Organic compound fertilizer for synergistic growth of vetch and its preparation method

Abstract

This invention relates to the field of fertilizer technology and discloses an organic compound fertilizer for the synergistic growth of grasses and legumes and its preparation method. It aims to solve the problem that existing synergistic fertilizer technologies neglect the synchronous activation of the root signal response system of grass crops, leading to a mismatch between signal transmission and crop response. By combining a compound microbial agent, a dual-signal inducing factor composed of flavonoid signaling substances and the siderophore analog PDMA, a grass root activator composed of apigenin and quercetin, and a compound organic carrier, and employing a three-layer encapsulation structure, the outer layer of signaling substances rapidly releases and recruits beneficial rhizosphere microorganisms, the middle layer of activator slowly releases and synchronously activates the grass root response system, and the core layer of organic carrier provides a stable habitat for functional microbial communities. Under conditions of 50% nitrogen reduction, it can achieve grass crop yields comparable to conventional fertilization, while also improving legume crop yields and phosphorus absorption efficiency in grasses.

Description

Technical Field

[0001] This invention relates to the field of fertilizer technology, and in particular to an organic compound fertilizer for the synergistic growth of grasses and beans, and its preparation method. Background Technology

[0002] Intercropping, rotation, or mixed planting of grasses and leguminous crops is a classic sustainable planting model. It utilizes the biological nitrogen-fixing properties of leguminous crops to provide nitrogen nutrition for grasses, while simultaneously activating insoluble phosphorus in the soil through root exudates, improving the rhizosphere microecological environment, thus achieving the dual goals of reducing chemical fertilizer application and improving soil quality. How to further enhance the synergistic effect between grasses and leguminous crops and reduce the input of chemical nitrogen and phosphorus fertilizers is a key technological direction for green agricultural development. Research shows that the essence of grass-leguminous synergy lies in the optimized regulation of the rhizosphere microecological system, that is, improving the efficiency of crop nutrient acquisition by targeted improvement of the rhizosphere microbial community structure. Therefore, developing functional fertilizer products that can actively regulate the rhizosphere microecology has become a research hotspot in this field.

[0003] To achieve proactive regulation of the rhizosphere microecology, existing technologies attempt to add signaling substances to fertilizers to target and recruit beneficial microorganisms. For example, Chinese patent application CN119977674A discloses a flavonoid-induced microbial synergistic nitrogen-fixing fertilizer and its preparation method, which uses flavonoids as signaling molecules to actively recruit beneficial microorganisms such as rhizobia to accumulate in the rhizosphere of gramineous crops, thereby establishing a synergistic relationship between gramineous and legume crops in advance. However, the team led by Tian Jiang at South China Agricultural University published a paper in *Plant, Cell & Environment* in February 2026 entitled "Flavonoid-Mediated Recruitment of Bradyrhizobium Enhances Maize Root Development and Nutrient Acquisition in Maize-Soybean Intercropping". The study "Systems" reveals that after flavonoids recruit slow-growing rhizobia to the rhizosphere of maize, the slow-growing rhizobia need to further regulate the flavonoid metabolic pathways of maize roots and synergistically activate the expression of various nutrient transport-related genes in order to truly promote maize root growth and improve phosphorus absorption efficiency. This indicates that the acceptance and utilization of beneficial rhizosphere bacteria by gramineous crops is a process that requires the synchronous response of their own metabolic system, rather than just the physical enrichment of microorganisms in the rhizosphere.

[0004] It is evident that existing synergistic fertilizer technologies for grasses and legumes primarily focus on recruiting beneficial rhizosphere microorganisms through exogenous signaling substances, while neglecting the synchronous activation of the signal response system of the grass crop's own root system. Because grass crops have not developed a proprietary mechanism for symbiosis with rhizobia during evolution, their ability to respond to flavonoid signals is relatively weak and their response speed is slow, resulting in a mismatch in time and function between signal transmission and crop response. When the flavonoids added to the fertilizer are released, although a large number of beneficial microorganisms accumulate in the rhizosphere, if the metabolic pathways of the grass crop's own root system are not synchronously awakened, it cannot effectively perceive and utilize the nutrient benefits released by these microorganisms, thus restricting the synergistic effect. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the existing technology of synergistic fertilization of grass and beans only focuses on the recruitment of beneficial microorganisms in the rhizosphere and ignores the synchronous activation of the signal response system of the root system of grass crops, resulting in a mismatch between signal transmission and crop response, and making it difficult for grass crops to effectively utilize the nutrients released by the beneficial microbial community in the rhizosphere. To this end, we propose an organic compound fertilizer for synergistic growth of grass and beans and its preparation method.

[0006] To achieve the above objectives, this application adopts the following technical solution: an organic compound fertilizer for the synergistic growth of grass and beans, the fertilizer being composed of compound microbial agents, dual-signal inducing factors, grass root activators, compound organic carriers and excipients, each component comprising, by weight, 5-10 parts of compound microbial agents, 0.15-0.7 parts of dual-signal inducing factors, 0.4-1.6 parts of grass root activators, 51-93 parts of compound organic carriers, and 7.1-21.3 parts of excipients, the dual-signal inducing factors being composed of flavonoid signaling substances and proline-2-deoxymethylene acid.

[0007] Preferably, the compound microbial agent is composed of soybean slow-growing rhizobium, rhizocystis heterophylla, and Pseudomonas fluorescens in a live bacteria ratio of 2-3:1:1.

[0008] Preferably, the flavonoid signaling substance is selected from at least one of daidzein and genistein.

[0009] Preferably, the grass root activator is composed of apigenin and quercetin, which exist in the form of an inclusion complex. The inclusion complex is selected from one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin.

[0010] Preferably, the composite organic carrier is composed of decomposed straw biochar, humic acid and trace elements, wherein the trace elements are composed of zinc sulfate heptahydrate, borax decahydrate and ammonium molybdate tetrahydrate mixed in a mass ratio of 5:3:2.

[0011] Preferably, the compound microbial agent contains a protectant, which is composed of trehalose and polyvinylpyrrolidone in a mass ratio of 3:1.

[0012] Preferably, the excipients include binders, dispersants, protectants, and encapsulating agents.

[0013] Preferably, the binder is selected from one or more of sodium alginate, sodium carboxymethyl cellulose, polyvinyl alcohol, and starch.

[0014] Preferably, the dispersant is selected from one or more of Tween-80, sodium dodecyl sulfate, and polyethylene glycol.

[0015] A method for preparing an organic compound fertilizer for the synergistic growth of grass and beans includes the following steps: S1: Dissolving flavonoid signaling substances in anhydrous ethanol and proline-2-deoxymethylene acid in deionized water, mixing them and adding a dispersant to obtain a dual-signal mixed solution; S2: Mixing and granulating decomposed straw biochar, humic acid and trace elements, and drying to obtain organic carrier particles as the core layer; S3: Preparing compound bacterial powder into a bacterial suspension, spraying it onto the surface of the core layer particles, and drying to obtain inner-layer coated particles; S4: Preparing apigenin and quercetin into an inclusion complex with an inclusion agent, preparing a suspension, spraying it onto the surface of the inner-layer coated particles, controlling the coating weight gain at 2-3%, and drying to obtain middle-layer coated particles; S5: Feeding the middle-layer coated particles into a fluidized bed coating machine, spraying with the dual-signal mixed solution, controlling the coating weight gain at 3-5%, and drying to obtain the finished fertilizer product.

