Polydopamine-loaded spinosad nano pesticide preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biopesticides, and particularly relates to a polydopamine-loaded spinosad nano pesticide preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) adding polydopamine nanoparticles into a spinosad solution, stirring, and drying to obtain polydopamine-loaded spinosad nanoparticles; the preparation method comprises the following steps: dispersing spinosad-loaded polydopamine nanoparticles in a PBS (Phosphate Buffer Solution) to obtain a polydopamine-loaded spinosad nanoparticle dispersion liquid; and (2) mixing the nano-particle dispersion liquid of the polydopamine loaded spinosad obtained in the step (1) and the Harpin403 protein dispersion liquid, and centrifuging to obtain the nano-pesticide preparation of the polydopamine loaded spinosad. The nano pesticide preparation has lasting and stable performance and excellent control effect on cnaphalocrocis medinalis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological pesticides, and in particular relates to a polydopamine-loaded spinosad nanopesticide preparation and a preparation method and application thereof. Background Art

[0002] Spinosad, also known as spinosad, is a new natural insecticide with high contact and feeding toxicity. Investigations have found that spinosad can not only effectively control Lepidoptera, Diptera, and Thysanoptera pests, but also some leaf-feeding pests in Coleoptera and Orthoptera. Spinosad is widely used in the field of biopesticide technology because it has both the safety of biological pesticides and the rapid effect of chemical drugs. To date, spinosad has been used in more than 250 crops in 73 countries. However, with the widespread use of spinosad, pest resistance has gradually increased, resulting in a decrease in control effect. In addition, spinosad has poor photostability and will decompose quickly in sunlight, resulting in poor persistence of the product. In order to solve the problem of persistence, researchers have modified the chemical structure of spinosad to produce ethyl spinosad. At the same time, by using it in combination with other pesticide ingredients, such as emamectin benzoate, it can also delay the development of resistance and improve the control effect. However, this method is relatively expensive and difficult to achieve ideal results.

[0003] With the development of science and technology, nanomaterials are gradually being used in various fields. Studies have found that the use of nanomaterial carriers can not only protect the active ingredients of pesticides from the influence of harsh environmental conditions and significantly improve the chemical stability of the active ingredients of pesticides, but also improve the dispersibility and wettability of the active ingredients of pesticides and reduce the risk of organic solvents flowing into the environment. As a new type of pesticide carrier, polydopamine has a unique degradability and excellent adhesion, showing extremely high application potential. The multifunctional modification of dopamine has become a key strategy to solve many challenges in the use of pesticides.

[0004] Harpin protein can activate the innate immune system of plants and improve their resistance to harmful organisms, while spinosad directly targets pests to exert its insecticidal effect. Based on the above background, the present invention provides a novel Harpin protein-modified polydopamine nanopesticide, which aims to enhance the control effect of traditional pesticides. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for preparing a nano pesticide preparation of polydopamine loaded with spinosad, and the preparation process is simple.

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

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

[0008] (1) adding polydopamine nanoparticles to a spinosad solution and stirring, and drying to obtain polydopamine-loaded spinosad nanoparticles; dispersing the polydopamine-loaded spinosad nanoparticles in a PBS solution to obtain a polydopamine-loaded spinosad nanoparticle dispersion;

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

[0010] Furthermore, the nucleotide sequence of the Harpin403 protein 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 the polydopamine-loaded spinosad nanoparticles in step (2), Harpin403

[0045] The volume ratio of the protein dispersion is 1:1-1.5; the concentrations of the dispersion of the polydopamine-loaded spinosad nanoparticles 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 PBS solution.

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

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

[0049] Furthermore, the preparation process of the polydopamine nanoparticles in step (1) is as follows: adding dopamine hydrochloride to a mixed solution of Tris-HCl buffer and isopropanol, stirring and reacting for 24-28 hours at room temperature in the dark, and after the reaction is completed, purifying the reaction solution to obtain polydopamine nanoparticles; the concentration of the dopamine hydrochloride in the mixed solution is 0.5-0.8 mg / mL, and the volume ratio of the Tris-HCl buffer and isopropanol is 1:(0.2-0.4).

[0050] The second object of the present invention is to provide a polydopamine-loaded spinosad nanopesticide preparation, which has a long-lasting and stable performance and an excellent effect of preventing and controlling rice leaf folders.

[0051] To achieve the above object, the present invention adopts the following technical solutions:

[0052] A polydopamine-loaded spinosad nanopesticide preparation is prepared by the above-mentioned preparation method.

