PH-responsive nano-selenium-loaded carbendazim nano-pesticide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano-pesticides, and particularly relates to a pH-responsive nano-selenium-loaded carbendazim nano-pesticide as well as a preparation method and application thereof. The chitosan-nano-selenium-loaded carbendazim nanoparticles prepared by the preparation method have the characteristics of high drug loading rate, pH-responsive targeted release and excellent stability, and can realize efficient disease control under low dosage, significantly improve the drug utilization rate, reduce drug residues in non-target areas and reduce environmental risks. The research shows that the chitosan nano-selenium carrier and the carbendazim have a synergistic effect, and the treatment effect of a pot experiment is obviously superior to that of the same dosage of carbendazim and chitosan nano-selenium carrier treatment. Besides, the nanoparticles can promote peanut seed germination under low concentration, have no residual toxicity to non-target plants, are environment-friendly, are suitable for prevention and control of fungal diseases of crops, provide an efficient and environment-friendly nano-pesticide scheme for agriculture, and assist sustainable agricultural development.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanopesticides, and more specifically relates to a pH-responsive nano-selenium-loaded carbendazim nanopesticide, a preparation method thereof, and an application thereof. Background Art

[0002] As modern agriculture's demand for pesticides increases, the shortcomings of traditional pesticides are becoming increasingly prominent. For example, while carbendazim (CBZ) can effectively inhibit fungal diseases, its susceptibility to environmental degradation, low utilization rate (less than 30%), and the tendency to induce pathogen resistance limit its application. Furthermore, traditional formulations lack targeting, making them prone to environmental pollution and toxicity to non-target organisms.

[0003] In recent years, nanocarrier technology has provided new insights for increasing pesticide efficacy and reducing pesticide dosage. Selenium nanoparticles (Se NPs) have attracted considerable attention due to their low toxicity, high bioactivity, and environmental friendliness. However, their high surface energy and tendency to agglomerate result in poor stability, limiting their practical applications. Chitosan (CS) is commonly used as a stabilizer, but when used alone as a pesticide carrier, it suffers from drawbacks such as uncontrollable drug release and low loading efficiency (typically less than 3%).

[0004] Peanut stem and root rot, caused by Fusarium oxysporum, is a major agricultural disease, causing over $10 billion in losses annually. Existing control methods rely on high pesticide doses, which not only exacerbate pesticide resistance but also lead to soil residues. Therefore, developing a nanopesticide delivery system that responds to the pathogen's acidic microenvironment, synergistically enhances antibacterial efficacy, and achieves high loading efficiency has become an urgent technical challenge. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of existing carbendazim pesticides, such as easy decomposition, low utilization rate, high non-target toxicity and high environmental residual risk, and provide a chitosan-nanoselenium-loaded carbendazim nanoparticle that can achieve efficient disease prevention and control at extremely low dosage, has high drug utilization rate, and is environmentally friendly.

[0006] The first purpose of the present invention is to provide a chitosan-nanoselenium-loaded carbendazim nanoparticle and a preparation method and application thereof.

[0007] The third object of the present invention is to provide a method for preventing and controlling plant fungal diseases.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention utilizes positively charged chitosan as a surface modifier to reduce selenite to generate nanoselenium, which is then self-assembled into a stable spherical structure through electrostatic interactions and hydrogen bonds. Carbendazim (CBZ) is then coupled to the surface of the nanoparticles to construct a new type of nanoparticle (CBZ@Se CS Nps, CSC). Infrared spectroscopy analysis of the CSC particles shows that the C=N / C=O vibration peak of carbendazim is clearly visible, while the intensity of the amide I band of chitosan is significantly reduced, indicating that the two interact with each other. The CSC nanoparticles of the present invention have a certain degree of stability and can not only release drugs in response to acidic conditions in the fungal microenvironment, but also significantly enhance the antibacterial effect through synergistic effects, while reducing the premature release of pesticides and environmental pollution, providing a new solution for the prevention and control of plant fungal diseases. Therefore, this application claims protection for the following solutions:

[0010] The present invention provides a method for preparing chitosan-nanoselenium-loaded carbendazim nanoparticles, comprising the following steps:

[0011] S1. chitosan is added to the reducing agent solution to react, and then selenite is added to react and the solid reaction product is recovered;

[0012] S2. The solid reaction product obtained in step S1 is added to a carbendazim solution and soaked. After soaking, the solid is recovered to obtain nanoparticles.

[0013] As an optional embodiment, the concentration of the reducing agent solution is 1-3 mg / mL.

[0014] As an optional embodiment, in step S1, the final concentration of chitosan in the reducing agent solution is 1-5 mg / mL; the final concentration of selenite in the reducing agent solution is 0.5-1 mg / mL.

[0015] As an optional embodiment, in step S1, the final concentration of chitosan in the reducing agent solution is 3.3 mg / mL; the final concentration of selenite in the reducing agent solution is 0.725 mg / mL.

[0016] As an optional embodiment, the reducing agent is any one of vitamin C, glutathione, cysteine, and sodium thiosulfate; the selenite is any one of sodium selenite, potassium selenite, and ammonium selenite.

[0017] As an optional embodiment, the reducing agent is vitamin C.

[0018] As an optional embodiment, the selenite is sodium selenite.

[0019] As an alternative embodiment, the concentration of the carbendazim solution is 0.5-1.5 mg / mL.

[0020] As an alternative embodiment, the concentration of the carbendazim solution is 0.85 mg / mL.

[0021] As an optional embodiment, in step S2, the mass volume ratio of the solid reaction product to the carbendazim solution is (5-15) mg: (5-15) mL.

[0022] As an optional embodiment, in step S2, the soaking is performed at 20-35° C. for 8-16 hours.

[0023] As an optional embodiment, in step S2, the soaking is performed at 20-35° C. for 12 hours.

[0024] As an optional embodiment, in step S2, the mass volume ratio of the solid reaction product to the carbendazim solution is 10 mg:10 mL.

[0025] As an optional embodiment, in step S1, after adding chitosan, stirring is carried out for 20-40 minutes (preferably 30 minutes) at a stirring speed of 300-500 rpm (preferably 440 rpm).

[0026] As an optional embodiment, in step S1, the solid reaction product is recovered by centrifugation, washing, and drying.

[0027] Specifically, the centrifugation is refrigerated centrifugation at 8000-12000 rpm for 10-20 min (preferably centrifugation at 10000 rpm for 15 min); the washing solution is methanol or water; and the drying is freeze drying.

[0028] As an optional embodiment, in step S2, the solid is recovered by centrifugation, washing, dialysis, and drying.