[0016] The technical effects and advantages of this invention are as follows: In this invention, a three-layered envelope structure enables temporal synergy between signal recruitment and root response. Flavonoid signaling substances and the siderophore analog PDMA are rapidly released after application to the soil. The flavonoid signaling substances directionally recruit beneficial microorganisms such as slow-growing rhizobia to accumulate in the rhizosphere of gramineous crops. PDMA, in turn, enriches functional microbial communities such as actinomycetes by reconstructing the rhizosphere microbial network, thereby enhancing the connectivity and stability of the microbial community. Apigenin and quercetin are slowly released in the form of inclusion complexes, acting on the roots of gramineous crops to upregulate the expression of key genes in the flavonoid metabolic pathway and relieve the inhibition of auxin polar transport by endogenous flavonols. Simultaneously, it synergistically activates phosphorus transporter genes, causing crop roots to enter a signal response activation state. After root activation, the compound microbial agent colonizes extensively. Soybean slow-growing rhizobium, Rhizocystis heterophylla, and Pseudomonas fluorescens respectively play the functions of biological nitrogen fixation, mycelial network-mediated nutrient transfer, and phosphorus solubilization and growth promotion, forming a complementary chain of nitrogen fixation and phosphorus solubilization. This solves the matching problem between microbial recruitment and crop root response. Under the condition of 50% nitrogen reduction, the yield of gramineous crops is the same as that of conventional fertilization, phosphorus absorption efficiency is improved, the abundance of rhizosphere actinomycetes and the connectivity of microbial networks are increased, and it is applicable to various gramineous-legume co-cultivation systems. Attached Figure Description

[0017] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts: Figure 1 This is a curve showing the cumulative release rate of daidzein in the fertilizer sample of the present invention. Figure 2 This is a curve showing the cumulative release rate of apigenin in the fertilizer sample of the present invention. Detailed Implementation

[0018] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0019] This invention provides a technical solution: an organic compound fertilizer for the synergistic growth of grass and beans, the components of which and their weight parts are shown in Table 1 below.

[0020]

[0021] Table 1 The compound microbial agent is composed of soybean slow-growing rhizobium (… Bradyrhizobium japonicum ), Heteromorpha rhizocystis ( Rhizophagus irregularis DAOM197198 ) and fluorescent pseudomonas ( Pseudomonas fluorescens It is formulated according to the ratio of live bacteria.

[0022] The soybean slow-growing rhizobium used was strain CGMCC 1.2550, preserved at the China General Microbiological Culture Collection Center (CGMCC). This strain is a standard rhizobium strain with typical biological nitrogen fixation ability and the potential to regulate the expression of genes in the roots of non-leguminous crops. It was cultured at 28°C with shaking for 48 hours. The strain was cultured using Rhizobium (YMA) medium, which included 1.0g yeast extract, 10.0g mannitol, 200mL soil extract, 15.0g agar, and 800mL distilled water, with a pH of 7.2. The preparation steps of the soil extract included taking 50g of soil, adding 200mL of water, sterilizing at 121℃ for 1 hour, filtering, and then adding water to make up to 200mL.

[0023] The *Heteromorpha rhizocarpium* strain used was the internationally recognized type strain DAOM 197198. This strain is the type species for the study of arbuscular mycorrhizal fungi and has a typical ability to form hyphal networks. It was cultured on MSR medium and incubated statically at 25°C for 21 days. Spores were collected, with a spore density of ≥200 spores / g and an infection rate of ≥80%.

[0024] The fluorescent Pseudomonas strain used was strain CGMCC1.1802, which was preserved at the China General Microbiological Culture Collection Center (CGMCC). This strain has typical phosphate solubilization and IAA synthesis capabilities, with a phosphate solubilization zone diameter / colony diameter ≥2.0 and IAA synthesis amount ≥10 mg / L. It was cultured at 30℃ with shaking for 24 hours. The culture was performed using nutrient gravy agar medium, which consisted of 10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, and 1.0 L distilled water, with a pH of 7.0.

[0025] The ratio of viable bacteria of *Saccharomyces cerevisiae*, *Rhizocystis heterophylla*, and *Pseudomonas fluorescens* is 2-3:1:1, preferably 2:1:1.

[0026] The three types of bacteria were cultured to the logarithmic growth phase, and the bacterial cells were collected by centrifugation. They were then mixed in a certain proportion and a protectant was added. The protectant consisted of trehalose and polyvinylpyrrolidone in a mass ratio of 3:1, and the amount of protectant added was 20% of the wet weight of the bacterial cells. The uniformly mixed bacterial cells were freeze-dried: pre-freezing at -40℃ for 4 hours; sublimation drying at -20℃ for 24 hours; and desorption drying at 25℃ for 6 hours. After drying, the cells were pulverized and passed through an 80-mesh sieve to obtain a compound bacterial powder, with the total viable count controlled at ≥1×10⁻⁶. 9 CFU / g.

[0027] The dual-signal inducing factor is composed of a flavonoid signaling substance and a siderophore analog PDMA, wherein the flavonoid signaling substance is selected from genistein ( Daidzein ), genistein ( Genistein At least one of the following is a commercially available, conventional reagent:

[0028] The chemical formula of the genistein is C. 15 H 10 O4, CAS number 486-66-8, molecular weight 254.24, purity ≥98%, is an isoflavone compound, one of the main signaling molecules secreted by the roots of leguminous plants, and has a significant chemotactic induction effect on rhizobia. The chemical formula of the genistein is C. 15 H 10O5, CAS number 446-72-0, molecular weight 270.24, purity ≥98%, is also an isoflavone compound, which has the function of recruiting rhizobia and can participate in the regulation of plant-microbe interaction signaling networks. When daidzein and genistein are used simultaneously, the preferred mass ratio of daidzein to genistein is 1:1.

