Qinghai cold land bluegrass seed coating agent and preparation method thereof

Abstract

The invention belongs to the technical field of multifunctional composite seed coatings for ecological restoration of high and cold regions, discloses a Qinghai cold-region bluegrass seed coating agent and a preparation method thereof, and aims to solve the composite problems of component antagonism, time sequence mismatch, freeze-thaw failure and the like in the prior art. The coating agent is characterized in that a three-layer composite structure of a biological core layer, a nutrition buffer layer and a functional protection layer is sequentially coated on the surface of a seed from inside to outside. The biological core layer comprises complex microbial inoculant particles and a first adhesive; the nutrition buffer layer comprises a low-temperature germination inducer, a slow-release nutrition microcapsule and a second adhesive; the functional protection layer comprises an interpenetrating network polymer skeleton, high molecular weight sodium alginate, nano silicon dioxide particles subjected to surface modification by a silane coupling agent KH-570 and food-grade indigo dye. By means of the scheme, efficient germination and stable planting of the Qinghai cold land bluegrass seeds in the high and cold environment are achieved.

Description

Technical Field

[0001] This invention belongs to the field of multifunctional composite coating technology for seeds used in high-altitude ecological restoration. Specifically, it relates to a seed coating agent for Kentucky bluegrass in cold regions of Qinghai and its preparation method. This coating agent is particularly suitable for the treatment of special forage seeds for ecological restoration in high-altitude and cold regions such as Kentucky bluegrass in cold regions of Qinghai. Background Technology

[0002] Seed coating technology, a key component of modern agriculture and ecological engineering, aims to enhance seed vigor, resist adverse environmental stresses, control pests and diseases, and provide early nutrition by coating the seed surface with a protective film containing specific functional components, thereby ensuring sowing efficiency and healthy plant establishment. In traditional crop production, this technology is quite mature, typically using polymers as the base film-forming agent, combined with chemical fungicides, fast-acting fertilizers, and plant growth regulators, providing strong support for stable and high yields of major food crops in temperate regions. The design concept of these conventional coating agents is based on the physiological rhythms of rapid germination and growth of crops under temperate climates; its technological logic lies in providing an immediate and high-intensity protective and promoting environment.

[0003] However, with the deepening of the national strategy for ecological civilization construction, the focus of technological applications is gradually shifting to more demanding and fragile ecosystems, such as the restoration of degraded grasslands in high-altitude and cold regions like the Qinghai-Tibet Plateau and alpine meadows. In these areas, native grass species such as *Poa crymophila* are considered pioneers and core species for vegetation restoration and soil and water conservation due to their outstanding cold resistance, drought resistance, and tolerance to poor soil conditions. Their successful establishment has irreplaceable strategic significance for maintaining regional biodiversity and ensuring ecological security. However, at the same time, the inherent biological characteristics of *Poa crymophila*, such as its long seed dormancy period, low natural germination rate, slow seedling growth, and weak stress resistance, coupled with the environmental pressures unique to high-altitude and cold regions, including low temperatures, strong freeze-thaw cycles, poor soil, and frequent occurrences of specific soil-borne diseases, mean that conventional seed coating technology is not only ineffective in such scenarios but also exposes deep-seated contradictions and inherent limitations in its design principles.

[0004] The reason for this lies in the fact that existing technologies generally employ a "homogenized mixing" formulation strategy, which involves simply and physically blending various functional components into a single film-forming agent system. This approach, when addressing the complex needs of high-altitude ecosystems, triggers a series of antagonistic effects and functional mismatches. First, there is a fundamental conflict regarding the compatibility of bioactive components. To control diseases specific to high-altitude regions, such as Rhizoctonia solani, researchers have attempted to introduce beneficial microorganisms like Trichoderma and arbuscular mycorrhizal fungi into coating agents, hoping to inhibit pathogens and promote root development through biological antagonism. However, to achieve broad-spectrum fungicide effects, chemical fungicides such as carbendazim are often retained in the formulation. This "mixing" directly leads to chemical agents indiscriminately killing or inhibiting the survival and colonization of active microorganisms during the liquid-phase preparation, storage, and soil environment of the coating, rendering the initial purpose of biological control futile, and the addition of microbial agents almost entirely ineffective. Second, there is a temporal mismatch in the synergy of functional release. The low temperatures (often below 5℃) in high-altitude and cold regions significantly slow down the physiological metabolic processes of Kentucky bluegrass seeds in Qinghai. Germination promoters such as gibberellins, designed for temperate crops, exhibit sharply reduced activity at low temperatures, failing to effectively break deep dormancy. Meanwhile, the fast-acting nitrogen, phosphorus, and potassium fertilizers in the formulation release nutrients at rates far exceeding the slow absorption rate of seedlings, resulting in substantial nutrient loss into the soil before the critical germination period. This not only wastes resources but may also stress sensitive young roots due to excessively high local salt concentrations. Finally, there is a performance contradiction between the stability of the physical structure and the protective function of biological functions. To reduce costs, traditional coatings often use simple polymers or mineral carriers such as bentonite. The resulting coating layer is highly susceptible to cracking and peeling under the repeated freeze-thaw cycles caused by the drastic diurnal temperature variations in high-altitude regions, thus losing its physical protection for the seeds. Premature destruction of this structure exposes the already fragile bioactive components (such as microbial agents and enzymes) that have not yet played a role to environments such as ultraviolet light, dryness, and extreme temperatures, leading to their rapid inactivation.

