Pesticide product with pH response release function based on protein grafting coupling

Abstract

The invention discloses a pesticide product with a pH response release function based on protein grafting coupling. The pesticide product is prepared by loading a pesticide containing alcoholic hydroxyl groups on a polyethylene glycol modified protein nano-film by utilizing a grafting coupling technology and amyloid-like phase transformation of protein, the pesticide product can intelligently and slowly release the contained pesticide according to environmental conditions and plant infection conditions, the lasting period of the pesticide is prolonged, and the pesticide spraying frequency is reduced, so that the pesticide utilization rate is improved, and the problems of resource waste, environmental pollution and the like caused by pesticide loss are reduced. In addition, along with the decomposition of the protein nano-film, the pesticide on the crops can be effectively removed. The protein as the main component is safe, environment-friendly and high in biocompatibility, and has a wide prospect in practical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticides and biomaterials, and specifically involves modifying proteins through chemical methods to combine the proteins with pesticides to form stimulus-responsive chemical bonds. The pesticide molecules are then released according to their original chemical structure by changing the environmental pH factor. The release process has different release rates due to different environmental factors, thereby improving the utilization rate of the pesticide's effective period and preventing or reducing problems such as environmental pollution and resource waste caused by pesticide loss. Background Art

[0002] The use of pesticides is indispensable in agriculture. Statistics show that without pesticides, insect pests would cause losses of up to 78%, 54%, and 32% for fruits, vegetables, and grains, respectively. Pesticides effectively protect crops from insects and plant pathogens, ensuring the quality and yield of agricultural products, and thus providing an adequate food supply for the world's growing population. Currently, pesticides are widely applied to a wide range of plants, including crops, vegetables, and fruits. However, due to the hydrophobic / superhydrophobic properties of plant leaves, pesticide droplets often bounce or splash upon contact, resulting in over 50% loss of the pesticide. Only a small fraction of the pesticide actually reaches the plant surface, and this amount can be washed away by rain, evaporated, or washed into groundwater, air, and soil. This not only leads to excessive pesticide use but also causes serious environmental pollution, adversely affecting human health, endangered species, and ecosystems.

[0003] To improve the deposition of pesticide droplets on plant leaves, a commonly used method is to add polymer solutions, surfactants, or charge the droplets to the pesticide liquid. However, given the potential harm of surfactants to the environment and the possibility of secondary pollution, this method should be avoided as much as possible. In addition, this method does not fully consider other natural factors, such as the scouring effect of rain on pesticides. Other methods include using natural attapulgite modified by high-energy electron beams to control the loss of pesticides from plant surfaces, using polydopamine microcapsules to extend the retention time of pesticides on leaves, and developing nanopesticides. However, the universality of these methods in the plant kingdom and their excellent ability to fix pesticides still need further verification.

[0004] Slow-release pesticide technology is a key breakthrough in addressing inefficient pesticide use and environmental risks. Traditional pesticide formulations are limited by their rapid release properties. After application, over 60% of the active ingredients are lost through volatilization, photolysis, or rainwater washoff, resulting in short control cycles and frequent reapplications. This not only exacerbates the development of pesticide resistance but also causes serious non-point source pollution—approximately 1.3 million tons of pesticides enter water bodies worldwide annually, causing acute poisoning of aquatic organisms and imbalances in soil microbial communities. Slow-release technology significantly prolongs the active ingredient's lifespan by encapsulating pesticides with carrier materials and controlling their release. The key to achieving precise controlled release lies in the stable interface between the carrier and the crop. In recent years, amyloid protein films have garnered attention for their unique interfacial adhesion and environmental responsiveness. These proteins self-assemble into a β-pleated structure, forming a strongly adhesive nanofilm on crop surfaces. Hydrogen bonding and hydrophobic interactions securely attach the drug-carrying system to the wax layer of leaves, resulting in a 3-5 times higher retention rate in water compared to conventional formulations. At the same time, the dense network structure of the film can regulate the on-demand release of pesticide molecules: under humidity triggering, protein conformational changes can precisely control the release rate, so that the pesticide utilization rate exceeds 70%, and the effective period is extended to 2 to 3 times that of conventional dosage forms (H.Su, Y.Liu, Y.Gao, C.Fu, C.Li, R.Qin, L.Liang, P.Yang, Amyloid-Like Protein Aggregation Toward Pesticide Reduction. Adv.Sci. 2022, 9, 2105106.) This bionic adhesion and slow-release synergistic mechanism can not only reduce the diffusion of pesticides into the environment, but also enhance the control effect by prolonging the retention time on the leaves, providing an innovative path for the development of smart pesticides with both high efficacy and low environmental load.

