Seed coating for improving nitrogen fixation efficiency of legume-grass mixed sowing grassland and preparation method thereof

Abstract

The application relates to the field of agricultural planting and seed treatment technology, and discloses a seed coating for improving the nitrogen fixation efficiency of a legume-grass mixed sowing grassland and a preparation method thereof. Raw material components of the seed coating comprise iron pyrophosphate, ammonium molybdate tetrahydrate, L-malic acid, chitosan, naringenin, guar gum powder, diatomite and talc powder. In the application, in-situ electrostatic cross-linking occurs between chitosan and L-malic acid after coating water absorption, a polyion complex gel is generated, free L-malic acid is fixed, iron pyrophosphate is kept in a very difficult to dissolve state, and trace elements are prevented from being lost in advance under the action of precipitation. Chitinase secreted by legume rhizosphere can specifically degrade the complex gel, release L-malic acid and complex dissociate iron pyrophosphate, and targeted supply of trace nutrients to legume rhizosphere is realized. The application solves the problem of competition for nutrients of gramineae plants in the mixed sowing grassland from the source, cooperates with naringenin to synergistically induce soil rhizobium, and improves the nodule formation rate and nitrogen fixation efficiency of legume forage.

Description

Technical Field

[0001] This invention relates to the field of agricultural planting and seed treatment technology, specifically to a seed coating method for improving nitrogen fixation efficiency in mixed bean and grass grasslands. Background Technology

[0002] Legume-grass mixed planting is a planting pattern used to maintain grassland ecosystem balance and increase forage yield. In this pattern, legumes provide nitrogen to the grassland system through symbiotic nitrogen fixation, while grasses provide biomass. To improve seed germination rates and early-stage resilience, seed coating technology is widely used in forage establishment. Seed coating involves covering the seed surface with a film-forming material containing micronutrient fertilizers or microbial agents, providing nutrition and protection during seed germination and seedling growth.

[0003] Current methods for providing trace elements in forage seed coating generally involve adding readily soluble inorganic salts. Conventional techniques use readily soluble metal salts as sources of iron and molybdenum, physically mixing them with polymeric film-forming agents and inorganic fillers, and then coating the seed surface. After the coated seeds are sown, the coating layer absorbs soil moisture, and the readily soluble salts in the coating layer quickly dissolve, releasing metal ions for absorption and utilization by the plant roots.

[0004] In mixed sowing applications of leguminous and gramineous forage grasses, conventional seed coating technology suffers from the drawback of low nitrogen fixation efficiency in leguminous forage grasses due to interspecific nutrient competition. Gramineous forage grasses have faster root development than leguminous forage grasses and a stronger capacity for water and nutrient absorption. When the readily soluble iron and molybdenum salts in existing seed coatings rapidly dissolve upon contact with water and diffuse into the surrounding soil, the free micronutrients are easily competitively absorbed by the roots of the accompanying gramineous plants. This excessive absorption by gramineous plants leads to insufficient iron and molybdenum content in the rhizosphere microenvironment of leguminous plants when their roots develop to the stage of rhizobium infection. Conventional seed coating cannot prevent the competitive consumption of micronutrients by gramineous plants within the mixed sowing system, ultimately limiting the nodulation rate and nitrogen fixation efficiency of leguminous forage grasses. Summary of the Invention

[0005] The technical problem solved by this invention is that during the establishment of mixed legume and grass pastures, the easily soluble trace elements in conventional seed coatings are easily lost when exposed to water or react with specific ions in the soil to form insoluble substances that are fixed. At the same time, the roots of grass pastures develop faster and compete with legume pastures for the absorption of free iron and molybdenum, resulting in a deficiency of trace elements in the rhizosphere of legume pastures, which in turn limits the nodulation rate of rhizobia and the activity of nitrogenase.

[0006] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a seed coating that improves nitrogen fixation efficiency in mixed legume-grass grasslands, employing the following technical solution: A seed coating for improving nitrogen fixation efficiency in mixed bean and grass grasslands comprises the following raw material components in parts by weight: 5.0-8.0 parts ferric pyrophosphate; 1.0-2.0 parts ammonium molybdate tetrahydrate; 10.0-15.0 parts L-malic acid; 5.0-8.0 parts chitosan; 0.1-0.2 parts naringenin; 15.0-20.0 parts guar collagen powder; 40.0-50.0 parts diatomaceous earth; and 10.0-15.0 parts talc.

