Pesticide self-assembly system, preparation method and application in prevention and treatment of soil-borne diseases

Abstract

The application discloses a pesticide self-assembly system, a preparation method and application in prevention and treatment of soil-borne diseases, and belongs to the technical field of nano-pesticides. The pesticide self-assembly system has a particle size of 40 nm, is positively charged, has a significantly reduced surface tension value, has good absorption of leaves and seed coats, has strong light degradation resistance, can effectively improve the utilization rate of pesticides and the added effective period, has ultraviolet absorption performance and fluorescence effect, can be used as a visual tool for the action mechanism research of hydrophobic pesticides and precise pesticide application, has good crop safety, does not need to add any surfactant, emulsifier or dispersant, eliminates the pollution problems of organic solvents and aids in traditional preparations from the source, and has simple steps, mild conditions and low cost.

Description

Technical Field

[0001] This invention relates to the field of nanopesticide technology, and in particular to a pesticide self-assembly system, its preparation method, and its application in the prevention and control of soil-borne diseases. Background Technology

[0002] Pesticides, as an indispensable production material in modern agriculture, play a crucial role in ensuring food security. However: (1) Organic solvent dependence and adjuvant toxicity. Emulsifiable concentrate formulations extensively use organic solvents such as benzene, toluene, and xylene as pesticide solvents. These solvents themselves have high biotoxicity and environmental persistence. The stronger the lipophilicity of the adjuvant, the more easily it reacts with the bioactive sites, leading to its toxicity to non-target organisms. Cationic surfactants are more toxic to fish and algae than anionic surfactants. Surfactants containing alkylphenol groups have higher ecotoxicity and are not easily photodegraded, and can cause oxidative stress and apoptosis in the human liver.

[0003] (2) Low pesticide utilization and single mechanism of action. After spraying with traditional formulations, the deposition of pesticide solution on the leaf surface is constrained by factors such as wettability and adhesion, making it difficult to achieve effective spreading and penetration. More importantly, traditional formulations lack intelligent response release function. The active ingredients of pesticides are released rapidly after application, resulting in a short duration of effect, making it difficult to accurately match the dynamics of pest and disease occurrence. Most chemical pesticides only have direct bactericidal activity and lack the function of inducing the plant's own immune response. The single mechanism of action not only limits the control effect but also easily induces drug resistance in pathogens.

[0004] (3) Ecological safety risks and uncertainties. The ecotoxicity of different formulations varies significantly. Factors such as the toxicity of adjuvants, processing materials, and the dispersion state of active ingredients can all affect the environmental safety of the formulation. In addition, the poor photostability of traditional pesticide formulations is particularly prominent. Most pesticide technicals are rapidly photodegraded under ultraviolet light. The decomposition rate of prochloraz technical reaches 50% after 130 minutes of ultraviolet light irradiation. This not only leads to a decrease in efficacy, but its photodegradation products may also have higher environmental toxicity.

[0005] Nanopesticides have shown significant effectiveness in overcoming many shortcomings of traditional formulations. However, existing nanopesticide technologies suffer from drawbacks such as complex preparation processes, difficulties in large-scale production, limited functionality, lack of synergistic effects, and uncertain biocompatibility and environmental safety. Summary of the Invention

[0006] The purpose of this invention is to provide a pesticide self-assembly system and preparation method, as well as its application in the prevention and control of soil-borne diseases, in order to solve the problems of existing nano-pesticides having single functions, complex preparation processes, and uncertain biocompatibility and environmental safety.

[0007] To achieve the above objectives, the present invention provides a method for preparing a pesticide self-assembly system, comprising the following steps: Hydrophobic pesticides and polysalicylic acid are added to an organic solvent and sonicated until the components dissolve to obtain a mixture. The mixture is then added dropwise to deionized water, mixed well, and then dialyzed in a dialysis bag to obtain a pesticide self-assembly system.

[0008] Preferably, the polysalicylic acid is prepared as follows: Pyridine and thionyl chloride were mixed and stirred in an ice bath. The resulting mixture was added to salicylic acid, mixed well, and reacted at room temperature. Deionized water was then added until no more precipitate was formed. The precipitate was washed with 1% hydrochloric acid and then freeze-dried to obtain polysalicylic acid.

[0009] Preferably, the molar ratio of pyridine:thionyl chloride:salicylic acid is 4:1:2; the stirring is carried out at 400~800 rpm for 5~30 min; and the reaction is carried out at room temperature for 30 min~2 h.

[0010] Preferably, the polysalicylic acid is prepared as follows: Salicylic acid and acetic anhydride were mixed and heated to 100-200°C under a protective atmosphere and then stirred to react. After stirring, the mixture was vacuum distilled. The precipitate was washed successively with saturated sodium bicarbonate aqueous solution, deionized water and dichloromethane / methanol mixed solvent and then freeze-dried to obtain polysalicylic acid.

[0011] Preferably, the molar ratio of salicylic acid to acetic anhydride is 6.67:1; the heating rate is 2~3℃ / min; and the stirring reaction is carried out for 4~6h.

[0012] Preferably, the hydrophobic pesticide is imazalil; the organic solvent is N,N-dimethylformamide or dimethyl sulfoxide; and the ultrasonic conditions are 30~50KHz.

[0013] Preferably, the mass-to-volume ratio of the hydrophobic pesticide, polysalicylic acid, organic solvent, and deionized water is 1g:1~10g:0.1L:1~20L.

