Trichoderma fermentation liquor, preparation method and application thereof

Abstract

The present application provides a kind of trichoderma fermentation liquor and its preparation method and application, belong to trichoderma fermentation technical field.The present application inoculates trichoderma into PDA solid culture medium, culture, obtains activated trichoderma cake;The activated trichoderma cake is inoculated into PDA liquid culture medium, culture, obtains trichoderma seed liquid;Trichoderma seed liquid is inoculated into trichoderma fermentation culture medium, fermentation, obtains trichoderma fermentation liquor.The present application selects 2 acid-resistant disease-resistant functional trichoderma strains, with plant straw and other agricultural solid waste as main carbon and nitrogen source, by adjusting material pH to control the growth of miscellaneous bacteria, material does not need to be sterilized, in open environment, the scale of trichoderma is expanded, and the cost of trichoderma spore production and application is reduced.The trichoderma fermentation liquor prepared by the present application has certain growth-promoting effect on tobacco plant growth and development;It can antagonize tobacco plant pathogenic bacteria, reduce the incidence of tobacco plant, and improve the quality and quality of tobacco leaf.

Description

Technical Field

[0001] This invention belongs to the field of Trichoderma fermentation technology, and particularly relates to a Trichoderma fermentation broth, its preparation method, and its application. Background Technology

[0002] Trichoderma ( Trichoderma Fungi, as a highly valuable microbial resource, occupy an important position in fields such as agricultural biocontrol, enzyme production, and environmental remediation. Among them, *Trichoderma harzianum* (… Trichoderma harzianum Due to its strong ability to antagonize pathogens, promote plant growth, and degrade insoluble organic matter, *Trichoderma* is widely used in the research and development of biocontrol agents. The fermentation process is the core link in the industrial application of *Trichoderma*, and its fermentation conditions directly determine mycelial biomass, spore yield, and metabolite activity. Although solid-state fermentation has advantages in *Trichoderma* production such as low cost and strong process adaptability, it still faces many technical bottlenecks during industrial scale-up, mainly in the following aspects: 1. Reliance on large-scale sterilization equipment, resulting in high costs and energy consumption: Sterilization of solid substrates requires large-scale high-pressure steam sterilization equipment (such as sterilizers and autoclaves), especially during large-scale production where substrate processing volumes can reach several tons, leading to significant equipment investment and high energy consumption. Compared to liquid fermentation, solid substrates have lower heat and mass transfer efficiency, making them prone to "sterilization dead zones" during sterilization, resulting in incomplete sterilization of certain areas and increasing the risk of contamination by other microorganisms.

[0003] 2. Requires a strict, closed, sterile environment and is difficult to operate: The entire process, from substrate sterilization and inoculation to fermentation culture, must be carried out in a sterile environment. Small laboratories can achieve this with the help of laminar flow hoods and sterile culture rooms, but large-scale production requires the construction of a closed sterile fermentation workshop equipped with an air purification system (such as a high-efficiency filter), resulting in high infrastructure and maintenance costs. Operations such as turning the material and sampling during fermentation can easily disrupt the sterile environment, becoming a major pathway for contamination by microorganisms.

[0004] 3. High risk of contamination by other microorganisms, making it difficult to guarantee product purity: The substrate (agricultural waste) used for Trichoderma solid-state fermentation itself carries a large number of other microorganisms (bacteria, molds, yeasts). Even after sterilization, it is difficult to completely eliminate heat-resistant spores. During fermentation, if temperature, humidity, or ventilation is not properly controlled, these other microorganisms will multiply rapidly, competing with Trichoderma for nutrients and space. This not only reduces spore production but also leads to a decrease in product purity, and may even render the product unusable. Common contaminating microorganisms include Bacillus, Aspergillus, and Penicillium.

[0005] 4. Significant bottlenecks in large-scale production hinder continuous operation: ① Poor equipment adaptability: Currently, Trichoderma solid-state fermentation mostly adopts batch production (such as shallow tray fermentation and fermentation bag fermentation), lacking efficient continuous fermentation equipment. Large-scale fermentation devices (such as solid-state fermentation tanks) are difficult to design, resulting in problems such as uneven substrate mixing, low aeration efficiency, and large temperature gradients. ② Difficult process control: During large-scale fermentation, the temperature, humidity, and oxygen concentration of the substrate are difficult to control uniformly, leading to significant differences in Trichoderma growth and sporulation in different areas, resulting in unstable product quality. ③ High labor intensity: Batch production processes such as loading, inoculation, turning, and unloading rely heavily on manual operation, with low automation levels, making it difficult to meet the needs of large-scale industrial production.

[0006] 5. Poor spore stability and short shelf life: Trichoderma conidia powder produced by solid-state fermentation is easily affected by factors such as temperature, humidity, and light during storage and transportation, leading to a decrease in spore viability. Even after drying, spore viability typically decreases significantly within 3-6 months under normal temperature storage conditions, limiting the product's market circulation. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a Trichoderma fermentation broth, its preparation method and application.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing Trichoderma fermentation broth, comprising the following steps: (1) Inoculate Trichoderma onto PDA solid medium and culture to obtain activated Trichoderma mycelium cake; (2) The activated Trichoderma mycelium cake was inoculated onto PDA liquid culture medium and cultured to obtain Trichoderma seed liquid; (3) Inoculate the Trichoderma seed liquid onto the Trichoderma fermentation medium and ferment to obtain Trichoderma fermentation broth.

