A microbial inoculant for controlling peanut root rot and its preparation method

Abstract

This invention discloses a microbial inoculant for controlling peanut root rot and its preparation method, belonging to the field of microbial materials technology. This invention screened and obtained a *Trichoderma harzianum* strain with highly efficient inhibitory effects against the pathogen causing peanut root rot. Firstly, the strain itself exhibits antagonistic activity against the peanut pathogen. Secondly, it was prepared as a spore suspension and fermented in solid-state with rice husks and wheat bran. During the solid-state fermentation process, the metabolites produced by *Trichoderma harzianum* promote the growth and development of plant roots, increasing plant biomass and yield. This microbial inoculant effectively controls peanut root rot, exhibiting good inhibitory effects against the pathogen, thus improving peanut quality and disease resistance. Furthermore, it improves soil ecological activity to a certain extent, making it worthy of application and promotion.

Description

Technical Field

[0001] This invention belongs to the field of microbial materials technology, specifically relating to a microbial agent for preventing and controlling peanut root rot and its preparation method. Background Technology

[0002] Peanuts (Arachis hypogaea) are the world's fourth largest oilseed crop and the thirteenth largest food source for humans, and are also an important source of high-quality plant protein and oil. Peanuts are a significant economic and oilseed crop, rich in fat, protein, dietary fiber, and various micronutrients, and are widely enjoyed.

[0003] Peanut root rot, also known as "rat's tail" or "root rot," is a fungal, soil-borne disease that severely impacts peanut yield and quality. It is primarily caused by fungi of the genus *Fusarium*, such as *Fusarium solani* and *Fusarium oxysporum*. Root rot can occur throughout the peanut's growth cycle, mainly affecting the roots and vascular bundles. The roots turn brown and rot, leading to the vascular bundles turning brown and rotting, resulting in poor plant growth and eventually the death of the entire plant. Later infection can damage the pods, causing pod rot, leading to the death of most or all of the plant and resulting in missing seedlings and gaps in the rows. This causes significant economic losses and severely restricts the development of the peanut industry.

[0004] Currently, the prevention and control of peanut root rot mainly relies on chemical pesticides, which not only leads to drug resistance in pathogens but also damages the entire agricultural ecosystem, causing enormous harm.

[0005] For example, Chinese patent application CN201810904875.2 discloses a medicament for preventing and controlling peanut root rot, along with its preparation and application methods. This medicament comprises a traditional Chinese medicine antibacterial component and a trace element nutrient solution component. The traditional Chinese medicine antibacterial component includes honeysuckle, turmeric, dried tangerine peel, cinnamon, clove, borneol, perilla, artemisia, sophora flavescens, saposhnikovia divaricata, cardamom, cocklebur, pinellia ternata, coptis chinensis, and platycodon grandiflorus, among other traditional 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 remainder water. The combination of the traditional Chinese medicine antibacterial component and the trace element nutrient solution component in this invention enables peanuts to successfully overcome the high-risk period for root rot, thereby reducing the incidence of peanut root rot.

[0006] Chinese patent application CN202410879433.2 discloses a special agent for controlling peanut root rot and its preparation method, belonging to the field of crop cultivation technology. The special agent for controlling peanut root rot of this invention, by weight, comprises the following raw materials: 20-30 parts of 50% carbendazim, 10-15 parts of volatile oil, and 20-30 parts of plant extract; the raw materials for preparing the volatile oil include dandelion and grapefruit peel; the raw materials for preparing the plant extract include soapberry, asparagus fern, ginkgo leaves, and houttuynia cordata. The special agent for controlling peanut root rot of this invention can significantly reduce the incidence of peanut root rot and increase peanut yield.

[0007] Most of these existing technologies use large amounts of traditional Chinese medicine or chemical drug ingredients, which easily leads to crop resistance and causes serious pollution to the soil environment, resulting in poor practical application effects.

[0008] Therefore, developing technologies that utilize functional microorganisms to control plant diseases in a way that is environmentally friendly and safe for human health has gradually become a key focus for researchers and is also a technical problem that urgently needs to be solved. Summary of the Invention

[0009] This invention addresses the problems existing in the prior art by screening a highly effective antibacterial and growth-promoting Trichoderma harzianum from strawberry rhizosphere soil. After solid-state fermentation, it is combined with biocontrol bacteria to form a microbial preparation, which can effectively control peanut root rot pathogens, enhance peanut resistance, regulate soil ecological activity, and effectively improve peanut quality.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0011] A microbial inoculant for controlling peanut root rot comprises the following raw materials: fungal ferments, biocontrol bacteria, and regulator components.

