Preparation method and application of double-response controlled-release pesticide

Abstract

The invention provides a preparation method and application of a double-response type controlled-release pesticide. The method comprises the following steps: S1, preparing modified biochar; s2, preparing a drug carrier by using the modified biochar; and S3, preparing the double-response controlled-release pesticide by using the drug carrier. The problems that an existing controlled-release pesticide cannot meet the controlled-release function easily, the preparation process is complex, and large-scale preparation is difficult are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant pathology, and particularly relates to a preparation method of a dual-response controlled-release pesticide and application thereof. Background Art

[0002] Fire blight caused by the amylovora pathogen E. amylovora is a devastating disease worldwide. The amylovora pathogen has a wide host range and can infect more than 220 species of plants in more than 40 genera. The amylovora pathogen mainly infects pome fruit trees and Rosaceae plants such as pears, apples, and Pyracantha. The pathogen can infect almost all above-ground tissues of the host plant, including flowers, twigs, trunks, main trunks, fruits, and rootstocks. Especially in the apple and pear industries, fire blight can cause a large number of fruit trees to die and cause serious economic losses. At present, chemical control is the most commonly used method to control the spread of fire blight. Spraying antibiotics is an effective method for preventing and controlling fire blight, but antibiotic resistance is a naturally occurring phenomenon. The irrational use of antibiotics accelerates the emergence of drug-resistant strains. Once bacteria acquire drug resistance, it is passed on to the next generation through vertical gene transfer or through other bacterial horizontal gene transfer processes, and the prevention and control of drug-resistant bacteria is more difficult.

[0003] Slow-release pesticides can reduce environmental damage while prolonging the efficacy of pesticides by slowly releasing the characteristics of pesticides. In addition, the rational use of slow-release pesticides can effectively inhibit the production of drug-resistant strains. However, most of the current pesticide controlled-release materials only provide a simple dissolution-regulated release behavior for pesticides, which makes it difficult to truly meet the controlled-release function in complex environments. Therefore, it is necessary to give the carrier environmental stimulus response characteristics to develop intelligent controlled-release pesticide systems. At present, pH, temperature, redox, enzymes and other stimulus response systems have entered the field of vision of researchers. Compared with traditional pesticides, intelligent controlled-release systems can prolong the shelf life of pesticides, reduce the amount of pesticides used, and improve the efficiency of pesticide use. However, the preparation process of existing intelligent controlled-release pesticide systems is relatively complicated and difficult to prepare and use on a large scale. Therefore, it is necessary to find a simple and effective method to control the release of pesticides. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of a dual-responsive controlled-release pesticide to solve the problems that existing controlled-release pesticides are difficult to meet the controlled-release function, have a complex preparation process, and are difficult to prepare on a large scale.

[0005] The technical solution adopted by the present invention is a method for preparing a dual-responsive controlled-release pesticide, comprising the following steps:

[0006] S1: Preparation of modified biochar;

[0007] S2: Preparation of drug carriers using modified biochar;

[0008] S3: Preparation of dual-responsive controlled-release pesticides using drug carriers.

[0009] Furthermore, the S1 includes:

[0010] S11: prepare a Na2SO4 solution, and add the gray jujube leaf powder into the Na2SO4 solution;

[0011] S12: stirring and centrifuging the mixed solution to separate the modified gray jujube leaf powder;

[0012] S13: drying the separated modified gray jujube leaf powder and then performing a pyrolysis treatment;

[0013] S14: cleaning the pyrolysis product, and performing suction filtration, drying, and sieving operations to obtain modified biochar.

[0014] Further, the S3 includes:

[0015] S31: mixing the drug carrier with the kasugamycin aqueous solution;

[0016] S32: adding 3-mercaptopropyltrimethoxysilane to the mixed solution of S31 and stirring, and centrifuging, washing, and drying to obtain KSM-loaded thiol-functionalized protein-biochar KSM-DM-YBC;

[0017] S33: dispersing the thiol-functionalized protein-biochar KSM-DM-YBC loaded with KSM in methanol, adding β-mercaptoethylamine and stirring, centrifuging, washing, and drying the obtained reaction solution to obtain amino-substituted protein-biochar particles KSM-DM-YBC-SS-NH2 loaded with KSM;

[0018] S34: dissolving pectin in deionized water to obtain a pectin aqueous solution, dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the pectin aqueous solution and stirring;

[0019] S35: Add amino-substituted protein-biochar particles KSM-DM-YBC-SS-NH2 and N-hydroxysuccinimide loaded with KSM to the mixed solution of S34 and stir, centrifuge, wash and dry the obtained reaction solution to obtain pectin-protein-biochar particles KSM-DM-YBC-SS-pectin loaded with KSM.

[0020] Another technical solution adopted by the present invention is:

[0021] The dual-response controlled-release pesticide prepared by the preparation method is used for the prevention and treatment of fire blight pathogen.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention uses biomass carbon material composite PTB nanofilm as a drug carrier, loads kasugamycin (KSM) and then connects the pectin shell with an amide bond to design a drug delivery system (KSM-DM-YBC-SS-pectin) that responds to both pH and pectinase stimulation. This design can achieve intelligent release of pesticide molecules, has rapid release capabilities under changes in pH and pectinase conditions, and can achieve precise release of KSM at the site of infection.

[0024] 2. Kasugamycin (KSM) has a good inhibitory effect on the prevention and treatment of fire blight. However, KSM is susceptible to photolysis, acid-base damage and oxidative degradation in the natural environment, which reduces its efficiency in production and application. The drug delivery system (KSM-DM-YBC-SS-pectin) prepared by the present invention can effectively prevent the photodegradation of KSM after 120h of ultraviolet radiation, increase its light stability by 2.8 times, and provide strong ultraviolet protection ability.

[0025] 3. At the same time, the material control effect of the drug delivery system (KSM-DM-YBC-SS-pectin) at a concentration of 400 mg / L is significantly better than that of KSM aqueous solution, and can significantly reduce the toxicity to pollen. In short, KSM-DM-YBC-SS-pectin exhibits excellent controlled release performance and light stability, and can safely and effectively control apricot fire blight at a concentration of 400 mg / L, providing a promising solution for the control of apricot fire blight in Xinjiang and similar environments.

[0026] 4. The present invention realizes the intelligent release of pesticide molecules and effectively prevents environmental pollution caused by pesticide leakage. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 These are the FT-IR spectra of pectin, kasugamycin KSM, 1.5hDM-YBC, KSM-1.5hDM-YBC and 1.5hDM-YBC-SS-pectin, wherein A is the spectrum of pectin and 1.5hDM-YBC-SS-pectin, and B is the spectrum of KSM, 1.5hDM-YBC and KSM-1.5hDM-YBC.

[0029] Figure 2 These are SEM images of M-YBC and 1.5hDM-YBC at different magnifications.

[0030] Figure 3 SEM images of M-YBC and 1.5hDM-YBC, C, N, O element distribution diagram, and EDS energy spectrum diagram.

[0031] Figure 4 These are the TGA charts of KSM, 1.5hDM-YBC and KSM-1.5hDM-YBC, wherein A is the TGA chart of KSM; B is the TGA chart of 1.5hDM-YBC; C is the TGA chart of KSM-1.5hDM-YBC; and D is the TGA chart of KSM, 1.5hDM-YBC and KSM-1.5hDM-YBC.

[0032] Figure 5 These are the N2 adsorption-desorption isotherms of M-YBC and 1.5hDM-YBC and their corresponding pore size distribution diagrams.

[0033] Figure 6 It is a graph showing the adsorption variation of M-YBC and 1.5hDM-YBC on KSM at 25°C, wherein A is the adsorption variation of M-YBC on KSM at 25°C, and B is the adsorption variation of M-YBC on KSM at 25°C.

[0034] Figure 7 These are the kinetic fitting curves of the adsorption of KSM by M-YBC and 1.5hDM-YBC, wherein A is the kinetic curve of the adsorption of KSM by M-YBC and 1.5hDM-YBC fitted by the pseudo-first-order adsorption kinetic equation; B is the kinetic curve of the adsorption of KSM by M-YBC and 1.5hDM-YBC fitted by the pseudo-second-order adsorption kinetic equation; C is the kinetic curve of the adsorption of KSM by M-YBC and 1.5hDM-YBC fitted by the intraparticle diffusion model.

[0035] Figure 8 It is a fitting graph of the adsorption amount of 1.5hDM-YBC obtained using the Langmuir model, wherein A is a fitting graph of the adsorption amount of 1.5hDM-YBC obtained using the Langmuir model, and B is a fitting graph of the adsorption amount of 1.5hDM-YBC obtained using the Freundlich model.