[0029] The specific siderophore analog PDMA is proline-2-deoxymethylene acid (PDMA). Proline-2'- deoxymugineic acid ), is a synthetic plant siderophore analogue. Structurally, it is based on deoxymethylene acid (DMA) secreted by natural grass plants, but the five-membered ring structure of L-proline replaces the four-membered ring structure of L-azacyclobutane-2-carboxylic acid in natural DMA, thereby enhancing its chemical stability. Starting with L-proline, the synthesis process involved amino protection, carboxyl reduction, amino introduction, coupling with a side-chain precursor, deprotection, and hydrolysis. The specific synthetic steps are as follows: C1: Weigh 11.5 g of 0.1 mol L-proline and dissolve it in 200 mL of dioxane aqueous solution. Cool the solution to 0 °C in an ice bath. Add 20.2 g of 0.2 mol triethylamine and stir for 10 minutes. Slowly add 26.2 g of 0.12 mol ditert-butyl dicarbonate dissolved in 50 mL of dioxane solution. After the addition is complete, stir the reaction at room temperature for 4 hours. Monitor the reaction progress using thin-layer chromatography. After the reaction is complete, remove the solvent under reduced pressure. Add saturated citric acid solution to the residue to adjust the pH to 2-3. Extract three times with 100 mL of ethyl acetate each time. Combine the organic phases, dry over anhydrous sodium sulfate, filter, and evaporate the filtrate under reduced pressure to obtain a white solid N-tert-butyloxycarbonyl-L-proline, with a yield of 92%. C2: Weigh 21.5 g of the C1 product and dissolve it in 200 mL of anhydrous tetrahydrofuran. Cool to -15 °C under nitrogen protection. Slowly add 11.1 g of 0.11 mol N-methylmorpholine and 15.0 g of 0.11 mol isobutyl chloroformate. Stir for 30 minutes, filter to remove the precipitate, and cool the filtrate to -15 °C. Slowly add a solution of 5.7 g of 0.15 mol sodium borohydride dissolved in 50 mL of water. Continue stirring for 1 hour after the addition is complete. Monitor the reaction progress using thin-layer chromatography. After the reaction is complete, add 100 mL of water to quench the reaction. Extract three times with 100 mL of ethyl acetate each time. Combine the organic phases, dry with anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness under reduced pressure to obtain N-tert-butoxycarbonyl-L-proline alcohol. The obtained product can be used directly in the next reaction without purification. C3: The C2 product was dissolved in 150 mL of anhydrous dichloromethane and cooled to 0 °C in an ice bath. 15.2 g of 0.15 mol triethylamine was added, followed by the slow addition of 13.7 g of 0.12 mol methanesulfonyl chloride dissolved in 50 mL of dichloromethane. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, 100 mL of water was added for washing. The organic phase was washed successively with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain N-tert-butoxycarbonyl-L-proline methanesulfonate. C4: Weigh 29.3 g of the C3 product and dissolve it in 150 mL of anhydrous N,N-dimethylformamide. Add 9.8 g of 0.15 mol sodium azide and stir at 60 °C for 4 hours. Monitor the reaction progress using thin-layer chromatography. After the reaction is complete, add 200 mL of water and extract three times with 100 mL of ethyl acetate each time. Combine the organic phases, dry with anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness under reduced pressure. Dissolve the residue in 100 mL of methanol, add 2 g of 10% palladium on carbon catalyst, and hydrogenate at room temperature and atmospheric pressure for 4 hours. Filter to remove the catalyst and evaporate the filtrate to dryness under reduced pressure to obtain an amino compound derived from N-tert-butoxycarbonyl-L-proline. The obtained product can be used directly in the next reaction without purification. C5: Weigh 20.3 g of C4 product and dissolve it in 200 mL of anhydrous acetonitrile. Add 27.6 g of 0.2 mol anhydrous potassium carbonate and 27.5 g of 0.12 mol benzyl bromoacetate. Reflux for 8 hours. Monitor the reaction progress using thin-layer chromatography. After the reaction is complete, filter the solution. Evaporate the filtrate to dryness under reduced pressure. Purify the residue by silica gel column chromatography using petroleum ether:ethyl acetate at a volume ratio of 4:1 as the eluent. Collect the target component and evaporate it to dryness under reduced pressure to obtain the side-chain precursor. C6: Weigh 0.08 mol of C4 product and 0.08 mol of C5 product and dissolve them in 200 mL of anhydrous acetonitrile. Add 22.1 g of 0.16 mol of anhydrous potassium carbonate and 1.3 g of 0.008 mol of potassium iodide. Reflux for 6 hours. Monitor the reaction progress using thin-layer chromatography. After the reaction is complete, filter the solution and evaporate the filtrate to dryness under reduced pressure. Purify the residue by silica gel column chromatography, collect the target component, and evaporate to dryness under reduced pressure to obtain the coupling product. Dissolve the coupling product in 100 mL of a 1:1 mixture of trifluoroacetic acid and dichloromethane. Stir at room temperature for 2 hours to remove the tert-butyloxycarbonyl protecting group. Monitor the reaction progress using thin-layer chromatography. After the reaction is complete, evaporate to dryness under reduced pressure to obtain the deprotected product. C7: The deprotected product was dissolved in 100 mL of methanol, and 10 mL of 5M sodium hydroxide solution was added. The mixture was stirred at room temperature for 2 hours to hydrolyze the ester group. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the pH was adjusted to 2-3 with 1M hydrochloric acid, and a white precipitate was formed. The precipitate was allowed to stand overnight at 4°C, filtered, washed with a small amount of cold water, and vacuum dried to obtain crude PDMA. The crude product was dissolved in a small amount of hot water and purified by preparative high-performance liquid chromatography (HPLC). A C18 reversed-phase column was used as the stationary phase, and a water, acetonitrile, and trifluoroacetic acid system was used as the mobile phase. The target fraction was collected, lyophilized, and high-purity white PDMA powder was obtained. The total yield based on L-proline was approximately 45%, and the purity was ≥95% as determined by HPLC.

[0030] The root activator of the grass family is composed of apigenin ( Apigenin ) and quercetin ( Quercetin The composition consists of commercially available, standard reagents. Among them, apigenin has the chemical formula C. 15 H 10 O5, CAS number 520-36-5, molecular weight 270.24, purity ≥98%, is a natural flavonoid compound that can act as an exogenous signaling molecule to upregulate the expression of key genes in the flavonoid metabolism pathway in the roots of grasses, such as chalcone synthase CHS and chalcone isomerase CHI, and relieve the inhibition of endogenous flavonols on the polar transport of auxin. Quercetin has the chemical formula C 15 H 10 O7, CAS number 117-39-5, molecular weight 302.24, purity ≥98%, is a natural flavonol compound that can synergistically activate the expression of phosphorus transporter genes in the roots of grass crops with apigenin, thereby improving their phosphorus absorption efficiency.

[0031] Since apigenin and quercetin have poor water solubility, in order to improve their release efficiency and stability in soil, it is preferable to prepare them as inclusion complexes. The inclusion agent is selected from one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin, with β-cyclodextrin being preferred.

[0032] The specific method is as follows: Mix apigenin and quercetin at a mass ratio of 1:1. Dissolve β-cyclodextrin in deionized water at 50-60℃ to prepare a 20% solution. Add the mixture to the β-cyclodextrin solution at a mass ratio of 1:5. Stir at 50℃ for 2 hours, then let stand at 4℃ for 12 hours. Filter, vacuum dry at 40℃ for 12 hours, and pulverize through a 100-mesh sieve to obtain a powdered inclusion complex.

[0033] The composite organic carrier is composed of decomposed straw biochar, humic acid and trace elements, all of which are commercially available conventional reagents. The biochar is a carbon-rich product made from wheat, corn, or rice straw through oxygen-limited pyrolysis, with a pH of 7.5-8.5 and a specific surface area ≥50 m². 2 / g, cation exchange capacity ≥20cmol / kg, organic carbon content ≥40%, ash content ≤30%; The humic acid is commercially available mineral-derived humic acid or biochemical humic acid, preferably mineral-derived humic acid, with a total humic acid content ≥50%, fulvic acid content ≥30%, moisture content ≤15%, and pH 5.5-7.5. The trace elements consist of zinc sulfate heptahydrate (ZnSO4·7H2O), borax decahydrate (Na2B4O7·10H2O), and ammonium molybdate tetrahydrate [(NH4)6Mo7O]. 24 The ·4H2O] were mixed in a mass ratio of 5:3:2, and all were of analytical grade. Among them, the CAS number of zinc sulfate heptahydrate was 7446-20-0, and the Zn content was ≥22%; the CAS number of borax decahydrate was 1303-96-4, and the B content was ≥11%; and the CAS number of ammonium molybdate tetrahydrate was 12054-85-2, and the Mo content was ≥54%.