[0009] Preferably, the PDA solid culture medium in step (1) comprises the following components at the following concentrations: potato extract powder 1~5 g / L, agar 10~20 g / L and glucose 10~30 g / L.

[0010] Preferably, the culture temperature in step (1) is 20~30℃ and the culture time is 48~96h.

[0011] Preferably, the PDA liquid culture medium in step (2) comprises the following components at the following concentrations: 150-250 g / L potato and 10-30 g / L glucose.

[0012] Preferably, the inoculation amount in step (2) is 1~10%; the culture temperature is 20~30℃; the culture rotation speed is 100~300r / min; and the culture time is 48~96h.

[0013] Preferably, the fermentation medium in step (3) comprises the following components at the following concentrations: KH2PO4 1~3g / L, NH4NO3 0.5~2g / L, FeSO4·7H2O 0.003~0.008g / L, MnSO4 0.001~0.002g / L, CaCl2 0.1~0.5g / L, NaCl 0.5~2g / L, MgSO4·7H2O 0.1~0.5g / L, corn flour 5~15g / L and soybean meal 3~8g / L; The corn flour and soybean meal have a mesh size of 60-150 mesh; The pH of the fermentation medium is 2.0 to 3.0, and the pH value is adjusted by phosphoric acid and nitric acid.

[0014] Preferably, the inoculation amount in step (3) is 0.5~5%; the fermentation device is a Trichoderma fermentation device, which includes: a fermentation container (1), a stirring mechanism, an air intake system and a pH adjustment system. The fermentation container (1) has a discharge port (11) at the bottom and a top cover (2) sealed to the top opening. The top cover (2) has a feeding port (21), an inoculation port (22), an exhaust port (23) and at least one pH meter port (24). The stirring mechanism is installed on the top cover (2) and extends into the fermentation container (1). The air intake system is connected to the inside of the fermentation container (1). The pH adjustment system is connected to the inside of the fermentation container (1). The fermentation container is made of any one of polypropylene, polyethylene or fiberglass. The fermentation temperature is 20~30℃, the fermentation speed is 100~300r / min, and the fermentation time is 60~96h.

[0015] Preferably, the Trichoderma includes Trichoderma harzianum and Trichoderma viride.

[0016] The present invention also provides a Trichoderma fermentation broth prepared by the aforementioned preparation method.

[0017] The present invention also provides the application of the aforementioned Trichoderma fermentation broth in promoting tobacco growth and preventing tobacco double black disease.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention screened two acid-resistant, disease-resistant, and growth-promoting Trichoderma strains. Using agricultural solid waste such as plant straw as the main carbon and nitrogen source, the growth of miscellaneous bacteria was controlled by adjusting the pH of the material. The material does not need to be sterilized. Trichoderma can be propagated on a large scale in an open environment, reducing the production and application costs of Trichoderma spores.

[0019] The Trichoderma fermentation broth prepared by this invention has a certain growth-promoting effect on the growth and development of tobacco plants; it can antagonize tobacco pathogens, reduce the incidence of tobacco diseases, and improve the quality and grade of tobacco leaves. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the Trichoderma fermentation apparatus; In the diagram: 1-Fermentation container; 11-Discharge port; 2-Top cover; 21-Feeding port; 22-Inoculation port; 23-Exhaust port; 24-pH meter socket; 25-Exhaust valve; 26-Pressure gauge; 31-Motor; 32-Agitator shaft; 33-Agitator paddle; 41-Air inlet pipe; 42-Air distributor; 43-Air filter; 51-Acid storage tank; 52-Acid addition pipe; 6-Online pH meter; 7-Jacket; 71-Media inlet; 72-Media outlet. Detailed Implementation

[0021] This invention provides a method for preparing Trichoderma fermentation broth, comprising the following steps: (1) Inoculate Trichoderma onto PDA solid medium and culture to obtain activated Trichoderma mycelium cake; (2) The activated Trichoderma mycelium cake was inoculated onto PDA liquid culture medium and cultured to obtain Trichoderma seed liquid; (3) Inoculate the Trichoderma seed liquid onto the Trichoderma fermentation medium and ferment to obtain Trichoderma fermentation broth.

[0022] In this invention, *Trichoderma* is inoculated onto a PDA solid culture medium and cultured to obtain activated *Trichoderma* mycelium cakes. The PDA solid culture medium comprises the following components at the following concentrations: potato extract 1-5 g / L, agar 10-20 g / L, and glucose 10-30 g / L; the amount of potato extract is preferably 2-4 g / L, more preferably 3 g / L; the amount of agar is preferably 12-18 g / L, more preferably 14 g / L; and the amount of glucose is preferably 15-25 g / L, more preferably 20 g / L. The culture temperature is 20-30°C, preferably 25-29°C, more preferably 28°C; and the culture time is 48-96 h, preferably 60-84 h, more preferably 72 h.

[0023] In this invention, activated Trichoderma mycelium cakes are inoculated onto PDA liquid culture medium and cultured to obtain Trichoderma seed culture. The PDA liquid culture medium comprises the following components at the following concentrations: 150-250 g / L potato and 10-30 g / L glucose; the amount of potato is preferably 180-220 g / L, more preferably 200 g / L; the amount of glucose is preferably 15-25 g / L, more preferably 20 g / L; the inoculation amount is 1-10%, preferably 3-8%, more preferably 5%; the culture temperature is 25-30℃, preferably 26-29℃, more preferably 28℃; the culture rotation speed is 100-300 r / min, preferably 150-250 r / min, more preferably 200 r / min; the culture time is 48-96 h, preferably 60-84 h, more preferably 72 h.

