Preparation method of compound microbial fermentation inoculant and application of compound microbial fermentation inoculant in prevention and treatment of root rot

Abstract

The invention discloses a preparation method of a compound microbial fermentation inoculant and application of the compound microbial fermentation inoculant in prevention and treatment of root rot, and belongs to the technical field of microorganisms, the method comprises the following steps: preparing a first single microbial fermentation inoculant, preparing a second single microbial fermentation inoculant, preparing mixed vermiculite powder, and mixing; the preparation of the first single microbial fermentation inoculant comprises the steps of primary activation, primary amplification culture and primary fermentation culture; the primary fermentation culture comprises the following steps: inoculating a paenibacillus polymyxa seed culture solution into 9-10L of a fermentation culture medium according to the inoculum size of 3-3.5%, culturing for 30-31h at the temperature of 29-31 DEG C, concentrating, adding vermiculite powder, and performing spray drying; when the compound microbial fermentation inoculant is prepared, the fermentation time is short, the prepared compound microbial fermentation inoculant has a good long-term control effect, the control effect and the shelf life are slightly influenced by temperature, and the compound microbial fermentation inoculant has a good short-term treatment effect on plants infected with root rot.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a preparation method of a composite microbial fermentation agent and application of the composite microbial fermentation agent in preventing and treating root rot. Background Art

[0002] Root rot is a soil-borne disease caused by the interaction of multiple pathogens and environmental factors. The types of pathogens are complex and diverse. The pathogens reported so far include Fusarium, Pythium, Phytophthora, Rhizoctonia and Pseudomonas. As for environmental factors, the high-temperature greenhouse method and chemical irrigation method commonly used in the planting process can easily lead to soil compaction, soil quality decline and plant resistance, which can also cause or aggravate root rot.

[0003] For root rot, traditional prevention and control methods include selecting seed varieties, disinfecting seeds, disinfecting seedbed soil, strengthening field management and chemical control. Among them, selecting seed varieties, disinfecting seeds, disinfecting seedbed soil and strengthening field management mainly play a role in early prevention, while chemical control can play both early prevention and treatment roles.

[0004] However, there are the following problems with chemical control: first, chemical residues can lead to soil, water and air pollution; second, chemical agents can easily cause poisoning in humans and animals; third, when using chemical agents, if they are used improperly, it is easy to cause drug damage; fourth, when using chemical agents, it is easy to kill beneficial microorganisms and cause pathogens to develop drug resistance.

[0005] As the key to the connection between plants and soil, rhizosphere soil plays a vital role in plant nutrition absorption and resistance to external environmental pressure. Studies have found that for plants that have been infected with root rot, the number and composition of microorganisms in the rhizosphere soil will change, which is specifically manifested in a decrease in the number and diversity of microorganisms, and also leads to a decrease in the uniformity of microorganisms in the rhizosphere soil. After chemical agents are used to treat root rot, they will affect the microorganisms in the rhizosphere soil, further reducing the number, diversity and uniformity of microorganisms in the rhizosphere soil, and reducing the treatment effect on root rot.

[0006] Biological control refers to reducing the survival and activity of pathogens through the action of other organisms, thereby reducing the occurrence of diseases. Biological control has the advantages of being environmentally friendly and harmless to humans and animals. Microbial control in biological control is the most commonly used biological control method for root rot. Studies have found that when using microbial control to control root rot, microorganisms can regulate the microorganisms in the rhizosphere soil while promoting plant growth. Currently, the public microorganisms mainly include two categories: biocontrol fungi and biocontrol bacteria. Biocontrol fungi include Trichoderma citrinodiflora, Trichoderma harzianum and Chaetomium, and biocontrol bacteria include Bacillus, Pseudomonas and Enzyme-producing Lysozyme.

[0007] Microbial fermentation agents contain microorganisms and their fermentation products. The fermentation products contain extracellular enzymes, carbon sources and nutrients. Microbial fermentation agents can activate the soil, provide nutrition for plants, optimize the community structure of microorganisms in the rhizosphere soil and prevent soil-borne diseases. Therefore, microbial fermentation agents can prevent and treat root rot from two aspects: inhibiting pathogens and improving environmental factors. However, considering the limited effect of a single microbial fermentation agent, more and more people are choosing compound microbial fermentation agents.

