Application of dual-controlled-release plant source nano pesticide with pH and oxidation reduction dual responsiveness to prevention and control of lissorhoptrus oryzophilus

Abstract

The invention discloses application of a double-controlled-release plant source nano-pesticide with pH and oxidation reduction double responsiveness to prevention and control of lissorhoptrus oryzophilus. The modified nano pesticide disclosed by the invention can continuously and effectively prevent and control the lissorhoptrus oryzophilus in a test field, so that the harm of the lissorhoptrus oryzophilus is reduced. By using the modified nano pesticide, the use amount of the raw pesticide can be effectively reduced, and the pollution and poison of the pesticide to the environment are reduced. Experiments also pay attention to the growth conditions of rice in a treatment group and a control group in each pesticide effect investigation day, no obvious difference exists in visual inspection of the rice and the control group, the phytotoxicity phenomena such as leaf rolling, leaf burning and yellowing do not exist, it is further found that the nano-pesticides NIC-coated MSN-SS-CMCS and MT-coated MSN-SS-CMCS have good safety to zebra fish, the toxicity of the nano-pesticides is far smaller than that of original drugs, and the nano-pesticides have good application prospects. The MSN-SS-CMCS nano-carrier has no adverse effect on growth and development of crops, and good safety of the MSN-SS-CMCS nano-carrier is confirmed.

Description

Technical Field

[0001] The present invention belongs to the field of nano pesticides, and particularly relates to the application of a dual-controlled release plant-derived nano pesticide with dual responsiveness to pH and redox in the control of rice water weevils. Background Art

[0002] Chemical pesticides play a crucial role in controlling crop pests and diseases and promoting agricultural production. However, in the past few decades, the abuse of chemical pesticides has caused serious environmental pollution, threatening the sustainable development of agriculture and human health. At present, the application of plant-derived pesticides in crop protection against pests and diseases has attracted increasing attention due to their low toxicity and high safety. However, plant-derived pesticides are easily affected by environmental factors (such as temperature, pH value, ultraviolet light, weather), resulting in slow action and easy degradation. Therefore, there is an urgent need to develop environmentally friendly means to promote the activity and stability of plant-derived pesticides, so as to effectively control pests in sustainable agriculture.

[0003] At present, mesoporous silica (nano particles) has been widely used in many fields such as agriculture, medicine and materials science as a nano carrier due to its surface modifiability, large surface area, large pore volume and stability. Research shows that carboxymethyl chitosan is a natural polymer with good biodegradability and environmental friendliness, and itself has acid-sensitive properties, which can be used as a gatekeeper for encapsulating nano particles. Specifically, first, the pesticide active ingredient is loaded into the pores of the nano particles, and then the pores of the nano particles are sealed with an encapsulant (such as chitosan) to improve the stability and controlled release of the pesticide active ingredient.

[0004] The rice water weevil (Lissorhoptrus oryzophitus) is a coleopteran pest and also the only quarantine pest on rice in China. It seriously threatens the yield of rice crops and can cause large-scale reduction or even failure of rice production. Its adults mainly feed on the upper epidermis and mesophyll of young rice leaves, seriously affecting the photosynthesis of rice leaves; the larvae feed on the roots of rice, resulting in root breakage and lodging of the plants. At present, the best control measure for rice water weevils is still chemical control. The best control period for rice water weevils is the adult stage, but the adults stay on the rice leaves for a short time of about 2-3 weeks, and then they successively enter the water to lay eggs on the roots of rice seedlings. In addition to two generations a year in Guangdong Province, rice water weevils in other provinces occur only once a year. The characteristics of a short control window, difficult to detect, and strong drug resistance make the control difficult greatly increase. Therefore, there is an urgent need for a continuous, green, efficient and precise drug for control.

[0005] In recent years, the progress of nanotechnology has provided new ways for the controlled release of pesticides. In particular, mesoporous silica nanoparticles (MSN) have become effective controlled release carriers due to their high loading efficiency, large specific surface area, and good biocompatibility. However, unmodified MSN cannot effectively control the drug release time and location. Chemically modified MSN can respond to environmental stimuli such as temperature, enzyme activity, pH, and glutathione to achieve precise drug release. The introduction of carboxymethyl chitosan (CMCS) enhances its solubility, pH sensitivity, biocompatibility, and biodegradability, further improving the environmental friendliness and application potential of the drug carrier. In this study, the characteristics of mesoporous silica and carboxymethyl chitosan were utilized to chemically modify MSN through the silane coupling agent and ion binding effect, effectively matching the microenvironment in the body of Lissorhoptrus oryzophilus Kuschel with the environmental stimuli of pesticides. Through characterization analysis such as SEM, TEM, FTIR, XPS, BET, TGA, and in vitro drug release performance testing, the successful construction of the prepared MSN-SS-CMCS nano-drug carrier and its dual responsiveness to pH and redox were confirmed, and the drug release rate could be adjusted according to environmental changes. A series of bioactivity assays and safety evaluation results showed that with the help of the environmentally friendly and sustainable pesticide controlled release carrier MSN-SS-CMCS, effective control of Lissorhoptrus oryzophilus Kuschel can be achieved, with good application prospects. Summary of the Invention

[0006] The object of the present invention is to provide the application of a dual-controlled release plant-derived nano-pesticide with dual responsiveness to pH and redox in the control of Lissorhoptrus oryzophilus Kuschel.

[0007] The dual-controlled release plant-derived nano-pesticide with dual responsiveness to pH and redox of the present invention is prepared by the following method:

[0008] Cetyltrimethylammonium bromide is dispersed and dissolved in water, then the pH value is adjusted to alkaline, and then tetraethyl orthosilicate and (3-mercaptopropyl)trimethoxysilane are added for reaction. After the reaction is completed, it is washed with methanol and deionized water in sequence, and freeze-dried to obtain a dry powder. The dry powder is redispersed in a methanol-hydrochloric acid mixed solution, and the CTAB template is removed by reflux reaction under N2 atmosphere, and then obtained MSN-SH after centrifugal washing and drying;

[0009] MSN-SH reacts with 2,2'-dithiopyridine under N2 atmosphere and acidic environment. The centrifuged precipitate is washed with methanol and deionized water to obtain MSN-SS-Py. MSN-SS-Py is dispersed in DMF containing acetic acid, and then 3-mercaptopropionic acid is added for reaction. The product is washed with methanol and deionized water to obtain MSN-SS-COOH;

[0010] MSN-SS-COOH was mixed and dispersed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in PBS for reaction to activate the hydroxyl group, and then carboxymethyl chitosan was added for further reaction. After centrifugation, washing and vacuum drying, MSN-SS-CMCS was obtained;

[0011] Then MSN-SS-CMCS was used to load pesticides to obtain a dual-controlled release plant-derived nano-pesticide.

[0012] Preferably, the pesticides can be various available pesticides, such as nicotine (NIC) or matrine (MT).

