Trichoderma sp. b2-1 and application thereof in prevention and treatment of plant diseases

Abstract

The application discloses a new Trichoderma hainanuensis B2-1 and application of the Trichoderma hainanuensis B2-1 as a biocontrol fungus in prevention and treatment of plant diseases, the strain B2-1 is preserved in the China General Microbiological Culture Collection Center, and a preservation registration number is CGMCC No.41610.The application separates and identifies a fungus B2-1 with inhibiting effects on multiple pathogenic bacteria from a tomato orchard rhizosphere soil, the fungus B2-1 is identified as a new Trichoderma hainanuensis, experiments prove that the B2-1 has a broad-spectrum inhibiting capacity and can inhibit growth of multiple plant pathogenic bacteria, and has a significant inhibiting effect on botrytis cinerea in particular.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism and plant disease prevention and control, and in particular to a new Trichoderma species B2-1 and its application as a biocontrol bacterium in preventing and controlling plant diseases. Background Art

[0002] Gray mold (Botrytis cinerea), also known as Botrytis cinerea, causes damping-off, leaf drop, blossom end rot, fruit rot, and cellar rot in plant seedlings, fruits, and storage organs. When humid, a large amount of gray mold (conidiophores and conidia) forms on the surface of the affected area, which is called gray mold. Botrytis cinerea is a broad-host pathogen that can cause a variety of plant diseases, such as pepper gray mold, eggplant gray mold, cucumber gray mold, tomato gray mold, grape gray mold, and strawberry gray mold. Tomato gray mold is one of the most important postharvest diseases of tomatoes, seriously affecting their quality and causing huge economic losses.

[0003] Currently, gray mold is primarily controlled with chemical pesticides. However, the overuse of these chemicals has led to a series of drawbacks, including environmental pollution, residual chemical residues, and the development of resistance in pathogens. Furthermore, the use of chemical pesticides makes it difficult to ensure the safety of cash crops. Therefore, there is an urgent need to explore cost-effective, environmentally friendly, and biocontrol methods.

[0004] Trichoderma spp. not only inhibits the growth of pathogens but also promotes plant growth. Furthermore, it produces abundant secondary metabolites, making it a promising biocontrol fungus. In the context of developing environmentally friendly agriculture, several microbial agents or fertilizers developed based on Trichoderma and its secondary metabolites have been successfully applied in the production of agricultural and cash crops. With the advancement of fungal genomics, the genomes of multiple Trichoderma strains have been sequenced, revealing a large number of secondary metabolite synthesis gene clusters. Only a small fraction of these clusters are conserved across species, and the functions of most genes in these secondary metabolite gene clusters remain unknown. Few Trichoderma secondary metabolites have been identified and have antifungal properties. Summary of the Invention

[0005] The purpose of the present invention is to address the above problems and provide a new Trichoderma species B2-1 and its use as a biocontrol bacterium in preventing and controlling plant diseases.

[0006] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a new species of Trichoderma hainanuensis B2-1, which is deposited in the General Microbiological Center of China Culture Collection of Microorganisms with a deposit registration number of CGMCC No.41610.

[0008] A second aspect of the present invention provides a bacterial agent, the active ingredient of which comprises the aforementioned Trichoderma hainanuensis B2-1 or its fermentation broth or a crude extract of the fermentation broth or its secondary metabolites.

[0009] The preparation method of the fermentation liquid is as follows: the Trichoderma hainanuensis B2-1 strain is activated and inoculated into a culture medium, and the fermentation liquid is obtained by shaking culture at 20-30° C., preferably for 1-12 days.

[0010] Preferably, the bacterial agent is shaken and cultured for 1 to 6 days or 3 to 5 days; and the culture medium is PDB culture medium.

[0011] The crude fermentation broth extract is prepared by filtering the fermentation broth of Trichoderma hainanuensis B2-1, collecting the bacterial broth, extracting it with an extractant, and subjecting the collected extract to rotary evaporation until the extractant and water are evaporated to dryness to obtain a crude fermentation broth extract.

[0012] Preferably, the extractant is ethyl acetate; and the rotary evaporation temperature is 45°C to 50°C.

[0013] Preferably, the secondary metabolites include methyl benzoate, acetophenone, 1-phenylethanol, 2-phenylethanol, p-cresol, dibenzyl disulfide and thymoquinone.

