Trichoderma reesei 5146-6, its inoculant and its application in the preparation of biocontrol agents.

Abstract

This invention belongs to the field of active microbial technology, specifically relating to a type of Trichoderma reesei ( Trichoderma reesei 5146-6. Inoculants and their application in the preparation of biocontrol agents. This invention provides a *Trichoderma reesei* ( Trichoderma reesei The strain 5146-6 has been verified to significantly inhibit the growth of plant pathogens such as Rhizopus in the environment, and its metabolites can inhibit a variety of clinical pathogens, which holds promise for further screening of broad-spectrum antibacterial active ingredients. In addition, the above-mentioned strain also has multiple extracellular enzyme secretion activities, including amylase and catalase, which is the first report in this field.

Description

Technical Field

[0001] This invention belongs to the field of active microbial technology, specifically relating to a type of Trichoderma reesei ( Trichoderma reesei )5146-6, its inoculants and their application in the preparation of biocontrol agents. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Litmus lichens, belonging to the genus Litmus, are branching lichens of the family Litmus, also known as Litmus tree lichens or stone flowers. Litmus lichens form a perfect symbiotic relationship between fungi and algae. The fungi provide shelter and water for the algae, while the algae provide nutrients for the fungi through photosynthesis. This mutually beneficial symbiotic relationship allows them to survive and reproduce together even in harsh environments, and also provides a unique micro-ecological environment for endophytic fungi.

[0004] Endophytic fungi are fungi that live within biological tissues and form a symbiotic relationship with the organism. Over a long evolutionary process, these fungi and their hosts have developed various mechanisms that promote the growth and metabolism of the host. Currently, the following biological activities of endophytic fungi have been confirmed: (1) Promoting plant growth: Endophytic fungi can produce plant hormones, such as indoleacetic acid (IAA), which promote plant growth (Li et al., 2025)(Nischitha, 2024) (Shah et al., 2018); (2) Enhancing stress resistance: Endophytic fungi can improve the plant's resistance to biotic and abiotic stresses, such as salt stress and drought stress (Li et al., 2025)(Nurrahma et al., 2024). They enhance plant adaptability by regulating the physiological and biochemical processes of plants, increasing the activity of antioxidant enzymes, and maintaining ion balance (Nurrahma et al., 2024); (3) Biocontrol: Endophytic fungi inhibit the growth of plant pathogens through competition, antagonism, and induction of systemic resistance (Sujoy, 2024)(Nasehi et al., 2023). For example, some endophytic fungi can produce secondary metabolites such as antibiotics and lysozymes, which directly inhibit pathogens (Wang et al., 2022)(Bogas et al., 2024)(Nasehi et al., 2023).

[0005] In addition, secondary metabolites of endophytic fungi have been shown to possess various biological activities, such as anticancer, antibacterial, antirheumatic, and insecticidal properties. Among them, Trichoderma ( Trichoderma) is a common type of endophytic fungus that has been extensively studied and has been shown to have significant potential in inhibiting plant pathogens and pests (Natsiopoulos et al., 2024). It can induce plant disease resistance through multiple pathways and has better sustainability and environmental friendliness compared to chemical biological control agents. Summary of the Invention

[0006] This invention provides the following technical solution:

[0007] Firstly, a *Trichoderma reesei* ( Trichoderma reesei Strain 5146-6 was deposited on July 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 42139.

[0008] The strain was isolated from litmus lichens, and its morphology met the characteristics of Trichoderma fungi. Gene sequencing comparison showed 100% identity with known Trichoderma fungi, thus identifying it as a Trichoderma species and naming it *Trichoderma reesei*. Trichoderma reesei 5146-6, abbreviated as 5146-6 Trichoderma reesei 5146-6, or strain 5146-6.

[0009] The above-mentioned Trichoderma reesei ( Trichoderma reesei )5146-6 has the ITS rDNA sequence shown in SEQ ID NO:1.

[0010] In a second aspect, the present invention provides a fungal agent comprising the *Trichoderma reesei* described in the first aspect. Trichoderma reesei ) 5146-6 and / or bacterial metabolites.