[0014] Preferably, the molecular weight cutoff of the dialysis bag is 500~3000D.

[0015] A pesticide self-assembly system prepared by the method described above.

[0016] The application of the pesticide self-assembly system described above in the control of soil-borne diseases in plants.

[0017] Therefore, the pesticide self-assembly system and preparation method provided by this invention, and its application in the prevention and control of soil-borne diseases, have the following specific technical effects: (1) The pesticide self-assembly system provided by the present invention uses polysalicylic acid as a carrier to load hydrophobic pesticides with a particle size of 40 nm, carrying a positive charge of +77.83 mV, having ultraviolet absorption properties and fluorescence effect, and can form a stable colloid with good dispersibility; it has a low surface tension value without adding surfactants. (2) The pesticide self-assembly system provided by the present invention can slowly degrade in the environment and continuously release low concentrations of salicylic acid monomers, which can activate the systemic acquired resistance of plants, induce the expression of disease-related proteins and enhance the activity of antioxidant enzyme systems, achieve the dual synergistic effect of direct bactericidal / nematicidal action and induction of plant immunity, significantly enhance the control effect and reduce the risk of plant resistance. (3) The pesticide self-assembly system provided by the present invention has good wetting and spreading, adhesion and rain washout resistance on the surface of plant leaves and seeds. It can be effectively absorbed by the seed coat and leaves, and can also effectively shield the degradation of pesticides by ultraviolet light, thereby effectively improving the utilization rate and field retention period of pesticides, and inhibiting the growth of pathogen mycelium for a long time. (4) The pesticide self-assembly system provided by the present invention can track the absorption, transport and distribution process of nano-pesticides in plants through fluorescence microscopy, providing a visualization tool for the study of the mechanism of action of hydrophobic pesticides and precise application; (5) The pesticide self-assembly system provided by the present invention has broad-spectrum bactericidal / nematicidal activity, causing severe twisting, bending, breakage, swelling and even hole in the mycelium of pathogens; the EC50 values ​​against Sclerotinia sclerotiorum, Verticillium dahliae and Fusarium oxysporum are 0.02, 0.04 and 0.04 μg / mL, respectively, which is about 2 times higher than the bactericidal activity of prochloraz technical, and the damage to the mycelium of pathogens is significantly more severe than that of the technical. (6) The pesticide self-assembly system provided by the present invention has good crop safety and has no significant effect on seed germination rate, relative chlorophyll content in leaves and growth performance; (7) The pesticide self-assembly system provided by the present invention does not require the addition of any surfactants, emulsifiers or dispersants, thus eliminating the pollution problems of organic solvents and adjuvants in traditional formulations from the source. The process is simplified, the conditions are mild and the cost is low.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This section presents the physicochemical characterization results of the PP NPs nano-self-assembled system in the effect test examples. A1 and A2 represent PSA under different magnification transmission electron microscopy (TEM); B1 and B2 represent PP NPs under different magnification TEM; C represents the 1H NMR spectra of SA and PSA; D represents the Fourier transform infrared (FTIR) spectra of PRO, PSA, and PP NPs; E represents the UV spectra of PRO, PSA, and PP NPs; F represents the Tyndall effect (a: SA, b: PRO, c: PSA, d: PP NPs); G represents the fluorescence phenomenon under 302nm UV illumination (a: SA, b: PRO, c: PSA, d: PP NPs); H represents the surface tension values ​​of water, PSA, and PP NPs; and I represents the Zeta potential values ​​of PRO, PSA, and PP NPs. Figure 2 These are the results of the plant systemic conductivity study of the PP NPs nano self-assembled system in the effect test examples; Figure 3 This is a test case of the effect of the PP NPs nano-self-assembled system on the bacterial load rate of sunflower kernels; where A is a representative photograph of the culture medium; and B is the statistical result of the bacterial load rate. Figure 4 This is a test result of the absorption characteristics of the PP NPs nano self-assembled system in the roots of sunflowers in the effect test case. Figure 5 These are the photostability results of the PP NPs nano self-assembled system in the effect test examples; Figure 6 These are the results of the sustained-release kinetics investigation of the PP NPs nano-self-assembled system in the effect test examples; where A is the zero-order kinetic model diagram; B is the first-order kinetic model diagram; C is the Higuchi kinetic model diagram; and D is the Ritger-Peppas kinetic model diagram. Figure 7 This is a partial effect test example of the PP NPs nano self-assembly system on the hyphal morphology of three pathogenic fungi; Figure 8 This is a partial effect test example of the PP NPs nano self-assembly system on the hyphal growth of three pathogenic fungi; Figure 9 This is a partial test example of the in vitro bactericidal activity of the PP NPs nano-self-assembled system against Sclerotium sclerotiorum mycelium; where A is CK; B is solvent control; C is 0.005 μg / mL; D is 0.01 μg / mL; E is 0.05 μg / mL; F is 0.1 μg / mL; G is 0.5 μg / mL; Figure 10This is a partial test example of the PP NPs nano-self-assembled system's in vitro bactericidal activity against Verticillium dahliae; where A is the control (CK); B is the solvent control; C is 0.005 μg / mL; D is 0.01 μg / mL; E is 0.05 μg / mL; F is 0.1 μg / mL; and G is 0.5 μg / mL. Figure 11 This is a partial test example of the in vitro bactericidal activity of the PP NPs nano-self-assembled system against Fusarium oxysporum; where A is the control (CK); B is the solvent control; C is 0.005 μg / mL; D is 0.01 μg / mL; E is 0.05 μg / mL; F is 0.1 μg / mL; and G is 0.5 μg / mL. Figure 12 This is a test case study of the safety of the PP NPs nano-self-assembled system on sunflower; where A is seed germination rate; B is plant height; C is stem diameter; D is chlorophyll content; E is fresh weight; and F is dry weight. Figure 13 This is a partial effect test example of the PP NPs nano self-assembled system on the incidence of sclerotinia stem rot in sunflowers; Figure 14 This is a partial effect test example of the PP NPs nano self-assembled system on the incidence of sunflower wilt; Figure 15 This is a partial effect test example of the PP NPs nano self-assembled system on the incidence of Verticillium wilt in sunflower. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0024] Example 1 The specific steps for preparing polysalicylic acid (PSA) are as follows: Pyridine (40 mmol, 3.22 mL) and thionyl chloride (10 mmol, 0.73 mL) were mixed (molar ratio of pyridine to thionyl chloride: 4:1). After stirring at 600 rpm for 15 min in an ice bath, salicylic acid (20 mmol, 2.76 g) was added directly to the mixture (molar ratio of salicylic acid to pyridine: 2:4), and the mixture was reacted at room temperature for 1 h. The product was then mixed with deionized water at a volume ratio of 2:8, shaken, and centrifuged at 6000 rpm / min. The supernatant was discarded, and the product was retained. The product was then washed five times with 1% hydrochloric acid to remove excess pyridine. Finally, the product was freeze-dried to obtain a white powder, which is polysalicylic acid.

