Trichoderma harzianum, biocontrol agent and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses an African Trichoderma harzianum, a biocontrol agent and application thereof, wherein the African Trichoderma harzianum is named as African Trichoderma harzianum (T10) with a preservation number of CGMCC No. 41709, and the strain is preserved in the China General Microbiological Culture Collection Center on December 11, 2024, and the preservation address is No. 3, Xili, Beichen West Road, Chaoyang District, Beijing. Trichoderma afroharzianum The active ingredient of the biocontrol agent is the African Trichoderma harzianum. The African Trichoderma harzianum T10 has inhibitory effects on apple tree rot disease and apple ring rot disease, can be used for preparing the biocontrol agent and applied to the prevention and treatment of apple tree rot disease and apple ring rot disease, has a growth promoting effect on fruit trees, can be used as an active ingredient to prepare a plant growth promoter, and can be applied to degradation of rot virus. The African Trichoderma harzianum T10 has the advantages of stable control effect and environmental friendliness.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Trichoderma harzianum, a biocontrol agent, and its application. Background Technology

[0002] Apple tree rot is a highly destructive disease in the apple industry, caused by the fungus *Hemibarbus thunbergii*. Valsa mali) This disease, caused by infection, seriously threatens the health of fruit trees and the yield and quality of fruit. It primarily affects branches and trunks, initially presenting as water-soaked, reddish-brown lesions that gradually expand into dark brown or black necrotic areas as the disease progresses, leading to bark cracking and peeling, ultimately causing branch dieback and even the death of the entire tree. The harm of apple tree canker is not only reflected in direct fruit yield reduction, but also in the weakened tree vigor leading to decreased fruit sugar content and reduced commercial value. Simultaneously, this disease forces fruit growers to invest heavily in scraping and treating lesions, chemical control, and tree repair, significantly increasing their management burden. Research shows that the pathogens can remain dormant through wounds for a long time, rapidly erupting when the tree vigor is weakened, forming a cyclical pattern of "spring expansion and autumn infection." Factors such as frost damage, excessive fruit load, and soil compaction can further exacerbate the disease. Due to the disease's strong latency and rapid spread, it is difficult to eradicate it using only a single control method. Achieving sustainable control of apple tree canker is of irreplaceable strategic significance for ensuring the economic benefits of orchards and maintaining the stable development of the industry.

[0003] Currently, the control of apple tree canker disease relies heavily on chemical pesticides. However, chemical control has significant drawbacks: continuous application easily leads to pesticide resistance in pathogens, insufficient pesticide penetration results in high recurrence rates, and organic solvent residues can cause soil compaction and ecological damage. More seriously, chemical control can only control the surface symptoms of the disease and cannot prevent latent infection of the pathogen within the xylem. Against this backdrop, biological control has become an important breakthrough direction, especially in line with the requirements of green agriculture and sustainable ecological development.

[0004] Compared to chemical pesticides, biocontrol agents offer multiple advantages. First, they are more environmentally compatible, as their active ingredients originate from natural microorganisms or metabolites, are easily degraded, and leave no residue, avoiding soil pollution and ecological damage. Second, their mechanisms of action are more comprehensive, often inhibiting pathogens through multiple pathways such as competition, antagonism, and parasitism, and their multi-target action significantly reduces the risk of pathogen resistance. Third, in addition to disease prevention, many biocontrol agents also promote growth, secreting growth hormones, activating soil nutrients, and enhancing root vitality, thereby improving the overall vigor and resilience of fruit trees, forming a virtuous cycle of "prevention promoting growth, and growth strengthening prevention." Furthermore, some agents can colonize the soil or plant body, exerting a sustained inhibitory effect on pathogens with a longer duration of effectiveness. These characteristics make biocontrol agents a key technology for promoting green pest control and sustainable production in fruit trees.

[0005] However, current biocontrol resources for apple tree canker remain limited and functionally singular. Few strains have been discovered that can effectively inhibit black rot fungi while also providing stable growth promotion, resulting in a scarcity of applicable biocontrol agents and inconsistent control efficacy. Existing biocontrol agents often suffer from weak colonization, poor environmental adaptability, and unstable efficacy in practical applications, making it difficult to completely replace chemical pesticides for large-scale field control. Therefore, screening and developing novel biocontrol strains that combine high antibacterial activity, significant growth promotion, and good environmental adaptability, along with the development of corresponding stable biocontrol agents, has become a critical technical challenge urgently needing breakthroughs in the field of microbial technology.

[0006] Therefore, existing technologies need further improvement. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a strain of Trichoderma harzianum T10, a biocontrol agent, and their applications. The Trichoderma harzianum T10 exhibits excellent antibacterial effects against apple tree rot pathogens and promotes the growth of fruit trees. It can be used as an active ingredient to prepare a biocontrol agent for the prevention and control of apple tree rot and ring rot, etc., and has the advantages of stable control effects and environmental friendliness.

[0008] To address the above problems, this application provides the following technical solution: Firstly, this application provides a strain of *Trichoderma harzianum*, which is named *Trichoderma harzianum* (…). Trichoderma afroharzianum T10, its accession number is CGMCC No.41709.

[0009] This strain was deposited on December 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41709. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] Secondly, this application also provides a biocontrol agent, the active ingredient of which is the aforementioned Trichoderma harzianum; the Trichoderma harzianum is a bacterial culture, fermentation filtrate or spore suspension of Trichoderma harzianum T10.

[0011] Optionally, the biocontrol agent may also include other biocontrol bacteria used in combination.