[0005] Therefore, the core issue is no longer simply finding a single, more cold-resistant alternative, but rather that existing seed coating technologies, based on a "homogenized, monomeric" design philosophy, cannot provide a spatiotemporally ordered and functionally synergistic microenvironment for diverse active components with varying functions and conflicting needs (such as living microorganisms requiring isolation and protection, nutrients requiring slow-release at low temperatures, and germination signaling molecules requiring immediate activation). This inherent structural deficiency leads to mutual constraints among components, and the overall efficiency is far from a simple additive sum of individual components, instead exhibiting systemic failure. Therefore, how to break through the framework of traditional homogenized coating and construct a novel coating system with multi-layered, heterogeneous structures possessing functional zoning, temporal release, and environmental response characteristics, enabling spatial isolation and temporal synergy among functional components, and solving the complex challenges of germination and establishment of Kentucky bluegrass in high-altitude and cold environments using a systems engineering approach, has become a key challenge and an urgent technical problem for those skilled in the art. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the complex technical difficulties encountered by existing seed coating technologies when applied to Kentucky bluegrass in the cold regions of Qinghai, due to the homogenized mixing strategy. These difficulties include antagonism between bioactive components, a severe mismatch between the timing of functional release and seed physiological rhythms, and rapid failure of the coating's physical structure under severe freeze-thaw cycles. The fundamental deficiency of existing technologies lies in their homogenized structure's inability to provide a spatiotemporally ordered and functionally synergistic microenvironment for diverse active components with different functions and conflicting needs (such as living microorganisms requiring isolation and protection, nutrients requiring slow-release at low temperatures, and germination signaling molecules requiring immediate activation).

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] This invention provides a seed coating agent for Kentucky bluegrass in cold regions of Qinghai suitable for high-altitude and cold environments. It forms a three-layer heterogeneous composite structure on the seed surface from the inside out, consisting of a biological core layer, a nutrient buffer layer, and a functional protective layer.

[0009] The biological core layer comprises composite microbial agent particles and sodium carboxymethyl cellulose as the first binder. The composite microbial agent particles have a core-shell structure, with the core consisting of a diatomaceous earth carrier and a composite microbial agent immobilized thereon. The composite microbial agent is a mixture of Trichoderma spores and arbuscular mycorrhizal fungal spores at a mass ratio of 2:1, with a total effective viable count of not less than 1 × 10⁻⁶. 8 CFU / g; its outer shell is a pH-responsive composite membrane formed by ionic crosslinking of chitosan and sodium alginate, with a thickness of 0.5-2.0 μm;

[0010] The nutrient buffer layer comprises a low-temperature germination inducer, slow-release nutrient microcapsules, and a second binder, polyvinyl alcohol. The low-temperature germination inducer is a composite of gibberellin and chitosan with a deacetylation degree of not less than 90% in a mass ratio of 1:50. The slow-release nutrient microcapsules have a core-shell structure. The core is composed of potassium dihydrogen phosphate, calcium nitrate, magnesium sulfate, and humic acid chelated iron in a mass ratio of 5:2:2:1. The outer shell is composed of ethyl cellulose and polylactic acid-glycolic acid copolymer in a dry weight mass ratio of 1:1. The molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25, and the outer shell thickness is 5-15 μm.

[0011] The functional protective layer comprises an interpenetrating polymer network backbone, high molecular weight sodium alginate, nano-silica particles modified with silane coupling agent KH-570, and food-grade indigo dye; the interpenetrating polymer network backbone is formed by crosslinking hydroxypropyl methylcellulose and polyvinyl acetate at a dry weight ratio of 3:2 under the condition of citric acid as a crosslinking agent; the original particle size of the nano-silica particles modified with silane coupling agent KH-570 is 20-30 nm;

[0012] The first adhesive, sodium carboxymethyl cellulose, has a mass fraction of 1%-3% in the biological core layer, and the second adhesive, polyvinyl alcohol, has a mass fraction of 5%-8% in the nutrient buffer layer.

[0013] Furthermore, the diatomaceous earth has a particle size of 10-50 μm and a specific surface area greater than 30 m². 2 / g.

[0014] Furthermore, the degree of substitution of the hydroxypropyl methylcellulose is 28%-30%, and its 2% aqueous solution has a viscosity of 4000 mPa·s at 20°C; the polyvinyl acetate is an emulsion with a solid content of 50%.

[0015] This invention also provides a method for preparing the seed coating agent for Kentucky bluegrass from Qinghai cold regions, comprising the following steps:

[0016] S1. Preparation of composite microbial agent particles: The composite microbial agent is dry-mixed with diatomaceous earth at a mass ratio of 1:5 to obtain a support carrier; the support carrier is dispersed in a 1.5% sodium alginate aqueous solution to form a suspension; the suspension is added dropwise to an acetic acid buffer solution with pH 4.5 containing 1.0% chitosan and 2.0% calcium chloride for gelation, and after solidification, it is washed and freeze-dried under vacuum to obtain the composite microbial agent particles;

[0017] S2. Preparation of nutrient buffer layer slurry: Polyvinyl alcohol is dissolved in water and heated to dissolve. After cooling, gibberellin-chitosan complex and sustained-release nutrient microcapsules are added and stirred to disperse evenly to obtain the nutrient buffer layer slurry.

[0018] S3. Preparation of functional protective layer slurry: Hydroxypropyl methylcellulose is dissolved in water to make a solution, and polyvinyl acetate emulsion, sodium alginate aqueous solution, modified nano silica dispersion and food-grade indigo dye are added in sequence and stirred evenly. Citric acid aqueous solution is added as a crosslinking agent before use to obtain the functional protective layer slurry.

[0019] S4. Three-layer coating: A bottom-spray fluidized bed coating machine is used to fluidize and preheat the Kentucky bluegrass seeds in cold regions.

[0020] S4-1. The composite microbial agent particles obtained in step S1 are mixed with sodium carboxymethyl cellulose aqueous solution and sprayed as the first coating liquid. The inlet air temperature is 45℃ and the atomization pressure is 0.15MPa to form a biological core layer.

