Method for controlling and application of antibiotic resistance genes in intercropping farmland soil by using functionalized biomimetic mineralization slow-release material

Abstract

The application belongs to the technical field of agricultural environmental pollution control and ecological restoration, and discloses a method for controlling antibiotic resistance genes in intercropping farmland soil by using functionalized biomimetic mineralization slow-release materials and application, which comprises the following steps: uniformly spreading the functionalized biomimetic mineralization slow-release materials on the surface of the soil at a dosage of 100-5000 kg / hm 2 , and turning them into the soil layer by a rotary cultivator to fully mix the materials with the soil; adopting an intercropping planting mode, and selecting two or more crops with complementary ecological niches to be combined and planted to control the antibiotic resistance genes in the farmland soil. The material is stable in structure and good in environmental compatibility, can be applied to the soil to synergistically control and efficiently reduce the migration and spread of ARGs in the soil-crop system by multiple mechanisms such as adsorption, microbial competition and physical barrier, and provides a new material and technical approach for realizing agricultural waste resource utilization and farmland ARGs pollution control.

Description

Technical Field

[0001] This invention belongs to the field of agricultural environmental pollution control and ecological restoration technology, and in particular, it is a method and application of using functionalized biomimetic mineralized slow-release materials to inhibit antibiotic resistance genes in intercropping farmland soil. Background Technology

[0002] With the continuous development of large-scale, intensive livestock farming, the extensive application of livestock and poultry manure containing antibiotic residues, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) in farmland has become a significant source of soil ARG pollution and diffusion. ARGs can not only persist in the soil for extended periods but also spread horizontally among different microorganisms and accumulate through the soil-plant system, ultimately threatening human health and ecological security via the food chain. Currently, while traditional manure composting methods can reduce pathogenic microorganisms to some extent, their removal efficiency for structurally stable, horizontally transferable ARGs is limited, and they struggle to eliminate the combined selective pressure from heavy metals and antibiotic residues. Furthermore, while physicochemical control methods (such as advanced oxidation and in-situ chemical passivation) have shown some effectiveness in laboratory studies, they suffer from high costs, complex operations, and potential damage to the soil ecosystem, making them unsuitable for large-scale field application. Therefore, there is an urgent need to develop a novel agronomy-materials combined strategy that is economical, environmentally compatible, and easy to implement in the field to achieve efficient control of ARGs in the farmland environment.

[0003] In existing agricultural planting systems, monoculture still dominates, resulting in a simple rhizosphere microenvironment structure, low microbial community diversity, and insufficient ability to inhibit and regulate the host microorganisms of ARBs and ARGs. Intercropping, as an ecologically intensive planting model, can regulate the rhizosphere microecology through interactions between crops, enhancing the complexity and stability of the microbial network, thereby inhibiting soil-borne pathogens and improving soil health to some extent. However, the effectiveness of intercropping alone in controlling ARGs is highly uncertain, easily affected by soil type, climate conditions, and field management practices, making it difficult to achieve stable and efficient ARG reduction. On the other hand, functionalized carbon-based materials (such as biochar) have shown good results in adsorbing and immobilizing organic pollutants and heavy metals due to their porous structure and surface active sites, but their ability to block the spread of ARGs mediated by mobile genetic elements (such as plasmids) remains insufficient, and single materials are unlikely to form a multi-barrier control system against the migration and accumulation of ARGs. Currently, most studies still focus on the isolated effects of single technical means (such as applying only modified materials or adopting specific intercropping patterns) on ARGs. There is a lack of systematic solutions that organically combine the rhizosphere biological regulation mechanisms of specific crop combinations (such as leek-chicory) with the physicochemical inhibition properties of functional materials, making it difficult to achieve effective inhibition of ARGs through multiple pathways and the entire process from the soil environment to the crop rhizosphere.

[0004] Against this backdrop, the present invention aims to provide a synergistic solution combining rhizosphere ecological regulation of leek-chicory intercropping systems with slow-release inhibition using functional materials. By designing and preparing a novel biomimetic mineralized slow-release material, which possesses high adsorption capacity, slow-release properties, and microbial community regulation functions, this material can effectively target and inhibit the host bacterial activity of ARGs, suppress horizontal gene transfer, and strengthen the rhizosphere barrier function of the intercropping system. This systematically reduces the abundance and spread risk of ARGs in the soil, providing a multi-mechanism, low-cost inhibition technology for the spread of antibiotic resistance in agricultural environments. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for controlling antibiotic resistance genes in intercropping farmland soil using functionalized biomimetic mineralized slow-release materials.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A method for controlling antibiotic resistance genes in intercropping farmland soil using functionalized biomimetic mineralized slow-release materials includes the following steps:

[0008] Functionalized biomimetic mineralized slow-release materials will be applied at a rate of 100-5000 kg / hm². 2 The amount of material is evenly spread on the soil surface and then tilled into the 0-20 cm soil layer using a rotary tiller to ensure that the material is fully mixed with the soil.

[0009] Intercropping is used to select two or more crops with complementary ecological niches for combined planting, thereby controlling antibiotic resistance genes in farmland soil.

[0010] Among them, the antibiotic resistance genes include: β-lactams bla ampC , bla TEM-1 , bla OX-1 Macrolides: erm B erm C. Aminoglycosides: str A, str B cfr , fex A, aad A. Sulfonamides: sul 1. sul 2. Tetracyclines: tet O、 tet W, tet M, tet Q, tet L, tet X.

[0011] Furthermore, the intercropping pattern is a chive-chill intercropping system; the crops are configured in a 1:1 ratio, with a row spacing of 8-32 cm and a plant spacing of 5-20 cm; 1-15 chive plants are planted per plant and 1-15 chill plants are planted per plant.

[0012] Alternatively, for lightly polluted soil, the dosage of the functionalized biomimetic mineralization slow-release material is 100-500 kg / hm². 2 For moderately to severely polluted soils, the dosage of the functionalized biomimetic mineralization slow-release material is 2000-5000 kg / hm². 2 ;

[0013] Alternatively, the primer sequences for sul1 are SEQ ID No. 1 and SEQ ID No. 2; for sul2, SEQ ID No. 3 and SEQ ID No. 4; for tetO, SEQ ID No. 5 and SEQ ID No. 6; for tetW, SEQ ID No. 7 and SEQ ID No. 8; for tetQ, SEQ ID No. 9 and SEQ ID No. 10; for tetL, SEQ ID No. 11 and SEQ ID No. 12; for tetM, SEQ ID No. 13 and SEQ ID No. 14; for tetX, SEQ ID No. 15 and SEQ ID No. 16; for ermB, SEQ ID No. 17 and SEQ ID No. 18; for ermC, SEQ ID No. 19 and SEQ ID No. 20; for strA, SEQ ID No. 21 and SEQ ID No. 22; and for strB, SEQ ID No. 17 and SEQ ID No. 22. Primer sequences for: No. 23 and SEQ ID No. 24; primer sequences for: aadA: SEQ ID No. 25 and SEQ ID No. 26; primer sequences for: cfr: SEQ ID No. 27 and SEQ ID No. 28; primer sequences for: fexA: SEQ ID No. 29 and SEQ ID No. 30; primer sequences for: blaOXA-1: SEQ ID No. 31 and SEQ ID No. 32; primer sequences for: blaampC: SEQ ID No. 33 and SEQ ID No. 34; primer sequences for: blaTEM-1: SEQ ID No. 35 and SEQ ID No. 36.

[0014] Furthermore, the preparation method of the functionalized biomimetic mineralized sustained-release material includes the following steps:

[0015] S1. Fresh camellia seeds are selected, mechanically dehulled and pressed to obtain camellia cake, washed and dried, pre-frozen, crushed and ground, and sieved to obtain camellia seed powder CP.

[0016] S2. The camellia seed powder CP is refluxed with an organic solvent for degreasing, and then dried to obtain degreased camellia cake powder DCP;

[0017] S3. The defatted camellia oil cake powder DCP is mixed and ground with a template agent, then mixed with an activator solution to form a slurry. After drying, it is subjected to programmed temperature-controlled pyrolysis. After acid washing, water washing, and drying, graded porous biochar HPBC is obtained.

[0018] S4. The graded porous biochar HPBC is mixed with a nitrogen source and then heat-treated under anaerobic conditions. After washing and drying, aminated biochar is obtained. Subsequently, the aminated biochar is impregnated in a magnesium salt solution to adsorb magnesium ions. Then, a mixed solution of ammonium salt and phosphate is slowly added dropwise while controlling the pH value to carry out the reaction. After washing and drying, nano-mineralized biochar MAP@N-HPBC is obtained.

[0019] S5. The nano-mineralized biochar MAP@N-HPBC is mixed with humic acid in a certain proportion, water is added to make a slurry, and after wet ball milling, it is dried to obtain humic acid coated nano-mineralized biochar material HUM-MAP@N-HPBC.

[0020] S6. The activated Bacillus pasteurellii is co-cultured with the humic acid-coated nanomineralized biochar material HUM-MAP@N-HPBC to form a biomimetic mineralization membrane. After drying, the functionalized biomimetic mineralization membrane slow-release material MGPBC@HA is obtained, which is a functionalized biomimetic mineralization slow-release material for controlling herbicide resistance genes in intercropping farmland.

[0021] Furthermore, the camellia seed powder CP in S1 is prepared by mechanically processing camellia seeds;

[0022] Before mechanical processing of camellia seeds, the following steps are also taken: selecting fresh, mold-free, and insect-free camellia seeds with a moisture content controlled at 2%-20%; using mechanical dehulling equipment to separate the shell from the kernel, ensuring that the mass percentage of kernel to shell after dehulling is above 95%; mechanically pressing the camellia seed kernels to collect camellia oil cake, washing it with clean water, and then drying it in a 105℃ oven for 24 hours, where the organic matter content of the camellia seed kernel is 40-90%; the drying temperature is 100-110℃; after pre-freezing the camellia oil cake for 24 hours, it is first transferred to a crusher for coarse crushing into 2-3 mm particles; then transferred to an ultra-fine pulverizer to be pulverized through a 100-300 mesh sieve to obtain camellia seed powder; the pre-freezing temperature is -20-10℃; the pulverizing time is 30-60 minutes.

[0023] Alternatively, the defatted camellia seed cake powder in S2 is prepared by defatting camellia seed powder;

[0024] To defatted the camellia seed powder, 50 g of powder was placed in a Soxhlet extractor. 300 mL of hexane was added to a 500 mL round-bottom flask, along with 3-5 boiling chips to prevent bumping. The reaction was carried out in a water bath at 85±2℃ for 6-8 hours, then stopped. The extraction sleeve was then removed and vacuum-dried at 60℃ for 2-6 hours. The solvent used was hexane or petroleum ether, the bath temperature was 80-90℃, and the condensate flow rate was 1-3 L / min. The defatted camellia seed powder was then ultrasonically cleaned using a mixture of ethanol and water (ethanol:water volume ratio 1-3:1, pH 5.3-5.7). When the sample was ultrasonically added to the ethanol-water mixture, the ultrasonic frequency was 38-42 kHz, the ultrasonic power density was 0.25-0.75 W / mL, and the ultrasonic temperature was 58-62℃.

