Method for regulating and controlling rhizosphere microbial community structure based on tea trees

Abstract

The invention relates to the technical field of agricultural biology, and discloses a method for regulating and controlling a rhizosphere microbial community structure based on a tea tree, and the core of the method is to prepare and apply a composite regulation and control preparation. The preparation method comprises the following steps: firstly, covalently immobilizing N-acyl homoserine lactonase on the surface of a mesoporous biochar carrier so as to intervene pathogenic bacteria quorum sensing; secondly, loading the plant system elicitor and the synergistic nutritional agent into the carrier through vacuum impregnation; and finally, coating a layer of pH-sensitive hydrogel composed of chitosan and sodium alginate on the outside through an ionic cross-linking method. The preparation structure ensures the long-term stability of surface enzyme, and meanwhile, the external hydrogel layer can respond to a local low-pH environment caused by root exudates of tea trees to trigger accurate release of internal active substances in a root zone. The method can effectively regulate and control rhizosphere microorganism balance, synergistically induce tea tree resistance and promote tea quality substance synthesis, and has the beneficial effects of high targeting property and high utilization rate.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a method for regulating the structure of the rhizosphere microbial community based on tea trees. Background Technology

[0002] As an important economic crop, the yield and quality of tea are significantly influenced by the rhizosphere microenvironment. The rhizosphere is the most active region for interaction between plant roots and soil, and a hotspot for the colonization and activity of numerous microorganisms. A healthy and balanced rhizosphere microbial community can effectively promote nutrient absorption, synthesis of key metabolites, and inhibition of soil-borne diseases in tea plants, playing a decisive role in the final yield and flavor quality of tea. Therefore, effectively regulating the structure of the rhizosphere microbial community in tea plants has become a key technical direction in modern tea cultivation and management.

[0003] Currently, common techniques for improving the rhizosphere environment of plants include the application of microbial inoculants, soil conditioners, or biostimulants. In disease control, in addition to traditional chemical pesticides, using biological methods to intervene in the quorum sensing system of pathogens has become an emerging strategy. However, existing technologies still have significant limitations in practical applications. When various beneficial bioactive substances (such as functional enzymes, microorganisms, and plant inducers) are directly applied to complex soil environments, their activity is easily affected by environmental factors (such as pH, temperature, and non-target microorganisms), resulting in a rapid decline in activity, short duration of action, and poor stability.

[0004] Furthermore, to improve the effectiveness of active substances, existing technologies often employ simple physical adsorption or mixing methods to combine them with carriers. However, this often leads to the premature and concentrated release of active ingredients after they enter the soil, failing to precisely match the actual needs of tea tree roots in time and space. This results in waste of active substances and low utilization rates, making it difficult to achieve the expected long-term regulatory effects. Simultaneously, existing technical solutions are often single-function, failing to effectively integrate multiple functions such as inhibiting harmful microorganisms, supporting beneficial microorganisms, and precisely supplying nutrients in response to plant needs. There is a lack of a comprehensive technical solution capable of achieving multi-functional synergy to systematically optimize the rhizosphere microecology of tea trees. Summary of the Invention

[0005] This invention aims to address the technical problems in existing tea tree cultivation and management, namely, the relatively singular and imprecise methods for regulating the rhizosphere microbial community, and the low utilization rate of nutrients and active substances. Specifically, existing technologies often face challenges in regulating the rhizosphere environment, such as easy loss of active ingredients, unclear target sites, and difficulty in achieving synergistic effects of multiple functions. Therefore, there is a need to provide a method that can precisely regulate the structure of the tea tree rhizosphere microbial community in multiple dimensions and in time and space to improve the rhizosphere microecology and indirectly affect the growth status of tea trees.

[0006] To address the aforementioned technical problems, this invention provides a method for regulating the rhizosphere microbial community structure based on tea trees. The method comprises the following steps: S1. Preparation of functionalized carriers: Mesoporous biochar carriers are provided, and pathogen quorum sensing inhibitors are covalently immobilized on their surface.

