Microbial agent for preventing and treating peanut root rot and preparation method thereof

Abstract

The invention discloses a microbial agent for preventing and treating peanut root rot and a preparation method thereof, and belongs to the technical field of microbial materials. A strain of trichoderma harzianum with an efficient inhibiting effect on pathogenic bacteria of peanut root rot is obtained through screening, firstly, the strain of trichoderma harzianum has an antagonistic effect on the pathogenic bacteria of peanut, secondly, the trichoderma harzianum is prepared into a spore suspension, and the spore suspension is subjected to solid fermentation with rice husks and wheat bran. Metabolite produced by the trichoderma harzianum in the solid-state fermentation process in the fermentation process can promote the growth and development of plant root systems and improve the biomass and yield of plants. On one hand, the microbial agent disclosed by the invention effectively prevents and treats peanut root rot, has a good inhibition effect on pathogenic bacteria of the peanut root rot, and improves the quality and disease resistance of peanuts; on the other hand, the ecological activity of soil is improved to a certain extent, and application and popularization are worthy.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial materials, and particularly relates to a microbial agent for preventing and treating peanut root rot and a preparation method thereof. Background Art

[0002] Peanut (Arachis hypogaea) is the world's fourth largest oil crop and the thirteenth largest food source for humans. It is also an important source of high-quality plant protein and oil. Peanut is one of the important economic and oil crops. It is rich in fat, protein, dietary fiber and a variety of micronutrients and is deeply loved by the public.

[0003] Peanut root rot, also known as "rat tail" or "rotten root", is a fungal soil-borne disease. It seriously affects the yield and quality of peanuts. Peanut root rot is mostly caused by infection with Fusarium fungi such as Fusarium solani and Fusarium oxysporum. Root rot can occur throughout the growth period of peanuts, mainly harming the roots and vascular bundles of peanuts, causing the roots to turn brown and rot, followed by the vascular bundles turning brown and rotting, causing poor plant growth, and then causing the whole plant to die. Late infection can harm the pods and cause fruit rot, resulting in the death of most or even all plants, resulting in missing seedlings and broken ridges. It causes serious economic losses and seriously restricts the development of the peanut industry.

[0004] At present, the prevention and control of peanut root rot mainly relies on chemical pesticides, which not only causes drug resistance of pathogenic bacteria, but also causes damage to the entire agricultural ecosystem, causing huge harm.

[0005] For example, Chinese patent application CN201810904875.2 proposes a medicament for preventing and treating peanut root rot, and its preparation method and use method, the medicament includes Chinese medicine antibacterial components and trace element nutrient solution components, the Chinese medicine antibacterial components include honeysuckle, turmeric, tangerine peel, cinnamon, cloves, borneol, perilla, wormwood, sophora flavescens, siler, cardamom, Xanthium sibiricum, pinellia, coptis root, platycodon and other Chinese medicine components; each 1L of the trace element nutrient solution component includes 0.8-1.2g ferrous sulfate, 0.8-1.2g zinc sulfate, 0.1-0.4g copper sulfate, and the rest of water. The Chinese medicine antibacterial components of the present invention are combined with the trace element nutrient solution components to enable peanuts to smoothly pass the high-risk period of root rot, thereby reducing the incidence of peanut root rot.

[0006] Chinese patent application CN202410879433.2 discloses a special agent for the prevention and treatment of peanut root rot and its preparation method, which belongs to the field of crop planting technology. The special agent for the prevention and treatment of peanut root rot of the present invention comprises the following raw materials by mass: 20-30 parts of 50% carbendazim, 10-15 parts of volatile oil, and 20-30 parts of plant extract; the raw materials for the preparation of volatile oil include: dandelion and grapefruit peel; the raw materials for the preparation of plant extract include: Gleditsia sinensis, Agaricus lemaneiformis, Ginkgo biloba leaves and Houttuynia cordata. The special agent for the prevention and treatment of peanut root rot of the present invention can significantly reduce the incidence of peanut root rot and increase the yield of peanuts.

[0007] Most of these existing technologies use a large amount of traditional Chinese medicine or chemical drug ingredients, which can easily cause crop resistance and cause serious pollution to the soil environment, and the actual application effect is not good.

[0008] Therefore, developing technologies that use functional microorganisms to control plant diseases that are environmentally friendly and safe for people's health has gradually become the focus and focus of attention of the majority of scientific researchers, and is also a technical problem that urgently needs to be solved. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention screens out a highly effective antibacterial and growth-promoting Trichoderma harzianum from the rhizosphere soil of strawberries, and solid-state ferments the Trichoderma harzianum together with biocontrol bacteria to form a microbial preparation, which can effectively prevent and control peanut root rot pathogens, enhance peanut resistance, regulate soil ecological activity, and effectively improve peanut quality.

