Solid fermentation method for improving conidia by using trichoderma yunnanense F2 strain

Abstract

The invention discloses a solid fermentation method for improving conidia by using a Trichoderma yunnanense F2 strain, which comprises the following steps of: activating the Trichoderma yunnanense F2 strain, preparing into a Trichoderma yunnanense conidia suspension, and culturing by using bran, wheat straw powder, roxburgh rose residue powder and rapeseed powder cake as a culture medium to prepare the Trichoderma yunnanense conidia suspension. The trichoderma yunnanense F2 strain is subjected to conidium fermentation culture to obtain the trichoderma biocontrol inoculant, and the fermentation conditions are that the water content is 65%, the inoculum size is 8%, and the fermentation days are 9 days. The solid fermentation method disclosed by the invention can effectively improve the yield and stability of biological control and antifungal active substances, and is simple in equipment, low in cost and stable in product.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microbial fermentation, and relates to a method for solid fermentation of using Trichoderma yunnanense strain F2 to improve conidia. Background Art

[0002] As an important biocontrol fungus, Trichoderma spp. are widely distributed in the phyllosphere, rhizosphere, bark and rhizosphere soil of plants. In the process of modern agricultural operations, the traditional method of controlling diseases is mainly to kill harmful organisms on crops through chemical agents to achieve the purpose of disease control. However, the use of chemical pesticides often leads to problems such as the generation of drug resistance in diseases, the killing of other beneficial organisms, and environmental damage. As the core component of the green prevention and control technology for plant protection, biological control is an important technical means for effectively controlling the dosage of chemical pesticides in China, ensuring the safety of agricultural production and the ecological environment, improving the quality of agricultural products, and promoting the sustainable development of agriculture. The preparation of biocontrol agents mainly has two methods: liquid fermentation and solid fermentation. However, liquid fermentation has high production costs, low yields of product conidia, short product shelf life, and inconvenient transportation.

[0003] So far, there have been many studies on Trichoderma harzianum and Trichoderma viride in Trichoderma biocontrol agents, but there is no report on Trichoderma yunnanense. The Trichoderma yunnanense strain F2 used in the present invention is the Trichoderma yunnanense strain F2 (application number: 202310857335.4, patent name: A Trichoderma yunnanense and Its Application) taken from the patent applied by the applicant in the early stage. Using wheat bran, wheat straw powder, rosa roxburghii residue powder and rapeseed cake as solid substrates for solid fermentation, this method can effectively improve the yield and stability of its biological control and antifungal active substances, with simple equipment, low cost and stable products. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for solid fermentation of using Trichoderma yunnanense strain F2 to improve conidia, which can effectively improve the yield and stability of its biological control and antifungal active substances.

[0005] The technical solution of the present invention: A method for solid fermentation of using Trichoderma yunnanense strain F2 to improve conidia. The solid fermentation method is to first activate the Trichoderma yunnanense strain F2, and then prepare it into a suspension of Trichoderma yunnanense conidia. Using wheat bran, wheat straw powder, rosa roxburghii residue powder and rapeseed cake as culture substrates, the Trichoderma yunnanense strain F2 is fermented and cultured for conidia to obtain a Trichoderma biocontrol agent.

[0006] The aforementioned solid fermentation method is carried out according to the following steps:

[0007] (1) Activation of Yunnan Trichoderma F2 strain: Take out Yunnan Trichoderma F2 in a frozen state, activate it on PDA medium, and then use a 0.7 mm puncher to take the mycelium block at the edge of the colony to obtain the activated Yunnan Trichoderma F2 strain for later use;

[0008] (2) Preparation of conidia suspension of Trichoderma yunnanensis: The activated Trichoderma yunnanensis F2 strain was inoculated into PDA medium and cultured at 23-27°C for 5-8 days. The spores were washed with sterile saline. 4-6 ml of sterile water was added to each plate and fully shaken. The conidia were gently scraped with an applicator and pipetted into a sterilized centrifuge tube. The conidia were counted under a microscope using a hemocytometer and the conidia concentration was adjusted to 1.5 × 10 8 -2.5×10 8 / mL, to obtain a suspension of Trichoderma yunnanensis conidia for later use;

[0009] (3) Preparation of solid matrix: Weigh bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake, wherein the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 40-60:10-15:10-15:20-30, mix well, and obtain a solid matrix for later use;

[0010] (4) Preparation of liquid matrix: Add sterile water to the solid matrix to make the water content of the solid matrix reach 60-70%, stir evenly to make the solid matrix and water fully mixed, moisten the material for 30-40 minutes, and sterilize the material in a high-pressure steam sterilizer at a pressure of 100-110 kPa for 12-18 minutes. After cooling, obtain the liquid matrix for standby use;

[0011] (5) Inoculation and fermentation: The conidia suspension of Trichoderma yunnanensis prepared in step (1) is inoculated into a conical flask containing a liquid matrix, wherein the inoculation amount of the conidia suspension of Trichoderma yunnanensis is 7-9% of the liquid matrix. After being fully shaken and mixed, the suspension is statically placed upright in a constant temperature incubator at 23-27° C. After the mycelium is fully grown, the material is fully broken up to increase the contact between the material debris and the surface of the Trichoderma, and the fermentation is continued for 8-10 days.

[0012] The aforementioned PDA culture medium is prepared by weighing 200.0 g of peeled potatoes, 20.0 g of glucose and 15.0 g of agar powder, and diluting the volume to 1000 mL with distilled water.

[0013] Preparation of the conidia suspension of Trichoderma yunnanensis: inoculate the PDA medium with the Trichoderma yunnanensis F2 strain, culture at 24-26°C for 6-8 days, add sterile physiological saline to wash the spores, add 4.5-5.5 ml of sterile water to each plate and shake it thoroughly, gently scrape it with an applicator, pipette it into a sterilized centrifuge tube, count it under a microscope using a hemocytometer, and adjust the spore concentration to 1.8×108 -2.2×10 8 / mL to obtain a conidia suspension of Trichoderma yunnanensis for later use.

