Application of novel disease-resistant and production-promoting biological agent

Abstract

The invention relates to the field of agriculture, and discloses an application of a novel disease-resistant and yield-promoting biological agent, a preparation method comprises the following steps: screening a microbial strain and performing gene editing, so that the microbial strain overexpresses heat shock protein and antifreeze protein genes; carrying out microcapsule embedding on the strain subjected to gene editing, and adding a low-temperature protective agent; carrying out vacuum freeze drying on the embedded strain; nano-particles are prepared by using a nanotechnology, and a plant growth regulator is loaded; and mixing the dried microcapsule particles and the nanoparticles loaded with the plant growth regulator, adding a buffering agent and an antioxidant, and packaging by adopting an intelligent packaging material. A polylactic acid-glycolic acid copolymer is utilized to form a physical barrier to wrap a strain, glycerin and trehalose are matched to reduce the freezing point and stabilize the cell structure, ice crystal damage and environmental impact are jointly resisted in a low-temperature environment, the problem that the strain is prone to inactivation in low-temperature storage and transportation of a biological preparation is solved, and the survival rate of the strain and the stability of the preparation are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of agriculture, and in particular to the application of a novel disease-resistant and growth-promoting biological preparation. Background Art

[0002] Agriculture, as a foundational industry of the national economy, cultivates plants, animals, and microorganisms, harnessing the laws of nature and scientific technology to produce food, industrial raw materials, and other agricultural products. It encompasses a wide range of sectors, including crop production, animal husbandry, fisheries, and forestry, and plays an irreplaceable role in ensuring food security, promoting economic development, and maintaining social stability. With the acceleration of agricultural modernization, traditional agricultural production methods face numerous challenges, including frequent pests and diseases, excessive pesticide use, and declining agricultural product quality. These challenges not only impact crop yield and quality but also pose potential threats to the ecological environment and human health. Therefore, the research and development and application of safe, efficient, and environmentally friendly new biological agents have become a crucial approach to addressing pest and disease control and promoting crop growth in agricultural production.

[0003] However, during the storage and transportation of traditional biological preparations, the active ingredients in them may degrade or deteriorate due to various factors, resulting in a decrease in the disease resistance of the biological preparations and a lower production rate. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides an application of a new disease-resistant and production-promoting biological preparation, which solves the problem that the active ingredients of traditional biological preparations may degrade or deteriorate due to various factors during storage and transportation, resulting in a decrease in the disease resistance of the biological preparation and a lower production promotion rate.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for preparing a novel disease-resistant and growth-promoting biological preparation comprises the following steps: Screening and gene editing of microbial strains to overexpress heat shock protein and antifreeze protein genes; The gene-edited strains are microencapsulated and cryoprotectants are added; The embedded strains were vacuum freeze-dried; Using nanotechnology to prepare nanoparticles and load them with plant growth regulators; The dry microcapsule particles and nanoparticles loaded with plant growth regulators are mixed, and a buffer and an antioxidant are added before packaging using intelligent packaging materials.

[0006] Preferably, the screening of microbial strains is carried out by collecting samples from hot springs with a temperature above 60°C, permafrost with a temperature below -15°C, salt lakes with a salt concentration greater than 20%, and acidic mine wastewater environments with a pH value less than 3 using a sterile sampling device, and separating the microbial strains on a solid culture medium using a dilution coating plate method and a streak separation method using sterile culture dishes, coating rods, and inoculation loops. Morphological observation is performed using Gram stain and a microscope, and preliminary purification is carried out based on the growth characteristics of the strains and the standards of their antagonistic ability to common plant pathogens to screen out strains with disease resistance and growth-promoting abilities.

[0007] Preferably, the gene editing adopts the CRISPR-Cas9 gene editing system, uses an ultra-clean workbench, a PCR instrument, and an electrophoresis device to design sgRNA targeting the promoter region of the heat shock protein HSP70, HSP90 and antifreeze protein AFP genes, and the PAM sequence is NGG, to perform site-specific modification on the genome of the screening strain.

[0008] Preferably, the microcapsule embedding adopts polylactic acid-glycolic acid copolymer with a molecular weight of 10000-50000Da and a lactic acid / glycolic acid molar ratio of 70-80:20-30, and is carried out by emulsification-solvent evaporation method using a magnetic stirrer and a separatory funnel. The oil phase includes dichloromethane, and the aqueous phase includes an aqueous solution containing 0.5% to 1.5% polyvinyl alcohol. The volume ratio of the oil phase to the aqueous phase is 1:3-1:5, and the emulsification is carried out for 15 to 25 minutes at a speed of 1500-2500 r / min using a magnetic stirrer.

[0009] Preferably, the cryoprotectant is 8% to 12% glycerol and 4% to 6% trehalose by mass, which are accurately weighed by an electronic balance and added to a magnetic stirrer. The magnetic stirrer is stirred at a stirring speed of 1000-1200 r / min for 10 to 15 minutes to mix evenly with the embedding system.

[0010] Preferably, the vacuum freeze drying process uses a vacuum freeze dryer, the pre-freezing temperature is controlled at -50°C to -40°C, the pre-freezing time is 2 to 4 hours, the vacuum degree is maintained at 15 to 25 Pa, and the drying time is 15 to 20 hours.

