A compound microbial agent for promoting seed germination of dendrobium candidum

Abstract

This invention relates to a compound microbial agent for promoting the germination of Dendrobium officinale seeds, belonging to the field of bio-fertilizer technology. It mainly includes Bacillus amyloliquefaciens, which dynamically constructs a constant microenvironment with a pH of 6.0 to 6.5 through metabolic synergy. Under acidic conditions, it induces carbonic anhydrase to generate calcium carbonate microcrystals to neutralize H⁺, and under alkaline conditions, it induces citrate synthase to secrete citrate and release H⁺, forming an "acid-base double buffer pair" with the mycelial network, thus controlling the pH fluctuation of the micro-region within ±0.3. This application also provides a method for preparing the microbial agent and its application technology, which significantly improves the germination rate and seedling quality of Dendrobium officinale seeds, while possessing advantages in ecological safety and economics, making it suitable for agricultural planting, ecological restoration, and other fields.

Description

Technical Field

[0001] This invention belongs to the field of bio-fertilizer technology, specifically relating to a compound microbial agent for promoting the germination of Dendrobium officinale seeds. Background Technology

[0002] The germination of Dendrobium officinale seeds is highly sensitive to soil pH. Existing compound microbial agents (such as mixtures containing Trichoderma and Rhizobium) show a significant decrease in germination rate outside the pH range of 5.5-6.5—less than 25% in acidic soils (pH < 5.5) and less than 30% in alkaline soils (pH > 7.0). This problem stems from three unresolved technical deficiencies: First, the pH buffering mechanism is unidirectional and passive. Mainstream microbial agents rely on single-function strains (such as lactic acid bacteria regulating acidity) and cannot cope with bidirectional pH fluctuations. When using Lactobacillus acidophilus to treat red soil, the effect is significantly weakened when the soil pH is >6.5; while using phosphate-solubilizing bacteria to improve alkaline soil exacerbates H⁺ accumulation under acidic conditions. More seriously, the buffering effect of these strains depends on a preset environment (such as lactic acid bacteria requiring pH <6.0 to start acid production), making it difficult to dynamically respond to real-time pH changes, resulting in a germination window shortened by more than 50%. Second, chemical buffers introduce safety risks. To compensate for insufficient biological buffering, some schemes add chemical substances (such as calcium carbonate powder), but this has three major drawbacks: ① Calcium carbonate precipitates and becomes ineffective in alkaline soils (solubility <0.001g / 100mL at pH>7.5), failing to neutralize OH⁻; ② Exogenously added sodium citrate releases excessive H⁺ in acidic soils, causing the micro-zone pH to plummet below 4.0 (measured pH fluctuations reach ±1.8); ③ Chemical residues inhibit Dendrobium root development (root length shortened by 35%). These schemes also suffer from poor germination rate stability (standard deviation >±20%) because the buffering substances do not synergize with the bacteria. Third, the ability to construct a microenvironment is lacking. Existing technologies only focus on improving the overall soil pH (such as adjusting 1 m³ of soil through microbial agents), but Dendrobium officinale seeds only require a stable micro-zone with a diameter of 1 mm for germination. Traditional microbial agents lack spatial constraint mechanisms, and the random distribution of microbial cells causes buffering substances to diffuse into ineffective areas (>90% of the buffering agent is wasted in non-germination areas), while the area around the seeds is still subject to external pH shocks. Summary of the Invention

[0003] To overcome the problems in the prior art, this application provides a compound microbial agent for promoting the germination of Dendrobium officinale seeds and its preparation method, which aims to dynamically construct a constant microenvironment with pH 6.0 to 6.5 within a range of 0.5 to 1 mm around the seeds through the metabolic synergistic effect of Bacillus amyloliquefaciens.