[0014] A third aspect of the present invention provides the use of the above-mentioned Trichoderma hainanuensis B2-1 or the above-mentioned bacterial agent in any of the following:

[0015] (1) Application in inhibiting Botrytis cinerea or preventing and treating plant gray mold;

[0016] (2) Application in the preparation of products for inhibiting Botrytis cinerea or preventing and treating plant gray mold.

[0017] In the application technology solution, the plants include peppers, eggplants, cucumbers, tomatoes, grapes, and strawberries.

[0018] A fourth aspect of the present invention provides the use of the above-mentioned Trichoderma hainanuensis B2-1 or the above-mentioned bacterial agent in any of the following:

[0019] (1) Use in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by said plant pathogenic fungi;

[0020] (2) Use in the preparation of products for inhibiting plant pathogenic fungi or preventing and treating plant diseases caused by said plant pathogenic fungi;

[0021] The plant pathogenic fungi include Curvularia fallax, the pathogen of banana long spot disease, Colletrichum litchi Trag, the pathogen of litchi anthracnose, Colletotrichum acutatum, the pathogen of mango / pepper anthracnose, Colletotrichum fragariae, the pathogen of strawberry anthracnose, Fusarium oxysporum (Schl.) F.sp cucumerinum Owen.anamorph, the pathogen of wheat fusarium wilt, FusaHum graminearum Sehw, and the pathogen of banana wilt, Fusarium oxysporum f.sp.cubense.

[0022] The beneficial effects of the present invention are:

[0023] The present invention isolates and identifies a fungus, Trichoderma hainanuensis B2-1, from rhizosphere soil of a tomato orchard that has an antibacterial effect on a variety of pathogens. Research has shown that the new species of Trichoderma, Trichoderma hainanuensis B2-1, is a new species of Trichoderma. Experiments have confirmed that Trichoderma hainanuensis B2-1 has a broad-spectrum antibacterial ability, capable of inhibiting the growth of a variety of plant pathogens, and particularly has a significant inhibitory effect on gray mold. Experiments have confirmed that B2-1, its fermentation broth, its crude extract, and its secondary metabolites all have significant anti-pathogenic effects and can be used as a biocontrol bacterium to prevent and control plant diseases, providing a new biocontrol option for plant disease prevention and control, reducing the use of chemical pesticides, being green and pollution-free, and protecting the environment. Furthermore, the problem of bacterial resistance caused by chemical pesticides can be reduced or avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the morphological characteristics of strain B2-1, where A and B are colony morphology (front and back), C is hyphae morphology, and D is spore morphology.

[0025] Figure 2 Bayesian phylogenetic diagram of strain B2-1 constructed based on TEF1.

[0026] Figure 3The ANI values ​​of the B2-1 strain based on the alignment of the Trichoderma Scaffold sequence and other species identified by KmerFinder were calculated (using the OrthoANI algorithm and BLAST calculations performed by the ANI tool OAT software).

[0027] Figure 4 These are the confrontation culture images of strain B2-1 and Botrytis cinerea. A is the plate confrontation image; B is the colony diameter image; C is the scanning electron microscope image.

[0028] Figure 5 This is the experimental result of the inhibitory effect of the sterile fermentation filtrate of strain B2-1 on gray mold.

[0029] Figure 6 This is the experimental result of the inhibitory effect of the crude extract of the fermentation broth of strain B2-1 on gray mold.

[0030] Figure 7 Screening of secondary metabolites produced by strain B2-1 that have inhibitory effects on Botrytis cinerea.

[0031] Figure 8 The inhibitory effect of volatile metabolites produced by strain B2-1 on tomato gray mold (MB: methyl benzoate, AC: acetophenone, 1-PE: 1-phenylethanol, 2-PE: 2-phenylethanol).

[0032] Figure 9 Schematic diagram of the antagonistic effect of strain B2-1 on various pathogenic fungi. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0035] The culture medium used in the examples is:

[0036] PDA plate culture medium (abbreviated as PDA plate): 200 g potatoes, 20 g glucose, 20 g agar, the balance is increased to 1 L with water, and autoclaved at 115°C for 30 min.

[0037] PDB liquid medium: 200 g potatoes, 20 g glucose, the balance is water to 1 L, and autoclave at 115°C for 30 min.

