Trichoderma harzianum, a biological inoculant thereof and application thereof

Abstract

The present application relates to a kind of trichoderma harzianum, its biological inoculant and application, belong to the field of microbial technology.Trichoderma harzianum (Trichoderma harzianum) TH2 strain, its preservation number is CCTCC NO:M20251938.The trichoderma harzianum TH2 strain of the present application can produce cellulase, protease and indole acetic acid, can be used for plant disease control, for controlling orobanche parasitizing in sunflower root, for promoting plant growth and for inhibiting pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to a Trichoderma harzianum with growth-promoting and broad-spectrum antibacterial capabilities, a compound biological agent containing the Trichoderma harzianum, and its applications, belonging to the field of microbial technology. Background Technology

[0002] Orobanche coerulescens, a holoparasitic seed plant, poses a serious threat to major sunflower-producing areas in northern my country. Its seeds germinate after sensing chemical signals such as strigolactones secreted by sunflower roots, forming haustoria that penetrate the host root cortex and connect to the vascular system to plunder water, nutrients, and organic matter. This leads to stunted growth, yellowing leaves, smaller flower heads, reduced yields, and even crop failure in sunflowers. Furthermore, orobanche seeds can survive in the soil for over 10 years, making control extremely difficult and a pressing problem for agricultural production.

[0003] Currently, there are various methods for controlling broomrape parasites, but each has its limitations. Agricultural control requires long-term crop rotation, continuous field cleaning and deep plowing, which is time-consuming and labor-intensive; physical and mechanical control relies on high temperatures and manual operation, which is costly and difficult to eradicate completely; chemical control is prone to phytotoxicity, environmental pollution, and may also affect soil ecology. Microbial control of broomrape has made some progress. The cell-free fermentation filtrate of Penicillium griseus CF3 can inhibit the germination and germ tube growth of broomrape seeds, and its powdered formulation can reduce the number of broomrape emerging from the soil and the dry weight of individual plants; in addition, the spore suspension of lily root rot fungus can reduce the germination rate and parasitism rate of serrated broomrape and branched broomrape, but some broomrape control fungi may be crop pathogens, posing a risk of causing disease in host crops; actinomycetes such as Streptomyces lilacinus and Streptomyces nigra can be used to control broomrape. Among them, the inoculant of Streptomyces nigra can reduce the number of broomrape emerging from the soil and increase the yield of tomatoes in field trials.

[0004] Trichoderma, as a type of filamentous fungus, shows potential in the field of biological control. Trichoderma species such as *Trichoderma harzianum* possess unique biological characteristics and functional advantages: diverse morphology, rapid reproduction, and the ability to quickly occupy ecological niches; wide environmental adaptability, surviving in various soil conditions, and long-lasting effects; and multiple functions including disease inhibition, growth promotion, and quality improvement. It inhibits pathogens through competition, antagonism, and hyperparasitism, while also improving soil and enhancing plant resistance. Its applications in the control of soil-borne diseases, foliar diseases, and fruit diseases align with the needs of green agricultural pest control. Given the urgent need for *Bacillus thuringiensis* control and the biocontrol advantages of *Trichoderma*, exploring its control potential against *Bacillus thuringiensis* is feasible. However, currently, there are no mature *Trichoderma* strains or technologies specifically designed for *Bacillus thuringiensis* control. Summary of the Invention

[0005] The purpose of this invention is to provide a Trichoderma harzianum, its biological agent, and its applications. The Trichoderma harzianum TH2 strain can produce cellulase, protease, and indoleacetic acid, and can be used for the prevention and control of plant diseases, for the control of broomrape parasitizing the roots of sunflowers, for promoting plant growth, and for inhibiting pathogens.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A strain of *Trichoderma harzianum* TH2, isolated from field soil in Weixian County, Xingtai City, China, has been deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20251938. The deposit date is September 1, 2025. The address of CCTCC is: Wuhan University, Wuhan, China.

[0008] The present invention also provides a biological agent, wherein the agent comprises the above-mentioned Trichoderma harzianum TH2 strain or the metabolites of the above-mentioned strain.

[0009] The present invention also provides a biological agent prepared from the said microbial agent.

[0010] The present invention also provides any of the following applications of the said biological agent:

[0011] 1) Used to produce cellulase;

[0012] 2) Used to produce proteases;

[0013] 3) Used to produce indoleacetic acid;

[0014] 4) Used for the prevention and control of plant diseases;

[0015] 5) Used to control broomrape parasitizing the roots of sunflowers;

[0016] 6) Used to promote plant growth;

[0017] 7) Used to inhibit pathogens.