[0024] In this invention, Trichoderma seed liquid is inoculated onto Trichoderma fermentation medium and fermented to obtain Trichoderma fermentation broth.The fermentation medium comprises the following components at the following concentrations: KH₂PO₄ 1~3 g / L, NH₄NO₃ 0.5~2 g / L, FeSO₄·7H₂O 0.003~0.008 g / L, MnSO₄ 0.001~0.002 g / L, CaCl₂ 0.1~0.5 g / L, NaCl 0.5~2 g / L, MgSO₄·7H₂O The following are the preferred concentrations of the following ingredients: 0.1-0.5 g / L of corn flour, 5-15 g / L of corn flour, and 3-8 g / L of soybean meal; the preferred concentration of KH₂PO₄ is 1.5-2.5 g / L, more preferably 2 g / L; the preferred concentration of NH₄NO₃ is 0.8-1.5 g / L, more preferably 1 g / L; the preferred concentration of FeSO₄·7H₂O is 0.004-0.007 g / L, more preferably 0.005 g / L; the preferred concentration of MnSO₄ is 0.0013-0.0018 g / L, more preferably 0.0016 g / L; and the preferred concentration of CaCl₂ is 0.2 g / L. The concentration of NaCl is preferably 0.8-1.5 g / L, more preferably 1 g / L; the concentration of MgSO4·7H2O is preferably 0.2-0.4 g / L, more preferably 0.3 g / L; the concentration of corn flour is preferably 8-12 g / L, more preferably 10 g / L; the concentration of soybean meal is preferably 4-7 g / L, more preferably 5 g / L; the mesh size of the corn flour and soybean meal is 60-150 mesh, preferably 80-120 mesh, more preferably 100 mesh; the pH of the fermentation medium is 2.0-2.0. 3.0, preferably 2.5; the pH value is adjusted by phosphoric acid and nitric acid, and the volume ratio of phosphoric acid and nitric acid is 1:1; the inoculum amount is 0.5~5%, preferably 1~3%, and more preferably 2%; the fermentation device is a Trichoderma fermentation device, which includes: a fermentation container (1), a stirring mechanism, an air intake system and a pH adjustment system. The fermentation container (1) has a discharge port (11) at the bottom and a top cover (2) sealed at the top opening. The top cover (2) has a feeding port (21), an inoculation port (22), an exhaust port (23) and at least one pH meter port (24); the stirring mechanism Installed on the top cover (2) and extending into the fermentation container (1); the air intake system is connected to the inside of the fermentation container (1); the pH adjustment system is connected to the inside of the fermentation container (1); the material of the fermentation container is any one of polypropylene, polyethylene or fiberglass; the fermentation temperature is 20~30℃, preferably 25~29℃, more preferably 28℃; the fermentation speed is 100~300r / min, preferably 150~250r / min, more preferably 200r / min; the fermentation time is 60~96h, preferably 72~84h, more preferably 72h.

[0025] In this invention, the Trichoderma includes Trichoderma harzianum and Trichoderma viride.

[0026] The present invention also provides a Trichoderma fermentation broth prepared by the aforementioned preparation method.

[0027] The present invention also provides the application of the aforementioned Trichoderma fermentation broth in promoting tobacco growth and preventing tobacco double black disease.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] Screening of acid-resistant functional Trichoderma strains

[0031] I. Experimental Objective

[0032] Acid-resistant strains from five Trichoderma strains (Trichoderma harzianum, Trichoderma echinospora, Trichoderma longifolia, Trichoderma viride, and Trichoderma hookeri) were screened to determine the acid resistance of each strain under different pH conditions (1-8, adjusted with sulfuric acid). The optimal acid resistance of Trichoderma harzianum and Trichoderma viride in the pH range of 2-3 was verified to provide experimental basis for subsequent applications of acid-resistant Trichoderma.

[0033] II. Experimental Materials and Instruments

[0034] Experimental strains: Trichoderma harzianum (China General Microbiological Culture Collection Center No. CGMCC23294), Trichoderma viride (China General Microbiological Culture Collection Center No. CGMCC41007), Trichoderma echinosporum, Trichoderma longifolium, and Trichoderma haptosporum were isolated from tobacco field soil. All of the above strains were purified active strains.

[0035] Separation method: Collect 1000g of soil around tobacco roots, add 5-10 liters of sterile water containing 0.01% Tween 80, stir for 1 hour, let stand for 3 hours, transfer the supernatant to a large-capacity centrifuge, centrifuge at 8000-10000 rpm at 20℃ for 20 minutes. Discard the supernatant and retain the precipitate. Resuspend the precipitate in 5 times its volume of sterile water containing 0.01% Tween 80, centrifuge at 1000 rpm at 20℃ for 5 minutes, discard the precipitate, transfer the supernatant to a clean small centrifuge tube, centrifuge at 10,000 rpm for 15 minutes at 20℃, and retain the precipitate. Resuspend the precipitate in 10 mL of pH 7.0 phosphate buffer. Then take 0.1 mL of Trichoderma screening medium (1g glucose, 1.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 1g ammonium chloride, 20g agar, 0.3g streptomycin, 0.2g pentachloronitrobenzene, 0.05g Bengal red).