[0038] Example 1 Isolation and identification of strain B2-1

[0039] 1. Isolation of strain B2-1

[0040] The rhizosphere soil containing Trichoderma was retrieved from the tomato orchard and brought back to the laboratory for subsequent isolation and purification of the fungus. 5.0 g of soil sample was weighed and placed in a 10 mL centrifuge tube, and the volume was made up to 10 mL with sterile water and mixed. The mixed stock solution was diluted to 10 mL by gradient dilution. -5 The original solution and the gradient dilutions were incubated in a shaker at 200 rpm at 28°C for 3 hours before being plated on PDA plates in a clean bench. The plate was then incubated in a 25°C incubator. Once colonies formed, purification was performed immediately. This process was repeated three times to obtain a purified strain (numbered B2-1). The strain was then stored in a glycerol freezer at -20°C and in a test tube freezer at 4°C.

[0041] 2. Taxonomic Identification of Strain B2-1

[0042] 1. Morphological identification

[0043] After activation, strain B2-1 was cultured in a dark incubator at 25°C for 5 days. The morphology of B2-1 colonies on PDA agar plates was observed. Mycelium was scraped from the edges of the B2-1 colonies and prepared into a suspension in 1% glucose solution. After preparation, the suspension was immediately placed under a microscope to observe its morphology and photograph it.

[0044] After the B2-1 strain was activated on PDA medium, the aerial hyphae grew rapidly. The colonies were round and the hyphae were white. The hyphae were filamentous, slender, and cotton-like, growing radially from the center. The conidia were spherical or ellipsoidal, single-celled, smooth, light green, and dark green when mature. Figure 1 ), which was tentatively identified as a species of Trichoderma.

[0045] 2. Molecular Biological Identification

[0046] DNA extraction: DNA of fungal sample B2-1 was extracted according to the operating instructions of the Tiangen Plant DNA Extraction Kit.

[0047] PCR amplification: DNA was amplified using primers for TEF1 (F: 5'-CGGTCACTTGATCTACAAGTG-3', SEQ ID NO. 1; R: 5'-CCTCGACACCAGTCACG-3', SEQ ID NO. 2) according to the sample loading system in Table 1 and the PCR program in Table 2. Finally, the amplified DNA samples were subjected to agarose gel electrophoresis.

[0048] Table 1 Reaction system

[0049]

[0050] Table 2 PCR program

[0051]

[0052] The amplified product was detected by 1% agarose gel electrophoresis and purified before being sent to a sequencing company for DNA sequencing. The sequence was entered into the NCBI website for sequence alignment, and base sequences with high homology coefficients (approximately 10-15) and foreign base sequences were downloaded. A phylogenetic tree was constructed using MEGA 11.

[0053] The TEF1 gene sequence of strain B2-1 (SEQ ID NO. 3) was compared with the NCBI homology results. Figure 2 As shown, strain B2-1 was found to have a 97% homology with Trichodema erinaceum CEN1558. A phylogenetic tree was constructed using MEGA 11 software, and the results showed that strain B2-1 and Trichodema erinaceum were clustered together, indicating that strain B2-1 may belong to the genus Trichoderma.

[0054] The TEF1 gene sequence of strain B2-1 (SEQ ID NO. 3) is as follows:

[0055] .

[0056] 3. Whole-genome identification

[0057] Since a single gene fragment is not sufficient to identify the fungal species, the whole genome of strain B2-1 was sequenced. Figure 3 As shown in the figure, the whole genome sequence of yeast was used as the outgroup, and the whole genome sequences of 8 strains adjacent to strain B2-1 were aligned to calculate their average nucleotide identity (ANI value). The results were ( Figure 3 ) showed that the strain B2-1 had the highest ANI value of Trichoderma endophyticum, which was 93%, lower than the same species threshold of 95%. This result indicated that strain B2-1 was a new species of Trichoderma, so the strain was named Trichoderma hainanuensis B2-1.

[0058] The deposit information of Trichoderma hainanuensis B2-1 is as follows:

[0059] Trichoderma hainanuensis B2-1 was sent to the General Microbiology Center of China Culture Collection Administration for Microorganisms (CGMCC) for preservation in November 2024. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is November 4, 2024, the deposit number is CGMCC No.41610, and the classification name is Trichoderma hainanuensis.

