Microcapsule for biological control of soybean cyst nematode to induce immune response of soybean

Abstract

The application relates to the field of biological control, and discloses a root-knot nematode biocontrol microcapsule for inducing soybean immune response. The microcapsule is a concentric spherical core-shell structure, and sequentially comprises an active core layer loaded with a nematicidal metabolite, an intermediate barrier layer composed of PLGA, a biocontrol bacteria carrier layer embedded with biocontrol bacteria spores, and a functional protective layer with pH response and light avoidance functions. Through the above structure, the application realizes rapid directional release of the nematicidal component and long-acting slow release of the biocontrol bacteria, builds a composite defense system composed of exogenous killing and endogenous immunity in the soybean rhizosphere, and improves the biological control efficiency and the plant stress resistance level.

Description

Technical Field

[0001] This invention belongs to the field of biological control, specifically relating to a root-knot nematode biocontrol microcapsule that induces an immune response in soybeans. Background Technology

[0002] Root-knot nematode infestation is one of the core biological stress factors currently restricting high and stable soybean yields. Root-knot nematodes pierce soybean roots with their specialized needle-like stylets, injecting various effector proteins into the host plant while feeding. This induces abnormal physiological transformations in root cells, forming typical giant cells and root knot structures, disrupting the anatomical structure of the root system, blocking the normal transport of water and mineral nutrients, and causing severe stunted plant growth, manifested in stunted plants, yellowing leaves, and flower and fruit drop in the field.

[0003] To control soybean root-knot nematodes, biocontrol strains, represented by Bacillus, can produce secondary metabolites with direct nematicidal activity during fermentation and metabolism, such as volatile organic compounds, antimicrobial proteins, and lipopeptides. These metabolites reduce nematode infection pressure by damaging the nematode body wall, inhibiting egg hatching, or interfering with chemotactic localization. Simultaneously, colonization in the plant rhizosphere can stimulate soybeans to develop induced systemic resistance. By regulating signaling pathways such as salicylic acid and jasmonic acid, they enhance the physical strength of plant cell walls and alter the chemical composition of root exudates, thereby constructing an endogenous immune barrier against nematode invasion.

[0004] However, the nematicidal active metabolites secreted by biocontrol bacteria are highly susceptible to soil pH fluctuations, microbial degradation, and ultraviolet radiation from sunlight, resulting in an extremely short effective half-life in the soil. This means that after the pesticide is applied to the soil, its active ingredients are degraded and inactivated before the nematodes enter their susceptible window, making it difficult to achieve a long-lasting and stable pest control effect.

[0005] Relying solely on immune induction mechanisms has a lag effect in early soybean growth stages or in severely diseased fields with high nematode pressure. The establishment of induced resistance requires physiological processes of signal transduction and resistance protein expression. Without timely exogenous nematode control, soybean roots may suffer large-scale nematode infection before the immune barrier is fully established, causing the beneficial effect of immune induction to be offset by severe physical damage in the early stages. Summary of the Invention

[0006] To address the technical challenges of existing biocontrol agents in controlling soybean root-knot nematodes, such as delayed efficacy due to single-immune induction, poor environmental stability of biocontrol bacterial metabolites, and a mismatch between the release rhythm of active ingredients and the nematode infection pattern, this invention provides a root-knot nematode biocontrol microcapsule that induces an immune response in soybean. The microcapsule, through a core-shell structure with multi-level response characteristics, achieves rapid and targeted release of nematicidal active metabolites and long-term sustained release of biocontrol bacterial spores, thereby constructing a composite defense system in the soybean rhizosphere consisting of exogenous killing and endogenous immunity.

[0007] The present invention discloses a root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans. The microcapsule is characterized by a regular concentric spherical structure, comprising, from the inside out, an active core layer, an intermediate barrier layer, a biocontrol bacteria carrier layer, and a functional protective layer. The overall particle size of the microcapsule is distributed between 50 μm and 120 μm, with the diameter of the active core layer accounting for 40% to 60% of the total particle size. The active core layer is composed of a porous hydrophobic mineral carrier adsorbing a high concentration of nematicidal active metabolites; the intermediate barrier layer is composed of a biodegradable polymer material, used to physically isolate the core layer from the outer components; the biocontrol bacteria carrier layer encapsulates highly active biocontrol bacteria spores and their nutrient matrix; and the functional protective layer is composed of a polymer material with pH-responsive properties and light barrier function.