[0036] Fig. 9These are the release curves of KSM from KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under the influence of different factors, wherein A is the release curve of KSM from KSM-1.5hDM-YBC at different pH values; B is the release curve of KSM from KSM-1.5hDM-YBC at different temperatures; C is the release curve of KSM from KSM-1.5hDM-YBC-SS-pectin at different pH values; D is the release curve of KSM from KSM-1.5hDM-YBC-SS-pectin at different temperatures; E is the release curve of KSM from KSM-1.5hDM-YBC-SS-pectin with or without the addition of pectinase.

[0037] Fig.10 The first-order release equation, Higuchi model and RP model were used to fit the KSM release results of KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under different pH, temperature and whether pectinase was added.

[0038] Fig.11 It is a comparison chart of the photostability of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin, wherein A is the stability chart of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under ultraviolet radiation, and B is the stability chart of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under visible light irradiation.

[0039] Fig.12 These are the minimum inhibition test result graphs of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin, where A is the minimum inhibition test result graph under light-proof conditions, and B is the minimum inhibition test result graph under ultraviolet radiation conditions.

[0040] Fig.13 This is a comparison chart of the control effects of kasugamycin technical and KSM-1.5hDM-YBC-SS-pectin on amylovora at different concentrations.

[0041] Fig.14 This is a comparison of the growth status of in vitro branches after being treated with different concentrations of KSM and KSM-1.5hDM-YBC-SS-pectin under greenhouse conditions, 0, 5, and 10 days later.

[0042] Fig.15This is a graph showing the effects of different concentrations of KSM and KSM-1.5hDM-YBC-SS-pectin on pollen germination rate, where A is the pollen germination under an electron microscope, and B is the pollen germination rate obtained by statistical calculation.

[0043] Fig.16 It is the adsorption amount of KSM by the modified biochar obtained in comparative example 1, M-YBC in Example 1, 1.5hDM-YBC, 2hDM-YBC in Example 3, 1hD-YBC in comparative example 2, and YBC in comparative example 3.

[0044] Fig.17 The release curves of KSM for KSM-M-YBC of Comparative Example 4, KSM-1.5hDM-YBC of Example 1, and KSM-2hDM-YBC of Example 3 are shown.

[0045] Fig.18 0.5M of Example 1 NaSO4 -YBC, 0.1M of Comparative Example 5 NaSO4 -The adsorption amount of YBC on KSM.

[0046] Fig.19 0.5M of Example 1 NaSO4 -YBC, 1M of Comparative Example 6 NaSO4 -The adsorption amount of YBC on KSM.

[0047] Fig. 20 It is the adsorption capacity of 0.5hDM-YBC of Comparative Example 7 and 1.5hDM-YBC of Example 1 for KSM.

[0048] Fig.21 The release curves of KSM of KSM-0.5hDM-YBC of Comparative Example 7 and KSM-1.5hDM-YBC of Example 1 are shown.

[0049] Fig. 22 It is the adsorption capacity of 3hDM-YBC of Comparative Example 8 and 1.5hDM-YBC of Example 1 for KSM.

[0050] Fig.23 The release curves of KSM for KSM-DM-YBC-SS-pectin of Comparative Example 10 and KSM-1.5hDM-YBC-SS-pectin of Example 1 are shown. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] The complete steps of the method of the present invention are as follows:

[0053] Materials: Gray jujube leaves were picked in the Jujube Resource Garden of Tarim University in September 2023, then dried, crushed and stored in a dry and cool place; Kasugamycin (98%) was purchased from Huaxia Chemical (China); tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 3-mercaptopropyltrimethoxysilane (MPTMS), β-mercaptoethylamine, sodium dodecyl sulfate (SDS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from McLean (Shanghai, China); pectinase (5ku) was purchased from Blue Season Technology Co., Ltd. (Shanghai, China); pectin was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China); bovine serum albumin (BSA) was purchased from Biotoda (Beijing, China).

[0054] S1: Preparation of modified biochar (M-YBC). The specific steps are described as follows:

[0055] S11: prepare 0.4-1 mol / L Na2SO4 modified solution, weigh jujube leaf powder according to the mass volume ratio of 1 / 30-1 / 20 and add it to the Na2SO4 modified solution;

[0056] S12: stirring the mixed solution with a magnetic force for 1.8 to 2.2 hours, and then centrifuging at a speed of 4800 to 5200 rpm for 4 to 6 minutes to separate the modified gray jujube leaf powder;

[0057] S13: drying the centrifuged sample at 78-82°C; pyrolysis treatment of the dried jujube leaf powder in a muffle furnace at 690-710°C for 2.8-3.2 hours;

[0058] S14: The pyrolysis product is washed with distilled water until it is neutral, and then vacuum filtered to remove water, and then dried. The dried biochar is then ground with an agate mortar and passed through a 200-mesh sieve, sealed and stored, named M-YBC, and stored in a desiccator for later use.

[0059] S2: Preparation of DM-YBC. The specific steps are as follows:

[0060] S21: prepare a bovine serum albumin (BSA) solution with a concentration of 9 to 11 mg / mL, prepare a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution with a concentration of 4.8×10-2 to 5.2×10-2 mol / L, and mix the two in a volume ratio of (0.8:1) to (1.2:1);

[0061] S22: Use sodium hydroxide solution to adjust the pH of the mixed solution to 4.4-4.6. This pH range is the critical value for the formation of starch-like aggregates, which can be observed by the naked eye through small amounts and multiple times.

[0062] S23: Take 95-105 mL of the pH-adjusted mixed solution and add 0.4-0.6 g of M-YBC. Use a magnetic stirrer to fully stir at room temperature for 1.4-2 hours to ensure uniform mixing, filter, and repeatedly wash the filter cake with deionized water to remove residual solvent and unbound reagent.

[0063] S24: The washed sample is placed in an oven at 55-65°C and dried to constant weight to obtain DM-YBC.

[0064] S3: Preparation of KSM-DM-YBC-SS-pectin microcapsules. The specific steps are as follows:

[0065] S31: 0.4-0.6 g DM-YBC and 95-105 mL of 0.9-1.1 mg / mL kasugamycin KSM aqueous solution were stirred and mixed;

[0066] S32: Add 0.65-0.90 mL of 3-mercaptopropyltrimethoxysilane MPTMS to the above system at a concentration of more than 98%, and stir at 58-62°C for 1.8-2.2 hours. Centrifuge the reaction solution at 5900-6100 rpm for 4-6 minutes, wash with deionized water, and dry at 58-62°C to obtain KSM-loaded thiol-functionalized protein-biochar KSM-DM-YBC;

[0067] S33: Disperse 0.8-1.2g of thiol-functionalized KSM-DM-YBC in a certain amount of AR analytical pure methanol, which can evenly disperse the biochar and react with mercaptoethylamine, about 20ml; add 1.8-2.2g of β-mercaptoethylamine and stir at room temperature for 23-25h to connect the amino group, which will subsequently form an amide bond with pectin. After centrifugation and washing, the reaction solution is centrifuged at the same speed and time as S32, and dried at 58-62℃ to constant weight to obtain amino-substituted protein-biochar particles KSM-DM-YBC-SS-NH2 loaded with KSM.

[0068] S34: dissolving pectin in deionized water to obtain a pectin aqueous solution with a mass volume ratio of (1.5:1):(2.5:1) g / L. Then dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC in 45-55 mL of the above pectin aqueous solution, controlling the concentration within 10-100 mM. The concentration within this range can usually obtain a reasonable cross-linking effect while preventing excessive cross-linking, and stirring at room temperature for 25-35 minutes;

[0069] S35: Add 0.1-0.3g KSM-DM-YBC-SS-NH2 microspheres to the above mixture to ensure that the pectin shell is of sufficient thickness; at the same time, add N-hydroxysuccinimide NHS and dissolve it in the above mixture. The concentration of NHS is controlled within 30-50mM, and the reaction is continued at room temperature for 23-25h. After centrifugation and washing, the reaction solution is dried at 58-62°C to constant weight at the same centrifugal speed and time as S32 to obtain KSM-loaded pectin-protein-biochar particles KSM-DM-YBC-SS-pectin.

[0070] Example 1

[0071] S1: Preparation of modified biochar (M-YBC). The specific steps are described as follows:

[0072] S11: Prepare 0.5 mol / L Na2SO4 modified solution, weigh 10 g of gray jujube leaf powder and add it to 250 mL of the modified solution;

[0073] S12: stirring the mixed solution with a magnetic force for 2 hours, and then centrifuging at a speed of 5000 rpm for 5 minutes to separate the modified gray jujube leaf powder;

[0074] S13: drying the centrifuged sample at 80°C; pyrolysis treatment of the dried jujube leaf powder was performed using a muffle furnace at 700°C for 3 hours;

[0075] S14: The pyrolysis product is washed with distilled water until it is neutral, and then vacuum filtered to remove water, and then dried. The dried biochar is then ground with an agate mortar and passed through a 200-mesh sieve, sealed and stored, named M-YBC, and stored in a desiccator for later use.

