Trichoderma fermentation broth and preparation method thereof

Abstract

The present application relates to a kind of Trichoderma fermentation liquor and preparation method thereof.The Trichoderma fermentation liquor is the fermentation liquor of Trichoderma sulphureum DH4-3, and the preservation number of Trichoderma sulphureum DH4-3 is CGMCC No.40704.The preparation method of the above-mentioned Trichoderma fermentation liquor includes the following steps: Trichoderma sulphureum DH4-3 is inoculated into culture medium and fermented.The Trichoderma fermentation liquor has inhibitory effect on 12 kinds of tested plant pathogenic fungi, such as poplar bark rot fungus, nursery wilt fungus, blueberry branch wilt fungus, apple rot fungus, cucumber sclerotium blight fungus, apple fruit rot fungus, melon vine cutting fungus, rice malignant worm fungus, red bean anthracnose fungus, tobacco brown spot fungus, tobacco target spot fungus and Pythium aphanidermatum, and has the advantages of good control effect, wide inhibitory spectrum and wide application range.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number was 202410127268.5, the application date was January 30, 2024, and the patent title at the time of application was: A Trichoderma strain and its application. Technical Field

[0002] This invention belongs to the field of microbial technology, and in particular relates to a Trichoderma fermentation broth and its preparation method. Background Technology

[0003] Damping-off in nurseries is a significant soil-borne disease primarily caused by *Fusarium oxysporum*, *Fusarium solani*, *Rhizoctonia solani*, *Pythium debaryanum*, and *P. aphaneridermatum*, and it is widely distributed globally. Currently, control methods mainly rely on chemical control and the breeding of resistant varieties, but both have drawbacks. On the one hand, resistant varieties often exhibit resistance degradation and low resistance levels; on the other hand, the extensive use of chemical pesticides easily leads to environmental pollution and disrupts the ecological balance. Therefore, research into sustainable control technologies based on ecosystem regulation is imperative. With societal progress, people are increasingly aware of the harm caused by the long-term and excessive use of chemical pesticides to the ecological environment and human health. Biological control can effectively overcome these drawbacks, thus biological pesticides are receiving increasing attention, with the application of antagonistic microorganisms in plant disease biological control attracting particular interest. In biological control, it is crucial to select appropriate strains for control. How to select suitable strains for the biological control of damping-off pathogens in nurseries is an urgent problem to be solved.

[0004] Trichoderma belongs to the phylum Fungi, subphylum Deuteromycetes, class Hyphomycetes, order Trichodermophytes, family Trichodermophyceae. It is a class of fungi widely used in biological control agents (BCAs). In 1932, Weindling first discovered that Trichoderma lignorum could parasitize two soil-borne plant pathogens, Pythium sp. and Rhizopus sp., a discovery only three years after Fleming's discovery of penicillin (Weindling R. Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi[J]. Phytopathology, 1932, 22: 837-845.). Bonicer discovered that *T. harzianum* can prevent *Trichoderma amylovory* from infecting cells by releasing an enzyme that breaks down the cell wall of the avocado blight pathogen (Lincoln University; Patent Issued for Methods and Compositions Comprising *Trichoderma Atroviride* for the Biological Control of Soil Borne Plant Pathogens and Promoting Plant Growth [J]. Journal of Engineering, 2013).Professor Liu Shiwang's team at Zhejiang University discovered that *Trichoderma viride* enhances plant resistance to pathogens by inducing the expression of plant resistance genes (Wan Chen L, Ting Chan L, Chia Ling C, et al. Complete Genome Sequences and Genome-Wide Characterization of Trichoderma Biocontrol Agents Provide New Insights into their Evolution and Variation in Genome Organization, Sexual Development, and Fungal-Plant Interactions.[J]. Microbiology spectrum, 2021, 9(3):e0066321). However, different species of *Trichoderma* can lead to differences in their antibacterial spectrum and control efficacy, making it crucial to provide new strains that can effectively control damping-off pathogens in nurseries. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a Trichoderma fermentation broth and its preparation method. The Trichoderma strain involved is discovered for the first time in this invention. Further verification has shown that the Trichoderma fermentation broth has the advantages of good prevention and control effect, broad antibacterial spectrum and wide application range, and can be used for biological control.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides a Trichoderma strain named *Trichoderma sulphureum* DH4-3, with accession number CGMCC No. 40704. This strain was deposited on June 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0008] This invention isolated seven Trichoderma strains from soil and host plants collected from forest areas in Jilin and Liaoning provinces. Through primary screening of viable bacteria and secondary screening of fermentation broth, one antagonistic strain that can effectively inhibit seedling damping-off pathogens was selected and named DH4-3. This invention represents a breakthrough in the field of Trichoderma in the control of seedling damping-off pathogens, providing a more efficient, broader-spectrum, and more applicable biocontrol strain for biological control (e.g., biological control of seedling damping-off).