[0010] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0011] A microbial agent for preventing and treating peanut root rot comprises the following raw materials: fungal fermentation products, biocontrol bacteria and regulator components.

[0012] Furthermore, the mass ratio of the fungal fermentation product, the biocontrol bacteria, and the regulator component is 1:1:(0.1-0.5).

[0013] Furthermore, the preparation method of the fungal fermentation product is:

[0014] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated on a PDA solid medium. After culturing for 5 days, 10 mL of a 0.9% NaCl solution was added to the surface of the plate. The spores on the surface of the plate were scraped 10 times with a sterile stick. The spores in the plate were then rinsed into a 150 mL conical flask containing glass beads with sterile water. The plate was placed in a shaker at 35-37° C. and 180-200 rpm for 30-40 min to break the mycelium. The suspension was then filtered using sterilized gauze and the filtrate was prepared to have a spore count of 1×10 9 cfu / mL of Trichoderma spore suspension for later use;

[0015] (2) Preparation of solid fermentation material: Rice husk and wheat bran are mixed evenly in a mass ratio of 4:6, and water is added, the amount of water added is 120% of the mass of the mixture, and the mixture is stirred evenly. The mixture is placed in a sterilizing pot at 121° C. for 30 min, and the mixture is naturally cooled after sterilization to obtain a base material. Corn flour and ammonium sulfate are added to the base material, and the mixture is mixed evenly to obtain a solid fermentation material.

[0016] (3) The Trichoderma spore suspension of step (1) was evenly sprayed on the solid fermentation material at an inoculum amount of 3%, stirred evenly, and placed in a 28-35° C. incubator for cultivation. Starting from the second day, water was added and the material was turned over every 24 hours until the Trichoderma spore production was greater than 5×10 9 cfu / g, the fermentation is ended and the fermented material can be dried at room temperature.

[0017] Furthermore, the preservation number of the Trichoderma harzianum described in step (1) is CGMCC No.40702, the preservation date is June 16, 2023, and it is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] The screening and isolation method of Trichoderma harzianum is as follows: take strawberry rhizosphere soil, weigh 100g of soil into a 1000mL triangular flask, add 500mL of sterile water, and incubate at 25-37℃ and 200r·min -1 After shaking for 30 minutes, centrifuge and collect the supernatant. Take 0.5 mL of the supernatant diluted 10 times and 100 times respectively, add it to the pre-prepared Trichoderma selection medium plate, and spread it evenly with a coating stick. Repeat 20 times for each dilution and culture at 28℃ for 3-5 days. Pick single fungal colonies of different shapes for purification and culture, repeat several times until a purified single colony is obtained, pick a small amount of mycelium, use 40% glycerol as a freezing solution and store it in a -80℃ refrigerator for later use.

[0019] The Trichoderma selection medium is sodium propionate medium: 200 g potato, 15-20 g agar powder, 20 g glucose, 0.05 g gentamicin, 0.05 g sodium propionate, and 1000 mL distilled water.

[0020] Plate confrontation method: Taking Fusarium solani, the pathogen of peanut root rot, as the target, the two-point confrontation culture method was adopted, and 5mm purified antagonistic Trichoderma and pathogen cakes were inoculated at symmetrical positions 2.50cm away from the center of the plate; 3 parallels were set up for each group, and a pathogen control group was set up at the same time. Cultured in a constant temperature incubator at 28-35℃, and the colonies were observed from the next day until the hyphae were about to touch the edge of the plate. Observe whether the inhibition zone appears, record and measure the strain radius of the experimental group and the control group, and calculate the inhibition rate: Inhibition rate = (pathogen radius of the control group - pathogen radius of the treatment group) / (pathogen radius of the control group - 2.5) × 100%, where 2.5 is the radius of the pathogen block of the control group. The strain with the largest inhibition rate is the target strain-Trichoderma harzianum. The antibacterial effect diagram is as follows Figure 1 As shown. This Trichoderma grows faster when cultured on PDA plates and can cover the entire plate on the third day. The hyphae are white, felt-like, and slightly raised. Figure 2 .

[0021] Furthermore, the PDA solid culture medium in step (1) is composed of the following components by mass: 150-200 g of potato, 15-20 g of glucose, and 10-15 g of agar.

[0022] Furthermore, in step (2), the amount of corn flour and ammonium sulfate added is 1% of the mass of the base material.

[0023] Furthermore, the biocontrol bacteria include Bacillus, Pseudomonas and Actinomycetes.

