Microbial preparation as well as preparation method and application thereof

Abstract

The invention discloses a microbial preparation and a preparation method and application thereof, the microbial preparation has active ingredients of trichoderma koningii Tr25 and trichoderma asperellum Tr85, the classification name of trichoderma koningii Tr25 is Trichoderma koningii Tr25 is Trichoderma koningii, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) 7.284; the classification name of the trichoderma asperellum Tr85 is Trichoderma asperellum, and the preservation number of the trichoderma asperellum Tr85 is CGMCC (China General Microbiological Culture Collection Center) 7.283. The trichoderma koningii Tr25 has a good nutrition space-occupying resistance induction effect, and the trichoderma asperellum Tr477 has good antagonism and hyperparasitism effects; trichoderma koningii Tr25 and Trichoderma asperellum Tr477 belong to the same trichoderma, and show good compatibility compared with the combination of different biocontrol bacteria, and the combination of the two strains has a good control effect on the pathogenic bacteria of the sorghum leaf spot disease.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial preparations, and in particular to a microbial preparation and a preparation method and application thereof. Background Art

[0002] Sorghum, an annual herbaceous plant in the Poaceae family, is one of the world's most important cereal crops, ranking fifth in cultivated area after wheat, rice, corn, and barley. It is currently a major food crop in my country, with yields ranking second in the world after those of the United States. Sorghum grains are primarily used for food and winemaking. In recent years, sorghum leaf spot has become a frequent disease in my country, occurring in sorghum-growing areas across the country and with increasing severity, severely hampering the development of the sorghum industry. Affected plants experience premature leaf withering, leading to yield losses. In severely affected fields, plants die prematurely. Economic losses from sorghum leaf spot range from 12% to 55%.

[0003] Currently, there are no registered microbial agents for the prevention and treatment of sorghum leaf spot in China; chemical pesticides such as carbendazim are commonly used in the field to control the disease. However, the unscientific and irrational use of chemical pesticides can lead to a series of problems, including environmental pollution, drug residues, increased bacterial resistance, and the elimination of beneficial microorganisms. With the continuous improvement of national economic and living standards, the demand for high-quality and organic sorghum is increasing. Furthermore, high-quality organic sorghum is an urgent need for high-end brewing by wineries.

[0004] Biocontrol fungi, Trichoderma species, can control a wide range of diseases through complex mechanisms, including nutrient occupancy, synergistic antagonism, resistance induction, and hyperparasitism. These include leaf diseases caused by pathogens such as Staphylococcus cinerea, Botrytis cinerea, and Rhizoctonia solani. Compared to chemical control, biological control is considered a safe and effective method for plant disease management. However, limited research has been conducted on the biological control of sorghum leaf spot. Summary of the Invention

[0005] The purpose of the present invention is to provide a microbial preparation and a preparation method and application thereof in response to the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect is to provide a Trichoderma koningiopsis Tr25, the classification name of the Trichoderma koningiopsis Tr25 is Trichoderma koningiopsis, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit date of May 15, 2025, and a deposit number of CGMCC7.284.

[0008] The second aspect is to provide a Trichoderma asperellum Tr85, the classification name of the Trichoderma asperellum Tr85 is Trichoderma asperellum, which has been deposited in the General Microbiology Center of the China Culture Collection Administration, with a preservation date of May 15, 2025 and a preservation number of CGMCC7.283.

[0009] The third aspect is to provide a microbial preparation having active ingredients, wherein the active ingredients are the above-mentioned Trichoderma koningii Tr25 and Trichoderma acanthosporum Tr85.

[0010] Furthermore, the microbial preparation contains at least 2×10 8 cfu of Trichoderma koningii Tr25 and Trichoderma aspergillus Tr477.

[0011] The fourth aspect is to provide a method for preparing the above-mentioned microbial preparation, comprising the following steps:

[0012] Step 1: taking Trichoderma koningii Tr25 and Trichoderma acanthosporum Tr85 and performing liquid fermentation;

[0013] Step 2: spore concentration and harvesting;

[0014] Step 3: Drying and preparation of microcapsule raw powder;

[0015] Step 4: Mix and package the preparations.

