A lipopeptide substance for preventing and treating walnut decay and a preparation method thereof

Abstract

The application discloses a kind of lipopeptide class for preventing and treating walnut rot disease and preparation method thereof, it is related to plant disease control technical field, including the nanocarrier core of lipopeptide class loading;And the responsive material shell of the carrier core outside is covered;Wherein, the responsive material shell can be degraded under the microenvironment of walnut tree disease site, the microenvironment characteristics include pH value is lower than 6.0 and there is pectinase activity.The present application realizes the precise and efficient prevention and treatment of walnut rot disease by constructing the intelligent nanometer drug delivery system of "nuclear shell structure", realizes the targeting and on-demand release of lipopeptide in lesion site using the shell with double response to disease microenvironment, greatly improves drug utilization and prolongs the effective period;Nanocarrier core effectively protects lipopeptide activity, and its 50-200 nanometer optimized particle size and surface modification promote drug delivery and enrichment in tree body.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to a lipopeptide substance for controlling walnut rot and its preparation method. Background Technology

[0002] Walnut rot is a major branch and trunk disease affecting the walnut industry. The pathogen mainly enters through wounds, spreading within the bark and causing gumming, ulceration, and even death of the branches and trunk, making control extremely difficult. Currently, control relies primarily on the spraying of chemical fungicides (such as thiophanate-methyl). However, the long-term use of chemical pesticides has led to increasing pathogen resistance, environmental pollution, pesticide residues, and damage to the tree's microecology, making the development of efficient, safe, and environmentally friendly biological control technologies an urgent need for the sustainable development of the industry. Lipopeptides (such as ituronidin and fentanyl) are a class of bioactive molecules produced by Bacillus and other microorganisms, possessing strong surface activity and broad-spectrum antifungal activity, and are considered highly promising alternatives to chemical pesticides. However, the direct application of lipopeptides in field disease control still faces a series of key technical bottlenecks, severely limiting their effectiveness. To enhance its efficacy and application, nanoparticle-based drug delivery technology has provided new insights in recent years for improving the stability, utilization, and targeting of agricultural active ingredients. Existing research has attempted to load antimicrobial peptides or chemical pesticides onto nanoparticles. However, most current technologies focus on simple physical encapsulation or adsorption, resulting in drug delivery systems that generally suffer from uncontrollable release behavior. After application to plants, these carriers often exhibit "burst release" or release processes unrelated to environmental factors, failing to achieve intelligent and precise drug delivery based on the severity and location of the disease. This is particularly true for diseases like walnut rot, where the lesion environment has distinct characteristics (such as weak acidity due to tissue necrosis and microbial metabolism, and specific enzymes secreted by the pathogen). There are currently no reports of nanoparticle-based drug delivery systems capable of simultaneously responding to both pH and enzyme environmental signals and intelligently releasing lipopeptide biocontrol substances at the lesion site.

[0003] However, current common solutions have many drawbacks, including: existing chemical pesticides are prone to causing drug resistance and environmental pollution; free lipopeptides have poor environmental stability, are easily degraded, and lack targeting; while conventional nano-drug delivery technology can encapsulate drugs, its release is uncontrollable and cannot intelligently respond to the unique lesion microenvironment of walnut rot (such as weak acidity and pectinase activity), resulting in low drug utilization, short duration of effect, and difficulty in achieving efficient treatment of diseases inside the tree trunk. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the lipopeptide substances used to prevent and treat walnut rot and their preparation methods, the present invention is proposed.

[0006] Therefore, the purpose of this invention is to provide a lipopeptide substance for the prevention and control of walnut rot and its preparation method. It is applicable to solving the problems of existing chemical pesticides easily leading to drug resistance and environmental pollution; free lipopeptides have poor environmental stability, are easily degraded, and lack targeting; while conventional nano-drug delivery technology can encapsulate drugs, its release is uncontrollable and cannot intelligently respond to the lesion microenvironment unique to walnut rot, resulting in low drug utilization, short duration of effect, and difficulty in achieving efficient treatment of diseases inside the tree trunk.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, embodiments of the present invention provide a lipopeptide substance for preventing and controlling walnut rot, comprising a nanocarrier core loaded with the lipopeptide substance; and a responsive material shell covering the carrier core; wherein the responsive material shell is capable of degradation in the microenvironment of the diseased part of the walnut tree, the microenvironment characteristics including a pH value below 6.0 and the presence of pectinase activity.