[0005] The invention patent with patent number ZL 202010132406.0 applied for based on this document fully demonstrates the fixation performance of amyloid-like protein products on fixtures such as coatings, pesticides, fertilizers, cosmetics, and medical drugs. It has the advantages of improving the utilization rate of fixtures, reducing resource waste and environmental pollution caused by the loss of target fixtures, and the target fixtures can be effectively removed as the protein membrane decomposes. The protein is safe, environmentally friendly, and has high biocompatibility. Despite the above advantages, the method in this patent is only a simple mixing of amyloid-like proteins and pesticide preparations. There are only common interaction forces such as van der Waals forces and hydrogen bonds between proteins and pesticides. The pesticides still work according to their own properties, and the protein is only used as an adjuvant to improve adhesion. There is no function of controlling the intelligent slow-release and controlled-release of pesticides, and it is impossible to release the target fixtures as required. Summary of the Invention

[0006] The purpose of the present invention is to reduce the frequency of pesticide application, improve the utilization rate of pesticides, combine the advantages of amyloid-like protein films, extend the effective period of pesticides, enhance the efficiency of pesticide control, and provide a pesticide product with pH-responsive release function based on protein graft coupling. The pesticide product has the advantages of good stability and can be removed with common detergents.

[0007] To achieve the above-mentioned purpose, the present invention provides a pesticide product with pH-responsive release function based on protein graft coupling, comprising: 50 to 300 parts of a pesticide protein product and 50 to 300 parts of a disulfide bond reducing agent; wherein the pesticide protein product is obtained by condensing a polyethylene glycol-modified protein with a pesticide containing an alcoholic hydroxyl group through a Steglich esterification reaction, followed by dialysis and freeze-drying.

[0008] The specific preparation method of the above-mentioned pesticide protein product is: dissolving the polyethylene glycol-modified protein, the pesticide containing alcoholic hydroxyl groups, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine in an organic solvent, stirring at room temperature for 18 to 24 hours, dialyzing with deionized water for 48 to 72 hours after the reaction is completed, and freeze-drying to obtain the pesticide protein product.

[0009] In the method for preparing the pesticide protein product, the mass ratio of the polyethylene glycol-modified protein to the pesticide containing alcoholic hydroxyl groups, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is preferably 10:5-30:5-20:5-30.

[0010] In the above-mentioned method for preparing the pesticide protein product, the organic solvent is any one of dichloromethane, dimethyl sulfoxide, methanol, ethanol, acetone, N,N-dimethylformamide, and ethyl acetate.

[0011] The preparation method of the above-mentioned polyethylene glycol-modified protein is as follows: polyethylene glycol and N,N'-succinimidyl carbonate are dissolved in dichloromethane, and an organic weak base is added. After stirring at room temperature for 6 to 10 hours, the activated polyethylene glycol is recrystallized to obtain the activated polyethylene glycol; the activated polyethylene glycol and protein are added to water, stirred at room temperature for 6 to 10 hours, dialyzed with deionized water for 48 to 72 hours after the reaction, and freeze-dried to obtain the polyethylene glycol-modified protein.

[0012] In the method for preparing the polyethylene glycol-modified protein, the mass ratio of the protein, polyethylene glycol, N,N'-succinimidyl carbonate, and organic weak base is preferably 10:50-200:5-20:5-20.

[0013] Furthermore, the above-mentioned protein is preferably any one or more of lysozyme, bovine serum albumin, human serum albumin, lactoferrin, whey albumin, insulin, α-lactalbumin, fibrinogen, β-lactoglobulin, ribonuclease A, cytochrome c, α-amylase, horseradish peroxidase, pepsin, myoglobin albumin, collagen, keratin, hemoglobin, DNA polymerase, casein, soy protein isolate, pea protein isolate, zein, gliadin, gluten, oat protein, potato protein, hemp kernel protein, walnut protein, rapeseed protein, hazelnut protein, quinoa protein, sunflower seed protein, pumpkin seed protein, black sesame protein, mushroom protein, red algae protein, legumin, barley protein, wheat protein, gluten, kidney bean protein, catalase, transferrin, and thyroglobulin.