[0018] The pathogens include: rice sheath blight fungus (Rhizoctonia solani), anthracnose fungus (Colletotrichum graminicola), grape rachis brown blight fungus (Alternaria viticola), strawberry root rot fungus (Fusarium solani), pineapple black rot fungus (Fusarium commune), sugarcane top rot fungus (Fusarium moniliforme), beet leaf spot fungus (Alternaria alternata), peanut fruit rot fungus (Fusarium proliferatum), peanut root rot fungus (Pythium myriotylum), lily root rot fungus (Fusarium oxysporum), beet root rot fungus (Fusarium redolens), and celery leaf spot fungus (Septoria apiicola Speg).

[0019] The beneficial effects of adopting the above technical solution are:

[0020] The Trichoderma harzianum strain TH2 of this invention can produce cellulase, protease, and indoleacetic acid, and can be used for the prevention and control of plant diseases, for the control of broomrape parasitizing the roots of sunflowers, for the promotion of plant growth, and for the inhibition of pathogens.

[0021] The Trichoderma harzianum TH2 strain of this invention has a good growth-promoting effect, which can increase the height of corn plants by 20.9%, the total root length of corn by 38%, and the fresh weight of corn roots by 31.9%.

[0022] The Trichoderma harzianum strain TH2 of this invention exhibits good tolerance to low to medium salinity environments, with a maximum tolerance salt concentration of 3%.

[0023] The Trichoderma harzianum strain TH2 of this invention is tolerant to pH values ​​between 3 and 8.

[0024] The average IAA yield of the Trichoderma harzianum TH2 strain of this invention was 0.129 mg / L on DF medium and 0.238 mg / L on DF+ medium.

[0025] The *Trichoderma harzianum* strain TH2 of this invention exhibited the highest inhibition rate against peanut fruit rot pathogens, reaching 71.12±3.13%; its inhibition rates against beet leaf spot pathogens and anthracnose pathogens were 66.55±2.37% and 66.31±3.83%, respectively, also showing good inhibitory effects; the inhibition rate of strain TH2 against most pathogens ranged from 50% to 65%, including peanut root rot pathogens (68.59±5.05%), sugarcane top rot pathogens (62.57±2.05%), and pineapple black rot pathogens (6...). The inhibitory effects of TH2 on *Strawberry root rot* (2.09±4.62%), *Strawberry rachis rot* (59.42±3.48%), *Strawberry rachis rot* (56.27±1.55%), *Strawberry sheath blight* (54.09±1.33%), *Strawberry root rot* (53.34±2.25%), and *Strawberry root rot* (51.66±3.61%) were observed. Among the 12 pathogens tested, TH2 showed the lowest inhibition rate against *Strawberry leaf spot*, at only 31.81±4.70%, with no complete lack of inhibition observed.

[0026] The TH2 strain metabolites showed the highest inhibition rate against peanut fruit rot pathogens, reaching 65.66±0.61%; the inhibition rate against pineapple black rot pathogens was 49.99±1.73%, close to 50%; and the inhibition rate against rice sheath blight pathogens was 48.88±4.39%. The inhibition rates of the metabolites against most pathogens were concentrated between 22% and 29%, including strawberry root rot pathogens (29.16±2.17%), sugarcane top rot pathogens (23.62±3.75%), beet leaf spot pathogens (23.42±2.60%), peanut root rot pathogens (22.80±2.87%), and anthracnose pathogens (22.22±0.69%).

[0027] The TH2 strain of this invention promotes the height of sunflower plants, with the average plant height reaching 116% of the control group. In terms of the number of orobanche survival, the control group had an average of 5 orobanche parasites per sunflower plant, while the Trichoderma treatment group had an average of only 1.8 parasites per plant, a reduction of 64% in the number of orobanche survival, which was significantly lower than the control. Attached Figure Description

[0028] Figure 1 This is a diagram showing the effect of Trichoderma solution on corn root development.

[0029] TH1-1, TH2-1, and TH3-1 indicate that the spore concentration of the bacterial culture in each treatment is 1×10⁻⁶. 5 spores / mL; TH1-2, TH2-2, TH3-2 indicate that the spore concentration of each treatment is 1×10⁻⁶. 6 per mL.

[0030] Figure 2 The graph shows the inhibitory effects of three Trichoderma strains on anthracnose fungus, strawberry root rot fungus, and lily root rot fungus.

[0031] The left side of each row of plates represents Trichoderma TH1, TH2, and TH3, while the right side represents the pathogenic fungi.

[0032] Figure 3 The graph shows the inhibitory effects of three Trichoderma strains on peanut fruit rot pathogens and peanut root rot pathogens.

[0033] The left side of each row of plates represents Trichoderma TH1, TH2, and TH3, while the right side represents the pathogenic fungi.

[0034] Figure 4 Morphological characteristics of Trichoderma TH2;

[0035] a represents pre-sporulation colonies, b represents post-sporulation colonies, and c represents hyphae and spores.