[0009] This invention provides a Trichoderma fermentation broth, which is the fermentation broth of Trichoderma DH4-3, and the preservation number of Trichoderma DH4-3 is CGMCC No. 40704.

[0010] The present invention provides a fermentation method for the above-mentioned Trichoderma or a method for preparing the above-mentioned Trichoderma fermentation broth, comprising the following steps: inoculating the above-mentioned Trichoderma into a culture medium for fermentation culture.

[0011] Furthermore, the culture medium can be potato glucose medium.

[0012] Furthermore, the fermentation culture temperature can be 28℃.

[0013] Furthermore, the fermentation culture time can be 5-7 days.

[0014] Furthermore, it also includes the step of obtaining the supernatant. For example, the supernatant can be obtained by centrifugation, filtration, or other methods.

[0015] The fermentation broth prepared by the above method can be used for biological control, and has the advantages of good control effect, broad antibacterial spectrum and wide application range.

[0016] This invention provides a microbial agent comprising the aforementioned *Trichoderma* and / or its fermentation product. This invention does not impose any particular limitation on the dosage form of the microbial agent; for example, the microbial agent can be a liquid formulation, a solid formulation, or other types of formulations. In addition to *Trichoderma*, components commonly used in the art for preparing biocontrol agents can also be added to facilitate their application.

[0017] The Trichoderma and its inoculant provided by this invention have the advantages of good prevention and control effects, broad antibacterial spectrum, and wide application range.

[0018] The present invention provides a method for preparing the above-mentioned microbial agent, comprising the following steps: fermenting the above-mentioned Trichoderma inoculation medium.

[0019] This invention provides the application of the above-mentioned Trichoderma and / or the above-mentioned fungal agents in the control of plant pathogens.

[0020] This invention provides the application of the fermentation broth of the above-mentioned Trichoderma in the control of plant pathogens. The fermentation broth of Trichoderma can be prepared by the following method: Trichoderma is inoculated into a culture medium and fermented at 28°C to obtain the fermentation broth.

[0021] Furthermore, the plant pathogens may be selected from one or a combination of several of the following: poplar bark rot fungus, nursery damping-off fungus, blueberry twig blight fungus, apple rot fungus, cucumber sclerotium rot fungus, apple fruit rot fungus, melon vine pruning fungus, rice seedling blight fungus, red bean anthracnose fungus, tobacco red spot fungus, tobacco target spot fungus, and melon and fruit pyridaben fungus.

[0022] This invention provides the application of the above-mentioned Trichoderma and / or the above-mentioned fungal agents in preventing and treating symptoms caused by the above-mentioned plant pathogens.

[0023] This invention provides the application of the above-mentioned Trichoderma fermentation broth in preventing and controlling symptoms caused by the above-mentioned plant pathogens. The plant pathogens are selected from one or more combinations of *Trichoderma*, *Dampness-causing bacteria*, *Bacillus subtilis*, *Bacillus thuringiensis*, *Bacillus rubrum*, *Bacillus sclerotiorum*, *Bacillus rubrum*, *Bacillus thuringiensis*, *Bacillus bakanae*, *Bacillus anthracis*, *Bacillus rubrum*, *Bacillus target spot*, and *Pythium spp.*.