[0016] Furthermore, in step 1, the specific method of the liquid fermentation is:

[0017] 1) Seed culture:

[0018] Spores were aseptically scraped from an eggplant-shaped culture flask and inoculated into a 100 L fermentation seed tank containing 75 L of sterile seed culture medium at 28° C., 280 RPM, 0.5-1 v / v, for 30-36 hours. The seed culture medium used was 1.5% potato flour and 1% glucose.

[0019] 2) Spore culture:

[0020] Aseptically transfer the culture to a 1000 L fermenter containing 750 L of sterilized seed culture medium at 25-32°C, 175 RPM, 0.5-1 v / v. Ferment for 72 hours, add a spore inducer, continue culturing for 96-122 hours, and harvest the fermentation broth. The spore-forming medium used is: 2.5% potato flour, 2% glucose, and 0.5% amino acids.

[0021] Furthermore, the specific method of step 2 is as follows: the fermentation liquid enters the centrifuge at a flow rate of 1000 L / h and a centrifuge speed of 30 Hz, the fermentation liquid is centrifuged and concentrated twice, and the concentrated liquid containing Trichoderma spores is collected.

[0022] Furthermore, the specific method of step three is as follows: mixing the spore concentrate with the wall material / protective agent dextran, gum arabic, Span 60, water-soluble starch, and white carbon black; transporting the mixture to a spray drying tower with an inlet air temperature of 75-110° C., an outlet air temperature of 40-60° C., and a feed rate of 1-2 L / min; and collecting the mixture by cyclone to obtain the spore raw powder.

[0023] Furthermore, the specific method of step four is to mix the raw powder, filler and additive in a double-screw mixer, and after passing the inspection, transport them to the 180 automatic packaging line, bag and box them.

[0024] The fifth aspect is to provide the use of the above-mentioned microbial preparation in preventing and treating sorghum leaf spot disease, wherein the sorghum leaf spot disease is caused by one or more pathogens selected from the group consisting of Alternaria kauri, Mycosphaerella kauri, and Curvularia lunata.

[0025] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0026] Corning Trichoderma Tr25 has a good nutrient occupancy resistance induction effect, and Trichoderma aspergillus Tr477 has a good antagonistic and heavy parasitic effect; Corning Trichoderma Tr25 and Trichoderma aspergillus Tr477 belong to the same genus Trichoderma. Compared with different types of biocontrol fungi, the combination shows good compatibility. The combination of the two species has a good control effect on the pathogen of sorghum leaf spot. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown are the morphological identification results of Trichoderma koningii Tr25.

[0028] Figure 2 Shown are the morphological identification results of Trichoderma aculeatus Tr85.

[0029] Figure 3 The results of confrontation culture of Trichoderma pseudokoningii Tr25 and Trichoderma aspergillus Tr477 with Alternaria sorghum SC02-4 are shown.

[0030] Figure 4 The results of confrontation culture of Trichoderma pseudokoningii Tr25 and Trichoderma acanthosporum Tr477 with Mycosphaera kaoliang were shown.

[0031] Figure 5 The results of confrontation culture of Trichoderma pseudokoningii Tr25 and Trichoderma acanthosporium Tr477 with the fungal pathogen Curvularia lunata are shown. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0033] Example 1

[0034] This embodiment provides a Trichoderma koningiopsis Tr25. The classification name of the Trichoderma koningiopsis Tr25 is Trichoderma koningiopsis, which has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit date of May 2025 and a deposit number of CGMCC7.284.

[0035] 1) Morphological identification ( Figure 1 )

[0036] The tested strain grew fast on MEA medium. After culturing at 25℃ in the dark for 5 days, the plate was filled with colonies that were white, flocculent, and produced sparse spores. The back was light brown and had no water-soluble pigment.

[0037] Conidiophores are slender, with smooth walls and unclear specialization. Branches are sparse and slender, with few secondary branches, and are 2.5-3.5 μm wide. A few phialobionts are clustered on the conidiophores or their branches, and are flask-shaped or lanceolate, 4.8-12.0×2.0-3.0 μm. Conidia are elliptical, with smooth or slightly rough walls, and are 2.5-4.1×2.0-3.0 μm. Thick-walled spores are common.