[0053] The third object of the present invention is to provide an application of a nano pesticide preparation of polydopamine loaded with spinosad, which has broad application prospects.

[0054] To achieve the above object, the present invention adopts the following technical solutions:

[0055] The application of the above-mentioned polydopamine-loaded spinosad nanopesticide preparation is used for preventing and controlling pests.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) The present invention mixes a dispersion of polydopamine-loaded spinosad nanoparticles with a dispersion of Harpin403 protein, and effectively combines the amino group of Harpin protein with the catechol group of dopamine to form highly efficient Harpin protein-modified polydopamine nanoparticles, which can improve the encapsulation efficiency and drug loading of polydopamine-loaded spinosad, while enhancing the adhesion of the preparation to plants, promoting the absorption and utilization of spinosad by plants, and achieving the purpose of highly efficient pest control.

[0058] (2) The polydopamine-loaded spinosad nanopesticide preparation prepared by the present invention has good stability, and the decomposition rate of spinosad after 20 days of illumination is less than 3%. In addition, the polydopamine-loaded spinosad nanopesticide preparation prepared by the present invention has excellent control effect of rice leaf roller, and the control effect of rice leaf roller 7 days after application reaches more than 88.72%, and the control effect of rice leaf roller 14 days after application reaches more than 93.45%. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0063] Figure 5 is a microscopic morphology of the polydopamine-loaded spinosad nanopesticide preparation obtained in Example 4;

[0064] Figure 6 This is a microscopic morphology of the polydopamine-loaded spinosad nanopesticide preparation obtained in Comparative Example 1. DETAILED DESCRIPTION

[0065] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0066] Example 1

[0067] Expression and purification of Harpin403 protein

[0068] Genewise was commissioned to synthesize the Harpin403 nucleotide sequence, and then the synthesized Harpin403 nucleotide sequence was integrated into the pET28a plasmid to obtain a 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 DH5a, and the positive clones were screened and sent for sequencing. The clones with correct sequencing were expanded and cultured, and the plasmids were extracted and transformed into E. coli BL21 (DE3) strains. PCR was used to identify the positive clones. The positive clones were the successfully constructed E. coli BL21 / Harpin403 engineering bacteria. The PCR verification results were as follows: Figure 1 shown.

[0069] The 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°C and 200 rpm for 10 h to obtain seed solution; the seed solution was inoculated into LB medium (containing 50 μg / mL kanamycin) at a 5% inoculum amount and cultured in a constant temperature shaker at 37°C and 200 rpm until OD 600 To 0.8, add IPTG at a final concentration of 1mM, and induce at low temperature for 4h. After the induction culture is completed, obtain the bacterial solution and store it at -80℃ for future use.

[0070] Thaw the bacterial solution stored at -80℃ on ice, add 5mL of lysis buffer per gram to resuspend the bacteria, add lysozyme to a final concentration of 1mg / mL, and place on ice for 30min. Centrifuge the culture medium at 8000rpm for 10min to collect the bacteria, break the cell wall with ultrasound, and centrifuge again at 8000rpm for 30min to obtain the supernatant, which is then stored on ice for later use.

[0071] After the supernatant was filtered through a 0.45 μm filter membrane, filler was added according to the ratio of 1 mL 50% Ni-NTA filler per 4 mL supernatant, incubated at 4°C for 60 min, loaded into the column, washed twice with wash buffer, 1 mL each time, and 4 times with elution buffer, 1 mL each time, and the eluate was collected. By SDS-PAGE detection, the target protein Harpin403 was clearly visible in lane 1, and the molecular weight corresponding to the band was about 45 kDa. The detection results are shown in Figure 2 shown.

[0072] Table 1 Recombination system

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

[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 / LTris-HCl buffer and isopropanol, wherein the volume ratio of Tris-HCl buffer to isopropanol in the mixed solution was 1:0.4, and the mixture was stirred and reacted 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 a speed of 15000 rpm for 15 min to obtain a precipitate, which was washed three times with deionized water to obtain polydopamine nanoparticles. According to the mass ratio of polydopamine nanoparticles to spinosad of 1:0.5, the above-mentioned polydopamine nanoparticles were added to the spinosad solution, and the pH of the reaction solution was maintained at 8.5 using Tris-HCl buffer. The reaction solution was stirred at room temperature for 12 hours, and polydopamine-loaded spinosad nanoparticles were obtained after purification by dialysis and vacuum freeze-drying. The polydopamine nanoparticles loaded with spinosad were dispersed in a 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: PBS, pH = 7.4) were mixed at room temperature for 12 h in a volume ratio of 1:1, centrifuged at 15000 rpm for 3 min, and washed to obtain a polydopamine loaded with spinosad nanopesticide preparation (Harpin-SP-PDA-NPs).