[0024] Furthermore, the Bacillus is Bacillus subtilis, the strain number is CGMCC No.1.7740, and the original deposit date is July 24, 209; the Pseudomonas is Pseudomonas fluorescens (

[0025] The present invention discloses a novel actinomycete, wherein the strain number of the present invention is CGMCC No. 1.4531, and the original deposit date is June 27, 2005; the actinomycete is Actinomadura chibensis, and the strain number of the present invention is CGMCC No. 4.6236, and the original deposit date is September 3, 2008; the volume ratio of the three is 2:1:3. The Bacillus subtilis, Pseudomonas fluorescens and Actinomadura chibensis used in the present application can be purchased through the preservation center without repeated biological preservation.

[0026] Furthermore, the preparation method of the biocontrol bacteria is: Bacillus subtilis, Pseudomonas fluorescens, and Actinomycetes chibaensis are inoculated into LB medium respectively, and cultured at 28-30°C with shaking until the bacterial concentration reaches OD 600 ≈2.0, and then mixed in a volume ratio of 2:1:3 and spray-dried to obtain the result.

[0027] Furthermore, the weight ratio of each component of the LB medium is: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride, 12-18 g of agar powder, and 1000 mL of water.

[0028] Furthermore, the regulator component comprises diatomaceous earth, dextrin, sodium humate and xanthan gum, and the mass ratio of the four is 10:1:3:1.

[0029] A method for preparing a microbial agent for preventing and controlling peanut root rot comprises the following preparation steps:

[0030] (1) preparing fungal fermentation products;

[0031] (2) preparing biocontrol bacteria;

[0032] (3) After the fungal fermentation product, biocontrol bacteria, and regulator components are fully mixed in a mass ratio of 1:1:(0.1-0.5), granulation is performed in a coating machine. The coating machine speed is set to 50 r / min and stirred for 30 minutes. After the materials are fully mixed, water or a binder is used to granulate the materials. After granulation, the materials are dried to obtain the target bacterial agent.

[0033] The binder is one or more of carboxymethyl cellulose, polyvinyl alcohol or attapulgite.

[0034] The effective live bacteria count of the microbial agent of the present invention is about 5-10 billion CFU / g. The method of use is to add an appropriate amount of the microbial agent to each sowing hole during sowing, and then cover the hole with soil. The dosage of the microbial agent is 5-10 kg / mu.

[0035] Beneficial effects:

[0036] (1) The present invention screened out a strain of Trichoderma harzianum that has a highly effective inhibitory effect on peanut root rot pathogens. First, the strain itself has an antagonistic effect on peanut pathogens. Secondly, we prepared it into a spore suspension and carried out solid fermentation with rice husks and wheat bran. The metabolites produced by Trichoderma harzianum during the solid fermentation process can promote the growth and development of plant roots and increase plant biomass and yield; and conidia can be produced in large quantities through solid fermentation. These spores can inhibit the growth of a variety of plant pathogens and inhibit the reproduction of harmful bacteria through mechanisms such as heavy parasitism and antimicrobial peptides. In addition, Trichoderma harzianum can also induce disease resistance in plants and improve the resistance of plants to pathogens;

[0037] (2) In combination with Trichoderma harzianum, the present invention also adds three biocontrol bacteria of different genera: the screened Bacillus subtilis of the genus Bacillus can inhibit the growth of pathogens by producing secondary metabolites such as antibiotics, enzymes, and iron carriers, and can also promote plant growth and induce plant systemic resistance; the screened Pseudomonas fluorescens of the genus Pseudomonas can produce a variety of volatile antibacterial substances, which have inhibitory effects on a variety of plant pathogens; and the screened Actinomycetes Madura chibae can enhance the resistance of plants to pathogens by inducing plant immune responses. For example, it can promote plant growth, reduce oxidative stress caused by diseases, and inhibit the growth of pathogens by changing plant immune responses. The three biocontrol bacteria of different genera are used together, in combination with the fermentation product of Trichoderma harzianum, to show a highly effective prevention and control effect on peanut root rot, promote the growth of peanut roots, improve the quality of peanuts, and improve the soil ecological environment;

[0038] (3) Use diatomaceous earth, dextrin, sodium humate and xanthan gum as microorganisms and soil conditioners. Diatomaceous earth can provide good microenvironmental conditions for soil microorganisms and promote their growth and reproduction; as a carbon source, dextrin can provide energy for microorganisms and promote their growth and metabolic activities; sodium humate can improve soil structure, increase soil permeability and water retention capacity, and thus provide a better living environment for rhizosphere microorganisms. Sodium humate can also promote soil microbial activity, increase the number of spontaneous nitrogen-fixing bacteria in the soil, enrich nitrogen nutrition, and improve the nutritional conditions of crop roots. Xanthan gum helps to improve the colonization ability and functional performance of microorganisms in the soil.