[0011] The above-mentioned microbial agents contain strains and / or fungal metabolites, wherein the strains include living or dead fungi, the metabolites represent the products of the strain's metabolism, the metabolic pathways include catabolic and anabololic metabolism, and the products include primary and secondary metabolites.

[0012] The above-mentioned microbial agents can be formulated in various forms, such as aqueous solutions, wettable powders, water-dispersible granules, suspensions, granules, oils, or capsules.

[0013] Applicable targets include insects, crops, soil, seeds, and environmental pest control.

[0014] Feasible application methods include spreading, spraying, coating, and landfilling.

[0015] In a third aspect, the present invention provides the application of the strain described in the first aspect and the microbial agent described in the second aspect in any of the following aspects:

[0016] (1) Application in the preparation of biocontrol agents;

[0017] (2) Applications in the food and feed processing industries;

[0018] (3) Applications in the preparation of pharmaceuticals or medical devices.

[0019] Examples of feasible biocontrol agents described in (1) above include pesticides, fertilizers, herbicides, insecticides, seed coating agents, disinfectants, baits, food preservatives, or antibacterial drugs.

[0020] According to the verification of the present invention, the above-mentioned strains can effectively inhibit Fusarium graminearum, Rhizopus, Alternaria, Blastomyces orientalis, and Botrytis cinerea in the living environment, especially showing outstanding inhibitory effect on Rhizopus. It is known in the art that Rhizopus is an opportunistic pathogen that can cause food spoilage. Therefore, in some embodiments of the present invention, the above-mentioned strains or agents are used to prepare food preservatives, which can effectively inhibit Rhizopus in the living environment and prevent food spoilage.

[0021] In addition, the metabolites of the above-mentioned strains also have broad-spectrum antibacterial effects, which can effectively inhibit common clinical pathogens such as Enterococcus faecalis, Staphylococcus aureus, Bacillus subtilis and Candida albicans. Therefore, in some embodiments, the metabolites or bacterial agents of the above-mentioned strains are used to prepare antibacterial drugs or disinfectants for the prevention, improvement or treatment of related diseases caused by harmful microorganisms, or for the elimination of pathogens in the environment.

[0022] The strains also possess extracellular secretory amylase and catalase activity, which can be used in bread baking, beer brewing, starch sugar production, decomposition of feed, and processing of starch in agricultural waste such as straw, based on the characteristic of amylase to break down starch into sugars, for use in food and feed processing.

[0023] It can also be used to improve digestive problems caused by insufficient amylase, and further, to prepare therapeutic drugs for digestive disorders of starchy foods; or, to remove hydrogen peroxide residues in medical devices and cosmetics, etc.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] In view of the above-mentioned research status, this invention provides an endophytic fungus of the genus *Trichoderma* from litmus lichens, named *Trichoderma reesei*. Trichoderma reeseiThis strain, 5146-6, significantly inhibits the growth of plant pathogens. Its metabolites exhibit broad-spectrum antibacterial activity, showing inhibitory effects against a variety of clinically pathogenic bacteria. More notably, this strain possesses multiple extracellular enzyme secretion activities, including amylase and catalase. Current research has not reported any endophytic fungi of the genus *Trichoderma* producing amylase and catalase. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 This is the maximum likelihood phylogenetic tree constructed based on ITS for the isolation and identification of strains in Example 1.

[0028] Figure 2 This is the neighbor-joining phylogenetic tree constructed based on ITS in the strain isolation and identification in Example 1.

[0029] Figure 3 Trichoderma reesei described in Example 1 ( Trichoderma reesei Morphology of 5146-6;

[0030] in, Figure 3 A in the middle is a lichen from the genus Litmus; Figure 3 B in the middle is Trichoderma reesei ( Trichoderma reesei Frontal view of 5146-6 grown on a PDA tablet after 5 days; Figure 3 The image in the middle (C) is the reverse side of the photo; Figure 3 D in the middle is Trichoderma reesei ( Trichoderma reesei Photograph of mycelium 5146-6; Figure 3 E in the middle is Trichoderma reesei ( Trichoderma reesei Photograph of spores from 5146-6.