[0076] S2: Preparation of 1.5hDM-YBC. The specific steps are as follows:

[0077] S21: prepare a bovine serum albumin (BSA) solution with a concentration of 10 mg / mL, prepare a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution with a concentration of 5×10-2 mol / L, and mix the two in a volume ratio of 1:1;

[0078] S22: Adjust the pH of the mixed solution to 4.5.

[0079] S23: Take 100 mL of the mixed solution and add 0.5 g of M-YBC. Use a magnetic stirrer to stir well for 1.5 hours at room temperature to ensure uniform mixing, filter, and repeatedly wash the filter cake with deionized water to remove residual solvent and unbound reagent.

[0080] S24: The washed sample was placed in a 60°C oven and dried to constant weight to obtain 1.5hDM-YBC.

[0081] S3: Preparation of KSM-1.5hDM-YBC-SS-pectin microcapsules. The specific steps are as follows:

[0082] S31: 0.5 g 1.5 hDM-YBC was mixed with 100 mL 1 mg / mL kasugamycin KSM aqueous solution;

[0083] S32: Add 0.7 mL of 3-mercaptopropyltrimethoxysilane MPTMS to the above system and stir at 60°C for 2 h. Centrifuge the reaction solution at 6000 rpm for 5 minutes, wash with deionized water, and dry at 60°C to obtain thiol-functionalized KSM-1.5hDM-YBC;

[0084] S33: Disperse 1.0 g of thiol-functionalized KSM-1.5hDM-YBC in a certain amount of methanol, add 2.0 g of β-mercaptoethylamine, and stir at room temperature for 24 h. Centrifuge and wash the reaction solution, and dry it at 60°C to constant weight to obtain KSM-1.5hDM-YBC-SS-NH2 microspheres.

[0085] S34: dissolving pectin in deionized water to obtain a pectin aqueous solution with a mass-to-volume ratio of (2:1) g / L. Then dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in 50 mL of the above pectin aqueous solution to a concentration of 30 mM, and stirring at room temperature for 30 min;

[0086] S35: Add 0.2 g KSM-1.5hDM-YBC-SS-NH2 microspheres to the above mixed solution, and add N-hydroxysuccinimide NHS at a concentration of 40 mM, and continue stirring and reacting at room temperature for 24 hours. After centrifugation and washing, the reaction solution is dried at 60°C to constant weight to obtain KSM-1.5hDM-YBC-SS-pectin.

[0087] Example 2

[0088] S1: Preparation of modified biochar (M-YBC). The specific steps are described as follows:

[0089] S11: prepare a 0.4 mol / L Na2SO4 modified solution, and add the gray jujube leaf powder to the Na2SO4 modified solution at a mass volume ratio of 1 / 30;

[0090] S12: stirring the mixed solution with a magnetic force for 1.8 hours, and then centrifuging at 4800 rpm for 6 minutes to separate the modified gray jujube leaf powder;

[0091] S13: drying the centrifuged sample at 78°C; pyrolysis treatment of the dried jujube leaf powder was performed using a muffle furnace at 690°C for 3.2 hours;

[0092] S14: The pyrolysis product is washed with distilled water until it is neutral, and then vacuum filtered to remove water, and then dried. The dried biochar is then ground with an agate mortar and passed through a 200-mesh sieve, sealed and stored, named M-YBC, and stored in a desiccator for later use.

[0093] S2: Preparation of 1.4hDM-YBC. The specific steps are as follows:

[0094] S21: prepare a bovine serum albumin (BSA) solution with a concentration of 9 mg / mL, prepare a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution with a concentration of 4.8×10-2 mol / L, and mix the two in a volume ratio of 0.8:1;

[0095] S22: Use sodium hydroxide solution to adjust the pH of the mixed solution to 4.4.

[0096] S23: Take 95 mL of the pH-adjusted mixed solution and add 0.4 g of M-YBC. Use a magnetic stirrer to stir well at room temperature for 1.4 hours to ensure uniform mixing, filter, and repeatedly wash the filter cake with deionized water to remove residual solvent and unbound reagent.

[0097] S24: The washed sample was placed in a 55°C oven and dried to constant weight to obtain 1.5hDM-YBC.

[0098] S3: Preparation of KSM-1.4hDM-YBC-SS-pectin microcapsules. The specific steps are as follows:

[0099] S31: 0.4 g 1.4hDM-YBC was mixed with 95 mL of 1.1 mg / mL kasugamycin KSM aqueous solution;

[0100] S32: Add 0.65 mL of 3-mercaptopropyltrimethoxysilane MPTMS to the above system and stir at 58°C for 2.2 h. Centrifuge the reaction solution at 5900 rpm for 6 minutes, wash with deionized water, and dry at 58°C to obtain thiol-functionalized KSM-1.4hDM-YBC;

[0101] S33: Disperse 0.8g of thiol-functionalized KSM-1.4hDM-YBC in a certain amount of methanol, add 1.8g of β-mercaptoethylamine, and stir at room temperature for 23h to connect the amino group and subsequently form an amide bond with pectin. Centrifuge and wash the reaction solution, and dry it at 58°C to constant weight to obtain KSM-1.4hDM-YBC-SS-NH2 microspheres.

[0102] S34: dissolving pectin in deionized water to obtain a pectin aqueous solution with a mass-to-volume ratio of (1.5:1) g / L. Then dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC in 45 mL of the above pectin aqueous solution to a concentration of 10 mM, and stirring at room temperature for 25 min;

[0103] S35: Add 0.1g KSM-1.4hDM-YBC-SS-NH2 microspheres to the above mixture to ensure that the pectin shell is of sufficient thickness; at the same time, add N-hydroxysuccinimide NHS and dissolve it in the above mixture to control the concentration at 30mM, and continue to stir and react at room temperature for 23h. After centrifugation and washing, the reaction solution is dried at 58°C to constant weight to obtain KSM-1.4hDM-YBC-SS-pectin.

[0104] Example 3

[0105] S1: Preparation of modified biochar (M-YBC). The specific steps are described as follows:

[0106] S11: prepare a 1 mol / L Na2SO4 modified solution, weigh the gray jujube leaf powder according to a mass volume ratio of 1 / 20 and add it to the Na2SO4 modified solution;

[0107] S12: stirring the mixed solution with a magnetic force for 2.2 hours, and then centrifuging at a speed of 5200 rpm for 4 minutes to separate the modified gray jujube leaf powder;

[0108] S13: drying the centrifuged sample at 82°C; pyrolysis treatment of the dried jujube leaf powder was performed in a muffle furnace at 710°C for 2.8 hours;

[0109] S14: The pyrolysis product is washed with distilled water until it is neutral, and then vacuum filtered to remove water, and then dried. The dried biochar is then ground with an agate mortar and passed through a 200-mesh sieve, sealed and stored, named M-YBC, and stored in a desiccator for later use.

[0110] S2: Preparation of 2hDM-YBC. The specific steps are as follows:

[0111] S21: prepare a bovine serum albumin (BSA) solution with a concentration of 11 mg / mL and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution with a concentration of 5.2×10-2 mol / L, and mix the two in a volume ratio of 1.2:1;

[0112] S22: Use sodium hydroxide solution to adjust the pH of the mixed solution to 4.6.

[0113] S23: Take 105 mL of the pH-adjusted mixed solution and add 0.6 g of M-YBC. Use a magnetic stirrer to stir well for 2 hours at room temperature to ensure uniform mixing, filter, and repeatedly wash the filter cake with deionized water to remove residual solvent and unbound reagent.

[0114] S24: The washed sample is placed in a 65°C oven and dried to constant weight to obtain 2hDM-YBC.

[0115] S3: Preparation of KSM-2hDM-YBC-SS-pectin microcapsules. The specific steps are as follows:

[0116] S31: 0.6 g of 2hDM-YBC was mixed with 105 mL of a 0.9 mg / mL kasugamycin KSM aqueous solution;

[0117] S32: Add 0.90 mL of 3-mercaptopropyltrimethoxysilane MPTMS to the above system and stir at 62°C for 1.8 h. Centrifuge the reaction solution at 6100 rpm for 4 minutes, wash with deionized water, and dry at 62°C to obtain thiol-functionalized KSM-2hDM-YBC;

[0118] S33: Disperse 1.2g of thiol-functionalized KSM-2hDM-YBC in a certain amount of methanol, add 2.2g of β-mercaptoethylamine, and stir at room temperature for 25h to connect the amino groups and subsequently form amide bonds with pectin. Centrifuge and wash the reaction solution, and dry it at 62°C to constant weight to obtain KSM-2hDM-YBC-SS-NH2 microspheres.