[0013] Preferably, the steps of loading pesticides with MSN-SS-CMCS are as follows:

[0014] MSN-SS-CMCS, nicotine or matrine were dissolved in absolute ethanol for reaction, and then centrifuged. The precipitate was washed with deionized water and absolute ethanol and vacuum dried to obtain NIC@MSN-SS-COOH. NIC@MSN-SS-COOH was mixed and dispersed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in PBS for reaction to activate the hydroxyl group, and then carboxymethyl chitosan was added for further reaction. After centrifugation, washing and vacuum drying, NIC@MSN-SS-CMCS or MT@MSN-SS-CMCS was obtained.

[0015] Preferably, the application of 6-6.5 and abundant glutathione in the body of Lissorhoptrus oryzophilus Kuschel in promoting the release of the dual-controlled release plant-derived nano-pesticide loaded with pesticides in the control of Lissorhoptrus oryzophilus Kuschel.

[0016] MSN-SS-CMCS has relatively excellent thermal stability performance, the drug loading situation and drug encapsulation situation of the nano-carrier are good, it has good wetting performance and adhesion performance, and has good pH responsiveness. The glutathione in the body of Lissorhoptrus oryzophilus Kuschel is more abundant and is more likely to promote the release of MT by MSN-SS-CMCS. Therefore, due to the permeability-promoting, adhesive and stable properties of CMCS, the loaded drugs can more easily penetrate the outer shell of the target pests, and then reach the effective insecticidal concentration through the dual environmental responsiveness to pH and GSH, improving the pesticide efficacy while avoiding unnecessary drug waste.

[0017] The modified nano-pesticide of the present invention can also continuously and effectively control the rice water weevil in the experimental field, reducing the damage caused by the rice water weevil. Using the modified nano-pesticide can effectively reduce the usage amount of the original drug, and reduce the pollution and toxicity of the pesticide to the environment. During the experiment, the growth conditions of the rice in the treatment group and the control group were also observed on each efficacy investigation day. There were no significant differences between the two visually, and there were no phytotoxicity phenomena such as leaf rolling, leaf burning, and chlorosis. It was further found that the nano-pesticides NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS had good safety for zebrafish. The toxicity of the nano-pesticide was much lower than that of the original drug, and the MSN-SS-CMCS nano-carrier had no adverse effects on the growth and development of crops, confirming its good safety. Description of the Drawings

[0018] Figure 1 It is a schematic diagram of the application and safety evaluation of the plant-derived nano-pesticide controlled release system;

[0019] Figure 2 It is the scanning electron microscope, transmission electron microscope and particle size distribution diagram of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS;

[0020] Figure 3 It is a characterization diagram of the nano-material;

[0021] Figure 4 It is the standard curve of NIC at different pH values;

[0022] Figure 5 It is the standard curve of MT at different pH values;

[0023] Figure 6 It is the static contact angle of NIC and MT;

[0024] Figure 7 It is the adhesion performance detection of NIC and MT;

[0025] Figure 8 It is the sustained release curve of NIC / MT@MSN-SS-CMCS under different pH and GSH environments;

[0026] Figure 9 It is the corrected mortality rate of the modified nano-carrier MSN-SS-CMCS, the ordinary nano-carrier MSN encapsulating nicotine and matrine against the rice water weevil under different concentrations for 96h. Note: Each data is the average value obtained after 3 repetitions. Different lowercase letters in the figure represent that there are significant differences in the mortality rate of the rice water weevil after loading drugs with MSN and MSN-SS-CMCS under the same concentration treatment by t-test;

[0027] Figure 10 It is the effect of MSN-SS-CMCS on the germination rate, root length and chlorophyll value (SPAD) of rice seeds;

[0028] Figure 11 The median lethal concentrations and fitted curves of NIC, NIC@MSN-SS-CMCS, MT, and MT@MSN-SS-CMCS at different concentrations on zebrafish, and the acute toxicity of MT@MSN-SS-CMCS to zebrafish at 96 h exposure time. After 96 h, the LC 50 values of NIC and NIC@MSN-SS-CMCS were 0.74 mg / L and 516.02 mg / L respectively, and the LC 50 values of MT and MT@MSN-SS-CMCS were 284.44 mg / L and 847.43 mg / L respectively. According to the acute toxicity classification of pesticides to fish, the LC 50 of NIC to zebrafish was below 1 mg / L at 96 h, showing high toxicity (high toxicity: LC 50 < 1 mg / L), while the LC 50 of NIC@MSN-SS-CMCS, MT, and MT@MSN-SS-CMCS to zebrafish at 96 h were all greater than 10 mg / L, indicating low toxicity (low toxicity: LC 50 > 10 mg / L). By comparing the LC 50 of the original drug and the nano-pesticide, it can be found that the toxicity of the nano-pesticide is much lower than that of the original drug, and the nano-pesticides NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS have good safety for zebrafish. Specific embodiments

[0029] The following examples are further illustrations of the present invention rather than limitations thereof.

[0030] The application and safety evaluation of the plant-derived nano-pesticide controlled release system are shown as Figure 1 shown. The specific preparation, application, and evaluation are as follows.

[0031] Example 1:

[0032] I. Materials and methods

[0033] 1.1 Synthesis method of MSN-SS-CMCS

[0034] 1.1.1 Synthesis of MSN and MSN-SH

[0035] 1.1.1.1 Synthesis of MSN

[0036] Add 500 mg of CTAB to 240 mL of deionized water, heat to 80 °C and stir to dissolve. Add 3.5 mL of 1.00 mol / L NaOH, and dropwise add 2.0 mL of TEOS at a rate of 0.2 mL per minute while continuously stirring. After stirring for 3 hours, freeze-centrifuge to separate the precipitate, and wash it three times with methanol and deionized water. Vacuum freeze-dry to obtain a powdery product. Subsequently, disperse the powder in 80 mL of methanol and 4.5 mL of 1.00 mol / L hydrochloric acid, and reflux at 70 °C for 24 hours under a N2 atmosphere to remove the CTAB template. After centrifugation, washing, and drying, pure MSN is obtained.

[0037] 1.1.1.1.2 Synthesis of MSN-SH

[0038] Add 500 mg of CTAB (cetyltrimethylammonium bromide) to 240 mL of deionized water, and heat the mixture to 80 °C and continuously stir to ensure complete dissolution and uniform dispersion of CTAB. Subsequently, add 3.5 mL of 1.00 mol / L NaOH to the solution to adjust the pH value to approximately 11. Under continuous stirring, slowly dropwise add 2.0 mL of TEOS (tetraethyl orthosilicate) at a rate of 0.2 mL per minute, and then add 0.5 mL of MPTMS ((3-mercaptopropyl)trimethoxysilane) at the same rate to ensure uniform mixing and introduction of functional groups. After the addition of MPTMS is completed, stir for another 3 h, use a refrigerated centrifuge (4 °C, 9000 r / min, 10 min) to separate the precipitate, and use an equal volume mixture of methanol and deionized water for three vortex washings, with each washing for 5 minutes to thoroughly remove all unreacted substances. After washing, vacuum freeze-dry the product to remove the solvent and obtain a dry powdery product.

[0039] Redisperse the dry powder in a mixed solution of 80 mL of methanol and 4.5 mL of 1.00 mol / L hydrochloric acid, and reflux at 70 °C for 24 h under a N2 atmosphere to completely remove the CTAB template through an acid-catalyzed hydrolysis reaction. After centrifugation, washing, and drying of the treated product, pure MSN-SH is obtained.