[0031] Figure 4 Trichoderma reesei described in Example 1 ( Trichoderma reesei The results of extracellular catalase activity assay for 5146-6; among which, Figure 4 In the middle A, Trichoderma reesei was not inoculated on the PDA plate. Trichoderma reesei ) 5146-6, but a negative control was added with hydrogen peroxide solution; Figure 4 B represents PDA plates inoculated with Trichoderma reesei ( Trichoderma reesei ) 5146-6, and the experimental group that added hydrogen peroxide solution.

[0032] Figure 5 Trichoderma reesei described in Example 1 ( Trichoderma reeseiThe results of extracellular amylase activity detection in 5146-6; among which, Figure 5 In the middle, A represents Trichoderma harzianum that was not inoculated on the PDA plate. Trichoderma reesei ) 5146-6, but a negative control with added iodine solution; Figure 5 B represents PDA plates inoculated with Trichoderma reesei ( Trichoderma reesei )5146-6, and the experimental group containing iodine solution; Figure 5 C represents Trichoderma harzianum that was not inoculated on the PDA plate. Trichoderma reesei )5146-6, but a negative control with iodine solution added to the well; Figure 5 D represents Trichoderma reesei inoculated on a PDA plate. Trichoderma reesei ) 5146-6, and the experimental group with iodine solution added to the well.

[0033] Figure 6 Trichoderma reesei described in Example 2 Trichoderma reesei Results of the confrontation experiment between 5146-6 and plant pathogens; among which, Figure 6 Plate A represents the control group, a Fusarium graminearum growth plate. Figure 6 B in the middle is Trichoderma reesei ( Trichoderma reesei ) 5146-6 confrontation plate with Fusarium graminearum; Figure 6 C represents the control group, a plate containing Rhizopus growth. Figure 6 D in the middle is Trichoderma reesei ( Trichoderma reesei ) 5146-6 confrontation plate with Rhizopus; Figure 6 E is the control group Alternaria growth plate; Figure 6 F is Trichoderma reesei ( Trichoderma reesei ) 5146-6 confrontation plate with Alternaria spp.; Figure 6 G represents the control group's rice blast fungus growth plate; Figure 6 H in the middle represents Trichoderma reesei ( Trichoderma reesei ) 5146-6 confrontation plate with rice blast fungus; Figure 6 Plate I represents the control group, a plate on which Botrytis cinerea grows. Figure 6 J represents Trichoderma reesei ( Trichoderma reesei ) 5146-6 Confrontation plate with Botrytis cinerea.

[0034] Figure 7 The strain Trichoderma reesei from Example 3 ( Trichoderma reesei The metabolites of 5146-6 inhibited clinically pathogenic bacteria; among them, Figure 7 In section A, the result shows the inhibition of Candida albicans CMCC98001 by the metabolite; in section B, the result shows the inhibition of Enterococcus faecalis ZDZA0109 by the metabolite. Figure 7In the middle, C represents the result of the metabolite inhibiting Staphylococcus aureus CMCC26003; Figure 7 D represents the result of the metabolite inhibiting Bacillus subtilis CMCC63501.

[0035] Strain preservation information:

[0036] The fungus is named Trichoderma reesei. Trichoderma reesei ) 5146-6, this strain was deposited on July 25, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The registration number of the collection center is CGMCC No. 42139. Detailed Implementation

[0037] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0039] In the context of this specification, the word "comprising" is considered to mean "especially including". It should not be interpreted as "consisting of only".

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0041] Example 1

[0042] In this embodiment, a strain of Trichoderma reesei is provided. Trichoderma reesei ) 5146-6, this strain was isolated from lichens of the genus Litmus. The isolation and identification process of this strain is as follows:

[0043] 1. Strains Isolation

[0044] Tissue blocks from different parts of Litsea lichens were collected from Lishui City, Jingning County, Wangdongyang Nature Reserve, and Baiyun Protection Station in Zhejiang Province. After disinfection, they were inoculated into PDA medium and cultured for 5-7 days until colonies grew around the tissue blocks. Mycelial cakes (5.3 mm in diameter) from the edge of the colonies were picked for isolation, purification, and preservation.

