Trichoderma new strain B2-1 and application of trichoderma new strain B2-1 as biocontrol bacterium in prevention and treatment of plant diseases

Abstract

The invention discloses a trichoderma new strain Trichoderma hainanensis B2-1 and an application of the trichoderma new strain Trichoderma hainanensis B2-1 as a biocontrol bacterium in prevention and treatment of plant diseases, the strain B2-1 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation registration number is CGMCC No.41610. The trichoderma new strain Trichoderma hainanensis B2-1 can be used for preventing and treating plant diseases. According to the invention, a fungus B2-1 with a bacteriostatic effect on various pathogenic bacteria is separated and identified from rhizosphere soil of a tomato orchard, the fungus B2-1 is identified as a new trichoderma strain, and experiments prove that the fungus B2-1 has broad-spectrum bacteriostatic ability, can inhibit the growth of various plant pathogenic bacteria, and especially has a remarkable inhibitory effect on botrytis cinerea.

Description

Technical Field

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

[0002] Botrytis cinerea, also known as gray grape spore, causes sudden wilting, leaf fall, blossom end rot, fruit rot and cellar rot in plant seedlings, fruits and storage organs. When it is humid, a large amount of gray mold (conidiophores and conidia) is produced on the surface of the diseased part, which is called gray mold. Botrytis cinerea is a broad-host pathogenic fungus 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 important post-harvest diseases of tomatoes, which seriously affects the quality of tomatoes after harvest and causes huge economic losses.

[0003] At present, gray mold is mainly controlled by chemical pesticides, but with the excessive use of chemical pesticides, a series of disadvantages have been caused, such as environmental pollution, chemical pesticide residues, and pathogens that are prone to drug resistance. In particular, the use of chemical pesticides is difficult to ensure the safety of cash crops. Therefore, it is urgent to explore an economical, effective, and environmentally friendly biological control method.

[0004] Trichoderma spp. can not only inhibit the growth of pathogens but also promote plant growth. In addition, it can produce abundant secondary metabolites, and is a type of biocontrol fungus with great potential. In the context of developing environmentally friendly agriculture, some microbial agents or fertilizer products developed based on Trichoderma and its secondary metabolites have been successfully applied to the production of crops and cash crops. With the development of fungal genomics, the sequencing of multiple Trichoderma genomes has been completed, and a large number of secondary metabolite synthesis gene clusters have been found, only a small part of which is conserved between different species, and the functions of most genes in the secondary metabolic gene clusters are unknown, while there are few identified and antifungal Trichoderma secondary metabolites. Summary of the invention

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

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

[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 Microorganism Culture Collection Administration with a deposit registration number of CGMCC No.41610.

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

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

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

[0011] The preparation method of the crude extract of fermentation broth is as follows: taking the fermentation broth of Trichoderma hainanuensis B2-1, collecting the bacterial liquid after filtering, extracting with an extractant, and rotary evaporating the collected extract phase until the extractant and water are evaporated to dryness, thereby obtaining a crude extract of fermentation broth.

[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] The 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 controlling 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 scheme, the plants include peppers, eggplants, cucumbers, tomatoes, grapes, and strawberries.

[0018] The 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 such plant pathogenic fungi;

[0020] (2) Use in the preparation of products for inhibiting plant pathogenic fungi or preventing and controlling 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 fusarium oxysporum f.sp.cubense.