[0077] Example 3

[0078] (1) According to the concentration of dopamine hydrochloride in the mixed solution of 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 and isopropanol in the mixed solution was 1:0.3, and the mixture was stirred at room temperature in the dark for 26 hours. After the reaction was completed, the reaction solution was placed in a centrifuge tube and centrifuged at a speed of 15000 rpm for 15 minutes to obtain a precipitate, which was washed three times with deionized water to obtain polydopamine nanoparticles. According to the mass ratio of polydopamine nanoparticles to spinosad of 1:0.2, the above-mentioned polydopamine nanoparticles were added to the spinosad solution, and the pH of the reaction solution was maintained at 8.5 using Tris-HCl buffer. The reaction solution was stirred at room temperature for 14 hours, and polydopamine-loaded spinosad nanoparticles were obtained after purification by dialysis and vacuum freeze-drying. The polydopamine-loaded spinosad nanoparticles were dispersed in a PBS solution (pH=7.4) to obtain a polydopamine-loaded spinosad nanoparticle dispersion 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: PBS, pH = 7.4) were mixed at room temperature for 13 h in a volume ratio of 1:1.2, centrifuged at 15000 rpm for 3 min, and washed to obtain a polydopamine-loaded spinosad nanopesticide preparation (Harpin-SP-PDA-NPs).

[0080] Example 4

[0081] (1) According to the concentration of dopamine hydrochloride in the mixed solution being 0.5 mg / mL, dopamine hydrochloride was added to a mixed solution of 0.1 mol / LTris-HCl buffer and isopropanol, wherein the volume ratio of Tris-HCl buffer to isopropanol in the mixed solution was 1:0.2, and the mixture was stirred and reacted 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 a speed of 15000 rpm for 15 min to obtain a precipitate, which was washed three times with deionized water to obtain polydopamine nanoparticles. According to the mass ratio of polydopamine nanoparticles to spinosad of 1:0.4, the above-mentioned polydopamine nanoparticles were added to the spinosad solution, and the pH of the solution was maintained at 8.5 using Tris-HCl buffer. The solution was stirred at room temperature for 13 hours, and polydopamine-loaded spinosad nanoparticles were obtained after purification by dialysis and vacuum freeze-drying. The polydopamine-loaded spinosad nanoparticles were dispersed in a 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: PBS, pH = 7.4) were mixed at room temperature for 14 h in a volume ratio of 1:1.5, centrifuged at 15000 rpm for 3 min, and washed to obtain a polydopamine-loaded spinosad nanopesticide preparation (Harpin-SP-PDA-NPs).

[0083] Comparative Example 1

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

[0085] Experimental Example 1

[0086] The microscopic morphology of the nanopesticide preparations 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 nanopesticide preparations prepared in Examples 2-4 and Comparative Example 1 were ultrasonically dispersed in ethanol to obtain nanopesticide preparation samples with a concentration of 100 μg / mL. 20 μL of each sample was dripped onto a copper mesh and naturally dried before observation. The results are as follows: Figure 3-6 shown.

[0088] Depend on Figure 3-6 It can be seen that the average dry particle size of the nanopesticide preparation of polydopamine loaded with spinosad in Example 2-4 is 203 nm. According to the results of TEM ( Figure 3-5 ), compared with unmodified PDA-NPs ( Figure 6 ), the periphery of the nanoparticles modified with Harpin403 protein showed an obvious film structure, which further proved the successful modification of Harpin403 protein.

[0089] Experimental Example 2

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

[0091] Table 2

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

[0093] The results in Table 2 show that the polydopamine-loaded spinosad nanopesticide preparation prepared in the present invention has a high encapsulation efficiency and drug loading capacity.

[0094] Experimental Example 3

[0095] The polydopamine-loaded spinosad nanopesticide preparations obtained in Examples 2-4 dispersed in water, the nanopesticide preparations obtained in Comparative Example 1, and a methanol solution of spinosad were placed in 25 mL quartz tubes, respectively. Two quartz tubes were set in each group, one group was directly sealed, and the other group was sealed and wrapped with tin foil to avoid 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 0d, 3d, 10d, and 20d after treatment and stored in a -4°C refrigerator. Then, the absorbance A of spinosad was measured using an ultraviolet / spectrophotometer (UV / Vis) meter, and the decomposition rate of spinosad in each group was calculated according to the following formula: decomposition rate = (1-Ai / A0) × 100%, Ai is the absorbance of spinosad after illumination, A0 is the absorbance of spinosad before illumination, and the results are shown in Table 3.