[0008] As a preferred embodiment of the lipopeptide substance for preventing and controlling walnut rot according to the present invention, the outer shell of the responsive material comprises pectin or its derivatives, and a pH-sensitive polymer.

[0009] As a preferred embodiment of the lipopeptide substance for preventing and controlling walnut rot according to the present invention, wherein: the pH-sensitive polymer is a polymer that undergoes dissolution or conformational change at pH below 6.0, and is selected from at least one of chitosan, sodium alginate, and Eudragit® series polymers.

[0010] As a preferred embodiment of the lipopeptide substance for preventing and controlling walnut rot according to the present invention, wherein: the nanocarrier core is formed by an amphiphilic block copolymer, and the lipopeptide substance is encapsulated or embedded in its hydrophobic core.

[0011] As a preferred embodiment of the lipopeptide substance for preventing and controlling walnut rot according to the present invention, wherein: the hydrophilic segment of the amphiphilic block copolymer is connected with a quaternary ammonium salt group or a polyethylene glycol chain.

[0012] As a preferred embodiment of the lipopeptide substance for preventing and controlling walnut rot according to the present invention, the average particle size of the lipopeptide substance is 50 nanometers to 200 nanometers.

[0013] Secondly, to further solve the above-mentioned technical problems, the present invention provides a method for preparing lipopeptide substances for preventing and treating walnut rot, comprising: preparing a nanocarrier core loaded with the lipopeptide substances; and coating the nanocarrier core with a responsive material shell to obtain a core-shell structured nanocomposition.

[0014] As a preferred embodiment of the method for preparing lipopeptide substances for preventing and controlling walnut rot according to the present invention, the nanocarrier core is formed by emulsification-solvent diffusion method, nanoprecipitation method or high pressure homogenization method, and is mixed and loaded with the lipopeptide substances during or after the formation process.

[0015] In a preferred embodiment of the method for preparing a lipopeptide substance for preventing and treating walnut rot according to the present invention, the responsive material shell is coated by ionogel method, layer-by-layer self-assembly method or spray drying method.

[0016] As a preferred embodiment of the method for preparing lipopeptide substances for preventing and controlling walnut rot according to the present invention, the method involves: preparing a nanocarrier core loaded with lipopeptides using an emulsification-solvent evaporation method or a thin film hydration method, and then coating the responsive material shell with the core core through electrostatic adsorption or cross-linking reaction.

[0017] The beneficial effects of this invention are as follows: By constructing a "core-shell structure" intelligent nano-drug delivery system, this invention achieves precise and efficient prevention and control of walnut rot disease. Utilizing the shell, which has dual responsiveness to the disease microenvironment (pH<6.0 and pectinase), it enables targeted and on-demand release of lipopeptides at the lesion site, greatly improving drug utilization and extending the duration of efficacy. At the same time, the nanocarrier core effectively protects the activity of lipopeptides, and its optimized particle size of 50-200 nanometers and surface modification promote drug delivery and accumulation within the tree. This technology ultimately achieves the comprehensive goals of reduced application, increased efficacy, delayed resistance development, and environmental friendliness. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a flowchart illustrating the implementation of the present invention in Example 1. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Example 1 Reference Figure 1 This is the first embodiment of the present invention, which provides a lipopeptide substance for preventing and treating walnut rot, comprising the following steps: S1: Nanocarrier core loaded with lipopeptides.

[0023] Preferably, the nanocarrier core is formed of an amphiphilic block copolymer, and lipopeptides are encapsulated or embedded in its hydrophobic core.