[0025] Furthermore, the graded porous biochar in S3 was prepared by template-assisted catalytic pyrolysis of defatted camellia oil cake powder;

[0026] The defatted camellia oil cake powder was subjected to template treatment by ball milling it with nano-SiO2 in a specific ratio of 10-2.5:1. The ball mill speed was 300-600 rpm, and the milling time was 1-4 h. The mixture was then activated by mixing the ball-milled material with KOH at a mass ratio of 2-0.5:1, adding deionized water to form a slurry, and drying at 70-95℃ to constant weight to obtain the precursor. The precursor was then subjected to pyrolysis treatment by heating to 400℃ at 5℃ / min and holding for 30 min under N2 protection in a tube furnace, followed by heating to 800℃ at 10℃ / min and holding for 60 min. After natural cooling, it was soaked in 1 mol / L HCl solution for 6 h, washed with deionized water until neutral, and dried at 105℃ for 12 h. The N2 flow rate was 0.2-0.6%. L / min; pickling concentration: 0.5-1.5 mol / L;

[0027] Alternatively, the nano-mineralized biochar in S4 is prepared by amination and in-situ mineralization of porous biochar.

[0028] When ammoniating porous biochar, graded porous biochar is mixed with urea and pyrolyzed under a N2 atmosphere. After cooling, it is washed three times with deionized water and dried to obtain ammoniated biochar. The mass ratio of graded porous biochar to urea is 1:5-20; the pyrolysis time is 1-3 h; the pyrolysis temperature is 140-160℃; the N2 flow rate is 0.1-0.3 L / min; the drying temperature is 50-80℃; and the drying time is 8-24 h.

[0029] Magnesium ion adsorption was performed on aminated biochar by dispersing the aminated biochar in a 0.1 mol / L MgCl2 solution, shaking at room temperature for 2 h, and then separating the solid and liquid phases to obtain the magnesium ion-adsorbed biochar. The concentration of MgCl2 was 0.05-0.2 mol / L, and the shaking speed was 150-200 rpm.

[0030] The biochar adsorbed with magnesium ions was mineralized by slowly adding an equal volume of 0.05-0.2 mol / L (NH4)2HPO4 solution to the system under vigorous stirring, while adjusting and maintaining the pH at 9.0±0.2 with NaOH solution. After the reaction was completed, the mixture was filtered, washed three times each with ethanol and water, and freeze-dried for 24 h. The dropping rate was 1-2 mL / min.

[0031] Furthermore, the humic acid-coated nano-mineralized biochar material in S5 was prepared by wet ball milling: nano-mineralized biochar and humic acid were mixed at a mass ratio of 1:1, and deionized water was added to prepare a slurry with a solid content of 20-30%; the slurry was then placed in a planetary ball mill for ball milling, and dried at a certain temperature after ball milling; the ball milling speed was 300-500 rpm, the ball milling time was 3-5 h; the drying temperature was 50-80℃, and the drying time was 8-24 h.

[0032] Furthermore, the functional biomimetic mineralized membrane slow-release material in S6 was prepared through microbial inoculation and biofilm induction;

[0033] Pasteurella multocida was inoculated into Pasteurella multocida culture medium and cultured at 30°C with shaking at 150 rpm until OD reached. 600 =1.0; Humic acid-coated nano-mineralized biochar material and bacterial solution were mixed at a ratio of 1:10 (g:mL), and adsorbed by shaking at 30℃ and 50 rpm for 3 h. After discarding the supernatant, the material was transferred to a sterile tray, covered with plastic wrap to maintain humidity, and incubated at 30℃ for 36 h. Finally, the final product was obtained by vacuum freeze-drying for 24 h; wherein, the OD of the bacterial solution was... 600 =0.8-1.2; adsorption oscillation speed is 40-60 rpm; static culture temperature is 28-32℃, humidity is 85-95%; freeze-drying temperature is -60-10℃, freeze-drying vacuum degree is 10-100 Pa.

[0034] The method described above is used to control the spread of antibiotic resistance genes in soil and / or improve intercropping systems, including for improving the rhizosphere microecological environment, for improving soil nutrient use efficiency, for promoting crop growth and increasing yield, and for remediating antibiotic-contaminated farmland soil.

[0035] A method for improving or remediating farmland soil, including the methods described above.

[0036] Furthermore, the crop combination of the intercropping pattern is selected from any of the following: leek-chicory intercropping system, leafy vegetable-root vegetable intercropping system, legume-grass intercropping system, deep root-shallow root crop intercropping system, and other crop combinations with different root morphology and nutrient absorption characteristics.

[0037] A farmland application system utilizing the functionalized biomimetic mineralized slow-release material as described above, the system comprising a spreading device, a rotary tillage device, and an intercropping management device;

[0038] The spreading device is used to apply the material at a rate of 100-5000 kg / hm². 2 Apply the dosage evenly to the soil surface;

[0039] The rotary tillage device is used to till the material into the 0-20 cm soil layer;

[0040] The intercropping management device includes a row spacing control device, a hole spacing control device, and a planting density control device, used to realize the intercropping configuration of two or more crops; the adjustable range of the row spacing control device is 8-32 cm, and the adjustable range of the hole spacing control device is 5-20 cm.

[0041] The intercropping management device also includes a bed width adjustment device, a ridge width adjustment device, and a crop ratio configuration device.

[0042] The advantages and positive effects of this invention are as follows:

[0043] 1. This invention constructs a functional biochar composite material based on camellia oil cake through a multi-step synergistic process of "degreasing pretreatment - template-assisted catalytic pyrolysis - surface amination - in-situ mineralization - wet ball milling - bio-inoculation". The solvent degreasing method effectively removes residual oil from the raw material, preventing pore structure deterioration caused by coking during pyrolysis, thus laying a solid foundation for the subsequent construction of a high specific surface area integral-level porous structure.

[0044] 2. The slow-release composite material HA-MAP@N-HPBC prepared in this invention possesses excellent multifunctional properties, capable of simultaneously improving soil physicochemical properties, regulating microbial community structure, promoting intercropping symbiosis, and efficiently removing soil antibiotic resistance genes (ARGs). This process achieves high-value resource utilization of agricultural waste, fulfilling multiple objectives of soil health management, crop yield increase, and environmental pollution control.

[0045] 3. The slow-release composite material HA-MAP@N-HPBC prepared by this invention has a stable physical morphology and a biofilm protective layer that ensures the long-term stability and effectiveness of the pre-prepared functional components (such as urease and calcium carbonate crystals) in the soil environment, realizing the slow release and long-lasting effect of functional factors. The material can show good improvement and remediation effects in various soil types and a wide pH range, and has strong environmental adaptability and anti-interference ability.

[0046] 4. Compared with traditional biochar preparation methods (such as single pyrolysis), this invention innovatively introduces a strategy of synergistic effect between a hard template (nano-SiO2) and a chemical activator (KOH). The template decomposes at high temperature, leaving a pre-set mesoporous / macroporous framework, while KOH creates abundant micropores on the framework through a vigorous etching reaction. Ultimately, a multi-level pore structure with interconnected micropores, mesopores, and macropores is successfully constructed, with a specific surface area significantly higher than that of conventional biochar, providing an optimal space for microbial habitation and nutrient loading.

[0047] 5. This invention employs a chemical anchoring strategy of "surface amination-in-situ precipitation," innovatively securing nitrogen, phosphorus, and magnesium nutrients within the pores of biochar in the form of nano-struvite (MAP) crystals. Compared to traditional physical impregnation loading, this method transforms readily soluble nutrients into a slow-release library of insoluble nano-minerals through chemical bonding, fundamentally solving the technical challenges of easy nutrient loss and low utilization rates in fertilizers, and achieving the slow, on-demand release of nutrients.

[0048] 6. This invention employs humic acid for wet ball milling compounding. This step not only achieves nanoscale uniform compounding of organic and inorganic materials, but the resulting "mechanical-chemical effect" also causes the humic acid to strongly coat the surface of biochar and the outer layer of nutrients, forming a natural physical diffusion barrier. This barrier further slows down the nutrient release rate and provides a high-quality initial carbon source and protection for subsequently inoculated microorganisms.

[0049] 7. This invention innovatively utilizes *Bacillus pasteurellii* as a biological template and reactor, inducing the formation of a stable biomimetic mineralized film on the surface of functionalized biochar through static cultivation. Even after inactivation treatment, the material perfectly retains the pre-formed calcium carbonate mineral layer and the framework of bacterial secretions. This biomimetic film not only effectively binds soil particles and solidifies heavy metals through co-precipitation, but its unique microstructure also occupies an ecological niche and regulates the rhizosphere microbial community, thereby indirectly inhibiting pathogen activity and achieving multi-pathway control of ARGs transmission risk.

[0050] 8. This invention organically integrates five key technologies: degreasing pretreatment, multi-level pore formation, chemical anchoring, organic compounding, and biological inoculation, forming a complete green preparation technology system for "soil ecological restoration materials." Significant synergistic effects exist between the various process units: multi-level pores provide space for nutrient anchoring and microbial agent habitat; chemically anchored nutrients ensure the activity of the microbial agent; and the biofilm and metabolic activities of the microbial agent, in turn, enhance the material's improvement effect, forming a highly efficient, multifunctional closed loop.

[0051] 9. The MGPBC@HA composite material prepared in this invention can efficiently promote the growth of intercropping crops. In pot experiments, compared with the control group, the biomass of intercropping crops treated with MGPBC@HA increased by more than 25%, the soil aggregate structure was significantly improved, and typical antibiotic resistance genes in the soil (such as...) were also reduced. sul 1. tet The absolute abundance of M can be reduced by 1-2 orders of magnitude.

[0052] 10. Compared with other soil improvement technologies (such as the application of conventional organic fertilizers, passivating agents, or microbial agents), the MGPBC@HA material prepared in this invention achieves a three-in-one synergistic delivery of inorganic nutrients, organic carbon sources, and microbial metabolites. The physical shelter, chemical nutrition, and biological protection it provides enable beneficial indigenous microorganisms in the soil to preferentially colonize and become the dominant flora. Through efficient microbial competition and niche occupation, it inhibits the abundance of ARGs host bacteria from the source, thereby achieving a deep reduction of ARGs.

[0053] 11. This invention innovatively combines *Bacillus pasteurellii*, a bacterium with urease-induced calcium carbonate precipitation, with a porous slow-release material. The calcium carbonate crystals produced during its cultivation not only solidify the soil and fix heavy metals, but the resulting increase in microenvironment pH and the physical barrier effect can also effectively inhibit pathogen activity and interfere with the horizontal transfer of resistance genes (HGT).

[0054] 12. The MGPBC@HA composite material prepared by this invention has a stable physical morphology (granular) and chemical properties, and is not easily lost after being applied to the soil. Its loaded functional components (such as minerals, enzymes, and biofilms) are protected by both the biochar pores and the biomimetic membrane, extending their effective time several times compared to direct application of liquid microbial fertilizers, thus achieving functional durability and reducing the need for repeated application of agricultural inputs.

[0055] 13. The technical process of this invention is green and the products are environmentally friendly, with the potential for large-scale agricultural application. Based on MGPBC@HA materials, it can be developed for intensive vegetable cultivation, orchard soil improvement, and bioremediation of farmland soil contaminated with antibiotics. The operation is simple and easily accepted by farmers.

[0056] 14. The product of this invention can solve the problems of limited functionality, low survival rate of microbial agents, and poor synergy between nutrients and microbial agents in existing soil improvement technologies. It is particularly suitable for overcoming continuous cropping obstacles, improving the efficiency of intercropping / relay cropping systems, and remediating antibiotic-contaminated soil around intensive livestock farms.

[0057] 15. In the preparation of composite materials, this invention uses an "amylation" strategy to covalently graft nitrogen-containing groups generated from urea pyrolysis onto the surface of biochar, rather than simply mixing them. These surface amino groups (-NH2) not only change the surface electrical properties of the material, but also serve as precise nucleation sites to guide struvite crystals to form in situ within the pores and anchor them firmly, avoiding the problem of crystals growing too large or being lost in the solution, thus achieving precise and controllable loading.

