Disease-preventing root-promoting microbial agent containing soybean phospholipid as well as preparation method and application of disease-preventing root-promoting microbial agent

Abstract

The invention discloses a soybean phospholipid disease-preventing and root-promoting microbial agent as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The microbial agent comprises a microbial composition, modified enzymolysis soybean phospholipid, a carrier and auxiliary components. According to the invention, the modified enzymolysis soybean phospholipid and microorganisms are combined to achieve a synergistic effect. According to the method, soybean phospholipid is modified and subjected to enzymolysis, and the steps of pretreatment, stepped low-temperature treatment, enzymolysis treatment, enzyme inactivation and purification and the like are carried out, so that the quality and performance of the soybean phospholipid are improved, the rhizosphere colonization rate of strains can be increased, the disease prevention and root promotion effects can be improved, the soil micro-ecological environment can be improved, and good growth conditions are created for plant root systems; and the disease-preventing and root-promoting effects of the microbial agent are further enhanced. The microbial agent disclosed by the invention can be used for remarkably reducing the morbidity of wheat sharp eyespot and reducing the use amount of chemical pesticides, can also be applied to planting of other crops, and can play a role in preventing and controlling various plant diseases and promoting root growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a soybean lecithin disease-preventing and root-promoting microbial agent, a preparation method and an application thereof. Background Art

[0002] Wheat sharp eyespot, also known as wheat sharp eyespot, is mainly caused by Rhizoctonia graminearum ( Rhizoctonia cerealis ) is a soil-borne fungal disease caused by infection with Rhizoctonia solani. In recent years, the incidence of wheat sheath blight has become increasingly serious, posing a major threat to wheat yield and quality (Xiao Qian, Yan Cuimei, Qi Yongzhi, et al., Research Progress on Chemical and Biological Control of Wheat Sheath Blight [J]. Pesticides, 2020, 59(9): 630.). Currently, the disease is mainly controlled by chemical means, but excessive use of chemical pesticides not only damages the environment, but also causes pathogens to develop drug resistance, and their residues are also harmful to human health. Therefore, there is an urgent need to develop natural and ecologically friendly biological pesticides to control wheat sheath blight.

[0003] As a biopesticide, microbial agents offer advantages such as environmental friendliness, safety, and sustainability, and are gradually becoming a research hotspot for plant disease control. For example, Chinese patent application CN202410855381.5 discloses a microbial composition for controlling wheat sheath blight, its preparation method, and its application. This composition, obtained by combining two specific Bacillus subtilis spore preparations, demonstrates significantly greater control over wheat sheath blight than single-bacterium spore preparations. Furthermore, when used in combination with chlorella, the combination achieves superior control over the full dose of chlorella, even with a halved dose. This achieves the goal of effectively controlling the disease while reducing pesticide use, making it suitable for widespread application.

[0004] For example, Chinese patent application CN202411153111.6 discloses Bacillus niger, metabolites, microbial agents, fertilizers and their applications. The Bacillus is named Bacillus sp. BCL1954, which has been deposited in the China Center for Type Culture Collection on July 12, 2024, with a deposit number of CCTCCM20241540. Bacillus, microbial agents and fertilizers have good effects in preventing and controlling plant diseases such as wheat stem rot, wheat fusarium rust, wheat sheath blight, tobacco black shank, and apple ring rot, solving the problem that existing biocontrol bacteria are not effective in preventing and controlling plant diseases.

[0005] However, the strains discovered so far have a single function and poor control effect. They also need to be compounded with chemical fungicides or have poor targeting. They do not have a particularly good control effect on the common wheat stripe blight, and their root colonization effect is poor. Therefore, it is difficult to exert a sustained and effective biocontrol effect. Summary of the Invention

[0006] The present invention addresses the problems of the existing technology by providing a soybean lecithin disease prevention and root promotion microbial agent, its preparation method, and application. This agent not only effectively prevents and treats wheat sheath blight but also promotes plant root growth, providing a new solution for plant disease prevention and growth promotion.

[0007] The microbial agent is applied to wheat planting and applied to the soil in a suitable dosage and manner, which can significantly reduce the incidence of wheat sheath blight and reduce the use of chemical pesticides. At the same time, the microbial combination and modified enzymatic soy lecithin contained therein work together to improve the soil microecological environment, create good growth conditions for plant roots, promote root growth and development, and improve wheat yield and quality. In addition, the microbial agent can also be applied to the planting of other crops and has broad application prospects in preventing and controlling various plant diseases and promoting root growth.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0009] Preferably, the microbial combination comprises Bacillus megaterium ( Bacillus megaterium ), Psychrotolerant Pseudomonas ( Pseudomonas psychrotolerans ) and Burkholderia bifida ( Burkholderia ambifaria ), wherein the Bacillus megaterium has a deposit number of CGMCC No. 27648 and a deposit date of June 16, 2023, deposited at the China National Center for Microbiological Culture Collection; the psychrotolerant Pseudomonas strain is numbered CGMCC No. 1.15631, and the bispecific Burkholderia strain is numbered CGMCC No. 1.10511. Both psychrotolerant Pseudomonas and bispecific Burkholderia were purchased from the China National Center for Microbiological Culture Collection. Both psychrotolerant Pseudomonas and bispecific Burkholderia are commercially available and do not require biological preservation.

[0010] The Bacillus megaterium was isolated from the rhizosphere soil of garlic sprouts in Lanling County, Linyi City, Shandong Province. The specific isolation method was as follows: 5 g of soil was weighed and added to a triangular flask containing 45 mL of sterile water, and the mixture was placed in a shaking incubator at 28°C and cultured at 200 r / min for 30 min; the supernatant was taken and diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5100 μL of each concentration was applied to LB medium. Incubated upside down in a 28°C incubator. After 3 days, a single colony was selected and shaken. After 24 hours, the colony was streaked and stored.