[0008] The porous hydrophobic mineral support in the active core layer is made of modified diatomaceous earth or modified montmorillonite. The modification process involves organically treating the mineral surface with long-chain alkyl quaternary ammonium salts, resulting in a pore size distribution between 20 nm and 100 nm and a specific surface area of ​​150 m². 2 / g to 300m 2 / g. The nematicidal active metabolite is loaded within the micropores of the mineral carrier, and its components include volatile organic compounds, lipopeptide antibiotics, and antimicrobial proteins. The volatile organic compounds are a mixture of dimethyl disulfide, 2-nonanone, and decanal in a mass ratio of 3:1:1; the lipopeptide antibiotics include surfactants, fentanyl, and ituronin, with a total content of 15% to 25% of the total mass of the core layer; the antimicrobial protein is a serine protease secreted by biocontrol strains, capable of degrading nematode body wall collagen, with an enzyme activity higher than 5000 U / g.

[0009] The active core layer also contains a chemotactic inducer, which is a mixed solution of L-glutamic acid and malic acid at a concentration of 0.05% to 0.15%. This chemotactic inducer utilizes the osmotic pressure difference inside and outside the microcapsule to diffuse outward through the micropores of the functional protective layer after being applied to the soil, mimicking the chemical signals of soybean root exudates. This induces secondary larvae of root-knot nematodes to aggregate towards the microcapsules, increasing the contact probability and killing efficiency of the nematicidal metabolites.

[0010] The intermediate barrier layer is composed of PLGA with a lactide to glycolide molar ratio of 75:25 and a molecular weight distribution ranging from 20kDa to 50kDa. The thickness of the intermediate barrier layer is 2μm to 5μm, and its function is to prevent volatile organic compounds in the active core layer from seeping outwards during storage, and to ensure that the core components can be released in a pulsed manner through the initial hydrolysis of the PLGA layer during the initial application to the soil.

[0011] The biocontrol bacterial carrier layer uses a cross-linked network of sodium alginate and chitosan as its framework, and uniformly embeds biocontrol bacterial spores within it. The biocontrol bacterial spores are Bacillus spores that have undergone induced sporulation treatment, and their viable count within the microcapsules is higher than 1.0 × 10⁻⁶. 10 CFU / g. The sodium alginate has a mass fraction of 2.0% to 3.5%, and the chitosan has a mass fraction of 1.0% to 2.0%, which are cross-linked by calcium ions to form a gel matrix with a three-dimensional network structure. This matrix is ​​also formulated with 1% to 3% potassium humate and 0.5% to 1% yeast extract as a nutrient source during the initial germination of the biocontrol bacteria. The degradation rate of the biocontrol bacteria carrier layer is designed for slow release in the soybean rhizosphere environment (pH 5.5 to 6.5), with a complete release period of 15 to 30 days, aiming to ensure continuous colonization of the biocontrol bacteria during the root growth of soybean seedlings.

[0012] The functional protective layer is composed of polymethyl methacrylate resin, nano-titanium dioxide, and nano-carbon black. The polymethyl methacrylate resin serves as the film-forming matrix and is pH sensitive, maintaining structural integrity under neutral or slightly acidic soil conditions while increasing permeability in locally acidic environments induced by root exudates. The nano-titanium dioxide and nano-carbon black are added at 0.5% and 1.2% of the polymer mass, respectively, to absorb and reflect ultraviolet light, protecting the internal biocontrol spores and antimicrobial proteins from inactivation by sunlight.