[0012] Thirdly, this application also provides a method for preparing the above-mentioned biocontrol agent, which includes the following steps: S1. The spores produced by the growth of Trichoderma harzianum colonies were separated by washing with sterile water to obtain the fermentation seed liquid of Trichoderma harzianum T10. Preferably, the concentration of the yeast seed liquid is 10. 7 cfu·mL 1 .

[0013] S2. Inoculate the fermentation seed liquid into the liquid culture medium for expansion culture, centrifuge and take the supernatant to obtain the fermentation broth of Trichoderma harzianum T10, which is a biocontrol agent. Alternatively, the aforementioned fermentation seed liquid can be inoculated into a solid fermentation medium for large-scale culture, the solid fermentation product can be washed with sterile water, and the spore suspension of Trichoderma harzianum T10 can be obtained by filtration, thus obtaining another biocontrol agent.

[0014] Optionally, the T10 colony is cultured as follows: *Trichoderma harzianum* T10 is inoculated into a solid culture medium and cultured to obtain activated fungal colonies. The culture conditions are: activation culture at 25℃ for 2 days.

[0015] Preferably, in step S2, the liquid culture medium is PDB, the culture temperature is 25°C, and the rotation speed is 160 rpm / min.

[0016] Preferably, in step S2, the solid fermentation medium is wheat bran and corn flour medium, the culture temperature is 23°C, and the inoculum size is 5%.

[0017] Preferably, the solid culture medium is PDA culture medium, the liquid culture medium is PDB culture medium, and the solid fermentation culture medium is wheat bran and corn flour culture medium.

[0018] Fourthly, this application also provides the application of the aforementioned Trichoderma harzianum or biocontrol agents as described above in the control of plant diseases.

[0019] Optionally, the application method is to spray the fruit tree branches with the fermentation liquid or spore suspension of Trichoderma harzianum T10 or the biocontrol agent, or to perform root irrigation on the fruit tree.

[0020] Experiments have shown that spraying with the Trichoderma harzianum T10 suspension of this invention can effectively inhibit lesions caused by apple tree canker, thus effectively controlling the disease. Therefore, Trichoderma harzianum T10 can be used to prepare biocontrol agents.

[0021] Optionally, in the application, the plant diseases include: apple and pear ring rot fungus (… Botryosphaeria dothidea ), pear tree rot pathogen ( Cytospora mali Apple tree rot pathogen ( Valsa mali Apple fruit spot disease ( ) Alternaria sp.、 Fusarium sp. Acremonium sclerotigenum ), apple core pathogen ( Trichothecium roseum ), Peach brown rot fungus ( Monilinia fructicola Apple anthracnose leaf blight fungus ( Glomerella cingulata Apple anthracnose bacteria ( Colletotrichum gloeosporioides Grape white rot fungus ( Coniella diplodiwlla ), gray mold of fruits and vegetables ( Botrytis cinerea Experiments have shown that Bacterial T10 has a good antibacterial effect against the above-mentioned pathogens.

[0022] Fifthly, this application also provides the application of the aforementioned Trichoderma harzianum or the aforementioned biocontrol agent in inhibiting the mycelial growth and spore germination of pathogens causing fruit tree diseases.

[0023] Sixthly, this application also provides the application of the aforementioned Trichoderma harzianum in promoting fruit tree growth.

[0024] Optionally, the fruit trees are apple trees, pear trees, peach trees, and grapevines.

[0025] Seventhly, this application provides a plant growth promoter, the active ingredient of which is the aforementioned Trichoderma harzianum.

[0026] Preferably, the plant growth promoter is made directly from the aforementioned Trichoderma harzianum T10 spore suspension or by adding excipients and auxiliaries to form different formulations.

[0027] Eighthly, this application also provides the application of the aforementioned *Trichoderma harzianum* or the aforementioned biocontrol agent in the degradation of putrefactive virin, wherein the putrefactive virin is p-hydroxybenzoic acid, trihydroxybenzoic acid, or protocatechuic acid. (Wang et al., 2014; Cui et al., 2025) Experiments showed that after 7 days of treatment, *Trichoderma harzianum* T10 exhibited a 96.6% degradation rate for p-hydroxybenzoic acid, a 90.3% degradation rate for trihydroxybenzoic acid, and an 86.3% degradation rate for protocatechuic acid. This demonstrates that *Trichoderma harzianum* T10 has a significant degradation effect on the main components of rot virins, mitigating the toxicity of these toxins to fruit trees.

[0028] The present invention has the following beneficial effects: 1. This invention provides a self-isolated Trichoderma harzianum strain ( Trichoderma afroharzianum T10, this bacterium has excellent antibacterial effect against apple tree rot pathogens, which can significantly inhibit the growth of apple tree rot mycelium and the germination of spores, and has a growth-promoting effect on fruit trees. It can be used as an active ingredient to prepare biocontrol agents and applied to the prevention and control of apple tree rot and ring rot, as well as to promote growth. It has the advantages of stable control effect and environmental friendliness.

[0029] 2. The spore suspension of strain T10 provided by this invention has excellent control effect on apple tree rot disease, with a control effect of over 70%; and strain T10 can induce an increase in the activity of defense-related enzymes in plant tissues and improve the expression level of disease resistance genes in plants; it has a growth-promoting effect on apple seedlings and can promote an increase in plant height, weight and chlorophyll content.