[0036] Figure 5 This is a diagram showing the growth status of TH2 at different salt concentrations.

[0037] Figure 6 The effect of different pH values ​​on TH2 biomass is shown in the figure.

[0038] Different letters indicate significant differences at the P<0.05 level.

[0039] Figure 7 The effect of TH2 producing protease.

[0040] Figure 8 A diagram showing the effect of TH2 producing cellulase.

[0041] Figure 9 The effect of TH2 on plate confrontation with 6 pathogens Figure 1 ;

[0042] 1-6 represent rice sheath blight fungus, anthracnose fungus, grape rachis brown blight fungus, strawberry root rot fungus, pineapple black rot fungus, and sugarcane top rot fungus, respectively.

[0043] Figure 10 The effect of TH2 on plate confrontation with 6 pathogens Figure 2 ;

[0044] 7-12 represent beet leaf spot fungus, peanut fruit rot fungus, peanut root rot fungus, lily root rot fungus, beet root rot fungus, and celery leaf spot fungus, respectively.

[0045] Figure 11 Comparison of antibacterial effects of TH2 strain and metabolites Figure 1 ;

[0046] The left side represents the strain's metabolites, and the right side represents the strain.

[0047] 1-4 represent rice sheath blight fungus, anthracnose fungus, grape rachis brown blight fungus, and strawberry root rot fungus, respectively.

[0048] Figure 12 Comparison of antibacterial effects of TH2 strain and metabolites Figure 2 ;

[0049] The left side represents the strain's metabolites, and the right side represents the strain.

[0050] 5-8 represent pineapple black rot fungus, sugarcane top rot fungus, beet leaf spot fungus, and peanut fruit rot fungus, respectively.

[0051] Figure 13 Comparison of antibacterial effects of TH2 strain and metabolites Figure 3 ;

[0052] The left side represents the strain's metabolites, and the right side represents the strain.

[0053] 9-10 represent the root rot fungus of peanut and the root rot fungus of lily, respectively.

[0054] Figure 14 Figure showing the effect of TH2 treatment on sunflower biomass and broomrape parasitism.

[0055] * indicates that the difference is significant at the P<0.05 level.

[0056] Figure 15 This is an illustration of the effects of orobanche parasitism.

[0057] The left image shows the control group, and the right image shows the bacterial solution treatment.

[0058] Figure 16 This describes the spore production and viable bacteria status of Trichoderma during solid-state fermentation. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] Example 1: Isolation of Trichoderma harzianum TH2 strain

[0061] The strain was isolated from field soil in Weixian County, Xingtai. The specific procedure was as follows: Wearing sterile gloves, 100g of rhizosphere soil from a depth of 15cm was placed in a sterile polypropylene plastic bag to obtain a soil sample. In a clean bench, 30g of the soil sample was placed in an Erlenmeyer flask containing 100mL of sterile distilled water, the cap was sealed, and the flask was shaken at 140r / min for 10min to obtain the stock solution. Then, 5-fold, 25-fold, 50-fold, and 75-fold dilutions were prepared. The stock solution and... 200 μL of each diluted solution was applied to the surface of Bengal Red solid agar medium using a sterile spreader, five plates of each solution were spread, for a total of 25 plates. These plates were incubated at 25°C for 36 hours to obtain *Trichoderma* colonies. The obtained *Trichoderma* colonies were observed in a clean bench. Twenty colonies from each sample were transferred to PDA slant agar and incubated at 25°C for 5 days to isolate *Trichoderma*, obtaining pure cultures and completing the isolation of *Trichoderma* from the plant rhizosphere. During the incubation process, colony development was observed, and the three strains with the best growth were selected as target bacteria and named TH1, TH2, and TH3, respectively.

[0062] Example 2: Screening of strains

[0063] Validation of the growth-promoting ability of the strain

[0064] Test plant: maize;

[0065] Preparation of inoculum: The three isolated strains were cultured in PDB medium until the spore concentration reached 1×10⁻⁶. 6 Then dilute the bacterial suspension to a spore concentration of 1×10⁶ / mL or higher. 6 cells / mL and 1×10 5 Units / mL available for use;

[0066] Treatment setup: Seeds were disinfected with sodium hypochlorite for 30 seconds and then rinsed twice with sterile water. The experimental group was soaked in 200 mL of bacterial agent for 6 hours (the control group CK used sterile water) and then germinated. After germination, seeds with consistent germination levels were selected for hydroponics using Hoagland nutrient solution. Ten seedlings were planted in each hydroponic box. 500 mL of nutrient solution was added initially, and the solution was replenished to 500 mL every 5 days. Each treatment was repeated three times and cultured for two weeks.