[0008] The composite microbial fermentation agent is a composite of multiple single microbial fermentation agents. During preparation, a commonly used method is to culture the microorganisms separately to obtain a single microbial fermentation agent, and then directly mix the multiple single microbial fermentation agents. The composite microbial fermentation agent prepared by the above preparation method has the following problems: First, when using composite microbial fermentation agents to treat root rot, it mainly inhibits the spread of pathogens, improves the soil environment and induces plant resistance, but has the disadvantage of slow treatment effect. For plants already infected with root rot, the short-term treatment effect is worse than that of chemical agents; second, the microorganisms in the composite microbial fermentation agent are greatly affected by the environment and have a short shelf life. Even if Bacillus with strong stress resistance and reproduction ability is used, it is easily affected by temperature and soil pH, resulting in large differences in prevention and control effects under different environments. Although the environment can be reduced by adding carriers and protective agents, the effect of temperature and soil pH can be greatly affected. The impact of the environment on microorganisms can extend the shelf life of the composite microbial fermentation agent, but the microorganisms and extracellular enzymes are mainly bound to the carrier surface by physical adsorption, and the physical adsorption force is greatly affected by temperature. The higher the temperature, the smaller the physical adsorption force, which leads to the desorption of microorganisms and extracellular enzymes, and further leads to the prevention and control effect and shelf life of the composite microbial fermentation agent being greatly affected by temperature; third, although the composite microbial fermentation agent has a synergistic effect in preventing and controlling root rot, due to the nutritional competition between different microorganisms, there may be mutual influence in long-term prevention and control, resulting in a decrease in long-term prevention and control effect; fourth, when a single microbial fermentation agent is obtained after culturing the microorganisms, there is a problem of long fermentation time. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention provides a method for preparing a composite microbial fermentation agent and an application of the composite microbial fermentation agent in preventing and controlling root rot. When preparing the composite microbial fermentation agent, the fermentation time is short, the prepared composite microbial fermentation agent has good long-term prevention and control effect, the prevention and control effect and the shelf life are less affected by temperature, and for plants already infected with root rot, the short-term treatment effect is good.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing a composite microbial fermentation agent, comprising: preparing a first single microbial fermentation agent, preparing a second single microbial fermentation agent, preparing mixed vermiculite powder, and mixing; The preparation of the first single microbial fermentation agent includes: one activation, one expansion culture, and one fermentation culture; The primary activation comprises adding 0.5 mL of NA liquid culture medium into a freeze-dried tube of the polymyxa bacillus lyophilized powder, shaking until fully dissolved, and obtaining a polymyxa bacillus suspension; applying 0.2 mL of the polymyxa bacillus suspension on a NA plate culture medium, and culturing for 23-25 ​​hours at a temperature of 29-31° C. to obtain an activated polymyxa bacillus lyophilized powder; In the one-time activation, the strain number of the polymyxa bacillus is BNCC 138393, which is purchased from the market; The preparation method of the NA liquid culture medium is to take peptone, sodium chloride, beef extract and manganese sulfate monohydrate, add purified water, adjust the pH to 7-7.2, sterilize, and obtain the NA liquid culture medium; In the NA liquid culture medium, the concentration of peptone is 9-11 g / L, the concentration of sodium chloride is 4.8-5.2 g / L, the concentration of beef extract is 3-3.2 g / L, and the concentration of manganese sulfate monohydrate is 5-5.2 mg / L; The sterilization temperature is 121°C and the time is 15-20 minutes; The preparation method of the NA plate medium is to take agar, peptone, sodium chloride, beef extract and manganese sulfate monohydrate, add purified water, adjust the pH to 7-7.2, sterilize, and obtain the NA plate medium; In the NA plate medium, the concentration of agar is 18-22 g / L, the concentration of peptone is 9-11 g / L, the concentration of sodium chloride is 4.8-5.2 g / L, the concentration of beef extract is 3-3.2 g / L, and the concentration of manganese sulfate monohydrate is 5-5.2 mg / L; The sterilization temperature is 121°C and the time is 15-20 minutes; The primary expansion culture is to pick a single colony from the activated polymyxa lyophilized powder and inoculate it into 100-120 mL of seed culture medium, and culture it for 23-25 ​​hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm to obtain a polymyxa lyophilized polymyxa seed culture solution; In the primary expansion culture, the seed culture medium is prepared by taking sucrose, tryptone, sodium chloride, yeast extract, beef extract and manganese sulfate monohydrate, adding purified water, adjusting the pH to 7-7.2, and sterilizing to obtain a seed culture medium; In the seed culture medium, the concentration of sucrose is 9.5-10.5 g / L, the concentration of tryptone is 9.5-10.5 g / L, the concentration of sodium chloride is 9.5-10.5 g / L, the concentration of yeast extract is 9.5-10.5 g / L, the concentration of beef extract is 3-3.2 g / L, and the concentration of manganese sulfate monohydrate is 5-5.2 mg / L; The sterilization temperature is 121°C and the time is 15-20 minutes; The primary fermentation culture comprises inoculating the polymyxa bacillus seed culture solution into 9-10 L fermentation medium at an inoculation rate of 3-3.5%, culturing for 30-31 hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm, and vacuum concentrating the solution to 20-25% of the original volume to obtain a polymyxa bacillus concentrated solution, adding vermiculite powder to the polymyxa bacillus concentrated solution, and spray drying the solution to obtain a first single microbial fermentation agent; In the primary fermentation culture, the fermentation medium is prepared by taking corn flour, sucrose, soybean meal powder, polysaccharide particles, tryptone, yeast extract, beef extract, sodium chloride, disodium hydrogen phosphate dodecahydrate, calcium nitrate tetrahydrate and manganese sulfate monohydrate, adding purified water, adjusting the pH to 7-7.2, sterilizing, and obtaining a fermentation medium; In the fermentation medium, the concentration of corn flour is 60-65 g / L, the concentration of sucrose is 18-20 g / L, the concentration of soybean meal powder is 10-12 g / L, the concentration of polysaccharide particles is 6.4-6.6 g / L, the concentration of tryptone is 5-5.2 g / L, the concentration of yeast extract is 0.7-0.8 g / L, the concentration of beef extract is 0.45-0.5 g / L, the concentration of sodium chloride is 1.5-1.6 g / L, the concentration of disodium hydrogen phosphate dodecahydrate is 1.5-1.6 g / L, the concentration of calcium nitrate tetrahydrate is 0.35-0.4 g / L, and the concentration of manganese sulfate monohydrate is 0.05-0.06 g / L; The sterilization temperature is 121°C and the time is 15-20 minutes; The preparation method of the polysaccharide particles comprises the following steps: mixing chitosan and an acetic acid aqueous solution, stirring the mixture at a stirring speed of 200-300 rpm for 20-30 minutes at room temperature, and spray drying the mixture to obtain chitosan microspheres; mixing genipin and a first portion of water, stirring the mixture at a stirring speed of 300-400 rpm for 1.5-2 hours at 40-42° C., adding chitosan microspheres, continuing stirring for 4-5 hours, centrifuging, and obtaining a precipitate to obtain cross-linked chitosan microspheres; mixing L-serine, L-valine, and a second portion of water, stirring the mixture at a stirring speed of 200-300 rpm for 20-30 minutes at room temperature, adding cross-linked chitosan microspheres, continuing stirring for 2.5-3 hours, centrifuging, obtaining a precipitate, washing the mixture with deionized water, and vacuum drying the mixture to obtain polysaccharide particles; In the preparation of the polysaccharide particles, the amount ratio of chitosan, acetic acid aqueous solution, genipin, the first portion of water, L-serine, L-valine and the second portion of water is 9-10g:500-550mL:8-10g:700-800mL:12-13g:18-20g:700-800mL; The mass concentration of the acetic acid aqueous solution is 2%; The weight average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%; In the primary fermentation and the preparation of the polysaccharide particles, the particle size of the vermiculite powder is 400 meshes; The spray drying in the preparation of the polysaccharide particles has an inlet air temperature of 200-220°C and an outlet air temperature of 80-90°C; In the primary fermentation culture, the mass ratio of the Paenibacillus polymyxa concentrate to the vermiculite powder is 100:20-23; The inlet air temperature of the spray drying in the primary fermentation culture is 140-155°C, and the outlet air temperature is 70-80°C; The bacterial concentration of the first single microbial fermentation agent is 5.1×10 10 cfu / g-7.2×10 10 cfu / g; The preparation of the second single microbial fermentation agent includes: secondary activation, secondary expansion culture, and secondary fermentation culture; The secondary activation comprises adding 0.5 mL of NA liquid culture medium into a freeze-dried tube of Bacillus firmus freeze-dried powder, shaking until fully dissolved, and obtaining a Bacillus firmus suspension; applying 0.2 mL of the Bacillus firmus suspension onto a NA plate culture medium, and culturing at a temperature of 29-31° C. for 23-25 ​​hours to obtain an activated Bacillus firmus freeze-dried powder; In the secondary activation, the strain number of the Bacillus firmus is BNCC 132595, which is purchased from the market; The method for preparing the NA liquid culture medium is the same as the method for preparing the NA liquid culture medium in the step of preparing the first single microbial fermentation agent; The method for preparing the NA plate culture medium is the same as the method for preparing the NA plate culture medium in the step of preparing the first single microbial fermentation agent; The secondary expansion culture is to pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 100-120 mL of seed culture medium, and culture it for 23-25 ​​hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm to obtain a Bacillus firmus seed culture solution; In the secondary expansion culture, the method for preparing the seed culture medium is the same as the method for preparing the seed culture medium in the step of preparing the first single microbial fermentation agent; The secondary fermentation culture comprises inoculating the Bacillus firmus seed culture solution into 9-10 L fermentation medium at an inoculum amount of 3-3.5%, culturing for 34-35 hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm, and vacuum concentrating the solution to 20-25% of the original volume to obtain a Bacillus firmus concentrate, adding vermiculite powder to the Bacillus firmus concentrate, and spray drying the solution to obtain a second single microbial fermentation agent; In the secondary fermentation culture, the method for preparing the fermentation medium is the same as the method for preparing the fermentation medium in the step of preparing the first single microbial fermentation agent; The particle size of the vermiculite powder is 400 mesh; In the secondary fermentation culture, the mass ratio of the Bacillus firmus concentrate to the vermiculite powder is 100:22-26; The inlet air temperature of the spray drying in the secondary fermentation culture is 140-155°C, and the outlet air temperature is 70-80°C; The bacterial concentration of the second single microbial fermentation agent is 7.0×10 10 cfu / g-10.3×10 10 cfu / g; The mixed vermiculite powder is prepared by mixing tris(hydroxymethyl)aminomethane) and the first portion of water, stirring at a stirring speed of 100-300 rpm for 15-20 min at room temperature, adding concentrated hydrochloric acid to adjust the pH to 8.5-9, adding the first portion of dopamine hydrochloride, continuing to stir for 15-20 min, adding vermiculite powder, continuing to stir for 50-55 h, centrifuging, taking a precipitate, washing with deionized water, and vacuum drying to obtain modified vermiculite powder; L-methionine, L-valine, the second portion of dopamine hydrochloride and the second portion of water are mixed, stirring at a stirring speed of 100-300 rpm for 20-30 min at room temperature, adding modified vermiculite powder, continuing to stir for 1.5-2 h, centrifuging, taking a precipitate, washing with deionized water, and vacuum drying to obtain mixed vermiculite powder; In the preparation of the mixed vermiculite powder, the dosage ratio of tris(hydroxymethyl)aminomethane, the first portion of water, the first portion of dopamine hydrochloride, vermiculite powder, L-methionine, L-valine, the second portion of dopamine hydrochloride, and the second portion of water is 27-30 g:10 L:70-75 g:35-40 g:14-15 g:14-15 g:7-8 g:900-1000 mL; The particle size of the vermiculite powder is 400 mesh; The mass concentration of the concentrated hydrochloric acid is 36%; The mixing comprises uniformly mixing the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder to obtain a composite microbial fermentation agent; In the mixing, the mass ratio of the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder is 100:40-42:15-16.