[0030] 3. The biocontrol agent of this invention has the characteristics of stable control effect and environmental friendliness, making it suitable for large-scale production. The fermentation process is simple, the production cost is low, and it has a promising market prospect. It not only helps reduce dependence on chemical pesticides and lower environmental pollution, but will also provide important support for the green transformation and high-quality development of the apple industry by strengthening tree vigor and improving fruit quality. Attached Figure Description

[0031] Figure 1 A represents the colony morphology of strain T10 on PDA medium; Figure 1 B represents the morphology of the conidiophores of strain T10; Figure 1 C represents the spore morphology of strain T10; Figure 1 D is the phylogenetic tree of the ITS gene of strain T10; Figure 2 The results of the confrontation test between strain T10 and the putrefactive bacteria; Figure 3 The control effect of strain T10 on apple tree canker; Figure 4 The degradation ability of strain T10 against three toxins of apple tree rot disease; Figure 5 The colonization effect of strain T10 on detached healthy and diseased branches; Figure 6 The colonization effect of strain T10 on branches with different disease severity; Figure 7 Figure 1 shows the effect of temperature on T10; Figure 2 shows the effect of temperature on the colony diameter of strain T10; Figure 3 shows the effect of temperature on the sporulation of strain T10; Figure 4 shows the colonization effect of strain T10 at different temperatures. Figure 8 The colonization effect of strain T10 under different humidity conditions; Figure 9 The effects of seven commonly used pesticides on strain T10 are shown in Figure A; the effects of seven commonly used pesticides on the colony morphology of strain T10 are shown in Figure B; the effects of seven commonly used pesticides on the spore production of strain T10 are shown in Figure C; and the effects of seven commonly used pesticides on the biomass of strain T10 are shown in Figure C. Figure 10The effects of fermentation broth of strain T10 on the growth of *Pseudomonas aeruginosa* are shown in Figure 1; A shows the effect of fermentation broth of strain T10 on the growth of *Pseudomonas aeruginosa*; B shows the effect of fermentation broth of strain T10 on the mycelial radius of *Pseudomonas aeruginosa*; C shows the effect of fermentation broth of strain T10 on the germination rate of conidia of *Pseudomonas aeruginosa*. Figure 11 A represents the growth-promoting effect of strain T10 treatment on apple seedlings; B represents the growth-promoting effect of strain T10 treatment on apple seedlings; C represents the effect of strain T10 treatment on the height, fresh weight, dry weight, and chlorophyll content of apple seedlings. Figure 12 The effect of strain T10 treatment on the activity of defense-related enzymes in apple leaf tissues; Figure 13 The effect of strain T10 treatment on the content of relevant defense substances in apple leaf tissues; Figure 14 The effect of treating apple seedlings with strain T10 on the expression of disease-related protein genes in apple leaves. Detailed Implementation

[0032] 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. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0033] The culture medium and its formula used in the experiment 1. PDA culture medium: After peeling the potatoes, weigh out 200g, cut them into small pieces, boil them in water for 15-20 minutes, filter them through four layers of gauze, add 20g of glucose and 15g of agar powder, bring the volume to 1000mL, set the pH to natural, and autoclave at 121℃ for 20 minutes.

[0034] 2. Solid fermentation culture medium Use a 1L glass bottle as the container, with a substrate thickness of 2cm. The substrate should consist of wheat bran and cornmeal in a 4:1 ratio. The moisture content of the culture medium should be 40%, and the temperature should be 23℃. The inoculum size should be 5%. Add 2% sucrose and 2% ammonium nitrate.

[0035] 3. Trichoderma selective culture medium 0.9g K₂HPO₄, 0.2g MgSO₄·7H₂O, 1.0g NH₄NO₃, 0.15g KCl, 3.0g glucose, 0.15g Bengal red, 15g agar, and 1L of Grade I water. After sterilization, add 0.25g chloramphenicol, 0.03g streptomycin, and 0.2g pentachloronitrobenzene.

[0036] 4. YEPD medium Formula: 10g peptone, 3g yeast extract, 20g glucose and 1L primary water, pH=6.7.

[0037] Example 1: Isolation and strain identification of Trichoderma harzianum T10 1. Isolation and screening of strains Apple branches infected with apple tree rot were collected from apple orchards. After surface disinfection, the branch tissue was ground and sterile water was added. The tissue was then isolated using a conventional gradient dilution spreader method and cultured on Trichoderma selective medium at 25°C. Colonies with significant morphological differences were selected, purified, and preserved on PDA medium. The fungal strain was then screened for antagonistic bacteria against the apple tree rot pathogen, and the strain was eventually identified as T10.

[0038] 2. Identification of strain T10 (1) Morphological identification A. Experimental Methods: The isolated strain was inoculated onto a PDA plate and cultured at 25°C. The morphology and color of the colonies formed were observed, as well as the morphology of the conidia and sporogenous cells produced by the strain.

[0039] B. Results and Analysis According to observations, such as Figure 1 As shown, the isolated strain forms white, circular colonies on PDA plates. With prolonged incubation, the colonies gradually turn green, indicating the formation of conidia (such as...). Figure 1 A), the sporogenous cells are flask-shaped (e.g., Figure 1 B), conidia are round (e.g. Figure 1 C); the morphological observation results indicate that this strain is a Trichoderma strain.

[0040] (2) Gene identification A. Experimental Methods: Genomic DNA was extracted from the hyphae and used as a template to amplify the strain's ITS using universal primers ITS1 and ITS4. The amplified fragments were sequenced and BLAST aligned, followed by MEGA(X) phylogenetic analysis.