[0029] Specifically, the centrifugation is refrigerated centrifugation at 8000-12000 rpm for 10-20 min (preferably 10000 rpm for 15 min); the washing solution is methanol or water; the dialysis is performed using a dialysis membrane; and the drying is freeze-drying.

[0030] The invention provides chitosan-nanoselenium-loaded carbendazim nanoparticles prepared by the above preparation method.

[0031] The present invention provides application of the chitosan-nanoselenium-loaded carbendazim nanoparticles in the preparation of pesticides.

[0032] The present invention provides application of the chitosan-nanoselenium-loaded carbendazim nanoparticles in preventing and treating plant fungal diseases.

[0033] The application also provides a method for preventing and treating plant fungal diseases, which uses the nanoparticles to treat plants.

[0034] The plant fungal diseases are diseases caused by Fusarium oxysporum, such as crop wilt.

[0035] The application has the following beneficial effects:

[0036] The application provides a kind of chitosan-nano selenium loaded carbendazim nanoparticles, which has high loading rate and excellent stability, can be used as nano pesticide, and provides a kind of efficient, environmentally friendly green prevention and control solution for modern agriculture. Experiments show that the nanoparticles have excellent performance in pH stability, salt ion stability and thermal stability, effectively solving the problem of instability and easy degradation of traditional carbendazim in the environment. At the same time, the carbendazim encapsulation rate of the nanoparticles is 20.13±0.87%, and the loading rate is 6.50%. High-efficiency disease prevention and control can be achieved at low dosage, which significantly improves the drug utilization rate and avoids the environmental pollution and drug resistance problems caused by large dosage of traditional pesticides.

[0037] In addition, the nanoparticles have pH-responsive targeted release characteristics and show significant drug accumulation release capacity under simulated fungal microenvironment (pH 5.0). The cumulative release amount of carbendazim after 144 hours is 79.85%, which is 26.8% higher than the release rate of 19% under neutral conditions (pH 7.4). This precise targeted delivery mechanism not only improves the drug efficacy, but also reduces drug residues in non-target areas and reduces environmental risks. The research also found that there is a synergistic effect between the chitosan nano selenium carrier and carbendazim. After loading carbendazim into the chitosan nano selenium carrier, the EC 50 value (0.139±0.014mg / L) is about 77.02% lower than that of free carbendazim (0.605±0.023mg / L). At the same time, the carrier itself (SC) has no significant toxicity at a concentration of 200ppm, ensuring its safety and providing a new idea for the development of efficient and low-toxicity pesticides.

[0038] In terms of environmental friendliness, the nanoparticles of the application can promote peanut seed germination at low concentrations (≤0.05ppm) and have no residual toxicity to non-target organisms (such as earthworms), which meets the green pesticide standard. Its excellent biocompatibility and environmental friendliness significantly reduce the negative impact on the ecosystem. More importantly, the nanoparticles of the application have the dual functions of treating and preventing plant fungal diseases, which are suitable for the prevention and treatment of crop fungal diseases and have wide application potential, providing a kind of efficient, environmentally friendly nano pesticide solution for agriculture and helping the sustainable agricultural development. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1Synthesis mechanism of chitosan-nano selenium loaded carbendazim nanoparticles.

[0040] Figure 2 Performance characterization of chitosan-nano selenium loaded carbendazim nanoparticles (A: particle size distribution of CSC and SC; B: Zeta potential of CSC and SC; C: thermogravimetric analysis results of CSC, SC, CBZ and CS; D: infrared spectroscopy analysis results).

[0041] Figure 3 Electron microscope observation results of SC and CSC (A: scanning electron microscope image; B: transmission electron microscope image).

[0042] Figure 4 XPS analysis results of different samples (A: XPS analysis results of CBZ, CS, SC and CSC; B, C, D, E are respectively 1s spectrum, N 1s spectrum, O 1s spectrum and Se spectrum of SC; F, G, H, I are respectively C1s spectrum, N 1s spectrum, O 1s spectrum and Se spectrum of CSC; J, K, L are respectively C1s spectrum, N 1s spectrum, O 1s spectrum of CBZ).

[0043] Figure 5 Cumulative release curves of chitosan-nano selenium loaded carbendazim nanoparticles under acidic and neutral conditions.

[0044] Figure 6 Dynamic contact angle and surface tension measurement results of nanomaterials on peanut leaves.

[0045] Figure 7 Plate inhibition experiment results of SC, CSC and CBZ (A: inhibition effect of CSC, SC, CBZ on Fusarium oxysporum; B: EC 50 value measurement results of CSC, SC, CBZ; inhibition rate of CSC, SC, CBZ).

[0046] Figure 8 Filter paper piece inhibition experiment results of CBZ and CSC (A: antibacterial effect of filter paper pieces of different treatments; B: inhibition rate of different treatments).

[0047] Figure 9 Spore germination inhibition experiment results of SC, CSC and CBZ (A: inhibition effect of SC, CSC and CBZ on spore germination; B: inhibition rate of SC; C: inhibition rate of CBZ and CSC; D: IC 50 value of spore germination inhibition).

[0048] Figure 10Results of the inhibition and destruction of biofilm (A: Inhibition effect of CSC and CBZ on biofilm; B: Destruction effect of CSC and CBZ on biofilm; C: EC50 of CSC and CBZ treated biofilm; D: Analysis results of inhibition rate and destruction rate). 50

[0049] Figure 11 Results of the treatment experiment of stem rot caused by Fusarium oxysporum infection on isolated peanut stems (A: Treatment mode; B: Prevention mode; C: Control efficiency of treatment mode; D: Control efficiency of prevention mode).

[0050] Figure 12 Results of the treatment experiment of Fusarium oxysporum infection on isolated peanut roots (A: Phenotype chart of the treatment effect of Fusarium oxysporum infection on isolated peanut roots; B: Disease control degree of CSC administration on isolated peanut roots).

[0051] Figure 13 Results of the potting prevention experiment of different drugs (A: Experimental photos of different drugs treating Fusarium oxysporum infection on peanut stems; B: Treatment efficiency and infection area).

[0052] Figure 14 Results of the safety evaluation experiment of different drugs on peanut seed germination (A: Seed germination rate; B: Comparison data of germination rate).

[0053] Figure 15 Results of the safety evaluation experiment of different drugs on peanut seed germination (A: Seed germination rate; B: Comparison data of germination rate).

[0054] Figure 16 Results of the risk evaluation of different drugs on non-target organisms. DETAILED DESCRIPTION

[0055] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0056] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0057] Chitosan, CS for short, was purchased from Shanghai Yinn Chemical Technology Co., Ltd., CAS: 9012-76-4.