[0119] S34: dissolving pectin in deionized water to obtain a pectin aqueous solution with a mass volume ratio of (2.5:1) g / L. Then dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC in the above 55 mL pectin aqueous solution, controlling the concentration within 100 mM, and stirring at room temperature for 35 min;

[0120] S35: Add 0.3g KSM-2hDM-YBC-SS-NH2 microspheres to the above mixture to ensure that the pectin shell is of sufficient thickness; at the same time, add N-hydroxysuccinimide NHS and dissolve it in the above mixture to control the concentration at 50mM, and continue to stir and react at room temperature for 25h. After centrifugation and washing, the reaction solution is dried at 62°C to constant weight to obtain KSM-2hDM-YBC-SS-pectin.

[0121] Comparative Example 1

[0122] The difference from Example 1 is:

[0123] In S11, other modification solutions are used to modify the biochar, for example, phosphoric acid solutions of different concentrations and diammonium phosphate solutions of different concentrations;

[0124] No S2 or S3.

[0125] Comparative Example 2

[0126] The difference from Example 1 is:

[0127] In S23, the stirring time is 1 h;

[0128] No S3.

[0129] Comparative Example 3

[0130] The difference from Example 1 is:

[0131] In S1, the gray jujube leaves were not modified;

[0132] No S2 or S3.

[0133] Comparative Example 4

[0134] The difference from Example 1 is:

[0135] No S21;

[0136] No S3.

[0137] Comparative Example 5

[0138] The difference from Example 1 is:

[0139] In S11, weigh the jujube leaf powder and add it to the 0.1M Na2SO4 modified solution;

[0140] No S2 or S3.

[0141] Comparative Example 6

[0142] The difference from Example 1 is:

[0143] In S11, the powder of jujube leaves was weighed and added into the 1M Na2SO4 modified solution;

[0144] No S2 or S3.

[0145] Comparative Example 7

[0146] The difference from Example 1 is:

[0147] In S23, the stirring time is 0.5 hours;

[0148] No S3.

[0149] Comparative Example 8

[0150] The difference from Example 1 is:

[0151] In S21, the stirring time is 3 hours;

[0152] No S3.

[0153] Comparative Example 9

[0154] The difference from Example 1 is:

[0155] In S34, the concentration of the EDC solution is 3 mM; at this time, the EDC concentration is too low to form a gel and a dual-response system cannot be formed.

[0156] Comparative Example 10

[0157] The difference from Example 1 is:

[0158] In S34, the concentration of EDC solution was 300 mM; if the concentration was too high, excessive gelation occurred, which hindered the subsequent sustained release of KSM.

[0159] Comparative Example 11

[0160] The difference from Example 1 is:

[0161] In S35, the NHS concentration was controlled at 1 mM.

[0162] Since the NHS concentration was too low, a dual-response system could not be formed.

[0163] Comparative Example 12

[0164] The difference from Example 1 is:

[0165] In S35, the NHS concentration was controlled at 300 mM.

[0166] Due to the excessively high NHS concentration and excessive gelation, the sustained release of KSM by KSM-DM-YBC-SS-pectin in Comparative Example 12 was hindered.

[0167] The composite material prepared in Example 1 was experimentally analyzed.

[0168] The structures and interactions of pectin, kasugamycin, M-YBC obtained from S1, 1.5hDM-YBC obtained from S2, KSM-1.5hDM-YBC obtained from S32, and 1.5hDM-YBC-SS-pectin obtained from S35 were analyzed by Fourier transform infrared spectrometer (FT-IR, Nicolet iS50, USA). The morphologies of 1.5hDM-YBC and M-YBC were characterized by scanning electron microscope-energy dispersive spectrometer (SEM-EDS, FEI APREO-S, USA). Brunauer-Emmett-Teller (BET, Micromeritics ASAP 2460, USA) was used to analyze the pore properties of the adsorbents, including specific surface area (SSA), total pore volume (TPV), and average pore size (APS). The temperature-mass change relationship of the substance was analyzed by a thermogravimetric analyzer (TG, TA TGA 550, USA), with a temperature range of 30-1000°C, a heating rate of 5°C / min, and a protective gas of N2.

[0169] Characterization results:

[0170] FT-IR spectra of pectin, KSM, 1.5hDM-YBC, KSM-1.5hDM-YBC and 1.5hDM-YBC-SS-pectin are shown in Figure 2. Figure 1 As shown in Figure 1, A is the spectrum of Pectin and 1.5hDM-YBC-SS-pectin, and B is the spectrum of KSM, 1.5hDM-YBC, and KSM-1.5hDM-YBC. From Figure A, we can see that the peaks of Pectin are at 2987, 1640, and 1050 cm -1 Characteristic absorption peaks appeared at 1658 cm-1, which correspond to CH stretching vibration, amide I band (C=O stretching vibration) and CO stretching vibration in the sugar ring, corresponding to the polysaccharide structure in the pectin molecular chain. -1 and 1620cm -1 The characteristic absorption peaks at correspond to the amino I band (NH stretching vibration) and the amino II band (NH bending vibration), respectively.

[0171] In the spectrum of the 1.5hDM-YBC complex, the -1 The characteristic peak at 2987 cm-1 is attributed to the stretching vibration of amide (C=O), which indicates that the PTB membrane and M-YBC are successfully combined. -1 、1640cm -1 、1050cm -1The characteristic absorption peak of KSM-1.5hDM-YBC belongs to pectin, indicating that 1.5hDM-YBC can successfully bind to pectin. -1 The characteristic absorption peaks attributed to NH bending vibration appeared, indicating that KSM was successfully grafted onto 1.5hDM-YBC. These results show that KSM-1.5hDM-YBC-SS-pectin has been successfully prepared.

[0172] The adsorption performance of biochar depends largely on its morphology and structure. Figure 2 As shown in the figure, the surface of M-YBC presents an irregular pore structure. Due to the lack of an ordered pore structure, M-YBC may not provide sufficient adsorption sites to effectively adsorb the target substance. In contrast, the surface of 1.5hDM-YBC is relatively smooth, with a small amount of rough small particles distributed, and its pore structure is arranged irregularly. This morphological structure indicates that 1.5hDM-YBC may have a certain adsorption capacity. However, since the regularity of its pore structure is still not very ideal, its adsorption performance may still be subject to certain limitations.

[0173] M-YBC modified with PTB exhibits a smoother surface structure. Figure 3 The first column is the SEM image, C, N, O element distribution map, and EDS energy spectrum of M-YBC; the second column is the SEM image, C, N, O element distribution map, and EDS energy spectrum of 1.5hDM-YBC.

[0174] Combined with the analysis results of the EDS spectrum, the contents of C, N and O elements in 1.5hDM-YBC increased significantly compared with M-YBC. This phenomenon can be attributed to the adhesion of PTB solution to biochar during the modification process, resulting in an increase in the contents of C, N and O. This effect will change the specific surface area and pore structure of biochar and improve the functional groups on the surface of biochar. Since kasugamycin contains a large number of hydroxyl and amino groups, the modified biochar is more likely to form electrostatic interactions and hydrogen bonds with kasugamycin.

[0175] Thermogravimetric analysis:

[0176] The loading capacity and thermal stability of 1.5hDM-YBC were analyzed by thermogravimetric analysis (TG). Figure 4 As shown, Figure 4Figure 1 is the TGA graph of KSM, 1.5hDM-YBC, and KSM-1.5hDM-YBC, wherein A is the TGA graph of KSM; Figure B is the TGA graph of 1.5hDM-YBC; Figure C is the TGA graph of KSM-1.5hDM-YBC; Figure D is the TGA graph of KSM, 1.5hDM-YBC, and KSM-1.5hDM-YBC. As can be seen from the figure, the weight loss observed in the range of 30°C to 211°C is attributed to the evaporation of adsorbed water in the sample. In Figure A, in the TG curve of KSM, the weight loss between 211°C and 400°C is due to the decomposition of KSM. For Figure B, the TG curve of 1.5hDM-YBC shows that the weight loss (10.77wt.%) in the range of 211°C to 472°C is due to the breakage of BSA side chains and non-covalent bonds. As the temperature increases, the main amino acid chain of BSA breaks, leading to dehydration and decarboxylation reactions, and the production of CO, CO2, and N2 gases. At 311 °C, the mass loss of KSM-1.5hDM-YBC was observed to be higher than that of 1.5hDM-YBC, which was caused by the decomposition of the loaded KSM at high temperatures. The KSM loading rate can be obtained based on 1.5hDM-YBC and KSM-1.5hDM-YBC, which is about 6.54wt.%.