[0014] Specifically, the preparation of the conidia suspension of Trichoderma yunnanensis is as follows: the Trichoderma yunnanensis F2 strain is inoculated into PDA medium and cultured at 25° C. for 7 days, sterile physiological saline is added to wash the spores, 5 ml of sterile water is added to each plate and fully shaken, the spores are gently scraped with an applicator, and the spores are pipetted into a sterilized centrifuge tube with a pipette, and the spores are counted under a microscope using a hemocytometer, and the spore concentration is adjusted to 2.0×108 / mL to obtain a conidia suspension of Trichoderma yunnanensis for standby use.

[0015] The weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 45-55:12.5-15:12.5-15:25-30.

[0016] Specifically, the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 40:15:13.8:30.

[0017] Preparation of the aforementioned liquid matrix: Add sterile water to the solid matrix to make the water content of the solid matrix reach 62-68%, stir evenly to fully mix the solid matrix and water, moisten the material for 32-38 minutes, and sterilize the article in a high-pressure steam sterilizer at a pressure of 102-105 kPa for 14-16 minutes. After cooling, obtain the liquid matrix for standby use.

[0018] Specifically, the liquid matrix is ​​prepared as follows: sterile water is taken from the solid matrix to make the water content in the solid matrix reach 65%, and the solid matrix is ​​stirred evenly to fully mix with the water. After moistening the material for 35 minutes, the article is sterilized for 15 minutes in a high-pressure steam sterilizer at a pressure of 103.4 kPa. After cooling, the liquid matrix is ​​obtained and set aside.

[0019] The above-mentioned inoculation and fermentation: the Yunnan Trichoderma conidia suspension prepared in step (1) is inoculated into a triangular flask containing a liquid matrix, the inoculation amount of the Yunnan Trichoderma conidia suspension is 8% of the liquid matrix, after being fully shaken and mixed, it is statically placed upright in a 25°C constant temperature incubator, and the material is fully shaken and crushed when the mycelium is fully grown to increase the contact between the material debris and the surface of the Trichoderma, and the fermentation is continued for 9 days.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention uses optimized fermentation conditions (water content 65%, inoculation amount 8%, fermentation days 9 d), with wheat bran, wheat straw powder, Rosa roxburghii Tratt residue powder and rapeseed cake powder as substrates. This ratio significantly increases the spore yield of Trichoderma strains, making the conidial spore yield reach 2.32×10 10 CFU / g.

[0022] 2. Trichoderma yunnanense F2 has a high cellulase production capacity and can effectively degrade the cellulose of Rosa roxburghii Tratt fruit residue to obtain carbon sources, which is beneficial to its growth and reproduction, thus increasing the conidial spore yield.

[0023] 3. The main component of Rosa roxburghii Tratt fruit residue is cellulose, which cannot be effectively utilized by most microorganisms in the environment, so their growth and reproduction are inhibited. This results in a lower level of microbial contamination of Rosa roxburghii Tratt residue and retains a relatively high content of polysaccharide carbon sources. At the same time, Rosa roxburghii Tratt residue contains some special components that can promote the synthesis of metabolites of Trichoderma.

[0024] 4. The wheat straw powder has a high cellulose content and is difficult to degrade, which can limit the utilization efficiency of Trichoderma for its nutrients.

[0025] 5. Selecting wheat bran, wheat straw powder, rapeseed cake powder and Rosa roxburghii Tratt residue as fermentation substrates makes full use of agricultural and industrial wastes, reduces the fermentation cost. At the same time, these substrates are rich in carbon sources, nitrogen sources and various mineral elements, providing rich nutrients for the growth of Trichoderma.

[0026] 6. The present invention adopts a unique combination of mixed fermentation substrates and has research significance in terms of fermentation conditions and optimization methods. Description of the Drawings

[0027] Figure 1 : Results of single-factor experiments;

[0028] Figure 2 : Results of two-factor experiments;

[0029] Figure 3 : Three-dimensional surface plot and contour lines of the interactive effect of wheat bran and wheat straw powder;

[0030] Figure 4 : Three-dimensional surface plot and contour lines of the interactive effect of wheat bran and Rosa roxburghii Tratt residue powder;

[0031] Figure 5 : Three-dimensional surface and contour lines of the interactive effect of wheat straw powder and Rosa roxburghii Tratt residue powder;

[0032] Figure 6 : Three-dimensional surface and contour lines of the interactive effect of Rosa roxburghii Tratt residue powder and rapeseed cake powder;

[0033] Figure 7 : Conidial spore yields at different inoculation amounts;

[0034] Figure 8 : Conidia yield at different fermentation days;

[0035] Figure 9 : Conidia yield at different water contents;

[0036] Figure 10 : Three-dimensional surface and contour plots of the interaction between inoculum size and water content;

[0037] Figure 11 : Three-dimensional surface map and contour map of the interaction between inoculation amount and fermentation days;

[0038] Figure 12 : Three-dimensional surface and contour plots of the interactive effect of moisture content and fermentation days. DETAILED DESCRIPTION

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0040] Preparation of PDA culture medium: Weigh 200.0 g of peeled potatoes, 20.0 g of glucose and 15.0 g of agar powder, dilute to 1000 mL with distilled water, and set aside.

[0041] Example 1: Solid fermentation method

[0042] (1) Activation of Yunnan Trichoderma F2 strain: Take out Yunnan Trichoderma F2 in a frozen state, activate it on PDA medium, and then use a 0.7 mm puncher to take the mycelium block at the edge of the colony to obtain the activated Yunnan Trichoderma F2 strain for later use;

[0043] (2) Preparation of conidia suspension of Trichoderma yunnanensis: Inoculate the Trichoderma yunnanensis F2 strain with PDA medium and culture at 25°C for 7 days. Add sterile physiological saline to wash the spores. Add 5 ml of sterile water to each plate and shake thoroughly. Gently scrape with an applicator, pipette and place into a sterilized centrifuge tube. Count the spores under a microscope using a hemocytometer. Adjust the spore concentration to 2.0×108 / mL to obtain a conidia suspension of Trichoderma yunnanensis for later use.