[0011] The invention discloses an application of the composite microbial fermentation agent prepared by the above-mentioned preparation method in preventing and treating root rot.

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation method of the composite microbial fermentation agent of the present invention comprises preparing a first single microbial fermentation agent, preparing a second single microbial fermentation agent, preparing mixed vermiculite powder and mixing, wherein polysaccharide particles are added in the fermentation and culturing steps of the first single microbial fermentation agent and the second single microbial fermentation agent, and the preparation method of the polysaccharide particles comprises spray drying an acetic acid aqueous solution of chitosan to obtain chitosan microspheres, and then adding the chitosan microspheres into a genipin aqueous solution and stirring, wherein the chitosan and genipin are chemically cross-linked to obtain cross-linked chitosan microspheres having a cross-linked network; then L-serine and L-valine are adsorbed on the surface and inside of the cross-linked chitosan microspheres to obtain polysaccharide particles; the polysaccharide particles can adsorb and fix Bacillus polymyxa and Bacillus firmus during fermentation, and then the L-serine, L-valine and chitosan on the surface of the polysaccharide particles can promote fermentation, The extracellular enzymes produced by Bacillus polymyxa and Bacillus firmus during fermentation can promote the decomposition of chitosan, the cross-linking network in the polysaccharide particles is destroyed, and the internal L-serine and L-valine are gradually released, so that the fermentation can be promoted while avoiding the side effects of excessive amino acid concentration on the fermentation; the decomposed chitosan can inhibit the pathogens of root rot, thereby improving the therapeutic effect of the composite microbial fermentation agent on root rot; when preparing the mixed vermiculite powder, firstly, a layer of polydopamine is combined on the surface of the vermiculite powder by utilizing the self-polymerization of dopamine, and then, the rich functional groups of polydopamine are combined with L-methionine, L-valine and dopamine hydrochloride, L-methionine, L-valine and dopamine hydrochloride can improve the stress resistance of Bacillus polymyxa and Bacillus firmus, reduce the influence of temperature on the prevention and control effect and shelf life, and improve the long-term prevention and control effect of the composite microbial fermentation agent; (2) The preparation method of the composite microbial fermentation agent of the present invention has a short fermentation time when preparing the composite microbial fermentation agent. In the fermentation culture of Paenibacillus polymyxa, after 30-31 hours of fermentation culture, the bacterial concentration of the first single microbial fermentation agent prepared is 5.1×10 10 cfu / g-7.2×10 10 cfu / g; in the fermentation culture of Bacillus firmus, after 34-35h of fermentation culture, the bacterial concentration of the first single microbial fermentation agent prepared was 7.0×10 10 cfu / g-10.3×10 10 cfu / g; (3) The composite microbial fermentation agent prepared by the present invention has a good short-term treatment effect on plants already infected with root rot. At 25°C, the antibacterial rate of the composite microbial fermentation agent of the present invention was tested, and the antibacterial rate against Fusarium solani could reach 84.0-86.7%, and the antibacterial rate against Fusarium solani could reach 86.7-89.0%; (4) The control effect of the composite microbial fermentation agent prepared by the present invention is less affected by temperature. At 45°C, the antibacterial rate of the composite microbial fermentation agent of the present invention was tested, and the antibacterial rate of Fusarium solani against Fusarium solani can reach 83.8-86.2%; (5) The shelf life of the composite microbial fermentation agent prepared by the present invention is less affected by temperature. After standing for 10 days under natural light at 45°C, the antibacterial rate of the composite microbial fermentation agent of the present invention was tested at 45°C, and the antibacterial rate against Fusarium solani could reach 82.1-86.4%; (6) The composite microbial fermentation agent prepared by the present invention has a good long-term control effect. 60 days after the ginger root irrigation treatment, the control rate of root rot can reach 65.9-69.4%. DETAILED DESCRIPTION

[0013] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0014] The Paenibacillus polymyxa freeze-dried powder and the Bacillus firmus freeze-dried powder used in Examples 1-6 were purchased from the market; the strain number of Paenibacillus polymyxa is BNCC 138393, and the strain number of Bacillus firmus is BNCC 132595.