[0060] Example 2: Confrontation culture of strain B2-1 and gray mold

[0061] The preserved strain B2-1 and tomato gray mold (Botrytis cinerea) were activated on PDA plates respectively, and holes were punched on the PDA culture medium with a yellow gun tip in a clean bench to obtain 3×3mm bacterial cakes. The two bacteria were transferred to new PDA plates and cultured in a 25°C incubator for 5 days. The PDA plates where strain B2-1 and gray mold were cultured for 5 days were taken out, photographed, and the colony diameter was measured using the cross method, and the inhibition rate was calculated. The contact position between strain B2-1 and gray mold was cut into small pieces of about 3×3mm in size with a knife, and then placed in a 2mL centrifuge tube containing 2.5% glutaraldehyde electron microscope fixative. After fixing at room temperature for 15 minutes, they were stored at 4°C and transported to Seville Co., Ltd. for scanning electron microscopy observation. The results are as follows Figure 4 As shown, after co-cultivation with the B2-1 strain, the diameter of the Botrytis cinerea colony decreased by 52.84 cm compared to the control. Scanning electron microscopy was used to examine the morphology of Botrytis cinerea hyphae in cultures co-cultured with and without the B2-1 strain. The results showed that the Botrytis cinerea hyphae in the CK group, which was not co-cultivated with the B2-1 strain, were thick and regular. In contrast, the Botrytis cinerea hyphae in the experimental group co-cultivated with B2-1 showed severe structural damage, including entanglement of the B2-1 strain with the Botrytis cinerea hyphae, resulting in shriveled and damaged hyphae.

[0062] Example 3 Fermentation activity of strain B2-1

[0063] The bacterial cake (3×3 mm) of the B2-1 strain was inoculated into PDB medium. Ten bacterial cakes were inoculated into a 50 mL triangular flask containing 30 mL of PDB medium and placed at 28°C for 200 min. -1After fermentation in a shaker for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days, the remaining bacterial liquid was filtered with a 0.22 μm sterile filter, and the fermentation broth of different days was inoculated onto a PDA culture medium with four holes. 3 mm gray mold was inoculated in the middle of the four holes. After culture in an incubator at 28 ° C for 5 days, photographs were taken, the diameter of the gray mold colony was measured, and the inhibition rate was calculated.

[0064] The results are as follows Figure 5 As shown, the active substances produced by strain B2-1 showed significantly higher antibacterial activity against Botrytis cinerea from days 1 to 6 than from days 7 to 12. During days 1 to 5, the active substances produced by strain B2-1 achieved an inhibition rate of over 50% against Botrytis cinerea. However, as fermentation days increased, the amount of active substances produced by strain B2-1 in the fermentation broth decreased, and the inhibition rate also decreased accordingly. On day 3, the active substances produced by strain B2-1 exhibited the highest antibacterial activity against Botrytis cinerea, reaching 100%. On days 7 and 9, strain B2-1 produced the least amount of active substances, resulting in the lowest inhibition rate against Botrytis cinerea. Therefore, the active substances produced by strain B2-1 exhibited the highest antibacterial activity against Botrytis cinerea on day 3.

[0065] Example 4 Activity of crude extract from fermentation broth of strain B2-1

[0066] Fermentation broth was obtained on day 3 according to the method of Example 3. The remaining bacterial broth was filtered through a double layer of sterile gauze, and 5 L of fermentation broth was collected for later use. Each 5 L of fermentation filtrate was extracted with 1 volume of ethyl acetate. The combined extracts were placed in a rotary evaporator and evaporated at 50°C with a vacuum pump until the organic solvent and water were evaporated to dryness. This yielded a crude fermentation broth extract of strain B2-1. The minimum effective concentration of the crude fermentation broth extract for antibacterial activity was then determined. After the PDA culture medium is prepared, it is sterilized under high pressure at 121°C. After cooling to room temperature, the crude fermentation broth extract prepared previously is added (culture medium with different concentrations of crude fermentation broth extract is prepared: 0.01, 0.02, 0.04, 0.1, 0.2, 0.4 mg / mL, and DMSO is added as a control). After mixing, pour it into a plate. After the culture medium solidifies, a 3mm gray mold is inoculated in the middle of the plate; each treated plate is placed in an incubator at a temperature of 25°C and inverted for 5 days, and the colony diameter is measured. The antibacterial rate of the plates containing crude fermentation broth extract at different concentrations is calculated based on the difference in colony diameter growth between each treatment and the control.

[0067] The results are as follows Figure 6As shown in the figure, when the concentration of fermentation broth crude extract was 0.02 mg / mL, the colony diameter of Botrytis cinerea was significantly smaller than that of the control group. As the concentration of fermentation broth crude extract continued to increase, the colony diameter of Botrytis cinerea continued to decrease and the inhibition rate continued to increase, indicating that the concentration of fermentation broth crude extract was negatively correlated with the colony morphology of Botrytis cinerea. Among them, the test results showed that the EC of fermentation broth crude extract on Botrytis cinerea was negatively correlated with the EC of fermentation broth crude extract on Botrytis cinerea. 50 =0.255 mg / mL, EC 50 =0.628 mg / mL. The crude extract of the fermentation broth of strain B2-1 has a significant inhibitory effect on Botrytis cinerea.