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

[0023] The present invention separates and identifies a fungus Trichoderma hainanuensis B2-1 having an antibacterial effect on multiple pathogenic bacteria from rhizosphere soil of a tomato orchard. It is found through research that the above-mentioned new species of Trichoderma fungus Trichoderma hainanuensis B2-1 is a new species of Trichoderma. It is confirmed through experiments that Trichoderma hainanuensis B2-1 has a broad-spectrum antibacterial ability and can inhibit the growth of multiple plant pathogenic bacteria, especially has a significant inhibitory effect on gray mold. Experiments have confirmed that B2-1, its fermentation liquid, fermentation liquid crude extract, and secondary metabolites all have significant anti-pathogenic bacteria effects, can be used as a biocontrol bacterium for preventing and controlling plant diseases, provide a new biocontrol option for the prevention and control of plant diseases, reduce the use of chemical pesticides, are green and pollution-free, protect the environment, and can reduce or avoid the problem of bacterial resistance caused by chemical pesticides. 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 This is the 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 similar to those 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 pictures of strain B2-1 and Botrytis cinerea. A is the plate confrontation picture; B is the colony diameter picture; C is the scanning electron microscope picture.

[0028] Figure 5 This is the experimental result of the inhibitory effect of the aseptic fermentation filtrate of strain B2-1 on Botrytis cinerea.

[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 Botrytis cinerea.

[0030] Figure 7 To screen for 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] Fig. 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 in conjunction with 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 (PDA plate for short): 200 g potatoes, 20 g glucose, 20 g agar, the balance is water increased to 1 L, and autoclaved at 115°C for 30 min.

[0037] PDB liquid culture medium: 200 g potatoes, 20 g glucose, the balance is water to increase 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 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 gradient diluted to 10 -5 The stock solution and gradient dilutions were placed in a 28°C shaker at 200 rpm for 3 hours and then coated with PDA plate medium in a clean bench. The coated plate was placed in a 25°C incubator for culture, and purification was performed immediately after the colonies were formed. The purification was performed three times to obtain a purified strain (numbered B2-1), which was stored in a -20°C refrigerator glycerol preservation method and a 4°C refrigerator test tube preservation method.

[0041] 2. Taxonomic identification of strain B2-1

[0042] 1. Morphological identification

[0043] After activation, strain B2-1 was placed in a biochemical incubator at 25°C in the dark for 5 days, and then the colony morphology of B2-1 on the PDA agar plate was observed. Mycelium at the edge of the B2-1 colony was scraped and made into a bacterial suspension with 1% glucose solution. After preparation, the suspension was immediately placed under a microscope to observe its morphological characteristics and take pictures.

[0044] After the B2-1 strain was activated on PDA medium, the aerial hyphae grew rapidly. The colony was 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 the genus Trichoderma.

[0045] 2. Molecular Biological Identification

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

[0047] PCR amplification: DNA was amplified using primers of 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, and 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 and sent to a sequencing company for DNA sequence determination. The measured sequence was entered into the NCBI website for sequence comparison, and base sequences with high homology coefficients (about 10-15) and exogenous base sequences were downloaded, and the evolutionary 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, and the results were as follows: Figure 2 As shown, it was found that the homology between strain B2-1 and Trichoderma erinaceum CEN1558 reached 97%. The phylogenetic tree was constructed using MEGA 11 software, and the results showed that strain B2-1 and Trichoderma 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 sequencing of strain B2-1 was performed. 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 compared to calculate their average nucleotide identity (ANI value). The results are ( Figure 3 ) showed that the strain B2-1 had the highest ANI value of Trichoderma endophyticum, which was 93%, which was 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 Microorganism Culture Collection (CGMCC) for preservation in November 2024. The address of the preservation unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is November 4, 2024, the preservation number is CGMCC No.41610, and the classification name is Trichoderma hainanuensis.

[0060] Example 2: Confrontation culture of strain B2-1 and Botrytis cinerea

[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 the 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 plate where strain B2-1 and gray mold were cultured for 5 days was taken out, photographed, and the colony diameter was measured using the cross method, and the inhibition rate was calculated. Use a knife to cut the contact position between strain B2-1 and gray mold into small pieces of about 3×3mm in size, and then put them in a 2mL centrifuge tube filled with 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 microscope observation. The results are as follows Figure 4 As shown, after co-culture of Botrytis cinerea with B2-1 strain, the diameter of Botrytis cinerea colony decreased by 52.84 cm compared with the control. Scanning electron microscopy was used to examine the morphology of Botrytis cinerea hyphae co-cultured with B2-1 strain and not co-cultured with B2-1. The results showed that the Botrytis cinerea hyphae of the CK group not co-cultured with B2-1 strain were thick and regular; in contrast, the Botrytis cinerea hyphae of the experimental group co-cultured with B2-1 showed severe structural damage, including B2-1 strain entangled with Botrytis cinerea hyphae, resulting in shriveling and hyphae destruction.