[0177] Specific surface characteristics analysis:

[0178] In order to analyze the specific surface characteristics of the material, the material was subjected to N2 adsorption-desorption test. The experimental results are as follows: Figure 5 As shown in Table 1, the isothermal adsorption-desorption curves of M-YBC and 1.5hDM-YBC are both type IV isotherms, which means that they have mesoporous and macroporous structures. In addition, the curves of M-YBC and 1.5hDM-YBC are convex in the low P / P0 region, indicating the presence of micropores; they rise rapidly in the high P / P0 region, showing the presence of mesopores and macropores. The desorption isotherms of M-YBC and 1.5hDM-YBC do not coincide with the adsorption isotherms, and there is a hysteresis loop, which indicates that there are a large number of mesopores in both. In addition, the sharp increase in adsorption amount at low relative pressure means the presence of micropores, indicating that all samples have a microporous structure.

[0179] Table 1 shows the structural parameters obtained by analyzing the N2 adsorption isotherms of M-YBC and 1.5hDM-YBC. The average pore sizes of M-YBC and 1.5hDM-YBC are 2.676nm and 2.556nm, respectively, which indicates that M-YBC and 1.5hDM-YBC are biochars with a large number of mesopores and micropores, and there are three types of pore structures: macropores, mesopores and micropores. It is worth noting that the S of M-YBC BET and total pore volume were 118.064 m higher than those of 1.5hDM-YBC. 2 / g and 0.088cm3 / g. This indicates that after the PTB membrane modification, the specific surface area and pore volume of 1.5hDM-YBC tend to decrease due to the coverage of PTB, and the interaction between PTB and M-YBC increases the density. The dense microstructure is conducive to confining the pesticide in the porous carbon material and controlling the release rate of the pesticide.

[0180] Table 1 Structural parameters obtained by analyzing the N2 adsorption isotherms of M-YBC and 1.5hDM-YBC

[0181]

[0182] Adsorption efficiency of pesticides on carbon-based composites:

[0183] Figure 6 The adsorption capacity of the two materials for KSM at 25°C is given respectively. As shown in the figure, due to the large specific surface area, rich pore structure and rich surface functional groups of biochar, M-YBC can adsorb up to 45.48 mg / mL in a short time, and its adsorption capacity is close to saturation after 15 minutes. Due to the PTB nanofilm covering, 1.5hDM-YBC improves the structure of biochar itself, making the carbon material more stable and the surface functional groups richer. The adsorption of KSM on 1.5hDM-YBC is fast in the early 100 minutes. After a period of time, due to the reduction of free adsorption sites, the adsorption rate is adjusted back. Within 240 minutes, both M-YBC and 1.5hDM-YBC reach adsorption equilibrium. Under the KSM adsorption equilibrium state, the adsorption capacity of M-YBC is 60.73 mg / mL, and the adsorption capacity of 1.5hDM-YBC is 60.68 mg / mL. The difference in the adsorption amount of M-YBC and 1.5hDM-YBC is not very obvious. This is because after PTB membrane covering, although the specific surface area and pore volume of 1.5hDM-YBC are reduced, the element distribution of C, N, and O is improved, the content of C, H, and O is increased, the functional groups are improved, and the protein membrane surface has a large number of functional groups, resulting in more active sites that can adsorb KSM.

[0184] Adsorption kinetics analysis:

[0185] Adsorption kinetics can reflect the adsorption rate of the adsorbent for the adsorbate, and can further reflect the adsorption efficiency of the adsorbent. In order to study the change trend of the adsorption capacity of M-YBC and 1.5hDM-YBC for KSM over time, M-YBC and 1.5hDM-YBC were selected as research materials, and the following time points were set: 10s, 20s, 30s, 1min, 2min, 5min, 10min, 15min, 20min, 30min, 40min, 50min, 60min, 80min, 100min, 120min, 150min, 180min, 210min and 240min. The adsorption capacity corresponding to each time point was calculated, and the adsorption data of different biochar materials under the same adsorption conditions were fitted using the pseudo-first-order adsorption kinetic equation, pseudo-second-order adsorption kinetic equation, and intraparticle diffusion model.

[0186]

[0187] q t =k i t 1 / 2+C (3)

[0188] In the formula, q e is the equilibrium adsorption capacity, mg / g; q t is the adsorption amount of KSM aqueous solution by the adsorbent at time t (min), mg / g; k1 is the rate constant of the pseudo-first-order kinetic equation, 1 / min; k2 is the rate constant of the pseudo-second-order kinetic equation, g / (mg·min); k i is the intraparticle diffusion rate constant, mg / g min 1 / 2 , C is another constant for the boundary layer thickness, mg / g.

[0189] Figure 7 are the kinetic fitting curves of M-YBC and 1.5hDM-YBC adsorbing KSM. Among them, A is the kinetic curve of M-YBC and 1.5hDM-YBC adsorbing KSM fitted by the pseudo-first-order adsorption kinetic equation; B is the kinetic curve of M-YBC and 1.5hDM-YBC adsorbing KSM fitted by the pseudo-second-order adsorption kinetic equation; C is the kinetic curve of M-YBC and 1.5hDM-YBC adsorbing KSM fitted by the intraparticle diffusion model. The experimental results show that with the extension of adsorption time, the adsorption capacity of KSM gradually increases and tends to equilibrium after about 60 minutes. In addition, from Figure 6It is not difficult to see from the equation parameters in the following paragraph that the adsorption process of KSM by M-YBC and 1.5hDM-YBC includes three stages: first, the surface diffusion stage, KSM molecules diffuse quickly to the surface, and the adsorption rate is the fastest; second, the intra-mesopore diffusion stage, the molecules diffuse through the mesopores to the inner surface, and the rate slows down; finally, the intra-micropore diffusion stage, the molecules diffuse into smaller pores, and the rate further decreases. Comparative analysis of these three adsorption stages reveals that the adsorption rate and adsorption resistance show unique characteristics in different stages. The surfaces of M-YBC and 1.5hDM-YBC are rich in various functional groups, including -OH, CC and CO. It can be inferred that on the surfaces of M-YBC and 1.5hDM-YBC, these functional groups actively interact with -NH2 and -OH in KSM molecules. These interactions include but are not limited to electrostatic interactions, hydrogen bonds, and chemical bonding, and different types of interactions jointly promote the adsorption process.

[0190] Equation parameters: In the pseudo-first-order adsorption kinetic equation, for M-YBC: k1 = 0.01, q e =5.058, R 2 is 0.76, for 1.5hDM-YBC: k1 = 0.01, q e =13.68, goodness of fit R 2 is 0.77; in the pseudo-first-order adsorption kinetic equation, for M-YBC: k2 = 0.00, q e =62.5, R 2 is 0.99, for 1.5hDM-YBC: k2 = 0.00, q e =66.67, R 2 is 0.99; in the intra-particle diffusion model, for M-YBC: k id1 is 6.44, the boundary layer thickness related constant C is 44.5, R 2 is 0.92, k id2 is 0.13, C is 59.3, R 2 is 0.84, for 1.5hDM-YBC: k id1 is 6.20, C is 10.09, R 2 is 0.98, k id2 is 0.41, C is 55.29, R 2 is 0.95.

[0191] Adsorption isotherm:

[0192] In order to study the adsorption equilibrium, the Langmuir and Freundlich models were selected for analysis according to the properties and types of the system. In the above two adsorption models, the Langmuir model assumes that the adsorbed substance is uniformly adsorbed on the surface in the form of a monolayer, while the Freundlich model focuses on describing the interaction between adsorbed molecules on heterogeneous surfaces. The Langmuir and Freundlich model fitting diagrams and fitting parameters of 1.5hDM-YBC are shown in Figure 8 and Table 2, which is a table of Langmuir and Freundlich adsorption isotherm model parameters. Figure 8 The adsorption amount fitting diagram of 1.5hDM-YBC obtained by Langmuir and Freundlich models, where A is the adsorption amount fitting diagram of 1.5hDM-YBC obtained by Langmuir and Freundlich models, and B is the adsorption amount fitting diagram of 1.5hDM-YBC obtained by Langmuir and Freundlich models. It can be seen from the results that as the temperature increases from 25°C to 45°C, the saturated adsorption amount of 1.5hDM-YBC on KSM shows an increasing trend. This phenomenon reveals that the adsorption process of KSM has endothermic characteristics. This improvement in adsorption performance can be attributed to two factors: First, the increase in temperature promotes certain chemical reactions between the active groups on the surface of 1.5hDM-YBC and the functional groups of KSM, thereby generating more active adsorption sites, thereby enhancing the adsorption effect. Therefore, increasing the adsorption temperature within an appropriate temperature range can help improve the adsorption performance of 1.5hDM-YBC for KSM. On the other hand, the increase in temperature provides more energy for the reaction, which helps to reduce the diffusion resistance of KSM in 1.5hDM-YBC and promotes adsorption.