[0021] S4-2. Spray the nutrient buffer layer slurry obtained in step S2 as the second coating liquid, with the inlet air temperature at 50°C and the atomization pressure maintained at 0.15MPa to form a nutrient buffer layer.

[0022] S4-3. Spray the functional protective layer slurry obtained in step S3 as the third coating liquid. The inlet air temperature is 60℃ and the atomization pressure is 0.20MPa.

[0023] S4-4. After all layers are sprayed, the seeds are fluidized and matured at 75°C for 30 minutes, and then cooled to obtain coated seeds.

[0024] Further, in step S4-1, the mass fraction of the sodium carboxymethyl cellulose aqueous solution is 2%.

[0025] Furthermore, in step S4-4, the ripening process causes citric acid to undergo an esterification crosslinking reaction with hydroxypropyl methylcellulose and polyvinyl acetate to form a stable interpenetrating polymer backbone.

[0026] The beneficial effects of this invention are:

[0027] (1) The present invention isolates microorganisms, chemical nutrients and germination regulators in space through a three-layer heterogeneous structure, thus avoiding their mutual inhibition.

[0028] (2) The present invention designs the degradation characteristics of each layer of materials so that microorganisms, germination signals and nutrients are released in sequence according to the physiological rhythm of seed germination and seedling growth, which matches the slow physiological process in high-altitude and cold environments.

[0029] (3) The IPN skeleton and nano-reinforcement structure of the functional protective layer of the present invention provide excellent freeze-thaw resistance, impact resistance and wear resistance, ensuring the long-lasting effectiveness of the coating in harsh environments.

[0030] (4) Through the above synergistic effect, the present invention ultimately achieves a germination rate of over 90% for coated seeds in experiments simulating high-altitude cold environments, and the seedling biomass is significantly better than that of traditional coated seeds. Detailed Implementation

[0031] This invention provides a seed coating agent for *Poa chinensis* seeds from Qinghai cold-region and its preparation method. The core technology lies in constructing a three-layered heterogeneous composite structure with clearly defined functional zones and temporal release characteristics on the surface of the original *Poa chinensis* seeds through a precisely controlled sequential coating process. This composite structure provides systematic support to the seed throughout the entire lifecycle of sowing, germination, and seedling establishment. Specifically, it includes a biological core layer directly coating the outer surface of the seed coat, a nutrient buffer layer surrounding it, and a functional protective layer serving as the outermost physical and chemical barrier. This three-layered structure achieves physical isolation and synergistic coupling of different functional components spatially, and orderly functional release that matches the seed's physiological rhythm temporally.

[0032] Specifically, the seed coating agent for Kentucky bluegrass from Qinghai cold-region and its preparation method are as follows:

[0033] 1. Composition and function of coating agents

[0034] (1) Biological core layer: This layer is the functional interface that directly contacts the seed coat and is designed to build a colony protection zone for beneficial microorganisms.

[0035] Compound microbial agent microparticles: have a core-shell structure.

[0036] Core: High-porosity diatomaceous earth (particle size distribution 10-50μm, specific surface area >30m²) 2 The composite microbial agent is immobilized on a carrier ( / g). The composite microbial agent is a uniform mixture of Trichoderma spp. spores and Arbuscular mycorrhizal fungi spores at a mass ratio of 2:1, with a total effective viable count of not less than 1×10⁻⁶. 8 CFU / g.

[0037] Shell: composed of chitosan-sodium alginate via iontophoresis (Ca... 2 The composite membrane formed by ⁺ and electrostatic complexation is controlled to a thickness of 0.5-2.0 μm. Furthermore, the shell is pH-responsive, dissolving when the seed roots secrete acidic substances, enabling precise, timed release of microorganisms.

[0038] The first adhesive is sodium carboxymethyl cellulose (CMC-Na), which has a mass fraction of 1-3% in the layer. It is used to firmly and evenly adhere the compound microbial agent particles to the seed surface, and has good film-forming properties and biocompatibility.

[0039] (2) Nutrient buffer layer: This layer is an intermediate functional layer responsible for responding to low temperature environment, providing germination signals and continuous nutrition.

[0040] Low-temperature germination inducer: a complex formed by gibberellin (GA3) and chitosan with a degree of deacetylation ≥90% at a mass ratio of 1:50. This design utilizes the adhesive and slow-release properties of chitosan to protect gibberellin and prolong its action time at low temperatures, effectively breaking seed dormancy.

[0041] Sustained-release nutrient microcapsules: also have a core-shell structure.

[0042] Core: A multinutrient mixture of potassium dihydrogen phosphate, calcium nitrate, magnesium sulfate and humic acid chelated iron in a mass ratio of 5:2:2:1, designed to meet the early needs of seedlings and address iron deficiency in high-altitude, cold soils.

[0043] The outer shell is composed of a blend of ethyl cellulose and polylactic-co-glycolic acid copolymer (PLGA). The molar ratio of lactic acid to glycolic acid in the PLGA is precisely set at 75:25, and the shell thickness is 5-15 μm. This formulation allows the microcapsules to slowly release nutrients through a dual diffusion-erosion mechanism in low-temperature (~5°C) and low-humidity environments, with a release cycle of 30-45 days.

[0044] The second adhesive is polyvinyl alcohol (PVA-1788), which has a mass fraction of 5-8% in the layer, providing stronger mechanical strength and film-forming properties to form a stable intermediate layer.

[0045] (3) Functional protective layer: This layer is the outermost physical barrier, providing comprehensive protection.