[0045] 2. Strain identification

[0046] (1) Molecular identification of strains

[0047] Genomic DNA was extracted from the above-mentioned strains according to the instructions of the bacterial genomic DNA rapid extraction kit. Using the extracted DNA as a template, PCR amplification and sequencing were performed using ITS universal primers. Geneious v.9.0.2 was used for sequence splicing and deletion of disordered sequences. The spliced ​​sequences were then compared with Blast. This strain is similar to *Trichoderma reesei* (…). Trichoderma reesei The sequences exhibited high similarity. Reference sequences with a similarity ≥98% were selected, aligned using MAFFT v.7, and pruned using Phylogeny.fr to ensure that only evolutionarily homologous sites were compared. A phylogenetic tree was constructed using MEGA X with maximum likelihood (ML) and neighbor-joining (NJ) methods, and 1000 bootstrap replication tests were performed on each node. Peziza griseorosea As an outgroup. Bootstrapping values ​​(BS) greater than or equal to 70% were considered significantly supported. ML and NJ phylogenetic trees showed that this fungus is related to *Trichoderma reesei* (…). Trichoderma reesei They converged into one group, with support rates of 95% and 99% respectively.

[0048] (2) Colony morphology and physiological and biochemical identification

[0049] Gram staining was performed on the isolated and preserved strain 5146-6 to observe the colony staining. The colony characteristics are as follows: the colonies are cottony, initially white and dense, later developing green spore clusters at the edges; the colonies appear yellow on the dorsal side of the culture medium. The hyphae are aseptate, white, and highly branched, with the mycelia intertwining to form hyphal clusters. The spores are usually elliptical or oval and often clustered together.

[0050] Physiological and biochemical characteristics were identified in accordance with the "Manual of Systematic Identification of Common Bacteria". The results are as follows: the optimal growth temperature is 24–30℃, the optimal pH is 4.5–5.5, it is strictly aerobic, uses sugars such as glucose as carbon source, and can produce catalase and amylase in vitro.

[0051] (3) Identification of extracellular enzyme activity

[0052] Trichoderma reesei was inoculated onto PDA medium. Trichoderma reesei5146-6 mycelial cakes (5.3 mm in diameter) were cultured for 3-5 days. Hydrogen peroxide was added to the surface of the culture medium, and the secretion of catalase was detected based on the generated bubbles. The test results are as follows. Figure 4 Figure B shows that the culture medium after inoculation with the above strains contained dense small air bubbles, proving that the above strains can secrete catalase.

[0053] Trichoderma reesei was inoculated onto a starch-containing PDA medium. Trichoderma reesei 5146-6 mycelial cakes (5.3 mm in diameter) were cultured for 3-5 days. Iodine solution was dropped onto the surface of the culture medium, and the ability of the strain to produce extracellular amylase was determined by whether the iodine solution turned blue. The test results are as follows: Figure 5 As shown in Figure B, the iodine solution on the culture medium inoculated with the above-mentioned strain did not turn blue, indicating that the strain produced extracellular amylase to degrade the starch in the culture medium, thus preventing the iodine solution from turning blue. Because... Figure 5 The B sample showed that the mycelial surface of this strain has a certain degree of hydrophobicity. To avoid false negatives caused by insufficient contact between the iodine solution and the culture medium, additional [treatment / treatment] was added. Figure 5 Experiments shown in C and D involve making small holes in the culture medium and injecting iodine solution into them, thus avoiding the hydrophobic film formed on the surface of the mycelium and achieving full contact between the iodine solution and the culture medium. Figure 5 As shown in C and D, the iodine solution in the control group turned blue, while the iodine solution in the experimental group remained brown, indicating that this strain can produce extracellular amylase.

[0054] Example 2

[0055] In this embodiment, Trichoderma reesei ( Trichoderma reesei The application of 5146-6 to inhibit plant pathogens (Fusarium graminearum, Rhizopus, Alternaria, Blastomyces oryzae, and Botrytis cinerea) was studied using the following methods:

[0056] Mark a 2cm length from the bottom of the petri dish using a ruler. Use a sterilized inoculation needle to inoculate *Trichoderma reesei* (…). Trichoderma reesei Mycelial discs containing 5146-6 and plant pathogens were inoculated at the marked locations, with a disc diameter of approximately 5.3 mm. A separate plate culture medium was inoculated only with the plant pathogens as a control. The plates were placed in an incubator and cultured upright for 24 hours, then inverted.