[0013] The present invention discloses a root-knot nematode biocontrol microcapsule that induces an immune response in soybeans. Its preparation method employs a stepwise microemulsion cross-linking method combined with layer-by-layer self-assembly technology. The specific steps are as follows:

[0014] The first step, core loading: The modified hydrophobic mineral carrier is placed in a mixed solution containing volatile organic compounds, lipopeptides and antimicrobial proteins, and physical adsorption is carried out under a vacuum atmosphere. Then, the residual liquid is removed by centrifugation to obtain a dry powder loaded with metabolites. The second step is the intermediate layer coating: the above dry powder is dispersed in a dichloromethane solution containing PLGA, and a primary microsphere with an intermediate barrier layer is formed by spray drying. The third step is the construction of the biocontrol layer: the primary microspheres are suspended in an aqueous solution containing biocontrol spores, nutrient matrix and sodium alginate, and the suspension is dripped into a calcium chloride solution through a precision extrusion device for hardening and solidification to form secondary microspheres; The fourth step is the modification of the protective layer: the secondary microspheres are placed in a fluidized bed containing chitosan solution, and the chitosan solution and polymethyl methacrylate resin dispersion are sprayed alternately. By utilizing the principle of charge interaction and solvent evaporation to form a film, a functional protective layer is formed on the surface of the microspheres. Step 5, post-processing: The formed microcapsules are vacuum freeze-dried at a low temperature of 35℃ to 40℃ to control the moisture content to be below 5%, and then packaged for later use.

[0015] The working logic of the microcapsules described in this invention in the control of soybean root-knot nematodes is as follows: On the first to third days after application to the soil (i.e., the initial stage of application), water penetrates through the functional protective layer, causing local swelling and degradation of the intermediate barrier layer PLGA. At this time, the high concentration of volatile organic compounds in the active core layer rapidly diffuses outward, forming a high-concentration biotoxic zone around the microcapsules. Due to the extremely strong penetrating power of components such as dimethyl disulfide, they can directly destroy the neural conduction system of secondary nematode larvae in the soil pores, causing them to lose their ability to move and eventually die. At the same time, the release of chemotactic inducers further attracts surrounding nematodes to approach the microcapsules, enhancing the initial mechanical killing effect.

[0016] Between the 3rd and 7th day after application to the soil (the peak infection period), lipopeptide antibiotics and antimicrobial proteins in the active core layer begin to be released on a large scale through the weakened intermediate layer. Lipopeptides, acting as biosurfactants, specifically adsorb onto the surface of nematode eggshells, altering shell permeability and causing osmotic pressure imbalance within the embryo. Serine proteases in the antimicrobial proteins degrade collagen, the main component of the nematode's body wall, disrupting its physical barrier and reducing its resistance to environmental stress and native predators in the soil. This stage of release primarily aims to reduce the number of nematode egg sacs and the initial number of hatching larvae in the soil.

[0017] Between 7 and 25 days after application to the soil (i.e., the root establishment and colonization period), as the sodium alginate-chitosan network of the biocontrol bacteria carrier layer slowly disintegrates, the biocontrol spores come into contact with rhizosphere exudates and begin to germinate in situ. Due to the presence of the nutrient substrate (potassium humate, yeast extract), the biocontrol bacteria can proliferate rapidly in the absence of competing rhizosphere sites. After germination, the biocontrol mycelia spread along the soybean root surface and form a stable biofilm. During colonization, the biocontrol bacteria, on the one hand, inhibit the near-root activity of residual nematodes by secreting secondary metabolites such as heparin and antibiotics; on the other hand, the colonization behavior of the biocontrol bacteria, as a typical biological signal, is recognized by pattern recognition receptors in soybean root epidermal cells, thereby activating the induced systemic resistance in soybeans.

[0018] The induced resistance system manifests as follows: colonization by biocontrol bacteria induces cross-talk between the salicylic acid and jasmonic acid / ethylene signaling pathways within soybean roots. By upregulating the gene expression levels of phenylalanine ammonia-lyase (PAL), polyphenol oxidase, and peroxidase, it promotes the accumulation of lignin and secondary metabolites (such as soybean isoflavones and phytoalexins) in soybean root tissues. The increase in lignin strengthens the physical strength of root cell walls, making it difficult for nematode stylets to penetrate; while changes in allelopathic substances reduce the attraction of root exudates to nematodes, and even produce a repellent effect. This internal-to-external immune enhancement allows soybean plants to limit the formation of giant cells, inhibit nematode development and maturation, and reduce root knot formation rate even when infested with a small number of residual nematodes.

