Preparation method of mesoporous selenium nano-pesticide

Abstract

The present application relates to a kind of mesoporous selenium nano pesticide preparation method, belong to the technical field of nano pesticide.The mesoporous selenium nano pesticide is black nanoparticle, and the effective particle size of nanoparticle is 150-200 nm, and drug loading rate is 47.49±2%, and encapsulation rate is 91.71±4%;For plant fungal disease.Preparation method: with mesoporous nano selenium as carrier, pyrithiobac-acid fungicide as loaded drug, and hydroxypropyl cellulose as end-capping agent.The present application is coupled with hydroxypropyl cellulose, and the oxalic acid produced by fungal metabolism and plant cell wall degrading enzyme are used as nano-carrier response switch, to control the release rate and release amount of active ingredient pyrithiobac-acid under the action of internal and external environment, specifically, after 96 h at pH=3, 5 and containing cellulase, the cumulative release rate of pyrithiobac-acid is 82.32%, 68.53% and 79.5% respectively.The active ingredient is released on demand during fungal infection process, and the inhibition rate of harmful bacteria is improved.The present application provides a new idea for the development of functionalized nano selenium in the treatment of plant fungal disease.

Description

Technical Field

[0001] This invention belongs to the field of nanopesticide technology and discloses a method for preparing mesoporous selenium nanopesticide particles and their application. Technical Background

[0002] Fungal diseases are the most numerous known plant diseases, accounting for approximately 80%-90% of all plant diseases. Among all types of diseases, fungal diseases exhibit the most diverse symptoms, appearing in various parts of the plant and severely impacting the growth and yield of vegetables, fruits, and flowers. They commonly affect over 6 billion acres annually. Anthracnose fungi, for example, use an in vitro oxalate-acetate hydrolase to convert oxalate-acetate into pyruvate and oxalic acid, acidifying the surrounding environment. Then, various extracellular enzymes secreted by their spores, including pectinase, protease, and laccase, are activated by oxalic acid, causing damage to the host plant. Although chemical pesticides are the primary treatment strategy for plant diseases, their utilization rate is generally only 10%, with approximately 90% remaining in the environment, causing pollution. Large amounts of released chemical pesticides volatilize into the air, flow into water bodies, and accumulate in the soil, contaminating agricultural, livestock, fishery, and fruit products. Furthermore, they can be transferred to humans through bioaccumulation in the food chain, posing a health risk.

[0003] Over the past few decades, nanotechnology has been extensively studied to assess its potential applications in promoting efficient and sustainable agriculture. Nanopesticides are considered a promising alternative to traditional chemical and biological pesticides because they can be designed as smart pesticides that respond to environmental stimuli, enabling on-demand pesticide release to control pests and diseases, thereby reducing the amount of chemical pesticides used and mitigating environmental risks. Studies have shown that nano-selenium can inhibit fungal spore germination, exhibiting antifungal activity, and the application of selenium in agriculture has been studied for decades; it is an element beneficial to plant growth and development. Therefore, to improve the targeting and bioavailability of pesticides, there is a need to develop biofriendly, responsive, high-load-bearing selenium nanopesticides. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a method for preparing a mesoporous selenium nanopesticide, which is a water-soluble black powdery solid particle.

[0005] Another objective of this invention is to provide the application of the above-mentioned mesoporous selenium nanopesticide in pesticides for treating plant fungal diseases.

[0006] A mesoporous selenium nanopesticide uses mesoporous selenium nanoparticles as a carrier, pyrimethanil fungicide as the loaded drug, and hydroxypropyl cellulose as a capping agent.

[0007] The mesoporous selenium nanopesticide is in the form of black nanoparticles with an effective particle size of 150-200 nm, a pesticide loading rate of 47.49±2%, and an encapsulation efficiency of 91.71±4%.

[0008] The mesoporous selenium nanopesticide is used for plant fungal diseases.

[0009] The plant fungal diseases mentioned include: anthracnose, sclerotinia rot, and sheath blight.

[0010] The preparation steps of a mesoporous selenium nanopesticide are as follows:

[0011] (1) Preparation of mesoporous selenium nanocarrier particles

[0012] Hexadecyltrimethylammonium bromide was dissolved in pure water at a weight-to-volume ratio of 1 g: 200 mL. Zinc powder, sulfur-modified polyethylene glycol, chitosan solution with a mass-to-volume concentration of 5 mg / mL, and sodium selenite were added, and the mixture was stirred thoroughly for 2 h. Ascorbic acid was added dropwise, and the pH was adjusted to 7.4 with sodium hydroxide. The mixture was stirred for 6 h. After the first centrifugation, the precipitate was placed in a 5% hydrochloric acid aqueous solution and refluxed at 80 °C for 12 h. After the second centrifugation at 10,000 rpm for 10 min, the precipitate was washed three times with pure water and freeze-dried to obtain mesoporous selenium nanocarrier particles.

