydopamine loaded with spinosad, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the polydopamine-loaded spinosad nanopesticide formulation according to claim 9, characterized in that, Used for pest control.

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