A soybean lecithin-based disease-preventing and root-promoting microbial agent, its preparation method and application

Abstract

This invention discloses a soybean lecithin-based microbial agent for disease prevention and root promotion, its preparation method, and its application, belonging to the field of microbial technology. The agent comprises a microbial ensemble, modified enzymatically hydrolyzed soybean lecithin, a carrier, and auxiliary components. This invention utilizes the synergistic effect of modified enzymatically hydrolyzed soybean lecithin and the microbial ensemble. The modified enzymatically hydrolyzed soybean lecithin undergoes pretreatment, stepwise low-temperature treatment, enzymatic hydrolysis, and enzyme inactivation and purification steps, improving its quality and performance. This enhances the colonization rate of the strain in the rhizosphere, improving not only disease prevention and root promotion effects but also improving the soil microecological environment, creating favorable growth conditions for plant roots, and further enhancing the disease prevention and root promotion effects of the agent. The agent of this invention significantly reduces the incidence of wheat sheath blight, reduces the use of chemical pesticides, and can also be applied to the cultivation of other crops, playing a role in the prevention and control of various plant diseases and promoting root growth.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a soybean lecithin disease-preventing and root-promoting microbial agent, its preparation method, and its application. Background Technology

[0002] Wheat sheath blight (wheat sharp eyespot), also known as wheat pointed eye disease, is mainly caused by Rhizoctonia graminearum (… Rhizoctonia cerealis Wheat sheath blight is a soil-borne fungal disease caused by infection. In recent years, the severity of wheat sheath blight has gradually increased, posing a significant 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, chemical control is the main method for this disease, but the overuse of chemical pesticides not only damages the environment but also leads to drug resistance in pathogens, and their residues are also detrimental to human health. Therefore, it is urgent to develop natural and environmentally friendly biological pesticides to control wheat sheath blight.

[0003] Microbial inoculants, as a type of biological pesticide, have advantages such as environmental friendliness, safety, and sustainability, and are gradually becoming a research hotspot in 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 microbial composition is obtained by specifically compounding two specific Bacillus subtilis spore preparations. The control effect on wheat sheath blight is significantly higher than that of single-spore preparations. Furthermore, when used in conjunction with Clonax, this composition achieves a better control effect than when Clonax is applied at half the dosage, thus achieving the goal of effectively controlling disease occurrence under conditions of reduced pesticide application, making it suitable for widespread application.

[0004] For example, Chinese patent application CN202411153111.6 discloses Bacillus niger, its metabolites, microbial agents, fertilizers, and their applications. The Bacillus niger is named Bacillus sp. BCL1954, which has been deposited at the China Center for Type Culture Collection on July 12, 2024, with accession number CCTCCM20241540. The Bacillus niger, microbial agents, and fertilizers have good effects in controlling plant diseases such as wheat stem base rot, wheat scab, wheat sheath blight, tobacco black shank, and apple ring rot, solving the problem of poor efficacy of existing biocontrol bacteria in controlling plant diseases.

[0005] However, the strains discovered so far have limited functions and poor control effects, requiring the addition of chemical fungicides. They also lack specificity and do not have a particularly good control effect on wheat sheath blight, which is prevalent in wheat. Furthermore, they do not colonize well in the roots, making it difficult to exert a sustained and effective biocontrol effect. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by providing a soybean lecithin-based microbial agent for disease prevention and root promotion, along with its preparation method and application. This agent not only effectively controls wheat sheath blight but also promotes plant root growth, offering a new solution for plant disease control and growth promotion.

[0007] When applied to wheat cultivation, this microbial agent, when administered to the soil at appropriate dosages and in appropriate methods, can significantly reduce the incidence of wheat sheath blight and decrease the use of chemical pesticides. Simultaneously, its microbial composition and modified enzymatically hydrolyzed soybean lecithin components synergistically improve the soil microecological environment, creating favorable growth conditions for plant roots, promoting root growth and development, and increasing wheat yield and quality. Furthermore, this microbial agent can also be applied to the cultivation of other crops, showing broad application prospects in the prevention and control of various plant diseases and the promotion of root growth.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0009] A soybean lecithin disease-preventing and root-promoting microbial inoculant includes a microbial combination, modified enzymatically hydrolyzed soybean lecithin, a carrier, and auxiliary components.

[0010] Preferably, the microbial ensemble comprises Bacillus megaterium (Betaminaria). Bacillus megaterium ), cold-resistant Pseudomonas ( Pseudomonas psychrotolerans ) and Burkholderia bifidum ( Burkholderia ambifaria The *Bacillus megaterium* was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 16, 2023, with accession number CGMCC No. 27648. The *Pseudomonas psychrophilus* strain number was CGMCC No. 1.15631, and the *Burkholderia bifidum* strain number was CGMCC No. 1.10511. Both *Pseudomonas psychrophilus* and *Burkholderia bifidum* were purchased from the CGMCC. Both *Pseudomonas psychrophilus* and *Burkholderia bifidum* are commercially available and do not require biopreservation.

[0011] The *Bacillus megaterium* was isolated from the rhizosphere soil of garlic scapes in Lanling County, Linyi City, Shandong Province. The specific isolation method was as follows: 5g of soil was weighed and added to an Erlenmeyer flask containing 45mL of sterile water. The flask was placed in a shaker at 28℃ and incubated at 200 r / min for 30min. The supernatant was then diluted to 10... -2 10 -3 10 -4 10 -5Each concentration was taken 100 μL to LB medium for coating. Inverted culture in 28℃ incubator, after 3d, single colony was selected for shaking culture, after 24h, streaked and preserved.

[0012] The soil sample was diluted 10 -5 times, the soil bacteria were evenly distributed, which was convenient for picking, so 10 -5 plates were selected for picking bacteria, and antagonistic test was carried out with Rhizoctonia cerealis as indicator bacteria

[0013] Screening: the bacteria obtained in the previous stage were activated, and Rhizoctonia cerealis, the pathogen of wheat sharp eyespot, was used as an indicator to screen bacteria with inhibitory effect by plate confrontation method. Four strains of bacteria were inoculated symmetrically at a distance of 2.5 cm from the mycelial block, and cultured for 2-3 days. If there was a transparent inhibition zone, it indicated that the bacteria had inhibitory effect. Five strains of bacteria were screened which inhibited Rhizoctonia cerealis, and the best one, Bacillus megaterium, was selected as the target strain. The strain was purified in LB medium ( Figure 1 ) and preserved. The target strain was round and opaque in LB medium, with luster, slightly raised surface, neat edge, and easy to be picked up by inoculation ring. It could produce pigment, which was white at the beginning and yellowish at the later stage. Figure 2 .

[0014] Preferably, the preparation method of the modified enzymatic soybean phospholipid is as follows:

[0015] (1) Pretreatment: soybean phospholipid and deionized water are mixed in a mass ratio of 1: (3-5), the pH is adjusted to 6.5-7.0, heated to 50-55℃ and stirred at constant temperature for 30 min to form a uniform emulsion:

[0016] (2) Stepwise low temperature treatment: the above uniform emulsion is first frozen at-5℃ for 1 hour, then reduced to-20℃ at a rate of 3℃ per hour, and kept at this temperature for 3 hours. Then, the frozen emulsion is stored at 4℃ for 2 hours, and then thawed at room temperature; through this stepwise low temperature treatment, the molecular structure and physical properties of soybean phospholipid can be changed, which is more conducive to the subsequent enzymatic reaction, so as to improve the quality and performance of the modified enzymatic soybean phospholipid, thereby enhancing the efficacy of the soybean phospholipid disease-preventing and root-promoting microbial agent;

[0017] (3) Enzymatic hydrolysis: lipase is added to the thawed emulsion, the amount of addition is 1.5 U / g of soybean phospholipid, and the reaction is carried out at 25℃ and pH 7.0 for 6-8 hours;

[0018] (4) Enzyme inactivation and purification: enzyme inactivation is carried out in a water bath at 20℃ for 30 min, then centrifugal treatment and ultrafiltration are carried out, and the <500 Da small molecule components are collected to obtain the modified enzymatic soybean phospholipid.