[0007] In one specific embodiment, the mesoporous biochar carrier is prepared from tea tree branches. The preparation process includes: Tea tree branches were heated to 650°C to 750°C under a nitrogen atmosphere at a heating rate of 8°C to 12°C to 1 min, and then subjected to pyrolysis treatment at this temperature for 1.5 to 2.5 hours.

[0008] In the above steps, the covalent fixation is achieved through chemical cross-linking. For example, activated mesoporous biochar is treated in a 2.0% to 3.0% (v / v) glutaraldehyde solution to form aldehyde groups on the biochar surface that can be fixed by enzymes, followed by the addition of a pathogen quorum sensing inhibitor to carry out the immobilization reaction.

[0009] S2, Loading Active Substances: Plant system inducers and plant microbial synergistic nutrients are loaded onto the carrier treated in step S1. The synergistic nutrients include precursors for the synthesis of tea quality substances and selective nutrients for beneficial rhizosphere microorganisms.

[0010] In one specific embodiment, the loading step is achieved by impregnation under a vacuum of -0.08 MPa to -0.095 MPa to ensure that the active material can enter the internal pore structure of the mesoporous biochar.

[0011] S3. Coating and Finished Product Preparation: A pH-sensitive hydrogel is formed by coating the surface of the carrier treated in step S2 using an ion crosslinking method, thereby obtaining the final composite regulatory formulation.

[0012] In one specific embodiment, the step involves uniformly dispersing carrier particles loaded with active substances in a sodium alginate solution, and then dropwise adding them to a curing solution. The curing solution contains 0.8% to 1.2% (w / v) chitosan and 1.5% to 2.0% (w / v) calcium chloride. The carrier particles are continuously cured in the curing solution for 30 to 50 minutes, forming composite formulation microspheres with a hydrogel-coated surface.

[0013] S4. Application: Apply the compound regulatory agent prepared in step S3 to the rhizosphere soil of the tea tree.

[0014] In one specific implementation, the application step is carried out before the spring shoots of tea trees sprout or during the autumn topdressing period. It can be done by trenching or hole application, applying the preparation to the main distribution area of ​​the tea tree root system.

[0015] The composite regulatory formulation used in the method of this invention has a specific composition for each component.

[0016] In one specific embodiment, the composite regulatory formulation comprises the following components in parts by weight: 75 to 85 parts of the mesoporous biochar carrier, 4.5 to 7.5 parts of the pH-sensitive hydrogel, 0.22 to 0.55 parts of the plant system inducer, 5.5 to 11.5 parts of the plant microbial synergist, and an effective amount of the pathogenic quorum sensing inhibitor.

[0017] In one specific embodiment, the plant system inducer is chitosan oligosaccharide or methyl salicylate.

[0018] In one specific embodiment, the precursor for the synthesis of the tea quality substances is L-glutamic acid 5-ethyl ester or L-phenylalanine, and the selective nutrient for the beneficial rhizosphere microorganisms is D amino acid or seaweed oligosaccharide.

[0019] In one specific embodiment, the pathogenic quorum sensing inhibitor is an N-acylhomoserine lactonease.

[0020] In one specific embodiment, the pH-sensitive hydrogel is a chitosan-sodium alginate interpenetrating network hydrogel.

[0021] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention constructs a structurally and functionally integrated compound regulatory agent through an ordered preparation process. The method first covalently fixes a pathogenic quorum sensing inhibitor, which requires high stability, onto the surface of a mesoporous biochar carrier, endowing it with long-term efficacy in complex soil environments. Subsequently, plant systemic inducers and synergistic nutrients are loaded into the internal pores of the carrier via vacuum impregnation. Finally, a pH-sensitive hydrogel is formed by external coating using ion crosslinking. This programmed preparation process ensures that each functional component is in a pre-defined structural position, achieving a unified overall structure and multiple functions of the agent, providing a material basis for subsequent precise regulation.