[0039] (4) In summary, the microbial agent of the present invention, on the one hand, effectively prevents and treats peanut root rot, has a good inhibitory effect on the pathogenic bacteria of peanut root rot, and improves the quality and disease resistance of peanuts. On the other hand, it improves the ecological activity of the soil to a certain extent, and is worthy of application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram showing the antibacterial effect of Trichoderma harzianum of the present invention;

[0041] Figure 2 This is a state diagram of Trichoderma harzianum of the present invention when it is cultured on a PDA plate;

[0042] Figure 3 The results of polyphenol oxidase assay in soils of different experimental groups;

[0043] Figure 4 The results of sucrase determination of soil in different experimental groups;

[0044] Figure 5 The results of sucrase determination of soil in different experimental groups;

[0045] Figure 6 These are the results of catalase determination in soils of different experimental groups. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0047] Example 1

[0048] A microbial agent for preventing and treating peanut root rot comprises the following raw materials: fungal fermentation products, biocontrol bacteria and regulator components.

[0049] The mass ratio of the fungal fermentation product, the biocontrol bacteria and the regulator component is 1:1:0.1.

[0050] The preparation method of the fungal fermentation product is as follows:

[0051] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated on a PDA solid medium. After culturing for 5 days, 10 mL of a 0.9% NaCl solution was added to the surface of the plate. The spores on the surface of the plate were scraped 10 times with a sterile stick. The spores in the plate were then rinsed into a 150 mL conical flask containing glass beads with sterile water. The plate was placed in a shaker at 35-37° C. and 180-200 rpm for 30-40 min to break the mycelium. The suspension was then filtered using sterilized gauze and the filtrate was prepared to have a spore count of 1×10 9 cfu / mL of Trichoderma spore suspension for later use;

[0052] (2) Preparation of solid fermentation material: Rice husk and wheat bran are mixed evenly in a mass ratio of 4:6, and water is added, the amount of water added is 120% of the mass of the mixture, and the mixture is stirred evenly. The mixture is placed in a sterilizing pot at 121° C. for 30 min, and the mixture is naturally cooled after sterilization to obtain a base material. Corn flour and ammonium sulfate are added to the base material, and the mixture is mixed evenly to obtain a solid fermentation material.

[0053] (3) The Trichoderma spore suspension of step (1) was evenly sprayed on the solid fermentation material at an inoculum amount of 3%, stirred evenly, and placed in a 28-35° C. incubator for cultivation. Starting from the second day, water was added and the material was turned over every 24 hours until the Trichoderma spore production was greater than 5×10 9 cfu / g, the fermentation is ended and the fermented material can be dried at room temperature.

[0054] The deposit number of the Trichoderma harzianum in step (1) is CGMCC No. 40702, the deposit date is June 16, 2023, and it is deposited in the General Microbiological Center of the China Culture Collection Administration, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0055] The screening and isolation method of Trichoderma harzianum is as follows: take strawberry rhizosphere soil, weigh 100g of soil into a 1000mL triangular flask, add 500mL of sterile water, and incubate at 25-37℃ and 200r·min -1 After shaking for 30 minutes, centrifuge and collect the supernatant. Take 0.5 mL of the supernatant diluted 10 times and 100 times respectively, add it to the pre-prepared Trichoderma selection medium plate, and spread it evenly with a coating stick. Repeat 20 times for each dilution and culture at 28℃ for 3-5 days. Pick single fungal colonies of different shapes for purification and culture, repeat several times until a purified single colony is obtained, pick a small amount of mycelium, use 40% glycerol as a freezing solution and store it in a -80℃ refrigerator for later use.

[0056] The Trichoderma selection medium is sodium propionate medium: 200 g potato, 15-20 g agar powder, 20 g glucose, 0.05 g gentamicin, 0.05 g sodium propionate, and 1000 mL distilled water.

[0057] Plate confrontation method: Taking Fusarium solani, the pathogen of peanut root rot, as the target, the two-point confrontation culture method was adopted, and 5mm purified antagonistic Trichoderma and pathogen cakes were inoculated at symmetrical positions 2.50cm away from the center of the plate; 3 parallels were set up for each group, and a pathogen control group was set up at the same time. Cultured in a constant temperature incubator at 28-35℃, and the colonies were observed from the next day until the hyphae were about to touch the edge of the plate. Observe whether the inhibition zone appears, record and measure the strain radius of the experimental group and the control group, and calculate the inhibition rate: Inhibition rate = (pathogen radius of the control group - pathogen radius of the treatment group) / (pathogen radius of the control group - 2.5) × 100%, where 2.5 is the radius of the pathogen block of the control group. The strain with the largest inhibition radius is the target strain-Trichoderma harzianum. The antibacterial effect diagram is as follows Figure 1 As shown. This Trichoderma grows faster when cultured on PDA plates and can cover the entire plate on the third day. The hyphae are white, felt-like, and slightly raised. Figure 2 .