[0019] Preferably, the carrier is a mixture of attapulgite and vermiculite, wherein the mass ratio of attapulgite to vermiculite is 1: (1-2). Attapulgite has good adsorption and ion exchange properties, can adsorb microbial combinations and modified enzymatic soybean phospholipids and other ingredients, and provides a stable carrier environment for them; vermiculite has a loose and porous structure, which helps to maintain the air permeability and water retention of the microbial agent, and promotes the growth and reproduction of microorganisms. This carrier combination can better exert the performance of the soybean phospholipid disease-preventing and root-promoting microbial agent, and improve its stability and effectiveness in the soil.

[0020] Preferably, the auxiliary ingredient is chitosan and sodium alginate, and the mass ratio of the two is 1: (0.5-1.5). Chitosan has good film-forming property and biocompatibility, can form a protective film on the surface of plant roots, reduce the infection of pathogenic bacteria, and stimulate the plant to produce a defense response, thereby enhancing the disease resistance of the plant. Sodium alginate is rich in various nutritional ingredients and bioactive substances, such as polysaccharides, amino acids, etc., which can provide nutrition for microorganisms, promote the growth and metabolism of microorganisms, and also improve the soil structure, increase the water and fertilizer retention capacity of the soil. The addition of chitosan and sodium alginate as auxiliary ingredients in the soybean phospholipid disease-preventing and root-promoting microbial agent can further improve the disease-preventing and root-promoting effect of the microbial agent, and improve the growth quality and yield of the plant.

[0021] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent, comprising the following preparation steps:

[0022] (1) preparing modified enzymatic soybean phospholipids;

[0023] (2) microbial liquid preparation:

[0024] a. strain activation: Bacillus megaterium, Pseudomonas psychrophila and Borkholderia bipapillosa were inoculated on LB plates and cultured at 30°C for 48 hours;

[0025] b. seed liquid culture: single colonies were picked and inoculated into 50mL seed culture medium, and cultured at 30°C with 180rpm shaking for 30 hours, to obtain three seed liquids with a viable bacterial count of 1×10 9 CFU / mL;

[0026] c. mixed fermentation: the three seed liquids were mixed in a volume ratio of 1:1:1, inoculated into a fermentation tank at a 6% inoculation amount, and cultured at 30°C with a gas flow of 1.0vvm and a stirring speed of 200rpm for 20 hours. Then, 5.0% modified enzymatic soybean phospholipids were added, and the fermentation was continued at 30°C for 24 hours to obtain a microbial liquid;

[0027] (3) carrier treatment: the attapulgite and the vermiculite are mixed in a mass ratio of 1: (1-2), dried at 120-150 DEG C for 2-3 hours to remove water and impurities, and then the dried mixture is crushed and sieved through a 100-200 mesh screen to obtain carrier powder with uniform particle size;

[0028] (4) mixing: the carrier powder and the auxiliary ingredients are mixed in a mass ratio of 10:0.3, then the microbial combined bacteria liquid is added according to a solid-liquid ratio of 1g:50mL, stirred at a speed of 100-120r / min at room temperature for 30-60 minutes to ensure that the components are fully mixed and uniform, and then left to stand for 3-5h;

[0029] (5) drying and molding: the mixture 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 for easy storage and use.

[0030] More preferably, the composition of the seed culture medium is: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride per liter, pH 7.0-7.2, and the balance is sterile water, which is used after sterilization.

[0031] More preferably, the fermentation tank is filled with a fermentation medium, and the composition of the fermentation medium is: 20g of glucose, 15g of yeast powder, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 5g of calcium carbonate per liter, pH 7.0-7.2, and the balance is sterile water, which is used after sterilization.

[0032] More preferably, the microbial bacteria liquid obtained in step (c) has an effective viable bacterial count of not less than 5x10 10 CFU / mL.

[0033] The solid microbial agent is used as follows:

[0034] Before sowing wheat, the soybean phospholipid disease-preventing and root-promoting microbial agent of the application is uniformly applied to the soil surface at a dosage of 10-15kg per mu, and then ploughed to mix the microbial agent with the soil.

[0035] Beneficial effects:

[0036] (1) The present application screens a bacillus megaterium with high antagonistic effect on the pathogen of wheat rice blast, the bacillus megaterium (CGMCC No. 27648) secretes lipopeptide antibacterial substances to directly destroy the cell membrane of rhynchosporium, and the pseudomonas putida produces siderophores to compete for iron elements of the pathogen, and the burkholderia bipapilata can secrete various extracellular enzymes such as chitinase to decompose the cell wall of the pathogen. The bacillus megaterium obtained by screening is combined with the pseudomonas putida and the burkholderia bipapilata, and has a significant control effect on wheat sharp eyespot. Meanwhile, the three have the effects of decomposing insoluble phosphorus and secreting auxin to promote growth, and can effectively colonize the crop roots and continuously exert the growth promotion effect.

[0037] (2) The present application adopts modified enzymatic soybean phospholipid combined with microorganisms to synergistically act. The modified enzymatic soybean phospholipid changes the molecular structure and physical properties of soybean phospholipid through pretreatment, stepwise low-temperature treatment, enzymatic hydrolysis treatment, enzyme inactivation and purification, so that it is more conducive to subsequent enzymatic reaction, and the quality and performance are improved. The modified enzymatic soybean phospholipid can improve the rhizosphere colonization rate of the strain, and the modified enzymatic soybean phospholipid combined with microorganisms can not only improve the disease prevention and root promotion effect, but also improve the soil microecological environment, create good growth conditions for plant roots, and further enhance the disease prevention and root promotion effect of the microbial agent.

[0038] (4) The microbial agent of the present application has a wide application prospect in agricultural production. It can not only be used for wheat planting to significantly reduce the incidence of wheat sharp eyespot, reduce the use amount of chemical pesticides, and improve the yield and quality of wheat, but also be applied to the planting of other crops, and play an important role in preventing and controlling various plant diseases and promoting root growth. Its environmental protection, safety and sustainability meet the needs of modern agricultural development, and provide a new solution for plant disease prevention and growth promotion. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is the growth state diagram of the bacillus megaterium of the present application in LB medium and the inhibition effect diagram of the bacillus megaterium on rhynchosporium, wherein (a) is the growth state diagram, and (b) is the inhibition effect diagram;

[0040] Figure 2 It is the colony morphology and gram staining of the bacillus megaterium of the present application;

[0041] Figure 3 It is the antagonistic effect diagram of the bacillus megaterium, the pseudomonas putida and the burkholderia bipapilata of the present application. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be further described below in combination with specific embodiments, but are not limited thereto.

[0043] Example 1

[0044] A soybean phospholipid disease-preventing and root-promoting microbial agent comprises a microbial combination, modified enzymatic soybean phospholipid, a carrier and auxiliary ingredients.