[0036] Fungal identification method: Trichoderma hyphae cultured for 3 days were used to extract total DNA using the CTAB method. The ITS region was amplified by PCR using universal fungal primers ITS (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO:1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO:2). The PCR products were sent to a biotechnology company for sequencing. Sequencing results were compared using BLAST in the NCBI database. Reference sequences with high homology were downloaded, and a phylogenetic tree was constructed using software. The taxonomic position of Trichoderma was determined based on sequence homology and phylogenetic tree branching.

[0037] Culture medium: PDA solid medium (3g potato, 20g glucose, 14g agar, distilled water added to 1000mL, natural pH, sterilized at 121℃ for 20min) was used for the culture and growth index determination of Trichoderma strains.

[0038] Reagents: concentrated sulfuric acid (analytical grade), sterile distilled water, pH meter calibration solution (4.00, 6.86 standard buffer solution).

[0039] Instruments and equipment: ultra-clean workbench, constant temperature incubator (28℃), pH meter (accuracy 0.01), electronic balance, autoclave, vernier caliper (accuracy 0.01mm), hemocytometer, microscope, alcohol lamp, inoculation loop, petri dish (90mm), conical flask, pipette, etc.

[0040] III. Experimental Design

[0041] pH gradient setting

[0042] The pH of the PDA medium was adjusted using concentrated sulfuric acid, and eight treatment groups were set up with gradients of 1, 2, 3, 4, 5, 6, 7, and 8, with three replicates for each treatment group.

[0043] Inoculation and culture of strains

[0044] Culture medium preparation: Prepare PDA solid culture medium according to conventional methods, add an appropriate amount of concentrated sulfuric acid to adjust to the target pH value, stir evenly, dispense into petri dishes, autoclave (121℃, 20min), and cool and solidify for later use.

[0045] Inoculation procedure: In a clean bench, use a punch (5mm in diameter) to take an equal amount of mycelial cake from the edge of the purified culture of each strain. Inoculate the mycelial cake into the center of PDA solid culture medium with different pH values, with the mycelial cake facing down, and gently press it to fit the culture medium.

[0046] Culture conditions: Place the inoculated culture dishes in a 28℃ constant temperature incubator and culture in the dark for 7 days, keeping the culture environment stable during the period.

[0047] IV. Measurement Indicators and Methods

[0048] Mycelial growth rate determination: On days 3, 5, and 7 of cultivation, the diameter of colonies in each petri dish was measured using the cross-sectional method, accurate to 0.01 mm, and the average value of each measurement was calculated. Mycelial growth rate (mm / d) = (final colony diameter - initial mycelial disc diameter) / number of cultivation days.

[0049] Determination of Trichoderma sporulation: On day 7 of culture, 10 mL of sterile distilled water was added to each petri dish. Spores on the surface of the colonies were gently scraped off with a sterile spreader to prepare a spore suspension. The suspension was then filtered through four layers of sterile gauze to remove hyphal fragments. The filtered spore suspension was serially diluted with sterile distilled water to an appropriate concentration. The spores were counted under a microscope using a hemocytometer. Each sample was counted three times, and the average value was taken. The number of spores per milliliter of suspension (spores / mL) was calculated as the sporulation yield of the strain.

[0050] Experimental results are shown in Table 1.

[0051] Table 1. Growth status of Trichoderma in culture media with different pH values

[0052] Note: The data in the table are mean ± standard deviation. The evaluation of acid resistance is based on mycelial growth rate and sporulation quantity, combined with statistical analysis results.

[0053] As shown in Table 1, the acid resistance test results of Trichoderma showed that Trichoderma harzianum and Trichoderma viride had the best acid resistance. In the pH range of 2 to 3, they inhibited the growth of miscellaneous bacteria and also had a relatively high yield of Trichoderma spores.

[0054] Example 2

[0055] Screening for suitable acids for Trichoderma fermentation

[0056] The pH of the Trichoderma fermentation broth was adjusted using five different types of acids: phosphoric acid, nitric acid, lactic acid, sulfuric acid, and a combination of phosphoric acid and nitric acid, to screen which type of acid was suitable for the growth of Trichoderma strains.

[0057] I. Experimental Objective

[0058] When the pH of the fermentation medium is adjusted using five acids (sulfuric acid, nitric acid, phosphoric acid, lactic acid, and a mixture of phosphoric acid and nitric acid in a 1:1 volume ratio), the effect on *Trichoderma harzianum* ( Trichoderma harzianum ) and dark green Trichoderma ( Trichoderma atrovirideThe effects of liquid fermentation on the two Trichodermas were investigated; the optimality of fermentation of the two Trichodermas was verified when the pH value was adjusted to 2-3 by phosphoric acid and nitric acid; and suitable acids and corresponding pH ranges for liquid fermentation of the two Trichodermas were screened with mycelial growth rate and sporulation quantity as the core evaluation indicators.

[0059] II. Experimental Materials and Instruments

[0060] Strains: Trichoderma harzianum (China General Microbiological Culture Collection Center No. CGMCC23294) and Trichoderma viride (China General Microbiological Culture Collection Center No. CGMCC41007), were preserved in the laboratory and activated in PDA medium for later use.

[0061] PDA solid medium: 3g potato, 20g glucose, 14g agar, add distilled water to 1000mL, natural pH, sterilize at 121℃ for 20min.

[0062] PDA liquid culture medium: 200 g / L potato and 20 g / L glucose, add distilled water to 1000 mL, pH at rest, sterilize at 121 °C for 20 min.

[0063] reagents

[0064] Concentrated sulfuric acid (98%, analytical grade), concentrated nitric acid (68%, analytical grade), phosphoric acid (85%, analytical grade), lactic acid (80%, analytical grade), sterile deionized water, 0.1 mol / L NaOH solution (for pH fine-tuning), hemocytometer counting solution, etc.