[0022] 2. This invention achieves multiple synergistic regulation of the tea tree-rhizosphere microbial system through a formulation. The pathogen quorum sensing inhibitor, fixed on the carrier surface, directly intervenes in the communication system of pathogens in the soil. The plant system inducer, protected by the hydrogel layer, can effectively induce the tea tree to produce systemic resistance after release. The plant microbial synergist provides precursors for the synthesis of quality substances in the tea tree and provides selective nutrition for the proliferation of beneficial microbial communities. These functional modules are different in spatial location and mode of action, but they cooperate with each other to form a complete and synergistic regulatory system.

[0023] 3. This invention features precise spatiotemporal release, significantly improving the utilization efficiency of active substances. After the compound regulatory agent is applied to the soil, its external pH-sensitive hydrogel layer can respond to the local soil pH decrease caused by tea tree root exudates, thereby undergoing structural changes. This allows the plant system inducers and nutrients encapsulated inside to be passively released in the most active root area. This on-demand release mechanism matches the release time of active substances with the root activity period and the release area with the target site, greatly improving the effective utilization rate of active substances and reducing the waste caused by their loss to non-rhizosphere areas. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to embodiments, comparative examples and test examples.

[0025] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0026] Glutaraldehyde solution, CAS No.: 111-30-8; N-acylhomoserine lactonease, EC3.1.1.81; Chitosan oligosaccharide, CAS No.: 9012-76-4; Methyl salicylate, CAS No.: 119-36-8; L-Glutamic acid 5-ethyl ester, CAS No.: 1499-55-4; L-Phenylanine, CAS No.: 63-91-2; Sodium alginate, CAS No.: 9005-38-3; Chitosan, CAS No.: 9012-76-4; Calcium chloride, CAS No.: 10043-52-4; N-Octayl-DL-homoserine lactone, CAS No.: 106983-30-6; Theanine, CAS No.: 3081-61-6.

[0027] Examples 1-3: Example 1: S1. Provide a mesoporous biochar carrier and covalently immobilize pathogenic quorum sensing inhibitors on its surface: Preparation of mesoporous biochar carrier: Fully dried and pulverized tea tree branches were placed in a tube furnace and heated to 650°C at a heating rate of 8°C / min under a nitrogen protective atmosphere. The mixture was then kept at this temperature for 1.5 hours for pyrolysis. After natural cooling to room temperature, the solid product was removed, ground, and sieved. Particles with uniform particle size were collected, which were then used as the mesoporous biochar carrier.

[0028] Covalently immobilized pathogen quorum sensing inhibitor: 75 parts by weight of the above-mentioned mesoporous biochar carrier were weighed, dispersed in deionized water, and filtered after ultrasonic treatment. The moistened carrier was then transferred to a 2.0% (v / v) glutaraldehyde solution and activated by stirring at room temperature for 2 hours. After the reaction, the carrier was repeatedly washed with phosphate buffered saline (PBS, pH = 7.4) to remove residual glutaraldehyde. Subsequently, the activated carrier was placed in a PBS solution of a predetermined concentration of N-acylhomoserine lactonease and immobilized at 4°C for 12 hours. After the reaction, the carrier was recovered and washed with PBS to remove physically adsorbed enzymes, thus obtaining a functionalized carrier with a pathogen quorum sensing inhibitor covalently immobilized on its surface.

[0029] S2. Load the plant system inducer and plant microbial synergist onto the carrier treated in step S1: Weigh 0.22 parts by mass of plant system inducers (chitosan oligosaccharide in this example) and 5.5 parts by mass of plant microbial synergists (a mixture of L-glutamic acid 5-ethyl ester and D amino acids in this example), and dissolve them in an appropriate amount of deionized water to form a homogeneous loading solution. Immerse the functionalized carrier prepared in step S1 in this loading solution, and then transfer the entire system to a vacuum drying oven and maintain it at a vacuum of -0.08 MPa for 2 hours to ensure that the loading solution fully penetrates the mesoporous structure of the carrier.