[0019] The technological advantage of the microcapsules described in this invention lies in the fact that their engineered release rhythm is highly compatible with the biological characteristics of soybean root-knot nematodes. Through a three-layered spatial functional partitioning structure, this invention solves the problem of nematicide metabolites being easily adsorbed and inactivated by soil. The porous mineral carrier's physical clamping effect on the metabolites reduces their volatilization rate, while the nanomaterial barrier in the functional protective layer blocks environmental factors from damaging the bioactive macromolecules. Simultaneously, this invention avoids the inhibitory interference of high-concentration metabolites on the colonization of biocontrol bacteria. Through the time-delaying effect of the intermediate barrier layer, the chemical killing process precedes the biological colonization process, creating an environment of low nematode pressure and low microbial competition for the successful colonization of biocontrol bacteria in the rhizosphere.

[0020] The microcapsules described in this invention have extremely high applicability. Due to their precisely controlled particle size, they can be adapted to existing mechanical sowing and fertilization systems, enabling simultaneous sowing and application of pesticides. Under soil moisture conditions, the physicochemical properties of the microcapsules remain stable, and the encapsulated biocontrol spores have a shelf life of over 12 months in a dormant state. The microcapsules ultimately degrade completely in the soil into inorganic mineral particles and small organic molecules that can be utilized by microorganisms, without producing any environmental pollution residues.

[0021] In the engineered control of soybean root-knot nematodes, the microcapsules described in this invention can maintain the root-knot nematode population density below the economic threshold during the critical 20-day protection period after soybean germination, and guide soybean plants to establish a lasting immune memory. This synergistic mode of rapid physicochemical killing and sustained biological colonization induction, compared with single biological agents, enhances the control efficacy in fields with continuous cropping, while also promoting the growth of soybean root biomass and improving the plant's absorption efficiency of mineral elements such as nitrogen and phosphorus.

[0022] To ensure the migration and colonization efficiency of microcapsules in soils of different textures, a small number of PEG molecular chains are grafted onto the outer surface of the functional protective layer through ion exchange. The molecular weight of the PEG chains is 2000 Da to 4000 Da, and the grafting density is controlled at 100 to 500 chains per square micrometer. This hydrophilic modification reduces the non-specific adsorption of microcapsules by soil colloidal particles, allowing the microcapsules to migrate moderately vertically and horizontally in the soil capillary pores with irrigation water or rainwater, thereby achieving a spatially uniform distribution of active ingredients in the soybean rhizosphere.

[0023] The screening and processing technology of the biocontrol spores described in this invention has been specially optimized. During fermentation, by controlling the carbon-nitrogen ratio to 15:1 to 20:1 and adding 0.5 mmol / L manganese ions as an inducing factor, the stress resistance of the spores and their germination rate in the soil are improved. The selected Bacillus strains can produce specific cyclic lipopeptides after colonization. These lipopeptides not only have direct nematode-killing effects but also act as signaling molecules to enhance plasmodesmata restriction in soybean root cortex cells, blocking the diffusion of effector proteins secreted by nematodes between plant cells.

[0024] The antimicrobial protein in the active core layer of this invention employs a microencapsulation protection scheme. Before being loaded onto a mineral carrier, the protein molecules are pre-encapsulated with a layer of nano-sized ethyl cellulose to prevent direct contact and degradation with proteases in the soil. This process ensures that the antimicrobial protein retains its biocatalytic activity against nematode collagen after being released into the soil, extending its effective period from the traditional 24 hours to more than 7 days.

[0025] The biocontrol microcapsules of this invention achieve a self-sealing effect on the microcapsule surface by adjusting the temperature curve of the fluidized bed drying process. In the final stage of drying, the polymer of the functional protective layer undergoes a slight glass transition, forming a dense, continuous film approximately 50 nm thick on the microcapsule surface, further enhancing the ability to lock in internal volatile components. The water absorption and swelling coefficient of this film after entering the soil is precisely calibrated to ensure that controllable microcracks are generated when the water absorption rate reaches 30%, thereby initiating subsequent chemical signal transduction and component release.