[0068] Example 5 Screening of secondary metabolites with antibacterial activity from strain B2-1

[0069] The fermentation broth of strain B2-1 on day 3 was obtained according to the method in Example 3, and the extracellular sample of strain B2-1 was collected by filtering through a 0.22 μm sterile filter. The mycelium of strain B2-1 fermented for 3 days was washed three times with PBS to prepare the intracellular sample of strain B2-1. PDB medium was used as a blank control. Non-targeted metabolomics analysis of the fermentation supernatant and intracellular samples of strain B2-1 was performed at Metrowell Biotechnology Co., Ltd. Analysis of the metabolome data revealed that strain B2-1 produces a large number of secondary metabolites inside and outside the cells. Considering its application in the prevention and control of post-harvest gray mold in tomatoes, we selected secondary metabolites with reasonable price and high safety for antibacterial activity testing by consulting literature and data. By reviewing the physicochemical properties of secondary metabolites produced inside and outside the cells, we selected 15 secondary metabolites. The 15 secondary metabolites were mainly divided into contact phase and non-contact phase for the subsequent experiments. Table 3 shows the 15 secondary metabolites after screening.

[0070] Table 3 15 significantly upregulated differential metabolites screened

[0071]

[0072]

[0073] Contact phase: Different active substances (succinic acid, 3,4,5-trimethoxybenzoic acid, p-quinone, 3-hydroxy-2-butanone, dibenzyl disulfide, thymoquinone, luteolin, sesamol, DL-3-phenyllactic acid, and vedilolactone) were prepared into different concentration gradients (DMSO, 4, 20, 40, and 200 g L -1) infected PDA plates were prepared and 3 mm Botrytis cinerea cakes were inoculated onto the infected plates. The plates were incubated at 25°C for 5 days and photographed. Non-contact phase: Volatile active substances (1-phenylethanol, 2-phenylethanol, dihydrocoumarin, methyl benzoate, acetophenone) were added to one side of the two plates (along with a piece of filter paper) at concentrations of 0, 0.10, 0.52, 1.04, 2.10, and 5.24 μL cm, respectively. -3 Place a 3mm Botrytis cinerea cake on the other side of the PDA medium, seal the two plates, and incubate at 25°C for 5 days. Then take out the plates and take photos.

[0074] The results are as follows Figure 7 As shown in Figure 2, since dihydrocoumarin, methyl benzoate, acetophenone, 1-phenylethanol and 2-phenylethanol are volatile, the fumigation inhibition activity of these substances against tomato gray mold was tested using two separate plates ( Figure 7 The results showed that methyl benzoate, acetophenone, 1-phenylethanol and 2-phenylethanol could inhibit Botrytis cinerea in the form of fumigation. When the application amount was 0.10 μL cm -3 When the application rate was 0.52 μL cm -3 When methyl benzoate, acetophenone, and 1-phenylethanol were added, the colony morphology of Botrytis cinerea was completely inhibited and the Botrytis cinerea did not grow. The remaining 10 secondary metabolites were prepared into poisonous plates with different concentrations and then inoculated with Botrytis cinerea and its growth was observed. The results showed that when the concentration was 40 μg L -1 When the concentration was 80 μg L -1 When thymoquinone was applied, Botrytis cinerea basically did not grow, while the inhibitory effects of p-cresol and dibenzyl disulfide on Botrytis cinerea were not as significant as that of thymoquinone.

[0075] Example 6 Inhibitory Effects of Volatile Secondary Metabolites on Tomato Gray Mold

[0076] Tomatoes of uniform size and without disease were washed and scratched with a sterile needle. The scratched area was inoculated with Botrytis cinerea cake (1 mm). A Petri dish was then placed at the bottom of a sealed glass desiccator. Sterile water, acetophenone, methyl benzoate, 1-phenylethanol, and 2-phenylethanol were added at 0.21 μL cm -3 Place 20 tomatoes in a sealed glass desiccator and seal it immediately. After 8 days, observe the lesions on the tomatoes and take photos.