[0011] Preferably, the nanoparticles are prepared by a nanoprecipitation method, using a magnetic stirrer and a constant pressure dropping funnel, and slowly adding a polyethylene glycol-polylactic acid PEG-PLA acetone solution with a mass concentration of 12-18 mg / mL to stirred deionized water, with a volume ratio of acetone to deionized water of 1:7-1:9, and using a magnetic stirrer at a stirring speed of 700-800 r / min to prepare nanoparticles with a particle size of 40-60 nm.

[0012] Preferably, the plant growth regulators are coronatine at a concentration of 2-4 mg / mL and brassinolide at a concentration of 3-5 mg / mL. The nanoparticles are loaded using an ultrasonic cell disruptor with an ultrasonic oscillation power of 280-320 W for 18-22 minutes. After loading, the loading rate of the plant growth regulators is tested using high-performance liquid chromatography, and the loading rate is required to be no less than 85%.

[0013] Preferably, the smart packaging material adopts a three-layer composite structure, the outer layer is a PET film, the middle layer is an EVOH gas barrier layer, and the inner layer is a PE film. The inner layer surface is coated with a temperature and humidity sensing coating and an integrated gas detection sensor, and a packaging machine is used for preparation packaging.

[0014] Preferably, a new type of disease-resistant and growth-promoting biological preparation is used. The biological preparation is used in the agricultural field and can be used for disease resistance and yield increase of rice, wheat, and corn crops. A seed mixer is used for seed mixing before sowing, and the mass ratio of the preparation to the seed is 1:200-1:400. A sprayer is used for foliar spraying during the growth period, and the dilution multiple is 800-1200 times. Irrigation equipment is used for root watering, and the dilution multiple is 600-1000 times.

[0015] The present invention provides a novel application of a disease-resistant and growth-promoting biological preparation. It has the following beneficial effects: 1. The present invention uses polylactic acid-glycolic acid copolymer to form a physical barrier to wrap the bacterial strain, and combines it with glycerol and trehalose to lower the freezing point and stabilize the cell structure, thereby jointly resisting ice crystal damage and environmental impact in a low-temperature environment, solving the problem of easy inactivation of bacterial strains during low-temperature storage and transportation of biological preparations, and significantly improving the survival rate of the strain and the stability of the preparation.

[0016] 2. The present invention screens strains with natural stress resistance potential from extreme environments such as high-temperature hot springs and low-temperature permafrost, and combines gene editing technology to target and enhance the expression of heat shock protein and antifreeze protein genes, giving the strains stronger high and low temperature adaptability, solving the problem of insufficient stress resistance of traditional strains, and laying the core foundation for biological preparations to maintain activity in complex environments.

[0017] 3. The present invention achieves water sublimation in a low-temperature vacuum environment, avoiding damage to the strain caused by high-temperature drying. Combining the physical protection of microcapsules and the chemical stabilization of cryoprotectants, it inhibits microbial contamination and component degradation, solves the problem of preparation failure caused by traditional drying processes, extends the shelf life of biological preparations and ensures long-term activity.

[0018] 4. The present invention utilizes the high specific surface area characteristics of nanoparticles to improve the dispersibility and targeted delivery ability of the regulator, and cooperates with stress-resistant strains to achieve the combination of disease resistance and production promotion functions; combined with the environmental isolation and dynamic monitoring of intelligent packaging, it prevents the oxidation and moisture absorption of ingredients, solves the problems of traditional preparations with single functions and uncontrollable storage environment, and improves agricultural application effects and product reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flow chart of a method for preparing a novel disease-resistant and growth-promoting biological preparation. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Please see the attached Figure 1 The present invention provides a method for preparing a novel disease-resistant and growth-promoting biological preparation, comprising the following steps: Screening and gene editing of microbial strains to overexpress heat shock protein and antifreeze protein genes; The gene-edited strains are microencapsulated and cryoprotectants are added; The embedded strains were vacuum freeze-dried; Using nanotechnology to prepare nanoparticles and load them with plant growth regulators; The dry microcapsule particles and nanoparticles loaded with plant growth regulators are mixed, and a buffer and an antioxidant are added before packaging using intelligent packaging materials.

[0022] Specifically, by screening microbial strains and performing gene editing, the strains are made to overexpress heat shock protein and antifreeze protein genes, giving the strains the ability to withstand high and low temperature environments, laying the foundation for biological agents to maintain activity under different temperature conditions; The gene-edited strains are microencapsulated and cryoprotectants are added. The microcapsules form a physical barrier to protect the strains, while the cryoprotectants further enhance the strains' ability to survive in low-temperature environments. The combination of the two solves the problem of strain inactivation during low-temperature storage and transportation of biological agents. The embedded strains are vacuum-freeze-dried. By controlling the pre-freezing temperature, time, vacuum degree and drying time, water sublimation is achieved in a low-temperature vacuum environment, avoiding damage to the activity of the strains caused by high-temperature drying. This, combined with microencapsulation, extends the storage period of biological preparations. Nanoparticles are prepared using nanotechnology and loaded with plant growth regulators. The properties of nanoparticles can improve the dispersibility and stability of plant growth regulators, making them easier for plants to absorb. Combined with stress-resistant strains, they can achieve the dual functions of disease resistance and production promotion. The dry microcapsule particles are mixed with nanoparticles loaded with plant growth regulators, and then a buffer and antioxidant are added before packaging using smart packaging materials. The buffer and antioxidant protect the internal components and the smart packaging materials monitor the environment in real time to ensure that the preparation is not disturbed by the external environment during storage and transportation. The above preparation method solves the problems of poor adaptability of biological preparations in different temperature environments, easy deterioration during storage and transportation, and single function through the synergistic effects of strain screening and gene editing, microcapsule encapsulation and low-temperature protection, vacuum freeze-drying, nanoparticle loading and intelligent packaging, thereby achieving the effect of improving the stability of the preparation and realizing disease resistance and production promotion.