[0067] Measurement indicators: Growth parameters such as stem length, root length, fresh weight, and dry weight are measured.

[0068] Table 1. Effects of bacterial suspension treatment on maize biomass.

[0069]

[0070] Note: Different letters indicate significant differences at the P<0.05 level; TH1-1, TH2-1, and TH3-1 represent spore concentrations of 1×10⁻⁶ in each treatment. 5spores / mL; TH1-2, TH2-2, TH3-2 indicate that the spore concentration of each treatment is 1×10⁻⁶. 6 per mL.

[0071] The effects of Trichoderma inoculum solution on corn root development are shown in the appendix. Figure 1 .

[0072] From the appendix Figure 1 It can be seen that, after treatment with the bacterial solution, the plant height of maize in each treatment group increased to varying degrees compared with the control, with the spore concentration being 1×10⁻⁶. 5 TH1 and TH2 bacterial cultures per mL and 1×10 6 Treatment with TH2 bacterial suspension at 1×10⁶ / mL resulted in plant height increases of 18%, 20.9%, and 19.1%, respectively, significantly higher than the control group; spore concentration was 1×10⁶ / mL. 5 Treatment with TH2 bacterial solution at a concentration of 1 × 10⁶ spores / mL significantly increased the total root length of maize, by 38%. There were no significant differences in fresh and dry weight of the aboveground parts compared to the control after treatment with any of the bacterial solutions. 5 Treatment with TH1 and TH3 bacterial solutions at concentrations of 1 × 10⁶ cells / mL slightly increased the fresh weight of corn roots. 6 Treatment with TH1 and TH3 bacterial solutions at concentrations of 1 × 10⁶ / mL resulted in a slight decrease in root fresh weight compared to the control. Treatment with both concentrations of TH2 bacterial solutions significantly increased root fresh weight, by 27.7% and 31.9% respectively compared to the control. The spore concentration was 1 × 10⁶ / mL. 5 Treatment with TH2 bacterial solution at 1×10⁶ cells / mL resulted in a slight increase in the dry weight of maize roots. 6 Treatment with TH1 and TH3 bacterial solutions at a concentration of 100 cells / mL resulted in a slight decrease in root dry weight, but neither was significant.

[0073] Validation of antibacterial ability of strains

[0074] Test pathogens: Five common plant disease pathogens were selected: *Colletotrichum graminicola*, *Fusarium haematococca*, *Fusarium oxysporum*, *Fusarium proliferatum*, and *Pythium myriotylum*. Pre-activation was performed.

[0075] Culture medium preparation: PDA culture medium is poured into plates;

[0076] Inoculation procedure: Trichoderma mycelium and pathogen mycelium with a diameter of 7.5 mm were inoculated on both sides of the plate 5 mm from the edge, respectively. The control group was inoculated only with pathogen mycelium.

[0077] Culture and determination: Incubate at 28℃ for 5-7 days, measure the inhibition distance from the pathogen colony to the Trichoderma colony, and calculate the inhibition rate: Inhibition rate (%) = (diameter of pathogen in control group - diameter of pathogen in treatment group) / diameter of pathogen in control group × 100%.

[0078] Table 2. Inhibitory effects of strains on common pathogens

[0079]

[0080] The inhibitory effects of three Trichoderma strains on five pathogens are shown in the appendix. Figure 2 and attached Figure 3 As shown in the attached figure and Table 2, the inhibition rate of strain TH2 against all five pathogens exceeded 50%, with the best inhibition effect against peanut fruit rot pathogen, reaching 73.09%. The average inhibition rate against the five pathogens reached 64.43%, and its overall performance was better than that of TH1 (47.23%) and TH3 (36.06%).

[0081] Based on preliminary verification of the growth-promoting and antibacterial experiments of the strain, it is believed that the TH2 strain has the potential for biocontrol applications.

[0082] Example 3: Identification of the strain

[0083] Morphological observations show that its characteristics include: slender, septate hyphae; multi-branched conidiophores; spherical conidia; and colonies on PDA medium that initially appear as white flocculent material, later turning dark green (see attached image). Figure 4 .

[0084] Molecular biological identification (ITS sequence analysis: DNA was extracted from the samples using the CTAB method and stored at -20℃ for later use. PCR amplification was performed using the universal ITS primers ITS1 / ITS4. The PCR reaction program was: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min, 32 cycles; 72℃ extension for 5 min, and storage at 4℃. The PCR products were sent to Sangon Biotech (Beijing) Co., Ltd. for sequencing, and the sequencing results were submitted to the NCBI website. The ITS sequence (SEQ ID NO.1) is shown in Table 3.

[0085] Table 3. ITS sequences of TH2 strains

[0086]

[0087] Based on morphological and molecular biological results, strain TH2 was identified as Trichoderma harzianum.