[0015] Example 1 A method for preparing a composite microbial fermentation agent is as follows: 1. Preparation of the first single microbial fermentation agent: (1) Primary activation: add 0.5 mL of NA liquid culture medium into the freeze-dried tube of Paenibacillus polymyxa freeze-dried powder, shake until fully dissolved, and obtain a suspension of Paenibacillus polymyxa; take 0.2 mL of the suspension of Paenibacillus polymyxa and spread it on NA plate culture medium, culture it at 29°C for 23 h, and obtain the activated Paenibacillus polymyxa freeze-dried powder; The preparation method of the NA liquid culture medium is as follows: 9 g of peptone, 4.8 g of sodium chloride, 3 g of beef extract and 5 mg of manganese sulfate monohydrate are added with 1000 mL of purified water, the pH is adjusted to 7, and the mixture is sterilized at 121° C. for 15 min to obtain the NA liquid culture medium; The NA plate medium is prepared by taking 18 g agar, 9 g peptone, 4.8 g sodium chloride, 3 g beef extract and 5 mg manganese sulfate monohydrate, adding 1000 mL purified water, adjusting the pH to 7, and sterilizing at 121° C. for 15 min to obtain the NA plate medium; (2) Primary expansion culture: Pick a single colony from the activated Bacillus polymyxa freeze-dried powder and inoculate it into 100 mL of seed culture medium. Cultivate it at a temperature of 29°C and a rotation speed of 150 rpm for 23 h to obtain a seed culture solution of Bacillus polymyxa. The preparation method of the seed culture medium is as follows: 9.5 g sucrose, 9.5 g tryptone, 9.5 g sodium chloride, 9.5 g yeast extract, 3 g beef extract and 5 mg manganese sulfate monohydrate are added with 1000 mL purified water, the pH is adjusted to 7, and the mixture is sterilized at 121° C. for 15 min to obtain a seed culture medium; (3) Primary fermentation culture: inoculate the polymyxa bacillus seed culture solution into 9 L fermentation medium at a 3% inoculation rate, culture for 30 h at a temperature of 29° C. and a rotation speed of 150 rpm, and vacuum concentrate to 20% of the original volume to obtain a polymyxa bacillus concentrate, add vermiculite powder to the polymyxa bacillus concentrate, control the mass ratio of the polymyxa bacillus concentrate to the vermiculite powder to be 100:20, spray dry, control the inlet air temperature to be 140° C., and the outlet air temperature to be 70° C., to obtain a first single microbial fermentation agent; The preparation method of the fermentation medium is as follows: 60g corn flour, 18g sucrose, 10g soybean meal powder, 6.4g polysaccharide particles, 5g tryptone, 0.7g yeast extract, 0.45g beef extract, 1.5g sodium chloride, 1.5g disodium hydrogen phosphate dodecahydrate, 0.35g calcium nitrate tetrahydrate and 0.05g manganese sulfate monohydrate are added, 1000mL purified water is added, the pH is adjusted to 7, and the mixture is sterilized at 121°C for 15min to obtain a fermentation medium; The preparation method of the polysaccharide particles is as follows: 9g of chitosan and 500mL of acetic acid aqueous solution with a mass concentration of 2% are mixed, stirred at a stirring speed of 200rpm for 20min at room temperature, spray-dried, and the air inlet temperature is controlled to be 200°C and the air outlet temperature is 80°C to obtain chitosan microspheres; 8g of genipin and 700mL of water are mixed, stirred at a stirring speed of 300rpm for 1.5h at 40°C, all the chitosan microspheres are added, stirring is continued for 4h, centrifuged at a centrifugal speed of 9000rpm for 5min, and a precipitate is taken to obtain cross-linked chitosan microspheres; 12g of L-serine, 18g of L-valine and 700mL of water are mixed, stirred at a stirring speed of 200rpm for 20min at room temperature, all the cross-linked chitosan microspheres are added, stirring is continued for 2.5h, centrifuged at a centrifugal speed of 9000rpm for 5min, a precipitate is taken, washed 3 times with deionized water, and vacuum dried to obtain polysaccharide particles; The weight average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%; In the primary fermentation and the preparation of polysaccharide particles, the particle size of the vermiculite powder is 400 meshes; The bacterial concentration of the first single microbial fermentation agent is 5.1×10 10 cfu / g; 2. Preparation of the second single microbial fermentation agent: (1) Secondary activation: add 0.5 mL of NA liquid culture medium into the freeze-dried tube of Bacillus firmus freeze-dried powder, shake until fully dissolved, and obtain a Bacillus firmus suspension; take 0.2 mL of the Bacillus firmus suspension and spread it on the NA plate culture medium, culture it at 29°C for 23 hours, and obtain the activated Bacillus firmus freeze-dried powder; The preparation method of the NA liquid culture medium is the same as the preparation method of the NA liquid culture medium in the step of preparing the first single microbial fermentation agent in step 1; The method for preparing the NA plate culture medium is the same as the method for preparing the NA plate culture medium in the step of preparing the first single microbial fermentation agent in step 1; (2) Secondary expansion culture: Pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 100 mL of seed culture medium. Cultivate it at a temperature of 29°C and a rotation speed of 150 rpm for 23 hours to obtain a Bacillus firmus seed culture solution. The method for preparing the seed culture medium is the same as the method for preparing the seed culture medium in the step of preparing the first single microbial fermentation agent in step 1; (3) Secondary fermentation culture: inoculate the Bacillus firmus seed culture solution into 9 L fermentation medium at a 3% inoculation rate, culture for 34 h at a temperature of 29° C. and a rotation speed of 150 rpm, and vacuum concentrate to 20% of the original volume to obtain a Bacillus firmus concentrate. Add vermiculite powder to the Bacillus firmus concentrate, control the mass ratio of the Bacillus firmus concentrate to the vermiculite powder to be 100:22, spray dry, control the inlet air temperature to be 140° C., and the outlet air temperature to be 70° C., to obtain a second single microbial fermentation agent; The method for preparing the fermentation medium is the same as the method for preparing the fermentation medium in the step of preparing the first single microbial fermentation agent in step 1; The polysaccharide particles are prepared in the same manner as the polysaccharide particles in the step of preparing the first single microbial fermentation agent in step 1; The particle size of the vermiculite powder is 400 mesh; The bacterial concentration of the second single microbial fermentation agent is 7.0×10 10 cfu / g; 3. Prepare mixed vermiculite powder: After mixing 27g tris(hydroxymethyl)aminomethane) and 10L water, stir at room temperature at a stirring speed of 100rpm for 15min, add concentrated hydrochloric acid to adjust the pH to 8.5, add 70g dopamine hydrochloride, continue stirring for 15min, add 35g vermiculite powder, continue stirring for 50h, centrifuge at a centrifugal speed of 4000rpm for 5min, take the precipitate, wash it 3 times with deionized water, and vacuum dry it to obtain modified vermiculite powder; After mixing 14g L-methionine, 14g L-valine, 7g dopamine hydrochloride and 900mL water, stir at room temperature at a stirring speed of 100rpm for 20min, add all the above modified vermiculite powder, continue stirring for 1.5h, centrifuge at a centrifugal speed of 4000rpm for 5min, take the precipitate, wash it 3 times with deionized water, and vacuum dry it to obtain mixed vermiculite powder; The particle size of the vermiculite powder is 400 mesh; The mass concentration of the concentrated hydrochloric acid is 36%; 4. Mixing: Evenly mix the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder in a mass ratio of 100:40:15 to obtain a composite microbial fermentation agent.

[0016] This embodiment also provides an application of the composite microbial fermentation agent prepared by the aforementioned preparation method in preventing and treating root rot.