[0023] Screening of microbial strains involves collecting samples using sterile sampling instruments from hot springs with temperatures above 60°C, permafrost below -15°C, salt lakes with salt concentrations greater than 20%, and acidic mine wastewater environments with a pH value less than 3. The samples are separated on solid culture media using the dilution spread plate method and the streak separation method using sterile culture dishes, spreader rods, and inoculation loops. Gram staining and a microscope are used for morphological observation, and preliminary purification is carried out based on the growth characteristics of the strains and their antagonistic ability to common plant pathogens to screen out strains with disease resistance and growth-promoting capabilities.

[0024] Specifically, by using sterile sampling instruments to collect samples from extreme environments such as hot springs with temperatures above 60°C, permafrost with temperatures below -15°C, salt lakes with salt concentrations greater than 20%, and acidic mine wastewater with a pH value less than 3, the microbial strains are separated on solid culture media using the dilution coating plate method, streak separation method, and sterile culture dishes, coating rods, inoculation loops and other instruments. The strains are then morphologically observed using Gram staining solution and a microscope, and preliminarily purified based on the growth characteristics of the strains and their antagonistic ability to common plant pathogens. Strains that naturally possess stress-resistant genes, such as high-temperature and low-temperature resistance genes, and have inhibitory effects on plant pathogens can be screened out under the highly selective conditions of extreme environments. At the same time, standardized sterile operations and multi-dimensional screening standards are used to ensure the purity and targeted functionality of the strains, providing core functional strains with both environmental adaptability and disease resistance and growth-promoting capabilities for subsequent gene editing to enhance stress resistance and preparation of preparations.

[0025] Gene editing uses the CRISPR-Cas9 gene editing system, an ultra-clean workbench, a PCR instrument, and an electrophoresis device to design sgRNA targeting the promoter region of the heat shock protein HSP70, HSP90, and antifreeze protein AFP genes, with a PAM sequence of NGG, to perform site-specific modification on the genome of the screened strain.

[0026] Specifically, the CRISPR-Cas9 gene editing system was used with the help of an ultra-clean workbench, a PCR instrument, and an electrophoresis equipment to design sgRNA targeting the promoter regions of the heat shock proteins HSP70, HSP90, and antifreeze protein AFP genes, with a PAM sequence of NGG. The genomes of the screened strains with disease resistance and growth-promoting abilities were site-specifically modified, which can accurately activate the expression of the strains' endogenous stress resistance genes. By enhancing the synthesis of heat shock proteins and antifreeze proteins, the protein structure stability and cell damage resistance of the strains in high and low temperature environments are improved. Compared with traditional mutagenesis technology, this method has the advantages of strong targeting, high editing efficiency, and good genetic stability, ensuring that biological agents can remain active and exert disease resistance and growth-promoting effects in extreme environments.

[0027] Microcapsule encapsulation uses a polylactic acid-glycolic acid copolymer with a molecular weight of 10,000-50,000 Da and a lactic acid / glycolic acid molar ratio of 70-80:20-30. The process is carried out by an emulsification-solvent evaporation method using a magnetic stirrer and a separatory funnel. The oil phase includes dichloromethane and the aqueous phase includes an aqueous solution containing 0.5% to 1.5% polyvinyl alcohol. The volume ratio of the oil phase to the aqueous phase is 1:3 to 1:5. The emulsification is carried out for 15 to 25 minutes using a magnetic stirrer at a speed of 1,500 to 2,500 r / min.

[0028] Specifically, a polylactic acid-glycolic acid copolymer with a molecular weight of 10,000-50,000 Da and a lactic acid / glycolic acid molar ratio of 70-80:20-30 is used. The emulsification-solvent evaporation method is used, using a magnetic stirrer and a separatory funnel, with dichloromethane as the oil phase and an aqueous solution containing 0.5% to 1.5% polyvinyl alcohol as the water phase. The microencapsulation is carried out at a speed of 1500-2500 r / min for 15 to 25 minutes. The biocompatibility and degradability of the polylactic acid-glycolic acid copolymer can be utilized to form a microcapsule wall material with uniform particle size and stable structure, and the gene-edited strain is encapsulated therein. Polyvinyl alcohol is used as an emulsifier to enhance the dispersion of the oil and water phases. By controlling the emulsification speed and time, the encapsulation efficiency and integrity of the microcapsules are ensured, providing a physical barrier for the strain to resist direct damage to the strain from the external environment. At the same time, in the subsequent vacuum freeze-drying process, the penetration damage of water to the strain is reduced, thereby improving the survival rate of the strain during storage, transportation and application.