[0046] Interpenetrating polymer network (IPN) backbone: This forms the main structure of the layers. It is formed by blending hydroxypropyl methylcellulose (HPMC, degree of substitution 28-30%, viscosity of 4000 mPa·s in 2% aqueous solution) and polyvinyl acetate (PVAc) emulsion (solid content 50%) at a dry weight ratio of 3:2, and reacting in situ with citric acid as a crosslinking agent. HPMC forms the first network and provides hydrophilicity, while PVAc interpenetrates within it, providing hydrophobicity and toughness. Citric acid esterification and crosslinking enhance network stability. This IPN structure endows the coating with extremely high freeze-thaw cycle resistance (integrity retention >95% after 50 cycles from -20℃ to 20℃) and forms a microporous structure of 50-100 μm, balancing protection with air and water permeability.

[0047] High molecular weight sodium alginate: Added with a molecular weight of 2.0 × 10⁻⁶ 5 Da's sodium alginate forms an amorphous vitrified hydrated gel at low temperatures, protecting embryonic cell membranes from ice crystal puncture.

[0048] Nano-silica with an original particle size of 20-30 nm, modified with silane coupling agent KH-570 (γ-methacryloyloxypropyltrimethoxysilane): significantly enhances the hardness, wear resistance and impact resistance of the protective layer through chemical bonding and physical anchoring.

[0049] Food-grade indigo dye: It serves to repel birds and for identification purposes.

[0050] 2. Preparation method of coating agent

[0051] S1. Preparation of compound microbial agent particles

[0052] Dry mixing: The compound microbial agent and diatomaceous earth carrier are placed in a V-type mixer at a mass ratio of 1:5 and mixed at a speed of 30 rpm for 30 minutes to obtain the compound microbial agent-supported carrier.

[0053] Suspension preparation: The above carrier is slowly added to a 1.5% (w / w) sodium alginate aqueous solution, with the solid-liquid mass ratio controlled between 1:10 and 1:15, and a uniform suspension is formed under low-speed stirring (200-400 rpm).

[0054] Droplet gelation: Using a nozzle with an inner diameter of 400 μm, the suspension is added dropwise at a flow rate of 50 mL / min to a gel bath with continuous gentle stirring (200 rpm). The gel bath is an acetate-sodium acetate buffer solution with pH=4.5 containing 1.0% (w / v) chitosan (degree of deacetylation ≥90%) and 2.0% (w / v) calcium chloride (CaCl2). The ratio of gel bath volume to suspension volume should be no less than 10:1 to ensure sufficient gelation reaction. After the addition is complete, continue the solidification reaction in the gel bath for 30 minutes.

[0055] Post-processing: The solidified microparticles were collected by filtering through a 200-mesh sieve and washed three times with deionized water to remove residues. Finally, the microparticles were freeze-dried under vacuum at 40°C and a vacuum degree below 100Pa for 12 hours to obtain the dried composite microbial agent microparticle product, which was then sealed and refrigerated for later use.

[0056] S2. Preparation of Nutrient Buffer Layer Slurry

[0057] Dissolution: Add the measured amount of polyvinyl alcohol (PVA-1788) to deionized water, heat and stir in a 90°C water bath for 2 hours until completely dissolved, then cool to room temperature.

[0058] Dispersion: Add the specified amount of gibberellin-chitosan complex and sustained-release nutrient microcapsules to the above PVA solution.

[0059] Homogenization: Transfer the mixture to a planetary mixer and stir at a low shear rate of 100 rpm for 30 minutes until a macroscopically homogeneous, agglomerated slurry is formed, with the final viscosity controlled at 500-800 mPa·s. After preparation, store the slurry at 4°C for later use.

[0060] S3. Preparation of functional protective layer slurry

[0061] Dissolving HPMC: Disperse the measured amount of HPMC in 10 times its total mass of hot water at 80°C, stir rapidly for 10 minutes, then immediately transfer to an ice-water bath and continue stirring for 30 minutes until a completely dissolved, clear, transparent, and viscous solution is formed.

[0062] Premixing: At room temperature (20-25℃), add measured amounts of PVAc emulsion, pre-dissolved sodium alginate aqueous solution, modified nano silica aqueous dispersion dispersed by ultrasonication for more than 30 minutes, and food-grade indigo dye solution to the HPMC solution in sequence.

[0063] Mix at low speed: Mix all components thoroughly at low speed (300 rpm).

[0064] Pre-crosslinking: Before starting the coating process, slowly add 20% (w / w) of a citric acid aqueous solution (8%-12% of the total mass of HPMC and PVAc (dry weight)) as a crosslinking agent. Increase the stirring speed to 700 rpm and continue stirring for 10 minutes to ensure the crosslinking agent is initially and evenly mixed before immediately applying it for coating. This slurry should be used within 2 hours of preparation to avoid excessive pre-crosslinking, which could affect spraying performance.

[0065] S4, a three-layer sequential fluidized bed coating process, is operated using a fluidized bed coating machine equipped with a Wurster bottom spray device.

[0066] Seed pretreatment and preheating: Selected Kentucky bluegrass seeds (purity ≥98%) are loaded into the fluidized bed hopper. The fluidizing fan is turned on, and the fluidizing air velocity is set to 15 m / s. After preheating the Kentucky bluegrass seeds to 35-40℃, spraying is performed according to the following steps:

[0067] S4-1. Spraying the biological core layer: The composite microbial agent particles obtained in step S1 are mixed with a 2% (w / w) sodium carboxymethyl cellulose (CMC-Na) aqueous solution at a mass ratio of 1:1, and this mixture is used as the first coating layer for spraying. The inlet air temperature of the fluidized bed is 45℃, and the atomization pressure is 0.15MPa. The coating solution is sprayed through the bottom atomizing nozzle at a flow rate of 10 g / min / kg of seeds using a peristaltic pump. Spraying continues until the coating weight gain reaches 3%-5% of the initial seed weight, forming the biological core layer.