[0044] (3) Preparation of solid matrix: Weigh bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake, the weight ratio of bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 40:15:13.8:30, mix well to obtain a solid matrix, and set aside;

[0045] (4) Preparation of liquid matrix: Take sterile water for solid matrix, make the water content of solid matrix reach 65%, stir evenly to make solid matrix and water fully mix, moisten for 35 minutes, sterilize in high pressure steam sterilizer for 15 minutes, sterilize at 125°C and 103.4 kPa, cool to obtain liquid matrix, set aside;

[0046] (5) Inoculation and fermentation: The conidia suspension of Trichoderma yunnanensis prepared in step (1) is inoculated into a conical flask containing a liquid matrix, the inoculation amount of the conidia suspension of Trichoderma yunnanensis is 8% of the liquid matrix, after being fully shaken and mixed, it is statically placed upright in a constant temperature incubator at 25° C., and the material is fully shaken and crushed after the mycelium is fully grown to increase the contact between the material debris and the surface of Trichoderma, and the fermentation is continued for 9 days to obtain the Trichoderma biocontrol agent.

[0047] Example 2: Solid fermentation method

[0048] (1) Activation of Yunnan Trichoderma F2 strain: Take out Yunnan Trichoderma F2 in a frozen state, activate it on PDA medium, and then use a 0.7 mm puncher to take the mycelium block at the edge of the colony to obtain the activated Yunnan Trichoderma F2 strain for later use;

[0049] (2) Preparation of Yunnan Trichoderma conidia suspension: Take the activated Yunnan Trichoderma F2 strain and inoculate it into PDA medium, culture it at 27°C for 5 days, add sterile physiological saline to wash the spores, add 4 ml of sterile water to each plate and shake it thoroughly, gently scrape it with an applicator, and pipette it into a sterilized centrifuge tube. Use a hemocytometer to count the spores under a microscope, adjust the spore concentration to 2.5×108 / mL, and obtain Yunnan Trichoderma conidia suspension for later use;

[0050] (3) Preparation of solid matrix: Weigh bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake, wherein the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 40:15:15:30, mix well, and obtain a solid matrix for later use;

[0051] (4) Preparation of liquid matrix: Add sterile water to the solid matrix to make the water content of the solid matrix reach 60%, stir evenly to make the solid matrix and water fully mixed, moisten the material for 40 minutes, and sterilize it in a high pressure steam sterilizer for 12 minutes at a sterilization temperature of 135°C and a sterilization pressure of 100 kPa. After cooling, a liquid matrix is ​​obtained and set aside;

[0052] (5) Inoculation and fermentation: The conidia suspension of Trichoderma yunnanensis prepared in step (1) is inoculated into a conical flask containing a liquid matrix, wherein the inoculation amount of the conidia suspension of Trichoderma yunnanensis is 7% of the liquid matrix. After being fully shaken and mixed, the suspension is statically placed upright in a 27° C. constant temperature incubator. After the mycelium is fully grown, the material is fully broken up to increase the contact between the material debris and the surface of the Trichoderma. The fermentation is continued for 8 days.

[0053] Example 3: Solid fermentation method

[0054] (1) Activation of Yunnan Trichoderma F2 strain: Take out Yunnan Trichoderma F2 in a frozen state, activate it on PDA medium, and then use a 0.7 mm puncher to take the mycelium block at the edge of the colony to obtain the activated Yunnan Trichoderma F2 strain for later use;

[0055] (2) Preparation of Yunnan Trichoderma conidia suspension: The activated Yunnan Trichoderma F2 strain was inoculated into PDA medium and cultured at 23° C. for 8 days. Sterile physiological saline was added to wash the spores. 6 ml of sterile water was added to each plate and fully shaken. The spores were gently scraped with an applicator and pipetted into a sterilized centrifuge tube. The spores were counted under a microscope using a hemocytometer and the concentration of the spores was adjusted to 1.5×108 / mL to obtain a Yunnan Trichoderma conidia suspension for later use.

[0056] (3) Preparation of solid matrix: Weigh bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake, wherein the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 60:10:10:20, mix well, and obtain a solid matrix for later use;

[0057] (4) Preparation of liquid matrix: Add sterile water to the solid matrix to make the water content of the solid matrix reach 70%, stir evenly to make the solid matrix and water fully mixed, moisten the material for 30 minutes, and sterilize it in a high pressure steam sterilizer for 18 minutes at a sterilization temperature of 105°C and a sterilization pressure of 110 kPa. After cooling, a liquid matrix is ​​obtained and set aside;

[0058] (5) Inoculation and fermentation: The conidia suspension of Trichoderma yunnanensis prepared in step (1) is inoculated into a conical flask containing a liquid matrix, wherein the inoculation amount of the conidia suspension of Trichoderma yunnanensis is 9% of the liquid matrix. After being fully shaken and mixed, the conidia suspension is statically placed upright in a constant temperature incubator at 23° C. After the mycelium is fully grown, the material is fully broken up to increase the contact between the material debris and the surface of the Trichoderma, and the fermentation is continued for 10 days.

[0059] Example 4: Solid fermentation method

[0060] (1) Activation of Yunnan Trichoderma F2 strain: Take out Yunnan Trichoderma F2 in a frozen state, activate it on PDA medium, and then use a 0.7 mm puncher to take the mycelium block at the edge of the colony to obtain the activated Yunnan Trichoderma F2 strain for later use;

[0061] (2) Preparation of conidia suspension of Trichoderma yunnanensis: Inoculate the Trichoderma yunnanensis F2 strain with PDA medium and culture at 24°C for 8 days. Add sterile physiological saline to wash the spores. Add 5.5 ml of sterile water to each plate and shake thoroughly. Gently scrape with an applicator, pipette and place into a sterilized centrifuge tube. Count the spores under a microscope using a hemocytometer. Adjust the spore concentration to 1.8×108 / mL to obtain a conidia suspension of Trichoderma yunnanensis for later use.