[0040] 1.1.2 Synthesis of MSN-SS-COOH

[0041] Adjust the pH value of the solution to 2.0 by adding 1100 μL of 1.00 M HCl to 100 mL of deionized water. Under a N2 atmosphere, add 500 mg of MSN-SH and 220 mg of 2,2′-dithiopyridine to this solution, and stir at room temperature for 24 h. After the reaction is completed, freeze-centrifuge to separate the precipitate product and wash it multiple times with methanol and deionized water to obtain MSN-SS-Py.

[0042] To further synthesize MSN-SS-COOH, 200 mg of MSN-SS-Py was dispersed in 15 mL of DMF containing 1.2 mL of acetic acid, and then 40 μL of 3-mercaptopropionic acid was added. After reacting at 40 °C for 24 h, MSN-SS-COOH nanoparticles were obtained, which were repeatedly washed with methanol and deionized water and stored at 4 °C in a refrigerator after drying.

[0043] 1.1.3 Synthesis of MSN-SS-CMCS

[0044] 100 mg of MSN-SS-COOH, 100 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and 80 mg of NHS (N-hydroxysuccinimide) were thoroughly mixed and dispersed in PBS buffer, and the whole system was sonicated for 30 min to activate the carboxyl groups. Then, 100 mg of carboxymethyl chitosan was added to 150 mL of 10% acetic acid aqueous solution, and the carboxymethyl chitosan was completely dissolved by shaking in a constant-temperature shaker. The prepared carboxymethyl chitosan solution was added to MSN-SS-COOH, and the whole system was reacted at room temperature for 24 h. Centrifugation was carried out at 9000 r / min for 10 min, washed several times, and vacuum dried for 24 h to obtain MSN-SS-CMCS.

[0045] 1.1.4 Synthesis of NIC@MSN-SS-CMCS

[0046] Weigh 100 mg of MSN-SS-COOH, and then add it to a solution of 100 mg of nicotine (Nicotine, NIC) in 50 mL of absolute ethanol solution, and stir for 24 h. Centrifugation was carried out at 9000 r / min for 10 min, and washed several times with deionized water and absolute ethanol to remove excess pesticide residues, and vacuum dried for 24 h to obtain NIC@MSN-SS-COOH. Then, according to 1.1.3 the steps were carried out to obtain NIC@MSN-SS-CMCS. Matrine (Matrine, MT) was carried out according to the same steps to obtain MT@MSN-SS-CMCS.

[0047] 1.1.5 Synthesis of NIC@MSN

[0048] Weigh 100 mg of MSN, and then add it to a solution of 100 mg of nicotine (Nicotine, NIC) in 50 mL of absolute ethanol solution, and stir for 24 h. Centrifugation was carried out at 9000 r / min for 10 min, and washed several times with deionized water and absolute ethanol to remove excess pesticide residues, and vacuum dried for 24 h to obtain NIC@MSN. Matrine (Matrine, MT) was carried out according to the same steps.

[0049] 1.1.6 The pH value in the body of the rice water weevil is acidic, ranging from 6 to 6.5

[0050] Grind the rice water weevil with an appropriate amount of PBS buffer solution with a pH of 7.0, and use a pH test paper to detect. The measured result shows that the pH is in the range of 6.0 - 6.5.

[0051] 1.1.7 The rice water weevil has abundant glutathione in its body.

[0052] According to the detection kit for the content of reduced glutathione (GSH), based on the concentration (x, μg / mL) and absorbance (ΔA standard, y) of the standard tubes, establish a standard curve. According to the standard curve, substitute ΔA (y, ΔA) into the formula to calculate the sample concentration (x, μg / mL). The glutathione standard curve equation is Y = 0.0026X - 0.00295.

[0053] Take 0.0025 g of rice water weevil and add 1 mL of reagent one for homogenization and grinding. After taking the supernatant, operate according to the determination steps of the kit. The calculated ΔA = A determination - A blank = 0.0338 is obtained by measuring with a 96 - well plate. For the standard curve y = 0.0026x - 0.00295, x = 13.65. Calculated according to the sample mass: GSH content (μg / g mass) = x ÷ W = 5460 μg / g mass.

[0054] Glutathione widely exists in animals and plants and plays an important role in organisms. Its content is very high in baker's yeast, wheat germ, and animal liver, reaching 1 - 10 mg / 1 g. In human blood, it contains 0.26 - 0.34 mg / 1 g, in chicken blood, it contains 0.58 - 0.73 mg / 1 g, and in pig blood, it contains 0.01 - 0.15 mg / 1 g. By comparison with the above, the glutathione content in the rice water weevil is 5.46 mg / g, indicating a relatively high content.

[0055] 1.2 Characterization of nanocarrier materials

[0056] 1.2.1 Scanning electron microscopy and transmission electron microscopy detection

[0057] Use a scanning electron microscope (SEM, Zeiss Gemini Sigma 300, Nova450; FEI - F50) and a transmission electron microscope (TEM, FEI - Talos F2000S) to observe the morphological structure of the nanoparticles, and use ImageJ software to measure the particle size of the nanomaterial samples in the SEM images.

[0058] 1.2.2 Fourier transform infrared spectroscopy detection

[0059] Using the potassium bromide tablet pressing method, the Fourier transform infrared spectrometer (FTIR, IRAffinity-1s) was used to analyze the changes in the functional groups of the carrier materials MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS, and the spectral range was 400-4000 cm -1 .

[0060] 1.2.3 X-ray photoelectron spectroscopy detection

[0061] The X-ray photoelectron spectrometer (XPS, Kratos Axis Supra TM) was used to detect the elements and atomic valence state compositions present in the nanomaterials MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS, and the change in the sulfur content of the elements on the sample surface was concerned.

[0062] 1.2.4 N2 adsorption-desorption specific surface area detection

[0063] Using a fully automatic specific surface area and porosity analyzer (BET-N2, Micromeritics ASAP2460 Version 3.01), N2 was used as the analysis gas and its saturation pressure was measured. By measuring the adsorption volume at different adsorption pressures, the nitrogen adsorption-desorption isotherm was obtained. Then, the specific surface area, pore size, and pore volume of the nanomaterial sample, as well as the pore volume and pore size distribution, were calculated using the Barrett-Emmett-Teller (BET) equation and the Barett-Joyner-Halanda (BJH) equivalent cylinder model.

[0064] 1.2.5 Thermogravimetric analysis test

[0065] Thermogravimetric detection was carried out using a thermogravimetric analyzer (TGA, Netzsch TG209) to investigate the modification rate and thermal stability of the carrier. The entire detection was carried out in an N2 environment, and the heating rate and temperature range were set to 10 °C / min and 25-800 °C, respectively.

[0066] 1.3 Nanopesticide performance evaluation

[0067] 1.3.1 Standard curve drawing

[0068] The ultraviolet spectrophotometer was used to detect the absorbance and concentration of two kinds of pesticide technicals, and a standard curve was established to prepare for the subsequent detection of the drug loading rate, encapsulation rate, and in vitro sustained release performance of the nanocarrier sample.