[0026] Targeting the physiological characteristics of soybean roots, the biocontrol microcapsules of this invention also integrate trace element regulation functions. In the biocontrol bacteria carrier layer, chelated trace element particles such as zinc, manganese, and boron are additionally and uniformly dispersed. These elements are slowly released during the colonization of the biocontrol bacteria, participating as cofactors in the synthesis of defense enzymes within the soybean plant. For example, zinc ions can promote the synthesis of tryptophan in soybeans, thereby regulating auxin levels and assisting in the repair of damaged root tissues; manganese ions, as peroxidase activators, directly participate in the biosynthesis of lignin. This integrated nutrition-defense design allows infected soybean plants to recover their growth vitality more quickly.

[0027] The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans described in this invention has undergone rigorous matching tests on the physicochemical properties of its components. The hydrophobicity of the active core layer carrier and the oleophobicity of the intermediate PLGA layer complement each other, ensuring the physical stability of the oil phase metabolites and aqueous bacterial solution during the production process. The molecular weight distribution of the polymethyl methacrylate resin in the functional protective layer is controlled within 1.2 to obtain a consistent pore opening threshold.

[0028] The microcapsules described in this invention are specifically optimized for the release flux of metabolites, targeting the unique infection habits of soybean root-knot nematodes. Nematode larvae are typically active when soil temperature and humidity are suitable (e.g., 20℃-28℃, soil moisture 60%-80%), and the chitosan-sodium alginate cross-linking system used in this invention is sensitive to temperature and humidity. When soil temperature rises and moisture is sufficient, the swelling pressure of the microcapsules increases, accelerating the release rate of the nematicidal components. This achieves synchronization between pesticide release and peak nematode activity, reducing pesticide waste and improving the economic efficiency of biological control.

[0029] In a preferred embodiment of the present invention, the biocontrol bacteria carrier layer further contains a biosurfactant enhancing factor, which is rhamnolipin at a mass fraction of 0.1%. The rhamnolipin can reduce the interfacial tension between the biocontrol bacteria spores and the soybean root surface, promoting rapid attachment of the strain to key infection sites such as the root hair zone and meristematic zone of soybean roots. This directional attachment mechanism ensures that the immune induction signal can be transmitted to the stele tissue of the plant root via the shortest path, shortening the induction period of the immune response.

[0030] In another preferred embodiment of the present invention, the outer side of the functional protective layer is further coated with a positively charged nano-sized humic acid film through electrostatic adsorption. This humic acid film not only enhances the chemical stability of the microcapsules in the soil, but also acts as a plant growth regulator in the early stages of soybean seed germination, directly promoting the root development of the taproot. This allows the soybean root system to cross the nematode-prone soil layer earlier, achieving pest-avoidance growth in a spatial dimension.

[0031] To further enhance the effectiveness of microcapsules in arid or semi-arid regions, the functional protective layer is also doped with 3% to 5% by mass of cross-linked polyacrylamide superabsorbent polymer (SAP) powder. These powders have a particle size of less than 5 μm and are uniformly distributed within the polymer matrix. Under drought stress, these superabsorbent powders retain localized moisture, providing a micro-humid environment for the internal biocontrol spores and slowing their inactivation rate. Simultaneously, when rainfall or irrigation occurs, the superabsorbent powders rapidly expand, acting as a physical pump to propel the internal active ingredients into the arid soil.

[0032] The root-knot nematode biocontrol microcapsule of this invention, which induces an immune response in soybeans, exhibits a synergistic effect between its inner layer metabolites and outer layer biocontrol bacteria through secondary transformation of metabolites. During their growth, the biocontrol bacteria colonizing the rhizosphere utilize some residual organic matter released from the core layer as a carbon source, transforming it into more active and polar secondary metabolites through their own metabolic pathways. This biotransformation process forms a relay-like defense chain over time, ensuring that even after the core active ingredient is depleted, the microcapsule can maintain its control pressure through the continuous metabolism of the biocontrol bacteria.