[0193] Table 2 Langmuir and Freundlich adsorption isotherm model parameters

[0194]

[0195] In vitro release studies:

[0196] Controlled release analysis of KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin:

[0197] 50 mg of KSM-1.5hDM-YBC or KSM-1.5hDM-YBC-SS-pectin were weighed and dispersed in 100 mL PBS. Samples were centrifuged at different time intervals, the concentration of kasugamycin in the supernatant was determined, the cumulative release rate of kasugamycin from the microcapsules was calculated, and the controlled release performance was evaluated. The release behavior of kasugamycin microcapsules under different pH values, temperatures and pectinase conditions was studied. In the enzyme addition test, 50 mg of kasugamycin microcapsules were dispersed in 100 mL PBS with a pH of 7, under different conditions: ① 0 mg / mL pectinase; ② 1 mg / mL pectinase, regular sampling, centrifugation, and detection. The content of KSM at 210 nm of each sample was measured by high performance liquid chromatography, and all experiments were repeated 3 times.

[0198]

[0199] Among them C t (mg / mL), V t (3mL) are the concentration and volume of the solution extracted at time t, V total (50 mL) is the concentration of the total solution, and m0 (mg) is the actual loading amount of the sustained-release agent.

[0200] Fig. 9The release curves of KSM from KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under different factors, where A is the release curve of KSM from KSM-1.5hDM-YBC at different pH; B is the release curve of KSM from KSM-1.5hDM-YBC at different temperatures; C is the release curve of KSM from KSM-1.5hDM-YBC-SS-pectin at different pH; D is the release curve of KSM from KSM-1.5hDM-YBC-SS-pectin at different temperatures; E is the release curve of KSM from KSM-1.5hDM-YBC-SS-pectin with or without the addition of pectinase. It can be seen from A and B that pH and temperature have no obvious promoting effect on the sustained release process of KSM-1.5hDM-YBC. As can be seen from Figure C, in PBS buffer solutions with pH values ​​of 6.0, 7.0 and 8.0, KSM-1.5hDM-YBC-SS-pectin material exhibits typical pH response performance and drug release capacity, and the release of KSM molecules shows typical pH-dependent increase characteristics. After incubation in PBS buffer solution with a pH value of 7.0 for 72 hours, the cumulative release of KSM in KSM-1.5hDM-YBC-SS-pectin material is very small, about 4.72wt.%; however, under pH conditions, the release of KSM increases significantly with time. After 72 hours, the release of KSM at pH values ​​of 6.0 and 8.0 reaches 36.99wt.% and 52.08wt.%, respectively, indicating that KSM-1.5hDM-YBC-SS-pectin material can stably exist under neutral conditions, and pH value can effectively regulate the release of KSM in KSM-1.5hDM-YBC-SS-pectin. Because under alkaline conditions, the carboxyl group ionizes into COO - The pectin macromolecular network unfolds through the electrostatic repulsion between the charged groups and forms a dissolved state in the solution. In an acidic environment, the amine group (-NH2) may be protonated to -NH3 + , increasing the electrostatic repulsion between molecules and accelerating the release of KSM. When the amylovora blight pathogen infects pear trees, it causes changes in the pH value of the infection site, allowing KSM to be released on demand, indicating that the KSM-1.5hDM-YBC-SS-pectin drug delivery system has good pH responsiveness.

[0201] As can be seen from E, the addition of pectinase also has a significant effect on the release of KSM from the KSM-1.5hDM-YBC-SS-pectin material. In the control group without pectinase, the release amount of KSM was about 0.4 wt.% after 12 h. After incubation for 12 h with the addition of pectinase, the release amount of KSM was about 60.15 wt.%. During the prevention and control of fire blight, the diseased parts need to be removed in time. The wounded areas are prone to infection by pathogens, and a large amount of pectinase will also be released at the wounded areas. The change in pectinase concentration will destroy the pectin shell on the material, achieving the preventive effect of KSM controlled release. These results confirm that KSM-1.5hDM-YBC has achieved a dual response to pectinase and pH value after adding a pectin shell.

[0202] Release kinetics analysis:

[0203] In order to better study the release mechanism of KSM in the composite material, three drug release kinetic models, namely the first-order release equation, the Higuchi model, and the Ritger-Peppas model, were used to perform nonlinear fitting on the drug release curve to clarify the release mechanism of KSM.

[0204]

[0205] In the formula, t is the release time, M t is the drug release amount at time t, M ∞ is the maximum drug release amount, M t / M ∞ is the cumulative drug release rate at time t, k is the drug release constant. n is a parameter used to characterize the release mechanism. If n < 0.45, it is Fickian diffusion; if the value of n is between 0.45 < n < 0.89, it means that the release belongs to non-Fickian diffusion, that is, the协同作用 of diffusion and matrix erosion; if n > 0.89, it is matrix erosion.

[0206] Three common kinetic models were used to describe the release behavior of KSM in different release media and different conditions (including the first-order, Higuchi, and R-P models). Fig.10The first-order release equation, Higuchi model, and RP model were used to fit the KSM release results of KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under different pH, different temperatures, and whether pectinase was added. It can be seen that in the KSM-1.5hDM-YBC material, under different pH and temperature conditions, the kinetic model fitting results showed that the Higuchi model had a high degree of fit, indicating that the drug release mainly followed the diffusion mechanism, and all n values ​​were far below 0.45, indicating the existence of the Fike diffusion mechanism. In the KSM-1.5hDM-YBC-SS-pectin material, under different pH and whether pectinase was added, the kinetic fitting conformed to the first-order release equation, indicating that the rate of drug release was proportional to the concentration of the remaining drug. Under different temperature conditions, it conformed to the Higuchi model, and under the conditions of pH=8 or the addition of pectinase, the n values ​​were all below 0.45, which also supported the conclusion of the Fike diffusion mechanism.

[0207] Light stability test:

[0208] In order to evaluate the photostability of KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin materials, a certain amount of KSM original drug, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin materials were dissolved in 5mL PBS buffer solution (pH=7.0) and added to a closed dialysis bag (MW: 3000Da), which was then immersed in 100mL PBS buffer solution (pH=7.0) and exposed to a 30W ultraviolet lamp at a distance of 20cm. 1mL of solution was taken from each sample within a predetermined time (1h, 2h, 4h, 8h, 12h, 24h, 32h, 40h, 48h, 72h, 96h, 120h) and supplemented with the same volume of PBS buffer solution. The content of KSM in the sample was measured by HPLC system. The sample incubated in the dark was set as the control group, and all experiments were repeated 3 times. The reduction of KSM in the sample is considered degradation. The degradation rate is calculated by the following formula:

[0209]

[0210] In the formula, A t is the concentration of KSM at irradiation time t, and A0 is the initial concentration of KSM.

[0211] Fig.11It is a comparison chart of the photostability of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin. Among them, A is the stability chart of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under ultraviolet radiation, and B is the stability chart of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin under visible light irradiation. As shown in A, after 24 hours of irradiation, the degradation rate of KSM original drug aqueous solution is as high as 52.36wt.%, indicating that KSM aqueous solution is sensitive to ultraviolet radiation and is easily degraded. After 120 hours of irradiation, the retention rate of KSM in KSM-1.5hDM-YBC is greater than 90wt.%, indicating that biochar can effectively reduce the degradation rate of KSM by ultraviolet radiation, and PTB film can also improve the ultraviolet protection of the material. KSM-1.5hDM-YBC-SS-pectin has stronger anti-ultraviolet ability than KSM-1.5hDM-YBC, and can effectively prevent the decomposition of KSM under ultraviolet radiation. It can be obtained from B that under natural light irradiation conditions, the degradation rate of KSM aqueous solution reaches 50wt.% in 12 hours, and the retention rate of KSM-1.5hDM-YBC-SS-pectin still reaches more than 95wt.% after 120 hours.

[0212] Minimum inhibitory test:

[0213] The minimum inhibitory concentration (MIC) was determined by the culture medium dilution method. 10 mL of LB liquid culture medium containing different concentrations of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin (50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 800 mg / L) was poured into a sterilized 50 mL centrifuge tube, and then 200 μL of activated amylovora bacteria solution (about 1×10 8 CFU·mL -1 ), and the LB liquid medium without drug was used as the blank control. Shake the culture medium at 28℃ and 220r / min for 18h, and spread 200μL of the culture medium on the LB solid plate. All experiments were set up in 3 parallel groups. After 24h of culture in a 28℃ constant temperature incubator, the minimum inhibitory concentration (MIC) of each treatment was determined based on the absence of visible colony growth.