[0029] The cryoprotectant is 8% to 12% glycerol and 4% to 6% trehalose by mass. It is accurately weighed by an electronic balance and added to a magnetic stirrer. The magnetic stirrer is stirred at a stirring speed of 1000-1200 r / min for 10 to 15 minutes to mix evenly with the embedding system.

[0030] Specifically, 8% to 12% by mass of glycerol and 4% to 6% by mass of trehalose are used as cryoprotectants. After being accurately weighed using an electronic balance, they are added to a magnetic stirrer and mixed at a stirring speed of 1000-1200 r / min for 10 to 15 minutes to make them evenly dispersed with the embedding system. Glycerol can lower the freezing point of the aqueous solution and reduce the physical damage of ice crystal formation to the strain cells. Trehalose replaces water molecules through hydrogen bonding to stabilize the cell membrane and protein structure. The two work together to form a protective hydration layer under low temperature conditions, and cooperate with the physical barrier of microcapsule embedding to further improve the survival rate of the strain during freeze-drying and low-temperature storage, thereby solving the problem of decreased activity of traditional biological preparations due to cell dehydration or ice crystal damage under low temperature conditions, and ensuring the effectiveness of the preparations during cold chain transportation and low-temperature environment.

[0031] Vacuum freeze drying treatment uses a vacuum freeze dryer, the pre-freezing temperature is controlled at -50°C to -40°C, the pre-freezing time is 2 to 4 hours, the vacuum degree is maintained at 15 to 25 Pa, and the drying time is 15 to 20 hours.

[0032] Specifically, a vacuum freeze dryer is used to pre-freeze at a pre-freezing temperature of -50°C to -40°C for 2 to 4 hours, and the vacuum degree is maintained at 15 to 25 Pa for 15 to 20 hours. The water in the embedded strain is frozen into solid ice crystals by pre-freezing, and the ice crystals are directly sublimated and removed under a high vacuum environment, avoiding the strain protein denaturation and activity loss caused by traditional high-temperature drying. At the same time, the low-temperature vacuum environment can inhibit microbial contamination and chemical reactions, and cooperate with the physical protection of microcapsule encapsulation and the chemical stabilization effect of the cryoprotectant to further extend the shelf life of the biological preparation, ensure that the strain remains active for a long time in a dry state, solve the problem of preparation failure caused by high temperature or oxidation in the traditional drying process, and provide reliable stability guarantee for the large-scale storage and transportation of preparations.

[0033] Nanoparticles were prepared by the nanoprecipitation method. A polyethylene glycol-polylactic acid PEG-PLA acetone solution with a mass concentration of 12-18 mg / mL was slowly added dropwise to stirred deionized water using a magnetic stirrer and a constant pressure dropping funnel. The volume ratio of acetone to deionized water was 1:7-1:9. Nanoparticles with a particle size of 40-60 nm were prepared using a magnetic stirrer at a stirring speed of 700-800 r / min.

[0034] Specifically, the nanoprecipitation method is adopted, using a magnetic stirrer and a constant pressure dropping funnel, and a polyethylene glycol-polylactic acid acetone solution with a mass concentration of 12-18 mg / mL is slowly added dropwise to deionized water. Nanoparticles with a particle size of 40-60 nm are prepared at a stirring speed of 700-800 r / min. The amphiphilicity of PEG-PLA can be used to self-assemble in the aqueous phase to form nanoscale carriers. By precisely controlling the solution concentration, phase volume ratio and stirring speed, the uniform particle size and good dispersion of the nanoparticles are ensured. The nanoparticles have high specific surface area and surface charge characteristics, providing an efficient carrier for the subsequent loading of plant growth regulators such as coronatine and brassinolide. The water solubility and targeted delivery ability of the regulators are enhanced through the nanoscale effect, making them easier to absorb and conduct in the plant body, synergistically acting with stress-resistant strains to achieve a combined enhancement of disease resistance and production promotion functions.

[0035] The plant growth regulators, coronatine at a concentration of 2 to 4 mg / mL and brassinolide at a concentration of 3 to 5 mg / mL, are loaded into the nanoparticles using an ultrasonic cell disruptor with an ultrasonic oscillation power of 280 to 320 W for 18 to 22 minutes. After loading, the loading rate of the plant growth regulators is tested using high-performance liquid chromatography, and the loading rate is required to be no less than 85%.

[0036] Specifically, an ultrasonic cell disruptor was used to load 2-4 mg / mL of coronatine and 3-5 mg / mL of brassinolide into the nanoparticles at a power of 280-320 W for 18-22 minutes. The cavitation effect of ultrasound created micropores on the surface of the nanoparticles, promoting the rapid embedding of the plant growth regulators into the particles or their adsorption on the surface. The high specific surface area of ​​the nanoparticles also improved the loading efficiency. After loading, the loading rate was tested using high-performance liquid chromatography to ensure the effective loading of coronatine and brassinolide. This makes the nanoparticles a composite carrier with both disease-resistant signal induction and growth regulation functions, synergizing with stress-resistant strains to ensure the effectiveness and stability of the biological agent in agricultural applications.