[0007] By adopting the above technical solution, and through the cross-linking reaction of ferric pyrophosphate, L-malic acid, and chitosan, combined with guar collagen powder, diatomaceous earth, and talc powder as a matrix and filler, the nitrogen fixation efficiency of leguminous forage grasses is improved while avoiding nutrient competition from gramineous forage grasses. The reaction mechanism and working process of this invention are as follows: The first step is the in-situ electrostatic cross-linking process. After sowing, the coated seeds absorb soil moisture. This moisture penetrates the coating layer, causing some L-malic acid to dissolve and dissociate, releasing protons and lowering the pH value inside the coating layer. In this slightly acidic environment, the amino groups on the chitosan molecular chain are protonated, generating positively charged amino ions. The protonated chitosan cross-links with the negatively charged malate anions through electrostatic interactions, forming an in-situ water-insoluble chitosan-malic acid polyionic composite gel. This composite gel network restricts the diffusion of free L-malic acid, preventing its loss to the external environment. Simultaneously, because ferric pyrophosphate is extremely insoluble in water, iron remains inert during water extraction, avoiding early loss and physical competitive absorption by symbiotic gramineous plants.

[0008] The second step is the enzymatic degradation process. During seed germination and as the taproot penetrates the seed coat, the legume roots and symbiotic rhizobia secrete chitinase and chitosanase into the rhizosphere. These enzymes degrade the chitosan β-(1,4)-glycosidic bonds in the polyionic composite gel, causing the gel network to disintegrate and releasing cross-linked L-malic acid into the rhizosphere. Because the rhizosphere of grasses lacks highly active chitinase and chitosanase secretion, the polyionic composite gel remains structurally stable around the roots of grasses.

[0009] The third step involves complexation and nodulation induction. The released L-malic acid undergoes a coordination complexation reaction with ferric pyrophosphate, promoting the dissociation of ferric pyrophosphate and generating a soluble malic acid-iron-molybdenum chelate. This malic acid-iron-molybdenum chelate is absorbed by the roots of leguminous plants, meeting the trace element requirements for the synthesis of iron-molybdenum cofactors in nitrogenase. Simultaneously, the slightly acidic nutrient environment, combined with the synchronous release of naringenin, induces the expression of nodulation genes in rhizobia in the soil, increasing the nodulation rate of leguminous plants.

[0010] Preferably, the raw material components include the following parts by weight: 6.5 parts ferric pyrophosphate, 1.5 parts ammonium molybdate tetrahydrate, 12.5 parts L-malic acid, 6.5 parts chitosan, 0.15 parts naringenin, 18.0 parts guar collagen powder, 45.0 parts diatomaceous earth, and 12.5 parts talc.

[0011] By adopting the above technical solution, the proportion of crosslinking precursor and trace element ratio is controlled within a specific molar ratio range. After L-malic acid meets the acidity required for the protonation crosslinking of chitosan, the remaining molar amount can provide sufficient coordination complexing driving force to ensure the stable release of iron.

[0012] Preferably, the chitosan has a degree of deacetylation of ≥85.0% and a weight-average molecular weight (Mw) in the range of 50kDa-150kDa; the ferric pyrophosphate has a particle size (D50) in the range of 2.0-5.0 μm.

[0013] By adopting the above technical solution, the degree of deacetylation and molecular weight of chitosan are limited, so that chitosan can be rapidly dissolved and protonated in the slightly acidic environment provided by L-malic acid, and is easily degraded by chitinase. The particle size of ferric pyrophosphate is limited, the specific surface area is increased, and the coordination complexation rate of L-malic acid on ferric pyrophosphate is improved, so as to meet the iron absorption rate requirements of legumes in the early stage of germination.

[0014] Preferably, the 1% aqueous solution of the guar collagen powder has a viscosity range of 3000-5000 mPa·s at 25°C and a fineness passing through a 200-mesh sieve; the diatomaceous earth is a calcined product with a specific surface area ≥ 20 m^2 / g and a porosity ≥ 80.0%.

[0015] By adopting the above technical solutions, the high viscosity of guar collagen powder ensures the structural stability of the coating layer after water absorption, providing a spatially confined matrix for the formation of polyionic composite gel; the high specific surface area and high porosity of diatomaceous earth improve the flowability and air permeability of the coating powder, preventing pore blockage from affecting seed radicle development.

[0016] Preferably, the naringenin and the guar gum powder exist in the form of a naringenin-preloaded modified guar gum matrix, which is prepared by uniformly spraying a naringenin-ethanol solution onto the surface of the guar gum powder and then vacuum drying to remove the solvent.

[0017] By adopting the above technical solution, ethanol solvent is used to penetrate into the pores of guar gum particles, so that the poorly soluble flavonoid compound naringenin is evenly dispersed in the matrix, avoiding uneven distribution caused by physical mixing and improving the stability of the signaling molecules that induce nodulation in rhizobia.