[0045] The microbial combination comprises Bacillus megaterium, Pseudomonas psychrophila and Burkholderia bipalis, wherein the Bacillus megaterium has a preservation number of CGMCC No. 27648 and was preserved on June 16, 2023 at the China General Microbiological Culture Collection Center; the Pseudomonas psychrophila has a strain number of CGMCC No. 1.15631, and the Burkholderia bipalis has a strain number of CGMCC No. 1.10511, and both the Pseudomonas psychrophila and the Burkholderia bipalis are purchased from the China General Microbiological Culture Collection Center. Both the Pseudomonas psychrophila and the Burkholderia bipalis are commercially available and do not need to be biologically preserved.

[0046] The Bacillus megaterium is isolated from the rhizosphere soil of garlic sprouts in Lanning County, Linyi City, Shandong Province, and the specific isolation method is as follows: 5 g of soil is weighed and added to a triangular flask containing 45 mL of sterile water, which is placed in a 28°C shaking incubator and shaken at 200 r / min for 30 min; the supernatant is diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 100 μL of each concentration is taken and spread on LB medium. Invert culture in a 28°C incubator, and after 3 days, select single colonies for shaking culture, and after 24 h, streak and store.

[0047] The soil sample is diluted 10 -5 times, and the soil bacteria are evenly distributed, which facilitates picking, so 10 -5 plates are selected for picking bacteria, and antagonistic test is performed using S. sylvaticum as an indicator

[0048] Screening: The bacteria obtained in the early stage are activated, and S. sylvaticum, a wheat brown leaf spot pathogen, is used as an indicator to screen bacteria with inhibitory effect by plate confrontation method. Four strains of bacteria are inoculated symmetrically at a distance of 2.5 cm from the mycelial block, and after 2-3 days of culture, it is observed whether there is a transparent inhibition zone. If there is a transparent inhibition zone, it indicates that the bacteria have inhibitory effect, and a total of 5 strains of bacteria are screened to inhibit S. sylvaticum, and the best bacteria with inhibitory effect are selected as the target strain Bacillus megaterium. The strain is purified in LB medium ( Figure 1 ), and is stored. The target strain has a round and opaque colony in LB medium, with a slight gloss, a slightly raised surface, a neat edge, and is easy to pick up with a loop. It can produce pigment, which is white initially and turns yellowish later ( Figure 2 ).

[0049] The method for preparing the modified enzymatically hydrolyzed soybean lecithin is as follows:

[0050] (1) Pretreatment: Mix soybean lecithin and deionized water at a mass ratio of 1:5, adjust the pH to 6.5-7.0, heat to 50-55℃ and stir at a constant temperature for 30 minutes to form a uniform emulsion.

[0051] (2) Step-by-step low temperature treatment: The above uniform emulsion is first frozen at -5°C for 1 hour, then lowered to -20°C at a rate of 3°C per hour, and held 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.

[0052] (3) Enzymatic hydrolysis: Add lipase to the thawed emulsion at a rate of 1.5 U / g soybean lecithin, and react at a constant temperature of 25°C and pH 7.0 for 6 hours with shaking.

[0053] (4) Enzyme inactivation and purification: Inactivate enzymes by water bath at 20℃ for 30 min, centrifuge and then ultrafilter, collect small molecule components <500Da to obtain modified enzymatically hydrolyzed soybean lecithin.

[0054] The carrier is a mixture of attapulgite and vermiculite, wherein the mass ratio of attapulgite to vermiculite is 1:1.

[0055] The auxiliary components are chitosan and sodium alginate, with a mass ratio of 1:0.5.

[0056] A method for preparing a soybean lecithin disease-preventing and root-promoting microbial inoculant includes the following preparation steps:

[0057] (1) Preparation of modified enzymatically hydrolyzed soybean lecithin;

[0058] (2) Preparation of microbial inoculum:

[0059] a. Strain activation: Bacillus megaterium, cold-resistant Pseudomonas aeruginosa, and Burkholderia bifidum were inoculated onto LB plates and incubated at 30°C for 48 hours.

[0060] b. Seed culture: Single colonies were picked and inoculated into 50 mL of seed culture medium, and cultured at 30 °C and 180 rpm for 30 hours with shaking, yielding viable bacterial counts of 1 × 10⁻³ for each of the three types of cultures. 9 CFU / mL seed culture;

[0061] c. Mixed fermentation: Mix the three seed liquids in a volume ratio of 1:1:1, inoculate them into the fermentation tank at a 6% inoculum, and culture at 30°C, aeration rate of 1.0 vvm, and stirring speed of 200 rpm for 20 hours. Add 5.0% of the modified enzymatic hydrolysed soybean lecithin by mass of the system, and continue fermentation at 30°C for 24 hours to obtain the microbial inoculum.

[0062] (3) carrier treatment: mix the attapulgite and the vermiculite at a mass ratio of 1:1, dry at 120-150°C for 2-3 hours to remove water and impurities, then crush the dried mixture through a 100-200 mesh sieve to obtain carrier powder with uniform particle size;

[0063] (4) mixing: mix the carrier powder and the auxiliary ingredients at a mass ratio of 10:0.3, then add the microbial combined bacteria liquid according to a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that the ingredients are fully mixed and uniform, then stand for 3h;

[0064] (5) drying and molding: dry the mixture obtained in step (4) to reduce the moisture content to 10%, then granulate the dried material to prepare a bacterial agent product for easy storage and use.

[0065] The composition of the seed culture medium is: 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride per liter, with a pH value of 7.0-7.2, and the rest being sterile water, which is used after sterilization.

[0066] The fermentation medium in the fermenter has the following composition: 20g of glucose, 15g of yeast powder, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, and 5g of calcium carbonate per liter, with a pH value of 7.0-7.2, and the rest being sterile water, which is used after sterilization.

[0067] The microbial bacteria liquid obtained in step (c) has an effective viable bacterial count of not less than 5×10 10 CFU / mL.

[0068] Determination of antagonism between strains of the complex microbial population:

[0069] The three single bacteria, Bacillus megaterium, Pseudomonas psychrophila, and Burkholderia bipapillosa, were cross-streaked on LB plates, and the culture state was observed. The culture results are shown in Table 1, and the three single bacteria can grow normally on the LB plates, with normal growth of the microbial population at the intersection of the colonies, without inhibition, so it can be determined that the three single bacteria do not have antagonistic effects on each other and can be compounded into a bacterial agent. Figure 3

[0070] Example 2

[0071] A soybean phospholipid disease-preventing and root-promoting microbial agent includes a microbial combination, modified enzymatic soybean phospholipid, a carrier, and auxiliary ingredients.

[0072] ​The microbial combination comprises Bacillus megaterium, Pseudomonas psychrophila and Burkholderia biphenylia, wherein the preservation number of Bacillus megaterium is CGMCC No. 27648, the preservation date is June 16, 2023, and the preservation is in the China General Microbiological Culture Collection Center; the strain number of Pseudomonas psychrophila is CGMCC No. 1.15631, and the strain number of Burkholderia biphenylia is CGMCC No. 1.10511, and Pseudomonas psychrophila and Burkholderia biphenylia are purchased from the China General Microbiological Culture Collection Center. Pseudomonas psychrophila and Burkholderia biphenylia are commercially available and do not need to be biologically preserved.

[0073] The isolation and screening method of the Bacillus megaterium is the same as that in Embodiment 1.