[0058] Step (1) PDA solid culture medium is composed of the following components by mass: 150-200 g of potato, 15-20 g of glucose, and 10-15 g of agar.

[0059] In step (2), the added amount of corn flour and ammonium sulfate is 1% of the mass of the base material.

[0060] The biocontrol bacteria include Bacillus, Pseudomonas and Actinomycetes.

[0061] The bacillus is Bacillus subtilis, with a strain number of CGMCC No. 1.7740 and an original deposit date of July 24, 2009; the pseudomonad is Pseudomonas fluorescens, with a strain number of CGMCC No. 1.4531 and an original deposit date of June 27, 2005; the actinomycete is Actinomadura chibensis, with a strain number of CGMCC No. 4.6236 and an original deposit date of September 3, 2008; and the volume ratio of the three is 2:1:3. The Bacillus subtilis, Pseudomonas fluorescens and Actinomadura chibensis used in this application can all be purchased through the preservation center without the need for repeated biological preservation.

[0062] The preparation method of the biocontrol bacteria is as follows: Bacillus subtilis, Pseudomonas fluorescens, and Actinomycetes chibaensis are inoculated into LB culture medium respectively, and cultured at 28-30° C. with shaking until the bacterial concentration reaches OD 600 ≈2.0, and then mixed in a volume ratio of 2:1:3 and spray-dried to obtain the result.

[0063] The weight ratio of each component of LB medium is: tryptone 8-12g, yeast extract 4-6g, sodium chloride 4-6g, agar powder 12-18g, water 1000mL.

[0064] The regulator component comprises diatomaceous earth, dextrin, sodium humate and xanthan gum, and the mass ratio of the four is 10:1:3:1.

[0065] A method for preparing a microbial agent for preventing and controlling peanut root rot comprises the following preparation steps:

[0066] (1) preparing fungal fermentation products;

[0067] (2) preparing biocontrol bacteria;

[0068] (3) After the fungal fermentation product, biocontrol bacteria, and regulator components are fully mixed in a mass ratio of 1:1:0.1, granulation is performed in a coating machine. The coating machine speed is set to 50 r / min and stirred for 30 minutes. After the materials are fully mixed, water or a binder is used to granulate the materials. After granulation, the materials are dried to obtain the target bacterial agent.

[0069] The binder is carboxymethyl cellulose.

[0070] Example 2

[0071] A microbial agent for preventing and treating peanut root rot comprises the following raw materials: fungal fermentation products, biocontrol bacteria and regulator components.

[0072] The mass ratio of the fungal fermentation product, the biocontrol bacteria and the regulator component is 1:1:0.5.

[0073] The preparation method of the fungal fermentation product is the same as that in Example 1.

[0074] The screening and separation method of Trichoderma harzianum in step (1) is the same as that in Example 1.

[0075] The selection and preparation methods of the biocontrol bacteria Bacillus subtilis, Pseudomonas fluorescens, and Actinomyces chibaensis are the same as those in Example 1.

[0076] A method for preparing a microbial agent for preventing and controlling peanut root rot comprises the following preparation steps:

[0077] (1) preparing fungal fermentation products;

[0078] (2) preparing biocontrol bacteria;

[0079] (3) After the fungal fermentation product, biocontrol bacteria, and regulator components are fully mixed in a mass ratio of 1:1:0.5, granulation is performed in a coating machine. The coating machine speed is set to 50 r / min and stirred for 30 minutes. After the materials are fully mixed, water or a binder is used to granulate the materials. After granulation, the materials are dried to obtain the target bacterial agent.

[0080] The binder is polyvinyl alcohol.

[0081] Comparative Example 1

[0082] In this comparative example, the strain type of Trichoderma harzianum in the fungal fermentation product was changed, and the remaining raw materials and process steps were the same as those in Example 2. That is:

[0083] A microbial agent for preventing and treating peanut root rot comprises the following raw materials: fungal fermentation products, biocontrol bacteria and regulator components.

[0084] The mass ratio of the fungal fermentation product, the biocontrol bacteria and the regulator component is 1:1:0.5.

[0085] The preparation method of the fungal fermentation product is as follows:

[0086] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated on a PDA solid medium. After culturing for 5 days, 10 mL of a 0.9% NaCl solution was added to the surface of the plate. The spores on the surface of the plate were scraped 10 times with a sterile stick. The spores in the plate were then rinsed into a 150 mL conical flask containing glass beads with sterile water. The plate was placed in a shaker at 35-37° C. and 180-200 rpm for 30-40 min to break the mycelium. The suspension was then filtered using sterilized gauze and the filtrate was prepared to have a spore count of 1×10 9 cfu / mL of Trichoderma spore suspension for later use;

[0087] (2) Preparation of solid fermentation material: Rice husk and wheat bran are mixed evenly in a mass ratio of 4:6, and water is added, the amount of water added is 120% of the mass of the mixture, and the mixture is stirred evenly. The mixture is placed in a sterilizing pot at 121° C. for 30 min, and the mixture is naturally cooled after sterilization to obtain a base material. Corn flour and ammonium sulfate are added to the base material, and the mixture is mixed evenly to obtain a solid fermentation material.