[0004] In a first aspect, this application provides a compound microbial agent for promoting the germination of Dendrobium officinale seeds, comprising the following technical features: 1. The core component of the compound microbial agent is Bacillus amyloliquefaciens (BNCC186043), whose cell membrane expresses the pH-sensing protein PhoR. When the external H⁺ or OH⁻ concentration exceeds a threshold, i.e., when the external pH is below 5.5 (acidic conditions) or above 6.5 (alkaline conditions), a dual-pathway metabolic activation mechanism is triggered. Acidic conditions induce carbonic anhydrase expression, while alkaline conditions induce citrate synthase expression. 2. Carbonic anhydrase catalyzes the formation of calcium carbonate microcrystals from CO2 in acidic soil. The microcrystals range in size from 1 to 5 micrometers. They are embedded in the gaps of the mycelial network and undergo a dissolution reaction upon contact with H⁺ (CaCO3 + 2H⁺ → Ca²⁺ + CO2 + H2O). Each milligram of microcrystal neutralizes 0.02 millimoles of H⁺. 3. Citrate synthase catalyzes the secretion of citric acid in alkaline soils. Its dissociation releases H⁺ (C6H8O7 → H⁺ + C6H7O7⁻), and each milligram of citric acid neutralizes 0.015 millimoles of OH⁻. 4. The hyphal network forms a selectively permeable membrane, allowing only H⁺ or OH⁻ to diffuse at a rate of less than 0.1 mm / min (equivalent to approximately 1.67 × 10⁻). 6 The slow diffusion (m / s) allows for a time window for metabolic regulation. By controlling the accumulation of metabolites in Bacillus amyloliquefaciens during the static culture phase, an intercalation distribution of 60%±5% microcrystals and 40%±5% citric acid in the micro-region volume is achieved, forming an "acid-base double buffer pair" and keeping the pH fluctuation range of the micro-region within ±0.3. Secondly, this application provides a method for preparing the above-mentioned compound microbial agent, comprising the following steps: S10: Inoculate Bacillus amyloliquefaciens into LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and incubate at 37°C with shaking for 16 hours until the logarithmic phase (OD600=0.9). S20: Collect bacterial cells by centrifugation (10,000 rpm, 10 min), wash three times with 10 mL pH 7.0 phosphate buffer, and resuspend in 5 mL of the same buffer to a final volume of 1×10⁻⁶. 9 CFU / mL. S30: Mix the bacterial solution with coconut coir carrier at a ratio of 1:5 (the carrier is pre-sterilized), and incubate at 28 degrees Celsius for 24 hours to allow the bacteria to form a complete hyphal network. S40: Freeze-dried (-50°C, 24 hours) to produce granular bacterial agent, with a particle size controlled at 0.5 to 1 mm and a viable count of not less than 5 × 10⁻⁶. 8 CFU per gram. Thirdly, this application provides specific application methods for the aforementioned compound microbial agent, including the following technical details: 1. Apply the inoculant 24 hours before sowing. Applying it earlier will activate the mycelial network in advance, while applying it later will result in a delayed response. 2. The mycelial network density was confirmed to be 1.5 × 10⁻⁶ by microscopic counting (400× magnification). 4 Up to 2.0×10 4 The density is controlled by adjusting the static incubation time; excessive density (greater than 2.5 × 10⁻⁶) can lead to problems. 4 This hinders seed respiration; excessively sparse (less than 1.0 × 10⁻⁶) 4 This leads to micro-region rupture. 3. The porosity of the coconut coir carrier must be greater than 65%, determined by mercury porosimetry (according to GB / T 1966-2021 standard, using a mercury porosimeter at 10MPa pressure), and verified before use. Perlite carrier may fail due to the adsorption of citric acid. 4. In acidic soils (pH below 5.5), calcium carbonate microcrystals dissolve and consume H⁺; in alkaline soils (pH above 6.5), citric acid dissociates and releases H⁺, keeping the pH dynamic within the micro-zone stable between 6.0 and 6.5. Fourthly, this application provides specific implementation effects of the above-mentioned compound microbial agent under different soil pH conditions: 1. Acidic conditions (pH 4.0): When the bacterial agent was placed in simulated red soil solution (containing 0.01 mol / L Al³⁺), calcium carbonate microcrystals were detected after 5 minutes (XRD peak position 29.4°), and the pH of the micro-area rose to 6.1 within 30 minutes. 2. Alkaline conditions (pH 8.5): When the bacterial agent is placed in simulated yellow soil solution (containing 0.02 mol / L Na2CO3), the citric acid secretion reaches 0.18 mg / g after 8 minutes, and the micro-zone pH drops to 6.4 within 30 minutes. 3. Dynamic stability: During the step change of pH from 4.0 to 8.5, the pH of the micro-area remained at 6.0 to 6.5 (fluctuation ±0.2), while the control group (conventional bacterial agent) fluctuated within a range of ±1.5. Fifthly, this application provides specific data supporting the field application of the aforementioned compound microbial agent: 1. Two experimental plots of 1 mu each were established at the Dendrobium officinale cultivation base in Gulinqing Township, Maguan County, Wenshan Prefecture, Yunnan Province (acidic red soil, pH 4.8) and the Dendrobium officinale cultivation base in Guangnan County, Wenshan Prefecture, Yunnan Province (alkaline yellow soil, pH 8.2). The experimental group substrate was mixed with the inoculant of this invention (5 grams per square meter), while the control group used conventional inoculant plus a chemical buffer (50 grams of calcium carbonate per square meter plus 30 grams of sodium citrate per square meter). 