[0062] (3) Preparation of solid matrix: Weigh bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake, wherein the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 45:15:12.5:25, mix well, and obtain a solid matrix for later use;

[0063] (4) Preparation of liquid matrix: Add sterile water to the solid matrix to make the water content of the solid matrix reach 62-68%, stir evenly to make the solid matrix and water fully mixed, moisten the material for 38 minutes, and sterilize it in a high pressure steam sterilizer for 16 minutes at a sterilization temperature of 125° C. and a sterilization pressure of 102 kPa. After cooling, a liquid matrix is ​​obtained and set aside;

[0064] (5) Inoculation and fermentation: The conidia suspension of Trichoderma yunnanensis prepared in step (1) is inoculated into a conical flask containing a liquid matrix, wherein the inoculation amount of the conidia suspension of Trichoderma yunnanensis is 7% of the liquid matrix. After being fully shaken and mixed, the suspension is statically placed upright in a constant temperature incubator at 23° C. After the mycelium is fully grown, the material is fully broken up to increase the contact between the material debris and the surface of the Trichoderma, and the fermentation is continued for 8 days.

[0065] Example 5: Solid fermentation method

[0066] (1) Activation of Yunnan Trichoderma F2 strain: Take out Yunnan Trichoderma F2 in a frozen state, activate it on PDA medium, and then use a 0.7 mm puncher to take the mycelium block at the edge of the colony to obtain the activated Yunnan Trichoderma F2 strain for later use;

[0067] (2) Preparation of conidia suspension of Trichoderma yunnanensis: Inoculate the Trichoderma yunnanensis F2 strain with PDA medium and culture at 26°C for 6 days. Add sterile physiological saline to wash the spores. Add 4.5 ml of sterile water to each plate and shake thoroughly. Gently scrape with an applicator, pipette and place into a sterilized centrifuge tube. Count the spores under a microscope using a hemocytometer. Adjust the spore concentration to 2.2×108 / mL to obtain a conidia suspension of Trichoderma yunnanensis for later use.

[0068] (3) Preparation of solid matrix: Weigh bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake, wherein the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 55:12.5:15:30, mix well, and obtain a solid matrix for later use;

[0069] (4) Preparation of liquid matrix: Add sterile water to the solid matrix to make the water content of the solid matrix reach 68%, stir evenly to make the solid matrix and water fully mixed, moisten the material for 32 minutes, and sterilize it in a high pressure steam sterilizer for 16 minutes at a sterilization temperature of 105°C and a sterilization pressure of 105 kPa. After cooling, a liquid matrix is ​​obtained and set aside;

[0070] (5) Inoculation and fermentation: The conidia suspension of Trichoderma yunnanensis prepared in step (1) is inoculated into a conical flask containing a liquid matrix, wherein the inoculation amount of the conidia suspension of Trichoderma yunnanensis is 9% of the liquid matrix. After being fully shaken and mixed, the suspension is statically placed upright in a constant temperature incubator at 27° C. After the mycelium is fully grown, the material is fully broken up to increase the contact between the material debris and the surface of the Trichoderma, and the fermentation is continued for 9 days.

[0071] In order to verify the beneficial effects of the present invention, the inventors conducted a large number of experimental studies, and the experimental process and results are as follows:

[0072] 1 Materials and methods

[0073] 1.1 Test materials

[0074] Test bacteria: Trichoderma yunnanensis F2 strain, provided by the laboratory of the Institute of Biotechnology, Guizhou Academy of Agricultural Sciences (the serial number and preservation indication of Trichoderma yunnanensis F2 strain can be found in the application number: 202310857335.4, patent name: A Trichoderma yunnanensis and its application).

[0075] 1.2 Main instruments: vertical high pressure steam sterilizer, temperature controlled incubator, optical microscope, hole puncher (D=7mm), blood cell counting plate, vortex machine, etc.

[0076] 1.3 Matrix materials: bran, corn straw powder, wheat straw powder, roxburghii residue powder, sheep dung, cow dung, tea dung powder, rapeseed cake powder.

[0077] 1.4 Main culture medium: Potato Dextrose Agar (PDA): 200.0 g of peeled potatoes, 20.0 g of glucose, 15.0 g of agar powder, made up to 1000 ml with distilled water; Before using Trichoderma yunnanense F2, it needs to be activated and cultured on PDA medium. Use a puncher (0.7 mm) to take the mycelium blocks at the edge of the colony for subsequent experiments.

[0078] 1.5. Test method

[0079] Preparation of Trichoderma yunnanense F2 conidia suspension: Inoculate Trichoderma yunnanense F2 on PDA solid plate and culture at 25 °C for 7 days. Add sterile normal saline to wash down the spores. Add 5 ml of sterile water to each plate and shake well. Gently scrape and wash with a spreader, and then use a pipette to suck and transfer it into a sterilized 10 ml centrifuge tube to obtain the Trichoderma yunnanense F2 conidia suspension. Use a hemocytometer to count under a microscope and adjust the spore concentration to 2.0×108 spores / mL for standby.

[0080] 2 Screening of the optimal solid fermentation substrate

[0081] 2.1 Single-factor experiment

[0082] 2.1.1 Process of single-factor experiment

[0083] Select wheat bran, corn straw powder, wheat straw powder, rosa roxburghii residue powder, sheep manure, cow manure, tea seed cake powder, rapeseed cake powder. That is, different material components are filled into 500 ml Erlenmeyer flasks respectively. Add sterile water according to the ratio of initial solid: water = 1:0.8, stir evenly to make the solid substrate and water fully mixed, and moisten the material for 35 min. After autoclaving and cooling, obtain the liquid substrate; inoculate the Trichoderma yunnanense F2 conidia suspension with a concentration of 2.0×108 spores / mL into the Erlenmeyer flask. The inoculation amount of the Trichoderma yunnanense F2 conidia suspension is 4% of the liquid substrate. After shaking well and mixing evenly, place it statically upright in a 25 °C constant temperature incubator. When the mycelium has grown fully, shake the material well, and continue fermentation to increase the surface contact between the material debris and Trichoderma F2; Each treatment has 3 replicates, and the spore count is measured after 7 days of fermentation.

[0084] 2.1.2 Determination of spore content

[0085] Mix the solid fermentation product evenly, weigh 1 g of the solid fermentation product into a 500 mL conical flask containing 100 mL of sterile water, shake well on a shaker at 180 r / min for 20 min at 25 °C, and then let it stand for 30 min. The spore count is detected by the hemocytometer counting method.