[0062] Example 3 Activity of fermentation broth 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 Erlenmeyer flask containing 30 mL 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 on a PDA medium with four holes, and 3 mm Botrytis cinerea was inoculated in the middle of the four holes. After culturing in an incubator at 28°C for 5 days, photographs were taken, the diameter of the Botrytis cinerea colony was measured, and the inhibition rate was calculated.

[0064] The results are as follows Figure 5 As shown in the figure, the antibacterial effect of the active substances produced by the B2-1 strain on Botrytis cinerea on days 1-6 was significantly higher than that on days 7-12. In days 1-5, the antibacterial rate of the active substances produced by the B2-1 strain on Botrytis cinerea reached more than 50%, but as the fermentation days increased, the active substances against Botrytis cinerea in the fermentation broth produced by B2-1 decreased, and the inhibition rate against Botrytis cinerea also decreased accordingly. On the third day, the active substances produced by the B2-1 strain had the highest antibacterial activity against Botrytis cinerea, with an inhibition rate of 100%. When the fermentation days were 7 and 9, B2-1 produced the least active substances, resulting in the lowest inhibition rate against Botrytis cinerea. Therefore, the active substances produced by the B2-1 strain with the strongest antibacterial effect on Botrytis cinerea were produced on the third day.

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

[0066] The fermentation liquid on the third day was obtained according to the method of Example 3, and the bacterial liquid was filtered with a double-layer sterile gauze, and 5L of fermentation liquid was collected in batches for standby use. 5L of fermentation filtrate was extracted with 1 volume of ethyl acetate respectively. The extract phases were collected together and placed in a rotary evaporator, and rotary evaporation was performed at a temperature of 50°C, and a vacuum pump was combined until the organic solvent and water were evaporated to dryness, and finally a crude extract of the fermentation liquid of strain B2-1 was obtained. Then the effective minimum concentration of the antibacterial activity of the crude extract of the fermentation liquid was determined. After the PDA medium is prepared, it is sterilized by high pressure at 121°C. After cooling to room temperature, the crude extract of fermentation broth prepared above is added (the culture medium with different concentrations of crude extract of fermentation broth is prepared: 0.01, 0.02, 0.04, 0.1, 0.2, 0.4 mg / mL, and DMSO is added as a control), mixed and poured into plates, and after the culture medium solidifies, 3 mm 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 plates containing crude extract of fermentation broth at different concentrations is calculated using 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 the 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 the 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 the fermentation broth crude extract was negatively correlated with the colony morphology of Botrytis cinerea. Among them, the test results showed that the EC of the fermentation broth crude extract on Botrytis cinerea 50 =0.255mg / 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 the 3rd day 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 3 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 carried out at Mattwell Biotechnology Co., Ltd. The metabolome data were analyzed and it was found that there were many secondary metabolites produced inside and outside the cells of strain B2-1. Considering the application to the prevention and control of gray mold after tomato harvest, the secondary metabolites with suitable price and high safety were selected for antibacterial activity test by consulting literature and materials. By consulting the physical and chemical properties of secondary metabolism produced inside and outside the cell, we selected 15 secondary metabolites, mainly divided the 15 secondary metabolites into contact phase and non-contact phase for the following test. Table 3 is 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, 200 g L -1) and inoculated 3 mm Botrytis cinerea cake on the infected plate, incubated in a 25°C incubator 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 (and a piece of filter paper), with the dosages of 0, 0.10, 0.52, 1.04, 2.10, and 5.24 μL cm -3 , place a 3mm Botrytis cinerea cake on the other side of the PDA medium, seal the two dishes, culture in a 25℃ incubator for 5 days, take out and take pictures.