[0065] instrument

[0066] Clean bench, constant temperature incubator (28℃), shaker (200r / min, 28℃), pH meter (accuracy 0.01), electronic balance (accuracy 0.001g), microscope (equipped with hemocytometer), ultraviolet spectrophotometer, centrifuge, pipettes (1mL, 5mL, 10mL), Erlenmeyer flask (250mL), sterile test tubes, etc.

[0067] III. Experimental Design

[0068] Variable settings

[0069] Independent variables: ① Acid type (sulfuric acid, nitric acid, phosphoric acid, lactic acid, mixed acid of phosphoric acid and nitric acid); ② pH gradient (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0), each pH value is adjusted with the corresponding acid, and the mixed acid of phosphoric acid and nitric acid is additionally set with three subdivided gradients of pH 2.0, 2.5, and 3.0 (to verify the optimal range).

[0070] Dependent variable: ① Mycelial growth rate (mg / (mL)) d)); ② Number of spores produced (spores / mL).

[0071] Other variables, such as fermentation temperature (28℃), shaking speed (200r / min), fermentation time (72h), culture medium volume (100mL in a 250mL Erlenmeyer flask), and inoculum size (5%, v / v), were kept constant.

[0072] Group design

[0073] The strains were grouped into two groups: Trichoderma harzianum (TH group) and Trichoderma viride (TA group).

[0074] Acid and pH grouping: Each group is divided into 5 acid subgroups, and each subgroup is set up with 8 treatment groups according to the pH gradient. The phosphate + nitrate subgroup has an additional pH 2.5 treatment group. Each treatment group has 3 replicates.

[0075] IV. Experimental Procedure

[0076] 1. Activation of the strain

[0077] Trichoderma harzianum and Trichoderma viride were inoculated onto PDA plates and incubated at 28°C for 72 hours. When the colony diameter reached 3-5 cm, mycelial cakes were collected from the edge of the colony using a sterile punch (5 mm in diameter) for later use.

[0078] 2. Culture medium preparation and pH adjustment

[0079] Prepare PDA liquid culture medium and dispense it into 250mL Erlenmeyer flasks (100mL per flask). Adjust the pH of the culture medium in each Erlenmeyer flask using five different acids: Single acid group: Slowly add the corresponding concentrated acid dilution solution (diluted at 1:10 to avoid local over-acidity) dropwise while stirring, monitor in real time with a pH meter, and fine-tune to the target pH value (error ±0.05).

[0080] Phosphoric acid + nitric acid mixed acid group: First mix phosphoric acid and nitric acid in a 1:1 volume ratio, dilute 10 times and add dropwise to adjust the pH value, and then fine-tune it to the target value.

[0081] After adjustment, sterilize all Erlenmeyer flasks at 121℃ for 20 minutes, cool to room temperature, and check the pH value again (if there is a deviation, fine-tune with sterile 0.1mol / L NaOH or the corresponding dilute acid).

[0082] 3. Vaccination

[0083] Five sterile bacterial discs (5 mm in diameter) were inoculated into each culture medium bottle, with an inoculation rate of 5% (v / v, converted from bacterial disc volume). The inoculated Erlenmeyer flasks were then placed on a shaker and incubated at 28°C and 200 rpm for 7 days.

[0084] 4. Indicator Measurement

[0085] Mycelial growth rate measurement (samples taken on days 3, 5, and 7 of culture): Sampling: Take 5 mL of fermentation broth from each replicate group, centrifuge at 1000 r / min for 10 min, and collect the mycelium.

[0086] Washing and drying: Wash the mycelium three times with sterile deionized water, blot dry the surface moisture with filter paper, and weigh it with an electronic balance (recorded as wet weight W1); then place the mycelium in a 60℃ oven to dry to constant weight and weigh it (recorded as dry weight W2).

[0087] Calculation: Mycelial growth rate (dry weight) = (W2 - initial mycelial dry weight) / (fermentation broth volume × number of days of culture), unit mg / (mL) d) The final growth rate after 7 days is taken as the evaluation index.

[0088] Sporulation count determination (sample taken on day 7 of culture): Sampling: Take 10 mL of fermentation broth from each replicate group and serially dilute with sterile physiological saline to the appropriate concentration (10 mL / mL). 7 ~10 8 times).

[0089] Counting: The number of spores was counted under a microscope using a hemocytometer. Three fields of view were counted for each sample, and the average value was taken.

[0090] Calculation: Sporulation count = Average count × Dilution factor × 10 4 (Conversion factor for volume of hemocytometer), unit: cells / mL.

[0091] V. Data Recording and Analysis

[0092] The mycelial growth rate (7-day dry weight method) and sporulation quantity (7-day count) of each treatment group were recorded in an Excel spreadsheet, and the mean and standard deviation of the three replicates for each treatment group were compiled.

[0093] Experimental results are shown in Tables 2 and 3.

[0094] Table 2. Effects of different acids and pH values ​​on the fermentation efficiency of Trichoderma harzianum.

[0095] Table 3. Effects of different acids and pH values ​​on the fermentation efficiency of Trichoderma viride.

[0096] Strongly acidic environments (pH≤1.0) strongly inhibit the growth and sporulation of Trichoderma harzianum and Trichoderma viride. All five acids cannot achieve effective fermentation at this pH value.