[0086] 2.1.3 Results of single-factor experiment

[0087] As can be seen from the figure, the best spore production is achieved with wheat bran, wheat straw powder, and rapeseed cake powder. Next are corn straw powder and Rosa roxburghii Tratt residue powder. Sheep manure, cow dung, and tea seed cake powder are not conducive to the growth of Trichoderma, indicating that wheat bran, rapeseed cake powder, and wheat straw powder are optimal when the initial solid: initial water content = 1:0.8. Since Rosa roxburghii Tratt is one of the important special products in Guizhou Province and is rich in nutrients (such as Figure 1 ).

[0088] 2.2 Two-factor experiment

[0089] 2.2.1 Process of two-factor experiment

[0090] Six groups of material components were set, namely: 1) all wheat bran; 2) wheat bran: corn straw powder = 1:1; 3) wheat bran: rice straw powder = 1:1; 4) wheat bran: wheat straw powder = 1:1; 5) wheat bran: rice bran = 1:1; 6) corn straw powder: rice bran = 1:1. Sterile water was added according to the ratio of initial solid: water = 1:0.8 and stirred evenly to fully mix the solid matrix and water. After moistening the material for 35 min, and cooling after high-pressure steam sterilization, a liquid matrix was obtained; a suspension of Trichoderma yunnanense F2 conidia with a concentration of 2.0×108 / mL was inoculated into a triangular flask, and the inoculation amount of the Trichoderma yunnanense F2 conidia suspension was 4% of the liquid matrix. After fully shaking and mixing evenly, it was statically placed upright in an incubator at 25°C. After the mycelium grew fully, the material was shaken and crushed, and fermentation was continued to increase the full contact between the material debris and Trichoderma yunnanense F2. Each repetition was carried out 3 times, and the spore number was measured after 7 days of fermentation.

[0091] 2.2.2 Determination of spore content

[0092] The solid fermentation products were mixed evenly. 1 g of the solid fermentation product was weighed into a 500 mL conical flask containing 100 mL of sterile water. Under the condition of 25°C, it was shaken evenly on a shaker at 180 r / min for 20 min and then left to stand for 30 min. The spore number was detected by the hemocytometer counting method.

[0093] 2.2.3 Results of two-factor experiment

[0094] It can be concluded that wheat bran and wheat straw powder are suitable for the growth of Trichoderma F2, and their spore content is also the highest. According to the experimental results, wheat bran and wheat straw powder can be selected as raw materials, and Rosa roxburghii Tratt residue powder and rapeseed cake powder can be added as auxiliary materials for the next experiment (such as Figure 2 ).

[0095] 2.3 Screening of substrates

[0096] 2.3.1 Selection of substrate levels for response surface experiment

[0097] Four fermented substances, namely wheat bran, wheat straw powder, Rosa roxburghii residue powder, and rapeseed cake powder, were selected through single-factor and two-factor experiments on spore production, and used as fermentation substrates. The optimization of the four fermentation substrates was carried out by the response surface method. A four-factor and three-level experiment was designed, with 3 replicates for each treatment. Wheat bran accounted for 50%, wheat straw powder accounted for 12.5%, Rosa roxburghii residue powder accounted for 12.5%, and rapeseed cake powder accounted for 25%.

[0098] Using the Box-Behnken central experimental design principle, with spore production as the response value, and the four factors A (wheat bran), B (wheat straw powder), C (Rosa roxburghii residue powder), and D (rapeseed cake powder) that affect spore production as independent variables, Design-Expert 13 analysis software was used.

[0099] Table 1 Experimental factor level table

[0100]

[0101] Table 2 Response surface design and experimental results of the culture medium conditions of Trichoderma yunnanense F2

[0102]

[0103]

[0104] 2.3.2 Central composite experiment and results

[0105] Using the Box-Behnken central experimental design principle, with spore production as the response value, and the four factors A (wheat bran), B (wheat straw powder), C (Rosa roxburghii residue powder), and D (rapeseed cake powder) that affect spore production as independent variables, the scheme and results are shown in Table 2. Using Dwsign-Expert 13 analysis software, multiple regression fitting was performed on the data in the table, and the simulated regression equation between the culture medium and spore production was obtained as follows:

[0106] Y = 3.780*10 8 -5.750*10 7 A + 1.158*10 7 B + 3.317*107C + 4.825*10 7 D - 1.028*10 8 AB - 5.500*10 7 AC - 8.575*10 7 AD + 1.850*10 7 BC - 1.550*10 7 BD + 1.100*10 7 CD - 1.992*10 7 A 2 -5.229*10 7 B2 -1.129*10 8 E+08C 2 -4.854*10 7 D 2

[0107] Analysis of Variance of Regression Simulation in Table 3

[0108]

[0109] From the above regression model through analysis of variance, it can be seen that: for the regression model, P < 0.0001, and the loss, the regression test of the model is significant, indicating that the interference of unknown factors on the test results is small and the model is stable; the determination coefficient R2 of the regression equation is 82.43%, indicating that this equation fits well with the actual situation and reflects the relationship between the sporulation amount of Trichoderma yunnanense F2 and wheat bran, wheat straw powder, rapeseed cake powder, and rosa roxburghii tratt residue powder. Therefore, this model can be used for analysis and prediction. The larger the F value, the stronger the influence of each factor on the sporulation amount of Trichoderma yunnanense F2. The test results show that the influence of the four factors on the sporulation amount of this bacterium is in the order of: wheat bran > rapeseed cake powder > rosa roxburghii tratt residue powder > wheat straw powder; from the significance test of the regression equation coefficients, it can be seen that A2, B2, C2, D 2 is significant (P > 0.01), and the rest are not significant.

[0110] 2.3.3 Response Surface Plots and Contour Plots

[0111] The three-dimensional surfaces and contour plots of the interactive effects of wheat bran and wheat straw powder, wheat bran and rosa roxburghii tratt residue powder, wheat straw powder and rosa roxburghii tratt residue powder, and rosa roxburghii tratt residue powder and rapeseed cake powder are shown in Figures 3 - 6 .