[0014] The polyethylene glycol is selected from any one or more of polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 6000.

[0015] The organic weak base is any one or more of triethylamine, diisopropylethylamine, pyridine, glycine, dimethylaminopyridine, quinuclidine, imidazole and piperidine.

[0016] The disulfide bond reducing agent is selected from any one or more of cysteine, tris(2-carboxyethyl)phosphine hydrochloride, reduced glutathione, dithiothreitol, and β-mercaptoethanol (2-mercaptoethanol).

[0017] The above-mentioned pesticide containing alcoholic hydroxyl group is selected from any one or more of tebuconazole, Jinggangmycin, blasticidin, kasugamycin, streptomycin, diniconazole, polyoxin, Ningnanmycin, chlorfenapyr, cyproconazole, hexaconazole, gibberellic acid, phenoxyethanol, flutriafol, bifenthrin, molluscicide ethanolamine salt, triacontanol, 14-hydroxybrassinosteroid, and curcumin.

[0018] When the pesticide product of the present invention is used, the pesticide protein product and the disulfide bond reducing agent are added to water or water containing a surfactant or an organic solvent, mixed evenly, and then sprayed on the surface of the target crop.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. The pesticide product of the present invention is composed of a pesticide protein product and a reducing agent. The pesticide protein product is composed of a protein and a pesticide molecule coupled by grafting. Under the action of the reducing agent, the protein rapidly undergoes a phase transition, forming a pesticide protein film with high adhesion properties on the surface of plant leaves. The film can stably exist on the surface interface of the plant leaves for a long time, has the performance of resisting rain erosion, and improves the utilization rate of the pesticide.

[0021] 2. In the pesticide product of the present invention, the protein and pesticide molecules are coupled via ester bonds. Experimental testing has shown that in a weakly acidic environment, the ester bond effectively breaks, allowing the pesticide to be slowly released at a high rate. In a neutral environment, the ester bond breaks more slowly, maintaining pesticide release at a lower level. In a healthy plant, its environment is weakly acidic. However, under external stresses such as infection, the plant secretes reactive oxygen species, making its environment weakly acidic. Therefore, the pesticide product of the present invention can intelligently control the release of the pesticide based on the plant's disease status, effectively preventing plant diseases.

[0022] 3. The pesticide product of the present invention has the function of prolonging the release of pesticides. According to experimental findings, compared with the tebuconazole technical and the commercially available tebuconazole preparation Fuliku, the pesticide product of the present invention can prolong the sustained-release time of the pesticide to more than 16 days, which can effectively reduce the number of times farmers spray pesticides and reduce production costs.

[0023] 4. The method of using the pesticide product of the present invention is simple. The pesticide protein product and the reducing agent are mixed with water to form a solution of the required concentration. The mixture is then evenly sprayed onto the plant surface using a common pesticide spray barrel.

[0024] 5. Compared with commercially available preparations, the pesticide product of the present invention has a certain synergistic effect on the efficacy of pesticides. Indoor bioassay experiments and field experiments found that, compared with commercially available tebuconazole preparations, the pesticide product of the present invention can increase the inhibitory effect of Fusarium graminearum by 10% to 20%, the inhibitory effect on wheat fusarium scab lesions by 2% to 10%, and the relative prevention effect on cucumber gray mold by about 10%.

[0025] 6. The pesticide product of the present invention can be removed by common surfactants, such as household detergents, baking soda water, etc., without any pesticide residue problem. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 3 is the standard curve of tebuconazole original drug solution (a) and the sustained-release curves of tebuconazole in different experimental groups (b).

[0027] Figure 2 The contact angles of water on a blank lotus leaf (a), water droplets on a lotus leaf treated with Fuliku and washed with simulated rain (b), water droplets on a lotus leaf treated with the pesticide protein product of Example 1 and washed with simulated rain (c), water droplets on a lotus leaf treated with the pesticide product of Example 1 and washed with simulated rain (d), and a photograph placed at 90° to the horizontal (e).

[0028] Figure 3 These are scanning electron microscope images of lotus leaves treated in each experimental group before and after simulated rainwater erosion.