[0024] The Trichoderma provided by this invention can be used to prepare biocontrol agents. These biocontrol agents can be used to control the aforementioned plant pathogens.

[0025] The above-mentioned Trichoderma fermentation broth provided by this invention can be used to prepare biocontrol agents for preventing and controlling the above-mentioned plant pathogens. The biocontrol agents are used to prevent and control diseases caused by plant pathogens; the plant pathogens are selected from one or more combinations of *Trichoderma rotense*, *Trichoderma hygrophila*, *Trichoderma truncatum*, *Trichoderma rotense*, *Trichoderma sclerotiorum*, *Trichoderma rotense*, *Trichoderma vine blight*, *Trichoderma bakanae*, *Trichoderma anthracnose*, *Trichoderma rubrum*, *Trichoderma target spot*, and *Pythium spp.*.

[0026] This invention provides the application of the above-mentioned Trichoderma and / or the above-mentioned biocontrol agents in the prevention and control of damping-off disease in seedlings.

[0027] This invention provides the application of the fermentation broth of the above-mentioned Trichoderma in the prevention and control of damping-off disease in seedlings.

[0028] This invention has the advantages of good prevention and control effect, broad antibacterial spectrum and wide application range.

[0029] This invention provides a biological control method, comprising the following steps: using the above-mentioned Trichoderma and / or the above-mentioned inoculant for biological control. Alternatively, the above-mentioned fermentation broth can be used for biological control. This method is used to control diseases caused by plant pathogens; the plant pathogens are selected from one or more combinations of *Trichoderma sclerotiorum*, *Trichoderma hygrophila*, *Trichoderma truncatum*, *Trichoderma rotense*, *Trichoderma sclerotiorum*, *Trichoderma rotense*, *Trichoderma vine blight*, *Trichoderma bakanae*, *Trichoderma anthracnose*, *Trichoderma rubrum*, *Trichoderma target spot*, and *Pythium spp.*.

[0030] The method provided by this invention has the advantages of good prevention and control effect, broad antibacterial spectrum, and wide application range. Attached Figure Description

[0031] Figure 1 The study aimed to investigate the inhibitory effect of live DH4-3 bacteria on seedling damping-off pathogens, with A representing the experimental group and B representing the control group.

[0032] Figure 2 The results of morphological observation of DH4-3 after 5 days of culture are shown. In the image, A is a photograph of DH4-3 observed on a plate, and B is a photograph of DH4-3 under an optical microscope.

[0033] Figure 3 The results are from the phylogenetic analysis of strain DH4-3 and related strains.

[0034] Figure 4 The inhibitory effect of live DH4-3 bacteria on pathogenic bacteria is shown in Figure 1. A represents DH4-3 against Sclerotinia sclerotiorum var. cucumberis, B represents DH4-3 against Tobacco target spot pathogen, and C represents DH4-3 against Pythium spp. var. melonis.

[0035] Figure 5 The antibacterial activity of DH4-3 fermentation broth against pathogenic bacteria is shown in Figure A, where DH4-3 fermentation broth is used against *Phyllostachys nigra* in nurseries, Figure B is used against *Phyllostachys nigra* in the control group, and Figure C is used against *Phyllostachys bark rot* in poplar trees. Detailed Implementation

[0036] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0037] This invention isolated 30 fungal species from forest soil and branches in various protected areas of Jilin Province and Dalian City. Using *Trichoderma sulphureum* as the target pathogen, a fungus strain DH4-3 was selected from healthy larch branches collected from Mudangang Forest Farm in Dunhua City, Jilin Province. Morphological observation and ITS-DNA sequence analysis confirmed the taxonomic position of strain DH4-3 as *Trichoderma sulphureum*. The antibacterial activity of live bacteria and fermentation broth of DH4-3 was further determined using the plate confrontation method and the cup-and-dish method, clarifying the biocontrol effect of this strain. This strain showed inhibitory activity against 12 tested plant pathogens, including *Trichoderma sulphureum*, exhibiting a broad spectrum of inhibition. It showed significant inhibitory effect against *Trichoderma sulphureum*, with the diameter of the pathogen in live bacteria confrontation being only 24.66 mm, and the inhibition zone diameter of the fermentation broth reaching 22.41 mm. This strain has promising application and development prospects and can be used as or used to prepare biocontrol agents. This invention is the first report of this strain and its fungicidal activity.