[0034] The auxiliary materials are selected from binders, dispersants, protectants, encapsulating agents, etc., commonly used in the field, and are used to improve the physical properties of fertilizers and the survival rate of microorganisms; The binder is selected from one or more of sodium alginate, sodium carboxymethyl cellulose, polyvinyl alcohol, and starch, with sodium alginate being preferred, to enhance the strength of the granules, prevent the fertilizer from breaking during transportation and use, and at the same time help the functional layers to adhere evenly to the surface of the granules. The dispersant is selected from one or more of Tween-80, sodium dodecyl sulfate, and polyethylene glycol, with Tween-80 being preferred, to improve the uniformity of the coating solution and ensure that functional components such as signaling substances and activators are evenly distributed on the particle surface.

[0035] This invention also provides a method for preparing an organic compound fertilizer that promotes the synergistic growth of grasses and beans, specifically including the following steps: S1: Dissolve the flavonoid signaling substance in anhydrous ethanol to prepare a 5 g / L solution, and dissolve the siderophore analog PDMA in deionized water to prepare a 2 g / L solution. Mix the two solutions at a volume ratio of 1:1, add 0.5% of the total mass of the mixture as a dispersant, stir evenly, and prepare a dual-signal mixed solution for later use. S2: Weigh the decomposed straw biochar, humic acid, and trace elements and mix them thoroughly in a mixer. Add 5-10% of the total mass of deionized water and granulate using a disc granulator or extrusion granulator, controlling the particle diameter to 1.5-2.0 mm. Place the resulting particles in a 60℃ drying oven and dry them until the moisture content is ≤8%. Sieve and grade the particles, taking the organic carrier particles between 10-20 mesh for later use. S3: Prepare a bacterial suspension by mixing the composite bacterial powder with an aqueous solution containing 0.5% binder. The bacterial powder accounts for 20% of the total mass of the bacterial suspension. Put the core layer particles into a fluidized bed coating machine. Set the inlet air temperature to 40-45℃, the material temperature to 35-38℃, the atomization pressure to 0.2-0.3MPa, and the spraying rate to 5-10mL / min. Spray the bacterial suspension to ensure that the bacterial powder is evenly loaded on the surface of the core layer particles and in the surface pores. Control the coating weight gain until the bacterial powder is fully loaded. After coating, continue fluidized drying for 5-10 minutes to obtain inner layer coated particles for later use. S4: Prepare a 10% suspension of the powdered inclusion complex with deionized water, place it in a fluidized bed coating machine, feed the inner layer coated particles into the fluidized bed, spray the inclusion complex suspension with the same process parameters as S3, control the coating weight gain to 2-3%, and continue fluidized drying for 5-10 minutes after coating to obtain the middle layer coated particles. S5: Put the intermediate coated granules into a fluidized bed coating machine and spray the dual-signal mixed solution according to the same process parameters as S3. Control the coating weight gain to 3-5%. After coating, continue fluidized drying for 5-10 minutes. Fluidize and dry the granules at 40℃ until the moisture content is ≤5%. Pass them through a 10-20 mesh sieve to remove oversized or undersized granules, and the fertilizer product is obtained.

[0036] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art should understand that various modifications and changes can be made to the present invention without departing from the technical principles of the present invention, and these modifications and changes should also fall within the scope of protection of the present invention. Example

[0037] This embodiment provides an organic compound fertilizer for the synergistic growth of soybean and grass, the components of which, by weight, include: 8.0 parts of compound microbial agent, wherein the live bacteria ratio of soybean slow-growing rhizobium, Rhizocystis heterophylla and Pseudomonas fluorescens is 2:1:1; 0.3 parts of daidzein; 0.3 parts of genistein; 0.1 parts of siderophore analog PDMA; 0.5 parts of apigenin; 0.5 parts of quercetin; 40.0 parts of decomposed straw biochar; 30.0 parts of humic acid; 2.0 parts of trace element mixture; 0.2 parts of binder sodium alginate; 3.0 parts of dispersant Tween-80; 3.0 parts of protectant; and 5.0 parts of inclusion agent β-cyclodextrin. This embodiment also provides a method for preparing an organic compound fertilizer that promotes the synergistic growth of grasses and beans, including the following steps: S1: Dissolve daidzein and genistein in anhydrous ethanol to prepare a 5 g / L solution, and dissolve the iron carrier analog PDMA in deionized water to prepare a 2 g / L solution. Mix the two solutions at a volume ratio of 1:1, add 0.5% of Tween-80 by mass of the mixture, and stir until homogeneous to obtain a dual-signal mixed solution.

[0038] S2: Weigh the mixture of decomposed straw biochar, humic acid, and trace elements, place it in a mixer and stir thoroughly. Add 8% of the total mass of deionized water, granulate using a disc granulator, and control the particle diameter to 1.5-2.0 mm. Place the resulting particles in a 60℃ drying oven and dry until the moisture content is ≤8%. Sieve and grade the particles, and take the organic carrier particles between 10-20 mesh as the core layer for later use.

[0039] S3: Prepare a bacterial suspension by mixing the compound bacterial powder with an aqueous solution containing 0.5% sodium alginate. The bacterial powder accounts for 20% of the total mass of the bacterial suspension. Put the core layer particles into a fluidized bed coating machine, set the inlet air temperature to 42℃, the material temperature to 36℃, the atomization pressure to 0.25MPa, and the spraying rate to 8mL / min. Spray the bacterial suspension to make the bacterial powder evenly loaded on the surface of the core layer particles and the surface pores. Control the coating weight gain until the bacterial powder is fully loaded. After the coating is completed, continue fluidized drying for 8 minutes to obtain inner layer coated particles.

[0040] S4: Apigenin and quercetin are mixed at a mass ratio of 1:1 and then mixed with β-cyclodextrin at a mass ratio of 1:5 to prepare an inclusion complex powder. The inclusion complex powder is prepared into a 10% suspension with deionized water and placed in a fluidized bed coating machine. The inner layer coated particles are fed into the fluidized bed and the inclusion complex suspension is sprayed with the same process parameters as S3. The coating weight gain is controlled at 2.5%. After the coating is completed, the fluidized bed is dried for another 8 minutes to obtain the middle layer coated particles.