2. Germination stage: In the red soil region, the germination rate of the experimental group was 42.6% on day 7 (compared to 18.3% in the control group), and the pH of the micro-area remained stable at 6.2±0.3. In the yellow soil region: the germination rate of the experimental group was 40.1% on day 7 (compared to 15.7% in the control group), and the pH of the micro-area remained stable at 6.4±0.2. 3. Seedling stage: index Red Soil Region Experimental Group control group in red soil region Yellow Soil Experimental Group Yellow soil area control group Seedling survival rate (30 days) 85.4% 43.7% 82.9% 41.2% Average seedling height 5.2 cm 3.1 cm 4.9 cm 2.8 cm Root system development 4.3 per seedling 2.1 per seedling 4.0 seedlings per seedling 1.9 per seedling 4. Ecological safety: The soil microbial diversity index (Shannon) of the experimental group reached 3.8 (2.1 in the control group), and the heavy metal detection results of the plants showed that the contents of Cd and Pb were lower than the pharmacopoeia standards (Cd less than 0.1 mg / kg, Pb less than 1.0 mg / kg). Sixthly, this application provides an economic analysis of the aforementioned compound microbial agent: 1. Cost of inoculant: The cost of preparing the inoculant of this invention increases by 18% (because the mycelial network culture needs to be extended by 8 hours), but because no chemical buffer is required, the input per mu is reduced by 220 yuan. 2. Output benefits: The increased germination rate increases seedling production by 1.8 times. Based on a market price of 30 yuan per seedling, this translates to an additional income of 27,000 yuan per mu. Seventhly, this application provides key process control points for the aforementioned compound microbial agent: 1. Mycelial network density: must be controlled at 1.5 × 10⁻⁶ 4 Up to 2.0×10 4 Strips per square millimeter. Too dense (greater than 2.5 × 10⁻⁶). 4 This hinders seed respiration; excessively sparse (less than 1.0 × 10⁻⁶) 4 This leads to micro-region rupture. 2. Carrier selection: The porosity of the coconut coir carrier must be greater than 65%, determined by mercury porosimetry (according to GB / T 1966-2021 standard, using a mercury porosimeter at 10MPa pressure), and verified before use. Perlite carriers may cause buffer failure due to the adsorption of citric acid. 3. Application timing: It must be applied 24 hours before sowing. Applying it in advance will activate the mycelial network in advance, while applying it in a later time will result in a delayed response. Eighthly, this application provides the following technical advantages of the aforementioned compound microbial agent: 1. Two-way dynamic regulation mechanism: Unlike one-way buffering schemes, this inoculant achieves two-way acid-base neutralization through metabolic switching of the same strain. In red soil with pH 4.0, the micro-zone pH rises to 6.2 within 30 minutes, with a germination rate of 81.3%; in yellow soil with pH 8.5, the micro-zone pH drops to 6.3, with a germination rate of 79.6%, while conventional inoculants have germination rates below 30% under the same conditions. 2. Micrometer-level precise microenvironment construction: Spherical microzones (0.5 to 1 mm in diameter) enclosed by mycelial networks cover only the seed body, with zero waste of buffering substances. Actual measurements show that the soil pH 5 mm outside the microzone remains at extreme values ​​(4.0 or 8.5), but the pH inside the microzone remains stable at 6.0 to 6.5, improving germination uniformity to 95%. 3. Complete bio-based safety guarantee: Contains only Bacillus amyloliquefaciens and its metabolites (calcium carbonate and citric acid are food-grade), with no chemical additive residues. The microcrystalline dissolution product Ca²⁺ can be absorbed and utilized by Dendrobium (Dendrobium is a calcium-loving plant), and has passed organic certification testing. Ninthly, this application provides further optimization directions for the above-mentioned compound microbial agent: 1. Strain improvement: Gene editing technology is used to enhance the expression efficiency of carbonic anhydrase and citrate synthase in Bacillus amyloliquefaciens, further shortening the response time. 2. Carrier optimization: Develop novel porous materials as carriers to improve the uniformity and stability of mycelial network formation. 3. Process improvement: Introduce automated equipment for the preparation and application of microbial agents to reduce human error and improve production efficiency. Tenthly, this application provides the application prospects of the above-mentioned compound microbial agent: 1. Agricultural field: In addition to Dendrobium officinale, it can also be applied to the seed germination of other crops that are sensitive to soil pH, such as orchids and Chinese medicinal herbs. 2. Ecological restoration: It can be used for localized improvement of acidic or alkaline contaminated soils and to restore soil ecological functions. 3. Scientific research value: It provides a new model system for studying the mechanism of microbial metabolic regulation and promotes the development of basic research in related fields. In summary, the compound microbial agent provided in this application, through the metabolic synergistic effect of Bacillus amyloliquefaciens and the spatial constraint ability of the mycelial network, solves the problems of the unidirectional and passive pH buffering mechanism, the safety risks introduced by chemical buffers, and the lack of microenvironment construction ability in the prior art. It achieves dynamic stability of pH in the micro-zone of Dendrobium officinale seed germination, and has significant technical innovation and practical application value. Detailed Implementation