[0077] The results are as follows Figure 8As shown in this study, fumigation treatment with volatile active substances (methyl benzoate, acetophenone, 1-phenylethanol, and 2-phenylethanol) was applied to tomato fruits. The results showed that fumigation with methyl benzoate, acetophenone, 1-phenylethanol, and 2-phenylethanol significantly inhibited gray mold in tomatoes compared to the control. These results demonstrate the potential of methyl benzoate, acetophenone, 1-phenylethanol, and 2-phenylethanol for the treatment of postharvest gray mold in tomatoes.

[0078] Example 7 Broad-spectrum antibacterial activity of strain B2-1

[0079] The strain B2-1 was inoculated with bacterial cakes of different pathogens according to the plate antagonism method. The pathogens used in the experiment are shown in Table 4. After culturing in a 25°C incubator for 5 days, the colony diameter was measured and the inhibition rate was calculated.

[0080] Table 4 Different types of pathogens

[0081]

[0082] The results are as follows Figure 9 As shown, strain B2-1 had a certain antagonistic effect on pathogenic bacteria isolated from pepper, banana, strawberry, cucumber, litchi, mango and wheat, and the inhibition rate reached more than 50%, indicating that the biocontrol fungus B2-1 has a broad-spectrum antibacterial activity.

[0083] In summary, strain B2-1, the fermentation broth, and the crude extract of the fermentation broth of the present invention all inhibit the mycelial growth of Botrytis cinerea, and the optimal number of days for the fermentation broth to inhibit Botrytis cinerea is the third day. Furthermore, strain B2-1 produces secondary metabolites (methyl benzoate, acetophenone, 1-phenylethanol, 2-phenylethanol-p-cresol, dibenzyl disulfide, and thymoquinone) that inhibit Botrytis cinerea and have promising application prospects. Furthermore, strain B2-1 exhibits significant inhibitory effects against a variety of pathogens, demonstrating broad-spectrum antibacterial activity.

Claims

1. A new species of Trichoderma hainanuensis B2-1 was deposited in the General Microbiology Center of China Culture Collection of Microorganisms with the registration number CGMCC No.41610.

2. A bacterial agent, the active ingredient of which comprises the Trichoderma hainanuensis B2-1 according to claim 1, or its fermentation broth or a crude extract of the fermentation broth; The fermentation broth is prepared by activating Trichoderma hainanuensis B2-1 strains and inoculating them into a culture medium, culturing them at 20-30° C. with shaking, and filtering them with a 0.22 μm sterile filter to obtain the fermentation broth. The preparation method of the crude fermentation broth extract comprises the following steps: taking the fermentation broth of Trichoderma hainanuensis B2-1, collecting the bacterial liquid after filtration, extracting with an extractant, and rotary evaporating the collected extract phase until the extractant and water are evaporated to dryness to obtain the crude fermentation broth extract, wherein the extractant is ethyl acetate.

3. The microbial agent according to claim 2, wherein: The shaking culture time is 1 to 12 days; and the culture medium is PDB culture medium.

4. The microbial agent according to claim 2, wherein: The rotary evaporation temperature is 45℃~50℃.

5. The microbial agent according to claim 2, wherein: The fermentation broth contains secondary metabolites, and the secondary metabolites include methyl benzoate, acetophenone, 1-phenylethanol, 2-phenylethanol, p-cresol, dibenzyl disulfide or thymoquinone.

6. Use of the Trichoderma hainanuensis B2-1 of claim 1 or the bacterial agent of claim 2 in any of the following: (1) Application in inhibiting Botrytis cinerea or preventing and treating plant gray mold; (2) Application in the preparation of products for inhibiting Botrytis cinerea or preventing and treating plant gray mold.

7. The use according to claim 6, characterized in that: The plant is selected from pepper, eggplant, cucumber, tomato, grape or strawberry.

8. Use of the Trichoderma hainanuensis B2-1 according to claim 1 in any of the following: (1) Use in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by said plant pathogenic fungi; (2) Use in the preparation of products for inhibiting plant pathogenic fungi or preventing and treating plant diseases caused by said plant pathogenic fungi; The plant pathogenic fungi are selected from Curvularia fallax, the pathogen of banana long spot disease, Colletrichum litchi Trag, the pathogen of litchi anthracnose disease, Colletotrichum acutatum, the pathogen of mango / pepper anthracnose disease, Colletotrichum fragariae, the pathogen of strawberry anthracnose disease, Fusarium oxysporum (Schl.) F.sp.cucumerinum Owen.anamorph, the pathogen of wheat fusarium wilt disease, FusaHum graminearum Sehw or the pathogen of banana fusarium oxysporum f.sp.cubense.

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