[0041] The amplification primer sequences for ITS are as follows: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' ITS4: 5'-TCCTCCGCTTATTGATATGC-3' B. Results and Analysis: Sequencing results showed that the ITS extended fragment was 490 bp in length, and its sequence is shown in the sequence listing SEQ ID NO: . The obtained sequence was compared with nucleic acid sequences in GenBank using BLAST software in the NCBI database (http: / / www.ncbi.nlm.nih.gov) and compared with Trichoderma harzianum ( Trichoderma afroharzianum The ITS sequences of isolate XD19 and isolate ZJ11 (Accession: ON045490.1 and ON649772.1) showed 99.9% homology. Phylogenetic analysis showed that this strain is related to... Trichoderma afroharzianum Clustered on the same branch ( Figure 1 D).

[0042] Based on the aforementioned morphological characteristics, physiological and biochemical characteristics, and sequence analysis results, strain T10 was identified as *Trichoderma harzianum* (African Trichoderma). Trichoderma afroharzianum ).

[0043] Example 2: Effect of strain T10 on mycelial growth of apple tree rot pathogen A. Experimental Methods: The inhibitory effect of Trichoderma T10 on the growth of two pathogens was evaluated using the plate confrontation method. The specific procedure was as follows: 5mm rot disease and Trichoderma mycelial blocks, pre-cultured for 3 days, were inoculated at two points on a 9cm diameter PDA agar plate, with a straight-line distance of 6cm between the two points. The control group consisted of only pathogenic mycelial discs and an equal volume of PDA blank block inoculated at the corresponding positions on the same plate. All treatments were incubated at 25℃ in the dark, and colony expansion was observed. Data was measured and recorded when the pathogen colony radius exceeded half the plate radius. Each treatment was repeated in triplicate. The inhibition rate was calculated as: [(Control group colony radius - Treatment group colony radius) / Control group colony radius] × 100%.

[0044] B. Results and Analysis: The inhibitory effect of Trichoderma T10 on the growth of *Trichoderma* pathogens was determined through a confrontation experiment. When the colony radius of the control group of *Trichoderma* pathogens reached 6.4 cm, the colony radius of the experimental group was only 1.65 cm. The inhibition rate of Trichoderma T10 on the growth of *Trichoderma* pathogens reached over 70% (e.g., ...). Figure 2 The results of this experiment show that Trichoderma T10 has a significant growth-inhibiting effect on decay pathogens.

[0045] Example 3: Control effect of strain T10 on apple tree canker. A. Experimental Methods: The control efficacy of Trichoderma T10 against apple tree canker was determined using a wound-inoculation method. The specific method was as follows: Trichoderma harzianum strain T10 was inoculated onto PDA medium and cultured at 25°C for 7 days. The colonies were then washed with purified water, conidia were collected, and the culture temperature was adjusted to 10⁻⁶. 7 The concentration was determined using CFU / mL. One- to two-year-old lateral branches were selected, both ends were sealed with wax, rinsed twice with tap water, disinfected with 75% alcohol, and rinsed three times with sterile water. Holes were then punched into the branch surface using a hole puncher. Subsequently, 20 μL of the previously treated *Trichoderma* spore suspension was inoculated into the holes. Two days later, apple tree rot fungal cakes were inoculated into the holes. The branches were then stored and cultured in a humidified container. Branches inoculated with PDA fungal cakes served as controls, with three replicates per group. Disease incidence was observed and recorded after 5 days, and the length and width of lesions were measured. The lesion area and control effect were calculated.

[0046] Lesion area = 1 / 4 × π × length × width.

[0047] Control effect = (Lesion area of ​​control group - Lesion area of ​​treatment group) / Lesion area of ​​control group × 100%.

[0048] B. Results and Analysis: The results are as follows Figure 3 As shown, the T10 treatment group exhibited significant antibacterial activity. Against apple tree canker and ring rot, the treatment group showed significant control effects. At 5 days, the length of canker lesions was 3 cm, while the lesion length in the T10 treatment group was only 0.7 cm, significantly smaller than that in the control group. This confirms that Trichoderma T10 has good control efficacy against apple tree canker.

[0049] Example 4: Degradation ability of strain T10 against putrefactive virus A. Experimental Methods: Preparation of toxin solution: Accurately weigh 0.1 g (accurate to 0.0001 g) of the target toxin standard using a 0.0001 g analytical balance. After ultrasonic-assisted dissolution in ultrapure water, transfer the solution to a 100 mL Grade A volumetric flask using a quantitative transfer method, and dilute to the mark to prepare a 1000 mg / L standard stock solution. Dilute the stock solution to 100 mg / L using a serial dilution method. After drawing the solution with a 2 mL sterile syringe, filter it through a 0.45 μm microporous membrane, and collect the filtrate into a 1.5 mL sample vial as the sample to be tested. The target toxins used in this example are p-hydroxybenzoic acid (Shanghai Sangon Biotech, CAS: 99-96-7), trihydroxybenzoic acid (also known as gallic acid, Tianjin Kemeio Chemical Reagent Co., Ltd.), and protocatechuic acid (Hebei Bailingwei Ultrafine Materials Co., Ltd., CAS: 99-50-3).