[0033] The physical strength of the microcapsules described in this invention has also been precisely designed. Their compressive strength is maintained between 2.0 MPa and 5.0 MPa. This strength ensures that the microcapsules do not break under the pressure during mechanized sowing, while also ensuring that they undergo structural disintegration under root pressure and microbial enzymatic hydrolysis in the soil, thereby releasing their internal functional components. This balance of strength is the technical foundation for achieving fully automated and efficient pesticide application in soybean fields.

[0034] In actual production, to improve the visual recognition and quality traceability of microcapsules, an edible-grade tracer dye, such as sodium copper chlorophyllin, can be added to the functional protective layer. This dye provides color identification and also acts as a secondary photosensitive oxidation inhibitor, capturing free radicals through the porphyrin ring in its molecular structure, further protecting the chemical stability of the internal biological components.

[0035] To address the potential for reactive oxygen species (ROS) bursts during soybean root growth, a natural antioxidant complex is incorporated into the biocontrol bacteria carrier layer. This complex consists of tea polyphenols and vitamin E acetate in a 2:1 ratio. This complex neutralizes oxidative stress induced by nematode infestation while protecting the biocontrol bacteria's cell membrane system from oxidative damage, thereby enhancing the biocontrol bacteria's survival ability and competitive advantage in the rhizosphere of infected plants.

[0036] In the preparation process of the microcapsules described in this invention, the uniformity of the coating thickness of the intermediate barrier layer (PLGA) is crucial for achieving programmed release. This invention employs high-speed centrifugal atomization combined with fluidized bed coating technology. By dynamically adjusting the spraying speed of the coating solution through real-time monitoring of exhaust air temperature and humidity, the thickness error of the barrier layer for each microcapsule is ensured to be controlled within ±0.2 μm. This high-precision process control guarantees a high degree of consistency in the release behavior of the microcapsules in field applications, avoiding localized phytotoxicity or periods of reduced efficacy due to release rhythm disruptions.

[0037] Compared with the prior art, the beneficial effects of the present invention are: This invention addresses the problem of biocontrol bacteria metabolites, such as lipopeptides and volatile organic compounds, being easily immobilized and deactivated by clay minerals in soil through multi-layered chemical and biological barriers via biocontrol microcapsules. The microenvironment within the microcapsules is regulated by a pH buffer (such as potassium dihydrogen phosphate) to maintain the most stable physiological state for the biocontrol bacteria spores. As the microcapsules gradually degrade with soybean growth, the sodium alginate and chitosan in their carrier materials, acting as biopolysaccharides, further improve the physical aggregate structure of the rhizosphere soil, enhancing its water and fertilizer retention capacity and providing a favorable physical environment for soybean growth recovery. This invention reduces the damage caused by soybean root-knot nematodes, improves the overall stress resistance of soybean plants, and ensures oilseed safety and ecological health. Detailed Implementation

[0038] This invention provides a root-knot nematode biocontrol microcapsule that induces an immune response in soybeans. Structurally, the microcapsule presents a highly ordered concentric spherical multi-layered system. Through precise control of the physicochemical properties of each layer, the spatiotemporal controlled release of the active ingredient within the soybean rhizosphere environment is achieved.