[0074] The preparation method of the modified enzymatic soybean phospholipid is as follows:

[0075] (1) Pretreatment: soybean phospholipid and deionized water are mixed at a mass ratio of 1:3, the pH is adjusted to 6.5-7.0, heated to 50-55℃ and constant temperature stirring for 30 min to form a uniform emulsion:

[0076] (2) Stepwise low temperature treatment: the above uniform emulsion is first frozen at -5℃ for 1 hour, then reduced to -20℃ at a speed of 3℃ per hour, and kept at this temperature for 3 hours, then the frozen emulsion is refrigerated at 4℃ for 2 hours, and then naturally thawed at room temperature;

[0077] (3) Enzymatic hydrolysis: add lipase to the thawed emulsion, the amount is 1.5 U / g of soybean phospholipid, constant temperature oscillation reaction at 25℃ and pH 7.0 for 6-8 hours;

[0078] (4) Enzyme inactivation and purification: enzyme inactivation in 20℃ water bath for 30 min, centrifugal treatment, ultrafiltration, collection of <500 Da small molecule components, and obtain modified enzymatic soybean phospholipid.

[0079] The carrier is a mixture of attapulgite and vermiculite, wherein the mass ratio of attapulgite to vermiculite is 1:2.

[0080] The auxiliary ingredient is chitosan and sodium alginate, and the mass ratio of the two is 1:1.5.

[0081] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent, comprising the following preparation steps:

[0082] (1) Preparation of modified enzymatic soybean phospholipid;

[0083] (2) Preparation of microbial liquid:

[0084] a. Strain activation: Bacillus megaterium, Pseudomonas psychrophila and Borkholderia bipapilata were inoculated on LB plates and cultured at 30°C for 48 hours;

[0085] b. Seed liquid culture: single colonies were picked and inoculated into 50 mL seed culture medium, and cultured at 30°C and 180 rpm for 30 hours to obtain three seed liquids with viable cell counts of 1×10 9 CFU / mL;

[0086] c. Mixed fermentation: the three seed liquids were mixed at a volume ratio of 1:1:1, inoculated into a fermenter at a 6% inoculation amount, and cultured at 30°C with aeration of 1.0 VVM and stirring speed of 200 rpm for 20 hours. Then, 5.0% modified enzymatic soybean phospholipid was added to the system, and the fermentation was continued at 30°C for 24 hours to obtain a microbial liquid.

[0087] (3) Carrier treatment: attapulgite and vermiculite were mixed at a mass ratio of 1:2, dried at 120-150°C for 2-3 hours to remove water and impurities, then the dried mixture was crushed and sieved through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size;

[0088] (4) Mixing: the carrier powder and auxiliary ingredients were mixed at a mass ratio of 10:0.3, then the microbial combined liquid was added according to a solid-liquid ratio of 1 g:50 mL, stirred at a speed of 100-120 r / min at room temperature for 30-60 minutes to ensure that the ingredients were fully mixed and uniform, and then allowed to stand for 3-5 hours;

[0089] (5) Drying and molding: the mixture obtained in step (4) was dried to reduce the moisture content to 15%, and then the dried material was granulated to produce a microbial agent product that is easy to store and use.

[0090] The composition of the seed culture medium is the same as in Example 1.

[0091] The fermentation medium was loaded into the fermenter, and the composition of the fermentation medium was the same as in Example 1.

[0092] The microbial liquid obtained in step (c) had an effective viable cell count of not less than 5×10 10 CFU / mL.

[0093] Comparative Example 1

[0094] 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 in Example 1. That is:

[0095] A soybean phospholipid disease-preventing and root-promoting microbial agent includes microorganisms, modified enzymatic soybean phospholipid, carriers, and auxiliary ingredients.

[0096] The microorganism is Bacillus megaterium, the preservation number is CGMCC No. 27648, and the preservation date is June 16, 2023, preserved in the China General Microbiological Culture Collection Center.

[0097] The Bacillus megaterium isolation and screening method is the same as that of Example 1.

[0098] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent, comprising the following preparation steps:

[0099] (1) Preparation of modified enzymatic soybean phospholipid;

[0100] (2) Microbial liquid preparation:

[0101] a. Strain activation: inoculate Bacillus megaterium on LB plates and culture at 30°C for 48 hours;

[0102] b. Seed liquid culture: pick single colonies and inoculate into 50 mL seed culture medium, and culture at 30°C, 180 rpm for 30 hours to obtain seed liquid with a viable cell count of 1×10 9 CFU / mL;

[0103] c. Mixed fermentation: inoculate the seed liquid into a fermenter at a 6% inoculation amount, and culture at 30°C, 1.0vvm aeration, and 200 rpm stirring speed for 20 hours, then add 5.0% modified enzymatic soybean phospholipid to the system, and continue to ferment at 30°C for 24 hours to obtain the microbial liquid;

[0104] (3) Carrier treatment: mix attapulgite and vermiculite at a mass ratio of 1:1, dry at 120-150°C for 2-3 hours to remove water and impurities, then crush the dried mixture and pass through a 100-200 mesh sieve to obtain carrier powder with uniform particle size;

[0105] (4) Mixing: mix the carrier powder and auxiliary ingredients at a mass ratio of 10:0.3, then add the microbial combined liquid according to a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min at room temperature for 30 minutes to ensure that the ingredients are fully mixed and uniform, and then stand for 3h;

[0106] (5) Drying and molding: dry the mixture obtained in step (4) to reduce the moisture content to 10%, then granulate the dried material to prepare an agent product that is easy to store and use.

[0107] The microbial liquid obtained in step (c) has an effective viable cell count of not less than 5×10 10 CFU / mL.

[0108] Comparative Example 2

[0109] The comparative example, in addition to using only the cold-tolerant Pseudomonas in the microbial combination, the remaining raw materials and process steps are the same as Example 1. That is:

[0110] A soybean phospholipid disease-preventing and root-promoting microbial agent includes microorganisms, modified enzymatic hydrolysis soybean phospholipids, carriers, and auxiliary ingredients.

[0111] The microorganism is cold-tolerant Pseudomonas, and the strain number of the cold-tolerant Pseudomonas is CGMCC No. 1.15631. The cold-tolerant Pseudomonas is purchased from the China General Microbiological Culture Collection Center. The cold-tolerant Pseudomonas is commercially available and does not need to be biologically preserved.

[0112] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent includes the following preparation steps:

[0113] (1) Preparation of modified enzymatic hydrolysis soybean phospholipids;

[0114] (2) Microbial liquid preparation:

[0115] a. Strain activation: inoculate the cold-tolerant Pseudomonas on an LB plate and culture at 30°C for 48 hours;

[0116] b. Seed liquid culture: pick single colonies and inoculate into 50 mL seed culture medium, and culture at 30°C with 180 rpm shaking for 30 hours to obtain seed liquid with a viable cell count of 1×10 9 CFU / mL;

[0117] c. Mixed fermentation: inoculate the seed liquid into a fermenter at a 6% inoculation amount, and culture at 30°C with aeration of 1.0 VVM and stirring speed of 200 rpm for 20 hours. Add 5.0% modified enzymatic hydrolysis soybean phospholipids to the system, and continue to ferment at 30°C for 24 hours to obtain the microbial liquid;

[0118] (3) Carrier treatment: mix the attapulgite and vermiculite at a mass ratio of 1:1, dry at 120-150°C for 2-3 hours to remove water and impurities, then crush the dried mixture and pass through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size;

[0119] (4) Mixing: mix the carrier powder and auxiliary ingredients at a mass ratio of 10:0.3, then add 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 the ingredients are fully mixed and uniform, and then stand for 3h;

[0120] (5) Drying and molding: dry the mixture obtained in step (4) to reduce the moisture content to 10%, then granulate the dried material to prepare a microbial agent product that is easy to store and use.

[0121] The microbial liquid obtained in step (c) has an effective viable cell count of not less than 5 x 10 10 CFU / mL.

[0122] Comparative Example 3

[0123] In this comparative example, the raw materials and process steps are the same as in Example 1, except that only B. ambifaria is used in the microbial combination.