[0069] Taking nicotine (NIC) as an example, the specific steps are as follows:

[0070] Weigh 4 mg of NIC, dissolve it in PBS buffer with different pH values (pH = 5.5 / 6.5 / 7.4) and make up the volume to 10 mL to prepare a stock solution with a concentration of 400 mg / L, and store it in the dark at 4 °C; then serially dilute the stock solution to prepare standard solutions with concentrations of 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / mL, and 12.5 mg / L. Using PBS buffer as a control, measure the absorbance at 259 nm with a UV-visible spectrophotometer, and plot the NIC standard curve with concentration (c, mg·L -1) as the abscissa and absorbance (A) as the ordinate to draw the standard curve, and fit to obtain the linear regression equation. The method for plotting the standard curve of MT is the same as the above steps, and the detection wavelength is 220 nm.

[0071] 1.3.2 Determination of drug loading rate and encapsulation efficiency

[0072] Using absolute ethanol as a solvent, prepare a 2 mg·mL -1 NIC solution. Inject 40 mg of MSN-SS-COOH into a sample bottle, add 20 mL of 2 mg·mL -1 NIC solution, and oscillate and adsorb it in a constant temperature water bath at room temperature for 24 h; centrifuge at 9 000 r·min -1 for 10 min, and use a UV-visible spectrophotometer to measure the concentration of unadsorbed NIC in the supernatant. MT is also detected according to the above steps, and the drug loading rate (%) and encapsulation efficiency (%) are calculated according to the following formulas.

[0073]

[0074] Note: In the formula, m0 is the added mass of NIC, g; m1 is the remaining mass of NIC in the solution, g; m2 is the mass of MSN-SS-COOH after loading NIC, g.

[0075] 1.3.3 Determination of wetting property and adhesion property

[0076] Use a contact angle measuring instrument ( DSA100) to evaluate the wetting property of the samples. Determine the wetting property of NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS on rice leaves. Fix the rice leaf firmly on a glass slide, and then drop 3 μL of NIC@MSN-SS-CMCS suspension on the surface of the rice leaf. Repeat each solution three times at different parts of the leaf, and record the change in contact angle. Operate MT@MSN-SS-CMCS, H2O, NIC@MSN, MT@MSN, NIC, and MT according to the above steps.

[0077] Weigh 5 mg each of NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS, and uniformly mix them with water to prepare an aqueous solution with a concentration of 100 mg / L. Take 20 mL as the solution required for the experiment. First, use an ultraviolet spectrophotometer to detect the absorbance A before spraying the drug. Secondly, take 10 mL of the drug solution and spray it on fresh, clean rice leaves of basically the same size. After natural air drying, soak the leaves in 10 mL of deionized water for 5 min, and use an ultraviolet spectrophotometer to measure the absorbance B of the pesticide in the water. Finally, calculate the residual rate according to the formula.

[0078]

[0079] In the formula: M A is the mass of NIC / MT at the start of spraying; M B is the mass of NIC / MT remaining on the leaves after washing with water.

[0080] 1.3.4 Effects of pH and GSH on the in vitro drug release performance

[0081] Weigh 5.0 mg of NIC@MSN-SS-CMCS and add it to a dialysis bag containing 3.0 mL of phosphate buffer solution (PBS, pH = 5.5 / 6.5 / 7.4) (the molecular weight cut-off is 8000 - 14000 Da). After placing the dialysis bag in a centrifuge tube, immediately add 30.0 mL of PBS (pH = 5.5 / 6.5 / 7.4) solution to the centrifuge tube. Finally, place the centrifuge tube containing NIC@MSN-SS-CMCS in a shaker at 200 r / min and oscillate it thermostatically for 96 h in the dark. At specified times, take out 3 mL of the release medium, measure the absorbance of NIC with an ultraviolet-visible spectrophotometer, and at the same time add 3.0 mL of fresh release medium to calculate the cumulative drug release percentage (CRP) of each sample.

[0082] Accurately weigh 5 mg of NIC@MSN-SS-CMCS and add it to a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da. Then add 3 mL of PBS buffer solution with different GSH contents (0 mM, 4 mM, and 10 mM) to it. Add the dialysis bag to 30 mL of phosphate buffer solution corresponding to the glutathione content, set the temperature at 30 °C, and take 3 mL of the sustained-release solution at fixed intervals for determination. At the same time, add 3 mL of phosphate sustained-release solution with the corresponding GSH value to detect the concentration of the pesticide in the release solution to calculate the cumulative drug release rate (CRP) of each sample. MT@MSN-SS-CMCS is subjected to pH and GSH sustained-release performance detection according to the above steps.

[0083]

[0084] Note: CRP: Cumulative release rate of the drug; Ve: Replacement volume of PBS; V0: Total volume of the release medium; C i : Concentration of the release solution at the i-th replacement sampling; M drug : Total mass of the drug carried by the nanoparticles; n: Number of times of replacing PBS.

[0085] 1.4 Toxicity determination and field application of nano-pesticides against Lissorhoptrus oryzophilus Kuschel

[0086] 1.4.1 Toxicity determination of nano-pesticides against Lissorhoptrus oryzophilus Kuschel

[0087] To determine the insecticidal activities of NIC@MSN-SS-CMCS nanoparticles and NIC@MSN against Lissorhoptrus oryzophilus Kuschel, the materials required to prepare NIC@MSN solution and NIC@MSN-SS-CMCS solution were taken to Wuhua County, Meizhou City, and prepared on-site on the day of the experiment. According to the results of the preliminary experiment, NIC@MSN-SS-CMCS and NIC@MSN were dispersed in water at five different concentrations of 800, 400, 200, 100, 50 mg / L and 1600, 800, 400, 200, 100 mg / L respectively. The rice water weevils starved for 24 h were then dispersed in petri dishes, with 10 rice water weevils and one rice seedling in each petri dish, and the rice seedlings were changed every 24 h. The rice water weevils were immersed in the solutions treated with different concentrations for 20 s using the dipping method. The control groups were treated with MSN-SS-CMCS and water. At 12 h, 24 h, 48 h, and 96 h, the rice water weevils were gently touched with a brush, and those with curled bodies or unable to move normally for 15 - 20 s were considered dead. The survival rates of adult rice water weevils were recorded, and each treatment was repeated 3 times. Finally, the corrected mortality rate of the rice water weevils after 96 h was determined and its LC 50 was calculated. MT@MSN-SS-CMCS was carried out according to the above steps.

[0088] 1.4.2 Field application of nano-pesticides

[0089] 1.4.2.1 General situation of the test site

[0090] The test was carried out in 2024 in a rice paddy field of a farmer in Nancun Village, Longcun Town, Wuhua County, Meizhou City, Guangdong Province. The test site was flat, with medium fertility, and surrounded by contiguous rice fields.

[0091] 1.4.2.2 Test agents

[0092] Table 1 Design of field efficacy test treatments for rice water weevils

[0093]

[0094] 1.4.2.2 Test methods

[0095] The experiment was designed by random block, with an area of ​​10m 2 There were 15 plots with obvious marks to distinguish them, including 4 pesticide treatments and 1 water control (the pesticide name and dosage design are shown in Table 1), and each treatment was repeated 3 times. In order to avoid marginal effects and reduce experimental errors, treatments 1-4 were numbered as one treatment, and the electric sprayer was used to apply pesticides starting on August 9, and the dosage is shown in Table 1.