[0038] In the examples, the pathogenic fungi of the tested plants: *Valsa sordida* (poplar bark rot), *Fusarium oxysporum* (nursery damping-off), *Valsa mali* (apple rot), and *Monilinia polystroma* (apple fruit rot) were isolated and preserved by the Forest Pathology Laboratory of the Jilin Provincial Academy of Forestry Sciences; *Sclerotinias cleroriorum* (cucumber sclerotium rot), *Fusarium oxysporium* (melon vine pruning), *Fusarium moniliforme* (rice seedling rot), *Colletotrichum sp.* (red bean anthracnose), *Alternaria alternata* (tobacco red spot), *Rhizoctonia solani* (tobacco target spot), and *Pythium aphanidermatum* (melon fruit rot) were donated by the Plant Virus Research Laboratory of Shenyang Agricultural University; *Neofusicoccum parvum* (blueberry twig blight) was donated by Associate Professor Xu Chengnan of Yan'an University; the public can obtain the examples described in this invention for non-commercial purposes only.

[0039] The test samples were collected from healthy larch branches from Mudangang Forest Farm in Dunhua City, Jilin Province; healthy red pine branches from Wangqing County in Yanji City, Jilin Province; soil from Chixi Nature Reserve in Changbai Mountain, Jilin Province; soil from birch forest in Hongshipizhou, Jilin City, Jilin Province; and soil from Mongolian oak forest in Jinhushan, Dalian City, Liaoning Province.

[0040] The test culture medium was Potato dextrose agar (PDA), which consisted of 20g glucose, 20g agar, 200g potato flour, and 1L of distilled water per liter, with a natural pH. The potato dextrose liquid fermentation medium was prepared by adding agar to the potato dextrose agar medium.

[0041] Test reagent: Ezup column-based fungal genomic DNA extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0042] Instrument: Olympus BX53 optical microscope purchased from Olympus Corporation.

[0043] If data analysis is involved in the embodiments, SPSS 23.0 software is used for statistical analysis, and the new complex range method is used for significance analysis of differences.

[0044] Unless otherwise specified, the experimental methods used in this invention are all conventional experimental methods in the field; the materials, reagents and instruments used are all conventional materials, reagents and instruments in the field, which can be obtained through commercial channels or prepared by conventional methods.

[0045] The following is a description through specific embodiments.

[0046] Example 1: Screening of Trichoderma strains

[0047] Healthy larch branches were collected from Mudangang Forest Farm in Dunhua City, Jilin Province; healthy red pine branches from Wangqing County in Yanji City, Jilin Province; soil from Chixi Nature Reserve in Changbai Mountain, Jilin Province; birch under forest in Hongshipizhou, Jilin City, Jilin Province; and Mongolian oak under forest in Jinhushan, Dalian City, Liaoning Province, between July and September 2022.

[0048] Strains were screened using a serial dilution plating method. 10g of twigs / soil were baked at 60℃ for 1 hour, then added to 100mL of sterile water. After shaking for 30 minutes, 1mL of the suspension was taken and serially diluted with sterile water, starting from 10g... -3 10 -4 and 10 -5 Take 0.1 mL from each dilution tube and add it to a PDA plate. Spread the plate evenly and then invert the petri dish in a 28°C incubator for 3-10 days. Observe and select strains with different colony morphologies and transfer them to PDA slant plates for culture. After repurifying the bacteria 3-5 times using the dilution separation method, number the samples and store them in a 4°C refrigerator for later use.

