Preparation method of a compound microbial fermentation inoculant and its application in preventing and controlling root rot

Abstract

The present invention discloses a preparation method of a compound microbial fermentation agent and its application in preventing and treating root rot, belonging to the technical field of microorganisms. The method includes: preparing a first single microbial fermentation agent, preparing a second single microbial fermentation agent, preparing a mixed vermiculite powder, and mixing; the preparation of the first single microbial fermentation agent includes: primary activation, primary scale-up culture, and primary fermentation culture; in the primary fermentation culture, the seed culture solution of Paenibacillus polymyxa is inoculated into 9-10 L of fermentation medium at an inoculation amount of 3-3.5%, cultured at a temperature of 29-31 °C for 30-31 h, concentrated, vermiculite powder is added, and spray drying is carried out; when preparing the compound microbial fermentation agent of the present invention, the fermentation time is short, the long-term prevention and treatment effect of the prepared compound microbial fermentation agent is good, the prevention and treatment effect and shelf life are less affected by temperature, and for plants already infected with root rot, the short-term treatment effect is good.

Description

Technical Field

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

[0002] Root rot disease is a soil-borne disease caused by the interaction of various pathogenic bacteria and environmental factors. The types of its pathogenic bacteria are complex and diverse. The pathogenic bacteria reported so far include Fusarium, Pythium, Phytophthora, Rhizoctonia and Pestalotiopsis, etc. For environmental factors, measures such as high-temperature soil steaming and chemical irrigation commonly used in the planting process are likely to cause problems such as soil compaction, soil quality decline and plant drug resistance, and will also cause or aggravate root rot disease.

[0003] For root rot disease, traditional prevention and treatment methods include selecting high-quality seed varieties, disinfecting seeds, disinfecting seedbed soil, strengthening field management and chemical agent control. Among them, selecting high-quality seed varieties, disinfecting seeds, disinfecting seedbed soil and strengthening field management mainly play a role in early prevention, and chemical agent control can not only play a role in early prevention, but also play a role in treatment.

[0004] However, chemical agent control has the following problems: First, chemical agent residues will cause soil, water source and air pollution; Second, chemical agents are easy to cause poisoning of humans and livestock; Third, when using chemical agents, if used improperly, it is easy to produce phytotoxicity; Fourth, when using chemical agents, it is easy to kill beneficial microorganisms and will also cause the pathogen to develop drug resistance.

[0005] Rhizosphere soil, as the key link between plants and soil, plays a crucial role in aspects such as plant nutrient absorption and resistance to external environmental pressures. Through research, it has been found that for plants already infected with root rot disease, the quantity and composition of microorganisms in the rhizosphere soil will change, specifically manifested as a decrease in the quantity and diversity of microorganisms, and will also lead to a decrease in the evenness of microorganisms in the rhizosphere soil. After applying chemical agents to treat root rot disease, the chemical agents will affect the microorganisms in the rhizosphere soil, further reducing the quantity, diversity and evenness of microorganisms in the rhizosphere soil and reducing the treatment effect on root rot disease.

[0006] Biological control refers to reducing the survival and activities of pathogenic bacteria through the action of other organisms, thereby reducing the occurrence of diseases. Biological control has the advantages of being pollution-free to the environment and harmless to humans and livestock. The microbial control method in biological control is the most commonly used biological control method for root rot. It has been found through research that when using the microbial control method to control root rot, the microorganisms can regulate the microorganisms in the rhizosphere soil while promoting the growth of plants. Currently, the publicly known microorganisms mainly include two categories: biocontrol fungi and biocontrol bacteria. Biocontrol fungi include Trichoderma citrinoviride, Trichoderma harzianum, and Chaetomium spp., etc. Biocontrol bacteria include Bacillus spp., Pseudomonas spp., and Lysobacter enzymogenes, etc.

[0007] Microbial fermentation inoculants contain microorganisms and their fermentation products. The fermentation products contain extracellular enzymes, carbon sources, and nutrients, etc. Microbial fermentation inoculants can play roles such as activating the soil, providing nutrients for plants, optimizing the microbial community structure in the rhizosphere soil, and preventing soil-borne diseases. Therefore, microbial fermentation inoculants can play preventive and therapeutic roles in root rot from two aspects: inhibiting pathogenic bacteria and improving environmental factors. However, considering the limited effect of a single microbial fermentation inoculant, more and more people choose compound microbial fermentation inoculants.

[0008] Compound microbial fermentation inoculants are composites of multiple single microbial fermentation inoculants. When preparing them, the commonly used method is to culture the microorganisms separately to obtain single microbial fermentation inoculants, and then directly mix multiple single microbial fermentation inoculants. The compound microbial fermentation inoculants prepared by the above preparation method have the following problems: First, when using compound microbial fermentation inoculants to treat root rot, it mainly inhibits the spread of pathogenic bacteria, improves the soil environment, and induces plants to produce resistance, but there is a disadvantage of slow onset of 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 compound microbial fermentation inoculants are greatly affected by the environment and have a short shelf life. Even when using Bacillus spp. with strong stress resistance and reproductive ability, they are easily affected by temperature and soil pH, resulting in large differences in control effects in different environments. Although the influence of the environment on microorganisms can be reduced and the shelf life of compound microbial fermentation inoculants can be extended by adding carriers and protectants, the microorganisms and extracellular enzymes are mainly bound to the surface of the carrier through physical adsorption force, and the physical adsorption force is greatly affected by temperature. The higher the temperature, the smaller the physical adsorption force, resulting in the desorption of microorganisms and extracellular enzymes, and further leading to the control effect and shelf life of compound microbial fermentation inoculants being greatly affected by temperature; Third, although compound microbial fermentation inoculants have a synergistic effect in controlling root rot, due to nutritional competition among different microorganisms, there may be mutual influences in long-term control, resulting in a decline in long-term control effects; Fourth, when culturing microorganisms to obtain single microbial fermentation inoculants, there is a problem of long fermentation time. Summary of the Invention

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

[0010] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0011] A method for preparing a compound microbial fermentation agent, comprising: preparing a first single microbial fermentation agent, preparing a second single microbial fermentation agent, preparing mixed vermiculite powder, and mixing.

[0012] The preparation of the first single microbial fermentation agent includes: primary activation, primary scale-up culture, and primary fermentation culture.

[0013] For the primary activation, 0.5 mL of NA liquid medium is added to the freeze-dried tube of the freeze-dried powder of Paenibacillus polymyxa, and shaken until fully dissolved to obtain a Paenibacillus polymyxa bacterial suspension; 0.2 mL of the Paenibacillus polymyxa bacterial suspension is spread on the NA plate medium and cultured at a temperature of 29 - 31 °C for 23 - 25 h to obtain the activated freeze-dried powder of Paenibacillus polymyxa.