[0096] 1.4.2.3 Drug efficacy investigation

[0097] A total of three surveys were conducted. The base number of rice water weevils was investigated one day before the application of pesticides (August 8), and the efficacy survey was conducted 1 day (August 10) and 7 days (August 16) after the application of pesticides. The number of live rice water weevils and the number of damaged rice leaves were counted. The survey method was parallel jump sampling. One row (or several rows) was taken every few rows in the rice field to investigate and record the number of rice water weevils and the number of damaged rice leaves. Ten clumps of rice were surveyed in each plot, and the growth of rice was observed at the same time. The insect population reduction rate, corrected control effect, rice leaf damage rate, and control effect were calculated and counted. After the percentage value of the corrected control effect was transformed by arcsine square root, the data were analyzed by multiple comparisons after Duncan's new multiple range method variance analysis using the DPS 7.05 software platform to analyze the significance of the differences between the results.

[0098]

[0099] 1.5 Safety evaluation of nanopesticides

[0100] 1.5.1 Effects of MNS-SS-CMCS on the growth of rice and seeds

[0101] The MSN-SS-CMCS nanocarriers were diluted with water to different concentrations (0 (CK), 100, 200, 400, 800, 1600 mg / L), and then filter paper and 20 seeds were coated in each culture dish. The rice seeds were soaked in MSN-SS-CMCS solution for 72 hours, with 3 replicates for each treatment. The blank control was deionized water without MSN-SS-CMCS, and the seed germination rate was checked after 72 hours. Subsequently, the root length of rice was measured after 2 weeks of hydroponics with MSN-SS-CMCS solution and deionized water of different concentrations. Similarly, rice seedlings were sprayed with MSN-SS-CMCS nanocarrier suspensions of different concentrations, and the chlorophyll content (SPAD) of each treatment was measured using a chlorophyll meter after 8 weeks.

[0102] 1.5.2 Acute toxicity test of nanopesticides on adult zebrafish

[0103] The zebrafish used were wild-type zebrafish reared in the laboratory, with a body length of 2.04 ± 1.0 cm, healthy and disease-free. Before the formal experiment, the experimental zebrafish were pre-cultured for 7 days under the same environmental conditions as during the experiment. The mortality rate of zebrafish during the pre-culture period was <5%. During the pre-culture, the zebrafish were fed once a day, and the debris in the fish tank was cleaned in time to ensure the safety of the living environment of the zebrafish. The daily light exposure was maintained for 12 h, and feeding was stopped 24 h before the formal experiment.

[0104] According to the preliminary experiment, the zebrafish were starved for 24 h before the experiment. According to the preliminary experiment, 10 zebrafish were respectively exposed to the water suspensions of NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS with concentrations of 1600, 800, 400, 200, and 100 mg·L -1 . The treatment concentrations of the NIC original drug were 0.125, 0.25, 0.5, 1.0, and 2.0 mg·L -1 , and the treatment concentrations of the MT original drug were 150, 120, 90, 60, and 30 mg·L -1 . At the same time, MSN-SS-CMCS exposure groups without NIC and MT and blank control groups were set at corresponding concentrations, and each treatment was repeated three times. 12 h after the start of the experiment, the poisoning symptoms and the number of dead fish of the experimental fish were observed and recorded, and the death conditions of the zebrafish at 12, 24 h, 48 h, 72 h, and 96 h were recorded. When the fish body showed no response when gently touched with a glass rod, it was considered dead. The dead zebrafish were quickly removed to reduce the pollution of the culture water by the dead fish. Finally, the corrected mortality rate and LC 50 of NIC, NIC@MSN-SS-CMCS and MT, MT@MSN-SS-CMCS in zebrafish at 96 h were determined.

[0105] 2. Result analysis

[0106] 2.1 Scanning electron microscopy and transmission electron microscopy analysis of nanocarriers

[0107] 2.1.1 Morphology and structure analysis

[0108] The observation results of scanning electron microscopy showed that the prepared MSN-SS-CMCS particles were generally spherical, with uniform particle size and rough surface structure, and had a dense mesoporous structure ( Figure 2 A-C). Further observation results of transmission electron microscopy showed that the particle size of MSN-SS-CMCS was relatively uniform, the mesoporous structure was clear, and the inner cavity space ( Figure 2 D-F). The average particle sizes of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS nanoparticles were 116 nm and 143 nm respectively. Due to further modification with disulfide bonds and carboxyl groups, the particle size further increased. Finally, after encapsulation with carboxymethyl chitosan, the diameter of the nanoparticles reached 168 nm ( Figure 2 G-I).

[0109] 2.1.2 Other characterization structure analysis

[0110] 2.1.2.1 Infrared spectroscopy analysis results

[0111] As can be seen from Figure 3 A, on the infrared spectrum of MSN-SH, the stretching and bending vibration absorption peaks of Si-OH are at 3392 cm -1 and 960 cm -1 , the stretching vibration absorption peaks of C-H are at 2926 cm -1 and 2858 cm -1 , and the vibration absorption peaks of Si-O-Si are at 1051 and 794 cm -1 , indicating the successful synthesis of mesoporous silica. In addition, the characteristic absorption peak of -SH is at 2565 cm -1 , indicating the successful modification of mercapto groups. As can be seen from the infrared spectrum of MSH-SS-COOH, the stretching and bending vibration absorption peaks of Si-OH are at 3425 cm -1 and 960 cm -1 , the stretching vibration absorption peak of C-H is at 2928 cm -1 . Due to the introduction of disulfide bonds and carboxyl groups, the mercapto characteristic peak disappears, and the stretching vibration absorption peak of C=O on the carboxyl group appears at 1627 cm -1 , and the vibration absorption peaks of Si-O-Si on mesoporous silica are at 1074 and 796 cm -1 .

[0112] As can be seen from the infrared spectrum of CMCS, the stretching vibration absorption peaks of N-H and O-H are at 3411 cm -1 , the stretching vibration absorption peak of C-H is at 2914 cm -1 , the stretching vibration absorption peak of C=O on the carboxyl group appears at 1618 cm -1 , the bending vibration absorption peak of -CH2- is at 1426 cm -1 , the stretching vibration absorption peak of C-O-C is at 1127 cm -1 , the in-plane bending vibration absorption peak of C-H is at 1059 cm -1 , and the out-of-plane bending vibration absorption peak of C-H is at 705 cm -1 .

[0113] As can be seen from the infrared spectrum of MSH-SS-CMCS, the stretching and bending vibration absorption peaks of Si-OH are at 3425 cm -1 and 959 cm -1 , and at the same time, at 3425, 2930 cm -1This is the stretching vibration absorption peak of C-H on MSH-SS-CMSS. 1657 cm -1 The absorption peak at this position is the stretching vibration of C=O (amide I band), 1573 cm -1 The absorption peak at this position is the stretching vibration of N-H (amide II band), 1413 cm -1 The absorption peak at this position is the stretching vibration of C-N (amide III band), indicating the successful introduction of CMCS. 1077 and 799 cm -1 This is the vibration absorption peak of Si-O-Si on mesoporous silica.