[0068] S4-2, Spraying the Nutrient Buffer Layer: Seamlessly switch the pipeline and spray the nutrient buffer layer slurry obtained in step S2 as the second coating liquid. Raise the inlet air temperature to 50℃ and maintain the atomization pressure at 0.15MPa. Spray at a flow rate of 12 g / min / kg seeds until the cumulative weight gain of the coating reaches 12%-18% of the initial seed weight, forming the nutrient buffer layer.

[0069] S4-3, Spraying the functional protective layer: Switch the pipeline again and spray the functional protective layer slurry obtained in step S3 as the third coating liquid. Increase the inlet air temperature to 60℃ and the atomization pressure to 0.20MPa. Spray at a flow rate of 15g / min / kg seeds until the cumulative weight gain of the coating reaches 28%-35% of the initial seed weight, forming the functional protective layer.

[0070] S4-4. Curing and Crosslinking: After all the slurry has been sprayed, stop spraying but maintain the fluidized state. Rapidly raise the inlet air temperature to 75°C and maintain the fluidized drying at this temperature for 30 minutes. This high-temperature curing process is a key step to ensure that citric acid and HPMC undergo sufficient esterification and crosslinking reactions to form a stable IPN framework.

[0071] Cooling and Discharging: After maturation, turn off the heating system and switch to cold air to continue fluidization, allowing the coated seeds to cool fully to room temperature (below 25℃). Discharge to obtain the finished coated seeds, which can be sealed and packaged or used directly for sowing.

[0072] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0073] Example 1

[0074] (1) Preparation of compound microbial agent particles

[0075] Weigh out Trichoderma spore powder (effective viable count ≥ 1 × 10⁻⁶) 10 200g of arbuscular mycorrhizal fungal spore powder (effective viable count ≥5×10⁻⁶ CFU / g) 9 100 grams of CFU / g were mixed with 1.5 kg of food-grade diatomaceous earth with a particle size of 30 micrometers and a specific surface area of ​​40 square meters / gram in a 50-liter V-type mixer and dry-mixed at a rate of 30 rpm for 30 minutes to obtain a composite microbial agent-supported carrier.

[0076] Separately prepare a suspension by slowly adding 20 liters of a 1.5% (w / v) sodium alginate aqueous solution to the above-mentioned carrier under low-speed stirring. Using a nozzle with an inner diameter of 400 micrometers, drop the suspension into a 100-liter acetic acid buffer solution containing 1.0% chitosan (92% degree of deacetylation), 2.0% anhydrous calcium chloride, and a pH of 4.5 at a flow rate of 500 ml / min, and allow it to solidify for 30 minutes.

[0077] The microparticles were collected by filtration through a 200-mesh sieve, washed three times with deionized water, and then freeze-dried under vacuum at 40°C and 80 Pa for 12 hours to obtain approximately 1.8 kg of the composite microbial agent microparticle product. Plate count analysis showed a total viable count of 1.5 × 10⁻⁶. 8 CFU / g.

[0078] (2) Preparation of nutrient buffer layer slurry

[0079] Weigh 600g of polyvinyl alcohol 1788 powder and add it to 9.4 liters of deionized water. Heat the water to 90°C and stir for 2 hours to dissolve it completely. Then cool it to room temperature.

[0080] Weigh 50 g of gibberellin-chitosan complex (gibberellin content 2%) and 2 kg of slow-release nutrient microcapsules with a core of potassium dihydrogen phosphate: calcium nitrate: magnesium sulfate: humic acid chelated iron = 5:2:2:1, an outer shell of ethyl cellulose: PLGA (75:25) = 1:1, and an average particle size of 100 micrometers, and add them to the above polyvinyl alcohol solution.

[0081] The mixture was stirred at a low shear rate of 100 rpm for 30 minutes in a planetary mixer to obtain a nutrient buffer slurry with a final viscosity of 650 mPa·s, which was then stored at 4°C for later use.

[0082] (3) Preparation of functional protective layer slurry

[0083] Weigh 900 g of HPMC (viscosity 4000 mPa·s), disperse it in 10 liters of hot water at 80°C, stir for 5 minutes, cool in an ice-water bath and continue stirring to form a transparent viscous solution.

[0084] 1.2 kg (600 g dry weight) of 50% solids content PVAc emulsion with a molecular weight of 2.0 × 10⁻⁶ was added sequentially. 5 100g of sodium alginate (pre-dissolved in 1.9 liters of water), 250g of modified nano silica aqueous dispersion (20% solid content), and 50g of food-grade indigo dye are stirred at low speed until homogeneous.

[0085] Just before coating, add 750 ml of 20% citric acid aqueous solution (containing 150 g of citric acid), stir quickly for 5 minutes, and obtain the functional protective layer slurry to be used.

[0086] (4) Three-layer sequential fluidized bed coating

[0087] Take 10 kg of Qinghai cold-region Kentucky bluegrass seeds with a purity of 98.5% and place them in a bottom-spray fluidized bed coating machine equipped with a Wurster plug. Set the fluidized air velocity to 15 m / s for preheating.

[0088] First layer (biological core layer): Mix 1.8 kg of composite microbial agent particles prepared in step (1) with 2% sodium carboxymethyl cellulose solution, spray at a flow rate of 100 g / min, with an inlet air temperature of 45°C and an atomization pressure of 0.15 MPa, until the coating weight gain is 0.4 kg.

[0089] Second layer (nutrient buffer layer): seamlessly switch to the nutrient buffer layer slurry prepared in step (2), spray at a flow rate of 120 g / min, inlet air temperature of 50°C, atomization pressure of 0.15 MPa, until the cumulative weight gain of the coating is 1.8 kg.