[0038] 2) rRNA gene sequence determination results

[0039] (including 18S rRNA fragments, full sequences of ITS1, 5.8S rRNA, ITS2 and 28S rRNA sequence fragments)

[0040] 5'-TCAACATTTCAGAAGTTGGGTGTTTTACGGACGTGGACGCGCCCG -3'

[0041] Example 2

[0042] This embodiment provides a Trichoderma asperellum Tr85, the classification name of the Trichoderma asperellum Tr85 is Trichoderma asperellum, which has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit date of May 2025 and a deposit number of CGMCC7.283.

[0043] 1) Morphological identification ( Figure 2 )

[0044] The strain tested grew rapidly on MEA medium. After culturing at 25°C in the dark for 5 days, the plate was filled with white colonies. Later, spores were produced that were light gray-green and flocculent. The underside was light brown and lacked water-soluble pigment. Conidia were produced in large quantities, with even distribution of the conidial area.

[0045] Conidiophores are tall, with smooth or slightly rough walls, usually with branches at opposite positions, and are 2.5-5.0 μm wide. A few conidiophores are clustered on the conidiophores or their branches, are flask-shaped, with curved necks, and are 5.8-11.7 × 2.5-3.5 μm. Conidia are broadly elliptical or nearly spherical, with smooth or slightly rough walls, and are 2.5-4.5 μm wide. Thick-walled spores are common.

[0046] 2) rRNA gene sequence determination results

[0047] (including 18S rRNA fragment, full sequence of ITS1, 5.8S rRNA, ITS2 and 28S rRNA sequence fragment)

[0048] 5'-AAAGTTGGGTGTTTTACGGACGTGGACGCGCCGCGCTCCCGGTGCG -3'

[0049] Example 3

[0050] This embodiment provides a method for preparing a microbial preparation, comprising the following steps:

[0051] Step 1: Take Trichoderma koningii Tr25 and Trichoderma acanthosporum Tr85 and perform liquid fermentation:

[0052] 1) Seed culture:

[0053] Spores were aseptically scraped from the eggplant-shaped culture flask and inoculated into a 100 L fermentation seed tank containing 75 L of sterile seed culture medium at 28°C, 280 RPM, 0.5-1 v / v, for 30-36 hours. The seed culture medium used was: 1.5% potato flour and 1% glucose.

[0054] 2) Spore culture:

[0055] Aseptically transfer the culture to a 1000 L fermenter containing 750 L sterilized seed culture medium at 25-32°C, 175 RPM, 0.5-1 v / v. Ferment for 72 hours, add a spore inducer, continue culturing for 96-122 hours, and harvest the fermentation broth. The spore-forming medium used is: 25% potato flour, 2% glucose, and 0.5% amino acids.

[0056] Step 2: The fermentation liquid enters the centrifuge at a flow rate of 1000 L / h and a centrifuge speed of 30 Hz, and the fermentation liquid is centrifuged and concentrated twice to collect the concentrated liquid containing Trichoderma spores.

[0057] Step 3: The spore concentrate is mixed with wall materials / protective agents such as dextran, gum arabic, Span 60, water-soluble starch, and white carbon black; the mixture is transported to a spray drying tower with an inlet air temperature of 75-110°C, an outlet air temperature of 40-60°C, and a feed rate of 1-2 L / min; and the spore raw powder is collected by cyclone drying.

[0058] Step 4: The raw powder, filler and additives are mixed in a double-screw mixer. After passing the inspection, they are transported to the 180 automatic packaging line for bagging and boxing.

[0059] The prepared microbial preparation has the active ingredients of Trichoderma koningii Tr25 and Trichoderma acanthosporum Tr85, and each gram contains 2×10 8 cfu Trichoderma sp.

[0060] Verification Example 1

[0061] Bacteria confrontation culture experiment:

[0062] Figure 3 The results of confrontation culture of Trichoderma pseudokoningii Tr25 and Trichoderma aspergillus Tr477 with Alternaria sorghum SC02-4 are shown.