[0039] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0040] Example 1: Active core layer (modified diatomaceous earth carrier, pore size 50nm, specific surface area 220m²) 2 / g; The nematicide metabolite contains dimethyl disulfide: 2-nonanone: decanal = 3:1:1, 20% lipopeptide antibiotics, and 5500 U / g antimicrobial protease activity. (Chemical chemotaxis inducer 0.1% L-glutamic acid + malic acid mixture); Intermediate barrier layer (PLGA lactide: glycolide = 75:25, weight average molecular weight 35kDa, thickness 3.5μm); Biocontrol carrier layer (sodium alginate 2.8%, chitosan 1.5%, Bacillus spores 1.2×10⁻⁶) 10CFU / g, potassium humate 2%, yeast extract 0.8%, rhamnolipid 0.1%, tea polyphenols: vitamin E acetate = 2:1, chelated zinc manganese boron granules). Functional protective layer (polymethyl methacrylate resin, nano titanium dioxide 0.5%, nano carbon black 1.2%, PEG2000 grafted, nano humic acid film, cross-linked polyacrylamide water-absorbing resin 4%). The total particle size of the microcapsules is 80 μm, and the diameter of the active core layer accounts for 50%. Preparation steps: S1: Core loading, modified diatomaceous earth is placed in a mixed solution of nematicide metabolites, vacuum adsorption at -0.09MPa for 5 hours, centrifugation is used to remove the residual liquid, and the loaded dry powder is obtained; S2: Intermediate layer coating, dry powder dispersed in PLGA dichloromethane solution, spray dried (air inlet 48℃) to form primary microspheres; S3: Construction of biocontrol microbial layer: Primary microspheres are suspended in an aqueous solution containing biocontrol microbial spores, nutrient matrix and sodium alginate, and then precisely squeezed and dripped into calcium chloride solution to harden, forming secondary microspheres; S4: Protective layer modification, secondary microspheres are placed in a fluidized bed, and chitosan solution, polymethyl methacrylate resin dispersion and functional modification component solution are sprayed alternately. The final drying stage is heated to the glass transition temperature of the resin to form a sealed film. S5: Post-processing, vacuum freeze-drying to 4% moisture content, to obtain the finished microcapsules.

[0041] Example 2: The total particle size of the microcapsules is 50 μm, the diameter of the active core layer accounts for 40%, and the remaining components and proportions are the same as in Example 1; Preparation steps: Adjust the spray drying and extrusion parameters, and the remaining steps are the same as in Example 1.

[0042] Example 3: The total particle size of the microcapsules is 120 μm, the diameter of the active core layer accounts for 60%, and the remaining components and proportions are the same as in Example 1; Preparation steps: Adjust the spray drying and extrusion parameters, and the remaining steps are the same as in Example 1.

[0043] Example 4: The active core layer contains 15% lipopeptide antibiotics, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0044] Example 5: The active core layer contains 25% lipopeptide antibiotics, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0045] Example 6: The sodium alginate content in the biocontrol bacteria carrier layer is 2.0%, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0046] Example 7: The sodium alginate content in the biocontrol bacteria carrier layer is 3.5%, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0047] Example 8: The cross-linked polyacrylamide water-absorbing resin content in the functional protective layer is 3%, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0048] Comparative Example 1: The active core layer contains only modified diatomaceous earth and chemical chemotactic inducers, without nematicide metabolites, and the other components are the same as in Example 1; Preparation steps: In the core loading stage, only chemotactic inducers are adsorbed, and the remaining process parameters and steps are the same as in Example 1.

[0049] Comparative Example 2: The biocontrol carrier layer was removed, and a functional protective layer was directly coated on the outside of the intermediate barrier layer. The remaining components were the same as in Example 1. Preparation steps: The biocontrol layer construction step is omitted, and the primary microspheres are directly modified with a protective layer. The remaining process parameters and steps are the same as in Example 1.

[0050] Test method: Nematode control efficacy test: Pot experiment to determine soybean root knot formation rate, nematode egg hatching inhibition rate and secondary larval mortality rate; Immunoinduction test: Determination of phenylalanine ammonia-lyase activity in soybean roots; Biocontrol colonization test: The number of biocontrol bacteria in the soybean rhizosphere was determined 30 days after application using the plate count method. Environmental stability test: Simulate soil pH, temperature and humidity conditions to determine the retention rate of nematicide metabolites and the survival rate of biocontrol bacteria within 30 days; Storage stability test: After 12 months of storage at room temperature, the decay rate of viable bacteria and the retention rate of nematicidal activity were measured. Soybean growth index test: measure soybean plant height, root fresh weight, root dry weight and yield.

[0051] The test data comparison tables are shown in Table 1 and Table 2.