[0050] Transfer 50 mL of the target toxin into 150 mL Erlenmeyer flasks, and inoculate each flask with 200 μL of a 1×10⁻⁶ toxin concentration. 6 A toxin-cell co-culture system was constructed using a CFU / mL Trichoderma spore suspension. The control group used an equal volume of sterile distilled water instead of the bacterial suspension. All treatment groups were simultaneously cultured in a 25℃ constant-temperature shaker (180 rpm), with samples taken at regular intervals on days 3 and 7. One mL of culture solution was filtered through a 0.45 μm microporous filter and collected in a 2 mL chromatographic vial for toxin quantification using high-performance liquid chromatography (HPLC). The degradation rate was calculated using the following formula: Degradation rate = (Concentration of Vm toxin culture medium in control group - Concentration of Vm toxin culture medium in degradation system) × 100% / Concentration of Vm toxin culture medium in control group B. Results and Analysis: Figure 4 The results showed that after 3 days of degradation of Vm toxin by *Trichoderma harzianum* T10, the content of p-hydroxybenzoic acid was 90.3 μg / mL, with a degradation rate of 82%; the content of trihydroxybenzoic acid was 72.3 μg / mL, with a degradation rate of 75.6%; and the content of protocatechuic acid was 73.08 μg / mL, with a degradation rate of 79.3%. After 7 days of degradation of Vm toxin by *Trichoderma harzianum* T10, the content of p-hydroxybenzoic acid was 3.6 μg / mL, with a degradation rate of 96.6%; the content of trihydroxybenzoic acid was 10.1 μg / mL, with a degradation rate of 90.3%; and the content of protocatechuic acid was 14.4 μg / mL, with a degradation rate of 86.3%. Therefore, *Trichoderma harzianum* T10 has a good degradation effect on the main toxins in rot virins, alleviating the toxicity of these toxins to fruit trees.

[0051] Example 5: Colonization effect of Trichoderma harzianum T10 on branches 5.1 Colonization effect of Trichoderma harzianum T10 on detached healthy and diseased branches A. Experimental Methods: To test the colonization effect of Trichoderma on detached healthy branches, healthy apple branches of uniform thickness were selected, cut to 5cm in size, and then rinsed three times with sterile water. After drying, the surface was disinfected by wiping with 75% alcohol and then dried for later use. Subsequently, a suspension of Trichoderma spores (10...) was... 7 Spray the inoculum (CFU / mL) onto the surface of the branches and place them in a humidification box for incubation. Use sterile water as a control, with three replicates per group. One week after inoculation, take samples: cut a 2cm² section of surface tissue from the branch surface, rinse with sterile water, and scrape off the green spores. Collect the spores into a 50 mL centrifuge tube and bring the volume to 10 mL to determine the spore concentration.

[0052] To test the colonization effect of Trichoderma on detached diseased branches, detached branches were first selected, both ends were sealed with wax, rinsed twice with tap water, disinfected with 75% alcohol, and rinsed three times with sterile water. Then, holes were punched in the surface of the branches, and *Trichoderma rot* and *Trichoderma ring rot* were inoculated at the punched sites. The branches were then placed in a humidity-controlled container for preservation and culture. After 5 days of disease development, the branches were removed, and a suspension of *Trichoderma* spores (10...) was collected. 7 The inoculum (CFU / mL) was sprayed onto the surface of the branches and placed in a humidity-controlled container for incubation. Three replicates were set up for each group, using sterile water and healthy branches as controls. One week after inoculation, samples were taken. A 2 cm² section of surface tissue was cut from the branch surface, rinsed with sterile water, and the green spores were scraped off. The collected spores were transferred to a 50 mL centrifuge tube and brought to a final volume of 10 mL to determine the spore concentration.

[0053] B. Results and Analysis: The colonization effect of Trichoderma T10 differs significantly between healthy and diseased branches in vitro. For example... Figure 5 As shown, when the number of spores on a healthy branch is 7.9 × 10⁻⁶ 6 At a concentration of CFU / mL, the spore concentration produced by colonization on diseased branches was 31.4 × 10⁻⁶. 6 CFU / mL. This shows that Trichoderma T10 can not only stably colonize the surface of fruit tree branches, but also has a better colonization effect on branches suffering from apple tree rot compared to healthy branches.

[0054] 5.2 Colonization effect of Trichoderma harzianum T10 on diseased branches under different disease severity A. Experimental Methods: Diseased detached apple branches were obtained using the method described in Example 5. One, three, five, and seven days after inoculation with the rot pathogen, a suspension of Trichoderma spores was sprayed onto the branch surface. The branches were then placed in a humidified culture box for further humidification, with three replicates per group. Samples were taken one week later to determine the spore concentration on the branch surface.

[0055] B. Results and Analysis: like Figure 6 As shown, Trichoderma T10 colonizes diseased branches better than healthy branches, and the colonization ability of Trichoderma T10 increases with the severity of the disease.

[0056] Example 6: Effects of different temperatures, humidity levels, and pesticides on Trichoderma harzianum T10 6.1 Effects of different temperatures on Trichoderma harzianum T10 (1) Experimental determination on a plate at different temperatures A. Experimental Methods: Collect mycelial fragments (5 mm in diameter) from the edge of Trichoderma colonies and inoculate them into the center of new PDA plates. Place the inoculated plates in constant temperature incubators at 10, 15, 20, 25, 30, 35, and 40°C, respectively, and incubate in the dark, with three replicates for each temperature group. Colony diameters are then measured every 24 hours (average value using the cross-hatching method) for 7 days. After 7 days of incubation, rinse the plate surface with 10 mL of sterile water to collect the spore suspension. After dilution, determine the spore concentration (CFU / mL) using a hemocytometer, and take the average of three replicates.