[0029] Figure 4 The fungal growth in the plate antibacterial experiment against Fusarium graminearum of the pesticide product in Example 1, tebuconazole technical and Fuliku at different concentrations is shown.

[0030] Figure 5 This is a comparison chart of the sterilization rates of the pesticide product in Example 1, tebuconazole technical, and Fuliku at different concentrations in a plate antibacterial test against Fusarium graminearum.

[0031] Figure 6 These are graphs showing the leaf segment bio-test sterilization experiments of Fuliku, the pesticide protein product in Example 1, and the pesticide product against wheat fusarium head blight in different solvent systems.

[0032] Figure 7 This is a bar graph showing the proportion of leaf lesion areas of Fuliku, the pesticide protein product in Example 1, and the pesticide product against wheat fusarium head blight in different solvent systems.

[0033] Figure 8 These are the cucumber fruit growth diagram (a) of different experimental groups in the large-scale field experiment on cucumber gray mold using the pesticide products in Example 1, the disease index statistics before inoculation (b), the statistics of the relative control results of four field surveys (c), and the statistics of the cucumber plant heights of different experimental groups (d). DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples.

[0035] Example 1

[0036] 5g of polyethylene glycol 2000 (PEG-2000) and 0.64g of N,N'-succinimidyl carbonate (DSC) were dissolved in 15mL of dichloromethane, and 0.6mL of triethylamine was added. The mixture was stirred at room temperature for 8 hours. After the reaction, activated PEG was recrystallized to obtain the activated PEG. The activated PEG and 0.5g of bovine serum albumin were added to 20mL of water and stirred at room temperature for 8 hours. After the reaction, the PEG-modified bovine serum albumin was dialyzed against deionized water for 72 hours and lyophilized to obtain the PEG-modified bovine serum albumin.

[0037] 3g of PEG-modified bovine serum albumin, 4g of tebuconazole stock, 3g of N,N'-dicyclohexylcarbodiimide (DCC), and 2g of 4-dimethylaminopyridine (DMAP) were dissolved in 100mL of DMSO and stirred at room temperature for 24 hours. After the reaction, the product was dialyzed against deionized water for 72 hours and lyophilized to obtain the pesticide protein product.

[0038] The pesticide product of this embodiment is composed of the obtained pesticide protein product and tris(2-carboxyethyl)phosphine hydrochloride. When used, the two are mixed in a mass ratio of 1:1, added to water at a material-liquid ratio of 1 mg:1 mL, gently stirred, and then the resulting mixture is evenly sprayed on the plant surface.

[0039] Example 2

[0040] In this example, PEG-modified whey protein was prepared according to the method of Example 1, and the PEG-modified whey protein was graft-coupled with tebuconazole technical to prepare a pesticide protein product according to the method of Example 1. The composition and use of the pesticide product were the same as those in Example 1.

[0041] Example 3

[0042] In this example, PEG-modified lysozyme was prepared according to the method of Example 1, and the PEG-modified lysozyme was graft-coupled with tebuconazole technical to prepare a pesticide protein product according to the method of Example 1. The composition and use of the pesticide product were the same as those in Example 1.

[0043] Example 4

[0044] In this example, PEG-modified soy protein isolate was prepared according to the method of Example 1, and the PEG-modified soy protein isolate was graft-coupled with tebuconazole technical according to the method of Example 1 to prepare a pesticide protein product. The composition and use method of the pesticide product were the same as those in Example 1.

[0045] Example 5

[0046] In this example, PEG-modified ovalbumin was prepared according to the method of Example 1, and the PEG-modified ovalbumin was graft-coupled with tebuconazole technical to prepare a pesticide protein product according to the method of Example 1. The composition and use of the pesticide product were the same as those in Example 1.

[0047] Example 6

[0048] In this example, PEG-modified human serum albumin was prepared according to the method of Example 1, and the PEG-modified human serum albumin was graft-coupled with tebuconazole technical to prepare a pesticide protein product according to the method of Example 1. The composition and use of the pesticide product were the same as those in Example 1.

[0049] Example 7

[0050] In this example, PEG-modified insulin was prepared according to the method of Example 1, and the PEG-modified insulin and tebuconazole technical were graft-coupled to prepare a pesticide protein product according to the method of Example 1. The composition and use of the pesticide product were the same as those in Example 1.