[0011] The soil samples were diluted 10 -5 When the soil bacteria are evenly distributed, it is easy to pick, so 10 -5 Pick bacteria from the plate and conduct antagonism test using Rhizoctonia graminearum as indicator bacteria Screening: The bacteria obtained in the early stage were evolved and activated, and the wheat sheath blight pathogen - Rhizoctonia graminearum was used as an indicator bacteria. The plate confrontation method was used to screen out bacteria with antibacterial effects. Four bacterial strains were symmetrically inoculated 2.5 cm around the mycelium block. After culturing for 2-3 days, the presence of a transparent inhibition zone was observed. If a transparent inhibition zone was present, it indicated that the bacteria had an antibacterial effect. A total of five strains were screened that inhibited Rhizoctonia graminearum. The bacteria with the best antibacterial effect were selected from them, which was the target strain Bacillus megaterium. The strain was purified in LB medium ( Figure 1 ) and preserve them. The colonies of the target strain in LB medium are round and opaque, shiny, slightly raised on the surface, with neat edges and easy to be picked up by the inoculation loop. It can produce pigments, which are white in the early stage and turn light yellow in the later stage ( Figure 2 ).

[0012] Preferably, the preparation method of the modified enzymatically hydrolyzed soybean lecithin is: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:(3-5), the pH was adjusted to 6.5-7.0, heated to 50-55°C and stirred at constant temperature for 30 minutes to form a uniform emulsion. (2) Step-by-step low-temperature treatment: The uniform emulsion is first frozen at -5°C for 1 hour, then cooled to -20°C at a rate of 3°C per hour, and maintained at this temperature for 3 hours. After that, the frozen emulsion is refrigerated at 4°C for 2 hours, and then thawed naturally at room temperature. Through this step-by-step low-temperature treatment, the molecular structure and physical properties of soybean lecithin can be changed, making it more conducive to the subsequent enzymatic hydrolysis reaction, thereby improving the quality and performance of the modified enzymatic hydrolyzed soybean lecithin, thereby enhancing the efficacy of the soybean lecithin disease prevention and root promotion microbial agent; (3) Enzymatic hydrolysis: add lipase to the thawed emulsion at a dosage of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6-8 hours; (4) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain modified enzymatically hydrolyzed soybean lecithin.

[0013] Preferably, the carrier is a mixture of attapulgite and vermiculite, with a mass ratio of attapulgite to vermiculite of 1:(1-2). Attapulgite has excellent adsorption and ion exchange properties, capable of absorbing the microbial composition and components such as modified enzymatically hydrolyzed soybean lecithin, providing a stable carrier environment. Vermiculite, with its loose and porous structure, helps maintain the inoculant's air permeability and water retention, promoting microbial growth and reproduction. This carrier combination can better utilize the properties of soybean lecithin-based disease prevention and root growth promotion microbial inoculant, improving its stability and effectiveness in the soil.

[0014] Preferably, the auxiliary ingredients are chitosan and sodium alginate, and the mass ratio of the two is 1:(0.5-1.5). Chitosan has good film-forming properties and biocompatibility, and can form a protective film on the surface of plant roots to reduce the invasion of pathogens. At the same time, it can also stimulate the plant to produce a defense response and enhance the plant's disease resistance. Sodium alginate is rich in various nutrients and bioactive substances, such as polysaccharides, amino acids, etc., which can provide nutrition for microorganisms, promote the growth and metabolism of microorganisms, and improve soil structure and increase the soil's water and fertilizer retention capacity. Adding chitosan and sodium alginate as auxiliary ingredients to soybean lecithin disease prevention and root promotion microbial inoculants can further enhance the disease prevention and root promotion effects of the inoculants, thereby improving plant growth quality and yield.

[0015] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of bacterial strains: Bacillus megaterium, Pseudomonas psychrotolerantus, and Burkholderia bispecificus were inoculated onto LB plates and cultured at 30°C for 48 hours; b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL seed culture medium. Cultivate with shaking at 30°C and 180 rpm for 30 hours to obtain three kinds of viable bacteria with a count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The three seed solutions were mixed in a volume ratio of 1:1:1, inoculated with 6% of the seed solution into a fermenter, cultured at 30°C, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm for 20 hours, and then 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added. The fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution. (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:(1-2), dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30-60 minutes to ensure that all components are fully mixed, and then let it stand for 3-5 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%-15%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0016] More preferably, the composition of the seed culture medium is as follows: per liter, it contains 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, with a pH value of 7.0-7.2, and the balance is sterile water, which is used after sterilization.

[0017] More preferably, the fermentation tank is filled with fermentation medium, and the composition of the fermentation medium is: 20g glucose, 15g yeast powder, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 5g calcium carbonate per liter, pH value is 7.0-7.2, and the balance is sterile water, which is used after sterilization.

[0018] More preferably, the effective viable count of the microbial solution obtained in step (c) is not less than 5×10 10 CFU / mL.

[0019] The usage of the solid bacterial agent of the present invention is: Before sowing wheat, the soybean lecithin disease prevention and root promotion type microbial agent of the present invention is evenly spread on the soil surface at a dosage of 10-15 kg per mu, and then the soil is plowed to fully mix the agent with the soil.

[0020] Beneficial effects: (1) The present invention screened out a strain of Bacillus megaterium that has a highly effective antagonistic effect on the pathogen of wheat blast. Bacillus megaterium (CGMCC No. 27648) secretes lipopeptide antimicrobial substances that directly destroy the cell membrane of Rhizoctonia graminearum. Pseudomonas psychrotrophica produces siderophores and other substances that competitively deprive the pathogen of iron. Burkholderia bifida can secrete a variety of extracellular enzymes, such as chitinase, which can decompose the cell wall of the pathogen. The screened Bacillus megaterium is used in combination with Pseudomonas psychrotrophica and Burkholderia bifida, and has a significant preventive and therapeutic effect on wheat sheath blight. At the same time, the three have the growth-promoting effects of decomposing insoluble phosphorus and secreting auxins. The three cooperate with each other to effectively colonize the roots of crops and continuously exert their growth-promoting effects.