[0090] The third layer (functional protective layer): seamlessly switch to the functional protective layer slurry prepared in step (3), spray at a flow rate of 150 g / min, with an inlet air temperature of 60°C and an atomization pressure of 0.20 MPa, until the cumulative weight gain of the coating is 3.0 kg.

[0091] After spraying, the spraying was stopped, and the inlet air temperature was raised to 75°C and maintained in a fluidized state for 30 minutes to mature. Finally, the mixture was cooled to room temperature with cold air, and the product was discharged, yielding approximately 13 kg of finished coated seeds.

[0092] Example 2

[0093] This implementation uses commercial seeds of Qinghai cold-region Kentucky bluegrass (purity 97.5%) from another batch and another production area. The preparation process is the same as in Example 1.

[0094] Comparative Example 1: Traditional Homogenized Coating

[0095] Take 10 kg of Qinghai cold-region Kentucky bluegrass seeds from the same source and in the same quantity as in Example 1. All the functional components used for each layer in Example 1 (i.e., 1.8 kg of compound microbial agent microparticles, 50 g of gibberellin-chitosan complex, 2 kg of slow-release nutrient microcapsules, 100 g of high molecular weight sodium alginate, 50 g of modified nano silica, and 50 g of indigo dye) and the binder (1.5 kg of polyvinyl alcohol 1788) are added to 20 liters of water at once and mixed in a high-speed disperser to prepare a single, homogenized coating slurry.

[0096] Using a conventional roller coating machine, the slurry was coated onto the seeds in one pass until the total weight gain of the coating reached 3.0 kg, the same as the final weight gain in Example 1. The seeds were then dried in an oven at 50°C to constant weight.

[0097] Comparative Example 2: Lack of a third functional protective layer

[0098] The preparation process is the same as in Example 1, but the third layer is not sprayed.

[0099] Comparative Example 3: Lack of a second nutrient buffer layer

[0100] The preparation process is the same as in Example 1, but the second layer of spraying is omitted. The functional components of the second layer (gibberellin-chitosan complex, sustained-release nutrient microcapsules) are directly added to the slurry of the third functional protective layer, mixed evenly, and then sprayed as the outermost layer.

[0101] Comparative Example 4: The first biological core layer is missing.

[0102] The preparation process is the same as in Example 1, but the first layer of spraying is omitted. The functional components of the first layer (composite microbial agent particles) are directly added to the nutrient buffer layer slurry of the second layer, mixed evenly, and then sprayed.

[0103] Performance testing and data comparison

[0104] The coated seeds obtained in Examples 1 and 2 and Comparative Examples 1-4 were subjected to the following performance tests. All experiments were performed in triplicate, and the data results are expressed as mean ± standard deviation and are summarized in Table 1.

[0105] (1) Freeze-thaw cycle resistance test: 100 grams of each of the five types of coated seeds were placed in a programmable temperature control chamber and subjected to rapid freeze-thaw cycles between -20℃ (4 hours) and +20℃ (4 hours). After 50 cycles, the shedding rate of the coating layer was determined by standard sieving method, and the coating integrity rate (1-shedding rate) was calculated.

[0106] (2) Microbial survival rate test after coating: Take the five kinds of finished coated seeds and use the dilution plate method to determine the effective viable number of compound microbial agents.

[0107] (3) Time-series release performance test: The cumulative release rate of phosphorus (P) from the five coated seeds was determined by using an in vitro simulated high-altitude soil environment (5℃, pH=6.0 PBS buffer). The release medium was 0.1 M PBS buffer (pH=6.0), the temperature was maintained at 5℃, and slow shaking (50 rpm) was applied to simulate the soil microenvironment. Samples were taken on days 1, 3, 5, 10, 20, 30, 45, and 60, and the cumulative release rate of phosphorus was determined by the molybdenum antimony spectrophotometric method.

[0108] (4) Low-temperature pot germination experiment: The soil environment in high-altitude cold regions in spring was simulated in an artificial climate chamber (average daily temperature 5℃, light intensity 10 hours / day). The five types of coated seeds were sown in seedling pots containing sterilized substrate. On the 60th day after sowing, the germination rate was counted, and the average plant height and average root length of the seedlings were measured.

[0109] Table 1

[0110] Test Project Example 1 Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Freeze-thaw cycle resistance (%) 96.8±1.2 95.6±1.5 45.2±3.1 58.1±2.8 92.5±1.8 95.0±1.0 Microbial survival rate after coating (CFU / g) <![CDATA[(1.3±0.2)×10 7 ]]> <![CDATA[(1.1±0.1)×10 7 ]]> <![CDATA[(8.0±1.5)×10 5 ]]> <![CDATA[(9.0±1.0)×10 5 ]]> <![CDATA[(1.1±0.3)×10 6 ]]> <![CDATA[<1.0×10 4 ]]> Germination rate (%) 92.5 91.8 65.0 75.3 71.2 88.5 Average plant height (cm) 8.5 8.2 4.2 6.8 5.5 7.3 Average root length (cm) 12.3 11.8 7.1 9.5 8.8 8.9

[0111] As shown in Table 1, the three-layer heterogeneous coating agent provided by this invention has the following significant advantages over traditional homogeneous coating or other defective structures in solving the problem of seed establishment in high-altitude and cold regions:

[0112] (1) Freeze-thaw cycle resistance (integrity retention rate): The coating integrity rate of Examples 1-2 was significantly higher than that of all comparative examples. Comparative Example 1 (homogeneous coating) had the worst integrity, indicating that simple mixing cannot withstand freeze-thaw stress. The integrity rate of Comparative Example 2 (lacking functional protective layer) was significantly lower than that of Example 1, directly proving that the functional protective layer is the key to resisting physical damage. Although the integrity rates of Comparative Examples 3 and 4 were relatively high, they still lagged behind that of Example 1, indicating that the integrity of the three-layer structure is crucial to maintaining the final performance.