[0058] Carbendazim, CBZ for short, was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., CAS: 10605-21-7.

[0059] ​Ascorbic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS: 50-81-7.

[0060] Sodium selenite was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., CAS: 10102-18-8.

[0061] Potato dextrose agar (PDA) was purchased from Beijing Solebaugh Technology Co., Ltd.

[0062] The preparation method of the Fusarium oxysporum spore suspension in Example 8 refers to Chinese patent CN114350521A.

[0063] The Fusarium oxysporum bacteria used in the experiment were stored in the inventor's laboratory.

[0064] Example 1 Preparation of chitosan-nanoselenium-loaded carbendazim nanoparticles

[0065] Synthesis mechanism of chitosan-nanoselenium-loaded carbendazim nanoparticles Figure 1 Chitosan anchors the nanoselenium particles through a Se-O conformational lock, forming a stable carrier structure. Its surface is rich in active hydroxyl and amino groups, providing binding sites for drug loading. CBZ is adsorbed onto the chitosan nanoselenium surface through hydrogen bonding and electrostatic interactions between carboxylic acid and amine groups. The coordination structure of the nanoselenium prevents rapid drug degradation at high temperatures or in ambient conditions through physical barriers and chemical stabilization.

[0066] Preparation method of chitosan solution: 2% chitosan solution: weigh 1g chitosan into a beaker, add 49mL 1% acetic acid to fully dissolve it, and transfer it to a plastic bottle for later use.

[0067] Preparation method of sodium selenite solution: 6 mg / mL sodium selenite solution: weigh 0.3 g of sodium selenite into a beaker, add 50 mL of distilled water to fully dissolve it, and transfer it to a plastic bottle for later use.

[0068] Preparation method of carbendazim solution: 0.85 mg / mL carbendazim solution: weigh 8.5 mg of carbendazim and dissolve it in 10 mL of glacial acetic acid. Slowly add it into 90 mL of deionized water, stir evenly, and transfer it to a plastic bottle for later use.

[0069] (1) Preparation of chitosan nanoselenium (Se@CS NPs, SC) carrier

[0070] 40 mg of vitamin C was dissolved in 20 mL of deionized water. Once completely dissolved, 4 mL of a 2% chitosan solution was added and magnetic stirring was performed at 440 rpm for 30 min. 3.3 mL of a 6 mg / mL sodium selenite solution was added dropwise, gradually changing the color from transparent to red. Subsequently, the resulting mixture was refrigerated centrifuged at 10,000 rpm for 15 min, and the sample was washed with methanol and water three times. Freeze-dried to obtain solid SC.

[0071] (2) Preparation of chitosan-selenium nanoparticles loaded with carbendazim (CBZ@Se CS NPs, abbreviated as CSC)

[0072] 10 mg of SC solid was added to 10 mL of 0.85 mg / mL carbendazim solution and activated at room temperature for 12 hours (temperature 27°C, humidity 75%). Subsequently, the resulting mixture was refrigerated centrifuged at 10,000 rpm / min for 15 minutes, washed with methanol and water, and repeated three times. After dialysis with a dialysis membrane, solid CSC was obtained by freeze-drying for 48 hours.

[0073] The Se concentration was determined by ICP-MS, and the encapsulation efficiency of carbendazim was determined by HPLC.

[0074] Example 2 Performance Characterization and Mechanism Verification of Chitosan-Nano-Selenium-Loaded Carbendazim Nanoparticles

[0075] 1. Experimental Methods

[0076] The SC and CSC samples prepared in Example 1 were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to analyze the size and shape of the nanomaterials. The particle size distribution of the nanomaterials was evaluated using a particle size analyzer (Zetasizer nano, Malvern Instruments). The sample size was calculated based on the dynamic light scattering (DLS) method. The scattered light was collected at a 90° angle by an optical fiber and converted into an electrical signal by an avalanche photodiode (APDS). The sample size was determined by Fourier transform infrared spectroscopy (FTIR) at 4000-500 cm -1 The presence of functional groups was confirmed in the spectral range of .Selenium components and elemental composition were observed by X-ray photoelectron spectroscopy.

[0077] 2. Experimental Results

[0078] (1) The performance characterization results show that the average particle sizes of SC and CSC are 102.82±4.86nm and 129.21±2.54nm ( Figure 2 The average particle size of CSC increased by about 26.39 nm compared with that of SC. The zeta potentials of SC and CSC were 70.6±3.83 mV and 43.1±0.9 mV, respectively. Figure 2Figure B shows that the zeta potential of CSC is about 27.5 mV lower than that of SC, which may be due to the interaction between the carboxylic acid group (-COOH) of CBZ and the protonated amino group (-NH3 + ) undergoes electrostatic neutralization, resulting in a decrease in the surface positive charge density, but it is still much higher than the critical stability value (usually ±30mV), indicating that CSC still has good stability in the dispersed system and is not prone to serious aggregation, making it suitable for application scenarios such as drug delivery.

[0079] (2) In order to study the thermal stability of SC and CSC materials, thermogravimetric analysis was performed on SC and CSC materials. The results are shown in the figure. Figure 2 As shown in Figure C, the results show that the thermal decomposition temperature range of CSC (250-450°C) is 50°C wider than that of free CBZ (250-400°C), and the residual content (15-20wt%) is between that of SC (30-40wt%) and CBZ (10-15wt%). The carbon barrier effect of the Se@CS core layer and the synergistic carbonization of the CBZ shell layer indicate that the core-shell interface delays thermal degradation through molecular confinement, significantly improving adaptability to high-temperature environments.

[0080] (3) Infrared spectroscopy (FTIR) analysis results are as follows Figure 2 As shown in Figure D, the results show that carbendazim (CBZ) is successfully loaded into the chitosan nanoselenium carrier. In the infrared spectrum of CSC, 3300-3500 cm -1 The broad OH / NH stretching vibration peak of chitosan (CS) and the NH characteristic peak of carbendazim are retained in the region, indicating the coexistence of the two functional groups; 1600-1700 cm -1 The C=N / C=O vibration peak of carbendazim is clearly visible in the range of 1500-1600 cm, while the intensity of the amide I band of chitosan is significantly reduced, indicating that the two interact with each other; -1 The C=C vibration peak of the benzene ring of CBZ did not shift or disappear in CSC, proving that the structure of carbendazim was intact and not destroyed. In addition, the OH peak of SC (3200-3500cm-1) shifted slightly to a lower wave number in CSC, and the peak shape became narrower. This may be due to the formation of a Se-O conformational lock coordination bond, which resulted in the restriction of hydroxyl vibration and the close intermolecular binding. It is also possible that the amino group (-NH2) of carbendazim formed a hydrogen bond or electrostatic interaction with the hydroxyl group (-OH) of chitosan. The vibration peak of Se-OC bond (such as selenized polysaccharide) is at 850-950cm -1 In summary, the infrared data verified the successful loading of carbendazim into the chitosan nanoselenium carrier at the molecular level through characteristic peak superposition, shift and interaction evidence.