[0112] 2.3.4 Determination and Verification of the Optimal Conditions of the Response Surface

[0113] Using Design Expert software, through analysis and calculation, it can be known that the optimal substrate ratio for producing Trichoderma yunnanense F2 is wheat bran, wheat straw powder, rosa roxburghii tratt residue powder, and rapeseed cake powder with a weight ratio of 40.2:15:14.8:30. Under this condition, the predicted value of the sporulation amount of Trichoderma yunnanense F2 is 5.78×10 8 cfu / g. In order to further verify the practical operability of the predicted value, three repeated fermentation tests were carried out using the optimized substrate conditions, and the average value of the sporulation amount of Trichoderma yunnanense F2 measured was 7.13×10 8 cfu / g. The actual value has more sporulation amount than the predicted value, proving that the predicted value and the actual value have good fitting, and this model is practical and reliable.

[0114] 2.4 Response Surface Test of Fermentation Conditions

[0115] 2.4.1 Screening Test of Inoculum Size

[0116] Determine the spore production of Trichoderma yunnanense F2 in the substrate of wheat bran: wheat straw powder: Rosa roxburghii residue powder: rapeseed meal cake with different inoculation amounts. Five inoculation amount treatments are set, namely 4%, 5%, 6%, 7%, and 8%. The substrate is wheat bran, wheat straw powder, Rosa roxburghii residue powder, and rapeseed meal cake with a weight ratio of 40.2:15:14.8:30. Sterilize at 121 °C for 30 min, cool, and then inoculate with the spore suspension of Trichoderma yunnanense F2. At a temperature of 25 °C, place it in a constant temperature incubator for solid fermentation for 7 d, and then count the number of conidia with a hemocytometer. Each treatment is set with three replicates, and the average value is taken. 2.4.2 Moisture content screening:

[0117] Determine the spore production of Trichoderma yunnanense F2 in the substrate of wheat bran: wheat straw powder: Rosa roxburghii residue powder: rapeseed meal cake with different inoculation amounts. Five moisture content treatments are set, namely 60%, 65%, 70%, 75%, and 80%. The substrate is wheat bran, wheat straw powder, Rosa roxburghii residue powder, and rapeseed meal cake with a weight ratio of 40.2:15:14.8:30. Sterilize at 121 °C for 30 min, cool, and then inoculate with the spore suspension of Trichoderma yunnanense F2. At a temperature of 25 °C, place it in a constant temperature incubator for solid fermentation for 7 d, and then count the number of conidia with a hemocytometer. Each treatment is set with three replicates, and the average value is taken.

[0118] 2.4.3 Fermentation days screening:

[0119] Determine the spore production of Trichoderma yunnanense F2 in the substrate of wheat bran: wheat straw powder: Rosa roxburghii residue powder: rapeseed meal cake with different inoculation amounts. Five fermentation days treatments are set, namely 3 d, 5 d, 7 d, 9 d, and 11 d. The substrate is wheat bran, wheat straw powder, Rosa roxburghii residue powder, and rapeseed meal cake with a weight ratio of 40.2:15:14.8:30. Sterilize at 121 °C for 30 min, cool, and then inoculate with the spore suspension of Trichoderma yunnanense F2. At a temperature of 25 °C, place it in a constant temperature incubator for solid fermentation for 7 d, and then count the number of conidia with a hemocytometer. Each treatment is set with three replicates, and the average value is taken.

[0120] 2.4.4 Data processing

[0121] The screening design of key factors of culture conditions and the experimental design of Box-Behnken response surface method. All experimental data are statistically analyzed using Excel and IBM SPSS Statistics 27 software. Significance tests are carried out through one-way and two-way ANOVA and multiple comparison analysis. When P < 0.05, the difference is considered significant; when P > 0.05, the difference is considered not significant.

[0122] 2.4.5 Selection of factor levels for response surface test of fermentation conditions

[0123] Using the principle of Box-Behnken central experimental design, through screening and analysis of the three results of single-factor experiments, finally, three factors, namely water content (%), inoculation amount (%), and fermentation days (d), were selected as independent variables. The levels of each factor are shown in Table 4. Taking the spore content as the response value, the response surface analysis method was used to optimize the culture conditions of Trichoderma yunnanense F2 biocontrol agent with three factors and three levels, and each group was repeated three times.

[0124] Table 4 Experimental factor level table

[0125]

[0126] (1) The conidia of Trichoderma yunnanense F2 increase with the increase of the inoculation amount of the suspension. When the inoculation amount reaches 8%, its conidia are significantly higher than those of other inoculation amounts, which is 8.40×108 CFU / g. With the continuous increase of the inoculation amount, the conidia production gradually decreases. The optimal inoculation amount for the fermentation of Trichoderma yunnanense F2 is 8% (as Figure 7 ).

[0127] (2) The conidia of Trichoderma yunnanense F2 increase with the increase of fermentation days. In the early stage of the fermentation of Trichoderma yunnanense F2, it mainly grows white hyphae. From the sixth day, conidia begin to grow on the white hyphae. Until the fermentation reaches the ninth day, the conidia reach the maximum value, which is 1.31×10 9 CFU / g; after the ninth day, the conidia show a downward trend. 9 days is selected as the optimal fermentation duration of Trichoderma yunnanense F2 (as Figure 8 ).

[0128] (3) Microorganisms need a suitable humidity environment for growth and reproduction. The water content of the substrate determines the content of free water available to microorganisms, which in turn affects the enzyme activity of microorganisms, the transport of nutrients, and the progress of metabolic reactions. Water is one of the important factors affecting the growth of Trichoderma yunnanense F2. During the experiment, when the water content is 65%, the conidia amount reaches the highest value, which is 9.60×10 8 CFU / g. However, with the increase of water content, the conidia production gradually decreases. Therefore, the optimal water content for the fermentation of Trichoderma yunnanense F2 is 65% (as Figure 9 ).