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

[0013] Furthermore, the method for preparing the fungal ferment is as follows:

[0014] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated onto PDA solid medium and cultured for 5 days. Then, 10 mL of 0.9% NaCl solution was added to the surface of the plate. The spores were scraped 10 times on the plate surface with a sterile stick. The spores in the plate were then washed with sterile water into a 150 mL Erlenmeyer flask containing glass beads. The flask was placed in a shaker at 35-37℃ and 180-200 rpm for 30-40 min to break up the hyphae. The solution was then filtered with sterile gauze to prepare a spore count of 1×10⁻⁶. 9 A suspension of Trichoderma spores at cfu / mL was prepared for later use.

[0015] (2) Preparation of solid fermentation material: Mix rice husks and wheat bran evenly at a mass ratio of 4:6, add water at 120% of the mass of the mixture, stir evenly, place in an autoclave at 121℃ for 30 minutes, and let cool naturally after sterilization to obtain the base material. Add corn flour and ammonium sulfate to the base material and mix evenly to obtain the solid fermentation material.

[0016] (3) Spray the Trichoderma spore suspension from step (1) evenly onto the solid fermentation material at an inoculation rate of 3%, stir well, and place in an incubator at 28-35℃ for cultivation. Starting from day 2, add water and turn the material every 24 hours until the Trichoderma spore production exceeds 5×10⁻⁶. 9 When the cfu / g reaches a certain level, stop fermentation and let the fermented material air dry at room temperature.

[0017] Furthermore, the Trichoderma harzianum mentioned in step (1) has the accession number CGMCC No.40702, the accession date is June 16, 2023, and it is deposited at the China General Microbiological Culture Collection Center, with the accession address at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] The screening and isolation method for Trichoderma harzianum is as follows: Take 100g of strawberry rhizosphere soil into a 1000mL Erlenmeyer 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, and add it to a pre-prepared Trichoderma selective medium plate, spreading it evenly with a spreader. Repeat each dilution 20 times and incubate at 28℃ for 3-5 days. Pick single colonies of fungi with different shapes for purification and culture, repeating the process multiple times until purified single colonies are obtained. Pick a small amount of hyphae and store them in a -80℃ freezer with 40% glycerol as the cryopreservation solution for later use.

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

[0020] Plate confrontation method: Using Fusarium harzianum, the pathogen of peanut root rot, as the target, a two-point confrontation culture method was adopted. Purified antagonistic Trichoderma and pathogenic fungal discs (5 mm in diameter) were inoculated symmetrically at positions 2.50 cm from the center of the plate. Each group had three replicates, with a pathogenic control group included. The plates were incubated at 28-35℃. Colony observation began on the second day, continuing until the hyphae touched the edge of the plate. The appearance of an inhibition zone was observed, and the radii of the strains in the experimental and control groups were recorded. The inhibition rate was calculated as follows: Inhibition rate = (Radius of pathogen in control group - Radius of pathogen in treatment group) / (Radius of pathogen in control group - 2.5) × 100%, where 2.5 is the radius of the pathogenic fungal disc in the control group. The strain with the highest inhibition rate was the target strain – Trichoderma harzianum. The inhibition effect is shown in the figure below. Figure 1 As shown. This Trichoderma grows rapidly when cultured on PDA plates, covering the entire plate by day 3. The hyphae are white, felt-like, and slightly raised. Colony diagram as shown. Figure 2 .

[0021] Furthermore, step (1) PDA solid culture medium consists of the following mass components: 150-200g potato, 15-20g glucose, and 10-15g 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*, strain number CGMCC No. 1.7740, with an original deposit date of July 24, 2008; the Pseudomonas is *Pseudomonas fluorescens*.

[0025] The *Pseudomonas fluorescens* strain, with CGMCC No. 1.4531, was originally deposited on June 27, 2005; the actinomycete is *Actinomadura chibensis*, with CGMCC No. 4.6236, originally deposited on September 3, 2008; 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 from the depository through open access, without the need for repeated biopreservation.