[0113] This invention utilizes the combined action of the outermost interpenetrating polymer network (IPN) skeleton and nano-silica particles modified with the silane coupling agent KH-570 to endow the coating layer with superior mechanical strength and toughness. This effectively resists drastic temperature changes and repeated freeze-thaw cycles in cold regions, preventing early cracking and peeling of the coating layer and providing long-lasting and stable physical protection for the internal active ingredients.

[0114] (2) Microbial survival rate after coating: The viable count of Examples 1-2 remained at 10. 7 The CFU / g level was significantly higher than that of Comparative Example 1. In Comparative Example 1, direct contact between chemical and biological components resulted in almost complete inhibition of microorganisms. Comparative Example 4 (lacking a biological core layer) had the lowest number of viable bacteria, demonstrating that without the protection of the core, a large number of microorganisms were inactivated during the preparation process. This data strongly supports the necessity of an independent biological core layer and its core-shell structure for microbial survival.

[0115] The biological core layer and its core-shell structure particles created in this invention physically isolate beneficial microorganisms from the external chemical environment (such as possible residual fungicides or harsh soil environment) through a pH-responsive shell, and are released only in the acidic rhizosphere environment after seed germination, thus achieving highly efficient protection of live bacteria agents.

[0116] (3) After 60 days of germination at low temperature, Examples 1-2 were significantly better than Comparative Example 1 in all indicators, including germination rate, plant height, and root length. Although Comparative Examples 2, 3, and 4 were better than Comparative Example 1 in all indicators, they were all lower than Examples 1-2. This shows that the three-layer structure of the present invention is an organic functional whole, and the absence of any layer will lead to a significant decrease in system performance, rather than a simple additive effect.

[0117] This invention creates an optimal microenvironment for seed germination and early growth through the combined protection of three major systems: physical protection, microbial activity, and slow-release nutrition. Ultimately, it significantly improves the success rate of seed establishment and seedling quality under high-altitude and cold adversity.

[0118] Table 2. Cumulative release rate of phosphorus from coated seeds at different sampling times

[0119] Time (days) Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 1 2.5% 25.8% 15.3% 28.5% 5.1% 3 5.1% 48.2% 28.7% 50.1% 10.5% 5 8.3% 65.5% 40.2% 68.8% 16.8% 10 15.7% 82.1% 60.5% 85.3% 30.2% 20 32.5% 95.0% 80.1% 96.8% 55.7% 30 55.8% 98.5% 90.5% 99.0% 75.3% 45 83.5% 99.2% 95.8% 99.5% 89.0% 60 89.0% 99.5% 97.2% 99.8% 92.5%

[0120] As shown in Table 2, Example 1 exhibits excellent slow-release characteristics: In the early stage (1-10 days), release is slow, with a cumulative phosphorus release rate of only 15.7% on day 10, effectively preventing nutrient loss before the critical seed germination period; in the middle stage (10-30 days), the release rate accelerates, reaching a cumulative release rate of 55.8% on day 30, matching the nutritional needs of seedlings in their early, slow growth phase; in the later stage (30-60 days), release is continuous and stable, reaching a cumulative release rate of 89.0% on day 60, providing sustained nutrient support for robust seedling establishment. This indicates that the structure of its nutrient buffer layer and the protective function of its protective layer play a crucial role.

[0121] Comparative Example 1 (homogeneous mixture) exhibited a completely different release behavior: nutrients were released explosively in the early stages, reaching a release rate of 65.5% by day 5 and 95.0% by day 20, approaching complete release. This release pattern is severely mismatched with the slow physiological processes of seeds in high-altitude and cold regions, leading to a large waste of nutrients and potentially causing salt stress on young roots. This mechanistically explains the poor germination and growth indicators of Comparative Example 1.

[0122] The release characteristics of Comparative Example 2 (lacking a functional protective layer) fall between those of Example 1 and Comparative Example 1. Its initial release rate was significantly faster than that of Example 1, reaching 40.2% by day 5, indicating that the functional protective layer is crucial for preventing excessive moisture penetration and maintaining the slow-release structure of the intermediate layer. The lack of this protective layer resulted in a significant decrease in slow-release performance.

[0123] The release behavior of Comparative Example 3 (lacking a nutrient buffer layer) was almost identical to that of Comparative Example 1, indicating that directly incorporating nutrient microcapsules into the outer slurry could not form an effective sustained-release structure. The rapid erosion or dissolution of the outer polymer layer led to the rapid exposure and release of the microcapsules, demonstrating the necessity of the presence of an independent nutrient buffer layer.

[0124] Although the initial release rate of Comparative Example 4 (lacking the biological core layer) was slower than that of Comparative Examples 1 and 3, it was still significantly faster than that of Example 1. This is because the lack of an inner layer coating allows nutrients to be closer to the seed surface or outer layer, thus contacting the medium earlier. However, its later release rate (92.5% at day 60) was still higher than that of Example 1, indicating that the layer-by-layer barrier effect formed by the three-layer structure is crucial for achieving optimal sustained-release performance.

[0125] The time-series release data intuitively demonstrates the design advantages of the three-layer heterogeneous structure of this invention. Only a complete "biological core layer - nutrient buffer layer - functional protection layer" structure can achieve precise time-series control of nutrient elements, matching their release pattern with the slow physiological rhythm of Kentucky bluegrass seeds in the cold-climate environment of Qinghai, thereby systematically solving the problems of premature nutrient loss and demand mismatch.