[0081] (4) The results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are as follows: Figure 3As shown in Figures A and B, SC is a monodisperse sphere, uniformly dispersed in the chitosan matrix through Se-N / O coordination; after drug loading, the surface roughness of CSC increases, the core-shell interface is clear (shell layer 20.3nm), and there is a significant positive correlation with the particle size increment (26.39nm). Surface potential analysis shows that the zeta potential of CSC (43.1±0.9mV) is lower than that of SC (70.6±3.83mV) ( Figure 2 Figure B), which may be due to the neutralization of the carboxylic acid-amino charge. Under 4°C storage conditions, the particle size change was <15% in 90 days, ensuring long-term colloidal stability ( Figure 2 Positively or negatively charged nanoparticles with potential amplitudes higher than 20 or 30 mV (Smoluchowski approximation) are more likely to be taken up by plant cells or chloroplast membranes, respectively.

[0082] (5) XPS analysis of the elemental composition and valence distribution of the SC and CSC samples prepared in Example 1 showed the following results: Figure 4 As shown, the SC main peak of 55.30eV corresponds to the Se-N / Se-O weak coordination bond, and the secondary peak of 56.15eV is attributed to the Se=O double bond ( Figure 4 The Se 3d binding energy is significantly higher than that of low-valence elemental Se (~54.5 eV), indicating that the selenium atoms may coordinate with highly electronegative N / O atoms (such as Se-OC or Se-NH2), resulting in a decrease in the electron cloud density; the OC=O characteristic peak (288.24 eV) in CSC matches the CBZ standard spectrum (ΔE=0.22 eV); the new C-NH-C component (399.10 eV) in the N 1s spectrum confirms that CBZ is anchored to the SC surface through electrostatic-hydrogen bonds and that the Se active sites (ΔE<0.1 eV) are not destroyed ( Figure 4 These characterizations collectively demonstrate that CBZ is precisely loaded through interfacial interactions while maintaining the structural integrity of the core layer, providing a structural basis for pH-responsive controlled release.

[0083] Example 3 Evaluation of drug loading performance of CSC

[0084] To obtain a standard curve for CBZ, 10 μg mL -1 Carbendazim standard stock solution was diluted with acetonitrile to obtain standard solutions with concentrations of 0.5, 2.5, 4.0, 6.0, 7.5, and 10 mg / L. The standard solutions were analyzed using high-performance liquid chromatography (HPLC, Shimadzu, LC-20AT) under specific conditions. The specific test conditions were as follows: a COSMOSIL C18 column (250 mm × 4.6 mm, 5 μm); a mobile phase of methanol and water (60:40, V / V); and a flow rate of 1.0 mL / min. -1; Detection wavelength: 281nm; column temperature: 35-38℃; injection volume: 20μL.

[0085] Chitosan nano-selenium was added to the carbendazim solution, stirred at room temperature for 12 hours, centrifuged (12000rpm / min, 15 / 20 / 30min), washed with a mixture of 50% ethanol and water and centrifuged again to obtain a supernatant. Subsequently, the precipitate was freeze-dried and weighed; the supernatant was filtered through a 0.22μm filter membrane and transferred to a 1.5mL brown injection bottle. The sample was then subjected to HPLC detection and analysis, and repeated 3 times to ensure the accuracy of the results. The standard curve was measured as A=0.03851C+0.00808(R 2 =0.999), and the encapsulation efficiency and loading rate in the nanocomposite material were calculated according to the following formulas.

[0086]

[0087] where M1 is the total amount of added CBZ, M2 is the free CBZ in the supernatant, and M0 is the weight of the nanoparticles.

[0088] The results showed that the encapsulation efficiency of CSC was 20.13±0.87% and the loading rate was 6.50%, meeting the basic requirements of a functional pesticide carrier.

[0089] Example 4 Controlled Release Kinetics of Chitosan-Nano-Selenium-Loaded Carbendazim Nanoparticles

[0090] To investigate the in vitro release characteristics of chitosan-nanoselenium-loaded carbendazim nanoparticles (CSC) in the slightly acidic environment of plants infected by Fusarium spp., the release kinetics of carbendazim were analyzed, the release model was fitted, and its potential as a pesticide controlled release system and its inhibitory effect on plant pathogenic fungi were evaluated.

[0091] The cumulative release rate of CBZ from CBZ@SeCS NPs (CSC) was studied under different pH conditions (pH 7.4 and pH 5) and in the presence or absence of chitosan nanoselenium. 1 mL of CSC solution was placed in a pre-prepared dialysis bag immersed in a beaker containing 150 mL of buffer solution (PBS: ethanol: Tween-80 = 70:29.5:0.5) and stirred at 440 rpm. The pH of the release medium was adjusted with NaOH and HCl solutions. 5 mL of solution was withdrawn from the beaker at 1, 2, 4, 6, 10, 14, 24, 36, 48, and 60 h, respectively, and 5 mL of the corresponding buffer solution was added to maintain a constant total volume.

[0092] In order to explore the release mechanism of CBZ@Se CS NPs, the zero-order, first-order, Ritger-Peppas, and Higuchi kinetic equation models were used to fit the cumulative release rate data of the drug. The regression coefficient in the model was R 2 The equation closest to 1 was considered the best fitting model. pH = 5, HPLC standard curve of carbendazim was measured as: y = 77947x + 4324.8, (R 2 =0.9999), pH=7.4, the HPLC standard curve of carbendazim is measured as: y=85549x+34360, (R 2 =0.9972).

[0093] The cumulative release rate was calculated using the following formula:

[0094]

[0095] Where: M t is the mass of CBZ in the solution absorbed at time t, and M is the amount of CBZ loaded in 1 mL of CSC.

[0096] The cumulative release curves of CSC at different pH values ​​are as follows: Figure 5 As shown in the results, CSC exhibited significant pH-responsive release characteristics in the acidic microenvironment (pH = 5) infected by Fusarium: under acidic conditions (pH = 5), the cumulative release of carbendazim reached 79.85% (first-order model), which was about 20% higher than that under neutral conditions (pH = 7.4), mainly attributed to the protonated swelling of chitosan accelerating drug diffusion.