[0096] Table 3

[0097]

[0098]

[0099] It can be seen from Table 3 that after 20 days of light irradiation, the decomposition rate of the polydopamine-loaded spinosad nanopesticide preparation obtained in the present invention is less than 3%. The above results show that the polydopamine-loaded spinosad nanopesticide preparation obtained in the present invention has excellent light stability.

[0100] Experimental Example 4

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

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

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

[0104] Table 4

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

[0106] As shown in Table 4, after washing, the residual rate of the ethanol solution of spinosad on the cabbage leaves was 32.85%, the residual rate of the nanopesticide preparation group of Comparative Example 1 on the cabbage leaves was 39.51%, and the residual rate of the nanopesticide preparation group of Examples 2-4 on the cabbage leaves was more than 46.24%. Through comparative analysis, it can be seen that the nanopesticide preparation of spinosad loaded with polydopamine obtained in the present invention has good adhesion performance to plants.

[0107] Experimental Example 5

[0108] A randomized block arrangement was used, and 6 treatments were set, namely, the polydopamine-loaded spinosad nanopesticide preparations prepared in Examples 2-4, the nanopesticide preparations of Comparative Example 1, the 5% spinosad suspension (control group), and the clean water (blank control group). Each treatment was repeated 3 times, with a total of 18 plots, and the plot area was 15 m 2 .

[0109] During the rice tillering period, when the 1-2-year-old larvae of rice leaf folder are at their peak, a Gongnong-16 sprayer was used to apply pesticides, with a water consumption of 30L per mu. After the damage of rice leaf folder stabilized, the number of rolled leaves was investigated once at 7d and 14d after application of pesticides, for a total of 2 investigations. Samples were taken at 5 points in each plot, for a total of 25 clumps, and the number of rolled leaves was recorded and the control effect was calculated. The control effect was calculated as follows:

[0110] Leaf curling rate (%) = number of curled leaves investigated / total number of leaves investigated × 100%;

[0111] Control effect (%) = (leaf curling rate of the clean water group - leaf curling rate of the pesticide preparation group after treatment) / leaf curling rate of the clean water group × 100%; see Table 5 for the results.

[0112] Table 5

[0113]

[0114] As shown in Table 5, compared with the polydopamine-loaded spinosad aqueous emulsion group, the polydopamine-loaded spinosad nanopesticide preparation prepared by the present invention has excellent control effect on rice leaf roller, wherein the control effect 7d after application is 88.72% to 89.57%, and the control effect 14d after application is 93.45% to 94.18%, which is significantly higher than the comparative group and the control agent group. The above results also show that the polydopamine-loaded spinosad nanopesticide preparation modified with Harpin403 protein has better persistence. In summary, the polydopamine-loaded spinosad nanopesticide preparation obtained by the present invention has better control effect and lasting and stable performance on the control object in practical application, and therefore has broad application prospects.

[0115] The above are only preferred embodiments of the present invention, and are not limited to the above examples. For those skilled in the art, various changes and variations are possible under the principle of the present invention. Any modifications and improvements made should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a nanopesticide preparation of polydopamine loaded with spinosad, characterized in that: The following steps are involved: (1) adding polydopamine nanoparticles to a spinosad solution and stirring, and drying to obtain polydopamine-loaded spinosad nanoparticles; dispersing the polydopamine-loaded spinosad nanoparticles in a PBS solution to obtain a polydopamine-loaded spinosad nanoparticle dispersion; (2) The polydopamine-loaded spinosad nanoparticle dispersion obtained in step (1) and the Harpin403 protein dispersion are mixed, and the mixture is centrifuged to obtain a polydopamine-loaded spinosad nanopesticide preparation.

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

1.

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

5. The method for preparing the polydopamine-loaded spinosad nanopesticide preparation 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 preparation 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 preparation according to claim 1, characterized in that: In step (1), the mass ratio of polydopamine nanoparticles to spinosad is 1:(0.2-0.5), and the stirring time is 12-14 hours.

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

9. A nano pesticide preparation of polydopamine loaded with spinosad, characterized in that: The preparation method is described in any one of claims 1 to 8.

10. The use of the polydopamine-loaded spinosad nanopesticide preparation according to claim 9, characterized in that: Used to control pests.

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