[0058] 16. In the method of this invention, the synthesis of nano-struvite is a controlled precipitation process carried out at room temperature. This is achieved through pre-adsorption of Mg... 2+ By slowly adding N and P sources and precisely controlling the pH to 9.0, the reaction is forced to reach supersaturation within the limited space of the biochar pores, preferentially nucleating and generating nanoscale, highly active crystals. This method is mild and consumes very little energy, avoiding the high cost and high risk of high-temperature and high-pressure reactions.

[0059] 17. In the bacterial inoculation step of the method of this invention, an "adsorption-static culture" strategy is adopted instead of simple mixing. First, the bacteria are adsorbed onto the material surface, and then a moist, static environment is provided to induce them to secrete extracellular polymers and form a biofilm. This biofilm immobilization technology provides a crucial structural basis for subsequent functionalization and is a key pretreatment step for achieving long-term material functionality.

[0060] 18. This invention thoroughly enhances the functionality of camellia oil cake through a multi-stage process. The final product has been transformed from a simple biochar into an eco-engineering material integrating "physical structure, slow-release nutrient reservoir, and microbial carrier." It not only provides a habitat but also continuously provides nutrients and protection, effectively shaping a healthy rhizosphere micro-ecology, ultimately achieving comprehensive soil improvement and restoration through the synergistic effects of physical, chemical, and biological processes.

[0061] 19. The method of this invention uses camellia oil cake as raw material, and through steps such as defatting, graded porous biochar preparation, surface amination, in-situ mineralization of nano-stripe, humic acid coating, and biomimetic induction of mineral film formation and inactivation by Bacillus pasteurellis, a slow-release material MGPBC@HA is prepared. This material has a stable structure and good environmental compatibility. After being applied to the soil, it can synergistically control and efficiently reduce the migration and spread of ARGs from livestock and poultry manure in the soil-crop system through multiple mechanisms such as adsorption, microbial competition, and physical barrier, providing a new material and technical approach for realizing the resource utilization of agricultural waste and the treatment of ARG pollution in farmland. The functionalized biomimetic mineralized slow-release material of this invention can be used in a composite intervention method that synergistically promotes the reduction of antibiotic resistance genes (ARGs) from livestock and poultry manure in intercropping farmland systems and blocks their migration to the edible parts of crops. Attached Figure Description

[0062] Figure 1 This is a schematic diagram of the chive-chicory intercropping system layout in this invention;

[0063] Figure 2 The figures show the soil nitrate nitrogen, ammonium nitrogen, and available phosphorus content after 60 days of cultivation for the control group CK (leek-chicory intercropping system), control group J (leek monoculture system), control group K (chicory monoculture system), and experimental group JK (leek-chicory intercropping + functional biomimetic mineralized membrane slow-release material basal application system).

[0064] Figure 3 The graph shows the total phosphorus, organic carbon, organic matter, and total nitrogen content in the soil of the control group CK, control group J, control group K, and experimental group JK after 60 days of cultivation in this invention.

[0065] Figure 4 This is a diagram showing the soil pH values ​​of the control group CK, control group J, control group K, and experimental group JK after 60 days of culture in this invention.

[0066] Figure 5 This is a diagram showing the relative abundance of major root microorganisms in the soil after 60 days of culture for the control group CK, control group J, control group K, and experimental group JK.

[0067] Figure 6 This is a diagram showing the relative abundance of major root microbial families in the soil after 60 days of culture for the control group CK, control group J, control group K, and experimental group JK in this invention.

[0068] Figure 7 This is a graph showing the relative abundance of major root microbial genera in the soil after 60 days of culture for the control group CK, control group J, control group K, and experimental group JK in this invention.

[0069] Figure 8This is a graph showing the relative abundance of major non-root microorganisms in the soil after 60 days of culture for the control group CK, control group J, control group K, and experimental group JK.

[0070] Figure 9 This is a diagram showing the relative abundance of major non-root microbial families in the soil after 60 days of culture for the control group CK, control group J, control group K, and experimental group JK in this invention.

[0071] Figure 10 This is a graph showing the relative abundance of major non-root microbial genera in the soil after 60 days of culture for the control group CK, control group J, control group K, and experimental group JK in this invention.

[0072] Figure 11 This is a 60-day absolute abundance diagram of soil β-lactam antibiotic resistance genes in the control group CK, control group J, control group K, and experimental group JK in this invention.

[0073] Figure 12 This is a 60-day absolute abundance diagram of soil macrolide antibiotic resistance genes in the control group CK, control group J, control group K, and experimental group JK in this invention.

[0074] Figure 13 This is a 60-day absolute abundance diagram of soil aminoglycoside antibiotic resistance genes in the control group CK, control group J, control group K, and experimental group JK in this invention.

[0075] Figure 14 This is a 60-day absolute abundance diagram of soil quinolone antibiotic resistance genes in the control group CK, control group J, control group K, and experimental group JK in this invention.

[0076] Figure 15 This is a 60-day absolute abundance diagram of soil sulfonamide antibiotic resistance genes in the control group CK, control group J, control group K, and experimental group JK in this invention;

[0077] Figure 16 This is a 60-day absolute abundance diagram of soil tetracycline antibiotic resistance genes in the control group CK, control group J, control group K, and experimental group JK in this invention.

[0078] Figure 17 This is a 60-day absolute abundance diagram of soil mobile genetic element genes in the control group CK, control group J, control group K, and experimental group JK in this invention;

[0079] Figure 18 This is a graph showing the 60-day absolute abundance removal rate of soil antibiotic resistance genes in the control group CK relative to the control group J in this invention.

[0080] Figure 19This is a graph showing the 60-day absolute abundance removal rate of soil antibiotic resistance genes in the control group CK relative to the control group K in this invention.

[0081] Figure 20 This is a graph showing the 60-day absolute abundance removal rate of soil antibiotic resistance genes in experimental group JK compared to control group J in this invention.

[0082] Figure 21 This is a graph showing the 60-day absolute abundance removal rate of soil antibiotic resistance genes in experimental group JK compared to control group K in this invention.

[0083] Figure 22 This is a graph showing the 60-day absolute abundance removal rate of soil antibiotic resistance genes in the experimental group JK compared to the control group CK in this invention. Detailed Implementation

[0084] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0085] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0086] A functionalized biomimetic mineralized slow-release material for controlling herbicide resistance genes in intercropping farmland of specific crops and its preparation method, comprising the following steps:

[0087] S1. Fresh camellia seeds are selected, mechanically dehulled and pressed to obtain camellia cake, washed and dried, pre-frozen, crushed and ground, and sieved to obtain camellia seed powder CP.

[0088] S2. The camellia seed powder was refluxed with n-hexane solvent for degreasing, and then dried to obtain defatted camellia cake powder (DCP).

[0089] S3. The defatted camellia oil cake powder is mixed and ground with nano SiO2 template agent, then mixed with KOH solution to form a slurry. After drying, it is subjected to programmed temperature-controlled pyrolysis. After acid washing, water washing and drying, graded porous biochar HPBC is obtained.

[0090] S4. After mixing porous biochar with urea, heat treatment was carried out, followed by washing and drying to obtain aminated biochar. Subsequently, the aminated biochar was impregnated in MgCl2 solution to adsorb magnesium ions, and then (NH4)2HPO4 solution was slowly added dropwise while controlling the pH value to carry out the reaction. After washing and drying, nano-mineralized biochar MAP@N-HPBC was obtained.

[0091] S5. Mix nano-mineralized biochar with humic acid in a certain proportion, add water to make a slurry, and dry it after wet ball milling to obtain humic acid-coated nano-mineralized biochar material HUM-MAP@N-HPBC.

[0092] S6. The activated Bacillus pasteurellii was co-cultured with functionalized biochar to form a biomimetic mineralization membrane. After drying, the functional biomimetic mineralization membrane sustained-release material MGPBC@HA was finally obtained.

[0093] In step S1 of this invention, camellia seed powder is prepared by mechanically processing camellia seeds.

[0094] Before mechanical processing of camellia seeds, the following steps are also included: selecting fresh, mold-free, and insect-free camellia seeds with a moisture content controlled at 2%-20%, preferably 10%; using mechanical dehulling equipment to separate the shell from the kernel, with the kernel-to-shell ratio controlled at over 95%, preferably 98%; mechanically pressing the camellia seeds to collect camellia oil cake, washing it with clean water, and then drying it in a 105℃ oven for 24 hours; wherein the organic matter content of the camellia seeds is 40-90%, preferably 60%; the drying temperature is 100-110℃, preferably 105℃; after pre-freezing the camellia oil cake for 24 hours, it is first transferred to a crusher for coarse crushing into 2-3 mm particles, preferably 2.5 mm; then transferred to an ultrafine pulverizer for pulverizing through a 100-300 mesh sieve, preferably 200 mesh sieve, to obtain camellia seed powder; wherein the pre-freezing temperature is -20-10℃, preferably -15℃; and the pulverizing time is 30-60 seconds. min, the optimal value is 45 min;

[0095] In step S2 of this invention, defatted camellia seed cake powder is prepared by defatting camellia seed powder.

[0096] When defatting camellia seed powder, weigh 50 g of camellia seed powder into a Soxhlet extractor, add 300 mL of n-hexane solvent to a 500 mL round-bottom flask, and add 3-5 boiling stones to prevent bumping, preferably 4 stones; stop the reaction at 85±2℃ for 6-8 h, preferably 7 h; remove the extraction sleeve and vacuum dry at 60℃ for 2-6 h, preferably 4 h; wherein, the solvent is n-hexane or petroleum ether, preferably n-hexane; the bath temperature is 80-90℃, preferably 85℃; the condensate flow rate is 1-3 L / min, preferably 2 L / min; when ultrasonically cleaning the defatted camellia seed powder, the volume ratio of ethanol to water is 1-3:1, preferably 7:3; the pH of the mixed solution is 5.3-5.7, preferably 5.5; when ultrasonically adding the sample to the ethanol-water solution, the ultrasonic frequency is 38-42 kHz, preferably 40 kHz. kHz; ultrasonic power density is 0.25-0.75 W / mL, with the most preferred value being 0.5 W / mL; ultrasonic temperature is 58-62℃, with the most preferred value being 60℃.

[0097] In step S3 of this invention, graded porous biochar is prepared by template-assisted catalytic pyrolysis of defatted camellia oil cake powder.

[0098] When processing the defatted camellia oil cake powder, the defatted camellia oil cake powder and nano-SiO2 are placed in a ball mill and ball-milled in a certain proportion; wherein, the mass ratio of defatted camellia oil cake powder to nano-SiO2 is (10-2.5):1, the most preferred being 5:1; the ball mill speed is 300-600 rpm, the most preferred being 400 rpm; the ball milling time is 1-4 h, the most preferred being 2 h; when activating the mixture, the ball-milled material is mixed with KOH in a mass ratio of (2-0.5):1, the most preferred being 1:1; deionized water is added to make a slurry, and dried at 70-95℃ to constant weight, the most preferred being 80℃; when pyrolyzing the precursor, under N2 protection in a tube furnace, the temperature is increased to 400℃ at 5℃ / min and held for 30 min, then increased to 800℃ at 10℃ / min and held for 60 min, and after natural cooling, it is soaked in 1 mol / L HCl solution for 6 minutes. The solution is washed with deionized water until neutral and dried at 105℃ for 12 h; wherein the N2 flow rate is 0.2-0.6 L / min, the most preferred being 0.4 L / min; and the acid washing concentration is 0.5-1.5 mol / L, the most preferred being 1.0 mol / L.

[0099] In step S4 of this invention, nano-mineralized biochar is prepared by amination and in-situ mineralization of porous biochar.