[0014] In the primary activation, the strain number of the Paenibacillus polymyxa is BNCC 138393, which is obtained by purchasing from the market.

[0015] The preparation method of the NA liquid medium is as follows: take peptone, sodium chloride, beef extract, and manganese sulfate monohydrate, add purified water, adjust the pH to 7 - 7.2, and sterilize to obtain the NA liquid medium.

[0016] In the NA liquid 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.

[0017] The sterilization temperature is 121 °C and the time is 15 - 20 min.

[0018] The preparation method of the NA plate medium is as follows: take agar, peptone, sodium chloride, beef extract, and manganese sulfate monohydrate, add purified water, adjust the pH to 7 - 7.2, and sterilize to obtain the NA plate medium.

[0019] 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;

[0020] The sterilization temperature is 121 °C and the time is 15 - 20 min;

[0021] For the primary scale-up culture, a single colony is picked from the freeze-dried powder of Paenibacillus polymyxa after activation and inoculated into 100 - 120 mL of seed medium, and cultured at a temperature of 29 - 31 °C and a rotation speed of 150 - 160 rpm for 23 - 25 h to obtain the Paenibacillus polymyxa seed culture solution;

[0022] In the primary scale-up culture, the preparation method of the seed medium is as follows: take sucrose, tryptone, sodium chloride, yeast extract, beef extract, and manganese sulfate monohydrate, add purified water, adjust the pH to 7 - 7.2, and sterilize to obtain the seed medium;

[0023] In the seed 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;

[0024] The sterilization temperature is 121 °C and the time is 15 - 20 min;

[0025] For the primary fermentation culture, the Paenibacillus polymyxa seed culture solution is inoculated into 9 - 10 L of fermentation medium at an inoculation amount of 3 - 3.5%, and cultured at a temperature of 29 - 31 °C and a rotation speed of 150 - 160 rpm for 30 - 31 h, then vacuum concentrated to 20 - 25% of the original volume to obtain the Paenibacillus polymyxa concentrate. Vermiculite powder is added to the Paenibacillus polymyxa concentrate, and spray drying is carried out to obtain the first single microbial fermentation agent;

[0026] In the primary fermentation culture, the preparation method of the fermentation medium is as follows: take 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, add purified water, adjust the pH to 7 - 7.2, and sterilize to obtain the fermentation medium;

[0027] 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;

[0028] The temperature for sterilization is 121 °C and the time is 15 - 20 min;

[0029] The preparation method of the polysaccharide particles is as follows: Chitosan and acetic acid aqueous solution are mixed and stirred at a stirring speed of 200 - 300 rpm at room temperature for 20 - 30 min, and then spray-dried to obtain chitosan microspheres; Genipin and the first portion of water are mixed and stirred at a stirring speed of 300 - 400 rpm at 40 - 42 °C for 1.5 - 2 h, then chitosan microspheres are added and stirring continues for 4 - 5 h, followed by centrifugation, and the precipitate is taken to obtain crosslinked chitosan microspheres; L-serine, L-valine and the second portion of water are mixed and stirred at a stirring speed of 200 - 300 rpm at room temperature for 20 - 30 min, crosslinked chitosan microspheres are added, and stirring continues for 2.5 - 3 h, followed by centrifugation, the precipitate is taken, washed with deionized water, and dried in vacuum to obtain polysaccharide particles;

[0030] In the preparation of the polysaccharide particles, the dosage 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 - 10 g : 500 - 550 mL : 8 - 10 g : 700 - 800 mL : 12 - 13 g : 18 - 20 g : 700 - 800 mL;

[0031] The mass concentration of the acetic acid aqueous solution is 2%;

[0032] The weight-average molecular weight of the chitosan is 150,000 and the degree of deacetylation is 90%;

[0033] In the first fermentation culture and the preparation of the polysaccharide particles, the particle size of the vermiculite powder is 400 mesh;

[0034] In the spray drying in the preparation of the polysaccharide particles, the inlet air temperature is 200 - 220 °C and the outlet air temperature is 80 - 90 °C;

[0035] In the first fermentation culture, the mass ratio of the Paenibacillus polymyxa concentrate to the vermiculite powder is 100 : 20 - 23;

[0036] The inlet air temperature for spray drying in the primary fermentation culture is 140 - 155°C, and the outlet air temperature is 70 - 80°C;

[0037] The bacterial concentration of the first single - microorganism fermentation inoculant is 5.1×10 10 cfu / g - 7.2×10 10 cfu / g;

[0038] The preparation of the second single - microorganism fermentation inoculant includes: secondary activation, secondary scale - up culture, and secondary fermentation culture;

[0039] For the secondary activation, add 0.5 mL of NA liquid medium into the freeze - dried tube of Bacillus firmus freeze - dried powder, shake until fully dissolved to obtain a Bacillus firmus bacterial suspension; take 0.2 mL of the Bacillus firmus bacterial suspension and spread it on an NA plate medium, and culture it at a temperature of 29 - 31°C for 23 - 25 h to obtain the activated Bacillus firmus freeze - dried powder;

[0040] In the secondary activation, the strain number of the Bacillus firmus is BNCC 132595, which is obtained by purchasing from the market;

[0041] The preparation method of the NA liquid medium is the same as that of the NA liquid medium in the steps of preparing the first single - microorganism fermentation inoculant;

[0042] The preparation method of the NA plate medium is the same as that of the NA plate medium in the steps of preparing the first single - microorganism fermentation inoculant;

[0043] For the secondary scale - up culture, pick a single colony from the activated Bacillus firmus freeze - dried powder and inoculate it into 100 - 120 mL of seed medium, and culture it at a temperature of 29 - 31°C and a rotation speed of 150 - 160 rpm for 23 - 25 h to obtain a Bacillus firmus seed culture solution;

[0044] In the secondary scale - up culture, the preparation method of the seed medium is the same as that of the seed medium in the steps of preparing the first single - microorganism fermentation inoculant;

[0045] For the secondary fermentation culture, inoculate the Bacillus firmus seed culture solution into 9 - 10 L of fermentation medium at an inoculation amount of 3 - 3.5%, and culture it at a temperature of 29 - 31°C and a rotation speed of 150 - 160 rpm for 34 - 35 h, then vacuum - concentrate it to 20 - 25% of the original volume to obtain a Bacillus firmus concentrated solution. Add vermiculite powder to the Bacillus firmus concentrated solution and perform spray drying to obtain the second single - microorganism fermentation inoculant;