[0037] The intelligent packaging material adopts a three-layer composite structure, with a PET film as the outer layer, an EVOH gas barrier layer as the middle layer, and a PE film as the inner layer. The inner layer is coated with a temperature and humidity sensing coating and integrated with a gas detection sensor, and a packaging machine is used for preparation packaging.

[0038] Specifically, a three-layer composite intelligent packaging material consisting of an outer PET film, an intermediate EVOH gas barrier layer, and an inner PE film is used. The inner layer is coated with a temperature and humidity sensing coating and integrated with a gas detection sensor. The preparation is packaged by a packaging machine. The PET film provides physical support and wear resistance, while the EVOH gas barrier layer effectively blocks oxygen and water vapor penetration, preventing oxidation or moisture absorption and deterioration of the preparation ingredients. The inner PE film monitors environmental temperature and humidity changes in real time through the temperature and humidity sensing coating applied. The integrated gas detection sensor can detect O2 and CO2 concentrations, enabling dynamic monitoring of the environment within the package. By synergizing multiple layers of material and combining them with the standardized packaging process of the packaging machine, full-cycle environmental isolation and quality monitoring are provided for biological preparations, resolving the problems of traditional packaging that cannot provide real-time feedback on storage environment changes and have limited protective performance. This ensures that the preparation is not disturbed by the external environment during transportation and storage, maintains the activity of the strain and the stability of the ingredients, and improves product reliability and user safety.

[0039] A new type of disease-resistant and growth-promoting biological preparation is used in the agricultural field. The biological preparation can be used to resist diseases and increase yields of rice, wheat, and corn crops. A seed mixer is used for seed mixing before sowing, with a mass ratio of the preparation to seeds of 1:200-1:400. A sprayer is used for foliar spraying during the growth period, with a dilution ratio of 800-1200 times. Irrigation equipment is used for root irrigation, with a dilution ratio of 600-1000 times. Example

[0040] First, sterile sampling instruments were used to collect samples from hot springs at a temperature of 61°C and salt lakes with a salt concentration of 21%. Microbial strains were isolated on solid culture media using the dilution spread plate method and streak separation method using sterile culture dishes, spreader rods, and inoculation loops. Morphological observations were performed using Gram stain and a microscope. Preliminary purification was carried out based on the growth characteristics of the strains and their antagonistic ability to common plant pathogens. Strains with disease resistance and growth-promoting abilities were screened. Then, the CRISPR-Cas9 gene editing system was used, along with a clean bench, PCR instrument, and electrophoresis equipment, to design sgRNAs targeting the promoter regions of the heat shock proteins HSP70 and HSP90 and antifreeze protein AFP genes with a PAM sequence of NGG. The genomes of the screened strains were site-specifically modified to overexpress the heat shock protein and antifreeze protein genes. Next, the gene-edited strain was microencapsulated using poly(lactic acid-co-glycolic acid) with a molecular weight of 10,000 Da and a lactic acid / glycolic acid molar ratio of 70:30. The emulsification-solvent evaporation method was performed using a magnetic stirrer and a separatory funnel. The oil phase was dichloromethane and the aqueous phase was an aqueous solution containing 0.5% polyvinyl alcohol. The volume ratio of the oil phase to the aqueous phase was 1:5. The emulsification was performed at a magnetic stirrer speed of 1,500 rpm for 15 minutes. Then, 8% by mass of glycerol and 4% by mass of trehalose were added as cryoprotectants, accurately weighed using an electronic balance, added to a magnetic stirrer, and stirred at a stirring speed of 1000 r / min for 10 minutes to mix evenly with the embedding system. The embedded strains were then freeze-dried using a vacuum freeze dryer, with the pre-freezing temperature controlled at -50°C for 2 hours, the vacuum degree maintained at 15 Pa, and the drying time for 15 hours. At the same time, nanoparticles were prepared using nanotechnology. The nanoprecipitation method was used. A 12 mg / mL polyethylene glycol-polylactic acid PEG-PLA acetone solution was slowly added dropwise to stirred deionized water using a magnetic stirrer and a constant pressure dropping funnel. The volume ratio of acetone to deionized water was 1:9. A magnetic stirrer was used at a stirring speed of 700 r / min to prepare nanoparticles with a particle size of 40 nm. Subsequently, 2 mg / mL of coronatine and 3 mg / mL of brassinolide were loaded into the nanoparticles using an ultrasonic cell disruptor, using an ultrasonic oscillation power of 280 W for 18 minutes. After loading, the loading rate of the plant growth regulators was tested using high-performance liquid chromatography, requiring a loading rate of no less than 85%. Finally, the dried microcapsule particles and nanoparticles loaded with plant growth regulators are mixed, and after adding buffers and antioxidants, they are packaged using smart packaging materials. The smart packaging material adopts a three-layer composite structure, with a PET film as the outer layer, an EVOH gas barrier layer as the middle layer, and a PE film as the inner layer. The inner layer surface is coated with a temperature and humidity sensing coating and an integrated gas detection sensor. The preparation is packaged using a packaging machine. The biological preparation is used to resist diseases and increase yields of rice crops in the agricultural field. A seed mixing machine is used for seed mixing before sowing, with a preparation to seed mass ratio of 1:400. A sprayer is used for foliar spraying during growth, with a dilution multiple of 1200 times. Irrigation equipment is used for root watering, with a dilution multiple of 1000 times. Example