[0025] The reaction formula is: .

[0026] Example 2 The specific steps for preparing polysalicylic acid are as follows: Salicylic acid (50 mmol, 6.9 g) and acetic anhydride (7.5 mmol, 7.0 mL) (salicylic acid:acetic anhydride molar ratio of 6.67:1) were added to a 100 mL three-necked flask. The mixture was gradually heated from room temperature to 160 °C at a rate of 2.67 °C / min under a nitrogen atmosphere, and the mixture was magnetically stirred for 5 h. After the reaction was complete, volatile byproducts were removed by vacuum distillation. The product was removed from the three-necked flask and washed three times each with saturated sodium bicarbonate aqueous solution and deionized water to obtain the crude product. Subsequently, the crude product was washed with a dichloromethane / methanol mixed solvent (dichloromethane:methanol volume ratio of 1:9) to remove monomers and short-chain polymers. Finally, the white powder obtained by freeze-drying was the purified polysalicylic acid (PSA).

[0027] The reaction formula is: .

[0028] Example 3 The specific steps for preparing the polysalicylic acid-imidazolium nanoparticle self-assembled system (PP NPs) are as follows: 0.1 g of the fungicide prochloraz technical (PRO) and 0.038 g of PSA prepared in Example 1 (PRO:PSA mass ratio of 1:2) were dissolved by sonication (40 kHz) in 1.0 mL of dimethyl sulfoxide (DMSO) (PSA was added first, then PRO, and finally DMSO was added to dissolve). The resulting mixture was added dropwise to 99 mL of deionized water under slow stirring (600 rpm). The solution was transferred to a dialysis bag (molecular weight cutoff of 1000D), immersed in deionized water, and dialyzed for 24 h to complete purification, finally obtaining a nano-self-assembled system suspension (PP NPs).

[0029] Effect Test Case (1) The physicochemical properties of the PP NPs nano self-assembly system prepared in Example 3 were characterized.

[0030] ① Observation was performed using TEM, and the results are as follows: Figure 1 As shown, A1 and A2 are morphological images of PSA under transmission electron microscopy at different magnifications, and B1 and B2 are morphological images of PP NPs under transmission electron microscopy at different magnifications. It can be seen that PSA is a spherical particle with a rough surface and is a small nanoparticle with a size of about 40 nm. PP NPs have a smoother morphology than PSA and are also small in size.

[0031] ② Salicylic acid (SA) and PSA prepared in Example 1 1 H NMR spectrum as shown Figure 1 As shown in C, SA's 1 The H chemical shifts were 6.9–6.96, 7.5, and 7.79–7.81 ppm, respectively, with an area integral ratio of 2:1:1. In polymer PSA... 1 The chemical shifts of H were located at 7.30–7.55, 7.56–7.80, and 8.08–8.22 ppm, respectively. Specifically, the α-hydrogen absorption peak shifted from 7.5 ppm to 7.56–7.80 ppm, and the d-hydrogen absorption peak shifted from 7.79–7.81 ppm to 8.08–8.22 ppm. These changes in chemical shift indicate that an ester bond formed between the hydroxyl and carboxyl groups in SA, thus proving the successful preparation of PSA.

[0032] ③FTIR spectra of PRO (imazalil), PSA and PP NPs are as follows Figure 1 As shown in D, PSA is at 1745cm -1 The presence of typical ester bond (-COO-) stretching vibrations further confirms the successful polymerization of SA to PSA. PRO exhibits stretching vibrations at 1682 and 1205 cm⁻¹. -1 The peaks at these locations correspond to the characteristic absorptions of carbonyl groups (-C=O) and ether bonds (-O-), respectively. The characteristic absorption peaks of the self-assembled PP NPs are basically consistent with those of PRO and PSA.