[0046] In the secondary fermentation culture, the preparation method of the fermentation medium is the same as that of the fermentation medium in the step of preparing the first single microbial fermentation agent;

[0047] The particle size of the vermiculite powder is 400 mesh;

[0048] In the secondary fermentation culture, the mass ratio of the Bacillus firmus concentrate to the vermiculite powder is 100:22 - 26;

[0049] In the spray drying in the secondary fermentation culture, the inlet air temperature is 140 - 155 °C, and the outlet air temperature is 70 - 80 °C;

[0050] The bacterial concentration of the second single microbial fermentation agent is 7.0×10 10 cfu / g - 10.3×10 10 cfu / g;

[0051] In the preparation of the mixed vermiculite powder, after mixing tris(hydroxymethyl)aminomethane and the first portion of water, stir at a stirring speed of 100 - 300 rpm for 15 - 20 min at room temperature, add concentrated hydrochloric acid to adjust the pH to 8.5 - 9, add the first portion of dopamine hydrochloride, continue stirring for 15 - 20 min, add vermiculite powder, continue stirring for 50 - 55 h, centrifuge, take the precipitate, wash with deionized water, and vacuum dry to obtain modified vermiculite powder; after mixing L-methionine, L-valine, the second portion of dopamine hydrochloride and the second portion of water, stir at a stirring speed of 100 - 300 rpm for 20 - 30 min at room temperature, add the modified vermiculite powder, continue stirring for 1.5 - 2 h, centrifuge, take the precipitate, wash with deionized water, and vacuum dry to obtain mixed vermiculite powder;

[0052] 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;

[0053] The particle size of the vermiculite powder is 400 mesh;

[0054] The mass concentration of the concentrated hydrochloric acid is 36%;

[0055] In the mixing, mix the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder evenly to obtain a composite microbial fermentation agent;

[0056] 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.

[0057] Application of a composite microbial fermentation inoculant prepared by the aforementioned preparation method in preventing and treating root rot disease.

[0058] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0059] (1) The preparation method of the composite microbial fermentation inoculant of the present invention includes preparing a first single microbial fermentation inoculant, preparing a second single microbial fermentation inoculant, preparing a mixed vermiculite powder and mixing. Polysaccharide particles are added in the fermentation culture steps of both the first single microbial fermentation inoculant and the second single microbial fermentation inoculant. The preparation method of the polysaccharide particles is to spray-dry an acetic acid aqueous solution of chitosan to obtain chitosan microspheres, and then add the chitosan microspheres to a genipin aqueous solution and stir. Chitosan and genipin undergo chemical cross-linking to obtain cross-linked chitosan microspheres with 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 immobilize Paenibacillus polymyxa and Bacillus firmus during fermentation, and then L-serine, L-valine and chitosan on the surface of the polysaccharide particles can play a role in promoting fermentation. The extracellular enzymes produced by Paenibacillus polymyxa and Bacillus firmus during fermentation can promote the decomposition of chitosan, the cross-linked network inside the polysaccharide particles is damaged, and the internal L-serine and L-valine are gradually released, so that while promoting fermentation, the side effects of excessive amino acid concentration on fermentation can be avoided; the decomposed chitosan can inhibit the pathogens of root rot disease, thereby improving the treatment effect of the composite microbial fermentation inoculant on root rot disease; when preparing the mixed vermiculite powder, first use the self-polymerization of dopamine to bind a layer of polydopamine on the surface of the vermiculite powder, and then use the rich functional groups of polydopamine to bind L-methionine, L-valine and dopamine hydrochloride. L-methionine, L-valine and dopamine hydrochloride can improve the stress resistance of Paenibacillus polymyxa and Bacillus firmus, reduce the influence of temperature on the control effect and shelf life, and improve the long-term control effect of the composite microbial fermentation inoculant;

[0060] (2) In the preparation method of the composite microbial fermentation inoculant of the present invention, when preparing the composite microbial fermentation inoculant, the fermentation time is short. In the fermentation culture of Paenibacillus polymyxa, after 30 - 31 h of fermentation culture, the bacterial concentration of the prepared first single microbial fermentation inoculant is 5.1×10 10 cfu / g - 7.2×10 10 cfu / g; in the fermentation culture of Bacillus firmus, after 34 - 35 h of fermentation culture, the bacterial concentration of the prepared first single microbial fermentation inoculant is 7.0×10 10 cfu / g - 10.3×10 10 cfu / g;

[0061] (3) The compound microbial fermentation inoculant 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 compound microbial fermentation inoculant of the present invention was tested. The antibacterial rate against Fusarium solani can reach 84.0 - 86.7%, and the antibacterial rate against Fusarium solani can reach 86.7 - 89.0%;

[0062] (4) The compound microbial fermentation inoculant prepared by the present invention is less affected by temperature in terms of control effect. At 45°C, the antibacterial rate of the compound microbial fermentation inoculant of the present invention was tested. The antibacterial rate against Fusarium solani can reach 83.8 - 86.2%;

[0063] (5) The compound microbial fermentation inoculant prepared by the present invention is less affected by temperature in terms of shelf life. After standing for 10 days under natural light at 45°C, at 45°C, the antibacterial rate of the compound microbial fermentation inoculant of the present invention was tested. The antibacterial rate against Fusarium solani can reach 82.1 - 86.4%;

[0064] (6) The compound microbial fermentation inoculant prepared by the present invention has a good long-term control effect. When ginger was treated by root irrigation for 60 days, the control rate against root rot can reach 65.9 - 69.4%. Specific Embodiments

[0065] In order to have a clearer understanding of the technical features, objectives, and effects of the present invention, the specific embodiments of the present invention are now described.

[0066] The freeze-dried powder of Paenibacillus polymyxa and the freeze-dried powder of Bacillus firmus used in Examples 1 - 6 were all purchased from the market; among them, the strain number of Paenibacillus polymyxa is BNCC 138393, and the strain number of Bacillus firmus is BNCC 132595.