[0041] First, samples were collected from hot springs at a temperature of 70°C and salt lakes with a salt concentration of 25%. Microbial strains were isolated on solid culture media using the dilution spread plate method and streak separation method, using sterile culture dishes, spreader rods, and inoculation loops. Morphological observations were performed using Gram stain and a microscope. Preliminary purification was carried out based on the growth characteristics of the strains and the antagonism against common plant pathogens. Strains with disease resistance and growth-promoting abilities were screened. Using the CRISPR-Cas9 gene editing system, a clean bench, a PCR instrument, and electrophoresis equipment, sgRNAs with NGG PAM sequences targeting the promoter regions of the heat shock proteins HSP70 and HSP90 and antifreeze protein AFP genes were designed. The genomes of the screened strains were then site-specifically modified to overexpress the heat shock protein and antifreeze protein genes. Next, the gene-edited strain was microencapsulated using poly(lactic acid-co-glycolic acid) with a molecular weight of 30,000 Da and a lactic acid / glycolic acid molar ratio of 75:25. The emulsification-solvent evaporation method was performed using a magnetic stirrer and a separatory funnel. The oil phase was dichloromethane and the aqueous phase was an aqueous solution containing 1.0% polyvinyl alcohol. The volume ratio of the oil phase to the aqueous phase was 1:4. The emulsification was performed at a magnetic stirrer speed of 2,000 rpm for 20 minutes. Then, 10% by mass of glycerol and 5% by mass of trehalose were added as cryoprotectants, accurately weighed using an electronic balance, added to a magnetic stirrer, and stirred at a stirring speed of 1100 r / min for 12 minutes to mix evenly with the embedding system. The embedded strains were then freeze-dried using a vacuum freeze dryer, with the pre-freezing temperature controlled at -45°C for 3 hours, the vacuum degree maintained at 20 Pa, and the drying time for 18 hours. At the same time, nanoparticles were prepared using nanotechnology. The nanoprecipitation method was used. A 15 mg / mL polyethylene glycol-polylactic acid PEG-PLA acetone solution was slowly added dropwise to stirred deionized water using a magnetic stirrer and a constant pressure dropping funnel. The volume ratio of acetone to deionized water was 1:8. A magnetic stirrer was used at a stirring speed of 750 r / min to prepare nanoparticles with a particle size of 50 nm. Subsequently, 3 mg / mL of coronatine and 4 mg / mL of brassinolide were loaded into the nanoparticles using an ultrasonic cell disruptor at 300 W of ultrasonic oscillation power for 20 minutes. After loading, the loading rate of the plant growth regulators was tested using high-performance liquid chromatography, with a requirement of no less than 85%. Finally, the dried microcapsule particles and nanoparticles loaded with plant growth regulators are mixed, and after adding buffers and antioxidants, they are packaged using smart packaging materials. The smart packaging material adopts a three-layer composite structure, with a PET film as the outer layer, an EVOH gas barrier layer as the middle layer, and a PE film as the inner layer. The inner layer surface is coated with a temperature and humidity sensing coating and an integrated gas detection sensor. The preparation is packaged using a packaging machine. The biological preparation is used to resist disease and increase yield of wheat crops in the agricultural field. A seed mixing machine is used for seed mixing before sowing, with a preparation to seed mass ratio of 1:300. A sprayer is used for foliar spraying during growth, with a dilution multiple of 1000 times. Irrigation equipment is used for root watering, with a dilution multiple of 800 times. Example