[0013] The mass ratio of hexadecyltrimethylammonium bromide to zinc powder is 8:1;

[0014] The mass ratio of hexadecyltrimethylammonium bromide to sulfur-modified polyethylene glycol is 16:1;

[0015] The mass ratio of hexadecyltrimethylammonium bromide to sodium selenite is 4:1;

[0016] The mass ratio of chitosan to sodium selenite is 1:40;

[0017] The mass ratio of ascorbic acid to sodium selenite is 4:1;

[0018] (2) Preparation of pyrimethanil-loaded mesoporous selenium particles

[0019] Mesoporous selenium nanoparticles were dispersed in acetone and stirred thoroughly for 30 min. 0.2 g of succinic anhydride was added, and the mixture was stirred for 24 h. The mixture was centrifuged for 10 min at 10,000 rpm for the first time, and the precipitate was washed three times with pure water and dried. The dried precipitate was dissolved in pure water, and pyrimethanil was added and stirred for 12 h. The mixture was centrifuged for 10 min at 10,000 rpm for the second time, and the supernatant was removed. The precipitate was washed three times with pure water and lyophilized to obtain pyrimethanil-loaded mesoporous selenium particles.

[0020] The mass-to-volume ratio of the mesoporous selenium nanocarrier particles to acetone was 4 mg: 1 mL.

[0021] The mass ratio of the mesoporous selenium nanocarrier particles to pyraclostrobin is 1:1.

[0022] The mass-to-volume ratio of the dried precipitate to pure water is 1 mg / 2 mL;

[0023] (3) Preparation of mesoporous selenium nanopesticides

[0024] The mesoporous selenium particles loaded with pyraclostrobin were dispersed in citrate buffer at pH 5.0 to obtain a suspension. 1 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide with a volume concentration of 1.915 mg / mL and 1 mL of N-hydroxysuccinimide with a volume concentration of 1.15 mg / mL were slowly added, and the mixture was stirred thoroughly for 4 h. Hydroxypropyl cellulose aqueous solution was added dropwise, and the mixture was stirred thoroughly for 24 h. The mixture was centrifuged at 10000 rpm for 10 min, the supernatant was removed, the precipitate was washed three times with pure water, and then lyophilized to obtain black nanoparticle-like mesoporous selenium nanopesticide.

[0025] The mass-to-volume ratio of the loaded pyrimethanil mesoporous selenium particles to the citrate buffer was 5 mg: 1 mL.

[0026] The mass ratio of the loaded pyrimethanil mesoporous selenium particles, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 10:1.915:1.15.

[0027] The mass ratio of the loaded pyrimethanil mesoporous selenium particles to hydroxypropyl cellulose is 1:2.

[0028] The further defined technical solution is as follows:

[0029] In step (1), the chitosan solution with a mass-volume concentration of 5 mg / mL is in the form of an aqueous solution of acetic acid with a volume concentration of 1%.

[0030] Compared with the prior art, the beneficial technical effects of the present invention are reflected in the following aspects:

[0031] 1. This invention utilizes mesoporous selenium nanoparticles as a carrier, pyrimethanil fungicide as the loaded drug, and hydroxypropyl cellulose as a capping agent to develop and synthesize novel mesoporous selenium nanoparticle pesticide particles. The mesoporous characteristics of the mesoporous selenium nanocarrier significantly improve pesticide utilization, primarily manifested in a drug loading rate of 47.49±2% and an encapsulation efficiency of 91.71±4%, which greatly reduces the amount of fungicide used. Furthermore, by coupling with hydroxypropyl cellulose, oxalic acid produced by fungal metabolism and plant cell wall degrading enzymes are used as nanocarrier response switches, controlling the release rate and amount of the active ingredient pyrimethanil under the influence of internal and external environments. Specifically, the cumulative release rates of pyrimethanil are 82.32%, 68.53%, and 79.5% at pH=3, 5, and after 96 h with cellulase, respectively. This achieves on-demand release of the active ingredient during fungal infection, improving the inhibition rate against harmful pathogens. This invention will provide new ideas for the development of functionalized nano-selenium in the treatment of plant fungal diseases.