[0214] To evaluate the residual antimicrobial properties of KSM, KSM-1.5hDM-YBC, and KSM-1.5hDM-YBC-SS-pectin against E. amylovora, MIC tests were performed. Fig.12The minimum inhibitory test results of KSM, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin are shown in Figure 1, where A is the minimum inhibitory test result under light-proof conditions, and B is the minimum inhibitory test result under ultraviolet radiation conditions. It can be seen from the figure that with the increase in the concentration of KSM and its complex, the density of E. amylovora decreases in a typical dose-concentration-dependent manner. In A, under the light-proof conditions in the laboratory, when the concentration of KSM is 400 mg / L, the growth of E. amylovora is completely inhibited, and when the concentration of KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin is 400 mg / L, the growth of E. amylovora is effectively inhibited, and when the concentration is 800 mg / L, the growth of E. amylovora is completely inhibited. In B, after UV treatment of the material and KSM, the growth inhibition of E. amylovora was weakened at a KSM concentration of 400 mg / L, indicating that UV accelerated the degradation of KSM. In contrast, even under UV irradiation, KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin were able to maintain complete inhibition of E. amylovora at a concentration of 800 mg / L, indicating that KSM-1.5hDM-YBC and KSM-1.5hDM-YBC-SS-pectin significantly slowed down the destruction of the active ingredients of the agent by UV.

[0215] In vitro branch control test:

[0216] Fig.13The figure is a comparison of the control effects of kasugamycin technical and KSM-1.5hDM-YBC-SS-pectin on Erwinia amylovora at different concentrations. The experimental results show that after 10 days of treatment, the disease index of all treatment groups was significantly reduced, indicating that both agents can effectively inhibit the infection of E. amylovora on pear tree leaves at all tested concentrations, and the effect increases with the increase of agent concentration. It should be pointed out that the control effect of KSM-1.5hDM-YBC-SS-pectin materials at different concentrations (100, 200, 400 and 800 mg / L) is better than that of kasugamycin technical at the corresponding concentrations. In particular, at a concentration of 400 mg / L, the control effect of KSM-1.5hDM-YBC-SS-pectin reached 68.23±3.34%, which is significantly higher than 42.29±3.90% of kasugamycin technical, which shows that the composite material has efficient control characteristics against Erwinia amylovora. This significant synergistic effect is derived from the special preparation process of KSM-1.5hDM-YBC-SS-pectin material, which enhances the stability and bioavailability of kasugamycin through microencapsulation and pectin coating. In addition, this preparation method may help the slow release of the agent in the plant, thereby achieving a higher control effect at a lower concentration.

[0217] In vitro branch control test:

[0218] The in vitro branch infusion solution was prepared by the "drug solution-bacteria solution co-culture method". The preserved strains were separated by streaking on the LB medium, cultured at a constant temperature of 28℃ for 48h, and secondary activation was performed to obtain single colonies. The single colony was transferred to the LB culture medium and shaken for 12h at 28℃ and 180r / min. The test bacterial suspension was obtained when the OD600 was 1.2, and it was placed in a refrigerated storage at 4℃ for standby use. Each fungicide was added to 50mL sterile water according to different dilution multiples to prepare a drug solution for standby use. And it was mixed according to the system of 20mL drug solution + 2mL bacterial suspension + 20mL LB culture solution, and the mixed drug suspension was prepared by shaking at 28℃ and 180r / min for 4h. The annual branch was inserted into the mixed drug suspension after sterile water and alcohol, and its disease was observed. Three parallel experiments were set up, and the lesions were observed and recorded after 10d. Referring to the method of Paprstein et al. and improving it, a disease grading standard for in vitro branches inoculated with the amylopectin pathogen was established, and the disease index and control effect were calculated.

[0219] Fig.14 This is a comparison of the growth status of the in vitro branches after they were treated with different concentrations of KSM and KSM-1.5hDM-YBC-SS-pectin for 0, 5, and 10 days under greenhouse conditions.

[0220] Pollen germination test:

[0221] In order to further determine the plant safety of KSM-1.5hDM-YBC-SS-pectin material on pollen germination, the inhibitory effect of KSM and KSM-1.5hDM-YBC-SS-pectin on pollen germination was evaluated by simulated field spraying test. Weigh 60g sucrose and 60mg boric acid and dissolve them in 600mL ultrapure water, stir and dissolve to prepare basic culture solution. Take 100mL basic culture solution in a conical flask and store at 4℃ for use. Weigh 7.5g agar powder and place it in the remaining 500mL basic culture solution to fully dissolve, sterilize at high temperature, and obtain germination medium. Weigh 0.5g pollen and put it into the prepared 100mL basic culture solution, shake for 30min, and prepare pollen: basic culture solution = 1:200 pollen solution. In a simulated field spraying test, agents containing different concentrations of KSM and KSM-1.5hDM-YBC-SS-pectin (100 mg / L, 200 mg / L, 400 mg / L and 800 mg / L) were sprayed on the surface of the sterilized germination medium, dried naturally for 2 hours, and then 0.8 mL of the diluted pollen solution was immediately applied to the solid germination medium plate, placed in a 25°C constant temperature incubator and incubated in the dark for 4 hours, and the pollen germination was recorded. Sterile water was used as a blank control. The five-point sampling method was used to observe and record the test results on an electron microscope, and all treatments were set up with 3 parallel experiments.

[0222] Pear fire blight mainly occurs during the flowering period. Since pollen germination is particularly sensitive to chemical agents, monitoring the pollen germination rate to assess plant toxicity is an effective method. Fig.15 The effect of different concentrations of KSM, KSM-1.5hDM-YBC-SS-pectin (100mg / L, 200mg / L, 400mg / L and 800mg / L) on pollen germination rate is shown. A is the pollen germination under an electron microscope, and B is the pollen germination rate obtained by statistical calculation.

[0223] The experimental results showed that at a concentration of 100 mg / L, the effects of the two agents on pollen germination were not significantly different from those of the blank control group, indicating that at this concentration the agents had no obvious toxicity to pollen germination. However, as the concentration of the agent increased, its toxic effect on pollen also increased. Specifically, KSM significantly inhibited pollen germination at concentrations of 400 mg / L and 800 mg / L, and the germination rate decreased by 12.87% and 21.72%, respectively. At a concentration of 800 mg / L, the growth of pollen tubes was also significantly inhibited.

[0224] On the other hand, the situation of KSM-1.5hDM-YBC-SS-pectin was slightly different. At a concentration of 800 mg / L, it caused a 9.34% decrease in pollen germination rate, but showed no significant effect on pollen tube length.

[0225] KSM-1.5hDM-YBC-SS-pectin had no effect on pollen germination at a concentration of 400 mg / L, indicating that materials within this concentration range can be safely used for the prevention and control of apricot fire blight during flowering. This finding has important practical significance for the formulation of apricot fire blight flowering management strategy.

[0226] Fig.16 The adsorption capacity of KSM by the modified biochar prepared by using phosphoric acid solution and diammonium phosphate solution of different concentrations in Comparative Example 1, M-YBC, 1.5hDM-YBC of Example 1, 2hDM-YBC of Example 3, 1hD-YBC of Comparative Example 2 and YBC of Comparative Example 3 is shown. It can be seen that the adsorption capacity of M-YBC, 1.5hDM-YBC of Example 1 and 2hDM-YBC of Example 3 is higher, while the adsorption capacity of the modified biochar prepared by phosphoric acid solution with concentrations of 10M, 7.5M and 5M, the modified biochar prepared by diammonium phosphate solution with concentrations of 10M, 7.5M and 5M and 1hD-YBC of Comparative Example 2 is lower.

[0227] This is because different modifying agents have different effects on the surface groups and pore structures of biochar, and the other solutions are not as effective as NaSO4. In addition, the stirring time in Comparative Example 2 is too short, which affects the adsorption amount.

[0228] Fig.17 The release curves of KSM from KSM-M-YBC in comparative example 4, KSM-1.5hDM-YBC in example 1, and KSM-2hDM-YBC in example 3 show that the sustained release amount of KSM-M-YBC is much lower than that of KSM-1.5hDM-YBC and KSM-2hDM-YBC modified by BSA and TCEP solutions during the sustained release process. This is because KSM-M-YBC in comparative example 4 cannot completely release KSM.

[0229] Fig.18 0.5M of Example 1 NaSO4 -YBC, i.e., the modified solution is 0.5M Na2SO4, the modified biochar prepared by comparative example 5 is 0.1M NaSO4 -YBC, i.e., the modified biochar prepared with 0.1M Na2SO4 as the modified solution, has a higher adsorption capacity on KSM. NaSO4 -YBC's adsorption capacity for KSM is greatly reduced. This is because different concentrations of the modifying agent have different effects on the pore structure of the material.

[0230] Fig.19 0.5M of Example 1 NaSO4 -YBC, i.e., the modified solution is 0.5M Na2SO4, the modified biochar prepared by comparative example 6 is 1M NaSO4 -YBC, i.e., the amount of KSM adsorbed by the modified biochar prepared with 1M Na2SO4 as the modified solution. NaSO4 -YBC's adsorption capacity for KSM decreased to a certain extent. Too high a concentration of the modifying agent has a negative impact on the pore structure of the material.