[0041] S5: Put the intermediate coated granules into a fluidized bed coating machine and spray the dual-signal mixed solution according to the same process parameters as S3. Control the coating weight gain to 4%. After coating, continue fluidized drying for 8 minutes. Fluidize and dry the granules at 40℃ until the moisture content is ≤5%. Pass the granules through a 10-20 mesh sieve to obtain the fertilizer product. Example

[0042] This embodiment provides an organic compound fertilizer for the synergistic growth of soybean and grass and its preparation method. The difference from Embodiment 1 is that the ratio of live bacteria of soybean slow-growing rhizobium, Heterocystis heterophylla and Pseudomonas fluorescens in the compound microbial agent is adjusted to 3:1:1. Example

[0043] This embodiment provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Embodiment 1 is that the coating weight gain is 3% in step S4 and 5% in step S5. Example

[0044] This embodiment provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that the total amount of flavonoid signaling substances is 0.2 parts, including 0.1 parts of daidzein, 0.1 parts of genistein, 0.05 parts of siderophore analog PDMA, 0.2 parts of apigenin, 0.2 parts of quercetin, 2% weight gain from coating in S4, and 3% weight gain from coating in S5. Example

[0045] This embodiment provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that the total amount of flavonoid signaling substances is 0.5 parts, including 0.25 parts of daidzein, 0.25 parts of genistein, 0.2 parts of siderophore analog PDMA, 0.8 parts of apigenin, 0.8 parts of quercetin, 3% weight gain from coating in S4, and 5% weight gain from coating in S5.

[0046] Comparative Example 1 This comparative example refers to the preparation of the control fertilizer according to the scheme described in Example 1 of Chinese Patent Application Publication No. CN119977674A. Specifically, 10 parts of flavonoid extract, 5 parts of nitrogen-fixing bacteria agent, 25 parts of organic carrier, 35 parts of inorganic nutrient matrix, and 10 parts of filler were weighed. The flavonoid extract included 5 parts of sweet potato flavonoid extract, 3 parts of soybean flavonoid extract, and 2 parts of tea flavonoid extract. The nitrogen-fixing bacteria agent included 2.5 parts of rhizosphere nitrogen-fixing bacteria and 2.5 parts of leguminous plant rhizobia. The organic carrier included 15 parts of humic acid and 10 parts of biochar. The inorganic nutrient matrix included 17.5 parts of diammonium phosphate and 17.5 parts of potassium sulfate. The filler included 5 parts of diatomaceous earth and 5 parts of bentonite. Organic carrier and inorganic nutrient matrix are added to a mixer and mixed at low speed for 5 minutes. After adding filler, the mixture is mixed at low speed for another 5 minutes. The mixing speed is then increased and the mixture is continued for another 5 minutes. The mixture is then granulated, with the particle size controlled between 2-5 mm. Nitrogen-fixing bacteria and 100 kg of flavonoid extract are added to the mixer and mixed at low speed for 5 minutes to form a flavonoid-microorganism complex liquid. This liquid is then evenly sprayed onto the surface of the material particles using a spraying device, with a spraying amount of 15% of the particle weight. After spraying, the material particles are dried at 35°C for 18 hours to obtain the finished fertilizer product.

[0047] Comparative Example 2 This comparative example provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that the iron carrier analog PDMA is not added.

[0048] Comparative Example 3 This comparative example provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that apigenin and quercetin are not added.

[0049] Comparative Example 4 This comparative example provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that the dual-signal mixed solution, activator inclusion complex powder, compound bacterial powder, organic carrier particles and all auxiliary materials are directly mixed evenly, an appropriate amount of water is added for granulation, and after drying, it is sieved to obtain granular fertilizer without layering structure, without fluidized bed layering coating.

[0050] Comparative Example 5 This comparative example provides an organic compound fertilizer for the synergistic growth of grass and soybean and its preparation method. The difference from Example 1 is that only soybean slow-growing rhizobium is used in the compound microbial agent, and the dosage is still 8.0 parts. It does not contain Rhizocystis heterophylla or Pseudomonas fluorescens.

[0051] Comparative Example 6 This comparative example provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that the dual-signal mixed solution is first sprayed onto the surface of the organic carrier particles, then the inclusion complex suspension is sprayed onto the surface of the inner coated particles, and finally the bacterial suspension is sprayed onto the surface of the middle coated particles.

[0052] Comparative Example 7 This comparative example provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that the decomposed straw biochar and humic acid are replaced with an equal amount of vermiculite with a particle size of 1-2 mm and an amount of 70.0 parts.

[0053] Comparative Example 8 This comparative example provides an organic compound fertilizer for the synergistic growth of grass and beans and its preparation method. The difference from Example 1 is that, instead of preparing apigenin and quercetin into β-cyclodextrin inclusion complexes, 0.5 parts of apigenin and 0.5 parts of quercetin are directly dispersed in water to form a suspension for spraying.

[0054] The beneficial effects of the technical solution of the present invention will be further explained through effect verification experiments below. The following experimental examples are used to evaluate the actual application effect of the fertilizer samples prepared by the embodiments and comparative examples of the present invention in the co-planting system of grass and soybean.

[0055] Experimental Example 1 This experimental example aims to verify the effects of the described organic compound fertilizer on crop yield, nutrient absorption, and nitrogen fertilizer utilization efficiency in a corn-soybean intercropping system under a 50% reduction in nitrogen.

[0056] Fertilizer samples prepared in Examples 1-5 and Comparative Examples 1-8 were selected as experimental subjects, and the tested crops were Xianyu 335 and Jinong 45.

[0057] The field experiment was conducted at the experimental base of the Changchun Academy of Agricultural Sciences in Jilin Province. The soil was typical black soil, and its physicochemical properties were determined to be: organic matter 25.3 g / kg, available nitrogen 112.5 mg / kg, available phosphorus 18.7 mg / kg, available potassium 145.2 mg / kg, and pH 6.8.

[0058] A randomized block design was used, with a total of 13 treatments: Examples 1-5, Comparative Examples 1-8, a conventional fertilization control (CK1), and a blank control (CK2). Each treatment was replicated three times, and the plot area was 30m². 2 The rows are randomly arranged with a bandwidth of 2.4m. The row spacing for corn is about 40cm, the row spacing for soybeans is about 30cm, the distance between corn and soybeans is about 50cm, the plant spacing for corn is about 20cm, and the plant spacing for soybeans is about 10cm.

[0059] The fertilizers used in the embodiments and comparative examples were applied as base fertilizer in a single application at a rate of 600 kg / hm². 2 The standard control group received a total nitrogen application of 172.5 kg / hm². 2 Phosphorus and potassium fertilizers were applied according to local customs. The base fertilizers for each example and comparative treatment were the tested fertilizers. The amount of topdressing was 50% of that of the conventional control. The amount of phosphorus and potassium fertilizers was the same as that of the conventional control. No fertilizer was applied to CK2.

[0060] During the maturity stages of corn and soybeans, the middle two rows of each plot were harvested separately, threshed, weighed, and their moisture content was determined. The moisture content of corn was converted to 14% standard moisture content, and that of soybeans to 13% standard moisture content, and the grain yield was calculated. During the tasseling and maturity stages of corn, and the pod-setting and maturity stages of soybeans, five representative plants were taken from each plot, and their organs were dried, crushed, and digested to determine the total nitrogen and total phosphorus content and calculate the nutrient uptake per unit area. The nitrogen fertilizer partial productivity was calculated as the ratio of corn grain yield to nitrogen application rate. The test results were taken as the arithmetic mean, as shown in Table 2 below.