[0063] Figure 4 The results of confrontation culture of Trichoderma pseudokoningii Tr25 and Trichoderma acanthosporum Tr477 with Mycosphaera kaoliang were shown.

[0064] Figure 5 The results of confrontation culture of Trichoderma pseudokoningii Tr25 and Trichoderma acanthosporium Tr477 with the fungal pathogen Curvularia lunata are shown.

[0065] It can be seen that Trichoderma pseudokoningii Tr25 and Trichoderma acanthosporum Tr477 can effectively inhibit the sorghum leaf spot pathogen in the in vitro confrontation experiment.

[0066] Verification Example 2: Field efficacy test for controlling sorghum leaf spot

[0067] Test crop: Sorghum (Xinhong No. 5)

[0068] Target of prevention and control: Sorghum leaf spot

[0069] Test agent: 200 million spores / gram Trichoderma wettable powder (prepared in Example 3)

[0070] Trial start time: 2023.08

[0071] Trial completion time: September 2023

[0072] Growth conditions: Sorghum was planted on May 22, 2023, covering an area of ​​3 mu. It was in the filling stage during the experiment; the recommended crop in the experimental field was sorghum.

[0073] Soil of experimental field: The type of experimental field is loam soil with pH of about 8.0 and no interplanted crops.

[0074] Water and fertilizer management in the experimental site: Fertilizer and water management conditions were uniform and consistent, water and fertilizer were integrated, the management level was medium, and routine farming operations were followed before and after the experiment.

[0075] Meteorological data: The first application of pesticide (August 28) was cloudy to sunny, with a northeast wind of 2, a temperature of 10.3-26.27℃, an average temperature of 17.9℃, and a relative humidity of 74%; the second application of pesticide (September 4) was sunny, with a northeast wind of 2, a temperature of 15.6-31.7℃, an average temperature of 23.2℃, and a relative humidity of 71%; during the test period, there were three effective rainfall days with a total rainfall of 135.4mm. The maximum rainfall day was September 9 with a precipitation of 12.3mm. The maximum temperature was 31.7℃ and the minimum temperature was 10.3℃. There was no disastrous climate that particularly affected the efficacy of the pesticide.

[0076] Information on drugs used to control other diseases and pests: No fungicides or insecticides were used during the trial, which had no effect on the test results.

[0077] The dosage of the test agents is shown in Table 1 below:

[0078] Table 1

[0079]

[0080] Plot arrangement: There were 5 treatments in the experiment, each with 4 plots, and the plots were arranged in random blocks, totaling 20 plots.

[0081] Cell area and repetition: Cell area, 6m×5m=30m 2 ; Number of repetitions: 4 repetitions.

[0082] Application period and method: Start applying the pesticide at the early stage of sorghum grain filling. Weigh the pesticide according to the dosage required for each treatment, add water and spray normally. The spray should be uniform and consistent, without double spraying or missing spray. The blank control should be sprayed with water.

[0083] Applicator instrument number: SP-F-010 Instrument model, backpack electric sprayer, UIV-10 type, Weifang Motong Machinery Manufacturing Co., Ltd.

[0084] Application time and frequency: This experiment applied the pesticide twice, the first time on August 28, 2023, and the second time on September 4, 2023. The application volume was 54 liters per mu.

[0085] The survey method, time and frequency refer to the "Guidelines for Pesticide Field Efficacy Tests (II): Fungicide Control of Corn Large and Small Leaf Spot" (GB / T17980.107-2004).

[0086] Survey time and number: The first survey was conducted on August 28, 2023, before the first application of pesticides. Sporadic lesions appeared in the field and the disease index was recorded as zero. The second survey was conducted on September 13, 2023, 9 days after the last application of pesticides, to investigate the prevention and control effects and record the disease incidence according to the grading standards.

[0087] Survey method: Randomly select five points in each plot for investigation, select 5 plants at each point to investigate all leaves, and conduct investigation based on leaves according to the following grading method.