[0088] Example 4: Determination of TH2 salt tolerance

[0089] Culture medium preparation: Add NaCl to PDA medium to prepare a series of culture media with salt concentrations of 0%, 1%, 2%, 3%, 4%, and 5%, and then sterilize them to prepare plate culture media.

[0090] Inoculation and culture: Take TH2 strain mycelial cakes (from the edge of colonies cultured for 7 days) using a 7.5 mm punch and inoculate them in the center of various concentrations of culture medium, and culture at 28°C for 5 days;

[0091] Measurement indicators: After 5 days of culture, the colony diameter was measured using the cross-cross method, and the inhibition rate at different salt concentrations was calculated as ((control diameter - treatment diameter) / control diameter * 100%).

[0092] Table 4 Effect of different salt concentrations on TH2

[0093]

[0094] Note: Different letters indicate that the difference is significant at the P<0.05 level.

[0095] The growth status of TH2 at different salt concentrations is shown in the appendix. Figure 5 From Table 4 and Appendix Figure 5 It was found that strain TH2 showed good growth at salt concentrations of 1% and 2%, with inhibition rates of 8.07% and 22.36%, respectively. At 3%, the relative inhibition rate reached 45.95%, and at 4%, the relative inhibition rate exceeded 50%, with a significant decrease in colony diameter and markedly limited growth. These results indicate that TH2 exhibits good tolerance to low to medium salinity environments, with a maximum tolerant salt concentration of 3%.

[0096] Example 5: Determination of TH2's resistance to acids and alkalis

[0097] PDB medium: 200g potato, 20g glucose, add water to 1000mL, autoclave at 121℃ for 30min.

[0098] Acid-base gradient culture medium: Adjust the pH of PDB medium to pH 1, pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10 with 1 mol / L NaOH and HCl; dispense into smaller portions for use.

[0099] Single colonies from *Trichoderma* agar plates were picked using a sterile inoculation loop and incubated on PDB liquid medium at different pH values ​​on a shaker at 30°C and 200 rpm / min. After 40 h, 10 mL of each treatment was aliquoted into clean centrifuge tubes, repeated three times, balanced, and centrifuged at 10,000 rpm / min for 10 min. The supernatant was discarded after centrifugation, and the remaining bacterial cells were weighed.

[0100] The results showed that after 40 hours of shaking culture, the accumulated TH2 biomass in the culture medium with pH between 3 and 8 ranged from 1.29 g / 10 ml to 1.33 g / 10 ml, with no significant difference in biomass among the groups. When the pH was below 3 or above 8, the TH2 biomass decreased significantly, and growth was significantly inhibited.

[0101] Example 6: Determination of TH2's IAA Production Capacity

[0102] culture medium

[0103] PDA medium: 200g potato, 20g glucose, 15-20g agar, add water to 1000mL, pH 7.0, autoclave at 121℃ for 30min.

[0104] DF medium: 5.00 g peptone, 1.50 g yeast extract, 1.50 g beef extract, 5.00 g NaCl, 1000 mL distilled water, pH 7.0, autoclaved at 121°C for 30 min.

[0105] DF+ medium: Add 0.50 g / L of tryptophan according to the specified ratio.

[0106] reagents

[0107] 10mM phosphoric acid (prepared with 85% concentrated phosphoric acid): The molar mass of 85% concentrated phosphoric acid is 14.48 mol / L. First, prepare a stock solution with a concentration of 1 mol / L, then dilute it 100 times to prepare the target concentration of 10 mmol / L.

[0108] Salkowkin colorimetric solution: 1 mL of 0.5 M FeCl3 dissolved in 50 mL of 35% HClO4.

[0109] 0.5M FeCl3: Weigh 1.3500g FeCl3·6H2O and dissolve it in 10mL of deionized water.

[0110] 35% HClO4: Measure 50 mL of 70% HClO4, add it to 50 mL of deionized water, and mix well.

[0111] Operating steps

[0112] 1. Plotting the standard curve

[0113] Weigh 0.01g of pure IAA (SIGMA, CAS: 87-51-4) and dissolve it in 1mL of anhydrous ethanol. After mixing, take 100μL of the solution and dilute it with deionized water to 1mL. After mixing, take another 100μL of the solution and dilute it with deionized water to 1mL to obtain a 100μg / ml IAA stock solution.

[0114] Prepare aqueous solutions of IAA at the following concentration gradients: 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, and 90 mg / L. Use deionized water as a blank control. Measure the absorbance of the IAA solutions at different concentrations at 530 nm and plot a standard curve for IAA content.

[0115] 2. Inoculate the strain into LB medium and incubate at 28°C and 150 rpm for 12 hours using a constant temperature shaker.