[0018] Secondly, the present invention provides a method for preparing seed coatings that improve nitrogen fixation efficiency in mixed legume-grass grasslands, employing the following technical solution: A method for preparing seed coatings to improve nitrogen fixation efficiency in mixed legume and grass grasslands includes the following steps: S1, Preparation of inorganic-organic biochemical composite dry powder: Ferric pyrophosphate, ammonium molybdate tetrahydrate, L-malic acid, chitosan, diatomaceous earth and talc are sequentially added into a three-dimensional motion mixer to obtain a uniformly mixed composite dry powder. S2, Preparation of coated powder: The composite dry powder obtained in step S1 is dry-mixed with naringenin preloaded modified guar gum matrix to obtain the final coated powder. S3, Synchronous Alternating Coating: Sterile pure water and the final coating powder prepared in step S2 are alternately sprayed onto the surface of the rotating seeds. S4, In-situ crosslinking and hot air curing: After the coating powder is added, the coating machine continues to run at low speed to carry out the in-situ reaction. Then, dry hot air is continuously blown and dried, cooled and discharged to obtain the finished coated seeds.

[0019] By adopting the above technical solution, the dry powder mixing step ensures that each component remains chemically inert in a dry state; the synchronous alternating coating process utilizes an appropriate amount of moisture to directly induce the electrostatic cross-linking reaction of L-malic acid dissociation and chitosan protonation inside the coating layer, forming a cross-linked structure; the drying hot air removes excess moisture to complete the coating layer curing, preventing seeds from germinating during storage, and realizing the batch preparation of responsive coated seeds.

[0020] Preferably, step S2 includes a pretreatment step of naringenin-modified guar gum matrix, specifically: weighing naringenin and adding it to anhydrous ethanol and stirring magnetically until a clear and transparent naringenin-ethanol solution is formed; using a high-pressure atomizing nozzle, the clear and transparent naringenin-ethanol solution is evenly sprayed onto the surface of the guar gum powder, and then transferred to a vacuum drying oven and dried for 2.0-4.0 hours at a drying temperature of 40-45℃ and a vacuum degree of -0.08MPa to -0.09MPa.

[0021] By adopting the above technical solution, the low-temperature high-vacuum drying environment allows the anhydrous ethanol solvent to evaporate rapidly, avoiding the reduction of naringenin activity caused by high temperature and ensuring the integrity of the physicochemical properties of the preloaded matrix.

[0022] Preferably, in step S1, the mixing operation is carried out under controlled conditions of relative humidity ≤ 30% and temperature ≤ 25°C.

[0023] By adopting the above technical solutions, the dew point and temperature of the mixing environment are strictly controlled to prevent free water in the environment from causing premature cross-linking and agglomeration of L-malic acid and chitosan, thus ensuring the powder flowability of the composite dry powder and the continuity of subsequent coating processes.

[0024] Preferably, step S3 is implemented as follows: the initial spraying volume is 3.0%-4.0% of the dry weight of the seeds with sterile pure water to evenly moisten the seed surface. Then, the spraying and powdering operations are repeated alternately for 3-5 cycles, and the total water added during the entire coating process is controlled to be 8.0%-12.0% of the dry weight of the seeds.

[0025] By adopting the above technical solution, the first quantitative water spray can bind the bottom powder, and the alternating cycle operation ensures that the coating layer thickness grows evenly. The total amount of water added can both trigger the interfacial cross-linking reaction between components and be lower than the germination threshold of seed water absorption and rupture, thus maintaining the dormancy state of the seeds.

[0026] Preferably, in step S4, the duration of the in-situ reaction is controlled to be 5.0-10.0 minutes; and the temperature of the introduced hot drying air is 35-40°C.

[0027] By adopting the above technical solutions, the in-situ reaction time is controlled to ensure that the protonation and electrostatic cross-linking processes are fully completed; the upper limit of the drying hot air temperature is limited to prevent thermal stress from damaging the activity of seed radicles and the structure of biological signal molecules.

[0028] Preferably, in step S4, the degree of drying is controlled so that the moisture content of the finished coated seeds is reduced to below 8.0%; the seeds in step S3 are alfalfa seeds or white clover seeds.

[0029] By adopting the above technical solution, the moisture content is reduced to below the safe storage threshold, the shelf life of the finished coated seeds is extended, and the industrial applicability of the process method of the present invention to leguminous forage seeds is established.

[0030] This invention provides a seed coating method for improving nitrogen fixation efficiency in legume-grass mixed-sowing grasslands, and its preparation method. It has the following beneficial effects: 1. This invention enables the targeted supply of trace elements to the rhizosphere of leguminous plants, effectively avoiding nutrient competition from gramineous plants in mixed grasslands. This invention utilizes the electrostatic cross-linking of chitosan and L-malic acid under slightly acidic conditions to form an insoluble composite gel, fixing free L-malic acid and keeping ferric pyrophosphate in a poorly soluble state. When the roots of leguminous plants and the symbiotic rhizobia specifically secrete chitinase to degrade the composite gel, L-malic acid is released and complexes with and dissociates ferric pyrophosphate, thus forming soluble trace elements only locally in the rhizosphere of leguminous plants. This solves the technical problem of trace element deficiency in leguminous plants caused by competitive absorption by gramineous plants.