[0088] Leaf grading method: Level 0, disease-free; Level 1, the area of ​​lesions accounts for less than 5% of the entire leaf area; Level 3, the area of ​​lesions accounts for 6% to 10% of the entire leaf area; Level 5, the area of ​​lesions accounts for 11% to 25% of the entire leaf area; Level 7, the area of ​​lesions accounts for 26% to 50% of the entire leaf area; Level 9, the area of ​​lesions accounts for more than 51% of the entire leaf area.

[0089] The efficacy calculation method and formula used are as follows: calculate the disease index and control effect of each plot, calculate the average control effect of each treatment, and use SPSS17.0 software Duncan's new multiple range method to analyze the significance of differences among treatments.

[0090]

[0091] Direct Effects on Crops: One, two, and three days after each application, no phytotoxicity was observed on sorghum plants or ears. Effects on Other Pests: The test agent had no effect on other pests. Effects on Other Non-Target Organisms and Surrounding Crops: The test agent had no effect on natural enemies or surrounding crops.

[0092] The test results and the results of the investigation on the prevention effect showed (Table 2):

[0093] The test agent, a 200 million spore / g wettable powder of Trichoderma spores, achieved control efficacies of 51.98%, 68.97%, and 72.61% at doses of 200 g / mu, 300 g / mu, and 400 g / mu, respectively. The control agent, a 10 billion spore / g wettable powder of Bacillus subtilis, achieved a control efficacy of 73.15% at a dose of 55 g / mu. The Duncan's new multiple range analysis showed that the control efficacy of the 200 million spore / g wettable powder of Trichoderma spores was significantly different from that of the control agent at both the 0.05 and 0.01 levels, but was not significantly different from that of the control agent. The control efficacy of the low concentration of the test agent was significantly different from that of the control agent.

[0094] Table 2

[0095]

[0096]

[0097] Conclusion: It is recommended that 200 million spores / gram of Trichoderma spore wettable powder be applied from the jointing to the grain filling stage of sorghum leaf spot, with 2-3 applications every 7-10 days, at a dosage of 300-400 grams / mu. The test dose was safe for sorghum plants and ears, and had no effect on surrounding crops or non-target organisms.

[0098] Verification Example 3: Field plot efficacy test for controlling sorghum leaf spot

[0099] Test subject: Sorghum leaf spot (Exserohilum rostratum)

[0100] Test crop: Sorghum (variety: Liaoza 37)

[0101] Test location: Daguben Town, Fuxin Mongolian Autonomous County, Liaoning Province

[0102] Planting and Water and Fertilizer Management at the Experimental Site: Located in northwestern Liaoning Province, this site is one of the province's primary sorghum-growing areas. Sorghum leaf spot, purple spot, and head smut are common diseases affecting local sorghum cultivation. Spring temperatures in this region are relatively low. Sorghum was sown on May 25th, with a plant spacing of 25 cm and a row spacing of 60 cm, for a total of approximately 4,450 plants per mu. Base fertilizer was 500 kg / mu of organic fertilizer and 50 kg / mu of compound fertilizer. Disease developed naturally, with conventional management. The previous crop was corn, and no other fungicides were applied in this experiment. On August 2nd, 10% deltamethrin emulsifiable concentrate (20 ml / mu) was applied to control sorghum aphids.

[0103] Main instruments: balance (SW108002), sprayer (SW208009), wind direction and speed meter (SW208004).

[0104] Soil information: The soil type is sandy loam with neutral pH and medium fertility.

[0105] Trial Design and Arrangement

[0106] Dosage of drug (Table 3):

[0107] Table 3

[0108]

[0109] Plot arrangement: The test agent, control agent and blank control treatments were arranged in random blocks.

[0110] Cell area: 30m per cell 2 , set a protection row in the middle, and repeat 4 times.

[0111] Preparation of medicine:

[0112] (1) Before spraying, determine the appropriate amount of spray solution for each plot based on the plant growth status and the spray equipment used by spraying solvent water. Test results show that a spray volume of 2.25 liters per plot (30 square meters) can achieve full coverage of the plants, equivalent to a spray volume of 50 liters per mu.