[0032] 2. In step (1) of preparing mesoporous selenium nanocarrier particles, the addition of chitosan enables the surface of the mesoporous selenium nanocarrier to have amino groups, thereby giving it a positive potential, which is conducive to electrostatic adsorption and connection with pyrimethanil, which has a negative potential. The precipitate obtained by centrifugation is placed in hydrochloric acid diluted with pure water to maintain the spherical morphology of sodium selenite, which is reduced by ascorbic acid, and prevent its lateral growth. In step (2) of preparing pyrimethanil-loaded mesoporous selenium particles, succinic anhydride is added to modify the mesoporous selenium nanocarrier particles. The modified carboxyl groups can form hydrogen bonds with pyrimethanil, giving the pyrimethanil-loaded mesoporous selenium particles good water solubility. In step (3) of preparing mesoporous selenium nanopesticides, hydroxypropyl cellulose is added as a capping agent for the pyrimethanil-loaded mesoporous selenium particles. This is because fungal metabolism produces oxalic acid and cell wall invertase, so under acidic conditions, hydroxypropyl cellulose can be cleaved by enzymatic hydrolysis and the ester bonds can be destroyed to release the pesticide and carrier particles. Attached Figure Description

[0033] Figure 1 Transmission electron microscopy (TEM) images of the mesoporous selenium nanocarrier particles, pyrimethanil-loaded mesoporous selenium particles, and mesoporous selenium nanopesticide particles obtained in Example 1.

[0034] Figure 2 The diagram shows the physicochemical properties of the traditional nano-selenium, mesoporous selenium nanocarrier particles, pyrimethanil-loaded mesoporous selenium particles, and mesoporous selenium nanopesticides obtained in Example 1.

[0035] Figure 3 The diagram shows the structural characteristics of the mesoporous selenium nanocarrier particles, the pyrimethanil-loaded mesoporous selenium particles, and the mesoporous selenium nanopesticide obtained in Example 1.

[0036] Figure 4This is a graph showing the pesticide loading of the mesoporous selenium nanopesticide obtained in Example 1.

[0037] Figure 5 The graph shows the release curves of the mesoporous selenium nanopesticide obtained in Example 1 under different conditions.

[0038] Figure 6 The diagram shows the mycelial growth experiment of different concentrations of mesoporous selenium nanocarrier particles, pyrimethanil-loaded mesoporous selenium particles, mesoporous selenium nanopesticides, and pyrimethanil against the fungicidal activity of cucumber anthracnose fungus obtained in Example 1.

[0039] Figure 7 The diagram shows the mycelial growth experiment of different concentrations of mesoporous selenium nanocarrier particles, pyrimethanil-loaded mesoporous selenium particles, mesoporous selenium nanopesticides, and pyrimethanil against the fungicidal activity of Sclerotinia sclerotiorum var. tobaccois obtained in Example 1.

[0040] Figure 8 The diagram shows the mycelial growth experiment of different concentrations of mesoporous selenium nanocarrier particles, pyrimethanil-loaded mesoporous selenium particles, mesoporous selenium nanopesticides, and pyrimethanil against the fungicidal activity of *Rhizoctonia solani* obtained in Example 1.

[0041] Figure 9 The graph shows the inhibition rates of different concentrations of mesoporous selenium nanocarrier particles, pyrimethanil-loaded mesoporous selenium particles, mesoporous selenium nanopesticides, and pyrimethanil against three fungi obtained in Example 1. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0043] Unless otherwise specified, all reagents used in the following examples are commercially available.

[0044] Determine the complete embodiment

[0045] Example 1

[0046] The mesoporous selenium nanopesticide particles in this embodiment 1 include the drug component pyrimethanil and mesoporous selenium nanocarrier material. The pyrimethanil is loaded in the mesoporous selenium nanocarrier particles and capped with hydroxypropyl cellulose.

[0047] The specific preparation steps are as follows:

[0048] (1) Preparation of mesoporous selenium nanocarrier particles:

[0049] Dissolve 80 mg of hexadecyltrimethylammonium bromide in 16 mL of pure water and stir thoroughly for 30 min. Add 10 mg of zinc powder, 5 mg of sulfur-modified polyethylene glycol, 100 μL of chitosan solution with a volume concentration of 5 mg / mL, and 20 mg of sodium selenite, and stir thoroughly for 2 h. Add 80 mg of ascorbic acid dropwise; adjust the pH to 7.4 with sodium hydroxide, and stir for 6 h. After the first centrifugation, place the precipitate in a 5% hydrochloric acid aqueous solution and reflux at 80 °C for 12 h to remove hexadecyltrimethylammonium bromide and zinc; centrifuge a second time at 10000 rpm for 10 min, wash the precipitate three times with pure water, and freeze-dry to obtain mesoporous selenium nanocarrier particles.