[0116] 3. After 12 hours of shaking incubation, the bacterial culture was inoculated into DF and DF+ medium at a 1% inoculum concentration and incubated for 7 days at 28°C and 150 rpm in a constant temperature shaker. This experiment was performed in 4 replicates.

[0117] IAA content determination

[0118] Seven days later, remove the centrifuge tubes and centrifuge at 12,000 rpm for 5 min. Take 500 μL of the supernatant into a 2 mL test tube, add 25 µL of 10 mM phosphoric acid and 1 mL of Alkowkin colorimetric solution, and react the mixture in the dark at room temperature for 25 min. Then, pipette 200 μL of each solution into a 96-well plate, detect the absorbance at 530 nm, and calculate the IAA yield using a standard curve.

[0119] The experimental results showed that the average yield of IAA on DF medium was 0.129 mg / L, and the average yield of IAA on DF+ medium was 0.238 mg / L.

[0120] Example 7: Determination of TH2 protein production capacity

[0121] Culture media and reagents

[0122] PDA medium: 200g potato, 20g glucose, 15-20g agar, add water to 1000mL, pH 7.0, autoclave at 121℃ for 30min.

[0123] Culture medium for testing: 5.00 g tryptone, 3.00 g yeast extract, 1.00 g glucose, 15.00 g agar, 1000 mL distilled water, pH 7.0, autoclaved at 121°C for 30 min. 100 mL skim milk.

[0124] Operating steps

[0125] 1. Transfer the strain to a PDA plate using a toothpick and incubate at 28°C for 24 hours in a biochemical incubator.

[0126] 2. When the sterilized test medium is cooled to about 50°C, skim milk is added to the medium at a ratio of 10%, mixed well, and poured into a petri dish. After cooling, the medium is inoculated.

[0127] 3. Inoculate the activated strain onto the test solid medium using a toothpick. Inoculate one strain per plate, with four replicates, and incubate at 28°C.

[0128] 4. Observe the formation of a clear zone around the colony two days later. The appearance of a clear zone indicates the production of protease.

[0129] From the appendix Figure 7 It can be seen that the culture medium below the central region of the strain produces a 17.2±0.5 mm clear zone, indicating that TH2 has the ability to produce proteases.

[0130] Example 8: Determination of TH2's ability to produce cellulase

[0131] PDA medium: 200g potato, 20g glucose, 15-20g agar, add water to 1000mL, pH 7.0, autoclave at 121℃ for 30min.

[0132] Culture medium for testing: 5.00g yeast extract, 10.00g tryptone, 10.00g NaCl, 0.25g MgSO4•7H2O, 0.50g K2HPO4, 10g carboxymethyl cellulose, 15.0g agar, 1000mL distilled water, pH 7.0, autoclaved at 121℃ for 30min.

[0133] reagents

[0134] Congo red staining solution: 0.02g Congo red dissolved in 100mL water.

[0135] 1M NaCl: 5.85g NaCl dissolved in 100mL of water.

[0136] Operating steps

[0137] 1. Transfer the strain to a PDA plate using a toothpick and incubate at 28°C for 24 hours in a biochemical incubator.

[0138] 2. After cooling the sterilized test medium to about 50°C, pour it into a petri dish and inoculate it after it has cooled down.

[0139] 3. The activated bacterial strain was inoculated onto the test solid medium using a toothpick. One strain was inoculated per plate, with three replicates per strain, and incubated at 28°C. This experiment had four replicates.

[0140] 4. After 7 days, add 5 mL of 0.2 mg / mL Congo red staining solution to each plate, stain for 1 hour, discard the Congo red staining solution, add 1 M NaCl to wash for 1 hour, discard the washing solution, and observe the formation of hydrolysis zones around the colonies. The appearance of hydrolysis zones indicates the production of cellulase.

[0141] From the appendix Figure 8 It can be seen that a transparent hydrolysis zone with a diameter of 33.8±0.6 mm appears around the colony, indicating that TH2 has the ability to produce cellulase.

[0142] Example 9: Determination of TH2 antibacterial ability

[0143] PDA medium: 200g potato, 20g glucose, 15-20g agar, add water to 1000mL, pH 7.0, autoclave at 121℃ for 30min, cool to 50℃ to prepare plate medium.

[0144] Test pathogens: Select target pathogens and fully activate them in advance on PDA medium.

[0145] Inoculation procedure: 7.5 mm Trichoderma mycelium and pathogen mycelium were inoculated on both sides of the plate at 5 mm from the edge, respectively. The control group was inoculated only with pathogen mycelium.

[0146] Culture and determination: Incubate at 28℃ for 5-7 days, measure the inhibition distance from the pathogen colony to the Trichoderma colony, and calculate the inhibition rate: Inhibition rate (%) = (diameter of pathogen in control group - diameter of pathogen in treatment group) / diameter of pathogen in control group × 100%.