[0021] (2) The present invention uses a modified enzymatically hydrolyzed soybean lecithin and a microbial combination to work synergistically. The modified enzymatically hydrolyzed soybean lecithin undergoes pretreatment, step-by-step low-temperature treatment, enzymatic hydrolysis, enzyme inactivation, and purification, thereby changing the molecular structure and physical properties of the soybean lecithin, making it more conducive to subsequent enzymatic hydrolysis reactions and improving its quality and performance. This modified enzymatically hydrolyzed soybean lecithin can increase the colonization rate of the strain in the rhizosphere. The modified enzymatically hydrolyzed soybean lecithin and the microbial combination cooperate with each other, not only improving the disease prevention and root promotion effect, but also improving the soil microecological environment, creating good growth conditions for plant roots, and further enhancing the disease prevention and root promotion effect of the microbial agent.

[0022] (4) The microbial agent of the present invention has broad application prospects in agricultural production. It can not only be used in wheat cultivation, significantly reducing the incidence of wheat sheath blight, reducing the use of chemical pesticides, and improving wheat yield and quality, but can also be applied to the cultivation of other crops, playing an important role in preventing and controlling various plant diseases and promoting root growth. Its environmentally friendly, safe, and sustainable characteristics meet the needs of modern agricultural development and provide a new solution for plant disease prevention and control and growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The diagrams are the growth state diagram of Bacillus megaterium in LB medium and the inhibitory effect diagram on Rhizoctonia graminearum, wherein (a) is the growth state diagram and (b) is the inhibitory effect diagram; Figure 2 The colony morphology and Gram staining of Bacillus megaterium of the present invention; Figure 3 This is a diagram showing the antagonistic effects of the present invention on Bacillus megaterium, Pseudomonas psychrotolerant, and Burkholderia bispecifica. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0025] Example 1 A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0026] The microbial combination comprises Bacillus megaterium, psychrotolerant Pseudomonas and bispecific Burkholderia, wherein the deposit number of Bacillus megaterium is CGMCC No. 27648, the deposit date is June 16, 2023, and it is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms; the strain number of the psychrotolerant Pseudomonas is CGMCC No. 1.15631, and the strain number of the bispecific Burkholderia is CGMCC No. 1.10511. Both psychrotolerant Pseudomonas and bispecific Burkholderia are purchased from the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms. Psychrotolerant Pseudomonas and bispecific Burkholderia are both commercially available and do not require biological preservation.

[0027] The Bacillus megaterium was isolated from the rhizosphere soil of garlic sprouts in Lanling County, Linyi City, Shandong Province. The specific isolation method was as follows: 5 g of soil was weighed and added to a triangular flask containing 45 mL of sterile water, and the mixture was placed in a shaking incubator at 28°C and cultured at 200 r / min for 30 min; the supernatant was taken and diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 100 μL of each concentration was applied to LB medium. Incubated upside down in a 28°C incubator. After 3 days, a single colony was selected and shaken. After 24 hours, the colony was streaked and stored.

[0028] The soil samples were diluted 10 -5 When the soil bacteria are evenly distributed, it is easy to pick, so 10 -5 Pick bacteria from the plate and conduct antagonism test using Rhizoctonia graminearum as indicator bacteria Screening: The bacteria obtained in the early stage were evolved and activated, and the wheat sheath blight pathogen - Rhizoctonia graminearum was used as an indicator bacteria. The plate confrontation method was used to screen out bacteria with antibacterial effects. Four bacterial strains were symmetrically inoculated 2.5 cm around the mycelium block. After culturing for 2-3 days, the presence of a transparent inhibition zone was observed. If a transparent inhibition zone was present, it indicated that the bacteria had an antibacterial effect. A total of five strains were screened that inhibited Rhizoctonia graminearum. The bacteria with the best antibacterial effect were selected from them, which was the target strain Bacillus megaterium. The strain was purified in LB medium ( Figure 1 ) and preserve them. The colonies of the target strain in LB medium are round and opaque, shiny, slightly raised on the surface, with neat edges and easy to be picked up by the inoculation loop. It can produce pigments, which are white in the early stage and turn light yellow in the later stage ( Figure 2 ).

[0029] The preparation method of the modified enzymatically hydrolyzed soybean lecithin is as follows: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:5, the pH was adjusted to 6.5-7.0, and the mixture was heated to 50-55°C and stirred at a constant temperature for 30 minutes to form a uniform emulsion. (2) Stepwise low-temperature treatment: the homogeneous emulsion was first frozen at -5°C for 1 hour, then cooled to -20°C at a rate of 3°C per hour and maintained at this temperature for 3 hours. Afterwards, the frozen emulsion was refrigerated at 4°C for 2 hours and then thawed naturally at room temperature. (3) Enzymatic hydrolysis: add lipase to the thawed emulsion at a dosage of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6 hours; (4) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain modified enzymatically hydrolyzed soybean lecithin.

[0030] The carrier is a mixture of attapulgite and vermiculite, wherein the mass ratio of attapulgite to vermiculite is 1:1.

[0031] The auxiliary components are chitosan and sodium alginate, and the mass ratio of the two is 1:0.5.

[0032] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of bacterial strains: Bacillus megaterium, Pseudomonas psychrotolerantus, and Burkholderia bispecificus were inoculated onto LB plates and cultured at 30°C for 48 hours; b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL seed culture medium. Cultivate with shaking at 30°C and 180 rpm for 30 hours to obtain three kinds of viable bacteria with a count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The three seed solutions were mixed in a volume ratio of 1:1:1, inoculated with 6% of the seed solution into a fermenter, cultured at 30°C, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm for 20 hours, and then 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added. The fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution. (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:1, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all components are fully mixed, and then let it stand for 3 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0033] The seed culture medium is composed of 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride per liter, with a pH value of 7.0-7.2 and the balance being sterile water, which is used after sterilization.