[0050] In a chitosan solution with a mass-volume concentration of 5 mg / mL, the solvent is an aqueous solution of acetic acid with a volume concentration of 1%.

[0051] (2) Preparation of pyrimethanil-loaded mesoporous selenium particles:

[0052] 10 mg of the mesoporous selenium nanoparticles were dispersed in 2.5 mL of acetone and stirred for 4 h. 0.2 g of succinic anhydride was added, and the reaction was stirred for 24 h. The mixture was centrifuged for 10 min at 10000 rpm, and the precipitate was washed three times with pure water and dried. The dried precipitate was dissolved in pure water, and pyrimethanil was added and stirred for 12 h. A second centrifugation was performed at 10000 rpm for 10 min, the supernatant was removed, and the precipitate was washed three times with pure water and lyophilized to obtain pyrimethanil-loaded mesoporous selenium particles.

[0053] (3) Preparation of mesoporous selenium nanopesticides

[0054] 10 mg of pyrimethanil-loaded mesoporous selenium particles were dispersed in citrate buffer at pH 5.0 to obtain a suspension. 1 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1.915 mg / mL) and 1 mL of N-hydroxysuccinimide (1.15 mg / mL) aqueous solution were added, and the mixture was stirred thoroughly for 4 h. 20 mg of hydroxypropyl cellulose aqueous solution was added dropwise, and the mixture was stirred thoroughly for 24 h. The mixture was centrifuged at 10,000 rpm for 10 min, the supernatant was removed, the precipitate was washed three times with pure water, and then freeze-dried to obtain black nanoparticle-like mesoporous selenium nanopesticide.

[0055] Characterization of mesoporous nano-selenium pesticide particles:

[0056] Transmission electron microscopy was performed on the mesoporous selenium nanoparticles, pyrimethanil-loaded mesoporous selenium nanoparticles, and mesoporous selenium nanoparticles prepared in Example 1.

[0057] See Figure 1 , Figure 1 In this context, A represents mesoporous selenium nanoparticles carrying drugs. Figure 1 In this context, B represents mesoporous selenium particles loaded with pyrimethanil. Figure 1 C in the text represents mesoporous selenium nanoparticles. From Figure 1 A and Figure 1 As shown in Figure B, the synthesized dendritic mesoporous selenium drug-loaded nanoparticles have a larger pore size, enabling better drug loading. Furthermore, as the drug is loaded, the pores of the mesoporous selenium drug-loaded nanoparticles are filled. However, after adding a hydroxypropyl cellulose sealing layer to the outer layer, the mesopores on the surface of the mesoporous selenium drug-loaded nanoparticles disappear. Figure 1 C in the middle.

[0058] See Figure 2 , Figure 2 In the diagram, A represents the ultraviolet absorption spectrum. Figure 2 B in the image represents the infrared absorption spectrum. Figure 2 C in the diagram represents the Zeta potential. Figure 2 D in the diagram represents the particle size distribution. See also... Figure 2 In Figure A, the UV results show that the characteristic peaks of traditional nano-selenium and mesoporous nano-selenium pesticide-loaded particles are around 270 nm, while the characteristic peak of pyrimethanil is at 227 nm. After loading with pyrimethanil and hydroxypropyl cellulose, the characteristic peaks of the mesoporous nano-selenium in the pyrimethanil-loaded mesoporous selenium particles and the mesoporous selenium nano-pesticide particles exhibit a blue shift, and the characteristic peak of pyrimethanil appears. See also... Figure 2 Further analysis of the infrared results reveals that traditional selenium nanoparticles and mesoporous selenium nanoparticles exhibit NH content at 1630 cm⁻¹. -1 The bending vibrations at the [location] indicate the successful preparation of amino-functionalized selenium nanoparticles. Compared with the spectra of mesoporous selenium nanoparticles loaded with drug, the [format] after loading with pyraclostrobin [is observed] at 1641-1057 cm⁻¹. -1 Multiple characteristic peaks of pyraclostrobin were observed at 3264 cm⁻¹. -1 3266 cm -1 The appearance of the characteristic peak is attributed to the stretching vibration of CH, indicating that pyrimethanil was successfully modified onto mesoporous selenium nanoparticles via electrostatic adsorption. The carboxyl-functionalized pyrimethanil-loaded mesoporous selenium particles have a peak size of 1710 cm⁻¹. −1 The expected C=O tensile vibration absorption was observed at approximately 2960 cm⁻¹, indicating that the functionalization of the carboxyl group in the loaded pyrimethanil mesoporous selenium particles was successful. Furthermore, due to the increased tensile vibration of CH in hydroxypropyl cellulose, the absorption at approximately 2960 cm⁻¹ was observed. −1 The absorption peak at this point increased significantly, indicating that hydroxypropyl cellulose was successfully coated onto the pyrimethanil-loaded mesoporous selenium particles, ultimately synthesizing mesoporous selenium nanoparticles. See also... Figure 2The C, Zeta potential results showed that, compared with traditional selenium nanoparticles (32.96 mV), mesoporous selenium nanoparticles (49.48 mV) had a higher potential. Furthermore, as pyraclostrobin was loaded onto the mesoporous selenium nanoparticles via electrostatic adsorption, its potential decreased (pyraclostrobin-loaded mesoporous selenium particles, 15.35 mV). Finally, after loading hydroxypropyl cellulose, the nanosystem became negatively potentialed (mesoporous selenium nanoparticles, -10.24 mV), which provided the conditions for its targeting of fungal cell membranes via electrostatic adsorption. See also... Figure 2 The particle size distribution results further indicate that with the loading of pyrimethanil and hydroxypropyl cellulose, the particle size of the nanosystem gradually increases, and the particle size of the prepared mesoporous selenium nanopesticide particles is around 194 nm.