[0067] Example 1

[0068] A preparation method of a compound microbial fermentation inoculant is as follows:

[0069] 1. Preparation of the first single microbial fermentation inoculant:

[0070] (1) Primary activation: Add 0.5 mL of NA liquid medium into the freeze-dried tube of the freeze-dried powder of Paenibacillus polymyxa, shake until fully dissolved to obtain a Paenibacillus polymyxa bacterial suspension; take 0.2 mL of the Paenibacillus polymyxa bacterial suspension and spread it on the NA plate medium, and culture it at a temperature of 29°C for 23 h to obtain the activated freeze-dried powder of Paenibacillus polymyxa;

[0071] The preparation method of the NA liquid medium is as follows: Take 9 g of peptone, 4.8 g of sodium chloride, 3 g of beef extract, and 5 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7, and sterilize at 121 °C for 15 min to obtain the NA liquid medium;

[0072] The preparation method of the NA plate medium is as follows: Take 18 g of agar, 9 g of peptone, 4.8 g of sodium chloride, 3 g of beef extract, and 5 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7, and sterilize at 121 °C for 15 min to obtain the NA plate medium;

[0073] (2) Primary expansion culture: Pick a single colony from the activated freeze-dried powder of Paenibacillus polymyxa and inoculate it into 100 mL of seed medium, and culture it at a temperature of 29 °C and a rotation speed of 150 rpm for 23 h to obtain the Paenibacillus polymyxa seed culture solution;

[0074] The preparation method of the seed medium is as follows: Take 9.5 g of sucrose, 9.5 g of tryptone, 9.5 g of sodium chloride, 9.5 g of yeast extract, 3 g of beef extract, and 5 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7, and sterilize at 121 °C for 15 min to obtain the seed medium;

[0075] (3) Primary fermentation culture: Inoculate the Paenibacillus polymyxa seed culture solution into 9 L of fermentation medium according to an inoculation amount of 3%, and culture it at a temperature of 29 °C and a rotation speed of 150 rpm for 30 h, and vacuum concentrate it to 20% of the original volume to obtain the Paenibacillus polymyxa concentrate. Add vermiculite powder to the Paenibacillus polymyxa concentrate, control the mass ratio of the Paenibacillus polymyxa concentrate to vermiculite powder to be 100:20, and perform spray drying, control the inlet air temperature to be 140 °C, and the outlet air temperature to be 70 °C to obtain the first single microbial fermentation inoculant;

[0076] The preparation method of the fermentation medium is as follows: Take 60 g of corn flour, 18 g of sucrose, 10 g of soybean meal powder, 6.4 g of polysaccharide particles, 5 g of tryptone, 0.7 g of yeast extract, 0.45 g of beef extract, 1.5 g of sodium chloride, 1.5 g of disodium hydrogen phosphate dodecahydrate, 0.35 g of calcium nitrate tetrahydrate, and 0.05 g of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7, and sterilize at 121 °C for 15 min to obtain the fermentation medium;

[0077] The preparation method of the polysaccharide particles is as follows: Mix 9 g of chitosan and 500 mL of acetic acid aqueous solution with a mass concentration of 2%, stir at a stirring speed of 200 rpm at room temperature for 20 min, perform spray drying, control the inlet air temperature at 200 °C and the outlet air temperature at 80 °C to obtain chitosan microspheres; Mix 8 g of genipin and 700 mL of water, stir at a stirring speed of 300 rpm at 40 °C for 1.5 h, add all the above chitosan microspheres, continue to stir for 4 h, centrifuge at a centrifugal speed of 9000 rpm for 5 min, take the precipitate to obtain crosslinked chitosan microspheres; Mix 12 g of L-serine, 18 g of L-valine and 700 mL of water, stir at a stirring speed of 200 rpm at room temperature for 20 min, add all the above crosslinked chitosan microspheres, continue to stir for 2.5 h, centrifuge at a centrifugal speed of 9000 rpm for 5 min, take the precipitate, wash it 3 times with deionized water, and dry it in vacuum to obtain polysaccharide particles;

[0078] The weight-average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%;

[0079] In the primary fermentation culture and the preparation of the polysaccharide particles, the particle size of the vermiculite powder is 400 mesh;

[0080] The bacterial concentration of the first single microbial fermentation inoculant is 5.1×10 10 cfu / g;

[0081] 2. Preparation of the second single microbial fermentation inoculant:

[0082] (1) Secondary activation: Add 0.5 mL of NA liquid medium into the freeze-dried tube of Bacillus firmus freeze-dried powder, shake until fully dissolved to obtain a Bacillus firmus bacterial suspension; Take 0.2 mL of the Bacillus firmus bacterial suspension and spread it on the NA plate medium, and culture it at a temperature of 29 °C for 23 h to obtain the activated Bacillus firmus freeze-dried powder;

[0083] The preparation method of the NA liquid medium is the same as that of the NA liquid medium in the first step of preparing the first single microbial fermentation inoculant;

[0084] The preparation method of the NA plate medium is the same as that of the NA plate medium in the first step of preparing the first single microbial fermentation inoculant;

[0085] (2) Secondary scale-up culture: Pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 100 mL of seed medium, and culture it at a temperature of 29 °C and a rotation speed of 150 rpm for 23 h to obtain Bacillus firmus seed culture solution;

[0086] The preparation method of the seed culture medium is the same as that of the seed culture medium in the step of preparing the first single microbial fermentation agent in Step 1;

[0087] (3) Secondary fermentation culture: Inoculate the Bacillus firmus seed culture solution into 9 L of fermentation medium at an inoculation amount of 3%, and culture it at a temperature of 29 °C and a rotation speed of 150 rpm for 34 h, then concentrate it in vacuo to 20% of the original volume to obtain a concentrated Bacillus firmus solution. Add vermiculite powder to the concentrated Bacillus firmus solution, control the mass ratio of the concentrated Bacillus firmus solution to vermiculite powder to be 100:22, and perform spray drying, controlling the inlet air temperature to be 140 °C and the outlet air temperature to be 70 °C to obtain the second single microbial fermentation agent;

[0088] The preparation method of the fermentation medium is the same as that of the fermentation medium in the step of preparing the first single microbial fermentation agent in Step 1;

[0089] The preparation method of the polysaccharide particles is the same as that of the polysaccharide particles in the step of preparing the first single microbial fermentation agent in Step 1;

[0090] The particle size of the vermiculite powder is 400 mesh;

[0091] The bacterial concentration of the second single microbial fermentation agent is 7.0×10 10 cfu / g;

[0092] 3. Preparation of mixed vermiculite powder: Mix 27 g of tris(hydroxymethyl)aminomethane and 10 L of water, stir at a stirring speed of 100 rpm at room temperature for 15 min, add concentrated hydrochloric acid to adjust the pH to 8.5, add 70 g of dopamine hydrochloride, continue stirring for 15 min, add 35 g of vermiculite powder, continue stirring for 50 h, centrifuge at a centrifugation speed of 4000 rpm for 5 min, take the precipitate, wash it 3 times with deionized water, and dry it in vacuo to obtain modified vermiculite powder; Mix 14 g of L-methionine, 14 g of L-valine, 7 g of dopamine hydrochloride and 900 mL of water, stir at a stirring speed of 100 rpm at room temperature for 20 min, add all the above modified vermiculite powder, continue stirring for 1.5 h, centrifuge at a centrifugation speed of 4000 rpm for 5 min, take the precipitate, wash it 3 times with deionized water, and dry it in vacuo to obtain mixed vermiculite powder;

[0093] The particle size of the vermiculite powder is 400 mesh;

[0094] The mass concentration of the concentrated hydrochloric acid is 36%;

[0095] 4. Mixing: Mix the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder evenly according to a mass ratio of 100:40:15 to obtain a composite microbial fermentation agent.