[0124] A soybean phospholipid disease-preventing and root-promoting microbial agent includes microorganisms, modified enzymatic hydrolysis soybean phospholipids, carriers, and auxiliary ingredients.

[0125] The microorganism is B. ambifaria, with strain number CGMCC No. 1.10511, which is commercially available from the China General Microbiological Culture Collection Center. B. ambifaria is commercially available and does not need to be biologically preserved.

[0126] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent includes the following preparation steps:

[0127] (1) Preparation of modified enzymatic hydrolysis soybean phospholipids;

[0128] (2) Preparation of microbial liquid:

[0129] a. Strain activation: inoculate B. ambifaria on an LB plate and culture at 30°C for 48 hours;

[0130] b. Seed liquid culture: pick single colonies and inoculate into 50 mL seed culture medium, and culture at 30°C with 180 rpm shaking for 30 hours to obtain seed liquid with a viable cell count of 1 x 10 9 CFU / mL;

[0131] c. Mixed fermentation: inoculate the seed liquid into a fermenter at a 6% inoculation amount, and culture at 30°C with 1.0vvm aeration and 200 rpm stirring for 20 hours. Then add 5.0% modified enzymatic hydrolysis soybean phospholipids to the system, and continue to ferment at 30°C for 24 hours to obtain the microbial liquid;

[0132] (3) Carrier treatment: mix attapulgite and vermiculite at a mass ratio of 1:1, dry at 120-150°C for 2-3 hours to remove water and impurities, then crush the dried mixture and pass through a 100-200 mesh sieve to obtain carrier powder with uniform particle size;

[0133] (4) Mixing: mix the carrier powder and auxiliary ingredients at a mass ratio of 10:0.3, then add the microbial combination liquid at a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min for 30 minutes at room temperature to ensure that the ingredients are fully mixed and uniform, and then stand for 3h;

[0134] (5) Drying and molding: the mixture 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 for easy storage and use.

[0135] The microbial solution obtained in step (c) has an effective viable cell count of not less than 5 x 10 10 CFU / mL.

[0136] Comparative Example 4

[0137] In this comparative example, the raw materials and process steps are the same as in Example 1, except that only Bacillus megaterium and Pseudomonas putida are used in the microbial combination.

[0138] A soybean phospholipid disease-preventing and root-promoting microbial agent includes a microbial combination, modified enzymatic soybean phospholipid, a carrier, and auxiliary ingredients.

[0139] The microbial combination includes Bacillus megaterium and Pseudomonas putida, wherein the Bacillus megaterium has a preservation number of CGMCC No. 27648 and was preserved on June 16, 2023 at the China General Microbiological Culture Collection Center; the Pseudomonas putida has a strain number of CGMCC No. 1.15631 and was purchased from the China General Microbiological Culture Collection Center. The Pseudomonas putida is commercially available and does not need to be biologically preserved.

[0140] The isolation and screening method of the Bacillus megaterium is the same as in Example 1.

[0141] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent includes the following preparation steps:

[0142] (1) Preparation of modified enzymatic soybean phospholipid;

[0143] (2) Preparation of microbial solution:

[0144] a. Strain activation: Bacillus megaterium and Pseudomonas putida were inoculated on LB plates and cultured at 30°C for 48 hours;

[0145] b. Seed liquid culture: single colonies were picked and inoculated into 50 mL seed culture medium, and cultured at 30°C with 180 rpm shaking for 30 hours to obtain two seed liquids with a viable cell count of 1 x 10 9 CFU / mL, respectively;

[0146] c. Mixed fermentation: the seed liquids of the two bacteria were mixed at a volume ratio of 1:1, inoculated into the fermenter at a 6% inoculation amount, cultured at 30°C with a ventilation amount of 1.0 VVM and a stirring speed of 200 rpm for 20 hours, 5.0% modified enzymatic soybean phospholipid was added to the system, and the fermentation was continued at 30°C for 24 hours to obtain a microbial liquid;

[0147] (3) Carrier treatment: mix attapulgite and vermiculite at a mass ratio of 1:1, dry at 120-150°C for 2-3 hours to remove water and impurities, then crush the dried mixture and pass through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size;

[0148] (4) Mixing: mix the carrier powder and auxiliary ingredients at a mass ratio of 10:0.3, then add the microbial combined liquid to the mixture 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 the ingredients are fully mixed and uniform, then let stand for 3h;

[0149] (5) Drying and molding: dry the mixture obtained in step (4) to reduce the moisture content to 10%, then granulate the dried material to produce a microbial agent product that is easy to store and use.

[0150] The microbial liquid obtained in step (c) has an effective viable cell count of not less than 5x10 10 CFU / mL.

[0151] Comparative Example 5

[0152] In this comparative example, except that only Bacillus megaterium and B. bifida were used in the microbial combination, the remaining raw materials and process steps were the same as in Example 1. That is:

[0153] A soybean phospholipid disease-preventing and root-promoting microbial agent includes a microbial combination, modified enzymatic soybean phospholipid, a carrier, and auxiliary ingredients.

[0154] The microbial combination includes Bacillus megaterium and B. bifida, wherein the Bacillus megaterium has a preservation number of CGMCC No. 27648 and was preserved at the China General Microbiological Culture Collection Center on June 16, 2023; the B. bifida has a strain number of CGMCC No. 1.10511 and was purchased from the China General Microbiological Culture Collection Center. The B. bifida is commercially available and does not need to be biologically preserved.

[0155] The method for isolating and screening the Bacillus megaterium is the same as in Example 1.

[0156] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent, comprising the following preparation steps:

[0157] (1) Preparation of modified enzymatic soybean phospholipid;

[0158] (2) Microbial liquid preparation:

[0159] a. Strain activation: Bacillus megaterium and Burkholderia biphenylia were inoculated on LB plates and cultured at 30°C for 48 hours;

[0160] b. Seed liquid culture: single colonies were picked and inoculated into 50 mL seed culture medium, and cultured at 30°C with 180 rpm shaking for 30 hours, obtaining two seed liquids with viable cell counts of 1×10 9 CFU / mL, respectively;

[0161] c. Mixed fermentation: the seed liquids of the two strains were mixed at a volume ratio of 1:1, inoculated into a fermenter at a 6% inoculation amount, and cultured at 30°C with aeration of 1.0 VVM and stirring speed of 200 rpm for 20 hours. Then, 5.0% modified enzymatic soybean phospholipid was added to the system, and the fermentation was continued at 30°C for 24 hours, obtaining a microbial liquid;

[0162] (3) Carrier treatment: attapulgite and vermiculite were mixed at a mass ratio of 1:1, dried at 120-150°C for 2-3 hours to remove water and impurities, then the dried mixture was crushed and sieved through a 100-200 mesh sieve to obtain a carrier powder with uniform particle size;

[0163] (4) Mixing: the carrier powder and auxiliary ingredients were mixed at a mass ratio of 10:0.3, then the microbial combined liquid was added according to a solid-liquid ratio of 1g:50mL, stirred at a speed of 100-120r / min at room temperature for 30 minutes to ensure that all ingredients were fully mixed and uniform, and then left to stand for 3h;

[0164] (5) Drying and molding: the mixture obtained in step (4) was dried to reduce the moisture content to 10%, and then the dried material was granulated to prepare an agent product for easy storage and use.

[0165] The microbial liquid obtained in step (c) has an effective viable cell count of not less than 5×10 10 CFU / mL.