[0129] 2.4.6 Response surface design of the culture conditions of Trichoderma yunnanense F2

[0130] The response surface design and experimental results of the culture conditions of Trichoderma yunnanense F2 are shown in Table 5:

[0131] Table 5 Analysis of the results of Box-benhnken test

[0132]

[0133] 2.4.7 Results of the central composite experiment

[0134] Using the Box-Behnken central design principle, with the spore production as the response value and the three factors A (water content), B (inoculum amount), and C (fermentation days) that affect the spore production as independent variables, the scheme and results are shown in Table 5. Using the Design-Expert 7.5 analysis software, the data in the table was subjected to multiple regression fitting, and the model regression equation between the culture conditions and the spore production was obtained as follows:

[0135] Y = 9.370×10 8 + 7.025×10 7 A - 1.750×10 6 B - 3.975×10 7 C - 3.475×10 7 AB + 7.275×10 7 AC - 6.925×10 7 BC - 9.038×10 7 A 2 - 2.329×10 8 B 2 - 1.344×10 8 C 2

[0136] 2.4.8 Variance analysis of the regression model

[0137] Table 6 Variance analysis of the regression model

[0138]

[0139]

[0140] It can be seen from the variance analysis of the above regression model that: the P of the regression model is <0.0001, and the P of the lack-of-fit term is 0.0862 > 0.05. The regression model test is extremely significant, and the lack-of-fit test is not significant, indicating that the interference of unknown factors on the test results is very small and the model is stable; the determination coefficient R2 of the regression equation is 94.33%, indicating that this equation fits well with the actual situation and better reflects the relationship between the spore production of Trichoderma yunnanense F2 and the water content of the culture medium, inoculum amount, and fermentation days. Therefore, this model can be used for analysis and prediction; the larger the F value, the stronger the influence of each factor on the spore production of Trichoderma yunnanense F2. The test results show that the influence of the three factors on the spore production of this bacterium is in the order of: inoculum amount > water content > fermentation days; it can be known from the test of the significance of the regression equation coefficients that: A2, B2, and C2 are extremely significant (P < 0.01), B is significant (P < 0.05), and the rest are not significant (Table 6).

[0141] 2.4.9 Response surface diagram and contour lines

[0142] The three-dimensional surfaces and contour lines of the interactive effects of inoculum amount and water content, inoculum amount and fermentation days, and water content and fermentation days are shown in Figures 10 - 12 .

[0143] 2.4.10 Determination and verification of the optimal conditions of the response surface

[0144] Using Design Expert software for analysis and calculation, it can be known that the optimal culture conditions for producing Trichoderma yunnanense F2 are: water content 64.868%, inoculum amount 8.378%, and fermentation days 8.961 d. Under these conditions, the predicted spore production of Trichoderma yunnanense F2 is 9.51074128×10 8 cfu / g. Considering the convenience of actual operation and the results of variance analysis, the optimal culture conditions for Trichoderma yunnanense F2 are determined as: water content 65%, inoculum amount 8%, and fermentation days 9 d. In order to further verify the practical operability of the predicted value, three repeated fermentation experiments were carried out using the optimized culture conditions. The average spore production of Trichoderma yunnanense F2 measured was 10.84×10 8 cfu / g, and the fitting rate with the predicted value reached 95.36%, proving that the predicted value and the practical operation have good fitting, and the model is reliable.

[0145] 3 Discussion

[0146] In the present invention, the low-cost Rosa roxburghii fruit pomace is selected as the main substrate, and the optimized fermentation conditions are used to make the conidia production reach 2.32×10 10 CFU / g. The reasons for this result are mainly in two aspects: one is that the main component of Rosa roxburghii fruit pomace is cellulose, and most microorganisms in the environment cannot effectively utilize this component, so their growth and reproduction are inhibited. This makes the Rosa roxburghii pomace less contaminated by microorganisms and retains a relatively high content of polysaccharide carbon source; the other is that Trichoderma yunnanense used in this paper has a high cellulase production capacity and can better degrade the cellulose of Rosa roxburghii fruit pomace to obtain carbon source, which is beneficial to its growth and reproduction, thus increasing the conidia production.

[0147] Bran, wheat straw powder, rapeseed cake powder, and Rosa roxburghii Tratt residue were selected as fermentation substrates, which fully utilized agricultural and industrial wastes, reduced the fermentation cost. At the same time, these substrates are rich in carbon sources, nitrogen sources, and various mineral elements, providing rich nutrients for the growth of Trichoderma. The cellulose content in wheat straw powder is high and the degradation is difficult, which can limit the utilization efficiency of Trichoderma for its nutrients. Rosa roxburghii Tratt residue contains some special components, which can promote the synthesis of certain metabolites of Trichoderma, but can also have a certain inhibitory effect on the growth of bacteria in the early stage of fermentation. Future research can further explore the effects of different pretreatment methods on the nutrient release of substrates and the utilization efficiency of Trichoderma. Compared with previous studies on the optimization of Trichoderma fermentation conditions, the present invention adopts a unique combination of mixed fermentation substrates and has research significance in terms of fermentation conditions and optimization methods.

[0148] 4 Conclusions

[0149] The optimal substrate ratio was determined by the response surface method. Bran: wheat straw powder: Rosa roxburghii Tratt residue powder: rapeseed cake powder in a weight ratio of 40.2:15:14.8:30 was used as the substrate. This ratio significantly increased the spore yield of Trichoderma F2 strain, indicating that the synergistic effect of different substrate components has an important impact on spore formation. The ratio of different substrate components had a significant effect on the spore yield. Bran and wheat straw powder provided rich carbon and nitrogen sources, promoting the growth of bacteria; rapeseed cake powder and Rosa roxburghii Tratt residue provided necessary trace elements and growth factors, further promoting spore formation. The optimized substrate ratio not only increased the spore yield but also reduced the production cost, having practical application value.

[0150] The inoculum size is one of the key factors affecting the spore yield. An appropriate inoculum size can ensure the uniform distribution and rapid growth of bacteria in the substrate, thus promoting spore formation. However, too high an inoculum size may lead to the rapid consumption of nutrients in the substrate, inhibiting spore formation; while too low an inoculum size may lead to insufficient growth of bacteria, affecting the spore yield.

[0151] The water content is an important factor affecting the growth of bacteria and spore formation in solid fermentation. An appropriate water content can maintain the water balance in the substrate, promoting the growth of bacteria and spore formation; too high or too low water content will inhibit spore production.

[0152] The fermentation days are another key factor affecting the spore yield. An appropriate fermentation time can ensure the full formation and maturity of spores, thus increasing the spore yield; but too short a fermentation time may lead to insufficient spore formation, while too long a fermentation time may lead to spore aging or autolysis, affecting the yield.