[0033] ④ Using DMF as a blank control, the UV-Vis absorption spectra of PRO, PSA, and PP NPs in the 200–400 nm range are as follows: Figure 1 As shown in Figure E, PRO exhibits weak characteristic absorption peaks at 240 and 279 nm, while PSA shows strong UV absorption at 251 nm. The peak shape of PP NPs is basically consistent with the characteristic absorption peaks of PRO and PSA, indicating that the PP NPs formed by PRO and PSA have composite properties.

[0034] ⑤ The Tyndall effect of SA, PRO, PSA and PP NPs under laser pointer illumination, such as Figure 1As shown in Figure F, at the same concentration, the PRO, PSA, and PP NPs liquids dissolved in DMF and diluted with deionized water all exhibit a good Tyndall effect, while SA does not. This indicates that SA is not a colloid, while the suspensions of PRO, PSA, and PP NPs are all well-dispersed and stable colloids.

[0035] ⑥ The fluorescence phenomenon under 302nm ultraviolet light irradiation is as follows Figure 1 As shown in G, PSA can significantly enhance the fluorescence effect of SA. Fluorescent PSA provides important support and advantages for subsequent experiments in many aspects, including drug distribution and metabolism in vivo, monitoring drug release behavior, determining pharmacokinetic parameters, and safety evaluation. Furthermore, PRO itself does not possess fluorescence, but the PP NPs formed after self-assembly with fluorescent PSA exhibit strong fluorescence, indicating that PRO retains good fluorescence performance after self-assembly with PSA.

[0036] ⑦ Surface tension values ​​of water, PSA, and PP NPs are as follows Figure 1 As shown in Figure H, water exhibits the highest surface tension (71.57 mN / m). At a concentration of 1000 μg / mL, the surface tensions of PSA and PP NPs are 69.83 mN / m and 46.07 mN / m, respectively. This indicates that the self-assemblies of PRO and PSA can significantly reduce the surface tension of water without the addition of surfactants, thereby allowing the drugs to adhere better to the leaf or seed surface.

[0037] ⑧ The Zeta potential values ​​of PRO, PSA, and PP NPs are as follows Figure 1 As shown in Figure I, it can be seen that PRO carries a positive charge of +54.83mV, PSA carries a negative charge of -39.97mV, and the self-assembled PP NPs carries a positive charge of +77.83mV, proving that a self-assembled system has been formed.

[0038] (2) Investigate the plant systemic conductivity of the PP NPs nano self-assembled system prepared in Example 3.

[0039] ① A PP NPs suspension containing 0.1% Tween-80 was prepared by adding Tween-80 to a PP NPs formulation containing 1000 μg / mL of active ingredient (PRO) (the drug addition amount was 0.2% of the seed weight). Edible sunflower LD5009 seeds were treated with the PP NPs suspension to ensure even distribution of the agent on the seeds. Untreated LD5009 seeds served as a control. Samples were taken at 1, 6, and 12 hours to prepare temporary specimens for microscopic observation. The results are as follows: Figure 2As shown, the amount of drug absorbed by the sunflower seed coat gradually increased with treatment time. This was evident at 6 hours of treatment, where fluorescent drug was clearly visible in some thicker seed coat tissues, and at 12 hours, the fluorescence intensity on the sunflower seed coat was significantly increased compared to before. These results indicate that PP NPs can be absorbed by the sunflower seed coat, and the amount absorbed gradually increases over time.

[0040] ② Sunflower kernel bacterial contamination rate.

[0041] Infected seeds collected from a sunflower field in Kober Town, Chahar Right Wing Middle Banner (41°17′45.9′′N, 112°34′23.8′′E) were treated with seed dressing using the method described in ①. Infected seeds that were not treated served as a control group. Twelve hours after treatment, the kernels of both the control group and the PP NPs-treated group were removed and gently placed on PDA medium for two days of incubation.

[0042] The results in ① indicate that PP and NPs can be absorbed inside the seed coat. Therefore, seeds with the pathogen were selected for seed dressing treatment, and the results were as follows: Figure 3 As shown, two days after seed dressing, the bacterial load in the control seeds was 85%, while the bacterial load in the treated seeds was 32.92%, significantly lower than that of the control seeds. Simultaneously, the mycelial growth of the bacteria-carrying seeds treated with PP NPs was clearly inhibited. This indicates that PP NPs can penetrate the seed interior and exert a bactericidal effect within the seed.

[0043] ③ Sunflower root absorption characteristics.

[0044] A 1cm thick layer of cotton was placed in a square petri dish. A piece of filter paper the size of the petri dish was cut and placed on top of the cotton. The rootstock of a two-leaf stage sunflower seedling was placed on top of the cotton and filter paper, and then another layer of filter paper was placed on top to construct a petri dish filter paper system. The petri dish filter paper system was moistened. 50mL of 100μg / mL PP NPs suspension was poured into each dish, with 50mL of sterile water added as a control group. The petri dishes were gently shaken to distribute the agent evenly on the culture medium. Samples were taken at 6, 12, and 24 hours after treatment to prepare temporary slides for observation under a microscope.