[0042] First, sterile sampling instruments were used to collect samples from hot springs at a temperature of 90°C and salt lakes with a salt concentration of 30%. Microbial strains were isolated on solid culture media using the dilution spread plate method and the streak separation method using sterile culture dishes, spreader rods, and inoculation loops. Morphological observations were performed using Gram stain and a microscope. Preliminary purification was carried out based on the growth characteristics of the strains and the standards for their antagonism to common plant pathogens. Strains with disease resistance and growth-promoting abilities were screened. Using the CRISPR-Cas9 gene editing system, an ultra-clean workbench, a PCR instrument, and electrophoresis equipment, sgRNAs with a PAM sequence of NGG targeting the promoter regions of the heat shock proteins HSP70 and HSP90 and antifreeze protein AFP genes were designed. The genomes of the screened strains were then site-specifically modified to overexpress the heat shock protein and antifreeze protein genes. Next, the gene-edited strain was microencapsulated using poly(lactic acid-co-glycolic acid) with a molecular weight of 50,000 Da and a lactic acid / glycolic acid molar ratio of 80:20. The emulsification-solvent evaporation method was performed using a magnetic stirrer and a separatory funnel. The oil phase was dichloromethane and the aqueous phase was an aqueous solution containing 1.5% polyvinyl alcohol. The volume ratio of the oil phase to the aqueous phase was 1:3. The emulsification was performed at a magnetic stirrer speed of 2,500 rpm for 25 minutes. Then, 12% by mass of glycerol and 6% by mass of trehalose were added as cryoprotectants, accurately weighed using an electronic balance, added to a magnetic stirrer, and stirred at a stirring speed of 1200 r / min for 15 minutes to mix evenly with the embedding system. The embedded strains were then freeze-dried using a vacuum freeze dryer, with the pre-freezing temperature controlled at -40°C, the pre-freezing time being 4 hours, the vacuum degree maintained at 25 Pa, and the drying time being 20 hours. At the same time, nanoparticles were prepared using nanotechnology. The nanoprecipitation method was used. A polyethylene glycol-polylactic acid PEG-PLA acetone solution with a mass concentration of 18 mg / mL was slowly added dropwise to stirred deionized water using a magnetic stirrer and a constant pressure dropping funnel. The volume ratio of acetone to deionized water was 1:7. A magnetic stirrer was used at a stirring speed of 800 r / min to prepare nanoparticles with a particle size of 60 nm. Subsequently, 4 mg / mL of coronatine and 5 mg / mL of brassinolide were loaded into the nanoparticles using an ultrasonic cell disruptor, using an ultrasonic oscillation power of 320 W for 22 minutes. After loading, the loading rate of the plant growth regulators was tested using high-performance liquid chromatography, requiring a loading rate of no less than 85%. Finally, the dried microcapsule particles and nanoparticles loaded with plant growth regulators are mixed, and after adding buffers and antioxidants, they are packaged using smart packaging materials. The smart packaging material adopts a three-layer composite structure, with a PET film as the outer layer, an EVOH gas barrier layer as the middle layer, and a PE film as the inner layer. The inner layer surface is coated with a temperature and humidity sensing coating and an integrated gas detection sensor. The preparation is packaged using a packaging machine. The biological preparation is used to resist disease and increase yield of corn crops in the agricultural field. A seed mixing machine is used for seed mixing before sowing, with a preparation to seed mass ratio of 1:200. A sprayer is used for foliar spraying during growth, with a dilution multiple of 800 times. Irrigation equipment is used for root watering, with a dilution multiple of 600 times. Comparative Example 1 differs from Example 1 in that no gene editing was performed, resulting in a strain survival rate of only 45% at a high temperature of 40°C, a decreased disease resistance effect in the field, and an insufficient yield increase rate; Comparative Example 2 differs from Example 1 in that there is no microcapsule embedding, resulting in a strain survival rate of only 38% after freeze-drying. After 3 months of storage, the degradation rate of the active ingredient increases and the antibacterial rate decreases. Comparative Example 3 differs from Example 2 in that no vacuum freeze-drying was performed. The strain survival rate was only 28%, the volatilization loss of coronatine was 60%, the antibacterial rate after foliar spraying was 60% lower than that of Example 2, and the yield increase was less than 12%. Comparative Example 4 is different from Example 2 in that the half-life of the regulator in the soil is only 2 days, the yield-promoting effect is reduced after root irrigation, and the antibacterial rate is reduced; Comparative Example 5 differs from Example 3 in that there is no intelligent packaging. As a result, after 6 months of storage, the oxygen concentration in the package increased from 0.5% to 12%, and the strain activity decreased by 45%; Comparative Example 6 differs from Example 3 in that there is no cryoprotectant, which causes the activity loss rate to be accelerated by 3 times during cold chain transportation, and cannot meet the application needs in northern winter.

[0043] Experimental Table 1 Experimental Table 2 Experimental Table 3 Data Description: Strain survival rate (-15°C): In Examples 1-3, the synergistic effects of gene editing + microencapsulation + cryoprotectant + vacuum freeze-drying resulted in strain survival rates exceeding 80% at -15°C. In Comparative Example 1 (no gene editing), the strain did not enhance antifreeze protein expression, resulting in a survival rate of only 45%. In Comparative Example 2 (no microencapsulation), the survival rate dropped to 38% due to direct cell damage from ice crystals due to the lack of a physical barrier. In Comparative Example 6 (no cryoprotectant), the survival rate was only 22% due to structural damage caused by freezing of intracellular water. Regulator loading rate: Examples 1-3 achieved loading rates of ≥85% by nanoprecipitation + ultrasonic loading. Comparative Example 4 (no nanoloading) directly mixed the regulator, which had poor dispersibility and easy degradation, resulting in a loading rate of only 50%. Comparative Example 3 (no vacuum freeze drying) experienced a 60% loss of the regulator due to volatilization due to high-temperature drying, resulting in a loading rate of only 40%. Activity retention after 6 months of storage: Examples 1-3 achieved activity retention rates >80% through gas barrier and temperature and humidity monitoring using smart packaging. Comparative Example 5 (without smart packaging) had an activity retention rate of only 55% due to oxygen permeation and environmental fluctuations. Comparative Example 2 (without microencapsulation) had an activity retention rate of only 55% due to the strain's easy inactivation. Disease resistance effect (inhibition rate): The inhibition rates of Examples 1-3 are greater than 80% due to the stress resistance of the gene-edited strains and the targeted inhibition of the nano-load regulator. The inhibition rates of Comparative Example 1 (without gene editing) and Comparative Example 4 (without nano-load) are less than 50% due to the weak disease resistance of the strains and the low utilization rate of the regulator, respectively.