[0097] The kinetic model fitting results of CBZ in CSC are shown in Table 1. The results show that the Ritger-Peppas model (R 2 =0.9813), indicating that the release mechanism of CSC is primarily Fickian diffusion, with chitosan swelling affecting the release rate. CSC achieves targeted rapid release in an acidic environment (0-10 hours for the rapid phase). Sustained release in a neutral environment not only reduces non-target exposure but also offers long-term efficacy (maintaining efficacy during the sustained release phase) and environmental friendliness (chitosan / nanoselenium are biodegradable).

[0098] Table 1 Kinetic model fitting results of CBZ in CSC

[0099]

[0100] Example 5 Leaf Wetting Performance Determination

[0101] 1. Experimental Methods

[0102] The dynamic wetting behavior of the nanomaterials on the peanut leaf surface was studied using a contact angle measuring instrument (SINDIN SDC-350, China) and a surface tension meter (KRUSS GmbH, Germany). The experiment was carried out in a constant temperature and humidity environment (25 ± 1°C), and 5 μL of 1.5 mg / L drug solution (aqueous solution of SC, CBZ and CSC) was vertically dropped on the peanut leaf surface cleaned by ethanol and naturally dried. The solid static drop method was used to record the evolution of the liquid drop contact angle with time (0-10 s). Each group of experiments was repeated 3 times, and the data was expressed as mean ± standard deviation. Single factor analysis of variance (ANOVA) was used to evaluate the significance of the difference between groups (p < 0.05).

[0103] II. Experimental results

[0104] The contact angle and surface tension test data showed that, as shown in Figure 6 the left and right contact angles of SC, CBZ and CSC were significantly different: after 10 seconds, in the left contact angle, SC showed a lower value (about 22.9°), CBZ was higher (about 73.8°), and CSC was in the middle (44.3°); the right contact angle trend was similar, the strong hydrogen bond between the protonated amino group on the SC surface and the water molecules accelerated the spreading of the liquid drop; the non-polar structure of the benzimidazole ring contained in CBZ limited the dynamic wetting (Δθ ≈ 0.5°), maintaining high hydrophobicity; the left and right contact angles of CSC were between the two, which may be because the carboxyl group of carbendazim (CBZ) partially neutralized the surface amino group, but the rough surface reduced the salt sensitivity, so it had better leaf surface wetting than CBZ. At the same time, the surface tension of CSC and SC was 213.33 ± 5 mN / m and 324 ± 10 mN / m, respectively, which was significantly better than the hydrophobic carbendazim technical material (73.8°, 18 ± 5 mN / m).

[0105] Example 6 Plate Bacteriostatic Experiment

[0106] I. Experimental method

[0107] The antifungal properties of the nanomaterials were determined using a PDA culture method. PDA culture medium was sterilized, and pre-designed concentrations of SC, CSC, and CBZ were added to the PDA medium to prepare PDA culture medium containing different concentrations of SC (0, 0.2, 0.4, 0.6, 1, 10, 100, 200, 300, and 400 mg / L), CSC, and CBZ (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg / L). A 6-mm-diameter filter cake was cut from a colony of Fusarium oxysporum grown on a PDA plate for 5 days and inoculated into the PDA medium. The filter cake was then concentrated in a warm incubator (28°C). Each component was treated three times, and an untreated group served as a blank control. Colony diameters of the treated components were measured using ImagieJ at pre-set time points (48, 72, 96, 120, and 144 hours). The following formula was used to calculate the inhibition rate:

[0108] inhibition rate (%) = [(D t～CK -D0)-(D t -D0)] / [(D t～CK -D0)]×100

[0109] Among them D t-CK is the diameter of the colony in the blank control group (mm), D t is the diameter of the bacterial colony in the experimental sample (mm), D0 is the initial diameter of the bacterial cake (6 mm), and t is the incubation time. 50 The value (the concentration corresponding to 50% inhibition rate) was obtained from the mycelial growth inhibition rate. GraphPad Prism was used to fit the concentration-effect curve and EC 50 The experiments were performed under sterile conditions throughout, and the data are presented as mean ± standard deviation.

[0110] 2. Experimental Results

[0111] The experimental results are as follows Figure 7 As shown in Figure 2, CSC showed a significant antibacterial advantage over SC and CBZ: CSC reached a 50% inhibition rate at 0.139 mg / L ( Figure 7 Figure B), compared with CBZ (0.605 mg / L, Figure 7 Figure C) reduced the dose requirement by 4.35-fold, while the EC of SC 50 As high as 73.61±0.920mg / L. The jump in efficacy is attributed to the intelligent drug delivery mechanism triggered by the acidic microenvironment (pH=5) - chitosan protonation promotes the targeted swelling of the carrier, and simultaneously releases nano-selenium and CBZ, forming a "ROS oxidation membrane rupture + microtubule inhibition" double killing effect ( Figure 7 Figure B).

[0112] At low concentration, nano-selenium catalyzes ROS to destroy the integrity of fungal membrane under acidic condition, forming a synergistic effect with CBZ microtubule inhibition; at high concentration, the carrier partially disintegrates to accelerate drug release, establishing a concentration gradient response mode of "rapid membrane damage amplification + sustained cell division inhibition". Compared with the non-targeted loss defects of traditional CBZ dosage forms, the drug utilization rate of CSC is increased by 16 times through pH-responsive positive charge targeting.

[0113] SC alone only relies on physical damage of nano-selenium (EC 50 = 73.61 ± 0.920 mg / L), confirming that the chitosan-selenium composite system mainly acts as an intelligent carrier to improve delivery efficiency by stabilizing drugs and enhancing membrane targeting, rather than directly participating in antibiosis. Plate experiments further reveal that CSC reduces the effective dose of CBZ from 0.605 mg / L to 0.139 mg / L through precise drug release.

[0114] Example 7 Filter paper antibacterial experiment

[0115] The filter paper experiment further demonstrates the inhibitory effect of the synthesized nano-pesticide on Fusarium oxysporum. Fusarium oxysporum was inoculated in PDA medium and incubated at 28°C for 5 days. Filter paper with a 1 cm edge was placed in the same size. Referring to the previous antibacterial experiment results, the filter paper was soaked in different concentrations of CBZ (CK, 6, 50, 100 mg / L), CBZ@Se-CSNps (CK, 1, 3, 6 mg / L) for 10 minutes, and sterile water was used as a blank control group. In the incubator, when the mycelium of the blank control group grew to the edge of the filter paper, the distance between the edge of each filter paper and the mycelium was measured and recorded to evaluate the antibacterial activity. GraphPad Prism was used to fit the concentration-effect curve. The whole experiment was conducted under sterile conditions, and the data was expressed as mean ± standard deviation.