[0166] Comparative Example 6

[0167] In this comparative example, except that only Pseudomonas psychrophila and Burkholderia biphenylia were used in the microbial combination, the remaining raw materials and process steps were the same as in Example 1. That is:

[0168] A soybean phospholipid disease-preventing and root-promoting microbial agent comprises a microbial combination, modified enzymatic soybean phospholipid, a carrier and auxiliary ingredients.

[0169] The microbial combination comprises Pseudomonas putida and Burkholderia bifermentans, the strain number of the Pseudomonas putida is CGMCC No. 1.15631, the strain number of the Burkholderia bifermentans is CGMCC No. 1.10511, and both the Pseudomonas putida and the Burkholderia bifermentans are purchased from the China General Microbiological Culture Collection Center.

[0170] A preparation method of a soybean phospholipid disease-preventing and root-promoting microbial agent comprises the following preparation steps:

[0171] (1) preparing modified enzymatic soybean phospholipid;

[0172] (2) microbial liquid preparation:

[0173] a. strain activation: inoculating Bacillus megaterium and Burkholderia bifermentans on LB plates respectively and culturing at 30°C for 48 hours;

[0174] b. seed liquid culture: picking single colonies and inoculating into 50 mL seed culture medium, and culturing at 30°C and 180 rpm for 30 hours to obtain two seed liquids with a viable cell count of 1×10 9 CFU / mL respectively;

[0175] c. mixed fermentation: mixing the seed liquids of the two strains in a volume ratio of 1:1, inoculating into a fermentation tank at a 6% inoculation amount, culturing at 30°C, aeration amount 1.0vvm and stirring speed 200 rpm for 20 hours, adding 5.0% modified enzymatic soybean phospholipid by mass of the system, and continuing to ferment at 30°C for 24 hours to obtain a microbial liquid;

[0176] (3) carrier treatment: mixing attapulgite and vermiculite in a mass ratio of 1:1, drying at 120-150°C for 2-3 hours to remove water and impurities, then crushing the dried mixture and passing through a 100-200 mesh sieve to obtain carrier powder with uniform particle size;

[0177] (4) mixing: mixing the carrier powder and auxiliary ingredients in a mass ratio of 10:0.3, then adding the microbial combination liquid in a solid-liquid ratio of 1g:50mL, stirring at a speed of 100-120r / min at room temperature for 30 minutes to ensure that the ingredients are fully mixed and uniform, and then standing for 3h;

[0178] (5) Drying and forming: drying the mixture obtained in step (4) to reduce the moisture content to 10%, and then granulating the dried mixture to obtain a microbial agent product for storage and use.

[0179] The microbial solution obtained in step (c) has an effective viable cell count of not less than 5 x 10 10 CFU / mL.

[0180] Comparative Example 7

[0181] In this comparative example, the raw materials and process steps are the same as in Example 1, except that no stepwise low-temperature treatment is performed.

[0182] A soybean phospholipid disease-preventing and root-promoting microbial agent includes a microbial combination, an enzymatic soybean phospholipid, a carrier, and an auxiliary ingredient.

[0183] The preparation method of the enzymatic soybean phospholipid is as follows:

[0184] (1) Pretreatment: soybean phospholipid and deionized water are mixed at a mass ratio of 1:5, the pH is adjusted to 6.5-7.0, heated to 50-55°C, and stirred at constant temperature for 30 min to form a uniform emulsion:

[0185] (2) Enzymatic treatment: lipase is added to the emulsion at an addition amount of 1.5 U / g of soybean phospholipid, and constant-temperature oscillation reaction is performed at 25°C and pH 7.0 for 6 hours;

[0186] (3) Enzyme inactivation and purification: enzyme inactivation is performed in a 20°C water bath for 30 min, followed by centrifugal treatment, ultrafiltration, collection of small molecule components <500 Da, and obtaining of the enzymatic soybean phospholipid.

[0187] Comparative Example 8

[0188] In this comparative example, the raw materials and process steps are the same as in Example 1, except that only a single-temperature low-temperature treatment is performed.

[0189] A soybean phospholipid disease-preventing and root-promoting microbial agent includes a microbial combination, a modified enzymatic soybean phospholipid, a carrier, and an auxiliary ingredient.

[0190] The preparation method of the modified enzymatic soybean phospholipid is as follows:

[0191] (1) Pretreatment: soybean phospholipid and deionized water are mixed at a mass ratio of 1:5, the pH is adjusted to 6.5-7.0, heated to 50-55°C, and stirred at constant temperature for 30 min to form a uniform emulsion:

[0192] (2) Low-temperature treatment: the uniform emulsion is frozen at -5°C for 6 hours, and then naturally thawed at room temperature;

[0193] (3) Enzymatic hydrolysis: add lipase to the thawed emulsion, the amount of addition is 1.5 U / g soybean phospholipid, constant temperature oscillation reaction at 25℃, pH 7.0 for 6 hours;

[0194] (4) Enzyme inactivation and purification: enzyme inactivation at 20℃ water bath for 30 min, centrifugal treatment, ultrafiltration, collect <500 Da small molecule components, and obtain modified enzymatic hydrolysis soybean phospholipid.

[0195] Comparative Example 9

[0196] In this comparative example, except for only single temperature low temperature treatment, the rest of the raw materials and process steps are the same as Example 1. That is:

[0197] A soybean phospholipid disease-preventing and root-promoting microbial agent, comprising a microbial combination, modified enzymatic hydrolysis soybean phospholipid, carrier and auxiliary ingredients.

[0198] The preparation method of the modified enzymatic hydrolysis soybean phospholipid is:

[0199] (1) Pretreatment: mix soybean phospholipid and deionized water according to the mass ratio of 1:5, adjust pH to 6.5-7.0, heat to 50-55℃ and constant temperature stirring for 30 min to form a uniform emulsion:

[0200] (2) Low temperature treatment: freeze the above uniform emulsion at -20℃ for 6 hours, and then thaw naturally at room temperature;

[0201] (3) Enzymatic hydrolysis: add lipase to the thawed emulsion, the amount of addition is 1.5 U / g soybean phospholipid, constant temperature oscillation reaction at 25℃, pH 7.0 for 6 hours;

[0202] (4) Enzyme inactivation and purification: enzyme inactivation at 20℃ water bath for 30 min, centrifugal treatment, ultrafiltration, collect <500 Da small molecule components, and obtain modified enzymatic hydrolysis soybean phospholipid.

[0203] Comparative Example 10

[0204] In this comparative example, except for only single temperature low temperature treatment, the rest of the raw materials and process steps are the same as Example 1. That is:

[0205] A soybean phospholipid disease-preventing and root-promoting microbial agent, comprising a microbial combination, modified enzymatic hydrolysis soybean phospholipid, carrier and auxiliary ingredients.

[0206] The preparation method of the modified enzymatic hydrolysis soybean phospholipid is:

[0207] (1) Pretreatment: mix soybean phospholipid and deionized water according to the mass ratio of 1:5, adjust pH to 6.5-7.0, heat to 50-55℃ and constant temperature stirring for 30 min to form a uniform emulsion:

[0208] (2) Low temperature treatment: the above homogeneous emulsion was refrigerated at 4°C for 6 hours, and then naturally thawed at room temperature;

[0209] (3) Enzymatic treatment: lipase was added to the thawed emulsion, the amount of addition was 1.5 U / g of soybean phospholipid, and the reaction was carried out at 25°C and pH 7.0 with constant temperature oscillation for 6 hours;

[0210] (4) Enzyme inactivation and purification: enzyme inactivation was carried out in a water bath at 20°C for 30 min, and after centrifugal treatment, ultrafiltration was carried out, the <500 Da small molecule components were collected, and modified enzymatic hydrolysis soybean phospholipid was obtained.