[0024] Furthermore, the hydrophilic segments of the amphiphilic block copolymer are attached with quaternary ammonium salt groups or polyethylene glycol chains.

[0025] It should be noted that quaternary ammonium groups can enhance the interaction with the negatively charged surface of plant xylem and promote conduction; polyethylene glycol chains can provide an "invisibility" effect and reduce non-specific adsorption.

[0026] Furthermore, the average particle size of lipopeptides ranges from 50 nanometers to 200 nanometers.

[0027] It should be noted that when the average particle size is less than 50 nanometers, the drug loading is significantly reduced; when it is greater than 200 nanometers, it is easy to settle in the viscous medium simulating plant xylem sap, and it is not conducive to the transport after injection into the tree. Therefore, a particle size range of 50-200 nanometers is selected to balance drug loading, stability and in vivo delivery efficiency.

[0028] Preferably, a nanocarrier core based on amphiphilic block copolymers is constructed. By encapsulating lipopeptides in its hydrophobic core, effective physical shielding and stable fixation of lipopeptide active molecules are achieved, significantly enhancing their ability to resist environmental degradation. Functional modification of hydrophilic segments (such as PEGylation or quaternization) further optimizes the surface properties of the particles, respectively prolonging the in vivo circulation time or enhancing the affinity with target tissues. The particle size is precisely controlled in the range of 50-200 nanometers. This is a creative optimization achieved based on systematic experiments. It cleverly balances the requirements of drug loading, colloidal stability and efficient transport in the plant vascular system, thereby overcoming the key problem of low transport efficiency of bioactive substances in plants and laying a physical basis for subsequent targeted action.

[0029] S2: and the responsive material shell covering the core of the carrier.

[0030] Preferably, the responsive material shell is capable of degradation in the microenvironment of the diseased parts of the walnut tree, the microenvironment being characterized by a pH value below 6.0 and the presence of pectinase activity.

[0031] Specifically, the responsive material shell contains pectin or its derivatives, as well as a pH-sensitive polymer.

[0032] Furthermore, the pH-sensitive polymer is a polymer that undergoes dissolution or conformational change at a pH below 6.0, and is selected from at least one of chitosan, sodium alginate, and Eudragit® series polymers.

[0033] It should be noted that Eudragit® is a pharmaceutical-grade acrylic resin.

[0034] Preferably, the nanocore is coated with a composite shell composed of pectin and pH-sensitive polymers, which creatively introduces an intelligent control mechanism with dual signal responses of "pH and pathogen enzymes". This design establishes a direct link between drug release behavior and disease activity: the shell will only undergo synergistic degradation in the weakly acidic microenvironment and rich in pectinase unique to walnut rot lesions. This triggering method based on "AND" logic gates enables precise on-demand drug release in space (lesion site) and time (disease active period), which greatly improves drug utilization and treatment specificity. The resulting high local drug concentration can effectively impact the established pathogen colony, while avoiding unnecessary exposure in healthy tissue. This is the core technology for achieving reduced application and increased efficacy and delaying the development of drug resistance.

[0035] For example, 100 mg of PLGA-PEG (50:50, molecular weight 15 kDa) and 20 mg of lipopeptide (itursin, purity >95%) were weighed and dissolved in 5 mL of dichloromethane… The above organic phase was injected into 40 mL of aqueous phase containing 1% (w / v) polyvinyl alcohol, and sheared at 10,000 rpm for 2 minutes in an ice bath… The resulting emulsion was subjected to magnetic stirring (500 rpm) to evaporate the organic solvent for 4 hours… The particles were collected by centrifugation (15,000 rpm, 20 min), washed three times with deionized water, and the nanocarrier core was obtained with an average particle size of (152±12) nm (determined by dynamic light scattering), a polydispersity index (PDI) of 0.18, and a lipopeptide encapsulation efficiency of (88.5±2.3)%.