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

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

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

[0029] A method for preparing a microbial inoculant for controlling peanut root rot includes the following preparation steps:

[0030] (1) Preparation of fungal ferments;

[0031] (2) Preparation of biocontrol bacteria;

[0032] (3) After thoroughly mixing the fungal ferment, biocontrol bacteria and regulator components in a mass ratio of 1:1:(0.1-0.5), the mixture is granulated using a coating machine. The coating machine speed is set to 50 r / min and stirred for 30 min to ensure that the materials are thoroughly mixed. Water or binder is then used to granulate the materials. After granulation, the mixture is dried to obtain the target microbial agent.

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

[0034] This invention relates to a microbial inoculant with an effective viable count of approximately 50-100 billion CFU / g. The method of application is to add an appropriate amount of the microbial inoculant to each sowing hole during sowing, followed by covering with soil. The dosage of the inoculant is 5-10 kg / acre.

[0035] Beneficial effects:

[0036] (1) This invention screened a Trichoderma harzianum strain that exhibits highly efficient inhibitory effects against peanut root rot pathogens. Firstly, the strain itself has antagonistic effects against peanut pathogens. Secondly, we prepared a spore suspension and fermented it with rice husks and wheat bran in a solid-state environment. During the solid-state fermentation process, the metabolites produced by Trichoderma harzianum can promote the growth and development of plant roots, increasing plant biomass and yield. Furthermore, solid-state fermentation can produce a large number of conidia, which can inhibit the growth of various plant pathogens and suppress the reproduction of harmful bacteria through mechanisms such as hyperparasitism and antimicrobial peptides. In addition, Trichoderma harzianum can induce disease resistance in plants, enhancing their resistance to pathogens.

[0037] (2) In addition to *Trichoderma harzianum*, this invention also adds three different genera of biocontrol bacteria. *Bacillus subtilis*, a selected *Bacillus* species, inhibits pathogen growth by producing secondary metabolites such as antibiotics, enzymes, and siderophores, while also promoting plant growth and inducing systemic resistance. *Pseudomonas fluorescens*, a selected *Pseudomonas* species, produces various volatile antibacterial substances that inhibit multiple plant pathogens. *Actinomyces madurae*, a selected *Actinomyces* species, enhances plant resistance to pathogens by inducing an immune response. For example, it promotes plant growth, reduces oxidative stress caused by diseases, and inhibits pathogen growth by altering the plant's immune response. The combined use of these three different genera of biocontrol bacteria, along with *Trichoderma harzianum* fermentation products, demonstrates highly effective control of peanut root rot, promotes peanut root growth, improves peanut quality, and simultaneously improves the soil ecological environment.

[0038] (3) Using diatomaceous earth, dextrin, sodium humate, and xanthan gum as microbial and soil conditioners, diatomaceous earth can provide favorable microenvironmental conditions for soil microorganisms, promoting their growth and reproduction; dextrin provides energy for microorganisms, promoting their growth and metabolic activities; sodium humate can improve soil structure, increase soil permeability and water retention capacity, thus providing a better living environment for rhizosphere microorganisms. Sodium humate can also promote soil microbial activity, increase the number of free-living 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 inoculant of the present invention effectively prevents and controls peanut root rot, exhibits good inhibitory effects on the pathogens causing peanut root rot, and improves peanut quality and disease resistance. On the other hand, it improves the ecological activity of the soil to a certain extent, making it worthy of application and promotion. Attached Figure Description

[0040] Figure 1 This is a diagram illustrating the antibacterial effect of the Trichoderma harzianum strain of this invention.

[0041] Figure 2 This is a diagram showing the state of *Trichoderma harzianum* during its cultivation on a PDA plate according to the present invention.

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

[0043] Figure 4 Results of sucrase assay in soil from different experimental groups;

[0044] Figure 5 Results of sucrase assay in soil from different experimental groups;

[0045] Figure 6 The results of catalase determination in soil from different experimental groups. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0047] Example 1

[0048] A microbial inoculant for controlling peanut root rot comprises the following raw materials: fungal ferments, biocontrol bacteria, and regulator components.