[0059] See Figure 3 , Figure 3 In the figure, A represents the thermogravimetric analysis curve. Figure 3 B in the diagram represents the X-ray diffraction pattern. Figure 3 C in the diagram represents nitrogen adsorption-desorption. Figure 3 D in the figure represents the pore size distribution. The grafting amount of hydroxypropyl cellulose, the loading of pyraclostrobin, and the thermal stability were estimated using thermogravimetric analysis (TGA). See [link to TGA]. Figure 3 Figure A shows the thermogravimetric curve after heating to 800℃. The mesoporous selenium nanoparticles exhibit good stability, showing an initial weight loss at 220℃ attributed to moisture evaporation, and a second weight loss at 350℃ attributed to carbon loss. The fact that 50% remained after heating to 800℃ demonstrates its good stability. Pyrimethanil, on the other hand, lost weight at 700℃, indicating poor stability. After grafting hydroxypropyl cellulose onto the carrier using mesoporous selenium nanoparticles, hydroxypropyl cellulose experienced an initial weight loss of approximately 12%, attributed to the grafting of hydroxypropyl cellulose, the mesoporous selenium nanoparticles, and the loss of pyrimethanil. The final weight remained at 23.5%, indicating that the mesoporous selenium nanoparticles can improve the stability of pyrimethanil. See also... Figure 3 B2X-ray diffraction results showed that, compared with mesoporous selenium nanoparticles loaded with pyraclostrobin, both the mesoporous selenium nanoparticles and the mesoporous selenium nanoparticles still exhibited characteristic diffraction peaks, with a slight decrease in intensity and a slight shift to higher angles. This verified that the pyraclostrobin groups were successfully modified on the mesoporous selenium nanoparticles without disrupting the ordered two-dimensional (2D) hexagonal pores. Furthermore, the changes in the pore structure of the nanoparticles before and after loading with pyraclostrobin were investigated using BET specific surface area and BJH pore size measurements. Nitrogen adsorption-desorption isotherms ( Figure 3 C in the middle) and pore size distribution ( Figure 3The results (D) show that the mesoporous selenium nanoparticles exhibit a type IV isotherm, indicating that the mesoporous selenium nanoparticles have a typical mesoporous structure. The surface area of ​​the mesoporous selenium nanoparticles is 483 m². 2 The narrow pore size distribution, with a maximum value of 3.17 nm, indicates that the mesoporous selenium nanoparticles possess abundant pores and a large surface area, which is beneficial for loading pyraclostrobin into these particles. For mesoporous selenium nanoparticles, due to hydroxypropyl cellulose grafting and pyraclostrobin filling of the pores, both the surface area and pore size are significantly reduced to 215 nm. 2 / g and 1.64 nm.

[0060] See Figure 4 , Figure 4 In this context, A represents the standard curve for pyrimethanil. Figure 4 B in the image represents the UV spectra of pyraclostrobin at different concentrations. Figure 4 In the figure, C represents the pesticide loading and encapsulation efficiency of the mesoporous selenium nanopesticide. The results showed that the pesticide loading rate of the mesoporous selenium nanopesticide particles was 47.49±2%, and the encapsulation efficiency was 91.71±4%.