[0005] The embodiments or implementations described in this specification adopt a progressive approach, with each embodiment focusing on its differences from other embodiments. In the description of this specification, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with an implementation or example that are included in at least one implementation or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same implementation or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more implementations or examples. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified. As described in the background section above, current methods for regulating soil pH during Dendrobium officinale seed germination mainly rely on chemical buffers. However, these buffers suffer from insufficient unidirectional regulatory capacity, high residual risk, and imprecise microenvironment construction. Therefore, this application provides a compound microbial agent for promoting Dendrobium officinale seed germination and its preparation method. The aim is to dynamically construct a constant pH microenvironment of 6.0 to 6.5 within a 0.5 to 1 mm radius around the seed through the synergistic metabolic action of Bacillus amyloliquefaciens. In one aspect, this application provides a compound microbial agent for promoting the germination of Dendrobium officinale seeds, comprising the following technical features: the core component of the compound microbial agent is Bacillus amyloliquefaciens (BNCC186043), whose cell membrane expresses the pH-sensing protein PhoR. When the external H⁺ or OH⁻ concentration exceeds a threshold, i.e., when the external pH is below 5.5 (acidic conditions) or above 6.5 (alkaline conditions), a dual-pathway metabolic activation mechanism is triggered. Acidic conditions induce the expression of carbonic anhydrase, and alkaline conditions induce the expression of citrate synthase. In acidic soil, carbonic anhydrase catalyzes the formation of calcium carbonate microcrystals from CO₂, with a particle size ranging from 1 to 5 micrometers. The microcrystals are embedded in the interstices of the mycelial network and undergo a dissolution reaction upon encountering H⁺ (CaCO₃ + 2H⁺ → Ca²⁺ + CO₂ + H₂O), with each milligram of microcrystals neutralizing 0.02 millimoles of H⁺. Citrate synthase catalyzes the secretion of citric acid in alkaline soils, which dissociates to release H⁺ (C₆H₈O₇ → H⁺ + C₆H₇O₇), neutralizing 0.015 mmol of OH⁻ per milligram of citric acid. The hyphal network forms a selectively permeable membrane, allowing only H⁺ or OH⁻ to diffuse at a rate below 0.1 mm / min (equivalent to approximately 1.67 × 10⁻). 6The slow diffusion (m / s) allows for a time window for metabolic regulation. By controlling the accumulation of metabolites in Bacillus amyloliquefaciens during the static culture phase, an intercalation distribution of 60%±5% microcrystals and 40%±5% citric acid in the micro-region volume is achieved, forming an "acid-base double buffer pair" and keeping the pH fluctuation range of the micro-region within ±0.3. Secondly, this application provides a method for preparing the above-mentioned compound bacterial agent, comprising the following steps: S10: Inoculating *Bacillus amyloliquefaciens* into LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl), and incubating with shaking at 37°C for 16 hours until the logarithmic phase (OD600 = 0.9). S20: Collecting bacterial cells by centrifugation (10,000 rpm, 10 min), washing three times with 10 mL pH 7.0 phosphate buffer, and resuspending in 5 mL of the same buffer to a final volume of 1×10⁻⁶. 9 CFU / mL. S30: Mix the bacterial culture with coconut coir carrier at a ratio of 1:5 (carrier pre-sterilized), and incubate at 28°C for 24 hours to allow the bacteria to form a complete hyphal network. S40: Freeze-dry (-50°C, 24 hours) to prepare granular bacterial agent, controlling the particle size to 0.5 to 1 mm, with a viable count of not less than 5 × 10⁻⁶. 8 CFU per gram. Thirdly, this application provides a specific application method for the aforementioned compound microbial agent, including the following technical details: the agent is applied 24 hours before sowing; early application leads to premature activation of the mycelial network, while delayed application results in a delayed response. Microscopic counting (400× magnification) confirmed the mycelial network density to be 1.5×10⁻⁶. 4 Up to 2.0×10 4 The density is controlled by adjusting the static incubation time; excessive density (greater than 2.5 × 10⁻⁶) can lead to problems. 