[0114] 2.1.2.3 X-ray photoelectron spectroscopy (XPS) spectrum analysis

[0115] Figure 3 It can be seen from the C 1s spectrum of B that there are mainly C-C (284.8 eV) and C-S (286.0 eV) bonds on MSH-SH. Among them, C-C mainly comes from the inevitable foreign contaminant carbon in the test, and the C-S bond comes from the grafting of -SH bond to MSN. Due to the introduction of COOH, a C=O bond appears at the 284.5 eV position in MSN-SS-COOH. Due to the modification of CMCS, a C-N bond (285.5 eV) appears on MSN-SS-CMCS.

[0116] Figure 3 It can be seen from the C S 2p spectrum that there is a C-S bond at 163.5 eV, and an R-SH bond (R refers to MSN) at 164.7 eV. After modification with 3-mercaptopropionic acid, the R-SH bond of MSN-SS-COOH disappears, and is replaced by an S=S bond at 164.9 eV.

[0117] Figure 3 It can be seen from the DXPS suvery that there are obviously four elements of Si, O, C and S on MSH-SH. The appearance of S proves the grafting of -SH to MSN. There is a peak of N 1s on MSN-SS-CMCS, which is mainly due to the introduction of N element by the modification of CMCS. These results indicate the success of the modification of the nanocarrier.

[0118] 2.1.2.4 BET-N2 adsorption and desorption result analysis

[0119] In order to determine the specific surface area and pore structure of the nanomaterials (Table 2), the synthesized nanomaterials MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS were characterized by BET. The N2 adsorption isotherm and pore size distribution of the nanomaterials are as Figure 3As shown in (E-F). From the N2 adsorption isotherm, it can be seen that for the N2 adsorption isotherms of different materials, the adsorption amount increases gently at low and medium pressure ratios, while at high pressure ratios (0.8 - 1.0), there is a sharp increase in the adsorption amount, and at the same time, an obvious adsorption hysteresis phenomenon appears. This belongs to a typical type V isotherm, indicating that micropores and mesopores exist in these nanomaterials. This phenomenon is particularly obvious in the MSN-SS-CMCS material, suggesting that its pore structure is more abundant. As can be seen from Table 3-1, the BET specific surface areas of MSN, MSN-SS-COOH, and MSN-SS-CMCS are 86.3769 m 2 / g, 37.8492 m 2 / g, and 120.1512 m 2 / g respectively, indicating that different synthesis methods have a significant impact on the specific surface area of the products. Among them, MSN wrapped with carboxymethyl chitosan has the largest specific surface area, which is beneficial to improving the performance of the nanomaterials.

[0120] Table 2 Specific surface area, pore volume, and average pore diameter of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS

[0121]

[0122] The results of the pore size distribution further show that the pore diameters of the MSN-SH and MSN-SS-COOH samples are mainly concentrated between 20 - 30 nm, mainly being mesoporous structures, while the pore diameters of the MSN-SS-CMCS sample are distributed below 20 nm, indicating that it has a hierarchical porous structure of micropores and mesopores, which is beneficial to improving the activity of the material. Moreover, from the distribution of their pore volumes, that of MSN-SS-CMCS is 0.4736 cm 3 / g, much higher than 0.1540 cm 3 / g of MSN-SS-COOH, indicating that MSN wrapped with carboxymethyl chitosan has a more abundant pore structure. From the analysis of the specific surface area and pore structure, it can be concluded that the existence of the rich pore structure and the large specific surface area of MSN-SS-CMCS are beneficial to improving the reaction activity of nano-pesticides.

[0123] 2.1.2.5 Results of thermogravimetric analysis

[0124] The thermal stabilities of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS were analyzed by a thermogravimetric analyzer as shown in Figure 3(G-H). It can be seen from the TG-DTG curves that mass loss occurred in all three samples in the temperature range of 25-150 °C, which was mainly caused by the evaporation of adsorbed water on their surfaces. When the temperature rose to 150 °C, all the above-mentioned moisture substances volatilized completely, and a small weight loss peak also confirmed this point. Continuing to increase the temperature caused further weight loss in the three samples. The continuous weight loss in the range of 150-500 °C was due to the decomposition of the substances grafted on the surfaces of the three samples. The relatively large decomposition rate of MSN-SH in the DTG curve may be directly related to the decomposition of the mercapto groups connected to its surface. The double peaks appearing in the DTG curve of MSN-SS-COOH were due to the decomposition of the disulfide bonds and carboxyl groups on its surface at high temperatures. The significantly reduced maximum decomposition rate of MSN-COOH indicated that the disulfide bonds had a positive effect on thermal stability. The maximum decomposition rate of MSN-SS-CMCS was significantly less than the previous two, which may be because CMCS is rich in carbon elements, and the formation of a dense carbon layer structure was promoted during the pyrolysis process, reducing the thermal-triggered mobility of the molecular chains and thus hindering thermal degradation. After 500 °C, the weight loss of the three samples was slow, which was because the solid coke and inert residues formed during the pyrolysis process were thermally stable. In addition, at 800 °C, the undegraded residues of the three samples were all above 58%, and MSN-SS-CMCS reached 71%. Therefore, MSN-SS-CMCS has excellent thermal stability. 2.2 Detection of nano-pesticide performance

[0125] 2.2.1 Standard curve and drug loading rate

[0126] The drug loading rate and encapsulation efficiency experiments were carried out through the standard curve ( Figure 4 、 5 ). The drug loading rate of NIC@MSN-SS-CMCS was measured to be 42.27%, and the encapsulation efficiency was 73.22%; the drug loading rate of MT@MSN-SS-CMCS was 41.86%, and the encapsulation efficiency was 71.99% (Table 3). The drug loading and drug encapsulation of the nano-carriers were good.

[0127] Table 3 Drug loading rate and encapsulation efficiency of nano-carriers

[0128]

[0129] 2.2.2 Wettability and adhesion performance

[0130] The wettability of pesticides is usually evaluated by the contact angle and adhesion performance, which is also an important index for evaluating whether plant-derived pesticides can be popularized and applied in agricultural practice. The contact angle is mainly used to judge whether a liquid can wet rice, and the adhesion performance test mainly simulates the proportion of pesticides remaining on rice leaves under rain washing.

[0131] At Figure 6Among them, the contact angle of water on rice leaves is 125.4°, and its contact angle is greater than 90°, indicating that the surface of rice leaves is a hydrophobic structure. The contact angle of the NIC aqueous solution with the leaves is 44.9°, and the nicotine solution can wet and adhere to the rice leaves. The contact angles of MSN@NIC and NIC@MSN-SS-CMCS on rice leaves are 83.3° and 85.8°, respectively, both within the range of 0° to 90°, indicating that the nano-carrier encapsulated nicotine can wet on rice leaves.

[0132] The contact angle of the MT aqueous solution with the leaves is 73.6°, indicating that the matrine aqueous solution can wet on rice leaves. The contact angles of MT@MSN and MT@MSN-SS-CMCS on rice leaves are 52.5° and 80.1°, respectively, both less than 90°, indicating that the use of nano-carriers to encapsulate nicotine can wet on rice leaves, but the contact angle increases slightly after the MSN-SS-CMCS carrier encapsulates MT.