[0088] (3) The Trichoderma spore suspension of step (1) was evenly sprayed on the solid fermentation material at an inoculum amount of 3%, stirred evenly, and placed in a 28-35° C. incubator for cultivation. Starting from the second day, water was added and the material was turned over every 24 hours until the Trichoderma spore production was greater than 5×10 9 cfu / g, the fermentation is ended and the fermented material can be dried at room temperature.

[0089] The Trichoderma harzianum described in step (1) was purchased from a collection center with a collection number of CGMCC No.5.1213.

[0090] Comparative Example 2

[0091] The same as in Comparative Example 1, the strain type of Trichoderma harzianum in the fungal fermentation product was changed, and the other raw materials and process steps were the same as in Example 2. That is:

[0092] The preparation method of the fungal fermentation product is as follows:

[0093] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated on a PDA solid medium. After culturing for 5 days, 10 mL of a 0.9% NaCl solution was added to the surface of the plate. The spores on the surface of the plate were scraped 10 times with a sterile stick. The spores in the plate were then rinsed into a 150 mL conical flask containing glass beads with sterile water. The plate was placed in a shaker at 35-37° C. and 180-200 rpm for 30-40 min to break the mycelium. The suspension was then filtered using sterilized gauze and the filtrate was prepared to have a spore count of 1×10 9 cfu / mL of Trichoderma spore suspension for later use;

[0094] (2) Preparation of solid fermentation material: Rice husk and wheat bran are mixed evenly in a mass ratio of 4:6, and water is added, the amount of water added is 120% of the mass of the mixture, and the mixture is stirred evenly. The mixture is placed in a sterilizing pot at 121° C. for 30 min, and the mixture is naturally cooled after sterilization to obtain a base material. Corn flour and ammonium sulfate are added to the base material, and the mixture is mixed evenly to obtain a solid fermentation material.

[0095] (3) The Trichoderma spore suspension of step (1) was evenly sprayed on the solid fermentation material at an inoculum amount of 3%, stirred evenly, and placed in a 28-35° C. incubator for cultivation. Starting from the second day, water was added and the material was turned over every 24 hours until the Trichoderma spore production was greater than 5×10 9 cfu / g, the fermentation is ended and the fermented material can be dried at room temperature.

[0096] The Trichoderma harzianum described in step (1) was purchased from a collection center with a collection number of CGMCC No.3.15684.

[0097] Comparative Example 3

[0098] In this comparative example, except that Bacillus subtilis is not used as the biocontrol bacteria, the remaining raw materials and process steps are the same as those of Example 1. That is:

[0099] A microbial agent for preventing and treating peanut root rot comprises the following raw materials: fungal fermentation products, biocontrol bacteria and regulator components.

[0100] The mass ratio of the fungal fermentation product, the biocontrol bacteria and the regulator component is 1:1:0.5.

[0101] The preparation method of the fungal fermentation product is the same as that in Example 1.

[0102] The screening and separation method of Trichoderma harzianum in step (1) is the same as that in Example 1.

[0103] The selection and preparation methods of the biocontrol bacteria Pseudomonas fluorescens and Actinomycetes chibaensis are the same as those in Example 1.

[0104] Comparative Example 4

[0105] In this comparative example, except that Pseudomonas fluorescens was not used in the biocontrol bacteria, the other raw materials and process steps were the same as those in Example 1. That is:

[0106] A microbial agent for preventing and treating peanut root rot comprises the following raw materials: fungal fermentation products, biocontrol bacteria and regulator components.

[0107] The mass ratio of the fungal fermentation product, the biocontrol bacteria and the regulator component is 1:1:0.5.

[0108] The preparation method of the fungal fermentation product is the same as that in Example 1.

[0109] The screening and separation method of Trichoderma harzianum in step (1) is the same as that in Example 1.

[0110] The selection and preparation methods of the biocontrol bacteria Bacillus subtilis and Actinomycetes chibaensis are the same as those in Example 1.

[0111] Comparative Example 5

[0112] In this comparative example, except that Actinomycetes chibaensis was not used in the biocontrol bacteria, the rest of the raw materials and process steps were the same as those in Example 1. That is:

[0113] A microbial agent for preventing and treating peanut root rot comprises the following raw materials: fungal fermentation products, biocontrol bacteria and regulator components.