[0036] In summary, this invention achieves precise and efficient control of walnut rot disease by constructing a "core-shell structure" intelligent nano-drug delivery system. Utilizing the shell, which has dual responsiveness to the disease microenvironment (pH < 6.0 and pectinase), it enables targeted and on-demand release of lipopeptides at the lesion site, significantly improving drug utilization and extending the duration of efficacy. Simultaneously, the nanocarrier core effectively protects the activity of the lipopeptides, and its optimized particle size of 50-200 nanometers and surface modification promote drug delivery and accumulation within the tree. This technology ultimately achieves the comprehensive goals of reduced application, increased efficacy, delayed resistance development, and environmental friendliness.

[0037] Example 2, an embodiment of the present invention, provides a method for preparing lipopeptide substances for preventing and treating walnut rot, comprising: preparing a nanocarrier core loaded with lipopeptide substances; By coating a responsive material shell around the core of a nanocarrier, a core-shell structured nanocomposite is obtained.

[0038] It should be noted that the nanocarrier core is formed by emulsification-solvent diffusion, nanoprecipitation, or high-pressure homogenization, and is mixed and loaded with lipopeptide substances during or after the formation process.

[0039] Specifically, the responsive material shell is encapsulated using ionogel, layer-by-layer self-assembly, or spray drying methods.

[0040] Furthermore, nanocarrier cores loaded with lipopeptides are prepared using emulsification-solvent evaporation or thin-film hydration methods, and then coated with responsive material shells via electrostatic adsorption or cross-linking reactions.

[0041] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A lipopeptide substance for preventing and treating walnut rot, characterized in that: include: Nanocarrier core loaded with lipopeptides; And a responsive material shell covering the core of the carrier; The responsive material shell can degrade in the microenvironment of diseased parts of walnut trees, and the microenvironment features include a pH value below 6.0 and the presence of pectinase activity.

2. The lipopeptide substance for preventing and treating walnut rot as described in claim 1, characterized in that: The responsive material shell comprises pectin or a derivative thereof, and a pH-sensitive polymer.

3. A lipopeptide substance for preventing and treating walnut rot as described in claim 2, characterized in that: The pH-sensitive polymer is a polymer that undergoes dissolution or conformational change at pH below 6.0, and is selected from at least one of chitosan, sodium alginate, and Eudragit® series polymers.

4. A lipopeptide substance for preventing and treating walnut rot as described in claim 1, characterized in that: The nanocarrier core is formed of an amphiphilic block copolymer, and the lipopeptide substances are encapsulated or embedded in its hydrophobic core.

5. A lipopeptide substance for preventing and treating walnut rot as described in claim 4, characterized in that: The hydrophilic block copolymer has quaternary ammonium salt groups or polyethylene glycol chains attached to its hydrophilic segments.

6. A lipopeptide substance for preventing and treating walnut rot as described in claim 1, characterized in that: The average particle size of the lipopeptides is 50 nanometers to 200 nanometers.

7. A method for preparing a lipopeptide substance for preventing and controlling walnut rot, based on the lipopeptide substance for preventing and controlling walnut rot as described in any one of claims 1 to 7, characterized in that: include, Prepare a nanocarrier core loaded with the lipopeptide substances; The responsive material shell is coated around the core of the nanocarrier to obtain a core-shell structured nanocomposite.

8. A method for preparing a lipopeptide substance for preventing and treating walnut rot as described in claim 7, characterized in that: The nanocarrier core is formed by emulsification-solvent diffusion, nanoprecipitation, or high-pressure homogenization, and is mixed and loaded with the lipopeptide substance during or after the formation process.

9. A method for preparing a lipopeptide substance for preventing and treating walnut rot as described in claim 7, characterized in that: The responsive material shell is encapsulated using ionogel, layer-by-layer self-assembly, or spray drying methods.

10. A method for preparing a lipopeptide substance for preventing and treating walnut rot as described in claim 7, characterized in that: The nanocarrier core loaded with lipopeptides is prepared by emulsification-solvent evaporation or thin film hydration, and then the responsive material shell is coated by electrostatic adsorption or cross-linking reaction.

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