[0133] MSN-SS-CMCS is a solid powder, which will increase the contact angle, but the carrier can effectively reduce the surface tension of water, thereby improving the adhesion performance of pesticides.

[0134] The results of the adhesion performance test show that ( Figure 7 ), the remaining rates of using MSN-SS-CMCS to load NIC and MT are increased from the original 4.93% and 5.36% to 56.25% and 26.39%, respectively, indicating that the MSN-SS-CMCS carrier can greatly improve the adhesion performance of pesticides on rice leaves and reduce the drift of pesticides caused by weather such as wind and rain and environmental factors.

[0135] 2.2.3 pH and GSH Sustained Release Performance

[0136] Figure 8 As shown, the sustained release curves of NIC / MT@MSN-SS-CMCS in different pH and GSH environments

[0137] By evaluating the cumulative release rate of NIC@MSN-SS-CMCS nanoparticles in different pH environments, it was found that pH value and GSH content play crucial roles in pesticide release. In the initial stage of drug release, the pore volume of the nano-silica particles is large. Due to the gradual decrease in the drug concentration difference, with the extension of time, the cumulative release rate first gradually increases and then tends to be stable. When pH = 5.5, the highest cumulative release rate of NIC is about 12.59%, and the initial release rate is significantly higher than that at pH = 6.8 and 7.4. At the same time, the highest cumulative release rate at pH values of 6.8 and 7.4 does not exceed 7.2%. For MT@MSN-SS-CMC within the first 96 hours of release, under different pH conditions (5.5, 6.5, 7.4), the cumulative release rates of MT are 27.29%, 9.51%, and 4.57% respectively. The drug release rate of MT is better than that of NIC under different pH environmental conditions. There are obvious differences in the same drug among different pH values, and this difference can be attributed to the protonation of the -NH2 groups in CMCS, resulting in the formation of -NH3 + groups. As the pH value decreases, the degree of protonation increases, resulting in an enhanced electrostatic repulsion between molecular chains, the mesoporous channels are opened, and the release of NIC is promoted. However, due to the gradual decrease in the drug concentration difference inside and outside the carrier, the drug release rate gradually slows down, and at the same time, as the disulfide bond breaks, the process gradually tends to equilibrium.

[0138] It can be seen from Figure 8 that in the initial stage of drug release, the cumulative release rate changes little. However, after 8 hours, the treatment group with a glutathione content of 10 mM has a higher cumulative release rate, up to 64.74% at most. This is because in the initial stage of drug release, the disulfide bond is not completely broken, and the groups contained in methyl chitosan are not reacted, resulting in a low release rate. But as time goes by, the disulfide bond gradually breaks, and the drug is gradually released. Without the addition of glutathione, the release rate of MT pesticide is even less than 10.45%. In contrast, after 4 mM and 10 mM of glutathione, the release rates of MT@MSN-SS-CMCS reach 51.84% and 61.29% respectively. Moreover, when the glutathione concentration increases to 4 mM and 10 mM, the release rate of MT instantaneously reaches more than 35% at the same time. The above results may be because in the presence of glutathione, a substitution reaction occurs between the disulfide bond grafted on the surface of MSN and GSH, further destroying the organosilicon skeleton and releasing a large number of MT molecules. It shows that the introduction of glutathione can improve the drug release rate, and at the same time, it also shows that the carrier has good redox properties. When the glutathione concentration increases to 10 mM, the release rate of MT is higher than that at 4 mM at the same time, indicating that a richer glutathione is more likely to release MT.

[0139] In summary, the drug-loading system has good pH responsiveness and excellent redox properties. Since the body of the rice water weevil is acidic and rich in GSH, this characteristic can be utilized for sustainable and precise insecticidal action, thus achieving a good insecticidal effect.

[0140] 2.3 Toxicity determination and field application of nano-pesticides against the rice water weevil

[0141] 2.3.1 Toxicity determination of nano-pesticides against the rice water weevil

[0142] Through t-test, it was found that the pesticides encapsulated by the modified nano-carriers ( Figure 9 ), namely NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS, had significantly higher insecticidal effects on the rice water weevil than the ordinary nano-carriers, namely NIC@MSN and MT@MSN, at the same concentration treatment. After 96 h, the corrected average mortalities of the two nano-pesticides, NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS, at a concentration of 800 mg / L were 82.59±10.9% and 85.93±5.13% respectively. After calculation and analysis by Graphpad Prism 8, the LC 50 of NIC@MSN-SS-CMCS was 117.6 mg / L, and the LC 50 of NIC@MSN = 453.6 mg / L. The efficacy of NIC loaded on the modified nano-carrier was 3.86 times higher than that of NIC loaded on ordinary MSN. The LC 50 of MT@MSN-SS-CMCS was 82.94 mg / L, and the LC 50 of MT@MSN = 557.4 mg / L. The efficacy of MT loaded on the modified nano-carrier was 6.72 times higher than that of MT loaded on ordinary MSN. From the above results, it can be seen that the effect of reducing the dosage and increasing the efficacy of the modified nano-carrier is very significant. Due to the promoting permeability, adhesiveness and stability of CMCS, the loaded drugs can more easily penetrate the outer shell of the target pests, and then reach the effective insecticidal concentration through the dual environmental responsiveness to pH and GSH, improving the efficacy of pesticides while avoiding unnecessary drug waste.

[0143] 2.3.1 Field application of nano-pesticides

[0144] According to Table 4, 1 day after pesticide application, the population reduction rate and corrected control efficacy of the NIC treatment group against Lissorhoptrus oryzophilus Kuschel were the highest, reaching 37.94% and 42.02% respectively. Followed by the MT@MSN-SS-CMCS treatment group, with a population reduction rate and corrected control efficacy of 36.67% and 40.95% respectively. The MT treatment group had the lowest population reduction rate and corrected control efficacy, which were 20.63% and 25.79% respectively. The corrected control efficacy of the MT treatment group was significantly lower than that of the MT@MSN-SS-CMCS and NIC treatment groups.

[0145] 7 days after pesticide application, the population reduction rate and corrected control efficacy of the NIC@MSN-SS-CMCS treatment group against Lissorhoptrus oryzophilus Kuschel were the highest, reaching 93.44% and 98.05% respectively; followed by the MT@MSN-SS-CMCS treatment group, with a population reduction rate and corrected control efficacy of 93.33% and 97.63% respectively; the NIC treatment group had the lowest population reduction rate and corrected control efficacy against Lissorhoptrus oryzophilus Kuschel, which were 77.46% and 76.04% respectively. By comparing each treatment group, it was found that the population reduction rate of the modified nano-pesticide was higher than that of the original drug group, and the corrected control efficacy of NIC@MSN-SS-CMCS was significantly higher than that of the original drug group (P < 0.05). There was no significant difference in the corrected control efficacy between the IMI@MSN-SS-CMCS and IMI treatment groups.

[0146] Thus, it can be seen that the modified nano-pesticide can also continuously and effectively control Lissorhoptrus oryzophilus Kuschel in the experimental field, reducing the damage of Lissorhoptrus oryzophilus Kuschel. Using the modified nano-pesticide can effectively reduce the usage amount of the original drug, and reduce the pollution and toxicity of pesticides to the environment.