[0030] S3. A pH-sensitive hydrogel is formed by coating the surface of the carrier treated in step S2 using an ionic crosslinking method, thereby obtaining the composite regulatory formulation: The carrier particles, processed in step S2 and fully adsorbed with the loading liquid, were uniformly dispersed in an aqueous sodium alginate solution. The sodium alginate mixture containing the carrier particles was added dropwise at a constant rate to a continuously stirred curing solution using a syringe pump. The curing solution contained 0.8% (w / v) chitosan and 1.5% (w / v) calcium chloride. Upon entering the curing solution, the droplets rapidly formed gel microspheres and remained in the curing solution for 30 minutes. After the reaction was complete, the microspheres were removed, washed several times with deionized water, and then freeze-dried to obtain the final composite regulating formulation. The mass of the pH-sensitive hydrogel in this composite regulating formulation was determined to be 4.5 parts.

[0031] S4. Apply the compound regulatory agent obtained in step S3 to the rhizosphere soil of tea trees: Before the spring shoots of tea trees sprout, apply the compound regulatory agent obtained in step S3 evenly to the soil in the main root distribution area at a certain distance from the main trunk of the tea tree using a trenching method, at a dosage of several grams per tea tree, and cover with soil.

[0032] Example 2: S1. Provide a mesoporous biochar carrier and covalently immobilize pathogenic quorum sensing inhibitors on its surface: Preparation of mesoporous biochar carrier: Fully dried and pulverized tea tree branches were placed in a tube furnace and heated to 700℃ at a heating rate of 10℃ / min under a nitrogen protective atmosphere. The mixture was then kept at this temperature for 2.0 hours for pyrolysis. After natural cooling to room temperature, the solid product was removed, ground, and sieved. Particles with uniform particle size were collected, which were the mesoporous biochar carrier.

[0033] Covalently immobilized pathogen quorum sensing inhibitor: 80 parts by weight of the above-mentioned mesoporous biochar carrier were weighed, dispersed in deionized water, and filtered after ultrasonic treatment. The moistened carrier was then transferred to a 2.5% (v / v) glutaraldehyde solution and activated by stirring at room temperature for 2 hours. After the reaction, the carrier was repeatedly washed with phosphate buffered saline (PBS, pH = 7.4) to remove residual glutaraldehyde. Subsequently, the activated carrier was placed in a PBS solution of a predetermined concentration of N-acylhomoserine lactonease and immobilized at 4°C for 12 hours. After the reaction, the carrier was recovered and washed with PBS to remove physically adsorbed enzymes, thus obtaining a functionalized carrier with a pathogen quorum sensing inhibitor covalently immobilized on its surface.

[0034] S2. Load the plant system inducer and plant microbial synergist onto the carrier treated in step S1: Weigh 0.4 parts by mass of plant system inducer (methyl salicylate in this example) and 8.5 parts by mass of plant microbial synergist (a mixture of L-phenylalanine and seaweed oligosaccharides in this example), and dissolve them in an appropriate amount of deionized water to form a homogeneous loading solution. Immerse the functionalized carrier prepared in step S1 in this loading solution, and then transfer the entire system to a vacuum drying oven and maintain it at a vacuum of -0.09 MPa for 2 hours to ensure that the loading solution fully penetrates the mesoporous structure of the carrier.

[0035] S3. A pH-sensitive hydrogel is formed by coating the surface of the carrier treated in step S2 using an ionic crosslinking method, thereby obtaining the composite regulatory formulation: The carrier particles, processed in step S2 and fully adsorbed with the loading liquid, were uniformly dispersed in an aqueous sodium alginate solution. The sodium alginate mixture containing the carrier particles was added dropwise at a constant rate to a continuously stirred curing solution using a syringe pump. The curing solution contained 1.0% (w / v) chitosan and 1.8% (w / v) calcium chloride. Upon entering the curing solution, the droplets rapidly formed gel microspheres and remained in the curing solution for 40 minutes. After the reaction, the microspheres were removed, washed several times with deionized water, and then freeze-dried to obtain the final composite regulating formulation. The mass of the pH-sensitive hydrogel in this composite regulating formulation was determined to be 6.0 parts.