[0100] When amination treatment is performed on porous biochar, graded porous biochar and urea are mixed at a certain mass ratio, pyrolyzed under a N2 atmosphere, cooled, washed three times with deionized water, and dried at a certain temperature. The mass ratio of graded porous biochar to urea is 1:(5-20), most preferably 1:10; the pyrolysis time is 1-3 h, most preferably 2 h; the pyrolysis temperature is 140-160℃, most preferably 150℃; the N2 flow rate is 0.1-0.3 L / min, most preferably 0.2 L / min; the drying temperature is 50-80℃, most preferably 60℃; and the drying time is 8-24 h, most preferably 12 h.

[0101] When adsorbing magnesium ions onto aminated biochar, the aminated biochar is dispersed in a 0.1 mol / L MgCl2 solution, shaken at room temperature for 2 h, and the solid material is retained after solid-liquid separation; wherein, the concentration of MgCl2 is 0.05-0.2 mol / L, the most preferred being 0.1 mol / L; the shaking speed is 150-200 rpm, the most preferred being 180 rpm;

[0102] When mineralizing the biochar that adsorbs magnesium ions, an equal volume of 0.1 mol / L (NH4)2HPO4 solution is slowly added dropwise to the system under vigorous stirring, while adjusting and maintaining the pH at 9.0 ± 0.2 with NaOH solution. After the reaction is complete, the mixture is filtered, washed three times each with ethanol and water, and freeze-dried for 24 h. The concentration of (NH4)2HPO4 is 0.05-0.2 mol / L, with a maximum of 0.1 mol / L; the dropping rate is 1-2 mL / min, with a maximum of 1.5 mL / min.

[0103] In step S5 of this invention, humic acid-coated nano-mineralized biochar material is prepared by wet ball milling.

[0104] Nano-mineralized biochar and humic acid are mixed at a mass ratio of 1:1, and an appropriate amount of deionized water is added to prepare a slurry with a solid content of 20-30%, most preferably 25%. The slurry is then placed in a planetary ball mill for ball milling and dried at a certain temperature. The ball milling speed is 300-500 rpm, most preferably 400 rpm; the ball milling time is 3-5 h, most preferably 4 h; the drying temperature is 50-80℃, most preferably 60℃; and the drying time is 8-24 h, most preferably 12 h.

[0105] In step S6 of this invention, the functional biomimetic mineralized membrane slow-release material is prepared by microbial inoculation and biofilm induction;

[0106] Pasteurella multocida was inoculated into Pasteurella multocida culture medium and cultured at 30°C with shaking at 150 rpm until OD reached. 600=1.0; Humic acid-coated nano-mineralized biochar material was mixed with bacterial solution at a ratio of 1g:10mL, and adsorption was carried out at 30℃ and 50 rpm for 3 h. After discarding the supernatant, the material was transferred to a sterile tray, covered with plastic wrap to maintain humidity, and incubated at 30℃ for 36 h. Finally, the final product was obtained by vacuum freeze-drying for 24 h; wherein, the OD of the bacterial solution was 1.0. 600 =0.8-1.2, most preferably 1.0; adsorption oscillation speed is 40-60 rpm, most preferably 50 rpm; static incubation temperature is 28-32℃, most preferably 30℃; humidity is 85-95%, most preferably 90%; freeze-drying temperature is -60-10℃, most preferably -45℃; freeze-drying vacuum degree is 10-100 Pa, most preferably 15 Pa.

[0107] In the preparation of the functional biomimetic mineralized membrane sustained-release material, the *Sporosarcina pasteurii* strain used was purchased from a standard bacterial culture center and was a second-generation pure culture. After activation and amplification in a specific culture medium, this strain was used to induce the formation of a biomimetic mineralized membrane on the surface of functionalized biochar. Its basic biological characteristics, culture conditions, and main uses are shown in Table 1.

[0108] Table 1. Main information on Bacillus pasteurellii used in this invention.

[0109]

[0110] Specifically, the composition of the Pasteurella multocida culture medium used is shown in Table 2:

[0111] Table 2. Pasteurella multocida culture medium used in this invention

[0112]

[0113] Example 1

[0114] A multi-level channel-biomimetic mineralization synergistic drug resistance gene blocking material and its preparation method, comprising the following steps:

[0115] S1. Select fresh, mold-free, and insect-free camellia seeds with a moisture content controlled at 10%. Use mechanical shelling equipment to separate the shell from the kernel. After shelling, the weight percentage of the camellia seed kernel to the shell should be controlled at 98%. After mechanically pressing the camellia seed kernel, collect camellia oil cake (CP). Wash it with clean water and dry it in a 105 ℃ oven for 24 h. The organic matter content of the camellia seed kernel should be 60%. After pre-freezing the camellia oil cake for 24 h, first transfer it to a crusher to coarsely crush it into 2.5 mm particles. Then transfer it to an ultra-fine pulverizer to grind it through a 200 mesh sieve to obtain camellia seed powder (CP). The pre-freezing temperature is -15 ℃ and the grinding time is 45 min.

[0116] S2. Weigh 50 g of camellia seed powder (CP) into a Soxhlet extractor. Add 300 mL of n-hexane solvent to a 500 mL round-bottom flask and add 4 boiling stones to prevent bumping. Stop the reaction at 85 ℃ for 7 h. Remove the extraction sleeve and dry it at 60 ℃ for 4 h. The solvent is n-hexane; the bath temperature is 85 ℃; the condensate flow rate is 2 L / min. After ultrasonic cleaning of the defatted camellia seed powder, defatted camellia cake powder (DCP) is obtained. The cleaning solution is 800 mL of ethanol-water solution (i.e., a mixture of ethanol and water with a volume ratio of 1-3:1), the pH of the cleaning solution is 5.5; the ultrasonic frequency is 40 kHz, the power density is 0.5 W / mL, and the temperature is 60 ℃.

[0117] S3. Defatted camellia oil cake powder (DCP) and nano-SiO2 were placed in a ball mill at a mass ratio of 5:1 and ball-milled at 400 rpm for 2 h. The ball-milled material was then mixed with KOH at a mass ratio of 1:1, and deionized water was added to form a slurry. The slurry was dried at 80℃ to constant weight. Under N2 protection in a tube furnace, the temperature was increased to 400℃ at 5℃ / min and held for 30 min, then increased to 800℃ at 10℃ / min and held for 60 min. After natural cooling, the slurry was soaked in 1 mol / L HCl solution for 6 h, washed with deionized water until neutral, and dried at 105℃ for 12 h. The N2 flow rate was 0.4 L / min. Hierarchical porous biochar (HPBC) was obtained.

[0118] S4. Graded porous biochar (HPBC) and urea were mixed at a mass ratio of 1:10, heated at 150 °C for 2 h under N2 atmosphere, cooled, washed 3 times with deionized water, and dried at 60 °C for 12 h; wherein the N2 flow rate was 0.2 L / min; aminated biochar (N-HPBC) was obtained.

[0119] Aminated biochar (N-HPBC) was dispersed in a 0.1 mol / L MgCl2 solution and shaken at room temperature for 2 h. After solid-liquid separation, the solid material was retained. The shaking speed was 180 rpm.

[0120] An equal volume of 0.1 mol / L (NH4)2HPO4 solution was slowly added dropwise to the system under vigorous stirring, while the pH was adjusted and maintained at 9.0 with NaOH solution. After the reaction was completed, the mixture was filtered, washed three times each with ethanol and water, and freeze-dried for 24 h. The dropping rate was 1.5 mL / min. Nano-mineralized biochar (MAP@N-HPBC) was obtained.

[0121] S5. Mix nano-mineralized biochar (MAP@N-HPBC) and humic acid at a mass ratio of 1:1, add an appropriate amount of deionized water to prepare a slurry with a solid content of 25%; place it in a planetary ball mill and ball mill at 400 rpm for 4 h, and then dry it at 60 ℃ for 12 h to obtain humic acid-coated nano-mineralized biochar material (HUM-MAP@N-HPBC).

[0122] S6. Inoculate *Bacillus pasteurellii* into *Bacillus pasteurellii* culture medium and incubate at 30°C with shaking at 150 rpm until OD. 600 =1.0; Humic acid-coated nano-mineralized biochar material (HUM-MAP@N-HPBC) was mixed with bacterial solution at a ratio of 1 g: 10 mL, and adsorbed by shaking at 30 ℃ and 50 rpm for 3 h. After discarding the supernatant, the material was transferred to a sterile tray, covered with plastic wrap to maintain humidity, and incubated at 30 ℃ for 36 h. Finally, the final product was obtained by vacuum freeze-drying for 24 h; among which, the OD of the bacterial solution was 1.0. 600 =1.0; adsorption oscillation speed was 50 rpm; static incubation temperature was 30℃; humidity was 90%; functional biomimetic mineralized membrane sustained-release material (MGPBC@HA) was obtained. The functional biomimetic mineralized membrane sustained-release materials used in the following examples were all prepared using the material prepared in Example 1.

[0123] Example 2

[0124] This embodiment systematically analyzes the dynamic changes in soil physicochemical indicators of different treatment groups, and elucidates in depth the soil improvement effect and multi-level mechanism of action of the functional biomimetic mineralization membrane slow-release material (MGPBC@HA) prepared in this invention in the leek-chicory intercropping system, including:

[0125] 1. Test site and soil background

[0126] The experiment was conducted at a vegetable base in Xiqing District, Tianjin. The tested soil type was alluvial soil. Due to the continuous application of chicken manure organic fertilizer for three years, significant antibiotic resistance gene contamination was observed in the soil, with a background value of 10. 4 -10 6 copies / g.

[0127] 2. Experimental Design

[0128] The experiment consisted of four treatment groups, each with three replicates, using a completely randomized block design.

[0129] Control group (CK): Intercropping of chives and chicory (no additional crops applied)

[0130] Control group J: Chives alone (no other ingredients, same area)

[0131] Control group K: Chicory planted alone (no materials applied, same area)

[0132] Experimental group JK: Leek-chicory intercropping + functional biomimetic mineralized membrane slow-release material base application

[0133] 3. Implementation Methods

[0134] Application of materials: 7 days before sowing, at a rate of 1000 kg / hm² 2 The MGPBC@HA material was evenly spread on the surface of the JK plot in the experimental group, and then tilled into the 0-20 cm soil layer using a rotary tiller to ensure that the material was fully mixed with the soil.

[0135] Planting pattern: The experimental groups JK and control group CK adopted the same intercropping pattern. Flatbed cultivation was used, with chives and chicory intercropped in a 1:1 ratio in the same row, with a row spacing of 20 cm. The spacing between chive plants was 8-10 cm, with 8-10 plants per plant; the spacing between chicory plants was 8-10 cm, with 5-8 plants per plant. The monoculture planting density in control groups J and K was consistent with the corresponding crops in the intercropping groups. Each plot area was 20 m². 2 (0.03 mu), total experimental area 0.5 mu.

[0136] Field management: All treatment groups adopted the same routine water and fertilizer management measures.

[0137] 4. Sample collection and testing

[0138] Sampling: Sixty days after planting, soil samples from the 0-20 cm soil layer of each plot were collected using a five-point sampling method and mixed to form a representative sample. Four independent biological replicates were obtained from each treatment group.

[0139] DNA Extraction: Approximately 0.5 g of soil sample was weighed and total genomic DNA was extracted from the soil using the FastDNA SPIN Kit for Soil (MPBiomedicals, USA). Three replicate extractions were performed for each soil sample to ensure accuracy.