[0052] Table 1. Comparison of reduced root knot formation rate, nematode mortality rate, and 30-day colonization rate of biocontrol bacteria. Table 2 Comparison of PAL activity enhancement, 30-day metabolite retention rate, soybean yield enhancement, and viable bacteria attenuation rate after 12 months of storage. Examples 1 to 8 utilize active core layer metabolites to achieve rapid initial nematode killing, a biocontrol bacterial carrier layer to induce immune colonization with slow-release strains, and a functional protective layer to ensure stability; these three elements synergistically construct a composite defense system. Comparative Example 1, lacking nematode-killing metabolites, showed a significantly reduced initial nematode-killing effect; Comparative Example 2, lacking a biocontrol bacterial carrier layer, suffered from ineffective immune induction, resulting in decreased root knot inhibition and yield enhancement.

[0053] Microcapsules with a particle size of 80μm to 120μm, a lipopeptide content of 20% to 25%, and a sodium alginate content of 2.8% to 3.5% exhibit superior nematicidal efficacy and immune-inducing ability. Among these factors, particle size affects the release rate and migration of the active ingredient, lipopeptide content determines the nematicidal intensity, and sodium alginate content controls the release cycle of the biocontrol bacteria. These three factors work synergistically to ensure the overall performance of the microcapsules.

[0054] Compared to Comparative Example 1 without metabolites, the root knot formation rate was reduced by more than 123%, the nematode mortality rate was increased by more than 102%, and the soybean yield was increased by more than 180%. Compared to Comparative Example 2 without biocontrol layer, the root knot formation rate was reduced by more than 62%, PAL activity was increased by more than 182%, and the yield was increased by more than 87%. Furthermore, the environmental stability and storage period were extended, meeting the green control requirements for soybean root knot nematodes.

[0055] In summary, this invention achieves simultaneous enhancement of efficient nematode killing and immune induction through the construction of a multi-level core-shell structure coupled with spatiotemporal release regulation. It exhibits excellent environmental stability and application adaptability, making it suitable for the control of soybean root-knot nematodes.

[0056] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A root-knot nematode biocontrol microcapsule that induces an immune response in soybeans, characterized in that, The biocontrol microcapsule has a regular concentric spherical structure and comprises the following components from the inside out: Active core layer; Intermediate barrier layer; Biocontrol bacteria carrier layer; Functional protective layer.

2. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 1, characterized in that, The active core layer is composed of a porous hydrophobic mineral carrier that adsorbs nematicidal active metabolites and chemotactic inducers.

3. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 1, characterized in that, The intermediate barrier layer is composed of biodegradable polymer material, which is used to physically isolate the active core layer from the biocontrol carrier layer and provide pulsed release control.

4. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 1, characterized in that, The biocontrol carrier layer encapsulates biocontrol spores, a nutrient matrix, and functional auxiliary components. The biocontrol carrier layer is constructed based on a cross-linked network of sodium alginate and chitosan.

5. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 1, characterized in that, The functional protective layer is composed of a polymer material with pH-responsive properties, light barrier function, and soil migration enhancement properties, and is wrapped around the outermost surface of the biocontrol carrier layer.

6. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 1, characterized in that, The overall particle size distribution of the biocontrol microcapsules is between 50 μm and 120 μm, of which the diameter of the active core layer accounts for 40% to 60% of the total particle size.

7. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 2, characterized in that, The porous hydrophobic mineral carrier in the active core layer is modified diatomaceous earth or modified montmorillonite; the modification process of the mineral carrier is: organic treatment of the mineral surface by long-chain alkyl quaternary ammonium salt; the nematicidal active metabolites are loaded into the micropores of the mineral carrier by physical adsorption.

8. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 2, characterized in that, The nematicidal active metabolites in the active core layer include: volatile organic compounds; lipopeptide antibiotics; and antimicrobial proteins. The antimicrobial proteins are pre-encapsulated in nanoscale with ethyl cellulose before being loaded onto the mineral carrier.

9. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 8, characterized in that, The volatile organic compound is a mixture of dimethyl disulfide, 2-nonanone and decanal in a mass ratio of 3:1:

1.

10. The root-knot nematode biocontrol microcapsule for inducing an immune response in soybeans according to claim 8, characterized in that, The lipopeptide antibiotics include surfactant, fentanyl, and iturin, and the total content of the lipopeptide antibiotics accounts for 15% to 25% of the total mass of the active core layer.

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