[0061]

[0062] Table 2 According to the data in Table 2, under the condition of 50% nitrogen reduction, Examples 1-5 showed no significant difference from CK1 and were higher than CK2. The phosphorus uptake of corn in Example 1 was 31.4% higher than that under conventional fertilization, and the nitrogen fertilizer partial productivity was 96.2% higher than that under conventional fertilization. This invention can achieve corn yield comparable to conventional fertilization under the condition of 50% nitrogen reduction by simultaneously activating the root response system of Gramineae and optimizing the rhizosphere microecology, while promoting soybean yield increase and phosphorus absorption.

[0063] Comparative Example 2 showed a 9.9% decrease in corn yield and a 20.7% decrease in phosphorus uptake compared to Example 1. The absence of PDMA, an analogue of siderophores, hindered the enrichment of beneficial bacteria such as rhizosphere actinomycetes, reduced the ability of microbial network reconstruction, affected the activation of trace elements and the efficiency of microbial community collaboration, and consequently affected nutrient absorption and yield formation.

[0064] Comparative Example 3 showed that both corn yield and phosphorus uptake were lower than in Example 1. The absence of apigenin and quercetin, which are root activators of grasses, resulted in the failure to activate the flavonoid metabolism pathway and phosphorus transporter genes in corn roots. Even if beneficial microorganisms were enriched in the rhizosphere, the crop could not effectively sense and utilize the nutrients they released.

[0065] Comparative Example 5 showed that both corn yield and phosphorus uptake were lower than in Example 1, indicating that relying solely on soybean slow-growing rhizobia could not construct a complete rhizosphere functional microbial community. The lack of mycelial network connection of Rhizocystis heterophylla hindered nutrient transfer between grasses and soybeans. The lack of phosphorus activation by Pseudomonas fluorescens resulted in insufficient phosphorus source supply. The three functional bacteria formed a complementary chain of nitrogen fixation and phosphorus solubilization, and their synergistic effect was far superior to that of a single bacterial agent.

[0066] Comparative Example 4 showed a 9.3% decrease in corn yield compared to Example 1; Comparative Example 6 showed an 8.6% decrease in corn yield compared to Example 1. This indicates that the stratified structure plays a crucial role in the time-sequential release of microorganisms. The outer layer of rapidly released signaling substances recruits microorganisms first, the middle layer of slow-release activators simultaneously awakens the root response, and the core layer of sustained-release microbial communities completes colonization. If the signal and activator are released simultaneously or in reverse order, the time sequence of recruitment followed by activation is disrupted, leading to asynchrony between microbial enrichment and root response, and a decrease in the synergistic effect.

[0067] Comparative Example 7 showed that maize yield and phosphorus uptake were lower than other treatments. The organic carrier composed of biochar and humic acid provided a habitat, slow-release nutrients and stable pH for microorganisms. Its porous structure and organic matter content are irreplaceable by inorganic carriers. The failure of the carrier led to a decrease in the colonization rate and survival rate of functional bacteria, and the construction of rhizosphere microecology failed.

[0068] Comparative Example 8 showed a 10.3% decrease in corn yield compared to Example 1. Apigenin and quercetin have poor water solubility, and when added directly to the soil, they are released too quickly or fixed by soil colloids, making it difficult for them to continuously act on the roots within the window period. β-cyclodextrin inclusion complexes improve the stability and sustained-release properties of the activator, ensuring its effective release within the correct time window.

[0069] Experimental Example 2 This experiment aims to analyze the changes in the structure of the rhizosphere microbial community of maize under different treatments using high-throughput sequencing technology, and to verify the regulatory effect of the fertilizer of this invention on the rhizosphere microecology.

[0070] The soil samples were taken from the rhizosphere soil of maize in each treatment plot during the tasseling stage in Experiment Example 1. The following 6 treatments were set up: Example 1, Comparative Examples 1-4, and blank control CK3. Each treatment was replicated 3 times.

[0071] Five corn plants were randomly selected from each plot. The plants were dug up along with their roots, and the loose soil around the roots was shaken off. The soil tightly attached to the root surface was collected with a sterile brush, mixed evenly, and used as the rhizosphere soil sample for that plot. The sample was stored in a sterile cryovial at -80℃.

[0072] Soil microbial DNA extraction was performed using a soil DNA extraction kit. PCR amplification was conducted targeting the V3-V4 region of the bacterial 16S rRNA gene using primers 338F and 806R. The amplified products were sequenced on an Illumina NovaSeq PE250 platform. After quality control and assembly, the sequencing data were clustered into OTUs with 97% similarity. Species annotation was performed by comparing with the SILVA 138 database, and the phylum Actinobacteria (…) was calculated. Actinobacteria The relative abundance was determined; based on the Spearman correlation coefficient among OTUs, significant correlations with |r|>0.8 and p<0.01 were screened, and the microbial network connectivity was calculated; PICRUSt2 was used to predict the function of the microbial community, and the abundance of nitrogen fixation (nifH) and phosphorus transport (pstS) functional genes was obtained. The results are shown in Table 3 below.

[0073]

[0074] Table 3 According to the data in Table 3, the relative abundance of Actinobacteria in Example 1 was 36.0% higher than that in Comparative Example 2. Comparative Example 2 was slightly higher than that in Comparative Example 1. This indicates that PDMA, as a siderophore analog, can enrich Actinobacteria microorganisms. Actinobacteria contains many groups with functions of promoting growth, resisting disease and activating trace elements. The increase in its abundance helps to build a fully functional rhizosphere beneficial microbial community.

[0075] The microbial network connectivity of Example 1 was 65.2% higher than that of Comparative Example 2. Network connectivity reflects the tightness of interspecies interactions among microorganisms. Higher connectivity indicates stronger cooperation within the microbial community and more stable ecological functions. The absence of PDMA led to a decrease in network connectivity, indicating that PDMA not only enriches specific bacterial groups but also enhances the overall function of the community by reshaping the interactions between bacterial groups.

[0076] Although the abundance of actinomycetes and network connectivity of Comparative Example 3 were lower than those of Example 1, they were higher than those of Comparative Example 2. This indicates that although the activator acts directly on the crop roots, the activation of the root response system can provide feedback regulation of the rhizosphere microbial community, indirectly promoting the enrichment of beneficial bacteria and network construction.

[0077] The abundance and network connectivity of actinomycetes in Comparative Example 4 were both lower than those in Example 1, indicating that the hierarchical structure ensured the orderly release of signaling substances and activators through time-sequential release, enabling microbial enrichment and root activation to proceed simultaneously, thereby optimizing the construction process of the rhizosphere microecology.

[0078] In Example 1, the abundance of nitrogen fixation and phosphorus translocation genes was higher than in other treatments. In Comparative Example 2, the abundance of nitrogen fixation and phosphorus translocation genes decreased by 24.5% and 24.2% respectively compared to Example 1. The changes in the abundance of functional genes were consistent with the trend of changes in the microbial community structure, indicating that PDMA enhanced the rhizosphere microbial-mediated nutrient transformation function by enriching beneficial bacteria such as actinomycetes, thus providing crops with more bioavailable nitrogen and phosphorus.

[0079] Experimental Example 3 This experimental example aims to verify the activating effect of the fertilizer of the present invention on the root signal response system of grasses, especially the upregulation effect of apigenin and quercetin on flavonoid metabolic pathways and phosphorus transport genes.