[0051] Example 8

[0052] In this example, PEG-modified α-lactalbumin was prepared according to the method of Example 1, and the PEG-modified α-lactalbumin was graft-coupled with tebuconazole technical to prepare a pesticide protein product according to the method of Example 1. The composition and use of the pesticide product were the same as those in Example 1.

[0053] In order to demonstrate the beneficial effects of the present invention, the pesticide product of Example 1 was subjected to performance tests, and the specific tests are as follows:

[0054] 1. Pesticide sustained-release performance of pesticide products

[0055] To compare the sustained-release properties of pesticide products, a Fuliku solution, a tebuconazole technical solution, and a pesticide product solution were prepared using 1× PBS as the solvent. The three solutions contained the same tebuconazole concentration. 10 mL of the solution was placed in a dialysis bag, which was then placed in a 100 mL blue-capped reagent bottle containing 90 mL of 1× PBS. The bottle was then placed in a shaker at 100 bpm and a constant temperature of 25°C. At regular intervals, 2 mL of the solution was removed from the bottle and replenished with 2 mL of 1× PBS. High-performance liquid chromatography (HPLC) samples were prepared using a polyethersulfone (PES) filter with a pore size of 0.45 μm. The tebuconazole concentration was measured on a Thermo-Vanquish HPLC instrument to obtain sustained-release curves for the samples.

[0056] To compare the effects of pH changes on the release of tebuconazole from pesticide products, the pH of the 1× PBS solution in a blue-capped reagent bottle was adjusted to pH = 5, pH = 7, and pH = 9 using 1 mM NaOH aqueous solution and 1 mM HCl aqueous solution, respectively. When supplementing with 1× PBS solution, 1× PBS solution of the corresponding pH also needed to be added.

[0057] Since the concentration of tebuconazole released in 1× PBS solution was low, we prepared tebuconazole technical standard solutions with concentration gradients of 0.5ppm, 1.0ppm, 2.5ppm, 5.0ppm, 10.0ppm, 15.0ppm, and 20.0ppm, and established a tebuconazole solution standard curve based on the obtained peak area and concentration (see Figure 1 a).

[0058] Subsequently, the total release rates of different experimental groups were calculated based on the measured tebuconazole peak areas of samples at different time periods. The specific calculation method is as follows:

[0059] The peak area of ​​the sample measured by HPLC is recorded as Y;

[0060] According to the standard curve Y = 0.13317 + 0.80724X, the concentration of the sample measured is

[0061] The concentration measured by the first sampling is recorded as X1, the concentration measured by the second sampling is recorded as X2, and the concentration measured by the nth sampling is recorded as X n .

[0062] When sampling for the nth time, the total mass of the sample in the entire 1×PBS solution system is m n =100X n +∑(2X n-1 +2X n-2 +…+2X1). Where 100 is the total volume of the solution. In this work, a 100mL blue-mouthed reagent bottle was used as the sustained-release container, and the total volume of the 1× PBS solution was 100mL. 2 is the pipetting volume for each sampling and addition of 1× PBS solution. In this work, the pipetting volume for each sampling was 2mL.

[0063] Therefore, when sampling for the nth time, the total release rate of the sample is Where 3580 is the mass of total tebuconazole in the sample. In this work, all experimental groups contained the same mass of tebuconazole, which was 3580 μg.

[0064] from Figure 1 As can be seen from the sustained-release curve of b, the original drug of tebuconazole reaches the sustained-release endpoint at 3.5 hours, and Fuliku reaches the sustained-release endpoint at 24 hours. However, the pesticide product of Example 1 under different pH conditions still does not reach the sustained-release endpoint at the last sampling, i.e., 384 hours, wherein the total release rate of the pesticide product under pH=5 is the highest, and the total release of the pesticide product under pH=7 and pH=9 varies substantially over time. Therefore, the release efficiency of the pesticide product of the present invention is the highest under weakly acidic conditions, which proves that the ester bond connecting the PEG-modified protein and the tebuconazole pesticide has conditional responsiveness to pH. When the plant is infected by a disease, its own protective mechanism causes the plant to secrete hydrogen peroxide, and the diseased area presents weak acidity. Moreover, the environmental pH value for the growth of most microorganisms is 5-9, so the pesticide product has good pH responsiveness for fungal disease control, and compared with the commercially available tebuconazole preparation Fuliku, there is a better sustained-release function.