[0034] The fermentation tank is filled with fermentation medium. The composition of the fermentation medium is as follows: 20g glucose, 15g yeast powder, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 5g calcium carbonate per liter, pH value is 7.0-7.2, and the balance is sterile water, which is used after sterilization.

[0035] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0036] Determination of antagonism between strains of complex bacterial flora: Bacillus megaterium, Pseudomonas psychrotolerantus and Burkholderia bispecificus were cultured in pairs on LB plates by cross-streaking. The culture status was observed. The culture results were as follows: Figure 3 As shown in the figure, the three strains of bacteria can grow normally on the LB plate, and the bacterial colony at the intersection of the colonies grows normally without inhibition. Therefore, it can be determined that there is no antagonistic effect between the three strains of bacteria and they can be compounded as a bacterial agent for use.

[0037] Example 2 A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0038] The microbial combination comprises Bacillus megaterium, psychrotolerant Pseudomonas and bispecific Burkholderia, wherein the deposit number of Bacillus megaterium is CGMCC No. 27648, the deposit date is June 16, 2023, and it is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms; the strain number of the psychrotolerant Pseudomonas is CGMCC No. 1.15631, and the strain number of the bispecific Burkholderia is CGMCC No. 1.10511. Both psychrotolerant Pseudomonas and bispecific Burkholderia are purchased from the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms. Psychrotolerant Pseudomonas and bispecific Burkholderia are both commercially available and do not require biological preservation.

[0039] The Bacillus megaterium isolation and screening method is the same as that in Example 1.

[0040] The preparation method of the modified enzymatically hydrolyzed soybean lecithin is as follows: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:3, the pH was adjusted to 6.5-7.0, heated to 50-55°C and stirred at constant temperature for 30 minutes to form a uniform emulsion: (2) Stepwise low-temperature treatment: the homogeneous emulsion was first frozen at -5°C for 1 hour, then cooled to -20°C at a rate of 3°C per hour and maintained at this temperature for 3 hours. Afterwards, the frozen emulsion was refrigerated at 4°C for 2 hours and then thawed naturally at room temperature. (3) Enzymatic hydrolysis: add lipase to the thawed emulsion at a dosage of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6-8 hours; (4) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain modified enzymatically hydrolyzed soybean lecithin.

[0041] The carrier is a mixture of attapulgite and vermiculite, wherein the mass ratio of attapulgite to vermiculite is 1:2.

[0042] The auxiliary components are chitosan and sodium alginate, and the mass ratio of the two is 1:1.5.

[0043] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of bacterial strains: Bacillus megaterium, Pseudomonas psychrotolerantus, and Burkholderia bispecificus were inoculated onto LB plates and cultured at 30°C for 48 hours; b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL seed culture medium. Cultivate with shaking at 30°C and 180 rpm for 30 hours to obtain three kinds of viable bacteria with a count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The three seed solutions were mixed in a volume ratio of 1:1:1, inoculated with 6% of the seed solution into a fermenter, cultured at 30°C, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm for 20 hours, and then 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added. The fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution. (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:2, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30-60 minutes to ensure that all components are fully mixed, and then let it stand for 3-5 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 15%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0044] The composition of the seed culture medium is the same as that of Example 1.

[0045] The fermentation tank is filled with fermentation medium, and the composition of the fermentation medium is the same as that in Example 1.

[0046] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0047] Comparative Example 1 In this comparative example, except that only Bacillus megaterium was used in the microbial combination, the remaining raw materials and process steps were the same as those in Example 1. A soybean lecithin disease prevention and root promotion type microbial agent comprises microorganisms, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0048] The microorganism is Bacillus megaterium, with a preservation number of CGMCC No. 27648 and a preservation date of June 16, 2023, and is deposited in the General Microbiology Center of the China Culture Collection Administration.

[0049] The Bacillus megaterium isolation and screening method is the same as that in Example 1.

[0050] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of the strain: Inoculate Bacillus megaterium onto an LB plate and incubate at 30°C for 48 hours; b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL of seed culture medium. Culture at 30°C and 180 rpm for 30 hours to obtain a viable count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The seed liquid was inoculated into a fermenter at a rate of 6%, cultured at 30°C, aeration 1.0 vvm, and a stirring speed of 200 rpm for 20 hours, and 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added. The fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution; (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:1, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all components are fully mixed, and then let it stand for 3 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0051] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0052] Comparative Example 2 In this comparative example, except that only psychrotolerant Pseudomonas was used in the microbial combination, the remaining raw materials and process steps were the same as those in Example 1. A soybean lecithin disease prevention and root promotion type microbial agent comprises microorganisms, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0053] The microorganism is psychrotolerant Pseudomonas, the strain number of which is CGMCC No. 1.15631, and is purchased from the General Microbiology Center of the China Culture Collection Administration of Microorganisms. Psychrotolerant Pseudomonas is commercially available and does not require biological preservation.

[0054] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of the strain: Inoculate the psychrotrophic Pseudomonas aeruginosa onto an LB plate and incubate at 30°C for 48 hours. b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL of seed culture medium. Culture at 30°C and 180 rpm for 30 hours to obtain a viable count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The seed liquid was inoculated into a fermenter at a rate of 6%, cultured at 30°C, aeration 1.0 vvm, and a stirring speed of 200 rpm for 20 hours, and 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added. The fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution; (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:1, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all components are fully mixed, and then let it stand for 3 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0055] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0056] Comparative Example 3 In this comparative example, except that only Burkholderia bifida was used in the microbial combination, the rest of the raw materials and process steps were the same as those in Example 1. A soybean lecithin disease prevention and root promotion type microbial agent comprises microorganisms, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0057] The microorganism is Burkholderia bifida, with a strain number of CGMCC No. 1.10511, purchased from the General Microbiology Center of the China Culture Collection Administration of Microorganisms. Burkholderia bifida is commercially available and does not require biological preservation.