[0096] This example also provides the application of a composite microbial fermentation inoculant prepared by the aforementioned preparation method in preventing and controlling root rot.

[0097] Example 2

[0098] A preparation method of a composite microbial fermentation inoculant is as follows:

[0099] 1. Prepare the first single microbial fermentation inoculant:

[0100] (1) Primary activation: Add 0.5 mL of NA liquid medium into the freeze-dried tube of Paenibacillus polymyxa freeze-dried powder, shake until fully dissolved to obtain a Paenibacillus polymyxa bacterial suspension; Take 0.2 mL of the Paenibacillus polymyxa bacterial suspension and spread it on an NA plate medium, and culture it at a temperature of 30 °C for 24 h to obtain the activated Paenibacillus polymyxa freeze-dried powder;

[0101] The preparation method of the NA liquid medium is: Take 10 g of peptone, 5 g of sodium chloride, 3.1 g of beef extract, and 5.1 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.1, and sterilize at 121 °C for 20 min to obtain the NA liquid medium;

[0102] The preparation method of the NA plate medium is: Take 20 g of agar, 10 g of peptone, 5 g of sodium chloride, 3.1 g of beef extract, and 5.1 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.1, and sterilize at 121 °C for 20 min to obtain the NA plate medium;

[0103] (2) Primary scale-up culture: Pick a single colony from the activated Paenibacillus polymyxa freeze-dried powder and inoculate it into 110 mL of seed medium, and culture it at a temperature of 30 °C and a rotation speed of 150 rpm for 24 h to obtain a Paenibacillus polymyxa seed culture solution;

[0104] The preparation method of the seed medium is: Take 10 g of sucrose, 10 g of tryptone, 10 g of sodium chloride, 10 g of yeast extract, 3.1 g of beef extract, and 5.1 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.1, and sterilize at 121 °C for 20 min to obtain the seed medium;

[0105] (3) Primary fermentation culture: Inoculate the Paenibacillus polymyxa seed culture solution into 9.5 L of fermentation medium at an inoculation amount of 3.5%, culture it for 30 h under the conditions of a temperature of 30 °C and a rotation speed of 150 rpm, concentrate it in vacuo to 20% of the original volume to obtain a Paenibacillus polymyxa concentrate. Add vermiculite powder to the Paenibacillus polymyxa concentrate, control the mass ratio of the Paenibacillus polymyxa concentrate to vermiculite powder to be 100:22, perform spray drying, control the inlet air temperature to be 150 °C and the outlet air temperature to be 75 °C to obtain the first single microbial fermentation inoculant;

[0106] The preparation method of the fermentation medium is as follows: Take 62 g of corn flour, 19 g of sucrose, 11 g of soybean meal powder, 6.5 g of polysaccharide particles, 5.1 g of tryptone, 0.75 g of yeast extract, 0.48 g of beef extract, 1.5 g of sodium chloride, 1.5 g of disodium hydrogen phosphate dodecahydrate, 0.38 g of calcium nitrate tetrahydrate, and 0.05 g of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.1, and sterilize it at 121 °C for 20 min to obtain the fermentation medium;

[0107] The preparation method of the polysaccharide particles is as follows: Mix 9.5 g of chitosan and 520 mL of acetic acid aqueous solution with a mass concentration of 2%, stir it at a stirring speed of 250 rpm at room temperature for 25 min, perform spray drying, control the inlet air temperature to be 210 °C and the outlet air temperature to be 85 °C to obtain chitosan microspheres; Mix 9 g of genipin and 750 mL of water, stir it at a stirring speed of 350 rpm at 41 °C for 2 h, add all the above chitosan microspheres, continue to stir for 4.5 h, centrifuge at a centrifugation speed of 9500 rpm for 6 min, take the precipitate to obtain crosslinked chitosan microspheres; Mix 12.5 g of L-serine, 19 g of L-valine and 750 mL of water, stir it at a stirring speed of 250 rpm at room temperature for 25 min, add all the above crosslinked chitosan microspheres, continue to stir for 3 h, centrifuge at a centrifugation speed of 9500 rpm for 6 min, take the precipitate, wash it 3 times with deionized water, and dry it in vacuo to obtain the polysaccharide particles;

[0108] The weight-average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%;

[0109] In the primary fermentation culture and the preparation of the polysaccharide particles, the particle size of the vermiculite powder is 400 mesh;

[0110] The bacterial concentration of the first single microbial fermentation inoculant is 5.8×10 10 cfu / g;

[0111] 2. Preparation of the second single microbial fermentation inoculant:

[0112] (1)Secondary activation: Add 0.5 mL of NA liquid medium into the freeze-dried tube of Bacillus firmus freeze-dried powder, shake until fully dissolved to obtain a Bacillus firmus bacterial suspension; take 0.2 mL of the Bacillus firmus bacterial suspension and spread it on the NA plate medium, and culture it at 30 °C for 24 h to obtain the activated Bacillus firmus freeze-dried powder;

[0113] The preparation method of the NA liquid medium is the same as that of the NA liquid medium in the first step of preparing the first single microbial fermentation agent;

[0114] The preparation method of the NA plate medium is the same as that of the NA plate medium in the first step of preparing the first single microbial fermentation agent;

[0115] (2)Secondary subculture: Pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 110 mL of seed medium, and culture it at 30 °C and a rotation speed of 150 rpm for 24 h to obtain a Bacillus firmus seed culture solution;

[0116] The preparation method of the seed medium is the same as that of the seed medium in the first step of preparing the first single microbial fermentation agent;

[0117] (3)Secondary fermentation culture: Inoculate the Bacillus firmus seed culture solution into 9.5 L of fermentation medium at an inoculation amount of 3.5%, and culture it at 30 °C and a rotation speed of 150 rpm for 35 h, and vacuum concentrate it to 20% of the original volume to obtain a Bacillus firmus concentrated solution. Add vermiculite powder to the Bacillus firmus concentrated solution, control the mass ratio of the Bacillus firmus concentrated solution to vermiculite powder to be 100:24, and perform spray drying, control the inlet air temperature to be 150 °C, and the outlet air temperature to be 75 °C to obtain the second single microbial fermentation agent;