[0017] Example 2 A method for preparing a composite microbial fermentation agent is as follows: 1. Preparation of the first single microbial fermentation agent: (1) Primary activation: add 0.5 mL of NA liquid culture medium into the freeze-dried tube of Paenibacillus polymyxa freeze-dried powder, shake until fully dissolved, and obtain a suspension of Paenibacillus polymyxa; take 0.2 mL of the suspension of Paenibacillus polymyxa and spread it on a NA plate culture medium, culture it at 30°C for 24 h, and obtain the activated Paenibacillus polymyxa freeze-dried powder; The preparation method of the NA liquid culture medium is as follows: 10 g of peptone, 5 g of sodium chloride, 3.1 g of beef extract and 5.1 mg of manganese sulfate monohydrate are added with 1000 mL of purified water, the pH is adjusted to 7.1, and the mixture is sterilized at 121° C. for 20 min to obtain the NA liquid culture medium; The NA plate medium is prepared by taking 20 g agar, 10 g peptone, 5 g sodium chloride, 3.1 g beef extract and 5.1 mg manganese sulfate monohydrate, adding 1000 mL purified water, adjusting the pH to 7.1, and sterilizing at 121° C. for 20 min to obtain the NA plate medium; (2) Primary expansion culture: a single colony was selected from the activated Paenibacillus polymyxa freeze-dried powder and inoculated into 110 mL of seed culture medium, and cultured at 30°C and 150 rpm for 24 h to obtain a Paenibacillus polymyxa seed culture solution; The preparation method of the seed culture medium is as follows: 10 g sucrose, 10 g tryptone, 10 g sodium chloride, 10 g yeast extract, 3.1 g beef extract and 5.1 mg manganese sulfate monohydrate are added with 1000 mL purified water, the pH is adjusted to 7.1, and the mixture is sterilized at 121° C. for 20 min to obtain a seed culture medium; (3) Primary fermentation culture: inoculate the polymyxa bacillus seed culture solution into 9.5 L fermentation medium at an inoculation rate of 3.5%, culture for 30 h at a temperature of 30° C. and a rotation speed of 150 rpm, and vacuum concentrate to 20% of the original volume to obtain a polymyxa bacillus concentrate, add vermiculite powder to the polymyxa bacillus concentrate, control the mass ratio of the polymyxa bacillus concentrate to the vermiculite powder to be 100:22, spray dry, control the inlet air temperature to be 150° C., and the outlet air temperature to be 75° C., to obtain a first single microbial fermentation agent; The preparation method of the fermentation medium is as follows: 62g corn flour, 19g sucrose, 11g soybean meal powder, 6.5g polysaccharide particles, 5.1g tryptone, 0.75g yeast extract, 0.48g beef extract, 1.5g sodium chloride, 1.5g disodium hydrogen phosphate dodecahydrate, 0.38g calcium nitrate tetrahydrate and 0.05g manganese sulfate monohydrate are added, 1000mL purified water is added, the pH is adjusted to 7.1, and the mixture is sterilized at 121°C for 20min to obtain a fermentation medium; The preparation method of the polysaccharide particles is as follows: 9.5g of chitosan and 520mL of acetic acid aqueous solution with a mass concentration of 2% are mixed, stirred at a stirring speed of 250rpm for 25min at room temperature, spray-dried, and the air inlet temperature is controlled to be 210°C and the air outlet temperature is 85°C to obtain chitosan microspheres; 9g of genipin and 750mL of water are mixed, stirred at a stirring speed of 350rpm for 2h at 41°C, all the chitosan microspheres are added, stirring is continued for 4.5h, centrifuged at a centrifugal speed of 9500rpm for 6min, and a precipitate is taken to obtain cross-linked chitosan microspheres; 12.5g of L-serine, 19g of L-valine and 750mL of water are mixed, stirred at a stirring speed of 250rpm for 25min at room temperature, all the cross-linked chitosan microspheres are added, stirring is continued for 3h, centrifuged at a centrifugal speed of 9500rpm for 6min, a precipitate is taken, washed with deionized water for 3 times, and vacuum-dried to obtain polysaccharide particles; The weight average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%; In the primary fermentation and the preparation of polysaccharide particles, the particle size of the vermiculite powder is 400 meshes; The bacterial concentration of the first single microbial fermentation agent is 5.8×10 10 cfu / g; 2. Preparation of the second single microbial fermentation agent: (1) Secondary activation: add 0.5 mL of NA liquid culture medium into the freeze-dried tube of Bacillus firmus freeze-dried powder, shake until fully dissolved, and obtain a Bacillus firmus suspension; take 0.2 mL of the Bacillus firmus suspension and spread it on the NA plate culture medium, culture it at 30°C for 24 hours, and obtain the activated Bacillus firmus freeze-dried powder; The preparation method of the NA liquid culture medium is the same as the preparation method of the NA liquid culture medium in the step of preparing the first single microbial fermentation agent in step 1; The method for preparing the NA plate culture medium is the same as the method for preparing the NA plate culture medium in the step of preparing the first single microbial fermentation agent in step 1; (2) Secondary expansion culture: Pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 110 mL of seed culture medium. Cultivate it at a temperature of 30°C and a rotation speed of 150 rpm for 24 h to obtain a Bacillus firmus seed culture solution. The method for preparing the seed culture medium is the same as the method for preparing the seed culture medium in the step of preparing the first single microbial fermentation agent in step 1; (3) Secondary fermentation culture: inoculate the Bacillus firmus seed culture solution into 9.5 L fermentation medium at a rate of 3.5%, culture for 35 h at a temperature of 30° C. and a rotation speed of 150 rpm, and vacuum concentrate to 20% of the original volume to obtain a Bacillus firmus concentrate. Add vermiculite powder to the Bacillus firmus concentrate, control the mass ratio of the Bacillus firmus concentrate to the vermiculite powder to be 100:24, spray dry, control the inlet air temperature to be 150° C., and the outlet air temperature to be 75° C., to obtain a second single microbial fermentation agent; The fermentation medium is prepared in the same manner as the fermentation medium in the step of preparing the first single microbial fermentation agent in step 1; The polysaccharide particles are prepared in the same manner as the polysaccharide particles in the step of preparing the first single microbial fermentation agent in step 1; The particle size of the vermiculite powder is 400 mesh; The bacterial concentration of the second single microbial fermentation agent is 8.1×10 10 cfu / g; 3. Prepare mixed vermiculite powder: After mixing 28g tris(hydroxymethyl)aminomethane) and 10L water, stir at room temperature at a stirring speed of 200rpm for 18min, add concentrated hydrochloric acid to adjust the pH to 8.5, add 72g dopamine hydrochloride, continue stirring for 18min, add 37g vermiculite powder, continue stirring for 52h, centrifuge at a centrifugal speed of 4500rpm for 6min, take the precipitate, wash 4 times with deionized water, and vacuum dry to obtain modified vermiculite powder; After mixing 15g L-methionine, 15g L-valine, 7.5g dopamine hydrochloride and 950mL water, stir at room temperature at a stirring speed of 200rpm for 25min, add all the above modified vermiculite powder, continue stirring for 2h, centrifuge at a centrifugal speed of 4500rpm for 6min, take the precipitate, wash 4 times with deionized water, and vacuum dry to obtain mixed vermiculite powder; The particle size of the vermiculite powder is 400 mesh; The mass concentration of the concentrated hydrochloric acid is 36%; 4. Mixing: Evenly mix the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder in a mass ratio of 100:41:16 to obtain a composite microbial fermentation agent.

[0018] This embodiment also provides an application of the composite microbial fermentation agent prepared by the aforementioned preparation method in preventing and treating root rot.