[0031] 2. This invention overcomes the shortcomings of traditional micronutrient coatings, such as easy loss and early ion osmotic stress. This method uses extremely insoluble ferric pyrophosphate instead of conventional easily soluble metal salts, thus avoiding the osmotic stress and toxicity of high-concentration metal ions to rhizobia in the early stages of seed germination. Combined with the chitosan-malic acid polyionic composite gel network formed in situ after the coating layer absorbs water, it forms a dual physical and electrostatic retention effect on the internal components of the coating, preventing premature dissolution and loss of micronutrients under rainfall or irrigation, and ensuring effective nutrient retention during the critical period of seed germination.

[0032] 3. The multi-component synergistic effect of this invention significantly improves the nodulation rate and nitrogen fixation efficiency of leguminous plants. Naringenin, as a specific biological signaling molecule, induces the expression of nodulation genes in rhizobia in the soil during seed germination. The simultaneously released high concentration of L-malic acid not only acts as a ligand to activate trace elements but also provides carbon source nutrition for the chemotactic movement of rhizobia. The activated iron and molybdenum elements are efficiently absorbed by the leguminous roots, directly meeting the elemental requirements for the synthesis of iron and molybdenum cofactors in nitrogenase. The above components form a suitable physicochemical microenvironment in the rhizosphere of leguminous plants, synergistically promoting the infection and colonization of rhizobia and the activity of nitrogenase. Attached Figure Description

[0033] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0034] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0035] Ferric pyrophosphate, CAS number 10058-44-3, molecular formula Fe4(P2O7)3, purity ≥98.0%, powder particle size D50 between 2.0-5.0μm.

[0036] Chitosan, CAS No. 9012-76-4, is a linear natural polymer composed of β-(1,4)-2-amino-2-deoxy-D-glucose residues, with a degree of deacetylation ≥85.0% and a weight-average molecular weight (Mw) ranging from 50kDa to 150kDa.

[0037] Naringenin, chemically known as 4',5,7-trihydroxyflavanone, CAS number 480-41-1, has the molecular formula C2. 15 H 12 O5, purity ≥98.0%.

[0038] Guar collagen powder, CAS number 9000-30-0, is a non-ionic galactomannan mainly composed of galactose and mannose structural units. The viscosity of a 1% aqueous solution at 25℃ ranges from 3000 to 5000 mPa·s, and the fineness passes through a 200-mesh sieve.

[0039] Diatomaceous earth, CAS No. 61790-53-2, calcined product, specific surface area ≥20m2 / g, porosity ≥80.0%.

[0040] Preparation Example 1: This preparation example provides a naringenin-preloaded modified guar gum matrix, including the following steps: Step 1: Weigh 0.1 parts of naringenin and add it to 4.0 parts of anhydrous ethanol. Stir magnetically at 25°C for 15 minutes until a clear and transparent naringenin-ethanol solution is formed.

[0041] Step 2: Place 15.0 parts of guar gum powder in a closed mixer equipped with a stirring paddle, turn on the mixer, and set the speed to 30 rpm. Using a high-pressure atomizing nozzle, spray the above naringenin-ethanol solution evenly onto the surface of the guar gum powder, controlling the spraying process to be completed within 10 minutes, and then continue mixing for 20 minutes.

[0042] Step 3: Transfer the above-mentioned moistened powder to a vacuum drying oven, set the drying temperature to 40℃, the vacuum degree to -0.08MPa, and dry for 2.0 hours.

[0043] Step 4: Take out the dried material, grind and break it up by mechanical grinding, and pass it through a 200-mesh sieve to obtain the naringenin preloaded modified guar gum matrix of this preparation example.

[0044] Preparation Example 2: This preparation example provides a naringenin-preloaded modified guar gum matrix, including the following steps: Step 1: Weigh 0.15 parts of naringenin and add it to 6.0 parts of anhydrous ethanol. Stir magnetically at 28°C for 18 minutes until a clear and transparent naringenin-ethanol solution is formed.

[0045] Step 2: Place 18.0 parts of guar gum powder in a closed mixer equipped with a stirring paddle, turn on the mixer, and set the speed to 40 rpm. Using a high-pressure atomizing nozzle, spray the above naringenin-ethanol solution evenly onto the surface of the guar gum powder, controlling the spraying process to be completed within 15 minutes, followed by continuous mixing for 25 minutes.

[0046] Step 3: Transfer the above-mentioned moistened powder to a vacuum drying oven, set the drying temperature to 42℃, the vacuum degree to -0.085MPa, and dry for 3.0 hours.

[0047] Step 4: Take out the dried material, grind and break it up by mechanical grinding, and pass it through a 200-mesh sieve to obtain the naringenin preloaded modified guar gum matrix of this preparation example.

[0048] Preparation Example 3: This preparation example provides a naringenin-preloaded modified guar gum matrix, including the following steps: Step 1: Weigh 0.2 parts of naringenin and add it to 8.0 parts of anhydrous ethanol. Stir magnetically at 30°C for 20 minutes until a clear and transparent naringenin-ethanol solution is formed.