4 This hinders seed respiration; excessively sparse (less than 1.0 × 10⁻⁶) 4 This leads to micro-zone rupture. The porosity of the coconut coir carrier needs to be greater than 65%, determined by mercury porosimetry (according to GB / T 1966-2021 standard, using a mercury porosimeter at 10 MPa pressure), and verified before use. Perlite carriers cause buffer failure due to citric acid adsorption. In acidic soils (pH below 5.5), calcium carbonate microcrystals dissolve and consume H⁺; in alkaline soils (pH above 6.5), citric acid dissociates and releases H⁺, keeping the pH dynamically stable within the micro-zone between 6.0 and 6.5. Fourthly, this application provides specific implementation effects of the above-mentioned compound microbial agent under different soil pH conditions: Acidic conditions (pH 4.0): When the agent was placed in simulated red soil solution (containing 0.01 mol / L Al³⁺), calcium carbonate microcrystals were detected after 5 minutes (XRD peak position 29.4°), and the micro-area pH rose to 6.1 within 30 minutes. Alkaline conditions (pH 8.5): When the agent was placed in simulated yellow soil solution (containing 0.02 mol / L Na₂CO₃), the citric acid secretion reached 0.18 mg / g after 8 minutes, and the micro-area pH dropped to 6.4 within 30 minutes. Dynamic stability: During the step change of pH from 4.0 to 8.5, the micro-area pH remained consistently between 6.0 and 6.5 (fluctuation ±0.2), while the control group (conventional agent) fluctuated within a range of ±1.5. Fifthly, this application provides specific data support for the field application of the aforementioned compound microbial agent: One mu (approximately 0.16 acres) experimental plots were established in two locations: an acidic red soil (pH 4.8) planting base in Gulinqing Township, Maguan County, Wenshan Prefecture, Yunnan Province, and an alkaline yellow soil (pH 8.2) planting base in Guangnan County, Wenshan Prefecture, Yunnan Province. The experimental group's substrate was mixed with the microbial agent of this invention (5 grams per square meter), while the control group used conventional microbial agents plus a chemical buffer (50 grams of calcium carbonate per square meter plus 30 grams of sodium citrate per square meter). Germination stage: Red soil area: The germination rate of the experimental group on day 7 was 42.6% (control group 18.3%), and the micro-plot pH remained stable at 6.2±0.3. Yellow soil area: The germination rate of the experimental group on day 7 was 40.1% (control group 15.7%), and the micro-plot pH remained stable at 6.4±0.2. Seedling stage: index Red Soil Region Experimental Group control group in red soil region Yellow Soil Experimental Group Yellow soil area control group Seedling survival rate (30 days) 85.4% 43.7% 82.9% 41.2% Average seedling height 5.2 cm 3.1 cm 4.9 cm 2.8 cm Root system development 4.3 per seedling 2.1 per seedling 4.0 seedlings per seedling 1.9 per seedling Ecological safety: The soil microbial diversity index (Shannon) of the experimental group reached 3.8 (2.1 in the control group), and the heavy metal detection results of the plants showed that the contents of Cd and Pb were lower than the pharmacopoeia standards (Cd less than 0.1 mg / kg, Pb less than 1.0 mg / kg). Sixthly, this application provides an economic analysis of the above-mentioned compound microbial agent: Agent cost: The preparation cost of the agent of this invention increases by 18% (due to the need to extend the mycelial network cultivation by 8 hours), but because no chemical buffer is required, the average input per mu is reduced by 220 yuan. Output benefits: The increased germination rate increases seedling yield by 1.8 times; calculated at a market price of 30 yuan per seedling, this translates to an increased income of 27,000 yuan per mu. Seventhly, this application provides key process control points for the aforementioned compound microbial agent: mycelial network density: must be controlled at 1.5 × 10⁻⁶. 4 Up to 2.0×10 4 Strips per square millimeter. Too dense (greater than 2.5 × 10⁻⁶). 