[0065] 2. Comparison of contact angles of pesticide products and the commercially available tebuconazole formulation Fuliku on super-hydrophobic lotus leaf surfaces

[0066] Lotus leaves treated with the pesticide protein product, pesticide product, and the tebuconazole formulation Fuliku (described in Example 1) and untreated lotus leaves were subjected to a rainwater flushing device with an impact force of 9.6 N, simulating a 2500 mm rainfall. The leaves were then dried in the shade. The treated leaves were then placed in a video optical contact angle meter to assess changes in the hydrophilicity and hydrophobicity of the leaves in the different experimental groups.

[0067] When ultrapure water drops fell on the three groups of lotus leaves that had been treated with simulated rainwater, the water drops on the Fuliku group and the pesticide protein product group still could not wet the lotus leaves, and the contact angle was about 143° ( Figure 2 b,c), which is similar to the contact angle of water droplets on untreated lotus leaves ( Figure 2 a) When the lotus leaf is tilted, the water droplets will slide down quickly and fall off the leaf. The contact angle of the water droplets of the pesticide product group on the lotus leaf is 128° ( Figure 2 d), and when the lotus leaf is placed vertically at 90° to the ground or even turned upside down, the water droplets can still adhere to the surface of the lotus leaf ( Figure 2 e).

[0068] 3. Comparative experiment on the flushing of pesticide products and the commercially available tebuconazole preparation Fuliku on super-hydrophobic lotus leaves

[0069] The above-mentioned pesticide products, the tebuconazole preparation Fuliku and untreated lotus leaves were taken, and the samples were attached to the sample stage with conductive adhesive. Gold ion sputtering was performed for 45 seconds to enhance the conductivity of the samples, and then the surface microstructure was observed.

[0070] In order to simulate the effect of rainy weather on pesticide spraying, the above-mentioned different lotus leaves were placed under a rainwater scouring device to simulate rainwater scouring. The impact force was 9.6N, and the lotus leaf surface was washed by simulating 2500mm rainfall. The lotus leaves were dried in the shade, and the microstructure of the lotus leaves was observed using the same method as above.

[0071] First, in order to test the changes in the surface morphology caused by pesticide products, we conducted scanning electron microscopy to compare the effects of pesticide products and Fuliku on the surface morphology of lotus leaves before and after simulated rainwater erosion. Figure 3 As shown, both lotus leaf groups had a well-coated surface before simulated rainwater scouring. After the simulated rainwater scouring, most of the coating on the Fuliku group's leaves was washed away, revealing the nanoscale branched structures on the leaf surface, with only a small amount of crystalline material remaining on the papillae. In contrast, after the simulated rainwater scouring, the pesticide product film remained on the nanoscale branched structures on the lotus leaf surface in the pesticide product experimental group, and the papillae were evenly coated with the pesticide product film.

[0072] 4. Verification of in vitro sterilization effect of pesticide products

[0073] In order to compare the fungicidal performance of tebuconazole technical, tebuconazole preparation Fuliku and pesticide products, tebuconazole solutions with concentrations of 10 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L and 800 mg / L were prepared, as well as Fuliku solutions and pesticide product solutions containing the same tebuconazole concentrations.

[0074] Take 100 μL of the solution and add it to 10 mL of uncooled potato dextrose agar medium (PDA medium) to prepare a drug-containing plate. Use a punch with an inner diameter of 6.5 mm to punch out uniformly sized circular fungal cakes on the outermost circle of the activated plant disease fungus culture dish and inoculate them in the center of the drug-containing plate. When the diameter of the fungus inoculated in the blank control group grows to a diameter close to that of the culture dish, the average diameter of the fungi in each drug-containing plate is counted to compare the fungal antibacterial effect of different experimental groups. Each experiment is treated with 5 parallel samples.

[0075] The target strain was selected as Fusarium graminearum, which is the pathogen of wheat head blight. Figure 4 and Figure 5 As shown, at a concentration of 0.25 ppm or less in the culture dish, Fuliku exhibited a higher inhibitory effect against the pathogen than tebuconazole technical. At a concentration of 0.5 ppm or greater, Fuliku exhibited a lower inhibitory effect than tebuconazole technical. Across each concentration gradient, the pesticide product demonstrated the highest inhibitory effect against the pathogen, with both exhibiting a 10% to 20% improvement in bactericidal efficacy, demonstrating its effectiveness in controlling wheat fusarium head blight.