[0044] Yield-promoting effect (yield increase rate): Examples 1-3 achieved a yield increase rate of >18% through efficient absorption of the nano-load regulator + strain metabolism to promote growth; Comparative Examples 1-2 and 4-6 had yield increases of <15% due to the lack of key processes (e.g., no gene editing, no nano-load).

[0045] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a novel disease-resistant and growth-promoting biological preparation, characterized in that: The following steps are involved: Screening and gene editing of microbial strains to overexpress heat shock protein and antifreeze protein genes; The gene-edited strains are microencapsulated and cryoprotectants are added; The embedded strains were vacuum freeze-dried; Using nanotechnology to prepare nanoparticles and load them with plant growth regulators; The dry microcapsule particles and nanoparticles loaded with plant growth regulators are mixed, and a buffer and an antioxidant are added before packaging using intelligent packaging materials.

2. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The screening of microbial strains is carried out by using a sterile sampling device to collect samples from hot springs with a temperature higher than 60°C, permafrost with a temperature lower than -15°C, salt lakes with a salt concentration higher than 20%, and acidic mine wastewater environments with a pH value lower than 3. The microbial strains are separated on a solid culture medium using a dilution coating plate method and a streak separation method using sterile culture dishes, coating rods, and inoculation loops. The morphology is observed using Gram stain and a microscope, and preliminary purification is carried out based on the growth characteristics of the strains and the standards of their antagonistic ability to common plant pathogens to screen out strains with disease resistance and growth promotion capabilities.

3. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The gene editing adopts the CRISPR-Cas9 gene editing system, uses an ultra-clean workbench, a PCR instrument, and an electrophoresis device to design sgRNA targeting the promoter region of the heat shock protein HSP70, HSP90 and antifreeze protein AFP genes, and the PAM sequence is NGG, to perform site-specific modification on the genome of the screening strain.

4. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The microcapsule embedding adopts polylactic acid-glycolic acid copolymer with a molecular weight of 10,000-50,000 Da and a lactic acid / glycolic acid molar ratio of 70-80:20-30, and is carried out by an emulsification-solvent evaporation method using a magnetic stirrer and a separatory funnel. The oil phase includes dichloromethane, and the water phase includes an aqueous solution containing 0.5% to 1.5% polyvinyl alcohol. The volume ratio of the oil phase to the water phase is 1:3-1:

5. The emulsification is carried out for 15 to 25 minutes at a rotation speed of 1,500 to 2,500 r / min using a magnetic stirrer.

5. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The cryoprotectant is 8% to 12% glycerol and 4% to 6% trehalose by mass, which are accurately weighed by an electronic balance and added to a magnetic stirrer. The magnetic stirrer is stirred at a stirring speed of 1000-1200 r / min for 10 to 15 minutes to mix evenly with the embedding system.

6. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The vacuum freeze drying process uses a vacuum freeze dryer, with the pre-freezing temperature controlled at -50°C to -40°C, the pre-freezing time being 2 to 4 hours, the vacuum degree maintained at 15 to 25 Pa, and the drying time being 15 to 20 hours.

7. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The nanoparticles are prepared by a nanoprecipitation method. A polyethylene glycol-polylactic acid PEG-PLA acetone solution with a mass concentration of 12 to 18 mg / mL is slowly added dropwise to stirred deionized water using a magnetic stirrer and a constant pressure dropping funnel. The volume ratio of acetone to deionized water is 1:7 to 1:

9. Nanoparticles with a particle size of 40 to 60 nanometers are prepared using a magnetic stirrer at a stirring speed of 700 to 800 r / min.

8. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The plant growth regulators, comprising coronatine at a concentration of 2 to 4 mg / mL and brassinolide at a concentration of 3 to 5 mg / mL, were loaded into the nanoparticles using an ultrasonic cell disruptor with an ultrasonic oscillation power of 280 to 320 W for 18 to 22 minutes. After loading, the loading rate of the plant growth regulators was tested using high-performance liquid chromatography, with a requirement that the loading rate be no less than 85%.

9. The method for preparing a novel disease-resistant and growth-promoting biological preparation according to claim 1, characterized in that: The smart packaging material adopts a three-layer composite structure, with an outer layer of PET film, a middle layer of EVOH gas barrier layer, and an inner layer of PE film. The inner layer surface is coated with a temperature and humidity sensing coating and integrated with a gas detection sensor, and a packaging machine is used for preparation packaging.

10. Application of a novel anti-disease and growth-promoting biological preparation, for use in a biological preparation prepared by the preparation method according to any one of claims 1 to 9, characterized in that: This biological agent is used in the agricultural field and can be used to resist diseases and increase yields of rice, wheat, and corn crops. It is mixed with seeds before sowing using a seed mixer with a mass ratio of 1:200-1:

400. It is sprayed on the leaves during the growth period using a sprayer with a dilution factor of 800-1200 times. It is used for root watering using irrigation equipment with a dilution factor of 600-1000 times.

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