[0057] B. Results and Analysis: like Figure 7 As shown, temperature significantly affects the colony growth, development, and sporulation capacity of *Trichoderma* T10. Regarding colony growth, *Trichoderma* T10 exhibits the fastest growth rate at 25℃ and 30℃, followed by growth rates at 20℃ and 15℃. When the temperature drops to 10℃, colony expansion is significantly affected; when the temperature rises above 35℃, colony expansion is significantly impacted, almost ceasing growth. In terms of sporulation, the spore concentration reaches its peak at 25℃ (1.4 × 10⁻⁶). 7 (CFU / mL) Sporulation significantly decreased when the temperature increased to 35℃ or decreased to 15℃. Both charts together reveal that 20-35℃ is the optimal temperature range for the synergistic optimization of Trichoderma T10 growth and sporulation.

[0058] (2) Experimental determination on detached branches at different temperatures A. Experimental Methods: Select healthy apple branches of uniform thickness, cut them to 5cm in size, then rinse the surface three times with sterile water, let them dry, and then disinfect them by wiping the surface with 75% alcohol. Let them dry again before use. Then, prepare a suspension of Trichoderma spores (10... 7 The solution (CFU / mL) was sprayed onto the surface of the branches, and then the humidity chambers were placed in incubators at 15, 20, 25, 30, 35, and 40°C for cultivation. Sterile water spraying served as a control, and each group was replicated three times. One week after inoculation, samples were taken. A 2cm² section of surface tissue was cut from the branch surface, rinsed with sterile water, and the green spores were scraped off. The collected spores were transferred to 50 mL centrifuge tubes, and the volume was adjusted to 10 mL to determine the spore concentration.

[0059] B. Results and Analysis: like Figure 7 As shown, the temperature experiment results differ slightly from those obtained with PDA plates. The spore concentration of *Trichoderma* on the branch surface showed a positive correlation with temperature; as the ambient temperature increased, its colonization level also increased. These results indicate that *Trichoderma* T10 is thermophilic, and its colonization ability increases with increasing temperature.

[0060] 6.2 Effects of different humidity levels on Trichoderma harzianum T10 A. Experimental Methods: Select healthy apple branches of uniform thickness, cut them to 5cm in size, then rinse the surface three times with sterile water, let them dry, and then disinfect them by wiping the surface with 75% alcohol. Let them dry again before use. Then, prepare a suspension of Trichoderma spores (10... 7 Spraying (CFU / mL) onto the surface of the branches was performed, and humidification boxes with relative humidity of 100%RH, 95%RH, 85%RH, and 75%RH were prepared using the saturated salt solution method. The branches were then placed in the humidification boxes and incubated at 25℃. Samples were taken after 7 days to determine the spore concentration.

[0061] B. Results and Analysis: The experimental results show that Trichoderma T10 can colonize under humidity conditions of 75% RH and above, and the colonization effect is optimal at higher humidity. Figure 8 ).

[0062] 6.3 Effects of different pesticides on Trichoderma harzianum T10 (1) Effects of pesticides on Trichoderma colony morphology and growth rate A. Experimental Methods: Seven pesticides (pyraclostrobin, carbendazim, mancozeb, anthraquinone, Bordeaux mixture, fluopyram, and difenoconazole) were added quantitatively to melted PDA medium at the highest field-recommended concentration. The mixture was shaken well and poured into Petri dishes to cool and solidify. Trichoderma mycelial blocks were cut using a sterile 5mm punch and vertically inoculated into the center of the pesticide-containing medium. PDA medium without pesticides was used as a blank control. Each experiment was performed in triplicate. The Petri dishes were incubated at 25°C. Colony growth characteristics and spore production were recorded after 5 days of inoculation.

[0063] B. Results and Analysis: like Figure 9 As shown in Figures A and 9B, several commonly used field fungicides all have some effect on Trichoderma T10. Carbendazim, mancozeb, and Bordeaux mixture have the greatest impact, almost completely inhibiting the growth of the strain. Difenoconazole and anthraquinone show significant differences in morphological characteristics and spore production between Trichoderma T10 and normally growing Trichoderma T10. Fluopyram has a relatively small effect on Trichoderma T10, showing good growth rate and morphological characteristics.

[0064] (2) Effects of pesticides on Trichoderma mycelium biomass A. Experimental Methods: 1000 ml of LPB liquid culture medium was dispensed into 1 L Erlenmeyer flasks, and seven test pesticides (pyraclostrobin, carbendazim, mancozeb, anthraquinone, Bordeaux mixture, fluopyram, and difenoconazole) were added to achieve the highest recommended field concentration. Trichoderma mycelial agar blocks with a diameter of 5 mm were prepared using a sterile punch and vertically inoculated into the culture medium of each treatment group. A blank control group without pesticide addition was also included. Each treatment group was subjected to three biological replicates. The inoculated culture system was placed in a constant temperature shaking incubator at 25℃ (180 r / min) for 10 days. After incubation, the mycelium was collected by filtration through four layers of filter paper, rinsed three times with distilled water, and then transferred to a constant temperature drying oven for dehydration for 12 hours. The final constant weight of the mycelium was determined.

[0065] B. Results and Analysis: like Figure 9 As shown in Figure C, after 10 days of fermentation in PDB medium containing seven pesticides, fluopyram and anthraquinone had relatively little effect on the biomass of *Trichoderma* T10, producing higher mycelial dry weight. Carbendazim and difenoconazole had a greater impact on *Trichoderma* T10, resulting in lower biomass. Pyraclostrobin, mancozeb, and Bordeaux mixture had the greatest impact, producing the lowest biomass.