[0076] 5. Verification of sterilization effect of pesticide products through indoor biological testing experiments

[0077] A 0.358 mg / mL Fuliku solution, a 1.33 mg / mL pesticide protein product solution, and a 1.33 mg / mL pesticide product solution (all three solutions contained 0.358 mg / mL tebuconazole) were prepared and evenly sprayed onto one-leaf wheat seedling pots using a spray bottle. To ensure the same amount of tebuconazole was applied to the pots, the volume of solution consumed, the angle, and the height of the spray were identical. After the solution dried naturally, the pots were inoculated with the target plant pathogen and cultured in a climate chamber at the corresponding disease-infecting temperature until the wheat seedlings reached the two-leaf stage. The disease grade and relative efficacy of the different experimental groups were then calculated.

[0078] Because wheat fusarium wilt only occurs during the wheat ear stage, indoor bioassay experiments used a leaf segment inoculation method to calculate the size of the bacterial plaques and evaluate the disease control efficacy of each agent. The specific method is: A Fuliku solution containing the same concentration of tebuconazole, a pesticide protein product solution with different solvent systems, and a pesticide product solution are evenly sprayed on one-leaf wheat seedlings. After the agent on the wheat leaves has dried naturally, wheat leaves of roughly uniform width are selected and cut into leaf segments of uniform length. These leaf segments are placed in a 10% agarose medium and a uniform-sized, 6mm-diameter PDA bacterial cake containing Fusarium graminearum is inoculated in the middle of the leaf segment. The leaves are placed in a humid, dark environment, and the experimental results are observed after 3 days.

[0079] Data processing methods: Infected wheat leaf segments were observed and photographed under a Wood's lamp. The resulting images were processed using Photoshop. The number of pixels in the entire leaf segment and the infected area was counted. The proportion of lesions was used to reflect the control effect of different experimental groups on wheat fusarium head blight. The lower the proportion of lesions, the better the control effect.

[0080]

[0081] like Figure 6 As shown in the figure, under the Wood's lamp, the healthy part of the wheat leaf segment appears purple-red, the inside of the diseased part appears black, and the outside appears white. Figure 7 It can be seen that methanol has a certain inhibitory effect on scab. Secondly, the incidence rate of the pesticide protein product group and the pesticide product group is lower than that of the Fuliku group, proving that protein has a certain synergistic effect on tebuconazole in preventing scab. In addition, the incidence rate of the pesticide product group is lower than that of the non-phase-transformed pesticide protein product group using the same solvent, and the incidence rate of the pesticide product group using methanol as the solvent is lower than that of the corresponding water solvent group. The above experimental results prove that the pesticide product of the present invention has a good effect of reducing the amount and increasing the effect on the prevention and treatment of scab.

[0082] 6. Verification of the sterilization effect of pesticide products in large-scale field experiments

[0083] Test object: cucumber gray mold.

[0084] Experimental crops: cucumber.

[0085] Experimental variety: Legend 778.

[0086] Experimental groups: blank group (CK), Fuliku group (FLK), pesticide protein product group (PEG-BSA-Teb), and pesticide product group (PEG-PTB-Teb).

[0087] Crop cultivation and environmental conditions: Managed according to farmers' conventional cultivation procedures. Clay soil texture, medium fertility.

[0088] Dosage method: Field spraying, 45L / mu, during the peak period of cucumber fruiting and the early stage of gray mold (relative disease index 2.76), spray three times in a row, and adjust the spraying interval to 5 days according to the field disease situation and the pattern of gray mold. The drug concentration is the recommended concentration for cucumber gray mold (tebuconazole active ingredient concentration 0.143 mg / mL).

[0089] Direct effects of the pesticide on crops: During the test period, each pesticide treatment had an inhibitory effect on plant height under the dosage applied, and no effect was found on other pests and non-target organisms (see Figure 8 a).

[0090] From the pre-drug morbidity index ( Figure 8 b) The disease is evenly distributed throughout the field, indicating the early stage of gray mold, and the test conditions are good.