[0019] Example 3 A method for preparing a composite microbial fermentation agent is as follows: 1. Preparation of the first single microbial fermentation agent: (1) Primary activation: add 0.5 mL of NA liquid culture medium into the freeze-dried tube of Paenibacillus polymyxa freeze-dried powder, shake until fully dissolved, and obtain a suspension of Paenibacillus polymyxa; take 0.2 mL of the suspension of Paenibacillus polymyxa and spread it on a NA plate culture medium, culture it at 31°C for 25 h, and obtain the activated Paenibacillus polymyxa freeze-dried powder; The preparation method of the NA liquid culture medium is as follows: 11 g of peptone, 5.2 g of sodium chloride, 3.2 g of beef extract and 5.2 mg of manganese sulfate monohydrate are added with 1000 mL of purified water, the pH is adjusted to 7.2, and the mixture is sterilized at 121° C. for 20 min to obtain the NA liquid culture medium; The NA plate medium is prepared by taking 22 g agar, 11 g peptone, 5.2 g sodium chloride, 3.2 g beef extract and 5.2 mg manganese sulfate monohydrate, adding 1000 mL purified water, adjusting the pH to 7.2, and sterilizing at 121° C. for 20 min to obtain the NA plate medium; (2) Primary expansion culture: a single colony was selected from the activated Paenibacillus polymyxa freeze-dried powder and inoculated into 120 mL of seed culture medium, and cultured at 31°C and 160 rpm for 25 h to obtain a Paenibacillus polymyxa seed culture solution; The preparation method of the seed culture medium is as follows: 10.5 g sucrose, 10.5 g tryptone, 10.5 g sodium chloride, 10.5 g yeast extract, 3.2 g beef extract and 5.2 mg manganese sulfate monohydrate are added with 1000 mL purified water, the pH is adjusted to 7.2, and the mixture is sterilized at 121° C. for 20 min to obtain a seed culture medium; (3) Primary fermentation culture: inoculate the polymyxa bacillus seed culture solution into 10 L fermentation medium at an inoculation rate of 3.5%, culture for 31 h at a temperature of 31° C. and a rotation speed of 160 rpm, and vacuum concentrate to 25% of the original volume to obtain a polymyxa bacillus concentrate. Add vermiculite powder to the polymyxa bacillus concentrate, control the mass ratio of the polymyxa bacillus concentrate to the vermiculite powder to be 100:23, spray dry, control the inlet air temperature to be 155° C., and the outlet air temperature to be 80° C., to obtain a first single microbial fermentation agent; The preparation method of the fermentation medium is as follows: 65g corn flour, 20g sucrose, 12g soybean meal powder, 6.6g polysaccharide particles, 5.2g tryptone, 0.8g yeast extract, 0.5g beef extract, 1.6g sodium chloride, 1.6g disodium hydrogen phosphate dodecahydrate, 0.4g calcium nitrate tetrahydrate and 0.06g manganese sulfate monohydrate are added, 1000mL purified water is added, the pH is adjusted to 7.2, and the mixture is sterilized at 121°C for 20min to obtain a fermentation medium; The preparation method of the polysaccharide particles is as follows: 10g of chitosan and 550mL of acetic acid aqueous solution with a mass concentration of 2% are mixed, and the mixture is stirred at a stirring speed of 300rpm for 30min at room temperature, spray-dried, and the air inlet temperature is controlled to be 220°C and the air outlet temperature is controlled to be 90°C to obtain chitosan microspheres; 10g of genipin and 800mL of water are mixed, and the mixture is stirred at a stirring speed of 400rpm for 2h at 42°C, all the chitosan microspheres are added, stirring is continued for 5h, centrifuged at a centrifugal speed of 10000rpm for 6min, and a precipitate is taken to obtain cross-linked chitosan microspheres; 13g of L-serine, 20g of L-valine and 800mL of water are mixed, and the mixture is stirred at a stirring speed of 200-300rpm for 20-30min at room temperature, all the cross-linked chitosan microspheres are added, stirring is continued for 3h, centrifuged at a centrifugal speed of 10000rpm for 6min, a precipitate is taken, washed 4 times with deionized water, and vacuum-dried to obtain polysaccharide particles; The weight average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%; In the primary fermentation and the preparation of polysaccharide particles, the particle size of the vermiculite powder is 400 meshes; The bacterial concentration of the first single microbial fermentation agent is 7.2×10 10 cfu / g; 2. Preparation of the second single microbial fermentation agent: (1) Secondary activation: add 0.5 mL of NA liquid culture medium into the freeze-dried tube of Bacillus firmus freeze-dried powder, shake until fully dissolved, and obtain a Bacillus firmus suspension; take 0.2 mL of the Bacillus firmus suspension and spread it on the NA plate culture medium, culture it at 31°C for 25 hours, and obtain the activated Bacillus firmus freeze-dried powder; The preparation method of the NA liquid culture medium is the same as the preparation method of the NA liquid culture medium in the step of preparing the first single microbial fermentation agent in step 1; The method for preparing the NA plate culture medium is the same as the method for preparing the NA plate culture medium in the step of preparing the first single microbial fermentation agent in step 1; (2) Secondary expansion culture: Pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 120 mL of seed culture medium. Cultivate it at a temperature of 31°C and a rotation speed of 160 rpm for 25 h to obtain a Bacillus firmus seed culture solution. The method for preparing the seed culture medium is the same as the method for preparing the seed culture medium in the step of preparing the first single microbial fermentation agent in step 1; (3) Secondary fermentation culture: inoculate the Bacillus firmus seed culture solution into 10 L fermentation medium at an inoculum rate of 3.5%, culture for 35 h at a temperature of 31° C. and a rotation speed of 160 rpm, and vacuum concentrate to 25% of the original volume to obtain a Bacillus firmus concentrate. Add vermiculite powder to the Bacillus firmus concentrate, control the mass ratio of the Bacillus firmus concentrate to the vermiculite powder to be 100:26, spray dry, control the inlet air temperature to be 155° C., and the outlet air temperature to be 80° C., to obtain a second single microbial fermentation agent; The fermentation medium is prepared in the same manner as the fermentation medium in the step of preparing the first single microbial fermentation agent in step 1; The polysaccharide particles are prepared in the same manner as the polysaccharide particles in the step of preparing the first single microbial fermentation agent in step 1; The particle size of the vermiculite powder is 400 mesh; The bacterial concentration of the second single microbial fermentation agent is 10.3×10 10 cfu / g; 3. Prepare mixed vermiculite powder: After mixing 30g tris(hydroxymethyl)aminomethane) and 10L water, stir at room temperature at a stirring speed of 300rpm for 20min, add concentrated hydrochloric acid to adjust the pH to 9, add 75g dopamine hydrochloride, continue stirring for 20min, add 40g vermiculite powder, continue stirring for 55h, centrifuge at a centrifugal speed of 5000rpm for 6min, take the precipitate, wash it 5 times with deionized water, and vacuum dry it to obtain modified vermiculite powder; After mixing 15g L-methionine, 15g L-valine, 8g dopamine hydrochloride and 1000mL water, stir at room temperature at a stirring speed of 300rpm for 30min, add all the above modified vermiculite powder, continue stirring for 2h, centrifuge at a centrifugal speed of 5000rpm for 6min, take the precipitate, wash it 5 times with deionized water, and vacuum dry it to obtain mixed vermiculite powder; The particle size of the vermiculite powder is 400 mesh; The mass concentration of the concentrated hydrochloric acid is 36%; 4. Mixing: Evenly mix the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder in a mass ratio of 100:42:16 to obtain a composite microbial fermentation agent.

[0020] This embodiment also provides an application of the composite microbial fermentation agent prepared by the aforementioned preparation method in preventing and treating root rot.

[0021] Example 4 The purpose of this example is to verify the effect of polysaccharide particles. Therefore, based on the preparation method of the composite microbial fermentation agent of Example 2, in the first step (3) of the first fermentation culture step in the step of preparing the first single microbial fermentation agent, the addition of polysaccharide particles is omitted in the preparation method of the fermentation medium. In the second step of preparing the second single microbial fermentation agent, in the second fermentation culture step (3), the addition of polysaccharide particles is omitted in the method for preparing the fermentation medium; The rest of the operations remain unchanged.

[0022] In this embodiment, the concentration of the first single microbial fermentation agent obtained in the step of preparing the first single microbial fermentation agent is 7.5×10 7 cfu / g; In this embodiment, the second single microbial fermentation agent obtained in the step of preparing the second single microbial fermentation agent in step 2 has a bacterial concentration of 1.0×10 8 cfu / g.

[0023] This embodiment also provides an application of the composite microbial fermentation agent prepared by the aforementioned preparation method in preventing and treating root rot.

[0024] Example 5 The purpose of this embodiment is to further verify the promoting effect of polysaccharide particles on fermentation on the basis of Example 4. Therefore, the fermentation time is extended on the basis of Example 4. Specifically, on the basis of the preparation method of the composite microbial fermentation agent of Example 4, in the first step of preparing the first single microbial fermentation agent in step 1, the fermentation time is extended from 30 hours to 45 hours in the first fermentation culture step (3); In the second step of preparing the second single microbial fermentation agent, in the second fermentation culture step (3), the fermentation time is extended from 35 hours to 52.5 hours; The rest of the operations remain unchanged.

[0025] In this embodiment, the concentration of the first single microbial fermentation agent obtained in the step of preparing the first single microbial fermentation agent is 5.2×10 10 cfu / g; In this embodiment, the second single microbial fermentation agent obtained in the step of preparing the second single microbial fermentation agent in step 2 has a bacterial concentration of 7.4×10 10 cfu / g.

[0026] This embodiment also provides an application of the composite microbial fermentation agent prepared by the aforementioned preparation method in preventing and treating root rot.

[0027] Example 6 The effect of this embodiment is to verify the effect of mixed vermiculite powder. Therefore, based on the preparation method of the composite microbial fermentation agent of Example 2, the third step of preparing mixed vermiculite powder is omitted, and in the fourth step of mixing, the addition of mixed vermiculite powder is omitted, and the fourth step of mixing is changed to: The first single microbial fermentation agent and the second single microbial fermentation agent were uniformly mixed in a mass ratio of 100:41 to obtain a composite microbial fermentation agent.