[0058] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of the strain: Inoculate the bispecific Burkholderia species onto LB plates and incubate at 30°C for 48 hours. b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL of seed culture medium. Culture at 30°C and 180 rpm for 30 hours to obtain a viable count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The seed liquid was inoculated into a fermenter at a rate of 6%, cultured at 30°C, aeration 1.0 vvm, and a stirring speed of 200 rpm for 20 hours, and 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added. The fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution; (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:1, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all components are fully mixed, and then let it stand for 3 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0059] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0060] Comparative Example 4 In this comparative example, except that only Bacillus megaterium and psychrotrophic Pseudomonas were used in the microbial combination, the remaining raw materials and process steps were the same as those in Example 1. A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0061] The microbial combination includes Bacillus megaterium and psychrotolerant Pseudomonas, wherein the Bacillus megaterium has a deposit number of CGMCC No. 27648 and a deposit date of June 16, 2023, deposited at the General Microbiology Center of the China Culture Collection Administration of Microorganisms; the psychrotolerant Pseudomonas strain number is CGMCC No. 1.15631, and psychrotolerant Pseudomonas was purchased from the General Microbiology Center of the China Culture Collection Administration of Microorganisms. Psychrotolerant Pseudomonas is commercially available and does not require biological preservation.

[0062] The Bacillus megaterium isolation and screening method is the same as that in Example 1.

[0063] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Bacterial activation: Bacillus megaterium and Pseudomonas psychrotolerant were inoculated onto LB plates and cultured at 30°C for 48 hours. b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL of seed culture medium. Culture at 30°C and 180 rpm for 30 hours to obtain two kinds of viable bacteria with a count of 1×10 9CFU / mL of seed solution; c. Mixed fermentation: The seed liquid of the two bacteria was mixed in a volume ratio of 1:1, and the inoculum was added to the fermenter at a 6% inoculum. The culture was carried out at 30°C, aeration volume of 1.0 vvm, and stirring speed of 200 rpm for 20 hours. 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added, and the fermentation was continued at 30°C for 24 hours to obtain a microbial culture liquid; (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:1, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all components are fully mixed, and then let it stand for 3 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0064] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0065] Comparative Example 5 In this comparative example, except that only Bacillus megaterium and Burkholderia bifida were used in the microbial combination, the remaining raw materials and process steps were the same as those in Example 1. That is: A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0066] The microbial combination includes Bacillus megaterium and Burkholderia bifida. Bacillus megaterium has a deposit number of CGMCC No. 27648 and a deposit date of June 16, 2023, deposited at the China National Center for the Administration of Culture Collection of Microorganisms and Microorganisms. The Burkholderia bifida has a strain number of CGMCC No. 1.10511 and was purchased from the China National Center for the Administration of Culture Collection of Microorganisms and Microorganisms. Burkholderia bifida is commercially available and does not require biological preservation.

[0067] The Bacillus megaterium isolation and screening method is the same as that in Example 1.

[0068] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of bacterial strains: Bacillus megaterium and Burkholderia bifida were inoculated onto LB plates and cultured at 30°C for 48 hours; b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL of seed culture medium. Culture at 30°C and 180 rpm for 30 hours to obtain two kinds of viable bacteria with a count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The seed liquid of the two bacteria was mixed in a volume ratio of 1:1, and the inoculum was added to the fermenter at a 6% inoculum. The culture was carried out at 30°C, aeration volume of 1.0 vvm, and stirring speed of 200 rpm for 20 hours. 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added, and the fermentation was continued at 30°C for 24 hours to obtain a microbial culture liquid; (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:1, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all components are fully mixed, and then let it stand for 3 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0069] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0070] Comparative Example 6 In this comparative example, except that only psychrotolerant Pseudomonas and bispecific Burkholderia were used in the microbial combination, the remaining raw materials and process steps were the same as those in Example 1. A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0071] The microbial combination includes psychrotolerant Pseudomonas and bispecific Burkholderia. The psychrotolerant Pseudomonas strain is numbered CGMCC No. 1.15631, and the bispecific Burkholderia strain is numbered CGMCC No. 1.10511. Both psychrotolerant Pseudomonas and bispecific Burkholderia were purchased from the General Microbiology Center of the China Culture Collection Administration. Both psychrotolerant Pseudomonas and bispecific Burkholderia are commercially available and do not require biological preservation.

[0072] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial agent comprises the following steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of bacterial strains: Bacillus megaterium and Burkholderia bifida were inoculated onto LB plates and cultured at 30°C for 48 hours; b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL of seed culture medium. Culture at 30°C and 180 rpm for 30 hours to obtain two kinds of viable bacteria with a count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The seed liquid of the two bacteria was mixed in a volume ratio of 1:1, and the inoculum was added to the fermenter at a 6% inoculum. The culture was carried out at 30°C, aeration volume of 1.0 vvm, and stirring speed of 200 rpm for 20 hours. 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added, and the fermentation was continued at 30°C for 24 hours to obtain a microbial culture liquid; (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:1, dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all components are fully mixed, and then let it stand for 3 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

[0073] The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

[0074] Comparative Example 7 In this comparative example, except that the step-by-step low-temperature treatment is not performed, the remaining raw materials and process steps are the same as those of Example 1. A soybean lecithin disease prevention and root promotion type microbial agent comprises a microbial combination, enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0075] The preparation method of the enzymatically hydrolyzed soybean lecithin is as follows: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:5, the pH was adjusted to 6.5-7.0, and the mixture was heated to 50-55°C and stirred at a constant temperature for 30 minutes to form a uniform emulsion. (2) Enzymatic hydrolysis: lipase was added to the emulsion at a dosage of 1.5 U / g soybean lecithin, and the reaction was carried out at a constant temperature of 25°C and pH 7.0 for 6 h. (3) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain enzymatically hydrolyzed soybean lecithin.