[0126] In summary, this invention overcomes the limitations of traditional homogeneous coating techniques by constructing a three-layer heterogeneous structure consisting of a biological core layer, a nutrient buffer layer, and a functional protective layer. It systematically solves three core challenges in seed establishment in high-altitude and cold regions: component antagonism, temporal mismatch, and physical failure. Through precise synergy between spatial isolation and temporal release, this design achieves a unified function of activity protection, slow nutrient release, and environmental stress resistance, significantly improving seed germination efficiency and seedling establishment quality under harsh conditions, providing an efficient and reliable technical solution for high-altitude ecological restoration.

Claims

1. A seed coating agent for Kentucky bluegrass in cold-climate areas of Qinghai suitable for high-altitude and cold environments, characterized in that, The seed surface is coated from the inside out with a three-layered heterogeneous composite structure consisting of a biological core layer, a nutrient buffer layer, and a functional protective layer. The biological core layer comprises composite microbial agent particles and sodium carboxymethyl cellulose as the first binder. The composite microbial agent particles have a core-shell structure, with the core consisting of a diatomaceous earth carrier and a composite microbial agent immobilized thereon. The composite microbial agent is a mixture of Trichoderma spores and arbuscular mycorrhizal fungal spores at a mass ratio of 2:1, with a total effective viable count of not less than 1 × 10⁻⁶. 8 CFU / g; its outer shell is a pH-responsive composite membrane formed by ionic crosslinking of chitosan and sodium alginate, with a thickness of 0.5-2.0 μm; The nutrient buffer layer comprises a low-temperature germination inducer, slow-release nutrient microcapsules, and a second binder, polyvinyl alcohol. The low-temperature germination inducer is a composite of gibberellin and chitosan with a deacetylation degree of not less than 90% in a mass ratio of 1:

50. The slow-release nutrient microcapsules have a core-shell structure. The core is composed of potassium dihydrogen phosphate, calcium nitrate, magnesium sulfate, and humic acid chelated iron in a mass ratio of 5:2:2:1, and the outer shell is composed of ethyl cellulose and polylactic acid-glycolic acid copolymer in a dry weight mass ratio of 1:

1. The molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25, and the outer shell thickness is 5-15 μm. The functional protective layer comprises an interpenetrating polymer network backbone, high molecular weight sodium alginate, nano-silica particles modified with silane coupling agent KH-570, and food-grade indigo dye; the interpenetrating polymer network backbone is formed by crosslinking hydroxypropyl methylcellulose and polyvinyl acetate at a dry weight ratio of 3:2 under the condition of citric acid as a crosslinking agent; the original particle size of the nano-silica particles modified with silane coupling agent KH-570 is 20-30 nm; The first adhesive, sodium carboxymethyl cellulose, has a mass fraction of 1%-3% in the biological core layer, and the second adhesive, polyvinyl alcohol, has a mass fraction of 5%-8% in the nutrient buffer layer.

2. The seed coating agent according to claim 1, characterized in that, The diatomaceous earth has a particle size of 10-50 μm and a specific surface area greater than 30 m². 2 / g.

3. The seed coating agent according to claim 1, characterized in that, The degree of substitution of the hydroxypropyl methylcellulose is 28%-30%, and its 2% aqueous solution has a viscosity of 4000 mPa·s at 20°C; the polyvinyl acetate is an emulsion with a solid content of 50%.

4. A method for preparing the seed coating agent for *Poa chinensis* seeds as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Preparation of composite microbial agent particles: The composite microbial agent is dry-mixed with diatomaceous earth at a mass ratio of 1:5 to obtain a support carrier; the support carrier is dispersed in a 1.5% sodium alginate aqueous solution to form a suspension; the suspension is added dropwise to an acetic acid buffer solution with pH 4.5 containing 1.0% chitosan and 2.0% calcium chloride for gelation, and after solidification, it is washed and freeze-dried under vacuum to obtain the composite microbial agent particles; S2. Preparation of nutrient buffer layer slurry: Polyvinyl alcohol is dissolved in water and heated to dissolve. After cooling, gibberellin-chitosan complex and sustained-release nutrient microcapsules are added and stirred to disperse evenly to obtain the nutrient buffer layer slurry. S3. Preparation of functional protective layer slurry: Hydroxypropyl methylcellulose is dissolved in water to make a solution, and polyvinyl acetate emulsion, sodium alginate aqueous solution, modified nano silica dispersion and food-grade indigo dye are added in sequence and stirred evenly. Citric acid aqueous solution is added as a crosslinking agent before use to obtain the functional protective layer slurry. S4. Three-layer coating: A bottom-spray fluidized bed coating machine is used to fluidize and preheat the Kentucky bluegrass seeds in cold regions. S4-1. The composite microbial agent particles obtained in step S1 are mixed with sodium carboxymethyl cellulose aqueous solution and sprayed as the first coating liquid. The inlet air temperature is 45℃ and the atomization pressure is 0.15MPa to form a biological core layer. S4-2. Spray the nutrient buffer layer slurry obtained in step S2 as the second coating liquid, with the inlet air temperature at 50°C and the atomization pressure maintained at 0.15MPa to form a nutrient buffer layer. S4-3. Spray the functional protective layer slurry obtained in step S3 as the third coating liquid. The inlet air temperature is 60℃ and the atomization pressure is 0.20MPa. S4-4. After all layers are sprayed, the seeds are fluidized and matured at 75°C for 30 minutes, and then cooled to obtain coated seeds.

5. The method according to claim 4, characterized in that, In step S4-1, the mass fraction of the sodium carboxymethyl cellulose aqueous solution is 2%.

6. The method according to claim 4, characterized in that, In step S4-4, the ripening process causes citric acid to undergo an esterification and crosslinking reaction with hydroxypropyl methylcellulose and polyvinyl acetate, forming a stable interpenetrating polymer backbone.