[0049] After purification, 30 purified strains with consistent morphology, size, and color were obtained.

[0050] Activated *Trichoderma wilt* pathogens from the nursery were inoculated onto PDA plates and cultured at 28°C for 7 days before use. All isolated strains were transferred to PDA plates for further cultivation. After vigorous growth, the antibacterial activity of the isolated *Trichoderma* strains was determined using the plate confrontation culture method, with *Trichoderma wilt* as the target bacterium. Experimental group: *Trichoderma* and *Trichoderma wilt* pathogens were each prepared into 5mm mycelial discs and placed on opposite sides of the center of a 90mm diameter PDA plate, with both *Trichoderma* and *Trichoderma wilt* strains 2.5cm from the center of the PDA plate. Control group: *Trichoderma wilt* pathogens were prepared into 5mm mycelial discs and placed 2.5cm from the center of the PDA plate.

[0051] After incubating both the experimental and control groups at a constant temperature of 28℃ for 72 hours, the diameter of the seedling damping-off pathogen was measured using the cross-shaped measurement method. Each treatment was repeated three times, and the inhibition rate was calculated.

[0052] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of experimental group) / Colony diameter of control group × 100%

[0053] The results of the plate confrontation test are shown in Table 1. It can be seen that after the confrontation culture of Trichoderma, a total of 7 strains of seedling damping-off pathogen with a diameter of less than 50 mm and 4 strains with a diameter of less than 35 mm were obtained. Among them, the strain with the best antibacterial effect was named DH4-3. The diameter of the pathogen of this strain was 24.66 mm and the inhibition rate reached 72.60%. Figure 1 The study investigated the inhibitory effect of live DH4-3 bacteria on *Dampness-causing bacteria*, with group A representing the experimental group and group B representing the control group. The results showed that DH4-3 exhibited a significant inhibitory effect on *Dampness-causing bacteria*. Therefore, DH4-3 was selected as the strain for the next stage of the experiment.

[0054] Table 1. Antifungal activity of Trichoderma against seedling damping-off pathogens.

[0055]

[0056] The data in the table are mean ± standard deviation.

[0057] Example 2: Morphological observation and ITS-DNA sequencing of DH4-3 strain

[0058] Morphological observation results of strain DH4-3 are as follows Figure 2 As shown.

[0059] On PDA plates, the colonies initially appear as white, fluffy colonies, later developing whorled hyphae and dense conidiophores, turning green or dark green in color. Conidiophores are arranged in a ring, with curved or wavy branches. Conidia are spherical. Strain DH4-3 grows vigorously on PDA medium.

[0060] The DH4-3 strain was inoculated onto PDA plates, and sterile coverslips were inserted at a 45° angle into the culture medium. After incubation at 28°C, the coverslips were removed and observed under an Olympus BX53 optical microscope. Microscopic observation showed that no spores were generated during incubation at 28°C for 1-3 days, mycelia were produced after 1-3 days, spores appeared after 5 days, and the spores spread radially after 7 days.

[0061] DNA was extracted from strain DH4-3 according to the Ezup column-based fungal genomic DNA extraction kit instructions. The ITS-DNA gene fragment was amplified, and the recovered fragment was sent to Takara Bio (Dalian) Engineering Co., Ltd. for bidirectional sequencing. Sequencing and assembly were successful, and the full-length ITS-DNA gene fragment of strain DH4-3 was 573 bp. After sequence analysis using software such as BioEdit 7.0.1 and manual correction, the sequence was compared with sequences of known similar type fungi downloaded from GenBank using NCBI's BLAST program for homology analysis. Neighbor-joining (NJ) was used in MEGA 6.0 software for sequence alignment, and a phylogenetic tree was constructed. Figure 3 A phylogenetic tree was constructed based on sequences with high homology to strain DH4-3. It can be seen that strain DH4-3 is a species of Trichoderma, namely Trichoderma sulfochromis.