[0028] This embodiment also provides an application of the composite microbial fermentation agent prepared by the aforementioned preparation method in preventing and treating root rot.

[0029] Test Example 1 Fusarium solani was isolated and screened from the root system of eggplant infected with root rot. Seven bacterial blocks at the edge of the colony of Fusarium solani were taken with a sterile cork puncher with a diameter of 4 mm, and were inoculated in the center of a PDA flat culture medium with a diameter of 9 cm, numbered as culture medium 1-7, and culture medium 1-6 was used as the test culture medium. 1 g of the composite microbial fermentation agent prepared in Example 1-6 was added to the upper side, lower side, left side, and right side of the central bacterial block in culture medium 1-6 at a distance of 1 cm from the edge of the culture dish, respectively. Culture medium 7 was used as the control culture medium without any treatment. Culture medium 1-7 was cultured under natural light at a temperature of 25° C. When the Fusarium solani in culture medium 7, i.e., the control culture medium, grew to the edge, the radius of the colony of Fusarium solani in culture medium 1-6, i.e., the test culture medium, was measured, and the inhibition rate was calculated. The calculation formula and the calculation results are as follows: Inhibition rate = (the radius of the Fusarium solani colony in the control medium - the radius of the Fusarium solani colony in the test medium) / the radius of the Fusarium solani colony in the control medium × 100%;

[0030] It can be seen from the above results that the antibacterial rate of the composite microbial fermentation agent prepared in Example 2 is higher than that of the composite microbial fermentation agents prepared in Examples 4-6, indicating that the composite microbial fermentation agent prepared in Example 2 has a better rapid antibacterial effect on Fusarium solani, and further illustrates that the composite microbial fermentation agent prepared in Example 2 has a good short-term treatment effect on plants already infected with root rot.

[0031] Test Example 2 On the basis of Experimental Example 1, the Fusarium solani was replaced with Fusarium solani isolated and screened from the root system of ginger infected with root rot, and then the method of Experimental Example 1 was used to conduct the test. The calculation results of the inhibition rate are as follows:

[0032] It can be seen from the above results that the antibacterial rate of the composite microbial fermentation agent prepared in Example 2 is higher than that of the composite microbial fermentation agents prepared in Examples 4-6, indicating that the composite microbial fermentation agent prepared in Example 2 has a better rapid antibacterial effect on Fusarium solani, and further illustrates that the composite microbial fermentation agent prepared in Example 2 can achieve good prevention and control effects on eggplant root rot and ginger root rot.

[0033] Test Example 3 On the basis of Test Example 1, the temperature was raised from 25°C to 45°C, and then the test was carried out using the method of Test Example 1. The calculation results of the antibacterial rate are as follows:

[0034] It can be seen from the above results that the antibacterial rate of the composite microbial fermentation agent prepared in Example 2 is higher than that of the composite microbial fermentation agents prepared in Examples 4-6, indicating that the control effect of the composite microbial fermentation agent prepared in Example 2 is less affected by temperature.

[0035] Test Example 4 The composite microbial fermentation agent prepared in Example 1-6 was placed under natural light at 45°C for 10 days to obtain a composite microbial fermentation agent after standing. Then, the composite microbial fermentation agent after standing was tested in an environment of 45°C according to the method of Test Example 1. The calculation results of the antibacterial rate are as follows:

[0036] It can be seen from the above results that the antibacterial rate of the composite microbial fermentation agent prepared in Example 2 is higher than that of the composite microbial fermentation agents prepared in Examples 4-6, indicating that the shelf life of the composite microbial fermentation agent prepared in Example 2 is less affected by temperature.

[0037] Test Example 5 A ginger planting field was selected in Weifang City, Shandong Province, and 7 acres were enclosed as an experimental ginger planting field. The experimental ginger planting field was divided into 7 plots, each with an area of ​​1 acre. They were numbered 1-7 and physically isolated from each other to avoid the spread of diseases.

[0038] Ensure that the planting time and planting density of ginger in ginger planting plots No. 1-7 are the same, and the fertilizer and water management after planting is also the same. Ginger planting plots No. 1-6 are used as experimental ginger planting plots. 6 weeks after ginger planting, the composite microbial fermentation agents prepared in Examples 1-6 are mixed with water to prepare dispersions with a mass concentration of 1%. Then, the dispersions prepared by the composite microbial fermentation agents in Examples 1-6 are used to perform root irrigation treatment on ginger planting plots No. 1-6. In the root irrigation treatment, the amount of dispersion used for each ginger plant is 500 mL. Ginger planting plot No. 7 is used as a control ginger planting plot, and no fungicide is used.

[0039] 60 days after the root irrigation treatment, the number of ginger plants with root rot in the No. 1-7 ginger planting areas was counted, the incidence of root rot was calculated, and then the control rate of root rot was calculated. The calculation formula and results of the control rate of root rot are as follows: The control rate of root rot = (the incidence of root rot in the control ginger planting area - the incidence of root rot in the experimental ginger planting area) / the incidence of root rot in the control ginger planting area × 100%;

[0040] It can be seen from the above results that the control rate of the composite microbial fermentation agent prepared in Example 2 is higher than that of the composite microbial fermentation agent prepared in Examples 4-6, indicating that the composite microbial fermentation agent prepared in Example 2 can continue to play a bactericidal role after root irrigation treatment, further indicating that the composite microbial fermentation agent prepared in Example 2 has good long-term control effect.

[0041] Unless otherwise specified, all percentages used in the present invention are by mass.

[0042] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a composite microbial fermentation agent, characterized in that: include: preparing a first single microbial fermentation agent, preparing a second single microbial fermentation agent, preparing mixed vermiculite powder, and mixing; The preparation of the first single microbial fermentation agent includes: one activation, one expansion culture, and one fermentation culture; The primary fermentation culture comprises inoculating the polymyxa bacillus seed culture solution into 9-10 L fermentation medium at an inoculation rate of 3-3.5%, culturing for 30-31 hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm, and vacuum concentrating to obtain a polymyxa bacillus concentrated solution, adding vermiculite powder to the polymyxa bacillus concentrated solution, and spray drying to obtain a first single microbial fermentation agent; In the primary fermentation culture, the fermentation medium is prepared by taking corn flour, sucrose, soybean meal powder, polysaccharide particles, tryptone, yeast extract, beef extract, sodium chloride, disodium hydrogen phosphate dodecahydrate, calcium nitrate tetrahydrate and manganese sulfate monohydrate, adding purified water, adjusting the pH to 7-7.2, sterilizing, and obtaining a fermentation medium; The mixed vermiculite powder is prepared by mixing tris(hydroxymethyl)aminomethane) and the first portion of water, stirring at room temperature, adding concentrated hydrochloric acid to adjust the pH to 8.5-9, adding the first portion of dopamine hydrochloride, continuing to stir, adding vermiculite powder, continuing to stir, centrifuging, taking precipitate, washing, vacuum drying to obtain modified vermiculite powder; and mixing L-methionine, L-valine, the second portion of dopamine hydrochloride and the second portion of water, stirring at room temperature, adding modified vermiculite powder, continuing to stir, centrifuging, taking precipitate, washing, vacuum drying to obtain mixed vermiculite powder.