[0140] qPCR assay: The absolute abundance of 16 high-risk ARGs was quantified using the QuantStudio 6 Pro real-time quantitative PCR system (Applied Biosystems). Target genes included:

[0141] β-lactams: bla ampC , bla TEM-1 , bla OX-1

[0142] Macrolides: ermB erm C

[0143] Aminoglycosides: str A, str B cfr , fex A, aad A

[0144] Sulfonamides: sul 1. sul 2

[0145] Tetracyclines: tet O、 tet W, tet M, tet Q, tet L, tet X

[0146] The qPCR reaction system consisted of 20 µL, containing 10 μL of TB Green Premix Ex Taq (TliRNase H Plus, Takara), 0.4 μL of ROX Reference Dye II, 0.4 μL each of the pre- and post-primers, 6.8 μL of nuclease-free PCR water, and 2 μL of sample DNA. Primer information is shown in Table 3. The qPCR amplification program consisted of: first, pre-denaturation at 95°C for 30 s; then, 40 cycles, each consisting of 5 s of denaturation at 95°C and 34 s of annealing at 60°C. For melting curve analysis, the temperature range was set between 55 and 95°C to ensure reaction specificity.

[0147] Table 3 qPCR primer sequence information

[0148]

[0149]

[0150] For applications involving lightly polluted soil or primarily aimed at soil improvement and yield increase, the dosage can be appropriately reduced to 100-500 kg / hm². 2 Because the MGPBC@HA material provided by this invention has extremely high functional group density and catalytic activity, it can significantly improve the rhizosphere microecology even at low dosages, and effectively inhibit the spread of ARGs through mechanisms such as competitive exclusion. For moderately to severely polluted soils, the dosage can be appropriately increased to 2000-5000 kg / hm². 2 This is to ensure that there are sufficient active sites to effectively control ARGs.

[0151] 5. Specific planting layout of the chive-chicory intercropping system

[0152] Figure 1 A schematic diagram of the field layout of this intercropping system is shown, and the specific implementation method is as follows.

[0153] The experiment adopted a standard flatbed cultivation model, with a bed width of 1.2 meters, a ridge width of 0.3 meters, and an effective planting area width of 0.9 meters. In the T1 group (intercropping + material) and the CK1 group (intercropping control), chives (Allium tuberosum) and chicory (Cichorium endivia) were intercropped in the same row at a ratio of 1:1. The planting rows were oriented north-south to ensure that the crops received uniform light conditions.

[0154] The specific arrangement for intercropping is as follows: leeks and chicory are planted alternately in each row, with a row spacing of 20 cm. The spacing between leek plants is 8-10 cm, with 8-10 seedlings planted per hole, ensuring a basic seedling count of 80,000-100,000 plants per acre. The spacing between chicory plants is also 8-10 cm, with 5-8 seedlings planted per hole, maintaining a basic seedling count of 50,000-80,000 plants per acre. This arrangement fully utilizes the complementarity of the two crops in terms of space and nutrient requirements.

[0155] The control groups included: Control J, which was monocultured with chives at the same planting density as the intercropped chives group; and Control K, which was monocultured with chicory at the same planting density as the intercropped chicory group. All treatments underwent identical cultivation and management practices to ensure comparability of the experimental results.

[0156] Each experimental plot had an area of ​​20 square meters (5 meters × 4 meters), equivalent to 0.03 acres. A total of 16 plots were set up for the entire experiment, with a total experimental area of ​​0.5 acres. A 1-meter-wide isolation zone was set up between plots to prevent interference between treatments. The experiment used a completely randomized block design, with each treatment replicated four times to ensure the statistical reliability of the experimental results.

[0157] The advantages of this intercropping arrangement are as follows: First, the shallow root system of chives and the deep root system of chicory create a three-dimensional root structure, improving soil resource utilization efficiency. Second, the differences in growth cycle and nutrient requirements between the two crops reduce interspecific competition. Finally, this configuration provides diverse ecological niches for functional microorganisms, which is conducive to the MGPBC@HA material exerting its dual function of promoting plant growth and inhibiting ARGs spread. The schematic diagram clearly shows the spatial arrangement of the intercropping, providing an intuitive reference for actual field operations.

[0158] Based on the above experimental design and sampling scheme, the obtained soil samples were systematically tested and analyzed, and the results are as follows. Figure 2 and Figure 3It can be seen that the forms and contents of soil nitrogen differed significantly among the treatment groups. Nitrate nitrogen (NO3) - The nitrogen (nitrate) content showed a gradient of > control group CK (159 mg / kg) > control group J (96.57 mg / kg) > control group K (47.34 mg / kg) > experimental group JK (27.28 mg / kg). This trend reveals the synergistic effect of MGPBC@HA material and intercropping system: the aminated biochar in the material fixes nitrate nitrogen through adsorption of functional groups, slowing down its conversion rate; the urease pre-loaded in the material catalyzes the decomposition of urea into ammonium nitrogen, directly providing nitrogen source for crops and reducing the formation and accumulation of nitrate nitrogen; at the same time, the differences in root exudates between the two crops create a diverse nitrogen transformation microenvironment, promoting the biological fixation and denitrification of nitrate nitrogen.

[0159] Ammonium nitrogen (NH4) + The changes in ammonium nitrogen (NH4PO4·6H2O) content showed that the experimental group JK (22.23 mg / kg) maintained an appropriate level, between that of the monoculture control group J (32.31 mg / kg) and the control group K (19.18 mg / kg). This result indicates that the nano-struvite (MgNH4PO4·6H2O) in the MGPBC@HA material plays a role in the slow-release of ammonium nitrogen. Its unique crystal structure allows it to slowly dissolve in the soil, continuously releasing ammonium nitrogen, thus meeting crop requirements while avoiding the volatilization loss of ammonium. Simultaneously, the humic acid component, by forming an ammonium-humic acid complex, reduces the nitrification of ammonium nitrogen and improves nitrogen use efficiency.

[0160] The total phosphorus content in the experimental group JK reached 1.3 g / kg, significantly higher than that in the control group CK (0.64 g / kg). This change was mainly attributed to the phosphorus release effect of nano-struvite in the MGPBC@HA material and the phosphorus activation effect of humic acid. Nano-struvite releases phosphorus in the soil through the following pathways: slow dissolution in the acidic rhizosphere microenvironment, directly releasing plant-available phosphate ions; decomposition promoted by organic acids produced by soil microbial metabolism; and biodegradation under the action of soil enzymes. This slow release characteristic synchronizes phosphorus supply with crop demand, greatly improving the utilization rate of phosphate fertilizer.

[0161] The available phosphorus content in the experimental group JK was 45.76 mg / kg, remaining within the ideal range. Humic acid played a crucial role in this process: its carboxyl and phenolic hydroxyl groups complexed with metal ions such as calcium, aluminum, and iron in the soil, reducing the chemical fixation of phosphorus; simultaneously, it formed a physical barrier by encapsulating phosphate fertilizer particles, delaying the phosphorus fixation process; furthermore, humic acid stimulated root development, expanding the space for phosphorus absorption. Pre-existing phosphatases in the material and introduced indigenous soil microorganisms promoted the mineralization of organic phosphorus and converted insoluble phosphorus into soluble phosphorus, further improving phosphorus availability.

[0162] The organic carbon content in the experimental group JK was 25.79 g / kg, which was slightly lower than that in the control group CK, but the carbon pool quality was significantly improved. The MGPBC@HA material optimizes the carbon-nitrogen balance through the following mechanisms: humic acid, as a stable organic carbon source, has an aromatic structure that is difficult to decompose rapidly, forming a persistent carbon pool; at the same time, humic acid forms an organic-inorganic complex with nitrogen, which slows down the mineralization rate of nitrogen and achieves the synchronization of carbon and nitrogen release.

[0163] The carbon-nitrogen ratio in experimental group JK was maintained at an optimal level of around 8.4. This balance is beneficial to the activity of soil microorganisms and nutrient cycling. A moderate carbon-nitrogen ratio promotes the diversity of microbial communities, accelerates the decomposition of organic matter and the release of nutrients, while avoiding nitrogen loss caused by nitrogen fixation due to an excessively high carbon-nitrogen ratio or nitrogen loss caused by an excessively low carbon-nitrogen ratio.

[0164] Depend on Figure 4 It was found that the pH value in experimental group JK was 8.68, remaining within the slightly alkaline range. This environment is conducive to the function of the MGPBC@HA material. The slightly alkaline environment promotes the solubility and reactivity of humic acid, enhancing its ability to bind with nutrients; at the same time, it is suitable for the growth and metabolism of urease-active microorganisms enriched in the soil, improving the ability of urea hydrolysis and calcium carbonate formation. The calcium carbonate crystals and biofilm matrix pre-formed on the material surface improve the soil environment through multiple pathways: its microstructure promotes the formation of soil aggregates, improving the soil's water and fertilizer retention capacity; its buffering effect helps regulate soil pH and maintain a suitable chemical environment; and its occupied ecological niche inhibits the growth of pathogens through competition, improving the microbial community structure.

[0165] The hierarchical porous structure of MGPBC@HA materials provides an ideal habitat for soil microorganisms. Macropores (>50 nm) provide living space and movement channels for microorganisms; mesopores (2-50 nm) facilitate the retention and transport of water and nutrients; and micropores (<2 nm) provide a huge specific surface area for the adsorption of nutrients and enzyme molecules. This multi-scale porous structure creates diverse microenvironments, promoting the coexistence and cooperation of different functional microorganisms.

[0166] The system offers significant environmental benefits: nitrate nitrogen is reduced by 83%, greatly minimizing the risk of leaching pollution; phosphorus utilization is increased by over 35%, reducing phosphate fertilizer input; organic matter quality is improved, enhancing soil carbon sequestration; and microbial diversity is increased, improving ecosystem stability. This improvement effect is long-lasting; the MGPBC@HA material has a half-life of over 180 days in the soil, providing long-term improvement benefits.

[0167] In summary, the MGPBC@HA material, through multiple mechanisms including physical adsorption, chemical slow release, and niche regulation, synergistically interacts with the leek-chicory intercropping system to jointly construct an efficient, stable, and sustainable soil nutrient management system, providing an effective approach to address non-point source pollution in intensive agriculture. Furthermore, it can be seen that in the method of using the functional biomimetic mineralized membrane slow-release material of this invention to control antibiotic resistance genes in the leek-chicory intercropping system, there is a synergistic effect between the functional biomimetic mineralized membrane slow-release material and the leek-chicory intercropping, which can synergistically improve the effectiveness of controlling antibiotic resistance genes in the intercropped farmland soil.

[0168] Example 3

[0169] This embodiment systematically analyzes the composition and structural changes of the root soil microbial community in different treatment groups using high-throughput sequencing technology, and elucidates the multi-level mechanism of MGPBC@HA material in regulating the microbial community in the leek-chicory intercropping system, including:

[0170] This experiment consisted of four treatment groups, with three biological replicates in each group. Experimental group JK received leek-chicory intercropping with basal application of functional biomimetic mineralized membrane slow-release material (MGPBC@HA); control group CK received leek-chicory intercropping (without material application); control group J received leek monoculture with basal application of MGPBC@HA; and control group K received chicory monoculture with basal application of MGPBC@HA. All field implementation details, including the application dosage and method of MGPBC@HA, soil tillage methods, planting spacing between leeks and chicory, planting density, and water and fertilizer management, were the same as in Example 2 for all treatment groups. Sixty days after planting, root soil samples were collected from each treatment group, and the microbial community structure was analyzed using 16S rRNA gene high-throughput sequencing technology. Data analysis was performed using QIIME2 software, and species annotation was performed using the Silva database. The root soil microbial data for each treatment group are shown below. Figure 5-7 As shown.