[0147] Table 4 Population reduction rate and control effect of Lissorhoptrus oryzophilus Kuschel before and after pesticide application

[0148]

[0149] Note: Each data is the average value obtained after 3 repetitions. The same marked letters after the data in the same column in the table indicate no significant difference, and different marked letters indicate significant difference. Among them, lowercase letters indicate the significant level (P < 0.05), and uppercase letters indicate the extremely significant level (P < 0.01).

[0150] According to Table 5, 1 day before application, the damage rate of rice water weevils on rice leaves was 29.70% to 34.53%, and the degree of damage was moderate (++). 1 day after application, the damage rate of rice water weevils on rice leaves did not change significantly, and the control effect of NIC@MSN-SS-CMCS was better, at 15.46%; NIC had the worst control effect, at 13.31%. 7 days after application, the damage rate of rice water weevils on leaves was 13.23% to 16.37%, and the degree of damage was reduced to slight (+). The control effects of NIC@MSN-SS-CMCS, NIC, MT@MSN-SS-CMCS, and MT on rice water weevils on leaves were 64.45%, 56.68%, 60.90%, and 54.86%, respectively, and there was no significant difference among the four treatments.

[0151] Comparison of the results 1 day before and 7 days after application showed that the rate of rice water weevils damaging rice leaves decreased significantly. The main reason is that spraying the pesticide curbed the damage of rice water weevils, resulting in a decrease in the number of damaged leaves. The experiment also paid attention to the growth of rice in the treatment group and the control group on each efficacy investigation day. There was no significant difference in visual observation between the two, and there was no phenomenon of leaf curling, leaf burning, yellowing, etc., which showed that the dosage of the above four groups of treatment pesticides was safe.

[0152] Table 5 Damage rate of rice water weevils leaves and control effect after application

[0153]

[0154] Note: All data are the average values ​​obtained after 3 repetitions. The same letters after the data in the same column in the table indicate that there is no significant difference, and different letters indicate that there is a significant difference. Lowercase letters indicate significant levels (P<0.05), and uppercase letters indicate extremely significant levels (P<0.01).

[0155] 2.4 Safety evaluation

[0156] 2.4.1 Rice crops

[0157] The determination of plant growth indicators that are sensitive to environmental changes can be used to evaluate the safety of the vector MSN-SS-CMCS on rice growth. Figure 10Shows the effects of MSN-SS-CMS on the germination rate (A) and root length (B) of rice. After 72 h of treatment, in the control group (0 mg / L), the germination rate of rice was 76.67%, and there was no significant difference compared with that of rice treated with different concentrations of MSN-SS-CMCS. After 2 weeks of treatment, the root length of rice in the control group was 4.87 cm, and the root length of rice in the treatment group was in the range of 4.53 - 5.53 cm, with no significant difference. The SPAD values (an index reflecting chlorophyll content) of rice treated with different concentrations of MSN-SS-CMCS were in the range of 36.77 - 39.7, and the SPAD value of rice in the control group was 37.87, showing no significant statistical difference. These data results indicate that the MSN-SS-CMCS nanocarrier has no adverse effects on the growth and development of crops, confirming its good safety.

[0158] 2.4.2 Aquatic organism zebrafish

[0159] In the environmental risk assessment of pesticides in water, zebrafish is an important biological test material for acute toxicity tests of pesticides on fish. Figure 11 Shows the acute toxicity of NIC and NIC@MSN-SS-CMCS, as well as MT and MT@MSN-SS-CMCS to zebrafish under 96 h of exposure time. After 96 h, the LC 50 values of NIC and NIC@MSN-SS-CMCS were 0.74 mg / L and 516.02 mg / L respectively, and the LC 50 values of MT and MT@MSN-SS-CMCS were 284.44 mg / L and 847.43 mg / L respectively. According to the acute toxicity classification of pesticides to fish, the LC 50 of NIC to zebrafish was less than 1 mg / L at 96 h, being highly toxic (high toxicity: LC 50 <1 mg / L), while the LC 50 of NIC@MSN-SS-CMCS, MT, and MT@MSN-SS-CMCS to zebrafish at 96 h was all greater than 10 mg / L, indicating low toxicity (low toxicity: LC 50 >10 mg / L). By comparing the LC 50 of the original drug and the nano-pesticide, we can find that the toxicity of the nano-pesticide is much less than that of the original drug, and the nano-pesticides NIC@MSN-SS-CMCS and MT@MSN-SS-CMCS have good safety for zebrafish.

Claims

1. Application of a dual-controlled release plant-derived nano-pesticide with dual responsiveness to pH and redox in controlling Lissorhoptrus oryzophilus Kuschel.

2. The application according to claim 1, wherein The dual-controlled release plant-derived nano-pesticide with dual responsiveness to pH and redox is prepared by the following method: Dissolve cetyltrimethylammonium bromide in water, adjust the pH value to alkaline, then add tetraethyl orthosilicate and (3-mercaptopropyl)trimethoxysilane for reaction. After the reaction is completed, wash with methanol and deionized water in sequence, and freeze-dry to obtain a dry powder. Redisperse the dry powder in a methanol-hydrochloric acid mixed solution, reflux and react under a N2 atmosphere to remove the CTAB template, and then obtain MSN-SH after centrifugation, washing and drying. React MSN-SH with 2,2′-dithiopyridine under a N2 atmosphere and in an acidic environment. Wash the centrifuged precipitate with methanol and deionized water to obtain MSN-SS-Py. Disperse MSN-SS-Py in DMF containing acetic acid, then add 3-mercaptopropionic acid for reaction. Wash the product with methanol and deionized water to obtain MSN-SS-COOH. Mix MSN-SS-COOH with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, disperse them in PBS for reaction to activate the hydroxyl group, then add carboxymethyl chitosan and react again. After centrifugation, washing and vacuum drying, obtain MSN-SS-CMCS. Then load the pesticide with MSN-SS-CMCS to obtain the dual-controlled release plant-derived nano-pesticide.

3. The application according to claim 2, characterized in that, The pesticide is nicotine (NIC) or matrine (MT).

4. The application according to claim 3, characterized in that The specific steps for loading the pesticide with MSN-SS-CMCS are as follows: Dissolve MSN-SS-CMCS, nicotine or matrine in absolute ethanol, react, then centrifuge. Wash the precipitate with deionized water and absolute ethanol, and vacuum dry to obtain NIC@MSN-SS-COOH. Mix NIC@MSN-SS-COOH with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, disperse them in PBS for reaction to activate the hydroxyl group, then add carboxymethyl chitosan and react again. After centrifugation, washing and vacuum drying, obtain NIC@MSN-SS-CMCS or MT@MSN-SS-CMCS.

5. The application according to claim 1, wherein The application of the dual-controlled release plant-derived nano-pesticide loaded with the pesticide in controlling Lissorhoptrus oryzophilus Kuschel promoted by the pH of 6 - 6.5 and abundant glutathione in the body of Lissorhoptrus oryzophilus Kuschel.

6. A dual-controlled release plant-derived nano-pesticide with dual responsiveness to pH and redox prepared by the preparation method described in claim 2.

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