[0147] Table 5. Antibacterial effect of TH2 on pathogens

[0148]

[0149] The data above show that strain TH2 exhibits varying inhibitory effects against the 12 pathogens, but overall demonstrates a strong broad-spectrum antibacterial ability.

[0150] The TH2 strain showed the highest inhibition rate against peanut fruit rot pathogens, reaching 71.12±3.13%; the inhibition rates against beet leaf spot pathogens and anthracnose pathogens were 66.55±2.37% and 66.31±3.83%, respectively, also demonstrating good inhibitory effects.

[0151] TH2 showed inhibition rates of 50%–65% against most pathogens, including peanut root rot fungus (68.59±5.05%), sugarcane top rot fungus (62.57±2.05%), pineapple black rot fungus (62.09±4.62%), strawberry root rot fungus (59.42±3.48%), grape rachis brown blight fungus (56.27±1.55%), rice sheath blight fungus (54.09±1.33%), lily root rot fungus (53.34±2.25%), and beet root rot fungus (51.66±3.61%). Among the 12 pathogens tested, TH2 showed the lowest inhibition rate against celery leaf spot fungus, at only 31.81±4.70%, with no cases showing complete lack of inhibition.

[0152] Example 10: Determination of the antibacterial activity of TH2 metabolites

[0153] Use a sterile inoculation needle to pick up Trichoderma and inoculate it onto a PDA plate. Incubate in a 25°C constant temperature incubator until the mycelium covers the entire plate.

[0154] Using a sterile inoculation loop, pick a single colony from a Trichoderma plate and transfer it to PDB liquid medium. Incubate on a shaker at 30°C and 200 rpm / min. After 20 hours of incubation, immediately transfer the colonies into clean 50 mL centrifuge tubes, balance the medium, and centrifuge at 10,000 rpm / min for 10 minutes. Aspirate the supernatant using a disposable syringe, filter the supernatant through a membrane to remove the bacterial cells, and collect the Trichoderma metabolites for later use.

[0155] First, microwave the sterilized PDA to boiling. After cooling to 50°C, add the test sample to the PDA culture medium at a 20-fold dilution in a clean bench. While still hot, pour the PDA containing the test sample onto a plate. Also, prepare a blank control (PDA without the test sample) and pour it onto a plate while still hot. Each treatment typically has three replicates. After the petri dishes have cooled, use a sterile punch to create 7.5mm diameter mycelial discs on the bacteria. Inoculate the mycelial discs in the center of each treatment petri dish. Finally, place all treatments in a 25°C incubator for 5 days.

[0156] Table 6. Inhibition of pathogens by TH2 metabolites

[0157]

[0158] From the data in the table above and the appendix Figure 11-13It is evident that the inhibitory effect of bacterial metabolites on different pathogens is generally weaker than that of direct bacterial inhibition, and the differences in inhibitory capacity among different pathogens are more pronounced. The highest inhibition rate was observed against *Phyllostachys edulis*, reaching 65.66±0.61%, close to the inhibition rate of direct bacterial inhibition (71.12±3.13%), indicating that the metabolites are the core inhibitory factor for this pathogen. The inhibition rate against *Phyllostachys pineapple* was 49.99±1.73%, close to 50%, and the inhibition rate against *Rhizoctonia solani* was 48.88±4.39%, all three demonstrating the strong inhibitory activity of the metabolites against these pathogens. The inhibition rates of metabolites against most pathogens were concentrated between 22% and 29%, including *Streptomyces cristatum* (29.16±2.17%), *Streptomyces cerevisiae* (23.62±3.75%), *Streptomyces cerevisiae* (23.42±2.60%), *Streptomyces cerevisiae* (22.80±2.87%), and *Streptomyces anthracnose* (22.22±0.69%). Although these rates were lower than the direct inhibitory effects of the strains, they still provided some growth inhibition through metabolites. The inhibition rates of metabolites against *Streptomyces cerevisiae* (9.73±0.64%) and *Streptomyces cerevisiae* (7.88±2.08%) were both below 10%, indicating weak inhibitory effects. No inhibitory effect was observed against *Streptomyces cerevisiae* (beetroot rot) or *Streptomyces cerevisiae* (celery leaf spot), suggesting that these pathogens have low sensitivity to the strains' metabolites.

[0159] Example 11: Determination of TH2's ability to alleviate the parasitic effect of orobanche on sunflower

[0160] Preparation of broomrape seeds: Collect mature broomrape seeds, sterilize the surface and set aside; mix them evenly into the soil at 1g / kg, and after slightly moistening the soil, form it into several 20g soil balls for later use.