[0045] The results are as follows Figure 4 As shown, the amount of drug absorbed by the sunflower roots gradually increased over time. At 6 hours, it was only present in the xylem; at 12 hours, the amount in the xylem increased and it also penetrated into some cortical cells; and at 24 hours, the drug fluorescence had penetrated into most of the root cell tissues. The experiment shows that PP NPs can be absorbed by plant roots, and the amount absorbed gradually increases over time.

[0046] (3) The photostability of the PP NPs nano self-assembled system prepared in Example 3 was investigated.

[0047] PRO and PP NPs were prepared into an aqueous suspension with a PRO concentration of 30 mg / L. The UV shielding performance of PP NPs was investigated under UV light (wavelength 254 nm, irradiation distance 20 cm). The PRO concentration was expressed as a function of time (…). t The variation curve of h) was fitted according to the first-order kinetic model (Equation 1), and the digestion half-life (DT) was calculated using Equation 2. 50 ): ; Equation 2); In the formula: C t The concentration of PRO in the release solution. C 0 The initial concentration of PRO in the release solution at the initial time (t=0) t For time, K DT is a first-order kinetic constant. 50 To reduce the half-life, It is the logarithm of 2 as the natural logarithm, with a value of approximately 0.693.

[0048] The effect of UV light irradiation on the photostability of PP NPs nanoparticles, such as Figure 5 As shown. DT of PRO active ingredient irradiated by UV light. 50 The time was 130 min, while the DT of PRO in PP NPs nanomaterials was... 50 The result was 223.33 min. The results indicate that PRO loaded onto PSA improved photostability by approximately 1.7 times, and PP NPs nanoparticles effectively reduced the photolysis of the active ingredient.

[0049] (4) Investigate the sustained-release kinetics of the PP NPs nano self-assembled system prepared in Example 3.

[0050] The release behavior of PRO in PP NPs was analyzed by dialysis. 2 mL of 500 mg·L⁻¹ PP NPs was used. -1 PP NPs were placed in dialysis bags (with a molecular weight cutoff of 3000 Da) and immersed in 98 mL of acetonitrile-water solution (80:20, pH 5.0, 7.0, and 9.0, respectively). 0.5 mL samples were taken periodically, and an equal volume of buffer was added. Release behavior was evaluated based on the FLU concentration in the release solution at different time points, and the cumulative release rate was calculated. CRR Calculate according to formula (3): Formula 3). In the formula: C t (mg·mL)-1 )for t PRO release concentration at any time; V t The volume of each sample taken is 0.5 mL. V total The total volume of the released liquid (100 mL). m 0 This represents the total drug loading of the PRO.

[0051] The release kinetics of PP NPs under different pH conditions were evaluated by fitting the release data to Zero-order (Equation 4), First-order (Equation 5), Higuchi (Equation 6), and Ritger-Peppas (Equation 7) models, respectively. Equation 4); Equation 5); Formula 6); Equation 7); in M t / M z Representing PP NPs in time t Release rate; k Represents the release rate constant; n Represents diffusion parameters; t For release time; b is the intercept constant of the model.

[0052] To evaluate the release behavior of PP NPs nanoparticles at different pH values, the cumulative release data of PRO in PP NPs were recorded. The results are as follows: Figure 6 As shown in the figure, the release behavior curves of PP NPs in PBS sustained-release solutions at different pH values ​​(pH=5, 7, 9) indicate that the cumulative release rate increases significantly with increasing pH, reaching 56.81%, 63.31%, and 73.44% at 84 h, respectively. The overall cumulative release rate increases significantly with increasing pH, with the highest cumulative release under alkaline conditions. PP NPs exhibit alkaline-responsive release capability, enabling more efficient release of PRO in alkaline environments. The drug release rate of PP NPs can be flexibly controlled by adjusting the ambient pH.

[0053] To further investigate the release kinetics of PP NPs at different pH values, the sustained-release kinetic parameters obtained by fitting the data of PP NPs releasing PRO under different pH conditions to four mathematical models are listed in Table 1. The first-order kinetic model was found to have Mt / Mz = 1 - e -ktIt exhibited the best fit under all pH conditions (R0). 2 >0.9633), suitable for describing drug release kinetics. The n value obtained from the Ritger-Peppas equation ranges from 0.10 to 0.13; therefore, PRO release follows Fickian diffusion. As pH decreases, T... 50 The release time increased from 2.40 hours at pH 9.0 to 3.41 hours at pH 5.0. In an alkaline environment, half the amount of drug was released more quickly; while in an acidic environment, the release rate was relatively slower. This indicates that the release of PRO from PP NPs is pH-dependent, with an alkaline environment being most conducive to its full and rapid release.

[0054] Table 1. Continuous-release kinetic parameters obtained by fitting data of PRO release from PP NPs under different pH conditions to four mathematical models.

[0055] (5) Investigate the effects of PRO and PP NPs on the mycelial morphology of the three pathogens.

[0056] ①Prepare a drug-containing culture medium.

[0057] Under sterile conditions, the active pharmaceutical ingredients of each agent were serially diluted to prepare stock solutions of different concentrations. First, the six active pharmaceutical ingredients (imidacloprid, fluopyram, prothioconazole, cyclophosphamide, flutriafol, and difenoconazole) were dissolved in methanol to prepare stock solutions with a concentration of 1×10⁴ μg / mL. Then, the test fungicides were serially diluted to suspensions of 1000 μg / mL, 100 μg / mL, 10 μg / mL, and 1 μg / mL. PP NPs were also diluted to suspensions of the same concentration as the active pharmaceutical ingredients and stored at 4°C for later use. After the sterilized PDA medium temperature dropped to 50°C–55°C, based on the results of previous preliminary experiments, the diluted fungicide solutions were mixed evenly with the PDA medium at the set concentration gradient in a clean bench to prepare drug-containing medium. An equal volume of sterile water replacing the fungicide suspension in the PDA medium served as a blank control. Each experiment was performed in triplicate.