[0036] S4. Apply the compound regulatory agent obtained in step S3 to the rhizosphere soil of tea trees: The compound regulatory agent prepared in step S3 is applied evenly to the soil in the main root distribution area at a certain distance from the main trunk of the tea tree during the autumn topdressing period, using a dosage of several grams per tea tree, and then covered with soil.

[0037] Example 3: S1. Provide a mesoporous biochar carrier and covalently immobilize pathogenic quorum sensing inhibitors on its surface: Preparation of mesoporous biochar carrier: Fully dried and pulverized tea tree branches were placed in a tube furnace and heated to 750℃ at a heating rate of 12℃ / min under a nitrogen protective atmosphere. The mixture was then kept at this temperature for 2.5 hours for pyrolysis. After natural cooling to room temperature, the solid product was removed, ground, and sieved. Particles with uniform particle size were collected, which were the mesoporous biochar carrier.

[0038] Covalently immobilized pathogen quorum sensing inhibitor: 85 parts by weight of the above-mentioned mesoporous biochar carrier were weighed, dispersed in deionized water, and filtered after ultrasonic treatment. The moistened carrier was then transferred to a 3.0% (v / v) glutaraldehyde solution and activated by stirring at room temperature for 2 hours. After the reaction, the carrier was repeatedly washed with phosphate buffered saline (PBS, pH = 7.4) to remove residual glutaraldehyde. Subsequently, the activated carrier was placed in a PBS solution of a predetermined concentration of N-acylhomoserine lactonease and immobilized at 4°C for 12 hours. After the reaction, the carrier was recovered and washed with PBS to remove physically adsorbed enzymes, thus obtaining a functionalized carrier with a pathogen quorum sensing inhibitor covalently immobilized on its surface.

[0039] S2. Load the plant system inducer and plant microbial synergist onto the carrier treated in step S1: Weigh 0.55 parts by weight of plant system inducers (chitosan oligosaccharide in this example) and 11.5 parts by weight of plant microbial synergists (a mixture of L-glutamic acid 5-ethyl ester and D amino acids in this example), and dissolve them in an appropriate amount of deionized water to form a homogeneous loading solution. Immerse the functionalized carrier prepared in step S1 in this loading solution, and then transfer the entire system to a vacuum drying oven and maintain it at a vacuum of -0.095 MPa for 2 hours to ensure that the loading solution fully penetrates the mesoporous structure of the carrier.

[0040] S3. A pH-sensitive hydrogel is formed by coating the surface of the carrier treated in step S2 using an ionic crosslinking method, thereby obtaining the composite regulatory formulation: The carrier particles, processed in step S2 and fully adsorbed with the loading liquid, were uniformly dispersed in an aqueous sodium alginate solution. The sodium alginate mixture containing the carrier particles was added dropwise at a constant rate to a continuously stirred curing solution using a syringe pump. The curing solution contained 1.2% (w / v) chitosan and 2.0% (w / v) calcium chloride. Upon entering the curing solution, the droplets rapidly formed gel microspheres and remained in the curing solution for 50 minutes. After the reaction was complete, the microspheres were removed, washed several times with deionized water, and then freeze-dried to obtain the final composite regulating formulation. The mass of the pH-sensitive hydrogel in this composite regulating formulation was determined to be 7.5 parts.

[0041] S4. Apply the compound regulatory agent obtained in step S3 to the rhizosphere soil of tea trees: Before the spring shoots of tea trees sprout, apply the compound regulatory agent obtained in step S3 evenly to the soil in the main root distribution area at a certain distance from the main trunk of the tea tree using a trenching method, at a dosage of several grams per tea tree, and cover with soil.

[0042] Comparative Examples 1-4: Comparative Example 1: Compared to Example 2, the difference lies in that the covalent fixation in step S1 and the hydrogel coating in step S3 are not performed. Instead, all components used in Example 2 (mesoporous biochar carrier, plant system inducer, synergist, N-acylhomoserine lactonease) are directly physically dry-mixed. The mass fractions of each component remain the same as in Example 2, and the remaining steps are not performed.