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

[0050] The method for preparing the fungal ferment is as follows:

[0051] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated onto PDA solid medium and cultured for 5 days. Then, 10 mL of 0.9% NaCl solution was added to the surface of the plate. The spores were scraped 10 times on the plate surface with a sterile stick. The spores in the plate were then washed with sterile water into a 150 mL Erlenmeyer flask containing glass beads. The flask was placed in a shaker at 35-37℃ and 180-200 rpm for 30-40 min to break up the hyphae. The solution was then filtered with sterile gauze to prepare a spore count of 1×10⁻⁶. 9 A suspension of Trichoderma spores at cfu / mL was prepared for later use.

[0052] (2) Preparation of solid fermentation material: Mix rice husks and wheat bran evenly at a mass ratio of 4:6, add water at 120% of the mass of the mixture, stir evenly, place in an autoclave at 121℃ for 30 minutes, and let cool naturally after sterilization to obtain the base material. Add corn flour and ammonium sulfate to the base material and mix evenly to obtain the solid fermentation material.

[0053] (3) Spray the Trichoderma spore suspension from step (1) evenly onto the solid fermentation material at an inoculation rate of 3%, stir well, and place in an incubator at 28-35℃ for cultivation. Starting from day 2, add water and turn the material every 24 hours until the Trichoderma spore production exceeds 5×10⁻⁶. 9 When the cfu / g reaches a certain level, stop fermentation and let the fermented material air dry at room temperature.

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

[0055] The screening and isolation method for Trichoderma harzianum is as follows: Take 100g of strawberry rhizosphere soil into a 1000mL Erlenmeyer 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, and add it to a pre-prepared Trichoderma selective medium plate, spreading it evenly with a spreader. Repeat each dilution 20 times and incubate at 28℃ for 3-5 days. Pick single colonies of fungi with different shapes for purification and culture, repeating the process multiple times until purified single colonies are obtained. Pick a small amount of hyphae and store them in a -80℃ freezer with 40% glycerol as the cryopreservation solution for later use.

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

[0057] Plate confrontation method: Using Fusarium harzianum, the pathogen of peanut root rot, as the target, a two-point confrontation culture method was adopted. 5 mm of purified antagonistic Trichoderma and pathogen mycelial cakes were inoculated symmetrically at positions 2.50 cm from the center of the plate. Each group had three replicates, with a pathogen control group included. The plates were incubated at 28-35℃. Colony observation began on the second day, continuing until the hyphae touched the edge of the plate. The appearance of an inhibition zone was observed, and the radii of the strains in the experimental and control groups were recorded. The inhibition rate was calculated as follows: Inhibition rate = (Radius of pathogen in control group - Radius of pathogen in treatment group) / (Radius of pathogen in control group - 2.5) × 100%, where 2.5 is the radius of the pathogen mycelial cake in the control group. The strain with the largest inhibition radius was the target strain – Trichoderma harzianum. The inhibition effect is shown in the figure below. Figure 1 As shown. This Trichoderma grows rapidly when cultured on PDA plates, covering the entire plate by day 3. The hyphae are white, felt-like, and slightly raised. Colony diagram as shown. Figure 2 .

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

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

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

[0061] The Bacillus species used in this application is *Bacillus subtilis*, strain number CGMCC No. 1.7740, with an original deposit date of July 24, 2008; the Pseudomonas species is *Pseudomonas fluorescens*, strain number CGMCC No. 1.4531, with an original deposit date of June 27, 2005; and the Actinomycete species is *Actinomadura chibensis*, strain number CGMCC No. 4.6236, with an original deposit date of September 3, 2008. The volume ratio of the three species is 2:1:3. All *Bacillus subtilis*, *Pseudomonas fluorescens*, and *Actinomadura chibensis* used in this application can be purchased through open access from the depository, eliminating the need for repeated biopreservation.

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

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

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

[0065] A method for preparing a microbial inoculant for controlling peanut root rot includes the following preparation steps:

[0066] (1) Preparation of fungal ferments;

[0067] (2) Preparation of biocontrol bacteria;

[0068] (3) After thoroughly mixing the fungal ferment, biocontrol bacteria and regulator components in a mass ratio of 1:1:0.1, the mixture is granulated using a coating machine. The coating machine speed is set to 50 r / min and stirred for 30 min to ensure that the materials are thoroughly mixed. Water or binder is then used to granulate the materials. After granulation, the mixture is dried to obtain the target bacterial agent.