[0097] Among single acids (sulfuric acid, nitric acid, phosphoric acid, and lactic acid), phosphoric acid has the best fermentation effect on the two Trichoderma species, with a suitable pH range of 3-6. Nitric acid (pH 4-5) and sulfuric acid (pH 4-5) are the next best, while lactic acid has the worst effect (pH 5-6).

[0098] The mixed acid of phosphoric acid and nitric acid (1:1) significantly promoted the fermentation of both Trichoderma species better than that of a single acid. Within the pH range of 2.0-6.0, both mycelial and sporulation yields of Trichoderma harzianum were better. Specifically, at pH 2.5, the mycelial growth rate of Trichoderma harzianum reached 3.56 mg / (mL). d) The number of spores produced reached 15.67 × 10 8 Cells / mL; Trichoderma viride mycelium growth rate reaches 3.15 mg / (mL) d) The number of spores produced reached 10.98 × 10 8 The number of cells / mL was significantly higher than that of other acids (P<0.05).

[0099] In summary, Trichoderma harzianum exhibits slightly better acid tolerance and fermentation performance than Trichoderma viride, but both show good fermentation results when the pH is adjusted to 2-3 with a mixture of phosphoric acid and nitric acid, demonstrating strong acid resistance.

[0100] Example 3

[0101] Trichoderma fermentation device for liquid fermentation

[0102] I. Experimental Materials

[0103] Basic ingredients: corn flour (100 mesh), soybean meal (100 mesh), and commercially available high-quality potatoes.

[0104] Strains: Trichoderma harzianum, Trichoderma viride

[0105] Reagents: Potassium dihydrogen phosphate (KH₂PO₄), ammonium nitrate (NH₄NO₃), ferrous sulfate heptahydrate (FeSO₄) 7H2O), manganese sulfate (MnSO4), calcium chloride (CaCl2), sodium chloride (NaCl), magnesium sulfate heptahydrate (MgSO4) 7H2O), potato starch, agar, and glucose were all purchased from Sinopharm Chemical Reagent Co., Ltd., and were of analytical grade. Sulfuric acid, nitric acid, lactic acid, and phosphoric acid were all analytical grade reagents.

[0106] Instruments and equipment: Trichoderma fermentation apparatus, constant temperature shaker, high pressure steam sterilizer, sterile ultra-clean workbench, snowball counter, biological microscope, electronic balance, pH meter, electric thermostatic incubator, etc.

[0107] II. Culture medium formulation

[0108] PDA solid culture medium: 3g potato extract powder, 14g agar, 20g glucose, add distilled water to 1000mL, sterilize at 121℃ for 30min, then pour into plates for later use.

[0109] PDA culture medium: 200g potato (cut into small pieces), 20g glucose, add distilled water to 1000mL, boil the potato for 15min and filter, add glucose to dissolve, add to a final volume of 1000mL, sterilize at 121℃ for 30min.

[0110] Trichoderma fermentation medium: KH2PO4 2.0g, NH4NO3 1.0g, FeSO4 7H2O 0.005g, MnSO40.0016g, CaCl20.3g, NaCl 1.0g, MgSO4 0.3g of 7H2O, 10g of corn flour (passed through a 100-mesh sieve), 5g of soybean meal powder (passed through a 100-mesh sieve), add distilled water to 1000mL, sterilize at 121℃ for 30min.

[0111] III. Experimental Methods

[0112] Trichoderma fermentation equipment (such as Trichoderma fermentation device) Figure 1 (As shown): Fermentation container 1, stirring mechanism, air intake system, and pH adjustment system. The fermentation container 1 is a cylindrical structure with an open top and a discharge port 11 at the bottom. The top opening is sealed with a top cover 2. The top cover 2 has a feeding port 21, an inoculation port 22, an exhaust port 23, and at least one pH meter socket 24. The stirring mechanism is installed on the top cover 2 and extends into the fermentation container 1. The air intake system is connected to the interior of the fermentation container 1. The pH adjustment system is also connected to the interior of the fermentation container 1. The feeding port 21 is used to add solid carbon and nitrogen sources such as corn flour and soybean meal powder, as well as water. The inoculation port 22 is used for aseptic inoculation of Trichoderma seed liquid. The exhaust port 23 is used to discharge waste gases such as carbon dioxide generated during fermentation. The pH meter socket 24 is used to seal and install an online pH meter 6. The probe of the online pH meter 6 extends below the liquid surface inside the fermentation container 1 to monitor the pH value of the fermentation liquid in real time. The top cover and the fermentation container can be connected by a flange with a sealing ring in the middle to ensure sealing and facilitate disassembly and cleaning.

[0113] The stirring mechanism includes a motor 31, a stirring shaft 32, and a stirring paddle 33. The motor 31 is fixedly installed above the top cover 2. The upper end of the stirring shaft 32 is connected to the output end of the motor 31, and the lower end of the stirring shaft 32 passes through the top cover 2 and extends into the interior of the fermentation container 1. The stirring paddle 33 is fixedly installed on the stirring shaft 32. The main function of the stirring mechanism is to mix the materials evenly, improve mass and heat transfer efficiency, and promote the contact between mycelium and oxygen. Since the culture medium contains solid particles (corn flour, soybean meal), the stirring paddle should have good agitation ability; a frame-type, anchor-type, or spiral-type stirring paddle is preferred. The motor speed should be adjustable to adapt to the dissolved oxygen and shear force requirements of Trichoderma at different growth stages; a variable frequency speed control motor with a speed range of 100-300 r / min is preferred.