[0049] Step 2: Place 20.0 parts of guar gum powder in a closed mixer equipped with a stirring paddle, turn on the mixer, and set the speed to 50 rpm. Using a high-pressure atomizing nozzle, spray the above naringenin-ethanol solution evenly onto the surface of the guar gum powder, controlling the spraying process to be completed within 20 minutes, followed by continuous mixing for 30 minutes.

[0050] Step 3: Transfer the above-mentioned moistened powder to a vacuum drying oven, set the drying temperature to 45℃, the vacuum degree to -0.09MPa, and dry for 4.0 hours.

[0051] Step 4: Take out the dried material, grind and break it up by mechanical grinding, and pass it through a 200-mesh sieve to obtain the naringenin preloaded modified guar gum matrix of this preparation example.

[0052] Reference Figure 1 Example 1: This embodiment provides a seed coating method for improving nitrogen fixation efficiency in soybean-grass mixed-sowing grasslands, including the following steps: Step 1: Preparation of the inorganic-organic biochemical composite dry powder. Under controlled conditions of 25% relative humidity and 20℃, 5.0 parts of ferric pyrophosphate, 1.0 part of ammonium molybdate tetrahydrate, 10.0 parts of L-malic acid, 5.0 parts of chitosan, 40.0 parts of diatomaceous earth, and 10.0 parts of talc were sequentially added to a three-dimensional motion mixer. The operating speed of the three-dimensional motion mixer was set to 15 rpm, and the mixture was continuously mixed for 30 minutes to obtain a uniformly mixed composite dry powder. The powder was then sealed in a moisture-proof bag and stored away from light for later use.

[0053] Step 2, preparation of the coating powder. Before use, the composite dry powder prepared above is dry-mixed with the naringenin preloaded modified guar gum matrix prepared in Preparation Example 1 to obtain the final coating powder.

[0054] Step 3, Synchronous Alternating Coating. Weigh 100 portions of alfalfa seeds and place them into the centrifugal rotary coating machine. Set the rotation speed of the rotary disc to 60 rpm. Spray sterile pure water onto the rotating seed surface through an atomizing nozzle. The initial water spray volume is 3.0% of the seed dry weight, ensuring the seed surface is evenly moistened. Then, evenly sprinkle the coating powder prepared in Step 2 into the machine using a quantitative screw feeder. Repeat the water spraying and powder sprinkling operation for 3 cycles. The total water added during the entire coating process is controlled to be 8.0% of the seed dry weight.

[0055] Step 4: In-situ crosslinking and hot air curing. After the coating powder is added, keep the coating machine running at a low speed, reducing the speed to 30 rpm, and continue running at 20℃ for 5.0 minutes to carry out the in-situ reaction. Then, turn on the hot air supply system of the coating machine, introduce dry hot air at a temperature of 35℃, control the air volume at 150 cubic meters / hour, and continue blowing and drying for 15 minutes. When the surface of the coating layer hardens and the moisture content of the finished coated seeds drops below 8.0% as measured by sampling, stop heating, continue to circulate room temperature natural air for cooling for 5.0 minutes, and then discharge the material to obtain the finished coated seeds.

[0056] Example 2: This embodiment provides a seed coating method for improving nitrogen fixation efficiency in soybean-grass mixed-sowing grasslands, including the following steps: Step 1: Preparation of the inorganic-organic biochemical composite dry powder. Under controlled conditions of 28% relative humidity and 22℃, 6.5 parts of ferric pyrophosphate, 1.5 parts of ammonium molybdate tetrahydrate, 12.5 parts of L-malic acid, 6.5 parts of chitosan, 45.0 parts of diatomaceous earth, and 12.5 parts of talc were sequentially added to a three-dimensional motion mixer. The three-dimensional motion mixer was set to a speed of 20 rpm, and the mixture was continuously mixed for 38 minutes to obtain a uniformly mixed composite dry powder. The powder was then sealed in a moisture-proof bag and stored away from light for later use.

[0057] Step 2, preparation of the coating powder. Before use, the composite dry powder prepared above is dry-mixed with the naringenin preloaded modified guar gum matrix prepared in Preparation Example 2 to obtain the final coating powder.

[0058] Step 3, Synchronous Alternating Coating. Weigh 100 portions of alfalfa seeds and place them into the centrifugal rotary coating machine. Set the rotation speed of the rotary disc to 70 rpm. Spray sterile pure water onto the rotating seed surface through an atomizing nozzle. The initial water spray volume is 3.5% of the seed dry weight, ensuring the seed surface is evenly moistened. Then, evenly sprinkle the coating powder prepared in Step 2 into the machine using a quantitative screw feeder. Repeat the water spraying and powder sprinkling operation for 4 cycles. The total water added during the entire coating process is controlled to be 10.0% of the seed dry weight.