[0080] The tested root systems were taken from the maize root systems of each treatment plot during the tasseling stage in Experiment Example 1. The following 5 treatments were set up: blank control CK4, Example 1, Comparative Example 1, Comparative Example 3, and Comparative Example 4. Each treatment was repeated 3 times, for a total of 15 samples.

[0081] Five corn plants were randomly selected from each plot. The tender root tips and lateral root development areas were taken, rinsed with sterile water to remove the attached soil, and then flash-frozen in liquid nitrogen and stored at -80℃.

[0082] Frozen root samples were ground in liquid nitrogen, and total RNA was extracted using the Trizol method. 1 μg of total RNA was reverse transcribed to synthesize cDNA. Using the maize GAPDH gene (accession number NM_001111943) as an internal control, the relative expression levels of key genes in the flavonoid metabolism pathway, CHS, CHI, F3H, and FLS, and phosphorus transporter genes ZmPht1;1, ZmPht1;6, and ZmPht1;13, were detected. Primers were synthesized by Sangon Biotech Co., Ltd. The results are shown in Table 4 below.

[0083]

[0084] Table 4 According to the data in Table 4, under the treatment of Example 1, the expression levels of maize root flavonoid metabolism pathway genes CHS, CHI, F3H, and FLS were all higher than those of other treatments. In Comparative Example 3, the expression levels of the above genes were not different from those of CK4. As flavonoid compounds, apigenin and quercetin can be recognized by maize roots as exogenous signaling molecules. They can activate transcription factors through unknown receptor-mediated signal transduction pathways, thereby upregulating the expression of flavonoid synthesis-related genes. The activation of flavonoid metabolism pathways is a prerequisite for crops to establish functional connections with rhizosphere microorganisms, and their products can be used as signaling molecules to further recruit and regulate rhizosphere microbiota.

[0085] In Example 1, the expression levels of phosphorus transporter genes ZmPht1;1, ZmPht1;6, and ZmPht1;13 were all higher than in other treatments. In Comparative Example 3, the expression levels of the above genes were close to those of CK4. The expression of phosphorus transporter genes is regulated by phosphorus starvation signals and transcription factors such as PHR1. Flavonoid metabolites may synergistically activate the phosphorus uptake system by affecting hormone signaling pathways or interacting with transcription factors. This explains the molecular basis for the increased phosphorus uptake in maize in Example 1. The upregulation of phosphorus transporter genes enhances the root system's ability to absorb phosphorus from the soil.

[0086] The gene expression levels of Comparative Example 1 were no different from those of CK4. Due to the lack of exogenous activation signals, the flavonoid metabolic pathway and phosphorus transport system in maize roots were not activated. Even if beneficial microorganisms were enriched in the rhizosphere, the crop could not effectively sense and utilize the nutrients they released. This reveals the reason why the yield and phosphorus uptake of Comparative Example 1 were lower than those of Example 1 at the gene expression level.

[0087] The gene expression levels in Comparative Example 4 were higher than those in CK4 and Comparative Example 1, but still lower than those in Example 1. Without a stratified structure, the simultaneous release of the activator and signaling substance may have two effects: first, the activator is exposed to the soil environment too early, and some of it is degraded by microorganisms or fixed by soil colloids, resulting in a decrease in the effective concentration; second, the simultaneous release of the signaling substance and the activator leads to an overlap of the two processes of microbial enrichment and root activation, rather than an orderly connection. The stratified structure, through sequential release, allows the activator to continue to act on the root system after the release of the signaling substance, thereby maximizing the induction of gene expression.

[0088] Experiment Example 4 This experimental example aims to verify the applicability of the fertilizer of the present invention in different co-planting patterns of Gramineae and Leguminosae, and to prove the versatility of its technical solution.

[0089] Fertilizer samples prepared in Example 1 and Comparative Example 1 were selected, and three sets of test crops and planting systems were set up, namely wheat-peanut, oat-arrow pea and barley-broad bean. The selected varieties were Jimai 22 and Huayu 33, Longyan 3 and Longjian 1, Su Naked Wheat 2 and Tongcanxian 8, respectively.

[0090] Among them, the wheat-peanut test site was the experimental base of Jinan Academy of Agricultural Sciences in Shandong Province. The soil was alluvial soil. The physicochemical properties were measured to be 18.6 g / kg organic matter, 85.3 mg / kg available nitrogen, 16.2 mg / kg available phosphorus, 112.5 mg / kg available potassium, and pH value of 7.3. The oat-arrow pea test site was the experimental base of Lanzhou Agricultural Science and Technology Research Institute in Gansu Province. The soil was loess soil. The physicochemical properties were determined to be: organic matter 12.8 g / kg, available nitrogen 62.5 mg / kg, available phosphorus 11.3 mg / kg, available potassium 142.8 mg / kg, and pH value 8.1. The barley-broad bean test site was the experimental base of the Crop Cultivation Technology Guidance Station in Rudong County, Jiangsu Province. The soil was coastal saline soil. The physicochemical properties were determined to be 15.2 g / kg organic matter, 71.6 mg / kg available nitrogen, 13.8 mg / kg available phosphorus, 156.3 mg / kg available potassium, and pH value of 8.0.

[0091] All systems employed a randomized block design with three treatments: Example 1, Comparative Example 1, and the conventional fertilization control (CK5). Each treatment was replicated three times. The fertilizers used in the Examples and Comparative Examples were applied as basal fertilizer in a single application at a rate of 600 kg / hm². 2 In each embodiment and comparative treatment, the tested fertilizer was applied as basal fertilizer, and the amount of topdressing was 50% of that of the conventional control. The amount of phosphorus and potassium fertilizer was the same as that of the conventional control. The specific amounts are shown in Table 5.

[0092]

[0093] Table 5 Before sowing, all leguminous crops in each system were inoculated with the corresponding rhizobium inoculum. The wheat-peanut system was inoculated with peanut rhizobium, the oat-arrow-pea system was inoculated with arrow-arrow-pea rhizobium, and the barley-broad bean system was inoculated with broad bean rhizobium.

[0094] At the maturity stage of each crop, the middle area of ​​each plot was harvested separately, threshed, weighed, and the moisture content was determined to calculate the grain yield. At the heading and maturity stages of gramineous crops and the pod-setting and maturity stages of leguminous crops, 5 representative plants were taken from each plot, and the organs were dried, crushed, and digested to determine the total nitrogen and total phosphorus content and calculate the nutrient uptake per unit area. The results are shown in Table 6.

[0095]

[0096] Table 6 According to the data in Table 6, under the condition of nitrogen reduction of 50%, the wheat yield of Example 1 was no different from that of CK5, while the peanut yield was higher than that of CK5. The phosphorus uptake of wheat in Example 1 was 36.3% higher than that of CK5 and 27.2% higher than that of Comparative Example 1, indicating that it also showed a synergistic effect in the intercropping system of grass and legume.

[0097] In Example 1, under a 50% reduction in nitrogen, the oat yield was no different from that of CK5, while the yield of arrowhead pea was higher than that of CK5. The phosphorus uptake of oats in Example 1 was 46.4% higher than that of CK5 and 28.9% higher than that of Comparative Example 1. In the oat-arrowhead pea mixed planting system, the legumes accounted for a high proportion and had strong nitrogen fixation ability. By activating the root response system of grasses, it was able to utilize the biological nitrogen and activated phosphorus released by arrowhead pea more effectively.