2. The method for preparing the composite microbial fermentation agent according to claim 1, characterized in that: The primary activation comprises adding 0.5 mL of NA liquid culture medium into a freeze-dried tube of the polymyxa bacillus lyophilized powder, shaking until fully dissolved, and obtaining a polymyxa bacillus suspension; applying 0.2 mL of the polymyxa bacillus suspension on a NA plate culture medium, and culturing for 23-25 ​​hours at a temperature of 29-31° C. to obtain an activated polymyxa bacillus lyophilized powder; In the one-time activation, the strain number of the polymyxa bacillus is BNCC 138393, which is purchased from the market; The preparation method of the NA liquid culture medium is to take peptone, sodium chloride, beef extract and manganese sulfate monohydrate, add purified water, adjust the pH to 7-7.2, sterilize, and obtain the NA liquid culture medium; In the NA liquid culture medium, the concentration of peptone is 9-11 g / L, the concentration of sodium chloride is 4.8-5.2 g / L, the concentration of beef extract is 3-3.2 g / L, and the concentration of manganese sulfate monohydrate is 5-5.2 mg / L; The preparation method of the NA plate medium is to take agar, peptone, sodium chloride, beef extract and manganese sulfate monohydrate, add purified water, adjust the pH to 7-7.2, sterilize, and obtain the NA plate medium; In the NA plate culture medium, the concentration of agar is 18-22 g / L, the concentration of peptone is 9-11 g / L, the concentration of sodium chloride is 4.8-5.2 g / L, the concentration of beef extract is 3-3.2 g / L, and the concentration of manganese sulfate monohydrate is 5-5.2 mg / L.

3. The method for preparing the composite microbial fermentation agent according to claim 2, characterized in that: The primary expansion culture is to pick a single colony from the activated polymyxa lyophilized powder and inoculate it into 100-120 mL of seed culture medium, and culture it for 23-25 ​​hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm to obtain a polymyxa lyophilized polymyxa seed culture solution; In the primary expansion culture, the seed culture medium is prepared by taking sucrose, tryptone, sodium chloride, yeast extract, beef extract and manganese sulfate monohydrate, adding purified water, adjusting the pH to 7-7.2, and sterilizing to obtain a seed culture medium; In the seed culture medium, the concentration of sucrose is 9.5-10.5 g / L, the concentration of tryptone is 9.5-10.5 g / L, the concentration of sodium chloride is 9.5-10.5 g / L, the concentration of yeast extract is 9.5-10.5 g / L, the concentration of beef extract is 3-3.2 g / L, and the concentration of manganese sulfate monohydrate is 5-5.2 mg / L.

4. The method for preparing the composite microbial fermentation agent according to claim 3, characterized in that: In the fermentation medium in the primary fermentation culture, the concentration of corn flour is 60-65 g / L, the concentration of sucrose is 18-20 g / L, the concentration of soybean meal powder is 10-12 g / L, the concentration of polysaccharide particles is 6.4-6.6 g / L, the concentration of tryptone is 5-5.2 g / L, the concentration of yeast extract is 0.7-0.8 g / L, the concentration of beef extract is 0.45-0.5 g / L, the concentration of sodium chloride is 1.5-1.6 g / L, the concentration of disodium hydrogen phosphate dodecahydrate is 1.5-1.6 g / L, the concentration of calcium nitrate tetrahydrate is 0.35-0.4 g / L, and the concentration of manganese sulfate monohydrate is 0.05-0.06 g / L; In the primary fermentation culture, the mass ratio of the Paenibacillus polymyxa concentrate to the vermiculite powder is 100:20-23; The bacterial concentration of the first single microbial fermentation agent is 5.1×10 10 cfu / g-7.2×10 10 cfu / g.

5. The method for preparing the composite microbial fermentation agent according to claim 1, characterized in that: The preparation method of the polysaccharide particles comprises the following steps: mixing chitosan and an acetic acid aqueous solution, stirring at room temperature, and spray drying to obtain chitosan microspheres; mixing genipin and a first portion of water, stirring at 40-42° C., adding chitosan microspheres, continuing stirring, centrifuging, and obtaining a precipitate to obtain cross-linked chitosan microspheres; and mixing L-serine, L-valine, and a second portion of water, stirring at room temperature, adding cross-linked chitosan microspheres, continuing stirring, centrifuging, obtaining a precipitate, washing, and vacuum drying to obtain polysaccharide particles.

6. The method for preparing the composite microbial fermentation agent according to claim 5, characterized in that: In the preparation of the polysaccharide particles, the amount ratio of chitosan, acetic acid aqueous solution, genipin, the first portion of water, L-serine, L-valine and the second portion of water is 9-10g:500-550mL:8-10g:700-800mL:12-13g:18-20g:700-800mL; The mass concentration of the acetic acid aqueous solution is 2%; The weight average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%.

7. The method for preparing the composite microbial fermentation agent according to claim 4, characterized in that: The preparation of the second single microbial fermentation agent includes: secondary activation, secondary expansion culture, and secondary fermentation culture; The secondary activation comprises adding 0.5 mL of NA liquid culture medium into a freeze-dried tube of Bacillus firmus freeze-dried powder, shaking until fully dissolved, and obtaining a Bacillus firmus suspension; applying 0.2 mL of the Bacillus firmus suspension onto a NA plate culture medium, and culturing at a temperature of 29-31° C. for 23-25 ​​hours to obtain an activated Bacillus firmus freeze-dried powder; In the secondary activation, the strain number of the Bacillus firmus is BNCC 132595, which is purchased from the market; The method for preparing the NA liquid culture medium is the same as the method for preparing the NA liquid culture medium in the step of preparing the first single microbial fermentation agent; The method for preparing the NA plate culture medium is the same as the method for preparing the NA plate culture medium in the step of preparing the first single microbial fermentation agent; The secondary expansion culture is to pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 100-120 mL of seed culture medium, and culture it for 23-25 ​​hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm to obtain a Bacillus firmus seed culture solution; In the secondary expansion culture, the method for preparing the seed culture medium is the same as the method for preparing the seed culture medium in the step of preparing the first single microbial fermentation agent.

8. The method for preparing the composite microbial fermentation agent according to claim 7, characterized in that: The secondary fermentation culture comprises inoculating the Bacillus firmus seed culture solution into 9-10 L fermentation medium at an inoculum amount of 3-3.5%, culturing for 34-35 hours at a temperature of 29-31° C. and a rotation speed of 150-160 rpm, vacuum concentrating to obtain a Bacillus firmus concentrate, adding vermiculite powder to the Bacillus firmus concentrate, spray drying, and obtaining a second single microbial fermentation agent; In the secondary fermentation culture, the method for preparing the fermentation medium is the same as the method for preparing the fermentation medium in the step of preparing the first single microbial fermentation agent; The mass ratio of Bacillus firmus concentrate to vermiculite powder is 100:22-26; The bacterial concentration of the second single microbial fermentation agent is 7.0×10 10 cfu / g-10.3×10 10 cfu / g.

9. The method for preparing the composite microbial fermentation agent according to claim 1, characterized in that: In the preparation of the mixed vermiculite powder, the dosage ratio of tris(hydroxymethyl)aminomethane, the first portion of water, the first portion of dopamine hydrochloride, vermiculite powder, L-methionine, L-valine, the second portion of dopamine hydrochloride, and the second portion of water is 27-30 g:10 L:70-75 g:35-40 g:14-15 g:14-15 g:7-8 g:900-1000 mL; The particle size of the vermiculite powder is 400 mesh; The mass concentration of the concentrated hydrochloric acid is 36%; The mixing comprises uniformly mixing the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder to obtain a composite microbial fermentation agent; In the mixing, the mass ratio of the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder is 100:40-42:15-16.

10. Use of the composite microbial fermentation agent prepared by the preparation method according to any one of claims 1 to 9 in preventing and treating root rot.

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