[0059] Step 4: In-situ crosslinking and hot air curing. After the coating powder is added, the coating machine continues to run at a low speed, reducing the speed to 35 rpm, and continues to run at 22℃ for 8.0 minutes to carry out the in-situ reaction. Then, the hot air supply system of the coating machine is turned on, and dry hot air at a temperature of 38℃ is introduced, with the air volume controlled at 180 cubic meters / hour, and continuous blowing and drying is carried out for 20 minutes. When the surface of the coating layer hardens and the moisture content of the finished coated seeds is measured to be below 8.0%, heating is stopped, and room temperature natural air is introduced for cooling for another 8.0 minutes. The material is then discharged to obtain the finished coated seeds.

[0060] Example 3: This embodiment provides a seed coating method for improving nitrogen fixation efficiency in soybean-grass mixed-sowing grasslands, including the following steps: Step 1: Preparation of the inorganic-organic biochemical composite dry powder. Under controlled conditions of 30% relative humidity and 25℃, 8.0 parts of ferric pyrophosphate, 2.0 parts of ammonium molybdate tetrahydrate, 15.0 parts of L-malic acid, 8.0 parts of chitosan, 50.0 parts of diatomaceous earth, and 15.0 parts of talc were sequentially added to a three-dimensional motion mixer. The operating speed of the three-dimensional motion mixer was set to 25 rpm, and the mixture was continuously mixed for 45 minutes to obtain a uniformly mixed composite dry powder. The powder was then sealed in a moisture-proof bag and stored away from light for later use.

[0061] Step 2, preparation of the coating powder. Before use, the composite dry powder prepared above is dry-mixed with the naringenin preloaded modified guar gum matrix prepared in Preparation Example 3 to obtain the final coating powder.

[0062] Step 3, Synchronous Alternating Coating. Weigh 100 portions of alfalfa seeds and place them into the centrifugal rotary coating machine. Set the rotation speed of the rotary disc to 80 rpm. Spray sterile pure water onto the rotating seed surface through an atomizing nozzle. The initial water spray volume is 4.0% of the seed dry weight, ensuring the seed surface is evenly moistened. Subsequently, evenly sprinkle the coating powder prepared in Step 2 into the machine using a quantitative screw feeder. Repeat the water spraying and powder sprinkling operation for 5 cycles. The total water added during the entire coating process is controlled to be 12.0% of the seed dry weight.

[0063] Step 4: In-situ crosslinking and hot air curing. After the coating powder is added, the coating machine continues to run at a low speed, reducing the speed to 40 rpm, and continues to run at 25°C for 10.0 minutes to carry out the in-situ reaction. Then, the hot air supply system of the coating machine is turned on, and dry hot air at a temperature of 40°C is introduced, with the air volume controlled at 200 cubic meters / hour, and continuous blowing and drying is carried out for 25 minutes. When the surface of the coating layer hardens and the moisture content of the finished coated seeds is measured to be below 8.0%, heating is stopped, and room temperature natural air is introduced for cooling for another 10.0 minutes. The material is then discharged to obtain the finished coated seeds.

[0064] Example 4: This embodiment provides a seed coating method for improving nitrogen fixation efficiency in soybean-grass mixed-sowing grasslands, including the following steps: The formula and process parameters in this embodiment are the same as those in Example 2, except that in step 3, 100 portions of white clover seeds are weighed to replace alfalfa seeds and placed into the centrifugal rotary coating machine for subsequent operations. This embodiment is used to verify the versatility of the process method of the present invention on different leguminous forage seeds.

[0065] Comparative Example 1: Compared to Example 2, the difference lies in the absence of ferric pyrophosphate, L-malic acid, and chitosan in the formulation. Instead, the iron source is replaced with an equimolar amount of readily soluble ferrous sulfate heptahydrate, and an equal amount of diatomaceous earth is used to compensate for the mass difference caused by the reduction of components. All other aspects remain the same. This comparative example represents the most conventional direct physical mixing and coating scheme for readily soluble micronutrient fertilizers in the art.

[0066] Comparative Example 2: Compared to Example 2, the difference lies in the absence of chitosan in the formulation; instead, an equal amount of diatomaceous earth is used to replenish the original chitosan mass fraction, while all other aspects remain the same. This comparative example decouples the "chemical lock" mechanism to verify whether organic acids are prematurely released upon contact with water in the absence of chitosan crosslinking.

[0067] Comparative Example 3: Compared to Example 2, the difference lies in that L-malic acid is not added to the formulation, and an equal amount of diatomaceous earth is used to make up the original mass fraction of L-malic acid; all other aspects are the same. This comparative example strips away the complexation driving force to verify whether insoluble trace elements and chitosan alone can provide sufficient soluble nutrients during seed germination.

[0068] Comparative Example 4: Compared to Example 2, the difference lies in replacing all talc powder in the formulation with an equal mass of light calcium carbonate; all other aspects remain the same. This comparative example is used to verify the destructive effect of misuse of conventional alkaline fillers on the slightly acidic in-situ crosslinking response system of the present invention.