[0062] On June 16, 2023, it was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. The deposit name is *Trichoderma sulphureum* DH4-3, and the accession number is CGMCC No. 40704.

[0063] Example 3: Determination of the antibacterial activity and antibacterial spectrum of Trichoderma.

[0064] Based on the results of detecting the diameter of the pathogen in Example 1, the strain DH4-3 with the best antagonistic effect was selected for determination of its antibacterial spectrum.

[0065] The following fungi were selected as test strains: poplar bark rot fungus, nursery damping-off fungus, blueberry branch blight fungus, apple rot fungus, cucumber sclerotium rot fungus, apple fruit rot fungus, melon vine pruning fungus, rice seedling blight fungus, red bean anthracnose fungus, tobacco red spot fungus, tobacco target spot fungus, and melon fruit pyridamus fungus. The inhibition spectrum of DH4-3 against the above test strains was determined by plate confrontation culture method, and the detection method is the same as in Example 1.

[0066] The experimental results are shown in Table 2. Table 2 shows that the antagonistic bacterium DH4-3 inhibited all 12 tested strains; it showed the strongest inhibitory effect against *Pythium spp.*, the pathogen's diameter after confrontation culture was only 24.66 mm, significantly different from other tested pathogens. It also showed a strong inhibitory effect against *Pythium spp.*, the pathogen causing ... Figure 4 C) Cucumber sclerotinia ( Figure 4 A) and *Tobacco Aster* also showed strong inhibitory effects, inhibiting pathogen growth. When the diameter of the control pathogen reached 90 mm, the diameters of the pathogens cultured in DH4-3 were 25.78 mm (*Pythium spp.*), 25.08 mm (*Sclerotinia sclerotiorum*), and 26.35 mm (*Tobacco Aster*). Compared with the above-mentioned tested strains, DH4-3 showed strong inhibitory effects on *Tobacco Target Spot* (*Pythium spp.*). Figure 4 B) The inhibitory effect on poplar bark rot fungus is relatively weak, but the diameter of the pathogen is only about 35mm.

[0067] Table 2. Antibacterial spectrum of antagonistic bacteria DH4-3 live bacteria

[0068]

[0069] Example 4: Preparation of fermentation broth and its antibacterial spectrum

[0070] Prepare 40 mL of potato glucose liquid fermentation medium and put it into 200 mL Erlenmeyer flasks. Add 6 DH4-3 mycelial cakes with a diameter of 5 mm to each flask. Incubate at 28℃ and 150 r / min for 6 days with constant temperature shaking. Then centrifuge at 7800 rpm and 4℃ for 10 min. Discard the precipitate and aspirate the supernatant.

[0071] The following strains were used as test strains: poplar bark rot fungus, nursery damping-off fungus, blueberry twig blight fungus, apple rot fungus, cucumber sclerotium rot fungus, apple fruit rot fungus, melon vine cutting fungus, rice seedling blight fungus, red bean anthracnose fungus, tobacco red spot fungus, tobacco target spot fungus, and melon and fruit pyridaben fungus.

[0072] The inhibition spectrum of DH4-3 fermentation broth was detected using the cup-and-dish method, which included the following steps: an Oxford cup was placed in the center of a PDA culture dish, and 200 μL of fermentation broth (i.e., the supernatant prepared by the above method) was added to the Oxford cup. Four pathogenic bacterial cakes with a diameter of 5 mm were placed 1.5 cm away from the Oxford cup at the top, bottom, left, and right. After incubation at 28℃ for 72 h, the diameter of the inhibition zone was measured using the cross-sectional method.