[0043] Comparative Example 2: Compared to Example 2, the difference is that after completing steps S1 (covalent fixation) and S2 (loading active substances), the hydrogel coating step S3 is not performed; instead, the product is directly freeze-dried. All other steps and parameters remain the same.

[0044] Comparative Example 3: Compared to Example 2, the difference lies in that, in step S1, the N-acylhomoserine lactonease is not covalently immobilized; instead, it is physically loaded in step S2 along with plant system inducers and plant microbial synergists via vacuum impregnation. All other steps and parameters remain the same.

[0045] Comparative Example 4: The difference from Example 2 is that, in step S4, no regulatory agent prepared by the method of this invention is applied; instead, a conventional compound fertilizer containing equivalent nitrogen, phosphorus, and potassium nutrients is applied. All other cultivation and management conditions remain the same.

[0046] Test Example 1-2: Test Example 1: In vitro performance testing of compound regulatory formulations Enzyme activity stability test: This test was used to evaluate the ability of different preparation methods to maintain the activity of N-acylhomoserine lactonease.

[0047] Equal masses of dried samples from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 3 were placed in Erlenmeyer flasks containing equal volumes of soil extract (pH = 7.0). All flasks were placed in a constant-temperature shaker and continuously shaken at 25°C. At time points of 0, 12, 24, 48, and 72 hours, equal volumes of suspension were taken from each flask, centrifuged, and the supernatant was collected. The enzyme activity in the supernatant was determined spectrophotometrically using N-octanoyl-DL-homoserine lactone as a substrate. The enzyme activity measured at 0 hours was taken as the initial activity (100%), and the percentage of remaining activity at subsequent time points was calculated. The test results are recorded in Table 1.

[0048] Table 1. Residual activity (%) of N-acylhomoserine lactonease in each sample sample 0h 12h 24h 48h 72h Example 1 100 96.2 91.5 88.1 84.7 Example 2 100 97.5 94.1 90.3 88.2 Example 3 100 98.1 95.8 92.6 90.4 Comparative Example 1 100 58.3 35.1 16.4 7.9 Comparative Example 3 100 72.6 51.7 33.8 21.5 Active substance sustained-release performance test: This test was used to evaluate the release behavior of the formulation of its internally loaded substances under different pH conditions.

[0049] Equal masses of dried samples from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 were placed in two series of dialysis bags. One series was treated with phosphate buffer solution (pH 7.0), and the other with acetate buffer solution (pH 5.5). The dialysis bags were then placed in beakers containing the corresponding buffer solutions. All beakers were placed in a constant-temperature shaker and continuously shaken at 25°C. At preset time points, equal volumes of release medium were drawn from the beakers, and equal volumes of fresh buffer solution were added. The concentration of chitosan oligosaccharides in the release medium was determined using high-performance liquid chromatography (HPLC), and its cumulative release rate was calculated. The test results are recorded in Table 2.

[0050] Table 2. Cumulative release rate (%) of chitosan oligosaccharides in different pH buffers for each sample Test Result Analysis: Table 1 shows that the N-acylhomoserine lactoneases prepared via step S1 in Examples 1, 2, and 3 maintained high residual activity after 72 hours. This is attributed to the covalent fixation achieved through glutaraldehyde crosslinking, which firmly connects the enzyme molecules to the surface of the mesoporous biochar support, effectively preventing their detachment and structural dissociation in the aqueous environment, thereby maintaining their biological activity. In contrast, the enzyme molecules in Comparative Example 1 (physical mixing) and Comparative Example 3 (physical adsorption) had weak binding forces to the support, resulting in rapid loss or inactivation in solution and a rapid decline in residual activity. This result indicates that the covalent fixation step in the method of the present invention is the structural basis for maintaining the long-term effectiveness of the inhibitor.