[0058] ② Vaccination and outcome survey.

[0059] A 5 mm diameter mycelial disc was obtained from the edge of the pre-cultured test colony using a punch and transferred to the center of the drug-containing PDA medium. Each treatment was repeated three times, with a control medium without the drug added. All cultures were incubated at a constant temperature of 25°C. When the control colonies reached 3 / 4 of the plate, the colony diameter was measured using the cross-sectional method, and Equation 8 was used to calculate the inhibition rate of each fungicide at different concentrations on the mycelial growth of the pathogen.

[0060] The mycelial morphology of *Sclerotinia sclerotiorum*, *Verticillium dahliae*, and *Fusarium oxysporum* was observed under a microscope after being cultured for several days on blank control medium (Mock), PDA medium containing PRO, and PDA medium containing PP NPs.

[0061] Preparing a temporary slide: Clean the glass slide and coverslip. Place a drop of water in the center of the glass slide. Transfer a small amount of mycelium taken from the edge of the colony into the droplet and spread it out. Cover the slide using a tilting method, ensuring the edge touches the droplet first, then slowly lower it to ensure a smooth coverage and eliminate air bubbles. The final step is to use filter paper to absorb excess water, thus completing the preparation of the temporary slide.

[0062] The effects of PRO and PP NPs on the hyphal morphology of three pathogenic fungi are as follows: Figure 7 As shown. In the control group (Mock), the mycelia of the three pathogens had smooth, rounded surfaces, were firm and plump, and grew uniformly. In contrast, some mycelia of *Sclerotinia sclerotiorum* grown on PRO and PP NPs medium showed twisting and bending. Similarly, some mycelia of *Verticillium dahliae* grown on PRO medium also showed twisting and bending, while those grown on PP NPs medium showed bending, breakage, swelling, and even holes. Some mycelia of *Fusarium oxysporum* grown on PRO medium showed breakage, but the overall effect was not significant, while those grown on PP NPs medium showed bending, breakage, and swelling.

[0063] The above results indicate that PRO and PP NPs all had a certain impact on the normal mycelial growth of Sclerotinia sclerotiorum, Verticillium dahliae, and Fusarium oxysporum.

[0064] (6) The inhibitory effect of PSA on the mycelial growth of three pathogens.

[0065] The method for determining PP NPs is the same as in (5), except that PP NPs are replaced with an equal amount of PSA. The results of the effects of different concentrations of PSA on the growth of the three pathogens are as follows: Figure 8 As shown, the growth of all three pathogens was not significantly inhibited, indicating that the polysalicylic acid material itself has no significant inhibitory effect on mycelial growth.

[0066] (7) The inhibitory effect of PP NPs on the mycelial growth of three pathogens.

[0067] The mycelial growth rate method was used to determine the effect of PP NPs on Sclerotinia sclerotiorum (PP NPs) on PDA solid medium. S. sclerotiorum X-8, Verticillium dahliae ( V.dahliae V89, Fusarium oxysporum ( F. oxysporumThe antibacterial activity of HJ1-2(A) was assessed. PP NPs were diluted with sterile water and ultrasonically dispersed into a stable suspension. The suspension was added to PDA medium at 50℃~55℃, shaken well, and then evenly dispensed into 9cm diameter petri dishes. The PDA plates were cooled until solidified. The concentrations of PRO in the PDA plates were 0.005, 0.01, 0.05, 0.1, and 0.5 μg / mL, respectively. A PDA plate containing only solvent and no drug was used as a blank control. A 5mm diameter mycelial disc was inoculated from the edge of a freshly activated test strain into the center of a PDA plate. The plates were incubated upside down at 25℃ in the dark for several days. The colony diameter was measured using the cross-sectional method. Each concentration treatment was repeated three times. Based on the colony diameter at each concentration, the inhibition rate (IR, %) and half-maximal effective concentration (EC50) of the drug against *Sclerotinia sclerotiorum*, *Verticillium dahliae*, and *Fusarium oxysporum* were calculated using Equation 8. 50 )value.

[0068] (Equation 8) Where D0 is the diameter of the control colony and D1 is the diameter of the treated colony. The logarithmic dose-response curve allows for the determination of the EC50 of fungal bioassays based on probability analysis. 50 .

[0069] The in vitro bactericidal activity of PP NPs against Sclerotinia sclerotiorum mycelium is as follows: Figure 9 As shown in Table 2, the inhibition rate reached as high as 97.62% at a concentration of 0.5 μg / mL. The virulence regression equation established by measuring and statistically analyzing the colony diameters of each treatment is shown in Table 3: y = 1.5363x + 7.5035, with an EC50 value of 0.02 μg / mL, which is lower than the EC50 value of 0.04 μg / mL for the original PRO drug.