[0211] Performance test

[0212] The strains Bacillus megaterium, Pseudomonas psychrophila and Burkholderia bipolaris were tested for their antibacterial performance. After activation, the target strains were cultured at 30°C and 200 r / min for 24h. Sterile filter paper pieces (Φ=6 mm) were placed at a distance of 2.5 cm from the center of the culture medium plate inoculated with the pathogenic bacteria (Rhynchosporium), and 3µL of culture solution was added to each filter paper piece. The plates were incubated in a 30°C incubator, and the diameter of the inhibition zone was measured after the control mycelium grew over the plate. The inhibition rate was calculated. The mycelial morphology was observed, and the normal growing mycelium was used as a control. The test was repeated three times. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter x 100%. The results are shown in Table 1:

[0213] Table 1 Inhibition effect of strains on Rhynchosporium

[0214]

[0215] From the data in Table 1, we can see that Bacillus megaterium, Pseudomonas psychrophila and Burkholderia bipolaris have good inhibition effect on Rhynchosporium.

[0216] Promoting ability test of Bacillus megaterium, Pseudomonas psychrophila and Burkholderia bipolaris:

[0217] Determination of the dissolution capacity of strains on insoluble phosphorus: single colonies of the strains to be tested were picked and activated overnight, and then inoculated into inorganic phosphorus culture medium containing 0.5% insoluble phosphate (calcium phosphate) at an inoculation amount of 2.0% (V / V). The experimental group without inoculation was used as a control, and the culture was incubated at 30°C and 180 r / min. After 24h, 5.0 mL of fermentation sample was collected, centrifuged at 10000 rpm for 5 min, and the fermentation supernatant was collected. Each treatment was set in triplicate, and the soluble phosphorus content in the fermentation supernatant was determined by the molybdenum-antimony anti-colorimetric method according to the above national standard method GB 11893-89.

[0218] The inorganic phosphorus culture medium is prepared by mixing 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·H2O and 5.0 g of calcium phosphate, and sterilizing at 121°C for 30 min.

[0219] Determination of the IAA-producing ability of the strains: single colonies of the strains to be tested were picked and activated overnight, and then inoculated into 500 mL conical flasks containing 200 mL of LB medium (containing 100 mg / L L-tryptophan) at a 2.0% (V / V) inoculation amount, and cultured at 30°C and 180 r / min. An un-inoculated experimental group was used as a control, and samples were taken at 0, 10, 15, 20, 24, 34, 44 and 56 h, 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 three times.

[0220] Table 2: Test of the growth-promoting ability of the strains

[0221]

[0222] Detection of the root colonization ability of the fermentation broth composed of different strains:

[0223] The microbial broth was prepared according to the method of Example 1 and Comparative Examples 1-10, and the different compositions of the fermentation broth were verified to have different effects on the rhizosphere colonization ability of the microorganisms. The preparation method of the microbial broth is as follows:

[0224] Preparation of the microbial broth

[0225] a. Activation of the strains: the strains of the examples or comparative examples to be tested were inoculated on LB plates and cultured at 30°C for 48 hours;

[0226] b. Seed broth culture: single colonies were picked and inoculated into 50 mL of seed culture medium, and cultured at 30°C and 180 rpm for 30 hours to obtain seed broth with a viable cell count of 1×10 9 CFU / mL;

[0227] c. Mixed fermentation: the seed broth was inoculated into a fermentation tank at a 6% inoculation amount, and cultured at 30°C, 1.0 L / min of aeration and 200 rpm of stirring speed for 20 hours. Then, 5.0% of modified enzymatic soybean phospholipid (or enzymatic soybean phospholipid) was added to the system, and the fermentation was continued at 30°C for 24 hours to obtain the microbial broth.

[0228] The seed of the exposed wheat was immersed with the prepared microbial liquid to be tested for 3 hours under greenhouse conditions, and was sown in the nutrient body containing sterile soil, and the test was set up for 3 times. The wheat seedling was investigated after 7 days, and was detected every 5 days. The root colonization bacteria were detected after 25 days. 3 pots of wheat seedlings were taken for detection each time, the roots were washed clean, dried in the clean bench, cut off, and weighed. The roots were placed in a 2 ml centrifuge tube with magnetic beads, oscillated and ground with a frozen grinder, centrifuged, and the supernatant was taken. 100 μl of tissue fluid was coated on each resistance plate, and the colonies with the same colony morphology were counted. The bacterial amount (cfu·g -1 )=(number of colonies x dilution factor x volume of sterile water required for suspension) / (amount of plate coating bacterial suspension x fresh weight of tissue used for separation).

[0229] Table 3 colonization ability of different microbial liquid in roots

[0230]

[0231] From the data in Table 3, it can be seen that the colonization ability of the fermentation broth composed of different strains in the roots of wheat is different. The microbial liquid prepared in the example has a relatively high colonization bacteria amount in the roots, which may be due to the synergistic effect of the microbial combination and the addition of modified enzymatic soybean phospholipid, which helps the strains to better colonize and grow in the roots. In each of the comparative examples, due to the change of the microbial combination, the absence of specific treatment (such as stepwise low temperature treatment) or the use of single temperature low temperature treatment, the colonization ability of the microbial liquid in the roots is different. Comparative examples 1-6 change the strain composition, and the colonization bacteria amount is lower than that of example 1, which is speculated to be that the synergistic balance between the three strains is broken, affecting the colonization effect. Comparative example 7 does not undergo stepwise low temperature treatment, which may cause the structure and performance of the modified enzymatic soybean phospholipid to be different from that of example 1, thereby affecting the colonization of the microorganisms in the roots. Comparative examples 8-10 use single temperature freezing treatment, and the colonization ability is also different, which may be due to the fact that single temperature low temperature treatment has a weak modification effect on the structure of soybean phospholipid, thereby affecting the subsequent enzymatic effect and weakening the promotion effect of the microbial liquid on the colonization ability. In summary, the microbial combination, the preparation process of the modified enzymatic soybean phospholipid and other factors have an important influence on the colonization ability of the microbial liquid in the roots of plants.

[0232] Field control effect:

[0233] Before sowing the wheat, the test agent was uniformly applied to the soil surface at a dosage of 10 kg per mu, and then ploughed to mix the agent with the soil.

[0234] The field plot area is 15 m 2Each test group was set 3 times, and was arranged randomly. The control agent was 27% benzoylurea thiacloprid flowable concentrate (Korlas), 200 mL per 100 kg seeds, produced by Syngenta Crop Protection Co., Ltd.

[0235] The fertility of the test field was at the medium level in the local area, and the wheat sharp eyespot disease occurred seriously in the past years. The soil type was moist soil, and the soil texture was medium soil. The previous crop was maize. The water and fertilizer management was in accordance with the conventional method. The wheat sharp eyespot disease was investigated at the milk stage, and 20 plants were randomly selected from each point in the plot. The disease incidence was investigated according to the Guidelines for Pesticide Field Trials. The specific grading standards were as follows:

[0236] 0 grade: no symptoms on leaf sheath and stem;

[0237] 1 grade: leaf sheath disease, or the lesion width on the stem was less than 1 / 4 of the stem circumference;

[0238] 2 grade: the lesion width on the stem was 1 / 4-1 / 2 of the stem circumference;

[0239] 3 grade: the lesion width on the stem was 1 / 2-3 / 4 of the stem circumference;

[0240] 4 grade: the lesion width on the stem was more than 3 / 4 of the stem circumference;

[0241] 5 grade: the diseased plants died early, and showed withered ear or withered white ear.