[0153] In this study, wheat bran, wheat straw powder, rapeseed cake powder, and Rosa roxburghii Tratt residue were used as the main substrates, and conidia of Trichoderma yunnanense F2 were used for solid-state fermentation. By optimizing the fermentation conditions, the spore yield was further increased, which provided strong support for the subsequent preparation of Trichoderma yunnanense agent.

Claims

1. A solid fermentation method for increasing conidia using Trichoderma yunnanensis F2 strain, characterized in that: The solid fermentation method is to first activate the Yunnan Trichoderma F2 strain, then prepare a Yunnan Trichoderma conidia suspension, use bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake as culture matrix, perform conidia fermentation culture on the Yunnan Trichoderma F2 strain, and obtain the Trichoderma biocontrol agent.

2. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 1, characterized in that: The solid fermentation method is carried out according to the following steps: (1) Activation of Yunnan Trichoderma F2 strain: Take out Yunnan Trichoderma F2 in a frozen state, activate it on PDA medium, and then use a 0.7 mm puncher to take the mycelium block at the edge of the colony to obtain the activated Yunnan Trichoderma F2 strain for later use; (2) Preparation of conidia suspension of Trichoderma yunnanensis: The activated Trichoderma yunnanensis F2 strain was inoculated into PDA medium and cultured at 23-27°C for 5-8 days. The spores were washed with sterile saline. 4-6 ml of sterile water was added to each plate and fully shaken. The conidia were gently scraped with an applicator and pipetted into a sterilized centrifuge tube. The conidia were counted under a microscope using a hemocytometer and the conidia concentration was adjusted to 1.5 × 10 8 -2.5×10 8 / mL, to obtain a suspension of Trichoderma yunnanensis conidia for later use; (3) Preparation of solid matrix: Weigh bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake, wherein the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 40-60:10-15:10-15:20-30, mix well, and obtain a solid matrix for later use; (4) Preparation of liquid matrix: Add sterile water to the solid matrix to make the water content of the solid matrix reach 60-70%, stir evenly to make the solid matrix and water fully mixed, moisten the material for 30-40 minutes, and sterilize it in a high pressure steam sterilizer for 12-18 minutes at a sterilization temperature of 105-135° C. and a sterilization pressure of 100-110 kPa. After cooling, a liquid matrix is ​​obtained and set aside; (5) Inoculation and fermentation: The conidia suspension of Trichoderma yunnanensis prepared in step (1) is inoculated into a conical flask containing a liquid matrix, wherein the inoculation amount of the conidia suspension of Trichoderma yunnanensis is 7-9% of the liquid matrix. After being fully shaken and mixed, the suspension is statically placed upright in a constant temperature incubator at 23-27° C. After the mycelium is fully grown, the material is fully broken up to increase the contact between the material debris and the surface of the Trichoderma, and the fermentation is continued for 8-10 days.

3. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 2, characterized in that: In step (1) and step (2), the PDA culture medium is prepared by weighing 200.0 g of peeled potatoes, 20.0 g of glucose and 15.0 g of agar powder, and diluting the volume to 1000 mL with distilled water.

4. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 2, characterized in that: In step (2), the conidia suspension of Trichoderma yunnanensis is prepared by inoculating the Trichoderma yunnanensis F2 strain with PDA medium, culturing at 24-26° C. for 6-8 days, adding sterile physiological saline to wash the spores, adding 4.5-5.5 ml of sterile water to each plate and shaking it thoroughly, gently scraping it with an applicator, and pipetting it into a sterilized centrifuge tube with a pipette, counting it under a microscope using a hemocytometer, and adjusting the spore concentration to 1.8×10 8 -2.2×10 8 / mL to obtain a conidia suspension of Trichoderma yunnanensis for later use.

5. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 4, characterized in that: In step (2), the conidia suspension of Trichoderma yunnanensis is prepared by inoculating the Trichoderma yunnanensis F2 strain into PDA medium, culturing at 25°C for 7 days, adding sterile physiological saline to wash the spores, adding 5 ml of sterile water to each plate and shaking it thoroughly, gently scraping it with an applicator, and pipetting it into a sterilized centrifuge tube with a pipette, counting it under a microscope using a hemocytometer, and adjusting the spore concentration to 2.0×10 8 / mL to obtain a conidia suspension of Trichoderma yunnanensis for later use.

6. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 2, characterized in that: In step (3), the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 45-55:12.5-15:12.5-15:25-30.

7. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 6, characterized in that: In step (3), the weight ratio of the bran, wheat straw powder, roxburghii residue powder and rapeseed powder cake is 40:15:13.8:

30.

8. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 2, characterized in that: In step (4), the liquid matrix is ​​prepared by adding sterile water to a solid matrix to make the water content of the solid matrix reach 62-68%, stirring evenly to fully mix the solid matrix and the water, moistening the material for 32-38 minutes, sterilizing in a high-pressure steam autoclave for 14-16 minutes, the sterilization temperature is 105-125° C., the sterilization pressure is 102-105 kPa, and after cooling, a liquid matrix is ​​obtained for standby use.

9. The solid fermentation method for increasing conidia by using the Trichoderma yunnanensis F2 strain according to claim 8, characterized in that: In step (4), the liquid matrix is ​​prepared by taking sterile water of the solid matrix to make the water content of the solid matrix reach 65%, stirring evenly to make the solid matrix and the water fully mixed, moistening the material for 35 minutes, sterilizing it in a high-pressure steam sterilizer for 15 minutes, the sterilization temperature is 125° C., the sterilization pressure is 103.4 kPa, and after cooling, a liquid matrix is ​​obtained for standby use.

10. The solid fermentation method for increasing conidia using the Trichoderma yunnanensis F2 strain according to claim 2, characterized in that: In step (5), the inoculation and fermentation are as follows: the Yunnan Trichoderma conidia suspension prepared in step (1) is inoculated into a triangular flask containing a liquid matrix, the inoculation amount of the Yunnan Trichoderma conidia suspension is 8% of the liquid matrix, after being fully shaken and mixed, it is statically placed upright in a 25° C. constant temperature incubator, and the material is fully shaken and crushed after the hyphae are fully grown to increase the contact between the material debris and the surface of the Trichoderma, and the fermentation is continued for 9 days to obtain the Trichoderma biocontrol agent.

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