[0069] The adhesive is carboxymethyl cellulose.

[0070] Example 2

[0071] A microbial inoculant for controlling peanut root rot comprises the following raw materials: fungal ferments, biocontrol bacteria, and regulator components.

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

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

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

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

[0076] A method for preparing a microbial inoculant for controlling peanut root rot includes the following preparation steps:

[0077] (1) Preparation of fungal ferments;

[0078] (2) Preparation of biocontrol bacteria;

[0079] (3) After thoroughly mixing the fungal ferment, biocontrol bacteria and regulator components in a mass ratio of 1:1:0.5, granulate them using a coating machine. Set the coating machine speed to 50 r / min and stir for 30 min to ensure the materials are thoroughly mixed. Then, use water or a binder to granulate the materials. After granulation, dry the materials to obtain the target bacterial agent.

[0080] The adhesive 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, while the remaining raw materials and process steps were the same as in Example 2. That is:

[0083] A microbial inoculant for controlling peanut root rot comprises the following raw materials: fungal ferments, biocontrol bacteria, and regulator components.

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

[0085] The method for preparing the fungal ferment is as follows:

[0086] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated onto PDA solid medium and cultured for 5 days. Then, 10 mL of 0.9% NaCl solution was added to the surface of the plate. The spores were scraped 10 times on the plate surface with a sterile stick. The spores in the plate were then washed with sterile water into a 150 mL Erlenmeyer flask containing glass beads. The flask was placed in a shaker at 35-37℃ and 180-200 rpm for 30-40 min to break up the hyphae. The solution was then filtered with sterile gauze to prepare a spore count of 1×10⁻⁶. 9 A suspension of Trichoderma spores at cfu / mL was prepared for later use.

[0087] (2) Preparation of solid fermentation material: Mix rice husks and wheat bran evenly at a mass ratio of 4:6, add water at 120% of the mass of the mixture, stir evenly, place in an autoclave at 121℃ for 30 minutes, and let cool naturally after sterilization to obtain the base material. Add corn flour and ammonium sulfate to the base material and mix evenly to obtain the solid fermentation material.

[0088] (3) Spray the Trichoderma spore suspension from step (1) evenly onto the solid fermentation material at an inoculation rate of 3%, stir well, and place in an incubator at 28-35℃ for cultivation. Starting from day 2, add water and turn the material every 24 hours until the Trichoderma spore production exceeds 5×10⁻⁶. 9 When the cfu / g reaches a certain level, stop fermentation and let the fermented material air dry at room temperature.

[0089] The Trichoderma harzianum mentioned in step (1) was purchased from the China Cultural Relics Center, with accession number CGMCC No. 5.1213.

[0090] Comparative Example 2

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

[0092] The method for preparing the fungal ferment is as follows:

[0093] (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum was inoculated onto PDA solid medium and cultured for 5 days. Then, 10 mL of 0.9% NaCl solution was added to the surface of the plate. The spores were scraped 10 times on the plate surface with a sterile stick. The spores in the plate were then washed with sterile water into a 150 mL Erlenmeyer flask containing glass beads. The flask was placed in a shaker at 35-37℃ and 180-200 rpm for 30-40 min to break up the hyphae. The solution was then filtered with sterile gauze to prepare a spore count of 1×10⁻⁶. 9 A suspension of Trichoderma spores at cfu / mL was prepared for later use.

[0094] (2) Preparation of solid fermentation material: Mix rice husks and wheat bran evenly at a mass ratio of 4:6, add water at 120% of the mass of the mixture, stir evenly, place in an autoclave at 121℃ for 30 minutes, and let cool naturally after sterilization to obtain the base material. Add corn flour and ammonium sulfate to the base material and mix evenly to obtain the solid fermentation material.

[0095] (3) Spray the Trichoderma spore suspension from step (1) evenly onto the solid fermentation material at an inoculation rate of 3%, stir well, and place in an incubator at 28-35℃ for cultivation. Starting from day 2, add water and turn the material every 24 hours until the Trichoderma spore production exceeds 5×10⁻⁶. 9 When the cfu / g reaches a certain level, stop fermentation and let the fermented material air dry at room temperature.