[0076] Comparative Example 8 In this comparative example, except for the low-temperature treatment at a single temperature, the rest of the raw materials and process steps are the same as those in Example 1. A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0077] The preparation method of the modified enzymatically hydrolyzed soybean lecithin is as follows: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:5, the pH was adjusted to 6.5-7.0, and the mixture was heated to 50-55°C and stirred at a constant temperature for 30 minutes to form a uniform emulsion. (2) Low temperature treatment: freeze the homogeneous emulsion at -5°C for 6 hours and then thaw naturally at room temperature; (3) Enzymatic hydrolysis: add lipase to the thawed emulsion at a dosage of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6 hours; (4) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain modified enzymatically hydrolyzed soybean lecithin.

[0078] Comparative Example 9 In this comparative example, except for the low-temperature treatment at a single temperature, the rest of the raw materials and process steps are the same as those in Example 1. A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0079] The preparation method of the modified enzymatically hydrolyzed soybean lecithin is as follows: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:5, the pH was adjusted to 6.5-7.0, and the mixture was heated to 50-55°C and stirred at a constant temperature for 30 minutes to form a uniform emulsion. (2) Low temperature treatment: freeze the homogeneous emulsion at -20°C for 6 hours and then thaw naturally at room temperature; (3) Enzymatic hydrolysis: add lipase to the thawed emulsion at a dosage of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6 hours; (4) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain modified enzymatically hydrolyzed soybean lecithin.

[0080] Comparative Example 10 In this comparative example, except for the low-temperature treatment at a single temperature, the rest of the raw materials and process steps are the same as those in Example 1. A soybean lecithin disease prevention and root promotion microbial agent comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

[0081] The preparation method of the modified enzymatically hydrolyzed soybean lecithin is as follows: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:5, the pH was adjusted to 6.5-7.0, and the mixture was heated to 50-55°C and stirred at a constant temperature for 30 minutes to form a uniform emulsion. (2) Low temperature treatment: refrigerate the homogeneous emulsion at 4°C for 6 hours and then thaw naturally at room temperature; (3) Enzymatic hydrolysis: add lipase to the thawed emulsion at a dosage of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6 hours; (4) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain modified enzymatically hydrolyzed soybean lecithin.

[0082] Performance Testing To test the antibacterial properties of Bacillus megaterium, psychrotrophic Pseudomonas aeruginosa, and Burkholderia bifida strains, the target strains were activated and cultured at 30°C, 200 rpm, and then set aside. A sterile filter paper disc (Ø = 6 mm) was placed 2.5 cm from the center of the culture plate containing the pathogen Rhizoctonia graminearum. 3 µL of culture medium was added to each disc. The plates were incubated in a 30°C incubator. After control mycelium had grown to full size, the diameter of the inhibition zone was measured and the inhibition rate was calculated. Mycelial morphology was observed, and normal mycelium was used as a control. The experiment was repeated three times. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%. The results are shown in Table 1: Table 1 Inhibitory effect of strains on Rhizoctonia graminearum From the data in Table 1, we can see that Bacillus megaterium, Pseudomonas psychrotolerant and Burkholderia bispecific all have good inhibitory effects on Rhizoctonia graminearum.

[0083] Growth promotion ability test of Bacillus megaterium, psychrotrophic Pseudomonas and bidirectional Burkholderia: Determination of the strain's ability to solubilize insoluble phosphate: Single colonies of the test strain were picked and activated overnight. They were inoculated into an inorganic phosphate culture medium containing 0.5% insoluble phosphate (calcium phosphate) at an inoculum size of 2.0% (V / V). The uninoculated experimental group was used as a control. The culture was shaken at 30°C and 180 rpm. After 24 hours, 5.0 mL of the fermentation sample was collected and the fermentation supernatant was collected by centrifugation at 10,000 rpm for 5 minutes. Three replicates were set for each treatment. The soluble phosphorus content in the fermentation supernatant was determined according to the above-mentioned national standard method GB 11893-89-Molybdenum Antimony Antigen Colorimetric Method.

[0084] The inorganic phosphorus culture medium is composed of: NaCl 0.3g, KCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O0.03, MnSO4·H2O 0.03, calcium phosphate 5.0 g, sterilized at 121℃ for 30min.

[0085] Determination of IAA production capacity of strains: A single colony of the strain to be tested was picked and activated overnight. The inoculum size was inoculated into a 500-mL conical flask containing 200 mL of LB medium (containing 100 mg / L L-tryptophan) at a 2.0% (v / v) inoculum. The culture was shaken at 30°C and 180 rpm. The uninoculated experimental group was used as the control. Samples were taken at 0, 10, 15, 20, 24, 34, 44, and 56 h, respectively. The supernatant was centrifuged at 10,000 rpm for 8 min, and the IAA content in the supernatant was calculated according to the IAA standard curve. Each treatment was repeated 3 times.

[0086] Table 2 Test of growth-promoting ability of strains Root colonization ability test of fermentation liquid composed of different strains: The microbial culture solution was prepared according to the method of Example 1 and Comparative Examples 1-10 to verify that different fermentation liquid compositions have different customization capabilities for the microbial rhizosphere. The preparation method of the microbial culture solution is as follows: Preparation of microbial culture solution: a. Bacterial activation: The strains to be tested in Example or Comparative Example were inoculated on LB plates and cultured at 30°C for 48 hours; b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL of seed culture medium. Cultivate with shaking at 30°C and 180 rpm for 30 hours to obtain a viable bacterial count of 1×109 CFU / mL of seed solution; c. Mixed fermentation: The seed liquid was inoculated into the fermentation tank at a rate of 6%, and cultured at 30°C, with a ventilation volume of 1.0 vvm and a stirring speed of 200 rpm for 20 hours. Modified enzymatically hydrolyzed soybean lecithin (or enzymatically hydrolyzed soybean lecithin) at a mass ratio of 5.0% was added, and the fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution.