4 This hinders seed respiration; excessively sparse (less than 1.0 × 10⁻⁶) 4This can lead to micro-zone rupture. Carrier selection: The porosity of coconut coir carrier must be greater than 65%, determined by mercury porosimetry (according to GB / T1966-2021 standard, using a mercury porosimeter at 10 MPa pressure), and verified before use. Perlite carriers may cause buffer failure due to citric acid adsorption. Application timing: It must be applied 24 hours before sowing. Early application activates the mycelial network, while delayed application results in a delayed response. Eighthly, this application provides the technical advantages of the aforementioned compound microbial agent: Two-way dynamic regulation mechanism: Unlike one-way buffering schemes, this agent achieves two-way acid-base neutralization through metabolic switching of the same strain. In red soil with pH 4.0, the micro-zone pH rises to 6.2 within 30 minutes, with a germination rate of 81.3%; in yellow soil with pH 8.5, the micro-zone pH drops to 6.3, with a germination rate of 79.6%, while conventional agents have germination rates below 30% under the same conditions. Micron-level precise microenvironment construction: Spherical micro-zones (0.5 to 1 mm in diameter) enclosed by a mycelial network cover only the seed itself, resulting in zero waste of buffering materials. Actual measurements show that the soil pH 5 mm outside the micro-zone remains at extreme values ​​(4.0 or 8.5), but the pH within the micro-zone remains stable at 6.0 to 6.5, improving germination consistency to 95%. Complete biological safety guarantee: Contains only Bacillus amyloliquefaciens and its metabolites (calcium carbonate and citric acid are food-grade), with no chemical additive residues. The microcrystalline dissolution product Ca²⁺ can be absorbed and utilized by Dendrobium (which is a calcium-loving plant), and has passed organic certification testing. Ninthly, this application provides further optimization directions for the above-mentioned compound microbial agent: Strain improvement: Enhancing the expression efficiency of carbonic anhydrase and citrate synthase in Bacillus amyloliquefaciens through gene editing technology to further shorten the response time. Carrier optimization: Developing novel porous materials as carriers to improve the uniformity and stability of mycelial network formation. Process improvement: Introducing automated equipment for the preparation and application of the microbial agent to reduce human error and improve production efficiency. Tenthly, this application provides the application prospects of the above-mentioned compound microbial agent: In agriculture: besides Dendrobium officinale, it can also be applied to the seed germination of other crops sensitive to soil pH, such as orchids and medicinal herbs. In ecological restoration: it can be used for the localized improvement of acidic or alkaline polluted soils, restoring soil ecological functions. In scientific research: it provides a new model system for studying the mechanisms of microbial metabolic regulation, promoting the development of basic research in related fields. The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially. Example 1 Preparation of compound microbial agents: S10: Inoculate Bacillus amyloliquefaciens into LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and incubate at 37°C with shaking for 16 hours until the logarithmic phase (OD600=0.9). S20: Collect bacterial cells by centrifugation (10,000 rpm, 10 min), wash three times with 10 mL pH 7.0 phosphate buffer, and resuspend in 5 mL of the same buffer to a final volume of 1×10⁻⁶. 9 CFU / mL. S30: Mix the bacterial solution with coconut coir carrier at a ratio of 1:5 (the carrier is pre-sterilized), and incubate at 28 degrees Celsius for 24 hours to allow the bacteria to form a complete hyphal network. S40: Freeze-dried (-50°C, 24 hours) to produce granular bacterial agent, with a particle size controlled at 0.5 to 1 mm and a viable count of not less than 5 × 10⁻⁶. 8 CFU per gram. Example 2 The preparation of the compound microbial agent is the same as in Example 1, except that the porosity of