[0171] At the phylum level, the microbial community composition of the different treatment groups showed significant differences. Figure 5 Proteobacteria were the dominant phylum in all groups, but their relative abundance in the JK group (24.3%) was significantly lower than that in the J group (26.2%) and the K group (27.1%), but similar to that in the CK group (24.8%). Acidobacteriota abundance in the JK group (19.2%) was higher than that in the J group (18.1%) and the K group (18.3%), but lower than that in the CK group (26.4%). Actinobacteriota abundance in the JK group (19.1%) was similar to that in the J group (19.2%) and the K group (18.4%), but significantly higher than that in the CK group (15.3%).

[0172] Notably, the abundance of Chloroflexi in group JK (12.5%) was significantly higher than in other groups, indicating that the MGPBC@HA material created and maintained a microenvironment favorable for the growth of this phylum in the intercropping system. The abundance of Bacteroidota in group JK (8.7%) was also higher than in the monoculture group, showing that the intercropping system was beneficial for the enrichment of this phylum.

[0173] At the level of family classification ( Figure 6 ), Fungiaceae ( Pyrinomonadaceae Sphingosomalidaceae was the most abundant family in all groups, but its abundance in group JK (7.2%) was slightly lower than that in group J (7.4%) and group K (7.3%), and similar to that in group CK (7.1%). Sphingomonadaceae The abundance of *Gymnospermia* in group JK (6.3%) was similar to that in group J (6.2%), but significantly lower than that in group K (8.1%) and group CK (11.2%). *Gymnospermia* family ( Gemmatimonadaceae The abundance of ) in the JK group (7.1%) was significantly higher than that in the J group (6.2%) and the CK group (6.3%), and similar to that in the K group (7.2%).

[0174] Of particular note is the presence of Nitrifying Spirulina family ( ) in group JK. Nitrospiraceae The abundance of nitrifying bacteria (3.2%) was significantly higher than that of other groups, indicating that the intercropping system combined with MGPBC@HA material optimized the nitrogen cycle microecology and promoted the enrichment of nitrifying bacteria, which is conducive to nitrogen conversion and utilization.

[0175] At the genus level ( Figure 7 ), Sphingosine Microbes ( Sphingomicrobium The abundance of *Gp6* in the JK group was the highest among all groups, but its abundance in the JK group (3.2%) was lower than that in the J group (4.1%) and the K group (4.3%), and similar to that in the CK group (3.3%). The abundance of *Gp6* in the JK group (4.2%) was similar to that in the J group (4.1%) and the K group (4.3%), but significantly higher than that in the CK group (3.5%). The abundance of *PSRF01* in the JK group (3.1%) was lower than that in the J group (3.3%) and the K group (3.2%), and similar to that in the CK group (3.0%).

[0176] Pseudomonas spp. in group JK ( Pseudomonas The abundance of *Bacillus* (2.8%) was significantly higher than in other groups. This genus contains various plant growth-promoting bacteria that can produce antibiotics to inhibit the growth of pathogens. Meanwhile, *Bacillus* genus (… BacillusThe abundance of *Bacillus* in the JK group (2.5%) was also significantly higher than that in the monoculture group, which is related to the suitable habitat and nutrient conditions provided by the MGPBC@HA material promoting the colonization and proliferation of indigenous Bacillus in the soil. The microbial community structure of each treatment group was clearly separated. The JK group and the CK group clustered together, while the J group and the K group formed independent clusters, indicating that the planting pattern had a greater impact on the microbial community structure than the material application.

[0177] The material's hierarchical porous structure provides diverse ecological niches for microorganisms. Macropores (>50 nm) serve as "refuges" for microorganisms, providing protective space for bacteria against protozoan predation and environmental stress; mesopores (2-50 nm) retain moisture through capillary action, creating a suitable micro-aqueous environment; micropores (<2 nm) possess a huge specific surface area (>2000 m² / g), adsorbing organic matter and enzyme molecules through π-π interactions and electrostatic interactions, providing abundant nutrient sources for microorganisms. This multi-scale porous structure forms a "microbial hotel," significantly improving the habitat efficiency and population density of microorganisms.

[0178] Nano-struck stone (MgNH4PO4·6H2O) achieves intelligent nutrient release through interfacial dissolution-recrystallization equilibrium. In the acidic rhizosphere microenvironment (pH = 5.5-6.5), H... + Attacking the mineral surface, prompting NH4 + and PO4 3- The ions are released slowly; however, in the neutral environment of the rhizosphere (pH=7.0-7.5), the supersaturated ions recrystallize, forming a dynamic equilibrium. This pH-responsive release mechanism synchronizes nutrient supply with plant needs, reducing nutrient loss and waste.

[0179] Humic acid forms coordination compounds with nutrient molecules through its abundant functional groups (-COOH, -OH, etc.). The carboxyl group reacts with NH4+. + This forms a stable ammonium-humic acid complex, reducing ammonium nitration and volatilization loss; the phenolic hydroxyl groups react with PO4. 3- Through hydrogen bonding, the phosphorus fixation process is slowed down; at the same time, humic acid, as an electron shuttle, promotes the extracellular respiration process of microorganisms, accelerating the decomposition of organic matter and the conversion of nutrients.

[0180] MGPBC@HA materials regulate the rhizosphere microenvironment through their unique physicochemical properties. Pre-existing urease activity and an alkaline microenvironment on the material surface promote urea decomposition to produce CO2 and NH3, with NH3 hydrating to generate NH4. + and OH - This causes a local pH increase, promoting Ca2+ uptake. 2+ and CO3 2-Calcium carbonate precipitates are formed. These precipitates fill soil pores, improve soil structure, and provide attachment sites for other microorganisms. This material inhibits pathogens by altering the rhizosphere niche. Its surface properties are conducive to the attachment and accumulation of beneficial microorganisms; it reduces the survival opportunities of pathogens through nutrient competition and space occupancy effects; and the humic acid and other components released by the material can stimulate plant systemic resistance, indirectly enhancing the plant's defense against pathogens.

[0181] The differences in the composition of root exudates between chives and chicory create diverse micro-niches. Chive roots secrete sulfur-containing compounds (such as diallyl disulfide), selectively promoting sulfur-oxidizing bacteria (such as...). Thiobacillus The growth of chicory; the roots secrete bitter terpenoids, attracting microorganisms with degradation capabilities (such as... Pseudomonas The root systems of these two crops are spatially intertwined, forming physical and chemical barriers that prevent the spread of pathogens.

[0182] MGPBC@HA materials create a nutrient gradient diffusion field in the rhizosphere. The micro-zones near the root surface have high nutrient concentrations, suitable for eutrophic microorganisms (such as...). Proteobacteria Growth; the nutrient concentration is low in areas far from the root surface, which is conducive to the growth of oligotrophic microorganisms (such as... Acidobacteria Survival. This spatial heterogeneity promotes the diversity of microbial communities and enhances the stability of the ecosystem.

[0183] The material inhibits the horizontal transfer of antibiotic resistance genes (ARGs) through multiple pathways. The surface properties of the nanomaterials can adsorb free DNA, reducing its environmental concentration and decreasing the frequency of transformation; the selective microenvironment created by the material helps suppress the abundance of ARG host bacteria; humic acid promotes the formation of soil aggregates, physically isolating microbial cells and reducing the chance of conjugation and transfer. These mechanisms work together to significantly reduce the risk of ARG transmission.

[0184] The improved microbial community exhibited enhanced ecological functions: the balance between nitrifying and denitrifying bacteria increased nitrogen conversion efficiency by 35%; phosphatase activity increased by 42%, promoting the mineralization of organic phosphorus; microbial biomass carbon increased by 28%, and soil respiration intensity increased by 31%, indicating a significant enhancement in microbial metabolic activity. These changes collectively constitute an efficient, stable, and sustainable soil ecosystem, providing a theoretical basis and technical support for green agricultural production.

[0185] MGPBC@HA materials influence soil microbial communities through multiple mechanisms: humic acid in the material provides carbon and energy sources for microorganisms, promoting their growth and metabolism; nano-struvite slowly releases nutrients such as nitrogen and phosphorus, maintaining the nutritional needs of microorganisms; and the hierarchical porous structure provides diverse habitat microenvironments for microorganisms. The rhizosphere microenvironment created by the material is conducive to the accumulation of beneficial microorganisms, while simultaneously inhibiting the growth of pathogenic microorganisms through resource competition.

[0186] Intercropping systems create diverse rhizosphere environments, with the root exudates of leeks and chicory complementing each other, providing suitable growth conditions for microorganisms with different functions. The spatially interwoven root systems of the two crops form a physical barrier, preventing the spread of pathogens while promoting spatial heterogeneity of microorganisms.

[0187] The results of this invention demonstrate that the combination of the leek-chicory intercropping system and the application of MGPBC@HA material significantly improves the soil microbial community structure, enhances microbial diversity, and promotes the enrichment of beneficial microorganisms. This improved microbial community helps enhance the stability and resilience of the soil ecosystem, improves nutrient use efficiency, and reduces disease occurrence. This technology provides an effective method for regulating soil microorganisms in green agricultural production and has significant value for widespread application. Furthermore, it can be seen that the application of the functional biomimetic mineralized membrane slow-release material in the leek-chicory intercropping system for controlling antibiotic resistance genes in intercropped farmland demonstrates a synergistic effect between the functional biomimetic mineralized membrane slow-release material and the leek-chicory intercropping, synergistically enhancing the multi-level regulation of the microbial community.

[0188] Example 4

[0189] This embodiment systematically analyzes the composition and structural changes of non-root soil microbial communities in different treatment groups using high-throughput sequencing technology, elucidating the multi-level mechanism of MGPBC@HA material in regulating microbial communities in the leek-chicory intercropping system, including:

[0190] This experiment consisted of four treatment groups, with three biological replicates in each group. Experimental group JK received leek-chicory intercropping with basal application of functional biomimetic mineralized membrane slow-release material (MGPBC@HA); control group CK received leek-chicory intercropping (without material application); control group J received leek monoculture with basal application of MGPBC@HA; and control group K received chicory monoculture with basal application of MGPBC@HA. All field implementation details, including the application dosage and method of MGPBC@HA, soil tillage methods, planting spacing between leek and chicory, planting density, and water and fertilizer management, were the same as in Example 2 for all treatment groups. Sixty days after planting, non-root soil samples (5-10 cm from the root surface) were collected from each treatment group, and the microbial community structure was analyzed using 16S rRNA gene high-throughput sequencing technology. Data analysis was performed using QIIME2 software, and species annotation was performed using the Silva database. The non-root soil microbial data for each treatment group are shown below. Figure 8-10 As shown.

[0191] At the phylum level, the microbial community composition of the different treatment groups showed significant differences. Figure 8 Proteobacteria was the most dominant phylum in all groups, with a significantly higher relative abundance in group JK (36.2%) than in groups J (30.1%) and K (27.3%), and also higher than in group CK (30.0%). This result indicates that the intercropping system combined with the application of MGPBC@HA material promoted the growth of Proteobacteria, a phylum containing many important species with functions in pollutant degradation and nutrient transformation.

[0192] The abundance of Acidobacteriota in group JK (17.3%) was lower than that in group J (22.1%) and group K (25.2%), but similar to that in group CK (17.8%). The abundance of Actinobacteriota in group JK (11.2%) was lower than that in group J (14.3%) and group K (15.1%), but similar to that in group CK (11.5%). Notably, the abundance of Chloroflexi in group JK (9.8%) was significantly higher than in other groups, indicating that the MGPBC@HA material promoted the growth of this phylum in the intercropping system.

[0193] At the level of family classification ( Figure 9 ), Fungiaceae ( Pyrinomonadaceae Sphingosomalidaceae was the most abundant family in all groups, but its abundance in group JK (9.2%) was lower than that in group J (13.1%) and group K (14.3%), and similar to that in group CK (9.5%). Sphingomonadaceae The abundance of ) in the JK group (8.3%) was lower than that in the J group (9.2%) and the K group (9.4%), but similar to that in the CK group (8.5%).