[0061] Release performance of mesoporous selenium nanoparticles for pesticides

[0062] The drug release assay for pyrimethanil was performed by detecting the absorbance of pyrimethanil molecules at different time intervals using UV-Vis spectroscopy to determine the release amount. In short, 1 mg of mesoporous selenium nanoparticles loaded with the pesticide were weighed and placed at the bottom of a MWCO 6000 dialysis bag. The bag was then immersed in beakers containing phosphate buffer solutions at pH 3, 5, and 7 at 25°C with stirring at 100 rpm to activate the hydroxypropyl cellulose control barrier. The maximum absorption wavelength of pyrimethanil was 227 nm. The amount of pyrimethanil released was determined using a standard curve constructed from the concentration of pyrimethanil dissolved in a phosphate buffer solution at pH 7 and the absorbance of pyrimethanil. Furthermore, pyrimethanil release assays were also conducted in buffer solutions with and without hydroxypropyl cellulose at pH 7.

[0063] See Figure 5 , Figure 5 In the figure, A represents the pyraclostrobin release curves at pH values ​​of 3, 5, and 7. Figure 5 Figure B shows the pyrimethanil release curves with and without cellulase. To detect the release behavior of mesoporous selenium nanoparticles under different pH conditions, the release medium was adjusted to pH = 3, 5, and 7, while maintaining a constant temperature at room temperature with a stirring rate of 100 rpm. Figure 5Figure A shows that after 96 h at pH 3, 5, and 7, the cumulative release rates of pyraclostrobin from mesoporous selenium nanoparticles were 82.32%, 68.53%, and 14.43%, respectively. These results indicate that mesoporous selenium nanoparticles can release pyraclostrobin in response to acidic pH. Furthermore, fungal metabolism produces oxalic acid and cell wall invertase, which, under acidic conditions, cleaves hydroxypropyl cellulose and breaks the ester bonds to release the pesticide. Therefore, the release behavior of mesoporous selenium nanoparticles in the presence or absence of cellulase was also investigated. Figure 5 As shown in B, in a solution containing cellulase, the cumulative release rate of mesoporous selenium nanopesticides was approximately 79.5% after 96 h, while in the absence of cellulase, the cumulative release rate was only 13.1% during the same time period.

[0064] By fitting the generalized model (M) t / M z =kt n Further analysis of the release data was conducted. As shown in Table 1, the correlation coefficients (r) ranged from 0.97 to 0.99 under different conditions, indicating a good correlation between the release data of the mesoporous selenium nanopesticide and the empirical equation. At pH 7.0, the n value for the mesoporous selenium nanopesticide was 0.46, less than 0.5, indicating that the release of pyrimethanil from the mesoporous selenium nanopesticide particles was controlled by Fickian diffusion. Under acidic conditions or in the presence of cellulase, the n values ​​were between 0.5 and 0.66, indicating that the release of pyrimethanil was driven by a combined diffusion / erosion process. The ester bond between the carboxyl groups of the pyrimethanil-loaded carboxylated mesoporous selenium particles and the hydroxyl groups of hydroxypropyl cellulose is unstable under acidic conditions, leading to the detachment of hydroxypropyl cellulose from the surface of the pyrimethanil-loaded mesoporous selenium particles; furthermore, hydroxypropyl cellulose can be hydrolyzed by cellulase into small glucose monomers, resulting in the release of pyrimethanil from the open pores. Clearly, the mesoporous selenium nanoparticles exhibit a pH / cellulase dual-stimulation response release behavior, which is a very useful feature for delivering pesticides to crops suffering from plant pathogens.

[0065]

[0066] Bioactivity of mesoporous selenium nanoparticles against plant pathogens

[0067] To determine the bioactivity of the prepared mesoporous selenium nanoparticles, pyrimethanil-loaded mesoporous selenium particles, and mesoporous selenium nanoparticles, *Sclerotinia sclerotiorum*, *Rhizoctonia solani*, and *Anthracnose causal agent of cucumber* were selected as test strains. The antifungal activity of the mesoporous selenium nanoparticles was determined using the mycelial growth rate method. Pyrimethanil technical grade was used as a control. Fungal cakes with a diameter of 8 mm were cut from the edge of 5-day-old colonies growing on potato dextrose agar plates and inoculated into the center of 90 mm diameter potato dextrose agar containing different concentrations of pyrimethanil, mesoporous selenium nanoparticles, pyrimethanil-loaded mesoporous selenium particles, and mesoporous selenium nanoparticles. The control group consisted of untreated medium. Each treatment was prepared in triplicate. The mycelia of each treatment group were grown in a constant temperature incubator at 25±1℃ under natural photoperiod. After different numbers of days of cultivation, the colony diameter of each treatment was measured using the cross-hatching method, and the fungicidal activity was determined using the relative inhibition percentage (%). The inhibition rate is calculated using the following equation, where C d It is the colony diameter of the control group, T d F is the diameter of the colonies in each treatment. d It is the diameter of the fungal filter cake.