[0113] (2) Medicine preparation equipment: measuring cylinder, measuring cup, bucket.

[0114] (3) Preparation method of the drug: dilute the weighed drug with 100 ml of water and stir evenly; add water to a fixed amount of 2.25 liters and then pour it into the sprayer.

[0115] Application method:

[0116] (1) Usage: Use the spray method to apply the pesticide, add water and spray evenly.

[0117] (2) Application equipment: knapsack sprayer, instrument number SW208009, working pressure: 0.15-0.4 MPa, flow rate 0.806 L / min, 0.815 L / min; nozzle: Φ15 mm, nozzle spray volume deviation within ±10%.

[0118] (3) Application time and frequency: August 21 and August 28, 2023, a total of 2 applications.

[0119] The first time the pesticide is applied is when the disease is in its early stages and the sorghum crop is in its jointing stage.

[0120] Survey, recording and statistical methods:

[0121] The experimental investigation was conducted according to the commissioning organization's experimental plan and in compliance with the SOP (SOP-G-081) "Field Efficacy Trial of Fungicides for Controlling Sorghum Leaf Spot" of the Institute of Biology and Medicine, Shenyang Research Institute of Chemical Industry Co., Ltd. The investigation period and frequency were as follows: The baseline disease status was investigated before the first application (August 21, 2023); efficacy investigations were conducted 7 days after the first application (August 28, 2023) and 10 days after the second application (September 7, 2023).

[0122] Survey sampling method and grading method: Randomly sample 5 points in each plot, survey 3 plants at each point, and survey all leaves of each plant. Grading is based on the percentage of the lesion area on each leaf to the total leaf area.

[0123] Leaf grading method: Level 0, disease-free; Level 1, the area of ​​lesions accounts for less than 5% of the entire leaf area; Level 3, the area of ​​lesions accounts for 6% to 10% of the entire leaf area; Level 5, the area of ​​lesions accounts for 11% to 25% of the entire leaf area; Level 7, the area of ​​lesions accounts for 26% to 50% of the entire leaf area; Level 9, the area of ​​lesions accounts for more than 51% of the entire leaf area.

[0124] The pharmacodynamic calculation method and formula used are as follows:

[0125] The disease index and control effect of each plot were calculated, the average control effect of each treatment was calculated, and the significance of differences among treatments was analyzed using SPSS17.0 software Duncan's new multiple range method.

[0126]

[0127] Compare the treated area with the blank control area to evaluate the percentage of damage. At the same time, accurately describe the symptoms of crop damage (stunting, chlorosis, deformity, etc.).

[0128] Test results and analysis:

[0129] Efficacy Survey Results: A disease index (DI) baseline survey conducted before application showed that within the sorghum leaf spot control trial area, the DI baseline ranged from 1.21 to 1.68 across plots, with an average DI baseline of 1.46. At the start of application, the DI baseline for sorghum leaf spot was relatively low, and the disease distribution was relatively uniform across the trial area. See Table 4 for details.

[0130] Table 4

[0131]

[0132] Effectiveness survey: Results from a seven-day follow-up study showed that the test agent, Trichoderma 200 million spores / g WP, demonstrated significant control efficacy against sorghum leaf spot. At dosages of 200 g / mu, 300 g / mu, and 400 g / mu, the average efficacy was 61.77%, 69.83%, and 73.01%, respectively. The efficacy of the test agent, Trichoderma 200 million spores / g WP at dosages of 300 g / mu and 400 g / mu, was comparable to that of the control agent, Bacillus subtilis 10 billion spores / g WP at a dosage of 55 g / mu (71.52%). Significant differences in efficacy were observed between the dosages of 200 g / mu, 300 g / mu, and 400 g / mu (p < 0.05). Detailed results are shown in Table 5.