[0066] Example 7: Effects of Trichoderma harzianum T10 fermentation broth on mycelial growth and spore germination of apple tree rot pathogen. (1) Effects of Trichoderma harzianum T10 fermentation broth on mycelial growth of apple tree rot fungus A. Experimental Methods: Mycelial blocks of *Trichoderma* strains were obtained using a sterile punch (5 mm diameter). Three mycelial blocks were inoculated into each 250 mL Erlenmeyer flask containing 100 mL of potato dextrose liquid (PDB). Three biological replicates were established for each strain. The flasks were sealed with breathable sealing film and placed in a 25°C constant-temperature shaker at 180 r / min for 7 days. After incubation, the fermentation product was first filtered through two layers of filter paper. Then, the fermentation broth was centrifuged at 8000 rpm for 20 minutes. The supernatant was collected and sterilely filtered through a 0.22 μm microporous membrane to obtain a *Trichoderma* fermentation filtrate free of mycelial cells.

[0067] Trichoderma T10 fermentation filtrate was thoroughly mixed with PDA medium cooled to approximately 50°C at different volume ratios (1:1, 1:2, 1:3, 1:4) and poured into Petri dishes. Subsequently, 5 mm diameter rot and ring spot fungal cakes were placed in the center of the plates. Each Trichoderma fermentation filtrate treatment group was replicated three times, with the group supplemented with PDB medium serving as a control. All Petri dishes were incubated at a constant temperature of 25°C. When the control group colonies were about to cover the entire plate, the colony diameter of each treatment group and the control group was measured using the cross-sectional method, and the inhibition rate was calculated according to the formula: Antibacterial rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100% B. Results and Analysis: like Figure 10 As shown in Figure A, compared with the treatment group without fermentation broth, the colony diameter of pathogens in the PDA medium containing fermentation broth was reduced to some extent. The inhibitory effect of the fermentation broth on pathogens increased with increasing concentration. When the colony radius of CK was 4 cm, the colony radius of rot pathogens in the PDA medium with a fermentation broth concentration of 30% was 0.3 cm, the colony radius in the 20% concentration treatment group was 0.93 cm, and the colony radius in the 10% concentration treatment group was 1.7 cm. The experimental results indicate that the fermentation filtrate of Trichoderma T10 has a strong inhibitory effect on the growth of apple tree rot.

[0068] (2) Effects of Trichoderma harzianum T10 fermentation broth on spore germination of apple tree rot pathogens A. Experimental Methods: The inhibitory effect of Trichoderma harzianum T10 fermentation filtrate on the germination of pathogenic fungal spores was evaluated using the slide method. The Trichoderma harzianum fermentation filtrate was mixed with a suspension of conidia of the decaying fungus (concentration 1×10⁻⁶). 5The spore suspension (CFU / mL) was prepared by mixing at a volume ratio of 1:9 and thoroughly shaking. 30 μL of the mixture was then added dropwise to the center of a clean glass slide. The slide was transferred to a petri dish lined with moistened filter paper and incubated at a constant temperature and humidity of 25°C. A mixture of equal volumes of sterile water and spore suspension served as a control group. After 12 hours of induction treatment in the incubator, the slide was removed and observed under an optical microscope to assess spore germination.

[0069] B. Results and Analysis: The results are as follows Figure 10 B and Figure 10 As shown in Figure C, the spore germination rate of the control group of *Trichoderma* T10 fermentation broth was 80.8%, while the spore germination rate of the treatment group was 36.0%. Compared with the control group, the fermentation broth treatment with *Trichoderma* T10 could inhibit the germination of conidia of *Trichoderma* to a certain extent, with an inhibition rate of 55.4%.

[0070] Example 8: Detection of the inhibitory rate of Trichoderma harzianum T10 fermentation broth against multiple plant pathogens. A. Experimental Methods: Fermentation broth of Trichoderma harzianum T10 was prepared, and PDA medium with a concentration of 20% was prepared. PDA without fermentation broth was used as a control. Then, activated and cultured pathogens were inoculated. The specific pathogens used in this example are shown in Table 1.

[0071] B. Results and Analysis: As shown in Table 1, the fermentation broth of Trichoderma harzianum T10 has a significant inhibitory effect on major plant pathogens such as apple tree rot fungus, apple ring rot fungus, pear tree rot, pear ring rot, apple bagged fruit spot disease, apple core rot, peach brown rot, and apple anthracnose leaf blight fungus, exhibiting good broad-spectrum antibacterial properties. Its inhibition rate is shown in Table 1.

[0072] Table 1. Antagonistic effect of Trichoderma harzianum T10 fermentation broth on pathogens causing fruit tree diseases.

[0073] Example 9: Effect of strain T10 treatment on apple growth promotion A. Experimental Methods: Apple seeds were sterilized and germinated, then potted in sterile soil. Experiments were conducted once seedlings emerged. The T10 strain was inoculated onto PDA medium and cultured at 25°C for 8 days. The colonies were then rinsed with purified water, conidia were collected, and the culture was adjusted to a concentration of 10... 7 After achieving a concentration of [number] spores / mL, the treatment group was irrigated with 50 mL of the conidial solution every week, while the control group was irrigated with an equal volume of purified water. After 4 weeks, the plant height, fresh weight, dry weight, chlorophyll content, and other indicators of the apple seedlings were measured.