[0070] PP NPs exhibit in vitro bactericidal activity against Verticillium dahliae, such as Figure 10 As shown in Table 2, the inhibition rate reached 83.61% at a concentration of 0.5 μg / mL. The colony diameters of each treatment were measured and statistically analyzed, and the established virulence regression equation is shown in Table 3: y = 0.8390x + 6.1457, EC 100%. 50 The value was 0.04 μg / mL, lower than the EC50 of the original PRO drug. 50 The value is 0.07 μg / mL.

[0071] PP NPs exhibit in vitro bactericidal activity against Fusarium oxysporum, such as Figure 11As shown in Table 2, the inhibition rate reached as high as 80.94% at a concentration of 0.5 μg / mL. The colony diameters of each treatment were measured and statistically analyzed. The established virulence regression equation, as shown in Table 3, is y = 0.8421x + 6.1423, with an EC50 value of 0.04 μg / mL. This is significantly lower than the EC50 value of the PRO original drug, which was 0.09 μg / mL.

[0072] PP NPs exhibited typical dose-dependent inhibition rates against the growth of *Sclerotinia sclerotiorum*, *Verticillium dahliae*, and *Fusarium oxysporum* colonies. Although different concentrations of PSA did not show growth inhibition against the pathogens, their combined application with PRO showed a synergistic effect. The nanoparticles formed by the amphiphilic PSA and PRO possessed excellent fungal cell membrane penetration; compared to PRO, the bactericidal activity of PP NPs against all three pathogens was approximately twice that of PP NPs.

[0073] Table 2. Inhibitory effects of PP and NPs on mycelial growth of three soil-borne diseases of sunflower.

[0074] Table 3. Virulence test results of PP NPs against three soil-borne pathogens of sunflower diseases.

[0075] (8) Safety test of PP NPs on sunflower.

[0076] Tween-80 was added to PP NPs with an active ingredient (PRO) concentration of 1000 μg / mL (0.2% of seed weight) to prepare a 0.1% Tween PP NP suspension. Healthy, rounded edible sunflower LD5009 seeds were rinsed with water and then soaked in the 0.1% Tween PP NP suspension, mixing thoroughly to ensure each seed was evenly coated. A control group (CK) was prepared by replacing the 0.1% Tween PP NP suspension with water. Seeds from both treatment groups (100 seeds per treatment) were placed in seedling trays lined with damp filter paper and germination was conducted at room temperature. Germination status was checked daily throughout the process, and water lost through evaporation was replenished to maintain optimal humidity conditions for seed germination. Germination rates were recorded on days 3 and 7 after germination. Each treatment was repeated three times.

[0077] Safety tests of PP NPs on sunflowers, such as Figure 12 As shown in Figure A, the PP NPs seed dressing treatment had no significant effect on the germination rate of sunflower seeds on days 3 and 7 after treatment. Specifically, on day 3 after treatment, the germination rates of untreated and treated seeds were 53.67% and 57%, respectively, and on day 7, the germination rates were 71.33% and 75.33%, respectively.

[0078] The effects of PRO, PSA, and PP NPs on the biological indicators of sunflower plants are as follows: Figure 12 As shown in the BF diagram, the plant height and stem diameter of the PP NPs seed treatment group were significantly higher than those of the other three treatment groups. Different treatments had no significant effect on the relative chlorophyll content of sunflower leaves. The dry weight and fresh weight of the PSA treatment group were significantly higher than those of the control group, and there was no significant difference between the PSA treatment group and the PP NPs treatment group. Spraying PSA alone can significantly increase the fresh weight of sunflower plants.

[0079] (9) Determination of the efficacy of seed dressing treatment against soil-borne diseases in sunflowers.

[0080] Tween-80 was added to PRO and PP NPs solutions with a concentration of 1000 μg / mL and PSA concentration of 380 μg / mL, respectively, to prepare 0.1% Tween suspensions. Seeds were treated with the three Tween suspensions to ensure even coating of each seed. Seeds without additives served as a blank control group. Ten pots were used for each treatment, with three plants per pot, and each treatment was repeated three times. After the plants reached the six-leaf stage, the chlorophyll concentration of the leaves (expressed as SPAD value) was measured using a TY-4N chlorophyll analyzer, and plant height, stem diameter, and the fresh and dry weight of the above-ground parts were recorded.

[0081] Under greenhouse conditions, the effects of PRO and PP NPs seed treatment on the control of different soil-borne diseases in sunflowers are shown in Table 4, and the results of growth index measurement are shown in Table 5.

[0082] Sunflower sclerotinia stem rot: such as Figure 13 As shown, the sunflower plants treated with distilled water did not exhibit any physiological abnormalities and were all healthy. However, the disease incidence rate of plants treated with PP NPs and then inoculated with Sclerotinia sclerotiorum was 43.13%, which was significantly lower than the disease incidence rate of 78.13% when inoculated with Sclerotinia sclerotiorum alone and the disease incidence rate of 49.38% when treated with PRO and then inoculated with Sclerotinia sclerotiorum. The relative control efficacy of PRO and PP NPs treatments was 36.80% and 44.62%, respectively, with the PP NPs treatment group being significantly better than the PRO treatment group.

[0083] Sunflower wilt disease: such as Figure 14 As shown, the sunflower plants treated with distilled water did not exhibit any physiological abnormalities and were all healthy. The disease index of Fusarium oxysporum treated with PP NPs before inoculation was 13.91, significantly lower than the disease index of 36.41 for those inoculated with Fusarium oxysporum alone, with a relative control efficacy of 61.84%. The disease index of Fusarium oxysporum treated with PRO before inoculation was 16.10, significantly lower than the disease index for those inoculated with Fusarium oxysporum alone, with a relative control efficacy of 55.65%.