[0069] Comparative Example 5: Compared to Example 2, the difference lies in the absence of naringenin; the matrix used in the preparation of the coating powder was simply guar collagen powder sprayed with anhydrous ethanol and dried, while all other aspects remained the same. This comparative example stripped the biological signal inducer to verify the effect of the absence of specific nodulation signaling molecules on the overall nitrogenase activity.

[0070] Test Example 1: In-situ crosslinking and leaching resistance test 50.0 g of each of the coated seeds prepared in Examples 1 to 3, Comparative Example 1, and Comparative Example 2 were weighed and placed in glass chromatography columns containing 500 mL of 25°C deionized water, with the water flow rate kept still. 10 mL samples were taken after 1 hour, 12 hours, and 24 hours of extraction, with an equal volume of deionized water added to the column after each sampling. The concentration of free L-malic acid in the water samples was determined by high-performance liquid chromatography (HPLC), and the concentration of iron in the water samples was determined by atomic absorption spectrometry (AAS). The cumulative dissolution rate of each component was calculated based on the initial coating loading.

[0071] Two phosphate buffer solutions with a pH of 6.5 were prepared, each with a volume of 200 mL. System A was a pure buffer solution, while System B consisted of a buffer solution supplemented with a commercially available multi-source chitinase mixture at a concentration of 50 U / L. 10.0 g of the coated seeds prepared in Example 2 were added to both System A and System B, and the solutions were placed in a constant-temperature shaking incubator at 25°C and shaken at 100 rpm. Samples were taken at 6, 24, and 48 hours, and the concentration of available complexed iron in the buffer solutions was determined using the o-phenanthroline spectrophotometric method.

[0072] Plastic pots filled with washed quartz sand were used, with 15 alfalfa seeds treated with different coatings and 15 uncoated awnless brome seeds evenly sown in each pot. The experimental groups included Example 2, Comparative Example 1, and Comparative Example 3. Iron-deficient Hoagland nutrient solution was periodically applied to the plastic pots to maintain moisture. After 35 days of growth under greenhouse conditions, the entire legume and grass plants were harvested, the quartz sand around the roots was rinsed with deionized water, and the plants were dried to constant weight. The iron accumulation in the aboveground parts of both plants was determined using microwave digestion-inductively coupled plasma atomic emission spectrometry (ICP-AES).

[0073] Slightly alkaline, nitrogen-poor weathered soil was used as the test soil for potting. Seeds from Examples 1 to 3, and Comparative Examples 1, 4, and 5 were sown. An equal volume of *Rhizobium sinense* suspension from the same batch of alfalfa was simultaneously inoculated with water at sowing. After 45 days of growth under standard water management, the plant entered its peak nodulation period. Samples were taken to count the number of effective nodules (pinkish interior) on the main root and lateral roots of each plant. Fresh roots with nodules were placed in sealed culture bottles with rubber septa, and acetylene gas was injected to fill 10% of the culture bottle volume. The plants were incubated at 28°C for 2 hours, and headspace gas was extracted and injected into a gas chromatograph to determine ethylene production to characterize nitrogenase activity.

[0074] Table 1. Test data on the cumulative dissolution rate of L-malic acid and iron in coated seeds.

[0075] Table 2. Test data on complexed iron release concentration in different buffer systems in Example 2.

[0076] Table 3. Test data on iron enrichment in the aboveground parts of different plants under mixed sowing conditions.

[0077] Table 4. Data on nodule count and nitrogenase activity per alfalfa plant in pot experiments.

[0078] Table 1 shows that in a deionized water environment without chitinase, the dissolution rates of L-malic acid and iron in Examples 1 to 3 were lower than those in Comparative Example 2. After the coating matrix came into contact with water, the internal pH value decreased, and the chitosan amino group protonated and reacted with the L-malate anion via electrostatic interaction to form an insoluble polyionic composite gel. Free organic acids were immobilized within the coating layer, blocking the reaction pathway for the organic acids to dissolve ferric pyrophosphate. In Comparative Example 2, no chitosan crosslinking precursor was added, and L-malic acid continued to dissolve within 24 hours. Comparative Example 1 used readily soluble iron salts, and iron was lost during pure water extraction.

[0079] Table 2 shows that in system A without chitinase, the concentration of released trace elements changed little over time. In system B with chitinase, the iron release concentration was higher than in system A. The enzymatic reaction degraded the polyionic composite gel, released electrostatic blockage, and released L-malic acid. The released L-malic acid formed a coordination complex with ferric pyrophosphate, converting the insoluble phase into a soluble complex.