[0231] Fig. 20 It is the adsorption capacity of KSM by 0.5hDM-YBC of comparative example 7 and 1.5hDM-YBC of example 1. It can be seen that the adsorption amount of KSM by 0.5hDM-YBC of comparative example 7 is slightly lower than that of example 1. This is because the time is too short, resulting in uneven protein loading and reduced adsorption effect of the material.

[0232] Fig.21 The release curves of KSM from KSM-0.5hDM-YBC of Comparative Example 7 and KSM-1.5hDM-YBC of Example 1 show that the sustained release amount of Comparative Example 7 is significantly lower than that of Example 1. This is due to uneven protein loading.

[0233] Fig. 22 The adsorption capacity of 3hDM-YBC in comparative example 8 and 1.5hDM-YBC in example 1 for KSM is shown in Figure 1. The adsorption capacity of KSM in comparative example 8 is greatly reduced. This is because the time is too long and the protein loading is too high, which will reduce its specific surface area and reduce the active sites for adsorption.

[0234] Fig.23 The release curves of KSM for KSM-DM-YBC-SS-pectin of Comparative Example 10 and KSM-1.5hDM-YBC-SS-pectin of Example 1 are shown.

[0235] It can be seen that due to the excessive EDC concentration and excessive gelation, the sustained release of KSM by KSM-DM-YBC-SS-pectin was hindered.

[0236] In summary, the present invention uses Na2SO4-modified jujube leaf biochar mixed with PTB solution to first prepare DM-YBC material, and then bonds it with pectin through amide bonds to prepare a material with dual response to pH value and pectinase. The material has excellent drug controlled release performance and can adjust drug release according to the pH value of the environment or the activity of pectinase.

[0237] KSM-1.5hDM-YBC-SS-pectin exhibited significant pH responsiveness in PBS buffer solutions at pH values ​​of 6.0, 7.0, and 8.0. At pH 7.0, the cumulative release of KSM within 72 hours was only 4.72wt.%, while at pH 6.0 and 8.0, the release increased significantly to 36.99wt.% and 52.08wt.%, respectively, indicating that the material was stable under neutral conditions and that the release of KSM could be regulated under different pH conditions. The acid-base environment affected the ionization state of the pectin network, thereby affecting the release rate of KSM. The addition of pectinase significantly increased the release of KSM from 0.4wt.% to 60.15wt.%, indicating that the material had dual responsiveness to pectinase and pH, effectively achieving the controlled release of KSM. The photostability test showed that KSM-1.5hDM-YBC-SS-pectin provided an effective UV shielding effect on kasugamycin, significantly reducing the degradation of KSM by UV radiation. The minimum inhibitory concentration (MIC) test showed that KSM-1.5hDM-YBC-SS-pectin at a concentration of 800 mg / L could completely inhibit the activity of E. amylovora, and the inhibitory effect remained unchanged after UV treatment. In the in vitro branch test, KSM-1.5hDM-YBC-SS-pectin at a concentration of 400 mg / L showed a high control effect of 68.23±3.34%. In addition, the pollen germination test also confirmed that at a concentration of 400 mg / L, the material had no adverse effect on pollen germination. This result highlights that the KSM-1.5hDM-YBC-SS-pectin material at a concentration of 400 mg / L can be safely and effectively used in the flowering period control of pear fire blight, and has important application value for the management of pear tree blight during the flowering period.

[0238] Each embodiment in this specification is described in a related manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0239] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A method for preparing a dual-response controlled-release pesticide, characterized in that: The following steps are involved: S1: Preparation of modified biochar; S2: Preparation of drug carriers using modified biochar; S3: Preparation of dual-responsive controlled-release pesticides using drug carriers.

2. The method for preparing a dual-responsive controlled-release pesticide according to claim 1, characterized in that: The S1 includes: S11: prepare a Na2SO4 solution, and add the gray jujube leaf powder into the Na2SO4 solution; S12: stirring and centrifuging the mixed solution to separate the modified gray jujube leaf powder; S13: drying the separated modified gray jujube leaf powder and then performing a pyrolysis treatment; S14: cleaning the pyrolysis product, and performing suction filtration, drying, and sieving operations to obtain modified biochar.

3. The method for preparing a dual-responsive controlled-release pesticide according to claim 2, characterized in that: In the S11, The concentration of Na2SO4 solution is 0.4~1mol / L; the mass volume ratio of gray jujube leaves and Na2SO4 solution is 1 / 30~1 / 20g / mL.

4. The method for preparing a dual-responsive controlled-release pesticide according to claim 2, characterized in that: In the S12, The stirring time is 1.8 to 2.2 hours; the centrifugal speed is 4800 to 5200 rpm; the centrifugal time is 4 to 6 minutes; In S13, the drying temperature is 78-82°C, the pyrolysis temperature is 690-710°C, and the pyrolysis time is 2.8-3.2h.

5. The method for preparing a dual-responsive controlled-release pesticide according to claim 1, characterized in that: The S2 includes: S21: mixing a bovine serum albumin solution and a tris(2-carboxyethyl)phosphine hydrochloride aqueous solution; S22: adjusting the pH of the mixed solution; S23: adding modified biochar to the mixed solution obtained in S22, and performing stirring, filtering, and washing operations; S24: Drying the washed sample to obtain the drug carrier DM-YBC.

6. The method for preparing a dual-responsive controlled-release pesticide according to claim 5, characterized in that: In S21, The concentration of bovine serum albumin solution is 9-11 mg / mL; the concentration of tris(2-carboxyethyl)phosphine hydrochloride aqueous solution is 4.8×10 -2 ~5.2×10 -2 mol / L; the volume ratio of bovine serum albumin solution and tris(2-carboxyethyl)phosphine hydrochloride aqueous solution is (0.8:1) to (1.2:1); In S22, Adjust pH to 4.4-4.6; In S23, according to the volume ratio, the volume of the mixed solution is 95-105 mL, the mass of the modified biochar is 0.4-0.6 g; the stirring time is 1.4-2 hours; In S24, the drying temperature is 55-65°C.

7. The method for preparing a dual-responsive controlled-release pesticide according to claim 1, characterized in that: The S3 includes: S31: mixing the drug carrier with the kasugamycin aqueous solution; S32: adding 3-mercaptopropyltrimethoxysilane to the mixed solution of S31 and stirring, and centrifuging, washing, and drying to obtain KSM-loaded thiol-functionalized protein-biochar KSM-DM-YBC; S33: dispersing the thiol-functionalized protein-biochar KSM-DM-YBC loaded with KSM in methanol, adding β-mercaptoethylamine and stirring, centrifuging, washing, and drying the obtained reaction solution to obtain amino-substituted protein-biochar particles KSM-DM-YBC-SS-NH2 loaded with KSM; S34: dissolving pectin in deionized water to obtain a pectin aqueous solution, dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the pectin aqueous solution and stirring; S35: Add amino-substituted protein-biochar particles KSM-DM-YBC-SS-NH2 and N-hydroxysuccinimide loaded with KSM to the mixed solution of S34 and stir, centrifuge, wash and dry the obtained reaction solution to obtain pectin-protein-biochar particles KSM-DM-YBC-SS-pectin loaded with KSM.

8. The method for preparing a dual-responsive controlled-release pesticide according to claim 7, characterized in that: In the S31, The drug carrier mass is 0.4-0.6 g; the concentration of the kasugamycin aqueous solution is 0.9-1.1 mg / mL; In S32, the volume of 3-mercaptopropyltrimethoxysilane is 0.65-0.90 mL; stirring is carried out at 58-62° C. for 1.8-2.2 h; The centrifugal speed is 5900-6100 rpm; the centrifugal time is 4-6 minutes; the drying temperature is 58-62°C; In S33, the mass of thiol-functionalized KSM-DM-YBC is 0.8-1.2 g, the mass of β-mercaptoethylamine is 1.8-2.2 g, the stirring time is 23-25 ​​h, and the drying temperature is 58-62°C.

9. The method for preparing a dual-responsive controlled-release pesticide according to claim 7, characterized in that: In the S34, The mass volume ratio of pectin to deionized water is (1.5:1):(2.5:1) g / L; 1-ethyl-(3-dimethylaminopropyl)carbodiimide is dissolved in 45-55 mL of pectin aqueous solution; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 10-100 mM; the stirring time is 25-35 min; In S35, the mass of KSM-DM-YBC-SS-NH2 microspheres is 0.1-0.3 g, the concentration of N-hydroxysuccinimide is 30-50 mM; the stirring time is 23-25 ​​h, and the drying temperature is 58-62 °C.

10. Use of the dual-responsive controlled-release pesticide prepared by the method for preparing the dual-responsive controlled-release pesticide according to any one of claims 1 to 9, characterized in that: Used for the prevention and treatment of fire blight.

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