[0084] Sunflower Verticillium wilt: such as Figure 15 As shown, the sunflower plants treated with distilled water did not exhibit any physiological abnormalities and were all healthy. The disease index of the seed treated with PP NPs and then inoculated with Verticillium dahliae was 26.56, significantly lower than the disease index of 57.35 for the seed treated with Verticillium dahliae alone, with a relative control efficacy of 56.18%. The relative control efficacy against Verticillium wilt in the PRO seed treatment group was lower, at 31.94%.

[0085] Seed treatment with PRO and PP NPs significantly reduced the incidence and disease index of three different soil-borne diseases in sunflower.

[0086] Measurements of various growth indicators of sunflower plants revealed that treatment with any of the three pathogens, individually, significantly inhibited sunflower growth. For example, after treatment with *Sclerotinia sclerotiorum*, the average plant height decreased from 35.75 cm in the control group to 22.94 cm; the average stem diameter decreased from 4.67 mm to 3.48 mm, demonstrating an inhibitory effect on plant growth. Treatments with *Fusarium wilt* pathogens and *Verticillium wilt* pathogens also inhibited plant growth indicators to some extent.

[0087] Both PRO and PP NPs treatments alleviated the growth-inhibiting effects of pathogens to varying degrees. For sclerotinia stem rot, both treatments restored plant height to a level not significantly different from the control group. For Fusarium wilt, PP NPs restored plant height to a level not significantly different from the control group, with stem diameter close to the control group, while PRO had no significant effect on plant height. After inoculation with Verticillium wilt, PP NPs treatment restored plant height to the control group level, with stem diameter even significantly higher than the control group, while PRO only partially restored plant height and stem diameter to the control group level.

[0088] Therefore, under greenhouse conditions, seed treatment with PP NPs can reduce the incidence of sunflower sclerotinia rot, wilt, and verticillium wilt to varying degrees, indicating that PP NPs have good control effects on these three soil-borne diseases in sunflowers. Furthermore, applying PP NPs can further reduce the impact of soil-borne diseases on sunflower plants, resulting in better plant growth.

[0089] Table 4. Effects of PRO and PP NPs seed treatments on the control of soil-borne diseases in sunflowers.

[0090] Table 5 Effects of PRO and PP NPs seed treatments on sunflower growth indicators

[0091] Therefore, the pesticide self-assembly system provided by this invention has a particle size of 40 nm, is positively charged, has a significantly reduced surface tension value, good absorption by leaves and seed coats, strong resistance to photodegradation, and can effectively improve the utilization rate and residual effect of pesticides; it has ultraviolet absorption properties and fluorescence effect, and can be used as a visualization tool for studying the mechanism of action of hydrophobic pesticides and for precision application; it has good crop safety; it does not require the addition of any surfactants, emulsifiers or dispersants, eliminating the pollution problems of organic solvents and adjuvants in traditional formulations from the source; the process is simplified, the conditions are mild, and the cost is low.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a pesticide self-assembly system, characterized in that, The steps are as follows: Hydrophobic pesticides and polysalicylic acid are added to an organic solvent and sonicated until the components dissolve to obtain a mixture. The mixture is then added dropwise to deionized water, mixed well, and then dialyzed in a dialysis bag to obtain a pesticide self-assembly system. The preparation method of the polysalicylic acid is as follows: Pyridine and thionyl chloride were mixed and stirred in an ice bath. The resulting mixture was added to salicylic acid, mixed well, and reacted at room temperature. Then, deionized water was added until no more precipitate was formed. The precipitate was washed with 1% hydrochloric acid and then freeze-dried to obtain polysalicylic acid. The molar ratio of pyridine:thionyl chloride:salicylic acid is 4:1:2; stirring is carried out at 400~800 rpm for 5~30 min; reaction is carried out at room temperature for 30 min~2 h; Alternatively, the preparation method of the polysalicylic acid is as follows: Salicylic acid and acetic anhydride were mixed and heated to 100-200℃ under a protective atmosphere and stirred to react. After stirring, the mixture was vacuum distilled. The precipitate was washed successively with saturated sodium bicarbonate aqueous solution, deionized water and dichloromethane / methanol mixed solvent and then freeze-dried to obtain polysalicylic acid. The molar ratio of salicylic acid to acetic anhydride is 6.67:1; the heating rate is 2~3℃ / min, and the reaction is stirred for 4~6h.

2. The method for preparing a pesticide self-assembly system according to claim 1, characterized in that: The hydrophobic pesticide is imazalil; the organic solvent is N,N-dimethylformamide or dimethyl sulfoxide; and the ultrasonic conditions are 30~50KHz.

3. The method for preparing a pesticide self-assembly system according to claim 1, characterized in that: The mass-to-volume ratio of the hydrophobic pesticide, polysalicylic acid, organic solvent, and deionized water is 1g:1~10g:0.1L:1~20L.

4. The method for preparing a pesticide self-assembly system according to claim 1, characterized in that: The molecular weight cutoff of the dialysis bag is 500~3000D.

5. A pesticide self-assembly system prepared by the method according to any one of claims 1 to 4.

6. The application of the pesticide self-assembly system as described in claim 5 in the control of soil-borne plant diseases.

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