[0118] The fermentation medium is the same as that of the fermentation medium in the first step of preparing the first single microbial fermentation agent;

[0119] The polysaccharide particles are the same as those of the polysaccharide particles in the first step of preparing the first single microbial fermentation agent;

[0120] The particle size of the vermiculite powder is 400 mesh;

[0121] The bacterial concentration of the second single microbial fermentation agent is 8.1×10 10 cfu / g;

[0122] 3. Preparation of mixed vermiculite powder: After mixing 28 g of tris (hydroxymethyl) aminomethane and 10 L of water, stir at a stirring speed of 200 rpm for 18 min at room temperature, add concentrated hydrochloric acid to adjust the pH to 8.5, add 72 g of dopamine hydrochloride, continue stirring for 18 min, add 37 g of vermiculite powder, continue stirring for 52 h, centrifuge at a centrifugal speed of 4500 rpm for 6 min, take the precipitate, wash it 4 times with deionized water, and dry it in vacuum to obtain modified vermiculite powder; After mixing 15 g of L-methionine, 15 g of L-valine, 7.5 g of dopamine hydrochloride and 950 mL of water, stir at a stirring speed of 200 rpm for 25 min at room temperature, add all the above modified vermiculite powder, continue stirring for 2 h, centrifuge at a centrifugal speed of 4500 rpm for 6 min, take the precipitate, wash it 4 times with deionized water, and dry it in vacuum to obtain mixed vermiculite powder;

[0123] The particle size of the vermiculite powder is 400 mesh;

[0124] The mass concentration of the concentrated hydrochloric acid is 36%;

[0125] 4. Mixing: Mix the first single microbial fermentation inoculant, the second single microbial fermentation inoculant and the mixed vermiculite powder evenly according to a mass ratio of 100:41:16 to obtain a compound microbial fermentation inoculant.

[0126] This example also provides an application of the compound microbial fermentation inoculant prepared by the foregoing preparation method in preventing and treating root rot.

[0127] Example 3

[0128] A preparation method of a compound microbial fermentation inoculant is as follows:

[0129] 1. Preparation of the first single microbial fermentation inoculant:

[0130] (1) Primary activation: Add 0.5 mL of NA liquid medium into the freeze-dried tube of Paenibacillus polymyxa freeze-dried powder, shake until fully dissolved to obtain a Paenibacillus polymyxa bacterial suspension; Take 0.2 mL of the Paenibacillus polymyxa bacterial suspension and spread it on the NA plate medium, and culture it at a temperature of 31 °C for 25 h to obtain the activated Paenibacillus polymyxa freeze-dried powder;

[0131] The preparation method of the NA liquid medium is: Take 11 g of peptone, 5.2 g of sodium chloride, 3.2 g of beef extract and 5.2 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.2, and sterilize it at 121 °C for 20 min to obtain the NA liquid medium;

[0132] The preparation method of the NA plate medium is as follows: Take 22 g of agar, 11 g of peptone, 5.2 g of sodium chloride, 3.2 g of beef extract, and 5.2 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.2, and sterilize at 121 °C for 20 min to obtain the NA plate medium;

[0133] (2) Primary expansion culture: Pick a single colony from the activated freeze-dried powder of Paenibacillus polymyxa and inoculate it into 120 mL of seed medium, and culture it at a temperature of 31 °C and a rotation speed of 160 rpm for 25 h to obtain the Paenibacillus polymyxa seed culture solution;

[0134] The preparation method of the seed medium is as follows: Take 10.5 g of sucrose, 10.5 g of tryptone, 10.5 g of sodium chloride, 10.5 g of yeast extract, 3.2 g of beef extract, and 5.2 mg of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.2, and sterilize at 121 °C for 20 min to obtain the seed medium;

[0135] (3) Primary fermentation culture: Inoculate the Paenibacillus polymyxa seed culture solution into 10 L of fermentation medium according to an inoculation amount of 3.5%, and culture it at a temperature of 31 °C and a rotation speed of 160 rpm for 31 h, and concentrate it in vacuo to 25% of the original volume to obtain the Paenibacillus polymyxa concentrate. Add vermiculite powder to the Paenibacillus polymyxa concentrate, control the mass ratio of the Paenibacillus polymyxa concentrate to vermiculite powder to be 100:23, and perform spray drying, control the inlet air temperature to be 155 °C, and the outlet air temperature to be 80 °C to obtain the first single microbial fermentation inoculant;

[0136] The preparation method of the fermentation medium is as follows: Take 65 g of corn flour, 20 g of sucrose, 12 g of soybean meal powder, 6.6 g of polysaccharide particles, 5.2 g of tryptone, 0.8 g of yeast extract, 0.5 g of beef extract, 1.6 g of sodium chloride, 1.6 g of disodium hydrogen phosphate dodecahydrate, 0.4 g of calcium nitrate tetrahydrate, and 0.06 g of manganese sulfate monohydrate, add 1000 mL of purified water, adjust the pH to 7.2, and sterilize at 121 °C for 20 min to obtain the fermentation medium;

[0137] The preparation method of the polysaccharide particles is as follows: After mixing 10 g of chitosan and 550 mL of acetic acid aqueous solution with a mass concentration of 2%, stir at a stirring speed of 300 rpm for 30 min at room temperature, and perform spray drying. Control the inlet air temperature at 220 °C and the outlet air temperature at 90 °C to obtain chitosan microspheres; After mixing 10 g of genipin and 800 mL of water, stir at a stirring speed of 400 rpm for 2 h at 42 °C, add all the above chitosan microspheres, continue stirring for 5 h, centrifuge at a centrifugal speed of 10000 rpm for 6 min, and take the precipitate to obtain crosslinked chitosan microspheres; After mixing 13 g of L-serine, 20 g of L-valine and 800 mL of water, stir at a stirring speed of 200 - 300 rpm for 20 - 30 min at room temperature, add all the above crosslinked chitosan microspheres, continue stirring for 3 h, centrifuge at a centrifugal speed of 10000 rpm for 6 min, take the precipitate, wash 4 times with deionized water, and dry in vacuum to obtain polysaccharide particles;

[0138] The weight-average molecular weight of the chitosan is 150,000, and the degree of deacetylation is 90%;

[0139] In the primary fermentation culture and the preparation of polysaccharide particles, the particle size of the vermiculite powder is 400 mesh;

[0140] The bacterial concentration of the first single microbial fermentation inoculant is 7.2×10 10 cfu / g;