[0116] The experimental results are shown in Figure 8 The results show that the antibacterial efficiency of 1 mg / L CSC is 57.08 ± 3.64%, while that of 6 mg / L CBZ is only 46.31 ± 4.82%. This indicates that CSC is not only more efficient in dosage, but also has stronger antibacterial effect. The filter paper experiment further verifies that the drug release concentration at low dose has met the demand for high-efficiency antibacterial effect, indicating that CSC significantly improves drug utilization rate.

[0117] Example 8 Spore germination inhibition experiment

[0118] I. Experimental method

[0119] Fusarium oxysporum spore suspension was prepared, washed and filtered with sterile deionized water, and adjusted to 10 6CFU / mL concentration. The spore solution of Fusarium oxysporum was dispensed into a biconcave slide (100 μL / concave), and 10 μL of gradient concentration drugs (SC: 0.1-100 mg / L; CSC: 0.05-2.0 mg / L; CBZ: 1.0-80 mg / L) were added respectively. Blank (CK) and solvent (B) controls were set up, and each group was repeated 3 times. After incubation at 28°C in the dark for 24 hours, 5 visual fields were randomly selected under an optical microscope (400×) to calculate the germination rate (number of germinated spores / total number of spores × 100%), and the relative inhibition rate (1-germination rate of treatment group / germination rate of blank group × 100%) was calculated. The concentration-effect curve and the concentration IC required to achieve 50% inhibition were fitted using GraphPad Prism. 50 The experiments were performed under sterile conditions throughout, and the data are presented as mean ± standard deviation.

[0120] 2. Experimental Results

[0121] The results of the spore germination inhibition experiments of SC, CSC and CBZ are as follows Figure 9 As shown in Figure 2, the results showed that SC, CSC and CBZ all exhibited concentration-dependent inhibitory effects on spore germination, among which CSC exhibited the best inhibitory activity ( Figure 9 Figure B). Its IC 50 The value (0.173±0.004mg / L) was nearly one order of magnitude lower (about 9 times) than that of CBZ (1.550±0.065mg / L) ( Figure 9 Figure C), and at a very low concentration of 0.1 mg / L, the germination rate was reduced to 53.33% ( Figure 9 Figure D). Especially at a high concentration of 2.0 mg / L, the germination rate of the CSC-treated group (6.67%) was significantly lower than that of the CBZ group (36.25%, p<0.01), revealing the synergistic effect of its composite structure. Combined with the slightly acidic response properties, CSC may release active ingredients rapidly at the target site, directly blocking morphological formation by inhibiting microtubule synthesis in the early stages of spore germination. In contrast, SC (IC 50 =39.810±1.080 mg / L) requires a concentration of 50 mg / L to achieve a germination rate <50%, indicating that CSC serves primarily as a carrier in the CSC nanosystem, indirectly regulating drug delivery through a physical encapsulation / sustained-release mechanism. This example demonstrates the outstanding advantages of CSC in achieving high-efficiency, low-dose antibacterial activity, with a dose-response relationship nearly 10-fold greater than that of traditional CBZ. This provides a novel structural template and theoretical support for the development of targeted antifungal nanomedicines.

[0122] Example 9 Biofilm Inhibition and Destruction Experiment

[0123] 1. Biofilm inhibition experimental method

[0124] Put 10 6A 1% inoculum of bacterial suspension (CFU / mL) was inoculated into 50 mL of PDB medium. The culture medium containing the bacterial suspension was transferred to a 96-well plate, 200 μL per well. CSC and CBZ were added at concentrations of 0.005, 0.01, 0.05, 0.1, 0.5, and 1 mg / L (diluted with sterile water), respectively. A blank PDB plate was set up, and the 96-well plate was placed in a 30°C incubator for static incubation. After 24 hours, the 96-well plate was removed from the incubator, excess bacterial suspension was aspirated, and after drying, it was washed once with 200 μL of 0.01% PBS buffer and dried. Anhydrous methanol was added (200 μL per well) and the biofilm was fixed for 15 minutes. Excess methanol was aspirated, and the plate was incubated at room temperature for 20 minutes, then inverted to dry. 0.01% crystal violet solution was added (220 μL per well) and stained for 20 minutes. The staining solution was aspirated, and the plate was inverted to dry. Add 220 μL of 0.01% PBS solution to wash twice, invert to dry; add glacial acetic acid, 220 μL per well, gently shake the plate, dissolve at room temperature for 3-5 minutes, and measure OD with a microplate reader. 570 nm values. GraphPad Prism fitted concentration-effect curves and EC 22 The experiments were performed under sterile conditions throughout, and the data are presented as mean ± standard deviation.

[0125] 2. Experimental Results

[0126] The effects of CSC and CBZ on the biofilm formation of Fusarium spp. Figure 10 Figure A) and the destruction of the formed biofilm ( Figure 10 (Figure B) effect.

[0127] Biofilm inhibition mechanism: CSCs exhibit ultrasensitive inhibitory properties, inhibiting ECs that form biofilms. 50 The value (0.020±0.001mg / L) was 70 times lower than that of CBZ (1.398±0.094mg / L) ( Figure 10 C), and at a concentration of 0.5 mg / L, the inhibition rate reached 91.67% (compared to 14.17% for CBZ, p < 0.01). Mechanistically, CSC triggers the protonation of chitosan through the acidic microenvironment (pH ≈ 5) at the site of Fusarium infection, resulting in the targeted release of high-concentration drugs, blocking bacterial adhesion and extracellular matrix (EPS) secretion. Simultaneously, nano-selenium catalyzes the production of reactive oxygen species (ROS), disrupting biofilm integrity and interfering with quorum sensing signal transmission, forming a dual inhibition mechanism of "physical barrier and signal interference," curbing biofilm formation at the source.

[0128] Biofilm degradation mechanism: CSC, with its nano-particle size (<200nm) and the positive charge of chitosan, efficiently penetrates the dense matrix of the biofilm, releasing CBZ and nano-selenium in the internal acidic microenvironment, achieving "outside-to-in" synergistic degradation: Nano-selenium decomposes β-1,3-glucan and membrane protein structures through ROS oxidation, while CBZ inhibits tubulin polymerization to eliminate deep-layer bacteria. The two work together to achieve a biofilm destruction rate of 92.55% for 0.5mg / L CSC (CBZ only 16.76%). At high concentrations, the carrier disintegrates and releases more active ingredients, simultaneously breaking through the physical barrier and drug resistance defense of the biofilm, and the removal efficiency is 5.5 times higher than that of traditional agents ( Figure 10 (Figure D), providing a new strategy for combating drug-resistant fungi.