[0114] The air intake system includes an air intake pipe 41 and an air distributor 42. One end of the air intake pipe 41 is connected to an external air source, and the other end passes through the side wall of the fermentation container 1 and communicates with the air distributor 42 located at the bottom of the fermentation container 1. The air distributor is responsible for evenly dispersing air into the fermentation liquid, forming microbubbles, and improving dissolved oxygen efficiency. The air distributor 42 is an annular porous pipe or a microporous aeration disc, and the annular porous pipe has multiple air outlet holes with a diameter of 1~3mm evenly distributed. To prevent impurities in the air from entering, the air intake pipe 41 is equipped with an air filter 43, and the filtration accuracy of the air filter 43 is 0.22μm or 0.45μm.

[0115] The pH adjustment system includes an acid storage tank 51 and an acid addition pipe 52. One end of the acid addition pipe 52 is connected to the outlet of the acid storage tank 51, and the other end passes through the side wall of the fermentation container 1 and communicates with the interior of the fermentation container 1. The acid addition pipe 52 is equipped with a flow regulating valve to control the amount of acid added. Since the pH value of *Trichoderma* fluctuates during fermentation, acid needs to be added automatically or manually to maintain it within the optimal pH range of 2.0 to 3.0. The added acid is preferably a mixture of phosphoric acid and nitric acid in a 1:1 volume ratio.

[0116] The discharge port 11 is equipped with a discharge valve 12; the exhaust port 23 is equipped with an exhaust valve 25 and a pressure gauge 26, which are used to adjust the pressure inside the fermentation container and maintain a slightly positive pressure, such as 0.01-0.05 MPa, to prevent external air from flowing back in.

[0117] The fermentation container 1 is fitted with a jacket 7, which has a medium inlet 71 and a medium outlet 72 for introducing constant temperature water or steam to regulate the fermentation temperature and maintain a constant fermentation temperature.

[0118] The fermentation container 1 is made of any one of polypropylene, polyethylene or fiberglass; the surfaces of the stirring shaft 32 and the stirring paddle 33 are coated with a polytetrafluoroethylene anti-corrosion layer.

[0119] Strain activation: Trichoderma harzianum and Trichoderma viride were inoculated onto PDA solid medium and cultured at 28°C for 72 hours. When the colony diameter reached 3-5 cm, the mycelial cakes were collected from the edge of the colony using a sterile punch (5 mm in diameter) for later use.

[0120] Seed culture preparation: Prepare 1000 mL of PDA culture medium, dispense 350 mL into 500 mL Erlenmeyer flasks, and autoclave at 121 °C for 30 min. After the culture medium cools to room temperature, inoculate the activated Trichoderma harzianum strain in a sterile laminar flow hood and incubate at 28 °C and 200 rpm for 72 h. The spore concentration is determined to be ≥1 × 10⁻⁶. 8 CFU / mL is prepared for later use.

[0121] Experiment on optimization of Trichoderma fermentation conditions: Culture medium pretreatment: Prepare Trichoderma fermentation medium, boil and cool to room temperature, and adjust the pH of the medium to two gradients of 2 and 3 with phosphoric acid + nitric acid (1:1).

[0122] Fermentation culture: Pour the pH-adjusted culture medium into the Trichoderma fermentation device, filling the container to 2 / 3 of its volume (30L), and inoculate with 600mL of Trichoderma seed liquid (inoculation amount of 2%). Culture at 28℃ and 200r / min for 72h.

[0123] Sporulation rate determination: After fermentation, the spore concentration in the fermentation broth was determined by snowball counting method. Each treatment was repeated 3 times and the average value was taken.

[0124] Experimental results are shown in Table 4.

[0125] Table 4. Effects of different initial pH conditions on sporulation yield of Trichoderma harzianum liquid fermentation

[0126] Table 5. Effects of different initial pH conditions on sporulation yield of Trichoderma viride liquid fermentation

[0127] Tables 4 and 5 show that there are significant differences in sporulation of Trichoderma under different initial pH conditions: when the initial pH is 3, the average sporulation concentration in the Trichoderma harzianum fermentation broth is 2.9 × 10⁻⁶. 9 The concentration of cells / mL is significantly higher than the 3.3 × 10⁻⁶ cells / mL at the initial pH of 2. 8 The average spore concentration in the *Trichoderma viride* fermentation broth was 1.3 × 10⁻⁶ spores / mL. 9The concentration of cells / mL is significantly higher than the 2.2 × 10⁻⁶ cells / mL at the initial pH of 2. 8 The sporulation rate was [number of sporotypes / mL]. This indicates that an acidic environment of pH 3 is more suitable for the growth and sporulation of the two Trichoderma strains. The lower sporulation rate at the initial pH of 2 is due to the excessively acidic environment inhibiting the activity of the Trichoderma strains, leading to hindered growth and metabolism, and reduced sporulation capacity. The small standard deviations of both treatment groups indicate good experimental repeatability and high data reliability.

[0128] Example 4

[0129] Datian Demonstration (Hanzhong)

[0130] This experiment was conducted from May to September 2025 in Yankou Town, Xixiang County, Hanzhong City, to investigate the effects of Trichoderma root irrigation on tobacco plant growth and soil-borne disease control. The tested flue-cured tobacco variety was Yunyan 99, a major locally grown variety provided by the Hanzhong Branch of Shaanxi Tobacco Company.

[0131] I. Experimental Treatment

[0132] CK: Water the roots with clean water.