[0133] Table 5

[0134]

[0135] Effectiveness survey: Ten days after the second application, the results showed that the 200 million spores / g WP of Trichoderma spores exhibited a significant control effect against sorghum leaf spot. At dosages of 200 g / mu, 300 g / mu, and 400 g / mu, the average control efficacy was 62.04%, 72.25%, and 76.49%, respectively. The control efficacy of the 200 million spores / g WP of Trichoderma spores at dosages of 300 g / mu and 400 g / mu was comparable to that of the control agent, 10 billion spores / g WP of Bacillus subtilis at a dosage of 55 g / mu (74.50%). There were significant differences (p < 0.05) in the efficacy of 200 million spores / g Trichoderma wettable powder against sorghum leaf spot at dosages of 200 g / mu, 300 g / mu, and 400 g / mu. Detailed results are shown in Table 6.

[0136] Table 6

[0137]

[0138] Conclusion: The test agent, Trichoderma 200 million spores / g wettable powder, showed good control efficacy against sorghum leaf spot, with an average control rate of over 72% at dosages of 300-400 g / mu. The control efficacy of the test agent, Trichoderma 200 million spores / g wettable powder at dosages of 300 g / mu and 400 g / mu, was comparable to that of the control agent.

[0139] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A Trichoderma koningii Tr25, characterized in that The classification name of the Trichoderma koningiopsis Tr25 is Trichoderma koningiopsis, which has been deposited in the General Microbiology Center of the China Culture Collection Administration Committee. The deposit date is May 2025 and the deposit number is CGMCC7.

284.

2. A Trichoderma acanthosporum Tr85, characterized in that The classification name of the Trichoderma asperellum Tr85 is Trichoderma asperellum, which has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms. The deposit date is May 2025 and the deposit number is CGMCC7.

283.

3. A microbial preparation, characterized in that The invention has active ingredients, wherein the active ingredients are the Trichoderma koningii Tr25 according to claim 1 and the Trichoderma acanthosporium Tr85 according to claim 2.

4. The microbial preparation according to claim 3, characterized in that The microbial preparation contains at least 2×10 8 cfu of Trichoderma koningii Tr25 and Trichoderma acanthosporum Tr85.

5. A method for preparing the microbial preparation according to any one of claims 3 to 4, characterized in that: The steps include: Step 1: taking Trichoderma koningii Tr25 and Trichoderma acanthosporum Tr85 and performing liquid fermentation; Step 2: spore concentration and harvesting; Step 3: Drying and preparation of microcapsule raw powder; Step 4: Mix and package the preparations.

6. The preparation method according to claim 5, characterized in that: Step 1: The specific method of liquid fermentation is: 1) Seed culture: Spores were aseptically scraped from the eggplant-shaped culture flask and inoculated into a 100 L fermentation seed tank containing 75 L of sterile seed culture medium at 28°C, 280 RPM, 0.5-1 v / v, for 30-36 hours. The seed culture medium used was: 1.5% potato flour and 1% glucose. 2) Spore culture: Aseptically transfer the culture to a 1000 L fermenter containing 750 L sterilized seed culture medium at 25-32°C, 175 RPM, 0.5-1 v / v. Ferment for 72 hours, add a spore inducer, continue culturing for 96-122 hours, and harvest the fermentation broth. The spore-forming medium used is: 25% potato flour, 2% glucose, and 0.5% amino acids.

7. The preparation method according to claim 5, characterized in that: The specific method of the step 2 is that the fermentation liquid enters the centrifuge at a flow rate of 1000 L / h and a centrifuge speed of 30 Hz, the fermentation liquid is centrifuged and concentrated twice, and the concentrated liquid containing Trichoderma spores is collected.

8. The preparation method according to claim 5, characterized in that: The specific method of step three is to mix the spore concentrate with the wall material / protective agent dextran, gum arabic, Span 60, water-soluble starch, and white carbon black; transport it to a spray drying tower with an inlet air temperature of 75-110° C., an outlet air temperature of 40-60° C., and a feed rate of 1-2 L / min; and collect the spore raw powder through a cyclone.

9. Use of the microbial preparation according to any one of claims 3 to 4 in preventing and treating sorghum leaf spot.

10. The use according to claim 9, characterized in that The sorghum leaf spot disease is caused by one or more pathogens selected from the group consisting of Alternaria kaoliang, Mycosphaerella kaoliang, and Curvularia lunata.

Images