[0051] Table 2 shows that in a neutral environment (pH 7.0), the chitosan oligosaccharide loaded inside Examples 1, 2, and 3 exhibits a slow release rate and limited release amount; however, in an acidic environment (pH 5.5), both the release rate and cumulative release amount of chitosan oligosaccharide are significantly increased. This is because the externally coated chitosan-sodium alginate hydrogel layer is pH sensitive. Under neutral conditions, the dense hydrogel network structure acts as a physical barrier against internal substances; under acidic conditions, the amino groups on the chitosan chains undergo protonation, and electrostatic repulsion causes the hydrogel network to swell, increasing the pore size and thus accelerating the diffusion and release of internal substances. Comparative Examples 1 and 2, which are not coated with hydrogel, exhibit rapid burst release behavior under both pH conditions and lack environmental responsiveness.

[0052] In summary, the method of this invention constructs a functionally partitioned core-shell structure through covalent fixation in step S1 and pH-sensitive hydrogel encapsulation in step S3. This structure enables the surface pathogen quorum sensing inhibitors to achieve high stability, while allowing the internal plant system inducers and synergists to be released in response to low pH signals in the rhizosphere microenvironment. This functionalized structure, obtained through a specific preparation sequence, directly results in high enzyme activity stability and controllable release of active substances, addressing the different functional and stability requirements of each component and achieving synergistic effects.

[0053] Test Example 2: Pot Experiment Effect Verification Test Description: This test is used to evaluate the comprehensive impact of the method of the present invention on the rhizosphere microbial community of tea trees and tea quality indicators in simulated actual applications.

[0054] Two-year-old tea seedlings with uniform growth were selected and transplanted into pots containing an equal volume of sterilized substrate. The pots were randomly divided into nine groups: Treatment Group 1 (Example 1), Treatment Group 2 (Example 2), Treatment Group 3 (Example 3), Treatment Group 1 (Comparative Example 1), Treatment Group 2 (Comparative Example 2), Treatment Group 3 (Comparative Example 3), Treatment Group 4 (Comparative Example 4), and a blank control group (treated with an equal volume of water only). Each group had 10 replicates. At the start of the experiment, the formulations or fertilizers prepared in each example and comparative example were applied once to the root zone of the potted soil according to the equivalent effective ingredient dosage. Routine maintenance and management were carried out in a greenhouse for 90 days.

[0055] After the experiment, samples were taken for analysis: Rhizosphere soil sampling: Non-rhizosphere soil adhering to the tea tree roots was shaken off, and soil tightly attached to the root surface was collected as rhizosphere soil samples. High-throughput sequencing was used to sequence the 16S rRNA and ITS gene amplicon sequences of soil DNA to analyze bacterial and fungal community composition. Microbial diversity indices were calculated, and the relative abundance of specific bacterial genera was statistically analyzed.

[0056] Tea sample collection: One bud and two leaves from the top shoots of tea trees in each treatment group were harvested as fresh leaf samples. The samples were processed into dried tea using standard processes such as fixation, rolling, and drying. The content of major quality components in the dried tea was determined using high-performance liquid chromatography (HPLC) and national standard chemical analysis methods.

[0057] The test results are recorded in Tables 3 and 4.

[0058] Table 3. Effects of each treatment on the rhizosphere soil microbial community of tea trees Table 4. Effects of each treatment on the content of major quality components in tea (mg / g) Processing group Theanine Total amino acid Tea polyphenols Example 1 25.1 41.8 315.6 Example 2 28.3 45.1 302.4 Example 3 26.9 43.6 309.7 Comparative Example 1 18.2 30.5 341.2 Comparative Example 2 19.5 32.8 335.8 Comparative Example 3 21.6 35.4 328.1 Comparative Example 4 16.5 28.9 350.3 Blank control 15.8 27.2 358.5 Test Result Analysis: Table 3 shows that the rhizosphere soils of treatment groups 1, 2, and 3 had significantly higher Shannon diversity indices than the comparative groups and the blank control group. Simultaneously, the relative abundance of harmful *Fusarium* species was suppressed to extremely low levels in these three examples, while the relative abundance of beneficial *Bacillus* species significantly increased. This indicates that the formulation constructed by the method of this invention, with its covalently fixed N-acyl homoserine lactonease, effectively intervenes in pathogen quorum sensing signals in the soil; while the beneficial microbial selective nutrients contained in the internal synergistic nutrient agent directionally promote the proliferation of beneficial bacteria after release. Together, these two factors reshape the structure of the rhizosphere microecology.