the coconut coir carrier is 50%. Example 3 The preparation of the compound microbial agent is the same as in Example 1, except that the mycelial network density is 2.5 × 10⁻⁶. 4 Each strip per square millimeter. Example 4 The preparation of the compound microbial agent is the same as in Example 1, except that the mycelial network density is 1.0 × 10⁻⁶. 4 Each strip per square millimeter. Comparative Example 1 The preparation of the compound microbial agent is the same as in Example 1, except that the carrier is perlite with a porosity of 70%. Comparative Example 2 The preparation of the compound microbial agent is the same as in Example 1, except that Bacillus amyloliquefaciens is not added, and only a chemical buffer (50 grams of calcium carbonate per square meter plus 30 grams of sodium citrate per square meter) is used. Comparative Example 3 The preparation of the compound microbial agent is the same as in Example 1, except that the mycelial network density is not controlled and is randomly distributed. Comparative Example 4 The preparation of the compound microbial agent is the same as in Example 1, except that the application time before sowing is not controlled and it is applied 48 hours in advance. The compound microbial agents obtained in the above examples and comparative examples were used in field trials on acidic red soil (pH 4.8) and alkaline yellow soil (pH 8.2), respectively. Germination rate, seedling rate, average seedling height, root development, and soil microbial diversity index were measured. The results are shown in the table below: index Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Germination rate (day 7) 42.6% 35.4% 30.1% 28.7% 20.3% 15.7% 25.4% 31.2% Seedling survival rate (30 days) 85.4% 72.3% 65.1% 60.3% 50.2% 41.2% 55.3% 62.4% Average seedling height 5.2 cm 4.5 cm 4.0 cm 3.8 cm 3.2 cm 2.8 cm 3.5 cm 4.1 cm Root system development 4.3 per seedling 3.8 per seedling 3.2 per seedling 3.0 per seedling 2.5 per seedling 1.9 per seedling 2.8 per seedling 3.3 per seedling Microbial biodiversity index 3.8 3.2 2.8 2.6 2.4 2.1 2.7 3.0 Based on the above results, Example 1 of this application exhibits higher germination rate, seedling rate, average seedling height, root development, and soil microbial diversity index compared to other examples and comparative examples. Example 2 suffers from weakened buffering effect due to the low porosity of the coconut coir carrier; Examples 3 and 4 suffer from inadequate control of mycelial network density, affecting the stability of the microenvironment; Comparative Example 1 suffers from buffering failure due to the adsorption of citric acid by perlite as the carrier; Comparative Example 2 does not use Bacillus amyloliquefaciens and relies entirely on chemical buffers, resulting in poor performance; Comparative Examples 3 and 4 suffer from inaccurate microenvironment construction due to the lack of strict control over mycelial network density and application time. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A compound microbial agent for promoting the germination of Dendrobium officinale seeds, characterized in that, Includes the following components: The core component is Bacillus amyloliquefaciens (BNCC186043), whose cell membrane expresses the pH-sensing protein PhoR; When the concentration of external hydrogen ions or hydroxide ions exceeds the threshold, a dual-pathway metabolic activation mechanism is triggered: acidic conditions induce the expression of carbonic anhydrase, and alkaline conditions induce the expression of citrate synthase. Carbonic anhydrase catalyzes the formation of calcium carbonate microcrystals with a particle size of 1 to 5 micrometers from carbon dioxide, and each milligram of microcrystal neutralizes 0.02 millimoles of hydrogen ions. Citrate synthase catalyzes the secretion of citrate, and each milligram of citrate neutralizes 0.015 millimoles of hydroxide ions; The hyphal network forms a selectively permeable membrane with a diffusion rate of less than 0.1 mm per minute. By controlling the accumulation of metabolites of Bacillus amyloliquefaciens during the static culture stage, an intercalation distribution of 60% ± 5% of microcrystals and 40% ± 5% of citric acid in the micro-region volume is achieved.

2. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, The *Bacillus amyloliquefaciens* was inoculated into LB medium containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, and cultured at 37°C with shaking for 16 hours to the logarithmic phase, with an OD600 value of 0.

9.

3. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, The microbial agent is prepared through the following steps: S10: Inoculate Bacillus amyloliquefaciens into LB medium and culture at 37°C with shaking for 16 hours to the logarithmic phase; S20: Collect bacterial cells by centrifugation (10,000 rpm, 10 min), wash three times with 10 mL pH 7.0 phosphate buffer, and resuspend in 5 mL of the same buffer to a final volume of 1×10⁻⁶. 9 CFU / mL; S30: Mix the bacterial culture with the coconut coir carrier at a ratio of 1:5 and incubate at 28 degrees Celsius for 24 hours. S40: Freeze-dried to produce granular bacterial agent, with a particle size controlled at 0.5 to 1 mm and a viable count of not less than 5 × 10⁻⁶. 8 CFU per gram.

4. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, The inoculum agent was applied 24 hours before sowing, with a mycelial network density of 1.5 × 10⁻⁶. 4 Up to 2.0×10 4 Each strip per square millimeter.

5. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, The porosity of the coconut coir carrier is greater than 65%.

6. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, In acidic soil, the compound microbial agent dissolves calcium carbonate microcrystals, consuming hydrogen ions; in alkaline soil, citric acid dissociates, releasing hydrogen ions, thus stabilizing the pH dynamic within the micro-zone at 6.0 to 6.

5.

7. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, The specific effects of the compound microbial agent under acidic conditions include: when the agent is placed in simulated red soil liquid, calcium carbonate microcrystals are detected after 5 minutes, and the pH of the micro-area rises to 6.1 within 30 minutes.

8. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, The specific effects of the compound microbial agent under alkaline conditions include: when the agent is placed in simulated yellow soil solution, the citric acid secretion reaches 0.18 mg / g after 8 minutes, and the micro-zone pH drops to 6.4 within 30 minutes.

9. The compound microbial agent for promoting the germination of Dendrobium officinale seeds according to claim 1, characterized in that, The specific data supporting the application of the compound microbial agent in the field include: in acidic red soil, the germination rate of the experimental group was 42.6% on day 7, and the micro-zone pH was stable at 6.2±0.3; in alkaline yellow soil, the germination rate of the experimental group was 40.1% on day 7, and the micro-zone pH was stable at 6.4±0.

2.

10. A method for promoting the germination of Dendrobium officinale seeds, characterized in that, The compound microbial agent described in any one of claims 1 to 9 shall be applied 24 hours before sowing, at a rate of 5 grams per square meter.