[0068]

[0069] See Figure 6-8 Among them, different concentrations of mesoporous selenium nanoparticles loaded with pesticide, mesoporous selenium nanoparticles loaded with pyrimethanil, and mesoporous selenium nanoparticles and pyrimethanil were obtained for their effects on cucumber anthracnose ( Figure 6 ), Sclerotinia sclerotiorum ( Figure 7 ) and sheath blight pathogen ( Figure 8 ) Experimental diagram of mycelial growth to demonstrate bactericidal activity. Figure 6 The figures show the mycelial growth of *Anthracnose causal agent* on cucumber at concentrations of 10 μg / mL and 50 μg / mL on days 2, 3, 4, 5, and 7. Figure 7 Figure 1 shows the mycelial growth of *Sclerotinia sclerotiorum* at 10 μg / mL and 50 μg / mL for 24, 36, 48, 60 and 72 hours. Figure 8 The figures show the mycelial growth of *Rhizoctonia solani* on days 3, 4, 5, 6, and 9 at concentrations of 1 μg / mL and 5 μg / mL, respectively.

[0070] See Figure 9 The inhibition rates of different concentrations of mesoporous selenium nanoparticles loaded with pesticide, mesoporous selenium nanoparticles loaded with pyrimethanil, and mesoporous selenium nanoparticles and pyrimethanil against three fungi were calculated. Figure 9 A in the text represents the cucumber anthracnose fungus. Figure 9 B in the text represents Sclerotinia sclerotiorum, the causal agent of tobacco diseases. Figure 9 C in the figure represents *Rhizoctonia solani*, the pathogen causing *Rhizoctonia solani*. Images of fungal colonies in different treatment groups at concentrations of 10 μg / mL and 50 μg / mL are shown below. Figure 7 As shown, the inhibition rate was calculated by measuring the colony diameter of cucumber anthracnose bacteria on the culture medium from day 2 to day 7. Figure 9 A. The results showed that pyraclostrobin, pyraclostrobin-loaded mesoporous selenium particles, and mesoporous selenium nanopesticide particles all exhibited excellent bioactivity, with their antifungal activity against cucumber anthracnose fungus showing a dose- and time-dependent effect, increasing with increasing concentration and time. Furthermore, under the same conditions, the mesoporous selenium nanopesticide showed significantly better antifungal activity against cucumber anthracnose fungus than pyraclostrobin. On day 7, when the concentration of the active ingredient reached 50 μg / mL, the inhibition rates of pyraclostrobin, pyraclostrobin-loaded mesoporous selenium particles, and mesoporous selenium nanopesticide particles were 77.91%, 77.73%, and 89.37%, respectively. Similarly, see [reference needed]. Figure 7 The antifungal effects of *Sclerotinia sclerotinia* showed that, at a concentration of 50 μg / mL of the active ingredient after 72 h, the inhibition rates of pyraclostrobin, pyraclostrobin-loaded mesoporous selenium particles, and mesoporous selenium nanopesticide particles were 78.69%, 82.71%, and 98.78%, respectively. (See [link to relevant documentation]). Figure 9 B in the middle; see also Figure 8 The antifungal effects of *Rhizoctonia solani* showed that on day 9, when the concentration of the active ingredient reached 50 μg / mL, the inhibition rates of pyraclostrobin, pyraclostrobin-loaded mesoporous selenium particles, and mesoporous selenium nanopesticide particles were 71.48%, 74.98%, and 90.29%, respectively. (See attached data). Figure 9 C in the middle.

[0071] The above research results indicate that, compared with pyraclostrobin, mesoporous selenium nanoparticles exhibit better and longer-lasting antifungal effects against various fungal diseases. This may be due to the dual-stimulation response mechanism of the mesoporous selenium nanoparticles, which allows pyraclostrobin to be released precisely on demand, prolonging its effective duration. This suggests that mesoporous selenium nanoparticles have practical application value in agricultural production and have the potential for further development and application.