[0194] Of particular note is the family Steroidobacteria ( Steroidobacteraceae The abundance of this bacterium in group JK (11.2%) was significantly higher than that in group J (4.1%) and group K (4.3%), and similar to that in group CK (11.5%). This family of bacteria has the ability to degrade complex organic matter, and its increased abundance indicates that intercropping systems are beneficial to the decomposition and transformation of soil organic matter.

[0195] At the genus level ( Figure 10 PSRF01 was the most abundant genus in all groups, but its abundance in group JK (6.2%) was lower than that in group J (7.3%) and group K (7.5%), but similar to that in group CK (6.4%). Sphingosine microbes ( Sphingomicrobium The abundance of Gp6 in the JK group (4.3%) was lower than that in the J group (6.2%) and the K group (6.4%), but similar to that in the CK group (4.5%). The abundance of Gp6 in the JK group (3.2%) was similar to that in the J group (3.3%) and the K group (4.1%), but lower than that in the CK group (3.4%).

[0196] Pseudomonas spp. in group JK ( Pseudomonas The abundance of *Bacillus* (3.1%) was significantly higher than in other groups, and this genus contains a variety of species with functions in pollutant degradation and plant growth promotion. Meanwhile, *Bacillus* (…) Bacillus The abundance of microorganisms in the JK group (2.8%) was also significantly higher than that in the monoculture group, which is related to the diffusion and colonization of functional microorganisms in the applied MGPBC@HA material. The microbial community structure of each treatment group was clearly separated. The JK group and the CK group clustered together, while the J group and the K group formed independent clusters, indicating that the planting pattern has a greater impact on the structure of non-root soil microbial community than the material application.

[0197] MGPBC@HA materials influence non-root soil microbial communities through multiple mechanisms. The nanoscale nutrient slow-release system within the material releases nutrients such as nitrogen and phosphorus slowly into non-root soil areas via concentration gradient diffusion. This slow release mode avoids drastic nutrient fluctuations, providing a stable growth environment for microorganisms. The humic acid component, through its colloidal properties, promotes lateral nutrient diffusion in the soil, ensuring sufficient nutrient supply for microorganisms far from the roots. The porous structure and surface chemistry of the material provide pathways and sites for microbial migration and colonization, facilitating the diffusion and distribution of soil functional microorganisms into non-root areas. During this process, the organic matter carried by the material and the pre-formed biofilm matrix alter the soil microstructure, creating a microenvironment conducive to the colonization of diverse microbial communities. The material's hierarchical pore structure not only provides habitat for microorganisms but also influences their distribution by altering the soil aggregate structure. Macropores facilitate the transport of microorganisms and nutrients, mesopores provide a protective microenvironment, and micropores enrich organic matter and enzyme molecules through adsorption. This multi-scale structural regulation maintains high activity and diversity of microbial communities in non-root soils. Secondary metabolites secreted by plant roots diffuse into non-root areas through the soil pore network, and these chemical signaling molecules regulate the structure and function of microbial communities. The humic acid component in the MGPBC@HA material acts as an electron shuttle, promoting electron transfer and metabolic cooperation among microorganisms, thereby enhancing the overall function of the microbial community.

[0198] The results of this invention demonstrate that the application of MGPBC@HA material in the leek-chicory intercropping system not only improves the rhizosphere microbial community but also positively impacts the non-root soil microbial community. This improvement effect helps enhance the ecological function of the entire soil profile, improve nutrient use efficiency, and reduce soil degradation. This technology provides an effective method for improving the ecological environment of farmland soil and has significant theoretical and practical value. Furthermore, it can be seen that the application of the functional biomimetic mineralized membrane slow-release material in the leek-chicory intercropping system for controlling antibiotic resistance genes in intercropped farmland exhibits a synergistic effect between the functional biomimetic mineralized membrane slow-release material and the leek-chicory intercropping, synergistically improving the ecological environment of farmland soil.

[0199] Example 5

[0200] This embodiment uses high-throughput quantitative PCR technology to analyze the abundance changes of antibiotic resistance genes (ARGs) in soil under different treatment groups, focusing on elucidating the overall inhibitory effect of MGPBC@HA material on various ARGs in the leek-chicory intercropping system, including:

[0201] This experiment set up 4 treatment groups, with 3 biological replicates in each group. Experimental group JK received leek-chicory intercropping with functional biomimetic mineralized membrane slow-release material (MGPBC@HA) as basal application; control group CK received leek-chicory intercropping (without material application); control group J received leek monoculture with MGPBC@HA as basal application; and control group K received chicory monoculture with MGPBC@HA as basal application. In all the above treatment groups, the application dosage and method of MGPBC@HA, soil tillage methods, planting spacing between leeks and chicory, planting density, and water and fertilizer management details were all the same as in Example 2. After 60 days of planting, the absolute abundance of soil ARGs in each treatment group was measured (…). Figure 11-17 The results show that different treatment methods have significantly different reduction effects on various ARGs.

[0202] like Figure 11 As shown, β-lactam ARGs bla ampC The experimental group JK had the highest gene abundance, and its abundance was significantly lower than that of the control groups, indicating that this treatment had a prominent inhibitory effect on these clinically relevant genes. Macrolides erm C gene ( Figure 12 The abundance of this gene was high in the monoculture chicory group (K), but significantly decreased in the JK group, indicating that intercropping and combined treatment effectively inhibited the spread of this type of gene. Aminoglycosides (ARGs) Figure 13 )middle str Gene A had the highest abundance across all treatments, but the JK group reduced its absolute abundance to the lowest level, significantly better than the single-crop supplementary material treatment groups (J, K).

[0203] It is worth noting that tetracycline ARGs ( Figure 16 The reduction effect was most pronounced with JK treatment, which significantly reduced the abundance of major genes such as tetX. Meanwhile, sulfonamides... sul 2 genes ( Figure 15 The abundance of this gene remained high in monoculture treatments but was significantly suppressed in the JK group. Of particular importance, the JK treatment significantly reduced the abundance of mobile genetic elements (…). Figure 17 In ) intI The gene 2 exhibits a strong reduction ability. As a key vector for the horizontal transfer of ARGs, the reduction in its abundance explains the mechanism by which JK treatment effectively controls the spread of ARGs, because it directly inhibits the gene's ability to transfer between different strains.

[0204] The 60-day absolute abundance removal rate of soil antibiotic resistance genes in the control group (CK) and experimental group (JK) is as follows: Figure 18-22As shown in the figure. The results indicate that the intercropping pattern adopted in this invention can significantly reduce the abundance of various ARGs in the soil. The reduction rates of β-lactams, macrolides, aminoglycosides, sulfonamides, and tetracyclines were 30.0-55.6%, 33.5-40.7%, 60.5-65.0%, 40.6-41.7%, and 34.5-69.5%, respectively. This suggests that the intercropping pattern adopted in this invention may affect the distribution and spread of ARGs by altering the soil microenvironment and microbial community structure.

[0205] Notably, the application of MGPBC@HA material further enhanced the removal of ARGs. In the intercropping system with material application as a base (JK group), the removal rates of various ARGs were significantly improved, with reductions of β-lactams, macrolides, aminoglycosides, sulfonamides, and tetracyclines by 80.2-87.3%, 60.9-65.1%, 62.6-66.8%, 54.6-55.1%, and 84.9-93.0%, respectively. This enhancement effect may stem from the multifunctional characteristics of MGPBC@HA material: First, the material's hierarchical porous structure provides a favorable habitat and physical protection for beneficial soil microorganisms, promoting the colonization and reproduction of functional microbial communities and effectively inhibiting the proliferation of drug-resistant bacteria through niche competition. Second, the pre-existing active components (such as urease and phosphatase) on the material surface and their unique chemical properties can directly interfere with the metabolic activities of drug-resistant bacteria, altering the rhizosphere microenvironment. Third, the nano-struvite and humic acid complex in the material continuously optimizes the soil chemical balance through intelligent slow-release of nutrients. This stable microenvironment is unfavorable for the preservation and horizontal transfer of ARGs. In addition, the humic acid carried by the material and the pre-formed biofilm matrix can act as electron shuttles, promoting interactions between microorganisms and strengthening the soil aggregate structure, physically isolating pathogens, thereby synergistically reducing the risk of ARG transmission through multiple pathways.

[0206] Comprehensive analysis showed that the combined application of leek-chicory intercropping and MGPBC@HA material (JK group) exhibited the most significant and comprehensive removal effect on all types of ARGs, with a removal rate significantly higher than any single treatment group. This indicates a significant synergistic effect between the intercropping system and the functionalized material. In contrast, the effects of single measures were relatively limited and selective: intercropping alone (CK group) had a certain removal effect on some ARGs (such as aminoglycosides), but its ability to control genes such as β-lactams was weak; while in the case of monocropping, the application of materials alone (J group or K group) showed some reduction potential for certain ARGs (such as tetracyclines), but the overall effect was not comprehensive, especially the removal rate of macrolides and sulfonamides was generally low.

[0207] Notably, the JK group, compared to the control group (CK group) without material application, still exhibited a significantly higher additional gain in the removal of β-lactam and tetracycline ARGs, fully demonstrating the core contribution of MGPBC@HA material in the intercropping system. This combined treatment strategy achieves efficient ARG control through multiple mechanisms: the multi-level porous structure of the material and its humic acid components effectively adsorb and fix antibiotic residues, reducing environmental selection pressure; the rhizosphere microenvironment and nutrient slow-release characteristics it creates optimize the microbial community structure, inhibiting the abundance of ARG host bacteria; simultaneously, the complementary effect of root exudates in the intercropping system further strengthens the synergistic physical-chemical-biological control network, thereby significantly reducing the risk of the spread of various ARGs in the soil environment.

[0208] Microbial community analysis revealed a correlation between changes in ARG abundance and changes in the abundance of specific microbial taxa. For example, the increase in the relative abundance of Proteobacteria was consistent with the decreasing trend of β-lactam ARGs, possibly because the proliferation of certain non-drug-resistant strains in Proteobacteria diluted the proportion of drug-resistant strains. Changes in the abundance of Acidobacteria were associated with a decrease in tetracycline ARGs, indicating that this phylum may contain a relatively large number of tetracycline-resistant bacteria. The enrichment of beneficial families such as Steroidobacteriaceae may have inhibited the proliferation of drug-resistant bacteria through a competitive mechanism. These findings are consistent with existing research on the relationship between microbial community structure and ARG distribution. Furthermore, it can be seen that in the method of using the functional biomimetic mineralized membrane slow-release material of this invention to control antibiotic resistance genes in intercropped farmland soil in a leek-chicory intercropping system, the functional biomimetic mineralized membrane slow-release material and the leek-chicory intercropping have a synergistic effect, which can synergistically improve the effect of controlling antibiotic resistance genes in intercropped farmland soil.

[0209] This invention also observed that the intercropping pattern itself already had a significant inhibitory effect on ARGs, and the application of MGPBC@HA material further enhanced this effect. This indicates that, in practical applications, intercropping can be combined with the application of functional materials to achieve more effective control of soil ARGs. This integrated management strategy not only helps reduce the environmental spread risk of ARGs but may also bring additional economic and environmental benefits by improving soil quality and increasing crop yields.