[0114] The mass ratio of the fungal fermentation product, the biocontrol bacteria and the regulator component is 1:1:0.5.

[0115] The preparation method of the fungal fermentation product is the same as that in Example 1.

[0116] The screening and separation method of Trichoderma harzianum in step (1) is the same as that in Example 1.

[0117] The selection and preparation methods of the biocontrol bacteria Bacillus subtilis and Pseudomonas fluorescens are the same as those in Example 1.

[0118] Performance Testing

[0119] Peanut planting experiment:

[0120] The peanut variety used for the test was “Luhua 14”.

[0121] Field trials were conducted in a peanut planting professional cooperative in Linyi City, Shandong Province, where root rot is serious.

[0122] The experiment set up 8 treatments, including the conventional fertilization group (CK), and applied the microbial agents obtained in Examples 1-2 of the present invention and Comparative Examples 1-5. The usage method was to add an appropriate amount of microbial agent to each sowing hole during sowing, and then cover the soil. The amount of microbial agent was 5kg / mu. :

[0123] CK: Local customary fertilization (conventional fertilizer: compound fertilizer 700kg·hm -2 , superphosphate 200kg·hm -2 , potassium sulfate 600kg·hm -2 );

[0124] A1: 50% conventional fertilizer + treatment of Example 1;

[0125] A2: 50% conventional fertilizer + Example 2 treatment;

[0126] A3: 50% conventional fertilizer + comparative example 1 treatment;

[0127] A4: 50% conventional fertilizer + comparative example 2 treatment;

[0128] A5: 50% conventional fertilizer + comparative example 3 treatment;

[0129] A6: 50% conventional fertilizer + comparative example 4 treatment;

[0130] A7: 50% conventional fertilizer + comparative example 5 treatment;

[0131] Each treatment was repeated three times and the results were averaged.

[0132] Index statistics: Statistics on yield, prevention effect and other indicators, observation of the root base of the plant, and statistics on peanut disease index. Peanut root rot incidence:

[0133] Level 0: There are no lesions on the stem base and main root of the plant; Level 1: There are a small number of lesions on the stem base and main root; Level 3: There are more lesions on the stem base and main root, and the lesion area accounts for 25%-50% of the total area of ​​the stem and root; Level 5: There are many and larger lesions on the stem base and main root, and the lesion area accounts for 50%-75% of the total area of ​​the stem base and main root; Level 7: The lesions on the stem base and main root are connected, forming a stem-wrapped phenomenon, but the root system is not dead; Level 9: The root system is necrotic, and the aboveground part of the plant wilts or dies.

[0134] Disease index = ∑ (number of diseased plants at each level × number of disease levels) (total number of plants × number of the highest disease level) × 100%;

[0135] Disease prevention effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0136] Table 1 Control effect

[0137]

[0138] During the peanut maturity period, the total sugar, crude fat, crude protein, fatty acid composition and protein composition of the peanut kernels were determined. The total sugar content was determined according to the copper reduction-iodine titration method according to GB / T37493-2019, the crude fat content was determined according to the residual method according to NY / T1285-2007, the protein content was determined according to the first method of GB5009.5-2016, and the protein component content was determined according to the ninhydrin post-column derivatization ion exchange chromatography according to GB5009.124-2016.

[0139] Table 2 Effect of different treatment groups on total sugar, crude fat and crude protein content in peanut kernels / %

[0140] Total sugar % Crude fat % Crude protein % Example 1 2.75 49.55 23.98 Example 2 2.89 50.12 23.01 Comparative Example 1 2.09 46.12 21.21 Comparative Example 2 2.12 47.25 21.05 Comparative Example 3 2.35 47.69 22.21 Comparative Example 4 2.24 46.88 22.45 Comparative Example 5 2.29 47.25 22.05 CK 2.01 45.11 20.45

[0141] From the data in Table 1-2, we can see that in the conventional fertilization experimental site, the incidence index of peanut root rot is high, and the root rot prevention and control effect is poor. However, the peanut yield and quality of Examples 1-2 of the present invention both showed a good improvement level, and the quality of peanuts was improved while achieving effective control of the disease. In Comparative Examples 1-5, which changed the microbial composition, both the root rot prevention and control effect and the peanut quality showed varying degrees of weakening. It can be proved that the Trichoderma harzianum screened by the present invention can cooperate with biocontrol bacteria of different genera to show obvious disease prevention and growth promotion effects. The selection of strains is an organic whole, each playing a corresponding role, and the effect is weak if one is missing.