[0141] 2. Preparation of the second single microbial fermentation inoculant:

[0142] (1) Secondary activation: Add 0.5 mL of NA liquid medium into the freeze-dried tube of Bacillus firmus freeze-dried powder, shake until fully dissolved to obtain a Bacillus firmus bacterial suspension; Take 0.2 mL of the Bacillus firmus bacterial suspension and spread it on the NA plate medium, and culture it at a temperature of 31 °C for 25 h to obtain the activated Bacillus firmus freeze-dried powder;

[0143] The preparation method of the NA liquid medium is the same as that of the NA liquid medium in the first step of preparing the first single microbial fermentation inoculant;

[0144] The preparation method of the NA plate medium is the same as that of the NA plate medium in the first step of preparing the first single microbial fermentation inoculant;

[0145] (2) Secondary expansion culture: Pick a single colony from the activated Bacillus firmus freeze-dried powder and inoculate it into 120 mL of seed medium, and culture 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;

[0146] The preparation method of the seed culture medium is the same as that of the seed culture medium in the first step of preparing the first single microbial fermentation agent;

[0147] (3) Secondary fermentation culture: Inoculate the Bacillus firmus seed culture solution into 10 L of fermentation medium at an inoculation amount of 3.5%, and culture it for 35 h at a temperature of 31 °C and a rotation speed of 160 rpm, and then concentrate it in vacuo to 25% of the original volume to obtain a concentrated Bacillus firmus solution. Add vermiculite powder to the concentrated Bacillus firmus solution, control the mass ratio of the concentrated Bacillus firmus solution to vermiculite powder to be 100:26, and perform spray drying, control the inlet air temperature to be 155 °C and the outlet air temperature to be 80 °C to obtain the second single microbial fermentation agent;

[0148] The preparation method of the fermentation medium is the same as that of the fermentation medium in the first step of preparing the first single microbial fermentation agent;

[0149] The preparation method of the polysaccharide particles is the same as that of the polysaccharide particles in the first step of preparing the first single microbial fermentation agent;

[0150] The particle size of the vermiculite powder is 400 mesh;

[0151] The bacterial concentration of the second single microbial fermentation agent is 10.3×10 10 cfu / g;

[0152] 3. Preparation of mixed vermiculite powder: Mix 30 g of tris(hydroxymethyl)aminomethane and 10 L of water, stir at a stirring speed of 300 rpm at room temperature for 20 min, add concentrated hydrochloric acid to adjust the pH to 9, add 75 g of dopamine hydrochloride, continue stirring for 20 min, add 40 g of vermiculite powder, continue stirring for 55 h, centrifuge at a centrifugation speed of 5000 rpm for 6 min, take the precipitate, wash it 5 times with deionized water, and dry it in vacuo to obtain modified vermiculite powder; Mix 15 g of L-methionine, 15 g of L-valine, 8 g of dopamine hydrochloride and 1000 mL of water, stir at a stirring speed of 300 rpm at room temperature for 30 min, add all the above modified vermiculite powder, continue stirring for 2 h, centrifuge at a centrifugation speed of 5000 rpm for 6 min, take the precipitate, wash it 5 times with deionized water, and dry it in vacuo to obtain mixed vermiculite powder;

[0153] The particle size of the vermiculite powder is 400 mesh;

[0154] The mass concentration of the concentrated hydrochloric acid is 36%;

[0155] 4. Mixing: Mix the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder evenly according to a mass ratio of 100:42:16 to obtain a composite microbial fermentation agent.

[0156] This embodiment also provides an application of the composite microbial fermentation inoculant prepared by the foregoing preparation method in preventing and treating root rot.

[0157] Example 4

[0158] The purpose of this embodiment is to verify the role of polysaccharide particles. Therefore, on the basis of the preparation method of the composite microbial fermentation inoculant in Example 2, in the (3) primary fermentation culture step in the first step of preparing the first single microbial fermentation inoculant, the addition of polysaccharide particles is omitted in the preparation method of the fermentation medium;

[0159] In the (3) secondary fermentation culture step in the second step of preparing the second single microbial fermentation inoculant, the addition of polysaccharide particles is omitted in the preparation method of the fermentation medium;

[0160] The remaining operations remain unchanged.

[0161] The bacterial concentration of the first single microbial fermentation inoculant obtained in the first step of preparing the first single microbial fermentation inoculant in this embodiment is 7.5×10 7 cfu / g;

[0162] The bacterial concentration of the second single microbial fermentation inoculant obtained in the second step of preparing the second single microbial fermentation inoculant in this embodiment is 1.0×10 8 cfu / g.

[0163] This embodiment also provides an application of the composite microbial fermentation inoculant prepared by the foregoing preparation method in preventing and treating root rot.

[0164] Example 5

[0165] 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 inoculant in Example 4, in the (3) primary fermentation culture step in the first step of preparing the first single microbial fermentation inoculant, the fermentation time is extended from 30 h to 45 h;

[0166] In the (3) secondary fermentation culture step in the second step of preparing the second single microbial fermentation inoculant, the fermentation time is extended from 35 h to 52.5 h;

[0167] The remaining operations remain unchanged.

[0168] The bacterial concentration of the first single microbial fermentation inoculant obtained in the first step of preparing the first single microbial fermentation inoculant in this embodiment is 5.2×10 10 cfu / g;

[0169] In this embodiment, the bacterial concentration of the second single microbial fermentation inoculant obtained in the second step of preparing the second single microbial fermentation inoculant is 7.4×10 10 cfu / g.

[0170] This embodiment also provides an application of the composite microbial fermentation inoculant prepared by the foregoing preparation method in preventing and treating root rot.

[0171] Example 6

[0172] The function of this embodiment is to verify the function of the mixed vermiculite powder. Therefore, on the basis of the preparation method of the composite microbial fermentation inoculant in Example 2, the third step of preparing the mixed vermiculite powder is omitted, and in the fourth step of mixing, the addition of the mixed vermiculite powder is omitted, that is, the fourth step of the mixing step is changed to:

[0173] Mix the first single microbial fermentation inoculant and the second single microbial fermentation inoculant evenly according to a mass ratio of 100:41 to obtain a composite microbial fermentation inoculant.

[0174] This embodiment also provides an application of the composite microbial fermentation inoculant prepared by the foregoing preparation method in preventing and treating root rot.