[0096] The Trichoderma harzianum mentioned in step (1) was purchased from the China Cultural Relics Center, with accession number CGMCC No.3.15684.

[0097] Comparative Example 3

[0098] This comparative example is identical to Example 1 in all raw materials and process steps, except that Bacillus subtilis is not used in the biocontrol bacteria. That is:

[0099] A microbial inoculant for controlling peanut root rot comprises the following raw materials: fungal ferments, biocontrol bacteria, and regulator components.

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

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

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

[0103] The selection and preparation methods of the biocontrol bacteria *Pseudomonas fluorescens* and *Actinomyces madurae* are the same as in Example 1.

[0104] Comparative Example 4

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

[0106] A microbial inoculant for controlling peanut root rot comprises the following raw materials: fungal ferments, biocontrol bacteria, and regulator components.

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

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

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

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

[0111] Comparative Example 5

[0112] In this comparative example, except for the omission of *Madula maculatus* in the biocontrol bacteria, the raw materials and process steps are the same as in Example 1. That is:

[0113] A microbial inoculant for controlling peanut root rot comprises the following raw materials: fungal ferments, biocontrol bacteria, and regulator components.

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

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

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

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

[0118] Performance testing

[0119] Peanut planting experiment:

[0120] The peanut variety tested was "Luhua 14".

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

[0122] The experiment included eight treatments, including a conventional fertilization group (CK), and treatments using the microbial agents obtained in Examples 1-2 and Comparative Examples 1-5 of this invention. The application method involved adding an appropriate amount of the microbial agent to each sowing hole at the time of sowing, followed by covering with soil. The dosage of the microbial agent was 5 kg / mu.

[0123] CK: Local fertilization practices (conventional fertilizer: 700 kg / hm² of compound fertilizer) -2 200 kg·hm of superphosphate -2 Potassium sulfate 600 kg·hm -2 );

[0124] A1: 50% conventional fertilizer + treatment as described in Example 1;

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

[0126] A3: 50% conventional fertilizer + treatment with comparison ratio 1;

[0127] A4: 50% conventional fertilizer + treatment in comparison ratio 2;

[0128] A5: 50% conventional fertilizer + treatment in comparison ratio 3;

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

[0130] A7: 50% conventional fertilizer + treatment with ratio 5;

[0131] Each treatment was repeated three times. The results were then averaged.

[0132] Indicator statistics: Statistics on yield, control efficacy, etc., observation of the base of the plant roots and stems, and statistical analysis of peanut disease index. Peanut root rot incidence rate:

[0133] Grade 0: No lesions on the base of the stem and the main root; Grade 1: A few lesions on the base of the stem and the main root; Grade 3: Many lesions on the base of the stem and the main root, covering 25%-50% of the total area of ​​the stem and roots; Grade 5: Many and large lesions on the base of the stem and the main root, covering 50%-75% of the total area of ​​the stem and roots; Grade 7: Lesions on the base of the stem and the main root merge together, forming a circling phenomenon, but the root system is not dead; Grade 9: Root necrosis, and the above-ground parts of the plant wilt or die.

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

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

[0136] Table 1 Prevention and control effects

[0137]

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

[0139] Table 2. Effects of different treatment groups on the content of total sugar, crude fat, and crude protein in peanut kernels (%)

[0140] Total sugar % Crude fat percentage 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] As shown in Table 1-2, the peanut root rot disease incidence index was high and the control effect was poor in the conventional fertilization experimental plots. However, Examples 1-2 of this invention showed significant improvements in both peanut yield and quality, effectively controlling the disease while enhancing peanut quality. Comparative Examples 1-5, which altered the microbial composition, showed varying degrees of weakening in both root rot control and peanut quality. This demonstrates that the *Trichoderma harzianum* strain selected in this invention, combined with biocontrol bacteria of different genera, can exhibit significant disease prevention and growth promotion effects. The selection of strains is an organic whole, with each strain playing a corresponding role; the absence of any one strain weakens the overall effect.