[0080] Table 3 shows that in Comparative Example 1, which used readily soluble micronutrient fertilizer, awnless bromegrass (a type of grass) absorbed the iron provided by the system, while alfalfa had a lower iron accumulation than awnless bromegrass. Example 2 utilized the chitinase secreted by the rhizosphere of leguminous plants to induce polyionic complex gel degradation when the leguminous roots came into contact with the coating; alfalfa had a higher iron content than awnless bromegrass. Comparative Example 3 did not contain L-malic acid, lacking the complexation and dissociation effects of organic acids; therefore, the iron absorption of both plants was lower than in Example 2.

[0081] Table 4 shows that the number of nodules and nitrogenase activity in Examples 1 to 3 were higher than those in Comparative Examples 1, 4, and 5. Comparative Example 4 used calcium carbonate filler, which neutralized the slightly acidic environment inside the coating, blocking the protonation cross-linking reaction, resulting in a decrease in the number of nodules and nitrogenase activity. Comparative Example 5 did not contain naringenin, thus lacking the nodulation-inducing signaling molecule, affecting the rhizobium infection efficiency, and the number of effective nodules per plant was lower than in Example 2.

Claims

1. A seed coating method for improving nitrogen fixation efficiency in mixed soybean-grass grasslands, characterized in that, The raw material components comprise the following parts by weight: 5.0-8.0 parts of ferric pyrophosphate; 1.0-2.0 parts of ammonium molybdate tetrahydrate; L-malic acid 10.0-15.0 parts; Chitosan 5.0-8.0 parts; Naringin 0.1-0.2 parts; Guar collagen powder 15.0-20.0 parts; 40.0-50.0 parts of diatomaceous earth; 10.0-15.0 parts of talc.

2. The seed coating according to claim 1, characterized in that, The chitosan has a degree of deacetylation ≥ 85.0% and a weight-average molecular weight (Mw) in the range of 50 kDa to 150 kDa; the iron pyrophosphate has a particle size (D50) in the range of 2.0 to 5.0 μm.

3. The seed coating according to claim 1, characterized in that, The 1% aqueous solution of the guar collagen powder has a viscosity range of 3000-5000 mPa·s at 25°C and a fineness passing through a 200-mesh sieve; the diatomaceous earth is a calcined product with a specific surface area ≥20 m² / g and a porosity ≥80.0%.

4. The seed coating according to claim 1, characterized in that, The naringenin and the guar gum powder exist in the form of a naringenin-preloaded modified guar gum matrix, which is prepared by uniformly spraying a naringenin-ethanol solution onto the surface of the guar gum powder and then vacuum drying to remove the solvent.

5. A method for preparing a seed coating to improve nitrogen fixation efficiency in soybean-grass mixed-sowing grassland as described in any one of claims 1-4, characterized in that, Includes the following steps: S1, Preparation of inorganic-organic biochemical composite dry powder: Ferric pyrophosphate, ammonium molybdate tetrahydrate, L-malic acid, chitosan, diatomaceous earth and talc are sequentially added into a three-dimensional motion mixer to obtain a uniformly mixed composite dry powder. S2, Preparation of coated powder: The composite dry powder obtained in step S1 is dry-mixed with naringenin preloaded modified guar gum matrix to obtain the final coated powder. S3, Synchronous Alternating Coating: Sterile pure water and the final coating powder prepared in step S2 are alternately sprayed onto the surface of the rotating seeds. S4, In-situ crosslinking and hot air curing: After the coating powder is added, the coating machine continues to run at low speed to carry out the in-situ reaction. Then, dry hot air is continuously blown and dried, cooled and discharged to obtain the finished coated seeds.

6. The preparation method according to claim 5, characterized in that, Step S2 includes a pretreatment step of naringenin-preloaded modified guar gum matrix, specifically: Weigh naringenin and add it to anhydrous ethanol and stir magnetically until a clear and transparent naringenin-ethanol solution is formed. Using a high-pressure atomizing nozzle, spray the clear and transparent naringenin-ethanol solution evenly onto the surface of guar collagen powder, and then transfer it to a vacuum drying oven and dry it for 2.0-4.0 hours at a drying temperature of 40-45℃ and a vacuum degree of -0.08MPa to -0.09MPa.

7. The preparation method according to claim 5, characterized in that, In step S1, the mixing operation is carried out under controlled conditions with relative humidity ≤30% and temperature ≤25℃.

8. The preparation method according to claim 5, characterized in that, The specific implementation method of step S3 is as follows: The initial spraying volume is 3.0%-4.0% of the seed dry weight of sterile pure water to evenly moisten the seed surface. Then, the spraying and powdering operations are repeated alternately for 3-5 cycles, controlling the total water added during the entire coating process to be 8.0%-12.0% of the seed dry weight.

9. The preparation method according to claim 5, characterized in that, In step S4, the duration of the in-situ reaction is controlled to be 5.0-10.0 minutes; the temperature of the incoming dry hot air is 35-40℃.

10. The preparation method according to claim 5, characterized in that, In step S4, the degree of drying is controlled so that the moisture content of the finished coated seeds drops below 8.0%; the seeds mentioned in step S3 are alfalfa seeds or white clover seeds.

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