[0133] T1: The Trichoderma harzianum fermentation broth prepared in Example 3 was used for root irrigation.

[0134] T2: The fermentation broth of Trichoderma viride prepared in Example 3 was used for root irrigation.

[0135] Each treatment group was replicated three times, with a plot size of 1 acre. Tobacco plants were transplanted on May 1st, with 1000 plants per acre. Agronomic traits were measured on the 7th day after topping (the topping stage of the tobacco plants), and the incidence of black shank disease and root rot was investigated. Economic traits were statistically analyzed after the curing and harvesting were completed in mid-September.

[0136] Trichoderma fermentation solution root irrigation method: Dilute the fermentation solution with water 500 times and irrigate the roots during three stages: tobacco seedling transplanting, tobacco bushing, and vigorous growth period. Use 2 kg of Trichoderma fermentation solution per acre for a single root irrigation.

[0137] The tobacco plants were observed and measured 60 days after transplanting in accordance with the standard YC / T 142—2010 "Methods for Surveying and Measuring Agronomic Traits of Tobacco". The plant height, number of leaves, stem circumference, internode distance, maximum leaf length, maximum leaf width, and maximum leaf area were investigated.

[0138] According to GB / T 23222—2008 "Classification and Survey Methods for Tobacco Diseases and Pests", the incidence rates of tobacco black shank and root rot in the experimental area were investigated, with 360 plants surveyed for each treatment. Incidence rate = number of diseased plants / total number of plants surveyed × 100%.

[0139] The economic traits of flue-cured tobacco were determined according to the national standard GB2635-1992 "Flue-cured Tobacco". The flue-cured tobacco was graded, and the yield of tobacco leaves of each grade for each treatment was statistically analyzed. The proportion of top-grade tobacco was statistically analyzed, and the output value and average price of different treatments were calculated according to the local purchase price.

[0140] II. Experimental Investigation

[0141] Table 6. Survey of agronomic traits of tobacco plants at the dome stage.

[0142] Table 7. Survey of soil-borne diseases during the dome-shaped stage of tobacco plants.

[0143] Table 8 Economic characteristics of each treatment

[0144] As shown in Tables 6, 7, and 8, the investigation and analysis of the experimental treatments revealed that both *Trichoderma harzianum* and *Trichoderma viride* strains had a certain growth-promoting effect on tobacco plants; at the same time, they antagonized tobacco pathogens, reduced the incidence of tobacco diseases, increased tobacco leaf yield, and ensured safe tobacco production.

[0145] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Trichoderma fermentation broth, characterized in that, Includes the following steps: (1) Inoculate Trichoderma onto PDA solid medium and culture to obtain activated Trichoderma mycelium cake; (2) The activated Trichoderma mycelium cake was inoculated onto PDA liquid culture medium and cultured to obtain Trichoderma seed liquid; (3) Inoculate the Trichoderma seed liquid onto the Trichoderma fermentation medium and ferment to obtain Trichoderma fermentation broth.

2. The preparation method according to claim 1, characterized in that, The PDA solid culture medium in step (1) comprises the following components at the following concentrations: potato starch 1~5 g / L, agar 10~20 g / L and glucose 10~30 g / L.

3. The preparation method according to claim 1, characterized in that, The culture temperature in step (1) is 20~30℃, and the culture time is 48~96h.

4. The preparation method according to claim 1, characterized in that, The PDA liquid culture medium in step (2) comprises the following components at the following concentrations: 150-250 g / L potato and 10-30 g / L glucose.

5. The preparation method according to claim 1, characterized in that, The inoculation amount in step (2) is 1~10%; the culture temperature is 20~30℃; the culture rotation speed is 100~300r / min; and the culture time is 48~96h.

6. The preparation method according to claim 1, characterized in that, The fermentation medium in step (3) comprises the following components at the following concentrations: KH2PO4 1~3g / L, NH4NO3 0.5~2g / L, FeSO4·7H2O 0.003~0.008g / L, MnSO4 0.001~0.002g / L, CaCl2 0.1~0.5g / L, NaCl 0.5~2g / L, MgSO4·7H2O 0.1~0.5g / L, corn flour 5~15g / L, and soybean meal 3~8g / L; The corn flour and soybean meal have a mesh size of 60-150 mesh; The pH of the fermentation medium is 2.0 to 3.0, and the pH value is adjusted by phosphoric acid and nitric acid.

7. The preparation method according to claim 1, characterized in that, The inoculation amount in step (3) is 0.5-5%; the fermentation device is a Trichoderma fermentation device, which includes: a fermentation container (1), a stirring mechanism, an air intake system and a pH adjustment system. The fermentation container (1) has a discharge port (11) at the bottom and a top cover (2) sealed at the top opening. The top cover (2) has a feeding port (21), an inoculation port (22), an exhaust port (23) and at least one pH meter port (24). The stirring mechanism is installed on the top cover (2) and extends into the fermentation container (1). The air intake system is connected to the inside of the fermentation container (1). The pH adjustment system is connected to the inside of the fermentation container (1). The fermentation container is made of any one of polypropylene, polyethylene or fiberglass. The fermentation temperature is 20-30℃, the fermentation speed is 100-300r / min, and the fermentation time is 60-96h.

8. The preparation method according to claim 1, characterized in that, The Trichoderma mentioned includes Trichoderma harzianum and Trichoderma viride.

9. The Trichoderma fermentation broth prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the Trichoderma fermentation broth according to claim 9 in promoting tobacco growth and preventing tobacco double black disease.

Images