[0161] Potting setup: Three sunflower plants were planted in each pot with 2.5 kg of soil. Before planting, the soil ball containing broomcorn seeds was placed under the sunflower seeds. The treatment group was inoculated with Trichoderma fungicide at a spore concentration of 1×10⁻⁶. 5 The number of sunflowers was 1 / mL, and each pot contained 500mL of sterile water. The control group was substituted with sterile water. The potted plants were grown in a greenhouse, with the temperature maintained at 28℃ during the 14-hour light period and 16℃ during the 10-hour dark period. All other management measures were the same for all treatments during the potted plant period, including appropriate watering. After the sunflowers grew to two pairs of needles, the seedlings were thinned, leaving one plant per pot. Each treatment was replicated 9 times.

[0162] Measurement indicators: After 60 days of cultivation, the number of sunflower roots that survived was counted, and the sunflower biomass (plant height, stem diameter, and flower head diameter) was measured.

[0163] As attached Figure 14 and attached Figure 15As shown, Trichoderma treatment promoted the height of sunflower plants, with the average plant height reaching 116% of the control group. There was no significant difference in stem diameter and flower head diameter compared with the control group. In terms of the number of orobanche surviving plants, the control group had an average of 5 orobanche surviving plants per sunflower, while the Trichoderma treatment group had an average of only 1.8 orobanche surviving plants per plant, a reduction of 64%, which was significantly lower than the control.

[0164] Example 12, TH2 Production Process

[0165] PDB medium: 200g potato, 20g glucose, add water to 1000mL, adjust pH to 5.5, autoclave at 121℃ for 30min.

[0166] Solid fermentation medium: Add an appropriate amount of amino acid solution to mushroom residue to adjust the pH to 3.5, and the moisture content to 65%. Autoclave at 121℃ for 30 minutes.

[0167] Using a sterile inoculation needle, *Trichoderma* was picked up and inoculated onto a PDA plate. It was then cultured in a 25°C incubator until the mycelium covered the entire plate. Mycelial cakes were created at the edge of the plate, and using a sterile inoculation loop, these cakes were transferred to PDB liquid medium and cultured on a shaker at 30°C and 200 rpm / min. After 30 hours of shaking culture, the seed culture from the shake flask was inoculated into a primary fermenter at a volume percentage of 0.3–0.5%. After 48 hours of incubation, the culture was transferred to a secondary fermenter and cultured at a constant temperature for 60 hours. Finally, the culture was transferred to solid medium at an inoculation rate of 100 mL / kg and thoroughly mixed under sterile conditions. The mixture was then evenly spread on the fermentation bed, maintaining a thickness of 3–4 cm. Fermentation was carried out at 30°C and 75% humidity. During solid-state fermentation, the growth and development of Trichoderma were continuously monitored. The sporulation and viable cell count of Trichoderma were counted using the hemocytometer method and the dilution plating method, respectively. In the early stage of fermentation, in order to avoid contamination of the fermentation material by other microorganisms, samples were randomly taken from three sampling points every 3 to 5 days.

[0168] From the appendix Figure 16 It can be seen that on the 10th day of solid-state fermentation, the number of viable bacteria reached 1×10⁻⁶. 9 The number of spores was counted per gram, and samples were taken daily thereafter. The results showed that between 10 and 14 days of fermentation, both the number of spores and the number of viable cells gradually increased with the fermentation time. At 14 days of fermentation, both the number of viable cells and the number of spores reached their peak values ​​of 2.7 × 10⁻⁶. 9 pcs / g and 8.3×10 9 The number of viable bacteria and spores decreased relative to the peak value for two consecutive days. Therefore, in actual production, to ensure maximum benefits, the solid-state fermentation time should not exceed 14 days.

[0169] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A Trichoderma harzianum TH2 strain, with accession number CCTCC NO:M20251938.

2. A biological agent, characterized in that: The inoculum contains the Trichoderma harzianum TH2 strain of claim 1 or a metabolite of the strain.

3. A biological agent prepared from the microbial agent of claim 2.

4. Any of the following applications of the biological agent according to claim 3: 1) Used to produce cellulase; 2) Used to produce proteases; 3) Used to produce indoleacetic acid; 4) Used for the prevention and control of plant diseases; 5) Used to control broomrape parasitizing the roots of sunflowers; 6) Used to promote plant growth; 7) Used to inhibit pathogens.

5. The application according to claim 4, characterized in that... The pathogens include: rice sheath blight pathogen, anthracnose pathogen, grape rachis brown blight pathogen, strawberry root rot pathogen, pineapple black rot pathogen, sugarcane top rot pathogen, beet leaf spot pathogen, peanut fruit rot pathogen, peanut root rot pathogen, lily root rot pathogen, beet root rot pathogen, and celery leaf spot pathogen.

Images