[0142] To further test the soil enzyme activity of each treatment group, the five-point sampling method was used to collect the rhizosphere soil of peanuts in each plot, mix it into a sample, put it into a sterile tube and store it at -4°C to determine the activity of polyphenol oxidase, sucrase, catalase and urease in each treatment. The enzyme activity was determined using the Solarbio enzyme activity detection kit. The test results are as follows Figure 3-6 The above results show that the use of the microbial agent of the embodiment of the present invention can improve the yield and quality of peanuts while increasing the soil enzyme activity of the planting soil. The increase in soil enzyme activity can accelerate the circulation and transformation of soil carbon, nitrogen, phosphorus and other elements, which is of great significance for maintaining the ecological function of the soil, improving agricultural production efficiency and protecting environmental health.

[0143] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A microbial agent for preventing and treating peanut root rot, characterized in that: Includes the following raw materials: fungal fermentation products, biocontrol bacteria, and regulator ingredients.

2. The microbial agent for preventing and treating peanut root rot according to claim 1, characterized in that: The mass ratio of the fungal fermentation product, the biocontrol bacteria and the regulator component is 1:1:(0.1-0.5).

3. The microbial agent for preventing and treating peanut root rot according to claim 2, characterized in that: The preparation method of the fungal fermentation product is as follows: (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated on a PDA solid medium. After culturing for 5 days, 10 mL of a 0.9% NaCl solution was added to the surface of the plate. The spores on the surface of the plate were scraped 10 times with a sterile stick. The spores in the plate were then rinsed into a 150 mL conical flask containing glass beads with sterile water. The plate was placed in a shaker at 35-37° C. and 180-200 rpm for 30-40 min to break the mycelium. The suspension was then filtered using sterilized gauze and the filtrate was prepared to have a spore count of 1×10 9 cfu / mL of Trichoderma spore suspension for later use; (2) Preparation of solid fermentation material: Rice husk and wheat bran are mixed evenly in a mass ratio of 4:6, and water is added, the amount of water added is 120% of the mass of the mixture, and the mixture is stirred evenly. The mixture is placed in a sterilizing pot at 121° C. for 30 min, and the mixture is naturally cooled after sterilization to obtain a base material. Corn flour and ammonium sulfate are added to the base material, and the mixture is mixed evenly to obtain a solid fermentation material. (3) The Trichoderma spore suspension in step (1) was evenly sprayed on the solid fermentation material obtained in step (2) at an inoculum amount of 3%, stirred evenly, and placed in an incubator at 28-35° C. for cultivation. Starting from the second day, water was added and the material was turned over every 24 hours until the Trichoderma spore production was greater than 5×10 9 cfu / g, the fermentation is ended and the fermented material can be dried at room temperature.

4. The microbial agent for preventing and treating peanut root rot according to claim 3, characterized in that: The deposit number of the Trichoderma harzianum in step (1) is CGMCC No. 40702, the deposit date is June 16, 2023, and it is deposited in the General Microbiological Center of the China Culture Collection Administration, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

5. The microbial agent for preventing and treating peanut root rot according to claim 3, characterized in that: In step (2), the added amount of corn flour and ammonium sulfate is 1% of the mass of the base material.

6. The microbial agent for preventing and treating peanut root rot according to claim 2, characterized in that: The biocontrol bacteria include Bacillus, Pseudomonas and Actinomycetes.

7. The microbial agent for preventing and treating peanut root rot according to claim 6, characterized in that: The Bacillus is Bacillus subtilis, with a strain number of CGMCC No.1.7740; the Pseudomonas is Pseudomonas fluorescens, with a strain number of CGMCC No.1.4531; and the actinomycetes is Actinomadura chibensis, with a strain number of CGMCC No.4.6236; and the volume ratio of the three is 2:1:

3.

8. The microbial agent for preventing and treating peanut root rot according to claim 7, characterized in that: The preparation method of the biocontrol bacteria is as follows: Bacillus subtilis, Pseudomonas fluorescens, and Actinomycetes chibaensis are inoculated into LB culture medium respectively, and cultured at 28-30° C. with shaking until the bacterial concentration reaches OD 600 ≈2.0, and then mixed in a volume ratio of 2:1:3 and spray-dried to obtain the result.

9. The microbial agent for preventing and treating peanut root rot according to claim 1, characterized in that: The regulator component comprises diatomaceous earth, dextrin, sodium humate and xanthan gum, and the mass ratio of the four is 10:1:3:

1.

10. A method for preparing the microbial agent for preventing and controlling peanut root rot according to any one of claims 1 to 9, characterized in that: The method comprises the following preparation steps: (1) preparing fungal fermentation products; (2) preparing biocontrol bacteria; (3) After the fungal fermentation product, biocontrol bacteria, and regulator components are fully mixed in a mass ratio of 1:1:(0.1-0.5), granulation is performed in a coating machine. The coating machine speed is set to 50 r / min and stirred for 30 minutes. After the materials are fully mixed, water or a binder is used to granulate the materials. After granulation, the materials are dried to obtain the target bacterial agent.

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