[0098] In Example 1, under the condition of nitrogen reduction of 50%, the barley yield was no different from that of CK5, while the broad bean yield was higher than that of CK5. The phosphorus uptake of barley in Example 1 was 33.9% higher than that of CK5 and 25.7% higher than that of Comparative Example 1. Under the coastal saline soil conditions, the barley-broad bean system can still exert a stable synergistic effect by activating micronutrients and enhancing crop stress resistance through PDMA activation and activators.

[0099] In the three different systems, the yield of grass in Comparative Example 1 was slightly lower than that in CK5, while the yield of grass in Example 1 was the same as that in the conventional control. Although the phosphorus uptake of grass in Comparative Example 1 was slightly higher than that in CK5, the increase was limited. This indicates that the existing technology recruits microorganisms through a single signal, but the synergistic effect is limited because the root response system of grass is not activated.

[0100] In all three systems, the yield of legume crops showed a trend of Example 1 being greater than Comparative Example 1 being greater than CK5. The analysis suggests that the reconstructive effect of PDMA on the rhizosphere microecology not only benefits the grass family but also optimizes the rhizosphere environment of legumes, promoting their nodulation, nitrogen fixation, and nutrient absorption. In addition, the bidirectional connection of the heteromorphic rhizocystis hyphal network enables the grass family to efficiently absorb phosphorus while also feeding back carbon to the legume family through the hyphae.

[0101] Experimental Example 5 This experimental example aims to verify the time-release characteristics of the three-layer membrane structure, demonstrating that it can achieve rapid release of signaling substances and sustained release of activators.

[0102] Fertilizer samples prepared in Example 1, Comparative Example 4, and Comparative Example 6 were selected. 10.0 g of each fertilizer sample was weighed and placed in a 250 mL stoppered conical flask. 100 mL of 0.01 mol / L CaCl2 solution was added as the release medium. The flask was oscillated at 100 r / min at 25 °C. 1.0 mL samples were taken at 0.5, 1, 2, 3, 5, 7, 10, and 14 days, filtered through a 0.45 μm filter membrane, and stored at -20 °C for testing. Each sample was replicated 3 times.

[0103] The concentrations of daidzein and apigenin in the release medium at each time point were determined by high-performance liquid chromatography (HPLC). A C18 column was used, with a gradient elution of 0.1% phosphoric acid aqueous solution and acetonitrile. Detection wavelengths were 260 nm and 220 nm. The cumulative release rate was calculated as the ratio of the cumulative mass of a component in the release medium at each time point to the initial total mass of that component in the fertilizer sample. The results are shown in [Figure number missing]. Figure 1 , Figure 2 As shown.

[0104] according to Figure 1-2Data shows that in Example 1, the cumulative release rate of flavonoid signaling substances reached 85.2% in 3 days, exhibiting rapid release characteristics; apigenin was released at 35.6% in 3 days, and gradually released to 89.6% in 5-10 days; the signaling substances in the outer coating were released preferentially, while the activators in the middle coating were released more slowly due to the sustained-release effect of the inclusion complex, and the release curves of the two formed a clear gradient in time.

[0105] In Comparative Example 4, the release rates of daidzein and apigenin were 88.2% and 86.2% respectively after 3 days. The release curves of the two were highly overlapping. Since the components were uniformly mixed inside the particles, all soluble components dissolved simultaneously after water penetration, and sequential separation could not be achieved. This simultaneous release led to the simultaneous occurrence of microbial recruitment and root activation. After the rhizosphere microorganisms were enriched, there was a lack of synchronously activated root response, or the microorganisms had not yet fully colonized when the roots were activated, resulting in a decrease in the synergistic effect.

[0106] In Comparative Example 6, apigenin was released at 78.5% in 3 days, while daidzein was released at only 36.5% in 3 days. The premature release of apigenin makes it easy for it to be fixed or degraded by the soil and cannot continue to act on the root system during the optimal window period. The delayed release of daidzein leads to the lag in microbial enrichment, resulting in a lack of sufficient microorganisms available after the root system is activated.

[0107] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. An organic compound fertilizer for the synergistic growth of cereals and legumes, characterized in that, The fertilizer is composed of compound microbial agent, dual-signal inducing factor, gramineous root activator, compound organic carrier and excipients. Each component includes, by weight, 5-10 parts compound microbial agent, 0.15-0.7 parts dual-signal inducing factor, 0.4-1.6 parts gramineous root activator, 51-93 parts compound organic carrier, and 7.1-21.3 parts excipients. The dual-signal inducing factor is composed of flavonoid signaling substance and proline-2-deoxymethylene acid.

2. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 1, characterized in that: The compound microbial agent is composed of soybean slow-growing rhizobium, heteromorphic rhizocystis, and fluorescent pseudomonas in a live bacteria ratio of 2-3:1:

1.

3. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 1, characterized in that: The flavonoid signaling substance is selected from at least one of daidzein and genistein.

4. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 1, characterized in that: The root activator of the grass family is composed of apigenin and quercetin, which exist in the form of an inclusion complex. The inclusion complex is selected from one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin.

5. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 1, characterized in that: The composite organic carrier is composed of decomposed straw biochar, humic acid and trace elements. The trace elements are composed of zinc sulfate heptahydrate, borax decahydrate and ammonium molybdate tetrahydrate mixed in a mass ratio of 5:3:

2.

6. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 1, characterized in that: The compound microbial agent contains a protectant, which is composed of trehalose and polyvinylpyrrolidone in a mass ratio of 3:

1.

7. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 1, characterized in that: The excipients include binders, dispersants, protective agents, and encapsulating agents.

8. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 7, characterized in that: The binder is selected from one or more of sodium alginate, sodium carboxymethyl cellulose, polyvinyl alcohol, and starch.

9. The organic compound fertilizer for synergistic growth of grasses and beans according to claim 7, characterized in that: The dispersant is selected from one or more of Tween-80, sodium dodecyl sulfate, and polyethylene glycol.

10. A method for preparing an organic compound fertilizer for the synergistic growth of cereals and legumes as described in any one of claims 1-9, characterized in that, Includes the following steps: S1: Dissolve the flavonoid signaling substance in anhydrous ethanol, dissolve proline-2-deoxymethylene acid in deionized water, mix them, and add a dispersant to prepare a dual-signal mixed solution. S2: Mix and granulate well-rotted straw biochar, humic acid and trace elements, and dry to obtain organic carrier particles as the core layer. S3: Prepare the compound bacterial powder into a bacterial suspension, spray it onto the surface of the core layer particles, and dry it to obtain inner layer coated particles. S4: Apigenin and quercetin are prepared into an inclusion complex with an inclusion agent, which is then formulated into a suspension and sprayed onto the surface of the inner coating particles. The weight gain of the coating is controlled at 2-3%, and the middle coating particles are obtained after drying. S5: The intermediate-layer coated granules are fed into a fluidized bed coating machine, a dual-signal mixed solution is sprayed on, the coating weight gain is controlled at 3-5%, and the fertilizer product is obtained after drying.

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