[0210] To objectively evaluate the technical advantages of this invention, it is compared with typical existing methods for controlling soil antibiotic resistance genes (ARGs), as shown in Table 4 below:

[0211] Table 4. Comparison of the present invention with existing soil ARGs control technologies

[0212]

[0213]

[0214] It is evident that the intercropping of leeks and chicory with growth-promoting materials constructs a highly efficient and green agricultural ecological control system. This innovative planting model not only effectively curbs the rampant spread and accumulation of antibiotic resistance genes from manure in the soil, reducing their potential threat to the stability of the soil ecosystem, but also plays a crucial role in ensuring the quality and safety of agricultural products and safeguarding human health. From the perspective of sustainable agricultural development, this model provides a highly feasible and scalable solution to the increasingly serious problem of antibiotic resistance gene pollution in farmland, and is expected to be widely applied in the vegetable planting industry, promoting the development of my country's agriculture towards a more ecological, environmentally friendly, and healthy direction.

[0215] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for controlling antibiotic resistance genes in intercropping farmland soil using functionalized biomimetic mineralized slow-release materials, characterized in that: Includes the following steps: Functionalized biomimetic mineralized slow-release materials will be applied at a rate of 100-5000 kg / hm². 2 The amount of material is evenly spread on the soil surface and then tilled into the 0-20 cm soil layer using a rotary tiller to ensure that the material is fully mixed with the soil. Intercropping is used to select two or more crops with complementary ecological niches for combined planting, thereby controlling antibiotic resistance genes in farmland soil. Among them, the antibiotic resistance genes include: β-lactams bla ampC , bla TEM-1 , bla OX-1 Macrolides: erm B erm C; Aminoglycosides: str A, str B cfr , fex A, aad A; Sulfonamides: sul 1. sul 2; Tetracyclines: tet O、 tet W, tet M, tet Q, tet L, tet X; The preparation method of the functionalized biomimetic mineralized sustained-release material includes the following steps: S1. Fresh camellia seeds are selected, mechanically dehulled and pressed to obtain camellia cake, washed and dried, pre-frozen, crushed and ground, and sieved to obtain camellia seed powder CP. S2. The camellia seed powder CP is refluxed with an organic solvent for degreasing, and then dried to obtain degreased camellia cake powder DCP; S3. The defatted camellia oil cake powder DCP is mixed and ground with a template agent, then mixed with an activator solution to form a slurry. After drying, it is subjected to programmed temperature-controlled pyrolysis. After acid washing, water washing, and drying, graded porous biochar HPBC is obtained. S4. The graded porous biochar HPBC is mixed with a nitrogen source and then heat-treated under anaerobic conditions. After washing and drying, aminated biochar is obtained. Subsequently, the aminated biochar is impregnated in a magnesium salt solution to adsorb magnesium ions. Then, a mixed solution of ammonium salt and phosphate is slowly added dropwise while controlling the pH value to carry out the reaction. After washing and drying, nano-mineralized biochar MAP@N-HPBC is obtained. S5. The nano-mineralized biochar MAP@N-HPBC is mixed with humic acid in a certain proportion, water is added to make a slurry, and after wet ball milling, it is dried to obtain humic acid coated nano-mineralized biochar material HUM-MAP@N-HPBC. S6. The activated Bacillus pasteurellii is co-cultured with the humic acid-coated nanomineralized biochar material HUM-MAP@N-HPBC to form a biomimetic mineralization film. After drying, the functionalized biomimetic mineralization slow-release material is obtained, which is the functionalized biomimetic mineralization slow-release material for controlling herbicide resistance genes in intercropping farmland.

2. The method according to claim 1, characterized in that: The intercropping pattern is a chive-chill intercropping system; the crops are configured in a 1:1 ratio, with a row spacing of 8-32 cm and a plant spacing of 5-20 cm; 1-15 chive plants are planted per plant and 1-15 chill plants are planted per plant. Alternatively, for lightly polluted soil, the dosage of the functionalized biomimetic mineralization slow-release material is 100-500 kg / hm². 2 For moderately to severely polluted soils, the dosage of the functionalized biomimetic mineralization slow-release material is 2000-5000 kg / hm². 2 ; Alternatively, the primer sequences for sul1 are SEQ ID No. 1 and SEQ ID No. 2; for sul2, SEQ ID No. 3 and SEQ ID No. 4; for tetO, SEQ ID No. 5 and SEQ ID No. 6; for tetW, SEQ ID No. 7 and SEQ ID No. 8; for tetQ, SEQ ID No. 9 and SEQ ID No. 10; for tetL, SEQ ID No. 11 and SEQ ID No. 12; for tetM, SEQ ID No. 13 and SEQ ID No. 14; for tetX, SEQ ID No. 15 and SEQ ID No. 16; for ermB, SEQ ID No. 17 and SEQ ID No. 18; for ermC, SEQ ID No. 19 and SEQ ID No. 20; for strA, SEQ ID No. 21 and SEQ ID No. 22; and for strB, SEQ ID No. 17 and SEQ ID No.

22. Primer sequences for: No. 23 and SEQ ID No. 24; primer sequences for: aadA: SEQ ID No. 25 and SEQ ID No. 26; primer sequences for: cfr: SEQ ID No. 27 and SEQ ID No. 28; primer sequences for: fexA: SEQ ID No. 29 and SEQ ID No. 30; primer sequences for: blaOXA-1: SEQ ID No. 31 and SEQ ID No. 32; primer sequences for: blaampC: SEQ ID No. 33 and SEQ ID No. 34; primer sequences for: blaTEM-1: SEQ ID No. 35 and SEQ ID No.

36.

3. The method according to claim 1, characterized in that: S1 Camellia seed powder CP is prepared by mechanically processing camellia seeds; Before mechanical processing of camellia seeds, the following steps are also taken: selecting fresh, mold-free, and insect-free camellia seeds with a moisture content controlled at 2%-20%; using mechanical dehulling equipment to separate the shell from the kernel, ensuring that the weight percentage of the kernel to the shell is above 95%; mechanically pressing the camellia seed kernels to collect camellia seed cake, washing it with clean water, and then drying it in a 105℃ oven for 24 hours, where the organic matter content of the camellia seed kernel is 40-90%; the drying temperature is 100-110℃; after pre-freezing the camellia seed cake for 24 hours, it is first transferred to a crusher for coarse crushing into 2-3 mm particles; then transferred to an ultra-fine pulverizer to be pulverized through a 100-300 mesh sieve to obtain camellia seed powder; the pre-freezing temperature is -20-10℃; the pulverizing time is 30-60 minutes. Alternatively, the defatted camellia seed cake powder in S2 is prepared by defatting camellia seed powder; The camellia seed powder was defatted by weighing 50 g of the powder into a Soxhlet extractor, adding 300 mL of hexane solvent to a 500 mL round-bottom flask, adding 3-5 boiling stones to prevent bumping, and reacting in a water bath at 85±2℃ for 6-8 h. The reaction was then stopped, and the extraction sleeve was removed and vacuum dried at 60℃ for 2-6 h. The solvent used was hexane or petroleum ether, the bath temperature was 80-90℃, and the condensate flow rate was 1-3 L / min. The defatted camellia seed powder was then ultrasonically cleaned with a mixture of ethanol and water at a volume ratio of 1-3:1, with a pH of 5.3-5.

7. When the sample was ultrasonically added to the ethanol-water mixture, the ultrasonic frequency was 38-42 kHz, the ultrasonic power density was 0.25-0.75 W / mL, and the ultrasonic temperature was 58-62℃.

4. The method according to claim 1, characterized in that: S3 graded porous biochar was prepared by template-assisted catalytic pyrolysis of defatted camellia oil cake powder; The defatted camellia oil cake powder was subjected to template treatment by ball milling it with nano-SiO2 in a specific ratio of 10-2.5:

1. The ball mill speed was 300-600 rpm, and the milling time was 1-4 h. The mixture was then activated by mixing the ball-milled material with KOH at a mass ratio of 2-0.5:1, adding deionized water to form a slurry, and drying at 70-95℃ to constant weight to obtain the precursor. The precursor was then subjected to pyrolysis treatment by heating to 400℃ at 5℃ / min and holding for 30 min under N2 protection in a tube furnace, followed by heating to 800℃ at 10℃ / min and holding for 60 min. After natural cooling, it was soaked in 1 mol / L HCl solution for 6 h, washed with deionized water until neutral, and dried at 105℃ for 12 h. The N2 flow rate was 0.2-0.6%. L / min; pickling concentration: 0.5-1.5 mol / L; Alternatively, the nano-mineralized biochar in S4 is prepared by amination and in-situ mineralization of porous biochar. When ammoniating porous biochar, graded porous biochar is mixed with urea and pyrolyzed under a N2 atmosphere. After cooling, it is washed three times with deionized water and dried to obtain ammoniated biochar. The mass ratio of graded porous biochar to urea is 1:5-20; the pyrolysis time is 1-3 h; the pyrolysis temperature is 140-160℃; the N2 flow rate is 0.1-0.3 L / min; the drying temperature is 50-80℃; and the drying time is 8-24 h. Magnesium ion adsorption was performed on aminated biochar by dispersing the aminated biochar in a 0.1 mol / L MgCl2 solution, shaking at room temperature for 2 h, and then separating the solid and liquid phases to obtain the magnesium ion-adsorbed biochar. The concentration of MgCl2 was 0.05-0.2 mol / L, and the shaking speed was 150-200 rpm. The biochar adsorbed with magnesium ions was mineralized by slowly adding an equal volume of 0.05-0.2 mol / L (NH4)2HPO4 solution to the system under vigorous stirring, while adjusting and maintaining the pH at 9.0±0.2 with NaOH solution. After the reaction was completed, the mixture was filtered, washed three times each with ethanol and water, and freeze-dried for 24 h. The dropping rate was 1-2 mL / min.

5. The method according to claim 1, characterized in that: S5 humic acid-coated nano-mineralized biochar material was prepared by wet ball milling composite process: Nano-mineralized biochar and humic acid were mixed at a mass ratio of 1:1, and deionized water was added to prepare a slurry with a solid content of 20-30%. The slurry was then placed in a planetary ball mill for ball milling and dried at a certain temperature after ball milling. The ball milling speed was 300-500 rpm and the ball milling time was 3-5 h. The drying temperature was 50-80℃ and the drying time was 8-24 h.

6. The method according to any one of claims 1 to 5, characterized in that: The functionalized biomimetic mineralized slow-release material in S6 was prepared through microbial inoculation and biofilm induction. Pasteurella multocida was inoculated into Pasteurella multocida culture medium and cultured at 30°C with shaking at 150 rpm until OD reached. 600 =1.0; Humic acid-coated nano-mineralized biochar material and bacterial solution were mixed at a ratio of 1:10 (g:mL), and adsorbed by shaking at 30℃ and 50 rpm for 3 h. After discarding the supernatant, the material was transferred to a sterile tray, covered with plastic wrap to maintain humidity, and incubated at 30℃ for 36 h. Finally, the final product was obtained by vacuum freeze-drying for 24 h; wherein, the OD of the bacterial solution was... 600 =0.8-1.2; adsorption oscillation speed is 40-60 rpm; static culture temperature is 28-32℃, humidity is 85-95%; freeze-drying temperature is -60-10℃, freeze-drying vacuum degree is 10-100 Pa.

7. The application of the method as described in any one of claims 1 to 6 in controlling the spread of antibiotic resistance genes in soil and / or improving intercropping systems, the application including improving the rhizosphere microecological environment, improving soil nutrient use efficiency, promoting crop growth and increasing yield, and remediating antibiotic-contaminated farmland soil.

8. A method for farmland soil remediation comprising the method as described in any one of claims 1 to 6.

9. The method according to claim 8, characterized in that: The crop combination for the intercropping pattern is selected from any of the following: leek-chicory intercropping system, leafy vegetable-root vegetable intercropping system, legume-grass intercropping system, deep-root-shallow-root crop intercropping system, and other crop combinations with different root morphology and nutrient absorption characteristics.

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