[0242] Disease index = ∑(number of plants in each disease grade x disease grade) = (total number of plants x highest disease grade) x 100;

[0243] Relative control efficiency = [(disease index of the control area - disease index of each treatment) / disease index of the control area] x 100;

[0244] Root morphological characteristics: at the heading stage of wheat, the “digging method” was used for sampling. The soil sample area was 900 cm 2 (30 cm x 30 cm) in each test plot, and then different soil layer samples were taken from each sample area. The sample type was root sample in 0-20 cm soil layer, and the sample volume was 18000 cm 3 (30 cm x 30 cm x 20 cm). After the samples in each layer were washed with the net bag and then put into the fresh-keeping bag for cold storage, the sample was scanned by using EPSON EU-88 scanner, and the WinRHIZO system software was used to analyze the wheat root characteristic parameters, including total root length and root dry weight. At the mature stage of wheat, the yield was measured, and the complete sample point was randomly selected from each test plot, and the sample point area was 6 m 2, threshing and weighing, then using grain moisture meter to measure the moisture content of wheat, calculating the actual yield of wheat in the field, according to the national grain storage safety moisture content standard 13% to calculate

[0245] Table 4 Field test results

[0246]

[0247] From the data in Table 4, we can see that the microbial agents of different treatments show obvious differences in field control effect, root morphological characteristics and wheat yield. In terms of field control effect, the microbial agent prepared in the embodiment shows good control effect on wheat sharp eyespot, with relatively high relative control effect, which may be related to the synergistic effect of the microbial combination and the addition of modified enzymatic soybean phospholipid. It can effectively inhibit the growth and infection of the pathogen. While each comparative example has reduced control effect due to changes in microbial combination, no special treatment or single low temperature treatment.

[0248] In terms of root morphological characteristics, the total length of wheat roots and root dry weight of the embodiment treatment are better than those of the comparative examples. This shows that the microbial agent of the embodiment helps to promote the growth and development of wheat roots, and enhances the absorption and fixation capacity of the roots, thereby providing better support for the growth of the plant.

[0249] In terms of wheat yield, the actual yield of wheat in the field of the embodiment treatment is significantly higher than that of the comparative examples. This further proves the effectiveness of the microbial agent of the embodiment in disease prevention and root promotion, which ultimately realizes the improvement of wheat yield by improving the health status of the plant and the function of the roots.

[0250] It should be noted that the above embodiments are only part of the preferred modes of implementing the present application, not all. Obviously, based on the above embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor should fall within the scope of the present application.

Claims

1. A soybean phospholipid disease-preventing and root-promoting microbial inoculant, characterized by, The microorganism combination comprises Bacillus megaterium (CGMCC No. Bacillus megaterium ), Pseudomonas syringae (CGMCC No. Pseudomonas psychrotolerans ), and Burkholderia bifermentans (CGMCC No. Burkholderia ambifaria ), wherein the Bacillus megaterium has a preservation number of CGMCC No. 27648 and a preservation date of June 16, 2023, and is preserved in the China General Microbiological Culture Collection Center; the Pseudomonas syringae has a strain number of CGMCC No. 1.15631, and the Burkholderia bifermentans has a strain number of CGMCC No. 1.10511, and the Pseudomonas syringae and the Burkholderia bifermentans are both purchased from the China General Microbiological Culture Collection Center; and the preparation method of the modified enzymatic soybean phospholipid is: (1) Pretreatment: soybean phospholipid and deionized water are mixed in a mass ratio of 1: (3-5), the pH is adjusted to 6.5-7.0, heated to 50-55℃ and constant temperature stirring for 30 min to form a uniform emulsion: (2) Stepwise low temperature treatment: the above uniform emulsion is first frozen at-5℃ for 1 hour, then reduced to-20℃ at a rate of 3℃ per hour, and kept at this temperature for 3 hours, then the frozen emulsion is stored at 4℃ for 2 hours, and then room temperature natural thawing; (3) Enzymatic hydrolysis: add lipase to the thawed emulsion, the amount is 1.5 U / g of soybean phospholipid, 25℃, pH 7.0 constant temperature oscillation reaction for 6-8 hours; (4) Enzyme inactivation and purification: enzyme inactivation in 20℃ water bath for 30 min, centrifugal treatment, ultrafiltration, collect <500 Da small molecule components, get modified enzymatic hydrolysis soybean phospholipid.

2. The soybean phospholipid disease-preventing and root-promoting microbial inoculant 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).

3. The soybean phospholipid disease-preventing and root-promoting microbial inoculant according to claim 1, characterized in that, The auxiliary component is chitosan and sodium alginate, and the mass ratio of the two is 1: (0.5-1.5).

4. The method for preparing the soybean phospholipid disease-preventing and root-promoting microbial inoculant according to any one of claims 1-3, characterized in that, The following preparation steps are included: (1) Preparation of modified enzymatic hydrolysis soybean phospholipid; (2) Microbial broth preparation: a. Strain activation: Bacillus megaterium, Pseudomonas psychrotolerans and Burkholderia ambifaria are inoculated on LB plates and cultured at 30℃ for 48 hours; b. Seed liquid culture: single colonies were picked and inoculated into 50 mL seed culture medium, respectively, and cultured at 30°C, 180 rpm for 30 hours. Three seed liquids with viable cell count of 1 x 10 9 CFU / mL were obtained, respectively. c. Mixed fermentation: mix the three seed liquids in a volume ratio of 1:1:1, inoculate the fermentation tank with a 6% inoculation amount, cultivate at 30℃ with aeration of 1.0vvm and stirring speed of 200rpm for 20 hours, add 5.0% of the modified enzymatic hydrolysis soybean phospholipid to the system, continue to ferment at 30℃ for 24 hours, and then obtain the microbial broth; (3) Carrier treatment: mix attapulgite and vermiculite in a mass ratio of 1: (1-2), dry at 120-150℃ for 2-3 hours to remove water and impurities, then crush the dried mixture, pass through a 100-200 mesh sieve, and obtain a carrier powder with uniform particle size; (4) Mixing: mix the carrier powder and the auxiliary component in a mass ratio of 10:0.3, then add the microbial combined broth to the mixture according to a solid-liquid ratio of 1g:50mL, stir at a speed of 100-120r / min for 30-60 minutes at room temperature to ensure that the components are fully mixed and uniform, and then stand for 3-5h; (5) Drying and shaping: dry the mixture obtained in step (4) to reduce the moisture content to 10%-15%, then granulate the dried material to prepare a microbial agent product for storage and use.

5. The method for preparing the soybean phospholipid disease-preventing and root-promoting microbial agent according to claim 4, characterized in that, The composition of the seed culture medium is: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride per liter, pH 7.0-7.2, and the rest is sterile water, which is used after sterilization.

6. The method for preparing the soybean phospholipid disease-preventing and root-promoting microbial agent according to claim 4, characterized in that, The fermentor is filled with a fermentation medium, the composition of the fermentation medium is: 20 g of glucose, 15 g of yeast powder, 2 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 5 g of calcium carbonate per liter, the pH value is 7.0-7.2, and the rest is sterilized water, which is used after sterilization.

7. The method for preparing the soybean lecithin disease-preventing and root-promoting microbial inoculant according to claim 4, characterized in that, The microbial liquid obtained in step (c) has an effective viable cell count of not less than 5 x 10 10 CFU / mL.

8. The use of the soybean phospholipid disease-preventing and root-promoting microbial inoculant according to any one of claims 1 to 3, characterized in that, It is applied to the prevention and treatment of wheat sharp eyespot.

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