[0129] Example 10 Peanut stem in vitro treatment

[0130] 1. Experimental Methods

[0131] A dual-mode control model for Fusarium oxysporum in peanut stems was established: homogenized peanut stems were rinsed with clean water to remove surface impurities, wiped with 75% ethanol for 30 seconds, soaked in 10% sodium hypochlorite solution for 2 minutes, rinsed four times with sterile water, and air-dried. A standardized wound (5 × 1 mm) was prepared in the middle of the stem using a sterile knife to avoid vascular damage. Fusarium oxysporum was cultured in polydimethylsiloxane (PDA) medium at 28°C for 5 days, and a 6 mm cake from the edge of the colony was applied to the wound. In the treatment mode, 20 μL of a drug solution (CSC / CBZ: 0.8 mg / L; control: sterile water) was injected into the stem 48 hours after inoculation. In the preventive mode, the stem was pre-injected with a drug solution (CSC / CBZ: 0.3 mg / L) for two consecutive days, followed by inoculation with the pathogen 48 hours later. Efficacy was evaluated under constant temperature incubation (28°C, 70% ± 2% relative humidity, 16 L:8 D photoperiod). The expansion of lesions was recorded daily, and the infected area was quantified by Image J. The control efficacy was calculated according to the formula:

[0132]

[0133] Among them, μ c and μ t are the average values ​​of the control group and the treated group after treatment, and and is the average value before treatment.

[0134] 2. Experimental Results

[0135] The results of the experimental treatment of stem rot caused by Fusarium oxysporum infection of isolated peanut stems are as follows Figure 11 shown.

[0136] In the treatment model ( Figure 11As shown in Figures A and C, CSC demonstrated a significant time-effectiveness advantage under the treatment model: its control efficacy (CE) continued to increase from 65.27% on day 2 to 77.8% on day 10, while the CE of the CBZ group decreased from 60.9% to 34.97% over the same period. This mechanism is hypothesized to arise from the slightly acidic microenvironment (pH 5.0-6.0) at the site of Fusarium infection, which triggers the disintegration of the nanoselenium carrier, simultaneously releasing carbendazim and selenium nanoparticles. Selenium-catalyzed production of reactive oxygen species (ROS) destroys the fungal cell membrane, creating a synergistic antibacterial "drug release-catalytic enhancement" mechanism. In contrast, CBZ, lacking carrier protection, experiences a sudden increase in concentration after application but is susceptible to environmental degradation, resulting in a significant decrease in efficacy over time.

[0137] In preventive mode ( Figure 11 (Figures B and D) On the second day after inoculation, CSC and CBZ had similar control efficiencies (27.10% vs. 26.98%), possibly reflecting a spatiotemporal competition between rapid spore invasion and drug penetration. By day 10, the CE of the CSC group reached 73.82%, slightly higher than the 71.20% of the CBZ group (p>0.05), but the difference was not significant. This suggests that plant resistance induction and initial drug exposure efficiency may dominate disease control in the preventive mode, partially masking the advantage of CSC's slow-release properties.

[0138] The experiment revealed the dual-mode action of CSC: in the therapeutic mode, its pH-responsive sustained release and selenium-carbendazim synergistic antibacterial mechanism effectively overcome the environmental degradation defect of traditional agents; while the equivalence in the preventive mode highlights the key regulatory role of the initial drug concentration gradient and host defense response, providing a spatiotemporal dynamic model reference for the design of intelligent drug delivery systems.

[0139] Example 11 Peanut root in vitro treatment

[0140] Peanut roots were used as the research object to systematically evaluate the therapeutic effect of CSC on Fusarium infection. Four treatment groups were set up in the experiment: CSC (1.5 mg / L), CBZ (1.5 mg / L), CK group (blank control, no drug added), and ddH2O group. Fusarium spore suspension (1×10 6 CFU / mL), the root phenotypic changes were recorded daily, and samples were taken for analysis from the second day (2D) to the seventh day (7D) after inoculation. The disease severity index (DSI) was used to quantify the degree of infection, and the calculation method was as follows:

[0141]

[0142] The disease grade was divided into 0-4 levels according to the mycelial coverage area (Table 2).

[0143] The root lesion area was segmented by threshold using Image J 1.54g software, and the absolute infection area (mm 2 The experiment was repeated three times for each group, and the data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to verify the significance of the differences among the groups (P < 0.05).

[0144] Table 2 Disease level

[0145] grade Mycelium coverage area Description of pathological characteristics 0 0％ No mycelium, healthy root system 1 1％-25％ Sparse hyphae, local infection 2 26％-50％ Moderate coverage of mycelium 3 51％-75％ Dense hyphae and browning of root surface 4 >75％ Completely covered with mycelium, root rot

[0146] 2. Experimental Results

[0147] The experimental results are as follows Figure 12 As shown, the CSC group maintained a DSI of 0% (no root infection) throughout the inoculation period, significantly outperforming the other treatments (P < 0.01). On the second day (2D) after inoculation, hyphae (DSI = 50%) were present in the CBZ group, while none were detected in the CSC group, indicating effective inhibition of initial hyphal colonization. By the seventh day, the control group had a DSI > 85% (root rot with hyphal proliferation), while the CSC group maintained intact roots, demonstrating sustained and stable rhizosphere drug concentration. There was no difference between the CBZ group and the original drug control (ddH2O) (P > 0.05), likely due to the rapid drop in effective concentration of the traditional formulation due to washout from the rhizosphere microenvironment. The zero-infection characteristic of CSC may be attributed to: ① the nanoselenium carrier enables targeted, sustained release of carbendazim, inhibiting hyphal spread; and ② continuous drug exposure blocks pathogen infection. In summary, CSC, through a carrier-mediated intelligent drug delivery system, transforms short-term antibacterial activity into long-term protection, providing a new technological path for root disease management.

[0148] Example 12 Potted Plant Prevention Effect

[0149] A dynamic model of plant-pathogen interactions was constructed to evaluate the efficacy of drugs against Fusarium infection. Six treatments (ddH2O control, FO suspension, 1.5 mg / L CBZ, 1.5 mg / L CSC, and 30 mg / L SC-SC) were used, with six biological replicates per group, encompassing a total of 36 peanut seedlings. A dynamic infection-treatment evaluation system was established using a standardized inoculation process and gradient drug treatment.