[0175] Test Example 1

[0176] Fusarium solani was isolated and screened from the eggplant roots infected with root rot. Seven mycelial blocks were taken from the edge of the Fusarium solani colony with a sterile punch with a diameter of 4 mm and inoculated in the center of a PDA plate medium with a diameter of 9 cm, numbered 1-7. The 1-6th media were used as test media. On the upper, lower, left, and right sides of the central mycelial block in the 1-6th media, 1 g of the composite microbial fermentation inoculant prepared in Examples 1-6 was added at a distance of 1 cm from the edge of the petri dish. The 7th medium was used as a control medium without any treatment. The 1-7th media were cultured under natural light at a temperature of 25°C. When the Fusarium solani in the 7th medium, that is, the control medium, grew to the edge, the radius of the Fusarium solani colony in the 1-6th media, that is, the test media, was measured, and the inhibition rate was calculated. The calculation formula and calculation results are as follows:

[0177] Inhibition rate = (radius of the Fusarium solani colony in the control medium - radius of the Fusarium solani colony in the test medium) / radius of the Fusarium solani colony in the control medium × 100%;

[0178]

[0179] It can be seen from the above results that the antibacterial rate of the compound microbial fermentation inoculant prepared in Example 2 is higher than that of the compound microbial fermentation inoculants prepared in Examples 4-6, indicating that the compound microbial fermentation inoculant prepared in Example 2 has a better rapid antibacterial effect against Fusarium solani, and further indicating that the compound microbial fermentation inoculant prepared in Example 2 has a good short-term treatment effect on plants already infected with root rot.

[0180] Test Example 2

[0181] On the basis of Test Example 1, Fusarium solani was replaced with Fusarium solani f. sp. cucurbitae isolated and screened from the ginger roots infected with root rot, and then the test was carried out by the method of Test Example 1. The calculation results of the antibacterial rate are as follows:

[0182]

[0183] It can be seen from the above results that the antibacterial rate of the compound microbial fermentation inoculant prepared in Example 2 is higher than that of the compound microbial fermentation inoculants prepared in Examples 4-6, indicating that the compound microbial fermentation inoculant prepared in Example 2 has a better rapid antibacterial effect against Fusarium solani f. sp. cucurbitae, and further indicating that the compound microbial fermentation inoculant prepared in Example 2 can achieve good control effects on eggplant root rot and ginger root rot.

[0184] Test Example 3

[0185] On the basis of Test Example 1, the temperature was increased from 25 °C to 45 °C, and then the test was carried out by the method of Test Example 1. The calculation results of the antibacterial rate are as follows:

[0186]

[0187] It can be seen from the above results that the antibacterial rate of the compound microbial fermentation inoculant prepared in Example 2 is higher than that of the compound microbial fermentation inoculants prepared in Examples 4-6, indicating that the control effect of the compound microbial fermentation inoculant prepared in Example 2 is less affected by temperature.

[0188] Test Example 4

[0189] The compound microbial fermentation inoculants prepared in Examples 1-6 were left standing for 10 d under natural light at 45 °C to obtain the compound microbial fermentation inoculants after standing treatment. Then, in an environment of 45 °C, the compound microbial fermentation inoculants after standing treatment were tested by the method of Test Example 1. The calculation results of the antibacterial rate are as follows:

[0190]

[0191] It can be seen from the above results that the antibacterial rate of the compound microbial fermentation inoculant prepared in Example 2 is higher than that of the compound microbial fermentation inoculants prepared in Examples 4-6, indicating that the shelf life of the compound microbial fermentation inoculant prepared in Example 2 is less affected by temperature.

[0192] Test Example 5

[0193] Select a ginger planting area in Weifang City, Shandong Province, and demarcate 7 mu as the test ginger planting area. Divide the test ginger planting area into 7 equal parts, with each part having an area of 1 mu. They are numbered as Ginger Planting Areas 1-7 respectively, and physical isolation is carried out between Ginger Planting Areas 1-7 to avoid the spread of diseases.

[0194] Ensure that the planting time and planting density of ginger in Ginger Planting Areas 1-7 are the same, and the fertilizer and water management after planting are also the same. Take Ginger Planting Areas 1-6 as the test ginger planting areas. Six weeks after ginger planting, mix the compound microbial fermentation inoculants prepared in Examples 1-6 with water to make a dispersion with a mass concentration of 1%. Then, use the dispersions prepared from the compound microbial fermentation inoculants in Examples 1-6 to carry out root irrigation treatment on Ginger Planting Areas 1-6. In the root irrigation treatment, the dosage of the dispersion for each ginger plant is 500 mL. Take Ginger Planting Area 7 as the control ginger planting area and do not use any fungicides.

[0195] Sixty days after the root irrigation treatment, count the number of diseased plants with root rot in Ginger Planting Areas 1-7, calculate the incidence rate of root rot, and then calculate the control rate against root rot. The calculation formula and calculation results of the control rate against root rot are as follows:

[0196] Control rate against root rot = (Incidence rate of root rot in the control ginger planting area - Incidence rate of root rot in the test ginger planting area) / Incidence rate of root rot in the control ginger planting area × 100%;

[0197]

[0198] It can be seen from the above results that the control rate of the compound microbial fermentation inoculant prepared in Example 2 is higher than that of the compound microbial fermentation inoculants prepared in Examples 4-6, indicating that the compound microbial fermentation inoculant prepared in Example 2 can continuously play a bactericidal role after root irrigation treatment, further indicating that the long-term control effect of the compound microbial fermentation inoculant prepared in Example 2 is good.

[0199] Unless otherwise specified, the percentages used in the present invention are all mass percentages.

[0200] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within 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 microorganism used in the preparation of the first single microorganism fermentation agent is Bacillus polymyxa, and the strain number is BNCC 138393; 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 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; mixing L-serine and 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; The microorganism used in the preparation of the second single microorganism fermentation agent is Bacillus firmus, and the strain number is BNCC 132595; 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 a precipitate, washing, vacuum drying, and obtaining modified vermiculite powder; L-methionine, L-valine, the second portion of dopamine hydrochloride and the second portion of water are mixed, stirring at room temperature, adding modified vermiculite powder, continuing to stir, centrifuging, taking a precipitate, washing, vacuum drying, and obtaining mixed vermiculite powder; The mixing is to uniformly mix the first single microbial fermentation agent, the second single microbial fermentation agent, and the mixed vermiculite powder to obtain a composite microbial fermentation agent.

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 primary activation, the NA liquid culture medium is prepared by taking peptone, sodium chloride, beef extract and manganese sulfate monohydrate, adding purified water, adjusting the pH to 7-7.2, and sterilizing to 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 concentrated solution of Paenibacillus polymyxa 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: 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%.

6. 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 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; 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 microorganism fermentation agent.

7. The method for preparing the composite microbial fermentation agent according to claim 6, 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.

8. 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%; 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.

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