[0059] The data in Table 4 show that, compared with the comparative groups and the blank control group, the total levels of theanine and amino acids in the tea leaves of the treatment groups in Examples 1, 2, and 3 were all higher. This result is attributed to the pH-sensitive release mechanism of the compound regulatory formulation. The hydrogel layer on the outside of the formulation responds to the acidic environment produced by the root exudates of the tea tree, allowing the precursors of tea quality substances (such as L-glutamic acid 5-ethyl ester or L-phenylalanine) loaded inside to be passively released in the active root area and efficiently absorbed and utilized by the tea tree, directly promoting the synthesis of nitrogen-containing quality compounds, represented by theanine.

[0060] Based on the results in Tables 3 and 4, the method of this invention provides a multifunctional and synergistic rhizosphere environment regulation scheme. The composite regulatory agent prepared by this method, with its specific core-shell structure and functional partitions, allows externally immobilized enzymes to pre-improve the rhizosphere microbial environment, providing tea tree roots with a low-disease-stress growth condition. On this basis, the precise release of internally active substances (plant systemic inducers and synergistic nutrients) triggered by root activity further induces the plant's own systemic resistance and supplies key precursors for quality synthesis. This synergistic effect of external environmental regulation and internal physiological promotion ultimately manifests as a dual technical effect of optimizing the rhizosphere microbial community and improving tea quality.

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for regulating the rhizosphere microbial community structure based on tea trees, characterized in that, Includes the following steps: S1. Provide a mesoporous biochar carrier and covalently immobilize pathogenic quorum sensing inhibitors on its surface; S2. Load the plant system inducer and plant microbial synergist onto the carrier treated in step S1. The synergist contains a precursor for the synthesis of tea quality substances and a selective nutrient for beneficial rhizosphere microorganisms. S3. A pH-sensitive hydrogel is formed by coating the surface of the carrier treated in step S2 with an ion crosslinking method, thereby obtaining a composite regulatory formulation. S4. Apply the compound regulatory agent obtained in step S3 to the rhizosphere soil of tea trees.

2. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, The compound regulatory formulation comprises the following components in parts by weight: The mesoporous biochar carrier consists of 75 to 85 parts, the pH-sensitive hydrogel consists of 4.5 to 7.5 parts, the plant system inducer consists of 0.22 to 0.55 parts, the plant microbial synergist consists of 5.5 to 11.5 parts, and an effective amount of the pathogenic quorum sensing inhibitor.

3. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, In step S1, the covalent fixation is achieved by a cross-linking reaction in a glutaraldehyde solution with a concentration of 2.0% to 3.0% (v / v).

4. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, The plant system inducers are chitosan oligosaccharide or methyl salicylate.

5. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, The precursors for the synthesis of the tea quality substances are L-glutamic acid 5-ethyl ester or L-phenylalanine, and the selective nutrients for the beneficial rhizosphere microorganisms are D amino acids or seaweed oligosaccharides.

6. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, The pathogenic quorum sensing inhibitor is N-acyl homoserine lactonease, and the pH-sensitive hydrogel is a chitosan-sodium alginate interpenetrating network hydrogel.

7. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, The mesoporous biochar carrier is prepared by pyrolyzing tea tree branches at a heating rate of 8°C / min to 12°C / min to a temperature of 650°C to 750°C for 1.5 to 2.5 hours.

8. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, In step S3, the formation conditions of the pH-sensitive hydrogel include: dropping the loaded carrier into a curing solution containing 0.8% to 1.2% (w / v) chitosan and 1.5% to 2.0% (w / v) calcium chloride, and curing for 30 to 50 minutes.

9. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, In step S4, the application is specifically carried out by trenching or hole application before the spring shoots of tea trees sprout or during the autumn topdressing period.

10. The method for regulating the rhizosphere microbial community structure based on tea trees according to claim 1, characterized in that, In step S1, the load is achieved by impregnation under a vacuum of -0.08MPa to -0.095MPa.

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