[0074] Improvement rate (%) = (Treatment group colony diameter - Control group colony diameter) / Control group colony diameter × 100% B. Results and Analysis: The results are as follows Figure 11 As shown in Table 2, compared with the control group, the experimental group treated with T10 conidial solution showed improvements in plant height, fresh weight, dry weight, and chlorophyll content. For example, plant height increased by 46.14%, and the above-ground dry weight increased by 59.16%. These results demonstrate that strain T10 has a good growth-promoting effect on apple seedlings.

[0075] Table 2. Growth-promoting effect of Trichoderma harzianum T10 spores on apple seedlings.

[0076] Example 10: Effects of strain T10 on the activity of defense-related enzymes and the content of defense substances in apple leaf tissues after treatment of apple seedlings. 10.1 Effects of strain T10 on the activity of defense-related enzymes in apple leaf tissues after treatment of apple seedlings A. Experimental Methods: Fresh leaves from apple seedlings obtained in Experiment 9 were used to determine the activities of defense-related enzymes in apple leaf tissue. The content and activity of POD (peroxidase) were detected using kit BC0090; the activity of SOD (superoxide dismutase) was determined using kit BC5165. Both kits were purchased from Beijing Solarbio Science & Technology Co., Ltd. B. Results and Analysis: Experimental results are as follows Figure 12 As shown, the activities of POD (peroxidase) and SOD (superoxide dismutase) in the leaf tissues of apple seedlings treated with spore suspension were increased to varying degrees compared with the control. This result indicates that *Trichoderma harzianum* T10 can induce host resistance to diseases by increasing the activity of defense-related enzymes in the leaves.

[0077] 10.2 Effects of strain T10 treatment on the content of relevant defense substances in apple leaf tissues of apple seedlings A. Experimental Methods: Fresh leaves from apple seedlings obtained in Experiment 9 were used to determine the content of defense-related substances in apple tissue. Lignin content was determined using kit BC4200, and total phenol content was determined using kit BC1340. Both kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0078] B. Results and Analysis: Experimental results are as follows Figure 13As shown, the lignin and total phenol contents in the leaf tissue of apple seedlings treated with spore suspension were increased to varying degrees compared with the control. This indicates that Trichoderma harzianum T10 can improve the host's resistance to diseases by increasing the content of related active substances in the leaves.

[0079] Example 11: Effect of strain T10 on the expression of disease-related protein genes in apple leaves after treatment of apple seedlings A. Experimental Methods: Fresh leaves from apple seedlings obtained in Experiment 9 were used to determine the expression of disease resistance-related protein genes in apple leaves. Total RNA was extracted from the leaves, reverse transcribed, and the expression of disease resistance-related protein genes in apple leaves was determined using RT-qPCR technology.

[0080] B. Results and Analysis: The results are as follows Figure 14 As shown, after apple seedlings were treated with T10 spore suspension, the expression levels of disease resistance-related protein genes MdPR1, MdPR2, MdPR5, and MdPR10 in their leaf tissues were significantly increased compared to the control. This indicates that Trichoderma harzianum T10 can induce the upregulation of disease-related protein genes in apple tissues, thereby improving the host's resistance to the disease.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

[0082] References Wang C, Li C, Li B, et al. Toxins Produced by Valsa mali var. mali andTheir Relationship with Pathogenicity [J]. Toxins, 2014, 6(3): 1139–1154. Cui X ,Wei L ,Zhang D , et al.Quercetin 2,3-dioxygenase genes VmQDO1and VmQDO2 of Valsa mali utilizes flavonoid to produce two key toxins forpathogenesis.[J]. International journal of biological macromolecules, 2025,337(P1): 149331.

Claims

1. A strain of Trichoderma harzianum ( Trichoderma afroharzianum T10, characterized in that, Its accession number is CGMCC No.41709.

2. A biocontrol agent, characterized in that, The active ingredient is Trichoderma harzianum as described in claim 1.

3. The method for preparing the biocontrol agent as described in claim 2, characterized in that, Includes the following steps: S1. The spores of Trichoderma harzianum were separated by washing with sterile water to obtain the fermentation seed liquid of Trichoderma harzianum T10. S2. Inoculate the fermentation seed liquid into the liquid culture medium for expansion culture, centrifuge and take the supernatant to obtain the fermentation broth of Trichoderma harzianum T10, which is a biocontrol agent. Alternatively, the aforementioned fermentation seed liquid can be inoculated into a solid fermentation medium for large-scale culture, the solid fermentation product can be washed with sterile water, and the spore suspension of Trichoderma harzianum T10 can be obtained by filtration, thus obtaining another biocontrol agent.

4. The application of *Trichoderma harzianum* as described in claim 1 or the biocontrol agent as described in claim 2 in the control of plant diseases, characterized in that... The application method is as follows: spray the apple tree branches with the fermentation liquid or spore suspension of Trichoderma harzianum T10 or the biocontrol agent, or drench the apple trees with the root drench.

5. The application according to claim 4, characterized in that, The plant diseases mentioned include: apple tree rot and apple ring rot.

6. The application of Trichoderma harzianum as described in claim 1 or the biocontrol agent as described in claim 2 in inhibiting mycelial growth and spore germination of pathogens causing apple tree diseases.

7. The application of Trichoderma harzianum as described in claim 1 in promoting apple tree growth.

8. A growth promoter for apple trees, characterized in that, The active ingredient includes Trichoderma harzianum as described in claim 1.

9. The application of Trichoderma harzianum as described in claim 1 or the biocontrol agent as described in claim 2 in the degradation of putrefactive virin, wherein the putrefactive virin is p-hydroxybenzoic acid, trihydroxybenzoic acid or protocatechuic acid.

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