[0057] Observe the growth of the colonies daily, until Trichoderma reesei ( Trichoderma reesei When the colonies of strains of plant pathogens (5146-6 or other plant pathogens) grow to the edge of the culture medium, the inhibition rate is calculated to evaluate the confrontation effect. The formula for calculating the inhibition rate is as follows: Measure the colony radius Rc (mm) of the plant pathogens in the control group, and the colony radius of the experimental group containing *Trichoderma reesei* (…). Trichoderma reesei)5146-6 The colony radius Rp (mm) of plant pathogens in opposite directions is calculated according to the following formula: Inhibition rate (%) = [(Rc-Rp)] / [Rc-5.3 / 2]×100.

[0058] The results of the confrontation experiment between the above strains and plant pathogens are as follows: Figure 6 As shown, Trichoderma reesei ( Trichoderma reesei 5146-6 exhibits excellent inhibitory effects against Fusarium graminearum, Rhizopus, Alternaria, blast fungi, and Botrytis cinerea. The inhibition rate against Fusarium graminearum is 76.04% (…). Figure 6 (B) showed an inhibition rate of 96.61% against Rhizopus. Figure 6 (D), with an inhibition rate of 85.8% against Alternaria spp. ( Figure 6 The inhibition rate against rice blast fungus was 82.35% (F). Figure 6 The inhibition rate of H) against Botrytis cinerea was 84.32% ( Figure 6 (J).

[0059] Example 3

[0060] In this embodiment, a Trichoderma reesei ( Trichoderma reesei Metabolites of 5146-6, and the application of said metabolites in inhibiting clinically pathogenic bacteria.

[0061] 1. Metabolite extraction

[0062] (1) Strain expansion: Trichoderma reesei ( Trichoderma reesei 5146-6 was inoculated into PBD liquid medium and cultured in a constant temperature shaking incubator at 25±0.5℃ (150 r / min) for 5-7 days.

[0063] (2) Metabolite extraction: Add an equal volume of ethyl acetate to the fermentation broth after the above-mentioned expansion culture, mix thoroughly immediately, let stand for 24 hours for extraction, transfer the upper ethyl acetate portion to a distillation flask, and concentrate the ethyl acetate portion using a rotary evaporator (40 °C, 0.08 MPa) to obtain crude extract.

[0064] 2. Evaluation of antibacterial effect

[0065] The product information for the four pathogens involved in this embodiment is as follows:

[0066] Enterococcus faecalis ( Enterococcus faecalis ZDZA 0109;

[0067] Staphylococcus aureus ( Staphylococcus aureus CMCC 26003;

[0068] Bacillus subtilis ( Bacillus subtilis 136;

[0069] Candida albicans ( Candida albicans CMCC 98001.

[0070] (1) The pathogen was inoculated onto LB agar plates using the three-zone streak method. After incubation at 37 °C for 24 h, single colonies grew. Several single colonies were picked and inoculated onto LB liquid medium. The plates were then incubated in a constant temperature incubator with shaking (37 °C, 150 r / min) until the logarithmic growth phase (OD). 600 The value is 0.6-0.8.

[0071] (2) Take 100 μL of the pathogenic bacterial fermentation suspension and spread it evenly on the surface of an MH agar plate. Use a sterile punch (pore diameter 7.5 mm) to prepare 5 equally spaced sample wells on the plate. Add 100 μL of sterile methanol to the control well as a negative control and 100 μL of 0.1 g / mL ampicillin solution as a positive control. The above metabolites are dissolved in methanol to prepare a concentration of 100 mg / mL, and 100 μL is added to each well.