[0072] The above embodiment 1 is a preferred embodiment of the present invention, but the embodiments of the present invention are not limited to the above embodiment. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a mesoporous selenium nanopesticide, characterized in that, The operation steps are as follows: (1) Preparation of mesoporous selenium nanocarrier particles Hexadecyltrimethylammonium bromide was dissolved in pure water at a weight-to-volume ratio of 1 g: 200 mL. Zinc powder, sulfur-modified polyethylene glycol, chitosan solution with a mass-to-volume concentration of 5 mg / mL, and sodium selenite were added, and the mixture was stirred thoroughly for 2 h. Ascorbic acid was added dropwise, and the pH was adjusted to 7.4 with sodium hydroxide. The mixture was stirred for 6 h. After the first centrifugation, the precipitate was placed in a 5% hydrochloric acid aqueous solution and refluxed at 80 °C for 12 h. After the second centrifugation at 10,000 rpm for 10 min, the precipitate was washed three times with pure water and freeze-dried to obtain mesoporous selenium nanocarrier particles. The mass ratio of hexadecyltrimethylammonium bromide to zinc powder is 8:1; The mass ratio of hexadecyltrimethylammonium bromide to sulfur-modified polyethylene glycol is 16:1; The mass ratio of hexadecyltrimethylammonium bromide to sodium selenite is 4:1; The mass ratio of chitosan to sodium selenite is 1:40; The mass ratio of ascorbic acid to sodium selenite is 4:1; (2) Preparation of pyrimethanil-loaded mesoporous selenium particles Mesoporous selenium nanoparticles were dispersed in acetone and stirred thoroughly for 30 min. 0.2 g of succinic anhydride was added, and the mixture was stirred for 24 h. The mixture was centrifuged for 10 min at 10,000 rpm for the first time, and the precipitate was washed three times with pure water and dried. The dried precipitate was dissolved in pure water, and pyrimethanil was added and stirred for 12 h. The mixture was centrifuged for 10 min at 10,000 rpm for the second time, and the supernatant was removed. The precipitate was washed three times with pure water and lyophilized to obtain pyrimethanil-loaded mesoporous selenium particles. The mass-to-volume ratio of the mesoporous selenium nanocarrier particles to acetone was 4 mg: 1 mL. The mass ratio of the mesoporous selenium nanocarrier particles to pyraclostrobin is 1:

1. The mass-to-volume ratio of the dried precipitate to pure water is 1 mg / 2 mL; (3) Preparation of mesoporous selenium nanopesticides The mesoporous selenium particles loaded with pyraclostrobin were dispersed in citrate buffer at pH 5.0 to obtain a suspension. 1 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide with a volume concentration of 1.915 mg / mL and 1 mL of N-hydroxysuccinimide with a volume concentration of 1.15 mg / mL were slowly added, and the mixture was stirred thoroughly for 4 h. Hydroxypropyl cellulose aqueous solution was added dropwise, and the mixture was stirred thoroughly for 24 h. The mixture was centrifuged at 10000 rpm for 10 min, the supernatant was removed, the precipitate was washed three times with pure water, and then lyophilized to obtain black nanoparticle-like mesoporous selenium nanopesticide. The mass-to-volume ratio of the loaded pyrimethanil mesoporous selenium particles to the citrate buffer was 5 mg: 1 mL. The mass ratio of the loaded pyrimethanil mesoporous selenium particles, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 10:1.915:1.

15. The mass ratio of the loaded pyrimethanil mesoporous selenium particles to hydroxypropyl cellulose is 1:

2.

2. The method for preparing a mesoporous selenium nanopesticide according to claim 1, characterized in that: In step (1), the chitosan solution with a mass-volume concentration of 5 mg / mL is in the form of an aqueous solution of acetic acid with a volume concentration of 1%.

3. A mesoporous selenium nanopesticide prepared according to the method for preparing a mesoporous selenium nanopesticide according to claim 1 or 2, characterized in that: The mesoporous selenium nanopesticide uses mesoporous selenium nanoparticles as a carrier, pyrimethanil fungicide as the loaded drug, and hydroxypropyl cellulose as a capping agent. The mesoporous selenium nanopesticide is in the form of black nanoparticles with an effective particle size of 150-200 nm, a pesticide loading rate of 47.49±2%, and an encapsulation rate of 91.71±4%. Used for plant fungal diseases.

4. The mesoporous selenium nanopesticide according to claim 3, characterized in that: The plant fungal diseases mentioned include: anthracnose, sclerotinia rot, and sheath blight.

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