[0150] A 5-day-old Fusarium cake (5mm in diameter) was precisely inoculated at the base of the second pair of true leaves of the plant; after inoculation, the plant was transplanted to sterilized nutrient soil, and drug treatment was initiated after the initial infection lesions were assessed; the drug administration mode was to spray 2mL of the corresponding concentration of drug solution in the rhizosphere area of ​​each plant root every day after 48h, and the intervention was continued for 3 days. On the 14th day after treatment, the plant root system was completely obtained, and phenotypic records were carried out after a standardized cleaning procedure. Image J software was used to perform threshold segmentation and pixel statistics on the root lesion area, and the absolute infection area (mm 2Throughout the experiment, the lesion area of ​​each peanut group was regularly observed and recorded, and photos were taken to record the infection status. The control efficacy of the drug infection degree in the isolated plant tissue was calculated using the following formula:

[0151]

[0152] Among them, μ c and μ t are the average values ​​of the control group and the treatment group after treatment, and The difference between the groups was significant (p<0.05) using one-way analysis of variance (ANOVA) combined with Tukey's multiple comparison test.

[0153] The experimental results are as follows Figure 13 The results showed that ten days after treatment with different drugs, at a dose of 1.5 mg / L, CSC achieved an 85% therapeutic efficacy through pH-responsive release and synergistic action with drug-nanoselenium, reducing the infected area by 66% compared to the positive control CBZ group, significantly outperforming treatment with the same dose of CBZ and SC. The therapeutic efficacy of SC (30 mg / L) was far lower than that of CSC and CBZ, indicating that chitosan nanoselenium assists the carrier, rather than directly participating in the antibacterial effect.

[0154] Example 13 Safety Assessment

[0155] (1) Seed and plant safety

[0156] The effects of CSC and SC on peanut seed germination and seedling growth were evaluated using a hydroponic system. Full seeds were selected, surface-sterilized with 75% ethanol for 10 minutes, rinsed three times with sterile water, and then grouped (n = 8 seeds / group × 3 replicates). Seeds were soaked in CSC, SC, CBZ (gradient concentrations), and sterile water (control) for 6 hours (15 mL / group) and then transferred to filter paper Petri dishes impregnated with the corresponding drug (2 mL of treatment solution / dish). Seeds were incubated in a light incubator (27°C, 16 / 8 h light / dark cycle, 75% humidity) for 4 days, with daily medium changes. Germination percentage (GP = number of germinated shoots / total number × 100%) was recorded when the embryo length reached ≥1 cm. Seedlings meeting the standards were transplanted into the hydroponic system, and root and stem length biomass parameters were measured on the 14th day.

[0157] The results of the safety evaluation experiment of different drugs on peanut seed germination are as follows Figure 14 The safety evaluation results of different drugs on peanut seedlings are shown in Figure 15As shown, the results showed that the core-shell sustained-release structure of CSC nanomaterials exhibited unique low-dose, high-efficiency and safe synergistic properties (within the effective concentration range of 0.5-1.5 mg / L, it precisely controlled the release kinetics of carbendazim to maintain a 100% peanut seed germination rate (an increase of ≥30% compared with the CBZ group at the same concentration), and the seedling growth parameters (stem length increased by 16.1%, root length increased by 27.4%, P<0.001) were not statistically different from the blank control. The dose-effect analysis revealed its significant safety threshold: when the concentration was 2 mg / L, the germination rate decreased in a dose-dependent manner. It is speculated that the toxicity originated from the sudden release of selenium caused by the disintegration of the core-shell structure.

[0158] (2) Risk assessment of non-target organisms

[0159] To assess the ecological risk of CSC nanomaterials to non-target soil organisms (annelids), healthy earthworms with reproductive annuli were selected. After 24 hours of intestinal decontamination with distilled water, they were exposed to a blank control (CK), carbendazim (CBZ) (0.1875, 0.375, 0.75, 1, and 1.5 mg / L), chitosan nanoselenium (SC) (37.5, 75, 150, and 300 mg / L), and selenium-chitosan carbendazim nanospheres (CSC) (0.1875, 0.375, 0.75, 1, and 1.5 mg / L). Ten earthworms were exposed to each group, with three replicates. The experiments were conducted under strict temperature and humidity control (20 ± 2°C, 80% humidity, and darkness). Mortality was monitored by needle prick reaction within 18, 24, 48, 72, and 96 hours.

[0160] The experimental results of the acute toxicity of the highest concentration of drugs in each treatment group to earthworms are as follows: Figure 16 The results showed that no individual died in the CSC treatment group at the highest exposure concentration (1.5 mg / L) within 96 h (survival rate 100%), confirming that CS had no acute toxicity to earthworms at environmentally relevant concentrations (96h LC 50 >1.5mg / L), and the ecological risk to key soil decomposers is low.

[0161] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing chitosan-nanoselenium-loaded carbendazim nanoparticles, characterized in that: The steps include: S1. chitosan is added to the reducing agent solution to react, and then selenite is added to react and the solid reaction product is recovered; S2. The solid reaction product obtained in step S1 is added to a carbendazim solution and soaked. After soaking, the solid is recovered to obtain nanoparticles.

2. The preparation method according to claim 1, characterized in that The concentration of the reducing agent solution is 1-3 mg / mL; the concentration of the carbendazim solution is 0.5-1.5 mg / mL.

3. The preparation method according to claim 1, characterized in that: In step S1, the final concentration of chitosan in the reducing agent solution is 1-5 mg / mL; the final concentration of selenite in the reducing agent solution is 0.5-1 mg / mL.

4. The preparation method according to claim 1, characterized in that The reducing agent is any one of vitamin C, glutathione, cysteine, and sodium thiosulfate; and the selenite is any one of sodium selenite, potassium selenite, and ammonium selenite.

5. The preparation method according to claim 1, characterized in that: In step S2, the mass volume ratio of the solid reaction product to the carbendazim solution is (5-15) mg: (5-15) mL.

6. The preparation method according to claim 1, characterized in that: In step S2, the soaking is performed at 20-35° C. for 8-16 hours.

7. Chitosan-nanoselenium-loaded carbendazim nanoparticles prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the chitosan-nanoselenium-loaded carbendazim nanoparticles according to claim 7 in the preparation of pesticides.

9. Use of the chitosan-nanoselenium-loaded carbendazim nanoparticles according to claim 7 in preventing and controlling plant fungal diseases.

10. A method for preventing and controlling plant fungal diseases, characterized in that: Plants are treated using the nanoparticles according to claim 7.

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