[0072] (3) Transfer the agar plates after sample addition to a 35℃ constant temperature incubator and incubate upright for 24 h. The diameter of the inhibition zone was measured using the cross-hatching method, with three biological replicates for each treatment. Based on the Quinto and Santos protocol, the zone of inhibition (ZOI) was classified into the following categories: inactive (ZOI < 10 mm), partially active (ZOI 10-12 mm), active (ZOI 13-19 mm), and highly active (ZOI > 19 mm). The inhibitory effects of the metabolites of strain 5146-6 on the pathogen are shown in Table 1 below. Figure 7 ( Figure 7 The other sample wells (showing other strains from the same batch of screening) are shown below:

[0073] Table 1 Antibacterial activity of strain metabolites

[0074]

[0075] Note: + (ZOI less than 10mm); ++ (ZOI between 10-12mm); +++ (ZOI between 13-19mm); ++++ (ZOI greater than 19mm)

[0076] Based on the above ZOI region results, strain 5146-6 has a good and significant inhibitory effect on Candida albicans, Enterococcus faecalis, Staphylococcus aureus and Bacillus subtilis (ZOI>13 mm), and is a broad-spectrum antibacterial active substance, especially with a more prominent inhibitory effect on Candida albicans.

[0077] Example 4

[0078] In this embodiment, a biocontrol agent is provided, which includes *Trichoderma reesei* isolated in Example 1. Trichoderma reesei ) 5146-6, or including the metabolites described in Example 3.

[0079] The above-mentioned biocontrol agents are used to control crop diseases caused by Fusarium graminearum, Rhizopus, Alternaria, Rice blast fungus and Botrytis cinerea, including wheat scab, sweet potato soft rot, tomato early blight, Chinese cabbage black spot, apple leaf spot, and rice leaf blast.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Trichoderma reesei ( Trichoderma reesei Strain 5146-6 was deposited on July 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 42139.

2. The Trichoderma reesei as described in claim 1 ( Trichoderma reesei 5146-6, characterized in that, The Trichoderma reesei ( Trichoderma reesei The ITS rDNA sequence of 5146-6 is shown in SEQ ID NO:

1.

3. The Trichoderma reesei as described in claim 1 ( Trichoderma reesei 5146-6, characterized in that, The morphological characteristics of the strain are as follows: Fungal characteristics: The hyphae are aseptate, white, and highly branched, with the hyphae intertwining to form hyphal clusters; the spores are elliptical or oval. Colony characteristics: The colonies are cotton-like, initially white and dense, later producing green spore clusters at the edges, and appear yellow when viewed from the back of the culture medium.

4. The Trichoderma reesei as described in claim 1 ( Trichoderma reesei 5146-6, characterized in that, The physiological and biochemical characteristics of the strain are as follows: suitable growth temperature 24-30℃, suitable pH 4.5-5.5, aerobic, using sugars as a carbon source, and capable of secreting catalase and amylase in vitro.

5. A microbial agent, characterized in that, The inoculum is *Trichoderma reesei* as described in any one of claims 1-4. Trichoderma reesei Metabolites of 5146-6; The method for extracting the metabolites is as follows: (1) Strain expansion: Trichoderma reesei ( Trichoderma reesei ) 5146-6 was inoculated into PBD liquid medium and cultured in a constant temperature incubator at 25±0.5℃ with a shaking speed of 150 r / min for 5-7 days to expand its scale-up. (2) Metabolite extraction: Add an equal volume of ethyl acetate to the fermentation broth after the above-mentioned expanded culture, mix thoroughly immediately, let stand for 24 hours for extraction, transfer the upper ethyl acetate portion to a distillation flask, and concentrate the ethyl acetate portion by rotary evaporator at 40 °C and 0.08 MPa to obtain the crude extract, which is the metabolite.

6. The microbial agent as described in claim 5, characterized in that, The dosage form of the microbial agent is selected from aqueous solutions, wettable powders, water-dispersible granules, suspensions, granules, oils, or encapsulated formulations. The objects to which the substance is applied include insects, crops, soil, and seeds; The application methods include spreading, spraying, coating, or landfilling.

7. The Trichoderma reesei as described in any one of claims 1-4 ( Trichoderma reesei 5146-6. The use of the microbial agent according to any one of claims 5-6 in the preparation of biocontrol agents; the biocontrol agents are used to inhibit Fusarium graminearum, Rhizopus, Alternaria, Blastomyces orientalis and Botrytis cinerea in the living environment, or to prevent, improve or treat related diseases caused by harmful microorganisms, wherein the harmful microorganisms are Enterococcus faecalis, Staphylococcus aureus, Bacillus subtilis or Candida albicans.

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