[0087] Under greenhouse conditions, the white wheat seeds were soaked in the prepared test microbial solution for 3 hours, and sown in the nutrient body filled with sterile soil. The experiment was repeated 3 times. The investigation began 7 days after the wheat seedlings emerged, and the test was carried out every 5 days. The culture was completed after 25 days to test the number of root colonization bacteria. Three pots of wheat seedlings were taken for testing each time. When testing, the roots of the wheat seedlings were first rinsed clean, blown dry in a clean bench, and the roots were cut off and weighed. The roots were placed in a 2ml centrifuge tube with magnetic beads, and ground with a frozen grinder. After removal, centrifugation was performed, and the supernatant was taken. 100μ of tissue fluid was applied to each resistance plate, and colonies with the same colony morphology were selected for counting. Bacterial quantity (cfu·g -1 ) = (number of colonies × dilution factor × volume of sterile water required for suspension) / (amount of bacterial suspension applied to the plate × fresh weight of tissue used for separation).

[0088] Table 3 Colonization ability of different microbial solutions on roots As can be seen from the data in Table 3, there are differences in the ability of fermentation broths composed of different strains to colonize wheat roots. The microbial culture prepared in the example has a relatively high amount of colonized bacteria in the roots, which may be due to the synergistic effect of its microbial combination and the addition of modified enzymatically hydrolyzed soybean lecithin, which helps the strains to better colonize and grow in the roots. In the various comparative examples, the colonization ability of the microbial culture in the roots is different due to factors such as changes in the microbial combination, the lack of specific treatment (such as step-by-step low-temperature treatment), or the use of a single temperature low-temperature treatment. Comparative Examples 1-6 changed the strain composition, and their colonization bacteria amount was lower than that of Example 1. It is speculated that the synergistic balance between the three bacteria was broken, affecting the colonization effect. Comparative Example 7 did not undergo step-by-step low-temperature treatment, which may have caused the structure and properties of the modified enzymatically hydrolyzed soybean lecithin to be different from those in Example 1, thereby affecting the colonization of microorganisms in the roots. Comparative Examples 8-10, each treated with a single temperature freezing treatment, showed varying colonization abilities. This may be due to the weak modification of the soy lecithin structure by the single temperature low-temperature treatment, which in turn affected the subsequent enzymatic hydrolysis and weakened the promoting effect on the colonization ability of the microbial solution. Overall, factors such as the microbial combination and the preparation process of the modified enzymatic hydrolyzed soy lecithin have a significant impact on the colonization ability of the microbial solution on plant roots.

[0089] Field control effect: Before sowing wheat, the test microbial agent was evenly spread on the soil surface at a rate of 10 kg per mu, and then the soil was plowed to fully mix the microbial agent with the soil.

[0090] Field plot area: 15m 2 Each experimental group was replicated three times in a randomized block arrangement. The control agent was 27% difenothiocarb suspension seed coating agent (Coolas), 200 mL per 100 kg of seeds, produced by Syngenta Nantong Crop Protection Co., Ltd.

[0091] The fertility of the test plot is average for the local area, and wheat sheath blight has been severe over the years. The soil type is fluvo-aquic soil with a medium soil texture. The previous crop was corn. Conventional water and fertilizer management was followed. Investigations into wheat sheath blight began at the milky stage. A five-point sampling method was used in the plot, with 20 plants randomly selected at each point. The sheath blight status was investigated in accordance with the "Guidelines for Pesticide Field Efficacy Tests." The specific grading standards are as follows: Level 0: no symptoms on the leaf sheaths and stems; Level 1: leaf sheath disease, or the width of the lesions on the stem is less than 1 / 4 of the stem circumference; Level 2: The width of the lesion on the stem accounts for 1 / 4 to 1 / 2 of the stem circumference; Level 3: The width of the lesion on the stem accounts for 1 / 2 to 3 / 4 of the stem circumference; Level 4: The width of the lesion on the stem accounts for more than 3 / 4 of the stem circumference; Level 5: diseased plants die prematurely, with dry ears or dry white ears.

[0092] Disease index = ∑ (number of plants at each disease level × disease level) = (total number of plants × highest disease level) × 100; Relative control effect = [(disease index of control area - disease index of each treatment) = disease index of control area] × 100; Root morphological characteristics: During the wheat heading period, the “excavation method” was used for sampling. Soil sampling areas were randomly selected from each experimental plot. The area of the soil sampling area was 900 cm 2 (30 cm long × 30 cm wide) Then, different soil layer samples were taken from each sampling area. The sample type was root samples in the 0-20 cm soil layer. The sample volume was 18000 cm 3 Each layer of samples was cleaned using a mesh bag washing method and then placed in a fresh-keeping bag for refrigeration. To determine the morphological characteristics of wheat roots, the samples were scanned using an EPSONE U-88 scanner, and WinRHIZO system software was used to analyze wheat root characteristic parameters, including total root length and root dry weight. Yield was measured at wheat maturity. Complete sampling points were randomly selected from each experimental plot, with a sampling area of 6 m2. 2, threshing and weighing, then using a grain moisture meter to measure the moisture content of the wheat, calculate the actual yield of wheat in the field, and calculate it according to the national grain storage safety moisture content standard of 13%. Table 4 Field experiment results The data in Table 4 show that the microbial agents treated differently exhibit significant differences in field efficacy, root morphology, and wheat yield. In terms of field efficacy, the microbial agents prepared in the Examples demonstrated excellent control of wheat sheath blight, with a high relative efficacy. This may be due to the synergistic effect of the microbial combination and the addition of modified enzymatically hydrolyzed soybean lecithin, which effectively inhibits the growth and infection of the pathogen. However, the control efficacy of the comparative examples was reduced due to factors such as changes in the microbial combination, the lack of specific treatment, or the use of a single, low-temperature treatment.