[0142] Further testing of soil enzyme activities in each treatment group was conducted. A five-point sampling method was used to collect rhizosphere soil samples from each peanut plot. The samples were mixed thoroughly, placed in sterile tubes, and stored at -4°C. The activities of polyphenol oxidase, sucrase, catalase, and urease in each treatment were measured using a Solarbio enzyme activity assay kit. The test results are as follows: Figure 3-6 As shown above, the results indicate that using the microbial inoculant of the present invention can improve peanut yield and quality while increasing soil enzyme activity. Increased soil enzyme activity accelerates the cycling and transformation of elements such as carbon, nitrogen, and phosphorus in the soil, which is of great significance for maintaining soil ecological functions, improving agricultural production efficiency, and protecting environmental health.

[0143] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A microbial inoculant for controlling peanut root rot, characterized in that, It includes the following raw materials: fungal ferment, biocontrol bacteria, and regulator components; the mass ratio of the fungal ferment, biocontrol bacteria, and regulator components is 1:1:(0.1-0.5). The fungus in the fungal fermentation product is *Trichoderma harzianum*, with accession number CGMCC No. 40702, accession date June 16, 2023, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the biocontrol bacteria include Bacillus, Pseudomonas, and Actinomycetes; the Bacillus is *Bacillus subtilis* (…). Bacillus subtilis The strain number is CGMCC No. 1.7740; the Pseudomonas is *Pseudomonas fluorescens* (…). Pseudomonas fluorescens The strain number is CGMCC No. 1.4531; the actinomycete is *Actinomyces madurae* (Cinomyces madurae). Actinomadura chibensis The strain number is CGMCC No. 4.6236; the volume ratio of the three components is 2:1:3; the preparation method of the biocontrol bacteria is as follows: Bacillus subtilis, Pseudomonas fluorescens, and Actinobacillus madurae are inoculated into LB medium and cultured with shaking at 28-30℃ until the bacterial concentration reaches OD. 600 The mixture is approximately 2.0, and then spray-dried after being mixed in a volume ratio of 2:1:

3. The regulator component includes diatomaceous earth, dextrin, sodium humate and xanthan gum, with a mass ratio of 10:1:3:

1.

2. The microbial inoculant for controlling peanut root rot according to claim 1, characterized in that, The method for preparing the fungal ferment is as follows: (1) Preparation of Trichoderma harzianum spore suspension: Trichoderma harzianum ( Trichoderma harzianum Inoculate the spores onto PDA solid medium and culture for 5 days. Then, add 10 mL of 0.9% NaCl solution to the surface of the plate. Use a sterile rod to scrape the spores off the surface of the plate 10 times. Rinse the spores from the plate with sterile water into a 150 mL Erlenmeyer flask containing glass beads. Place the flask in a shaker at 35-37℃ and 180-200 rpm for 30-40 minutes to break up the hyphae. Filter the solution using sterile gauze and prepare a solution with a spore count of 1×10⁻⁶. 9 A suspension of Trichoderma spores at cfu / mL was prepared for later use. (2) Preparation of solid fermentation material: Mix rice husks and wheat bran evenly at a mass ratio of 4:6, add water at 120% of the mass of the mixture, stir evenly, place in an autoclave at 121℃ for 30 min, and let it cool naturally after sterilization to obtain the base material. Add corn flour and ammonium sulfate to the base material and mix evenly to obtain the solid fermentation material. (3) Spray the Trichoderma spore suspension from step (1) evenly onto the solid fermentation material obtained in step (2) at an inoculation rate of 3%, stir well, and place in an incubator at 28-35℃ for cultivation. Starting from day 2, add water and turn the material every 24 hours until the Trichoderma spore production exceeds 5×10⁻⁶. 9 When the cfu / g reaches a certain level, stop fermentation and let the fermented material air dry at room temperature.

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

4. A method for preparing a microbial inoculant for controlling peanut root rot as described in any one of claims 1-3, characterized in that, The preparation steps include the following: (1) Preparation of fungal ferments; (2) Preparation of biocontrol bacteria; (3) After thoroughly mixing the fungal ferment, biocontrol bacteria and regulator components in a mass ratio of 1:1:(0.1-0.5), the mixture is granulated using a coating machine. The coating machine speed is set to 50 r / min and stirred for 30 min to ensure that the materials are thoroughly mixed. Water or binder is then used to granulate the materials. After granulation, the mixture is dried to obtain the target bacterial agent.

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