[0091] Significant growth inhibition was observed in all treatments after three applications, affecting both fruit size and plant height after the third application ( Figure 8 d), resulting in a shortened interleaf spacing. This phenomenon is caused by the fact that tebuconazole, as a triazole fungicide, inhibits the synthesis of endogenous gibberellins in plants, thereby inhibiting the plant's vegetative growth.

[0092] The results of the four surveys ( Figure 8 c) The control effect of pesticide products is better than that of Fuliku alone, and the antibacterial effect is improved by more than 10% to 20%.

Claims

1. A pesticide product with pH-responsive release function based on protein graft coupling, characterized in that The pesticide product comprises: 50 to 300 parts of a pesticide protein product and 50 to 300 parts of a disulfide bond reducing agent; The pesticide protein product is obtained by condensing a polyethylene glycol-modified protein with a pesticide containing an alcoholic hydroxyl group through a Steglich esterification reaction, followed by dialysis and freeze-drying.

2. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 1, characterized in that: The preparation method of the polyethylene glycol-modified protein comprises the following steps: dissolving polyethylene glycol and N,N'-succinimidyl carbonate in dichloromethane, adding an organic weak base, stirring and reacting at room temperature for 6 to 10 hours, and then recrystallizing to obtain activated polyethylene glycol; adding the activated polyethylene glycol and protein into water, stirring and reacting at room temperature for 6 to 10 hours, dialyzing with deionized water for 48 to 72 hours after the reaction is completed, and then freeze-drying to obtain the polyethylene glycol-modified protein.

3. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 2, characterized in that: The mass ratio of the protein, polyethylene glycol, N,N'-succinimidyl carbonate and organic weak base is 10:50-200:5-20:5-20.

4. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 2 or 3, characterized in that: The polyethylene glycol is any one or more of polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 6000.

5. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 2 or 3, characterized in that: The organic weak base is any one or more of triethylamine, diisopropylethylamine, pyridine, glycine, dimethylaminopyridine, quinuclidine, imidazole and piperidine.

6. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 1, characterized in that: The preparation method of the pesticide protein product comprises: dissolving a polyethylene glycol-modified protein, a pesticide containing an alcoholic hydroxyl group, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine in an organic solvent, stirring the mixture at room temperature for 18 to 24 hours, dialyzing the mixture with deionized water for 48 to 72 hours, and freeze-drying the mixture to obtain the pesticide protein product.

7. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 6, characterized in that: The mass ratio of the polyethylene glycol-modified protein to the pesticide containing alcoholic hydroxyl groups, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 10:5-30:5-20:5-30.

8. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 6, characterized in that: The organic solvent is any one of dichloromethane, dimethyl sulfoxide, methanol, ethanol, acetone, N,N-dimethylformamide and ethyl acetate.

9. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 1, characterized in that: The protein is selected from any one or more of lysozyme, bovine serum albumin, human serum albumin, lactoferrin, whey albumin, insulin, α-lactalbumin, fibrinogen, β-lactoglobulin, ribonuclease A, cytochrome c, α-amylase, horseradish peroxidase, pepsin, myoglobin albumin, collagen, keratin, hemoglobin, DNA polymerase, casein, soy protein isolate, pea protein isolate, zein, gliadin, gluten, oat protein, potato protein, hemp kernel protein, walnut protein, rapeseed protein, hazelnut protein, quinoa protein, sunflower seed protein, pumpkin seed protein, black sesame protein, mushroom protein, red algae protein, legumin, barley protein, wheat protein, gluten, kidney bean protein, catalase, transferrin, and thyroglobulin.

10. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 1, characterized in that: The disulfide bond reducing agent is selected from any one or more of cysteine, tris(2-carboxyethyl)phosphine hydrochloride, reduced glutathione, dithiothreitol, and β-mercaptoethanol (2-mercaptoethanol).

11. The pesticide product with pH-responsive release function based on protein graft coupling according to claim 1, characterized in that: The pesticide containing alcoholic hydroxyl groups is any one or more of tebuconazole, jinggangmycin, blasticidin, kasugamycin, streptomycin, diniconazole, polyoxin, ningnanmycin, chlorfenapyr, cyproconazole, hexaconazole, gibberellic acid, phenoxyethanol, flutriafol, bifenthrin, molluscicide ethanolamine salt, triacontanol, 14-hydroxybrassinosteroid, and curcumin.

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