[0093] In terms of root morphology, wheat treated with the Examples showed superior root length and root dry weight compared to the Comparative Example. This indicates that the microbial agents of the Examples help promote the growth and development of wheat roots, enhancing the root absorption and anchoring capacity, thereby providing better support for plant growth.

[0094] In terms of wheat yield, the actual yield of wheat fields treated with the Example was significantly higher than that of the Control Example. This further demonstrates the effectiveness of the Example microbial agent in preventing disease and promoting root growth, ultimately achieving an increase in wheat yield by improving plant health and root function.

[0095] It should be noted that the above embodiments are only some of the preferred embodiments of the present invention, and not all of them. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

Claims

1. A soybean lecithin disease prevention and root promotion microbial agent, characterized in that The invention comprises a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier and auxiliary components.

2. The soybean lecithin disease prevention and root promotion microbial agent according to claim 1, characterized in that The microbial combination comprises Bacillus megaterium ( Bacillus megaterium ), Psychrotolerant Pseudomonas ( Pseudomonas psychrotolerans ) and Burkholderia bifida ( Burkholderia ambifaria ), wherein the deposit number of Bacillus megaterium is CGMCC No.27648, the deposit date is June 16, 2023, and it is deposited in the General Microbiology Center of China Culture Collection Administration Committee; the strain number of the psychrotrophic Pseudomonas is CGMCC No.1.15631, and the strain number of the biphasic Burkholderia is CGMCC No. 1.10511. Both psychrotrophic Pseudomonas and biphasic Burkholderia were purchased from the General Microbiology Center of China Culture Collection Administration Committee.

3. The soybean lecithin disease prevention and root promotion microbial agent according to claim 1, characterized in that The preparation method of the modified enzymatically hydrolyzed soybean lecithin is as follows: (1) Pretreatment: Soybean lecithin and deionized water were mixed in a mass ratio of 1:(3-5), the pH was adjusted to 6.5-7.0, heated to 50-55°C and stirred at constant temperature for 30 minutes to form a uniform emulsion. (2) Stepwise low-temperature treatment: the homogeneous emulsion was first frozen at -5°C for 1 hour, then cooled to -20°C at a rate of 3°C per hour and maintained at this temperature for 3 hours. Afterwards, the frozen emulsion was refrigerated at 4°C for 2 hours and then thawed naturally at room temperature. (3) Enzymatic hydrolysis: add lipase to the thawed emulsion at a dosage of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6-8 hours; (4) Enzyme inactivation and purification: Inactivate the enzyme in a 20°C water bath for 30 min, centrifuge and ultrafilter to collect small molecular components <500 Da to obtain modified enzymatically hydrolyzed soybean lecithin.

4. The soybean lecithin disease prevention and root promotion microbial agent according to claim 1, characterized in that The carrier is a mixture of attapulgite and vermiculite, wherein the mass ratio of attapulgite to vermiculite is 1:(1-2).

5. The soybean lecithin disease prevention and root promotion microbial agent according to claim 1, characterized in that The auxiliary components are chitosan and sodium alginate, and the mass ratio of the two is 1: (0.5-1.5).

6. A method for preparing the soybean lecithin disease-preventing and root-promoting microbial agent according to any one of claims 1 to 5, characterized in that: The method comprises the following preparation steps: (1) Preparation of modified enzymatically hydrolyzed soybean lecithin; (2) Preparation of microbial culture solution: a. Activation of bacterial strains: Bacillus megaterium, Pseudomonas psychrotolerantus, and Burkholderia bispecificus were inoculated onto LB plates and cultured at 30°C for 48 hours. b. Seed liquid culture: Pick a single colony and inoculate it into 50 mL seed culture medium. Cultivate with shaking at 30°C and 180 rpm for 30 hours to obtain three kinds of viable bacteria with a count of 1×10 9 CFU / mL of seed solution; c. Mixed fermentation: The three seed solutions were mixed in a volume ratio of 1:1:1, inoculated with 6% of the seed solution into a fermenter, cultured at 30°C, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm for 20 hours, and then 5.0% of the system weight of modified enzymatically hydrolyzed soybean lecithin was added. The fermentation was continued at 30°C for 24 hours to obtain a microbial culture solution. (3) Carrier treatment: Attapulgite and vermiculite were mixed in a mass ratio of 1:(1-2), dried at 120-150 °C for 2-3 hours to remove moisture and impurities, and then the dried mixture was crushed and passed through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size; (4) Mixing: After mixing the carrier powder and auxiliary components at a mass ratio of 10:0.3, add them to the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30-60 minutes to ensure that all components are fully mixed, and then let it stand for 3-5 hours; (5) Drying and molding: The mixed material obtained in step (4) is dried to reduce the moisture content to 10%-15%, and then the dried material is granulated to prepare a microbial agent product that is easy to store and use.

7. The method for preparing the soybean lecithin disease-preventing and root-promoting microbial agent according to claim 6, characterized in that: The seed culture medium is composed of 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride per liter, with a pH value of 7.0-7.2 and the balance being sterile water, which is used after sterilization.

8. The method for preparing the soybean lecithin disease-preventing and root-promoting microbial agent according to claim 6, characterized in that: The fermentation tank is filled with fermentation medium. The composition of the fermentation medium is as follows: 20g glucose, 15g yeast powder, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 5g calcium carbonate per liter, pH value is 7.0-7.2, and the balance is sterile water, which is used after sterilization.

9. The method for preparing the soybean lecithin disease-preventing and root-promoting microbial agent according to claim 6, characterized in that: The effective viable count of the microbial culture obtained in step (c) is not less than 5×10 10 CFU / mL.

10. A use of the soybean lecithin disease prevention and root promotion microbial agent according to any one of claims 1 to 5, characterized in that: Used to prevent and control wheat stripe blight.

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