[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 experiment used two plates to test their fumigation inhibitory activity against tomato gray mold ( 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 thymoquinone.

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

[0076] Tomatoes of uniform size and without disease were washed and scratched with a sterile needle. Botrytis cinerea cake (1 mm) was inoculated at the scratched part. Then, a Petri dish was placed at the bottom of a sealed glass desiccator and 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. Observe the lesions on the tomatoes and take photos after the 8th day.

[0077] The results are as follows Figure 8As shown in the figure, active substances with volatile effects (methyl benzoate, acetophenone, 1-phenylethanol and 2-phenylethanol) were applied to tomato fruits by fumigation. The results showed that compared with the control group, tomato gray mold was significantly inhibited after fumigation with methyl benzoate, acetophenone, 1-phenylethanol and 2-phenylethanol. The above results show that the application of methyl benzoate, acetophenone, 1-phenylethanol and 2-phenylethanol to tomato postharvest gray mold has great potential.

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

[0079] The strain B2-1 was inoculated with bacterial cakes of different pathogenic bacteria according to the plate antagonism method. The pathogenic bacteria 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 Fig. 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, the strain B2-1, the fermentation liquid and the crude extract of the fermentation liquid of the present invention all have an inhibitory effect on the mycelial growth of Botrytis cinerea, and the optimal number of days for the fermentation liquid to inhibit Botrytis cinerea is the third day. In addition, the secondary metabolites (methyl benzoate, acetophenone, 1-phenylethanol, 2-phenylethanol p-cresol, dibenzyl disulfide and thymoquinone) produced by the strain B2-1 have an inhibitory effect on Botrytis cinerea and have good application prospects. The strain B2-1 has a significant inhibitory effect on a variety of pathogenic bacteria and has a broad-spectrum antibacterial activity.

Claims

1. A new species of Trichoderma, Trichoderma hainanuensis B2-1, was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration with the registration number of 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 its crude extract or its secondary metabolites.

3. The bacterial agent according to claim 2, characterized in that: The method for preparing the fermentation liquid is as follows: Trichoderma hainanuensis B2-1 is activated and inoculated into a culture medium, and the fermentation liquid is obtained by shaking culture at 20 to 30° C., preferably for 1 to 12 days.

4. The bacterial agent according to claim 3, characterized in that: Shake culture for 1 to 6 days or 3 to 5 days; The culture medium is PDB culture medium.

5. The bacterial agent according to claim 2, characterized in that: 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 filtering, extracting with an extractant, and rotary evaporating the collected extract phase until the extractant and water are evaporated to dryness, thereby obtaining the crude fermentation broth extract.

6. The bacterial agent according to claim 5, characterized in that: The extractant is ethyl acetate; The rotary evaporation temperature is 45℃~50℃.

7. The bacterial agent according to claim 2, characterized in that: The secondary metabolites include methyl benzoate, acetophenone, 1-phenylethanol, 2-phenylethanol, p-cresol, dibenzyl disulfide and thymoquinone.

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

9. The use according to claim 6, characterized in that: The plants include peppers, eggplants, cucumbers, tomatoes, grapes, and strawberries.

10. Use of Trichoderma hainanuensis B2-1 according to claim 1 or the bacterial agent according to claim 2 in any of the following: (1) Use in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by such plant pathogenic fungi; (2) Use in the preparation of products for inhibiting plant pathogenic fungi or preventing and controlling plant diseases caused by said plant pathogenic fungi; The plant pathogenic fungi include 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, wheat fusarium wilt pathogen FusaHum graminearum Sehw, banana wilt pathogen Fusarium oxysporum f.sp.cubense.

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