[0073] The antibacterial activity of the fermentation broth of the antagonistic strain DH4-3 was determined using the cup-and-dish method. The experimental results are shown in Table 3. The results indicate that the fermentation broth of DH4-3 maintained good antibacterial activity, and the fermentation broth of this strain showed the strongest antibacterial effect against *Dystrophus hygroscopicus*. Figure 5 A and B), with an inhibition diameter of 22.41 mm, also showed strong antagonistic effects against blueberry twig blight pathogens, with an inhibition zone diameter of 20.19 mm, and against poplar bark rot pathogens (… Figure 5 C) It also showed good inhibitory effects on tobacco red spot bacterium and tobacco target spot bacterium, with inhibition zone diameters of 18.36 mm, 18.53 mm and 17.83 mm, respectively. The fermentation broth of this antagonistic bacterium had a significant inhibitory effect on all tested pathogens, with the smallest inhibition zone diameter reaching 10.77 mm.

[0074] Table 3 Antibacterial spectrum of antagonistic bacteria DH4-3 fermentation broth

[0075]

[0076]

[0077] The data in the table are mean ± standard deviation. Different uppercase and lowercase letters after the data in the same column indicate significant differences at the P<0.01 and P<0.05 levels, as determined by Duncan's new multiple range test.

[0078] The DH4-3 strain provided by this invention has strong antifungal activity. Because this strain produces antifungal substances during its physiological metabolism, its live bacteria and fermentation broth show strong inhibitory effects on seedling damping-off pathogens. It can be used as a biological control agent for the prevention and control of pathogens.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Trichoderma fermentation broth, characterized in that, Trichoderma sulfide ( Trichoderma sulphureum The fermentation broth of DH4-3, the preservation number of Trichoderma sulfide DH4-3 is CGMCC No.40704.

2. The method for preparing the Trichoderma fermentation broth according to claim 1, characterized in that, Includes the following steps: Trichoderma sulfide DH4-3 was inoculated into the culture medium for fermentation.

3. The preparation method according to claim 2, characterized in that, The culture medium is potato glucose medium.

4. The preparation method according to claim 2, characterized in that, The fermentation temperature is 28℃; the fermentation time is 5-7 days.

5. The preparation method according to any one of claims 2-4, characterized in that, It also includes the step of obtaining the supernatant.

6. The application of the Trichoderma fermentation broth according to claim 1 in the control of plant pathogens; wherein the plant pathogen is selected from *Trichoderma*, the pathogen causing bark rot in poplar trees. Valsa sordida Nursery damping-off pathogen Fusarium oxysporum Apple rot bacteria Valsa mali Cucumber sclerotinia stem rot Sclerotinia scleroriorum Melon vine pruning pathogens Fusarium oxysporium Rice seedling blight pathogen Fusarium moniliforme Red bean anthracnose bacteria Colletotrichum sp. Tobacco red spot bacterium Alternaria alternata Tobacco target spot pathogen Rhizoctonia solani and Pythium spp. Pythium aphanidermatum One or more combinations of them.

7. The application of the Trichoderma fermentation broth according to claim 1 in preventing and treating symptoms caused by plant pathogens; wherein the plant pathogen is selected from *Trichoderma harzianum*, the pathogen causing bark rot in poplar trees. Valsa sordida Nursery damping-off pathogen Fusarium oxysporum Apple rot bacteria Valsa mali Cucumber sclerotinia stem rot Sclerotinia scleroriorum Melon vine pruning pathogens Fusarium oxysporium Rice seedling blight pathogen Fusarium moniliforme Red bean anthracnose bacteria Colletotrichum sp. Tobacco red spot bacterium Alternaria alternata Tobacco target spot pathogen Rhizoctonia solani and Pythium spp. Pythium aphanidermatum One or more combinations of them.

8. The application of the Trichoderma fermentation broth according to claim 1 in the preparation of biocontrol agents; the biocontrol agents are used to control diseases caused by plant pathogens; the plant pathogens are selected from *Trichoderma harzianum*. Valsa sordida Nursery damping-off pathogen Fusarium oxysporum Apple rot bacteria Valsa mali Cucumber sclerotinia stem rot Sclerotinia scleroriorum Melon vine pruning pathogens Fusarium oxysporium Rice seedling blight pathogen Fusarium moniliforme Red bean anthracnose bacteria Colletotrichum sp. Tobacco red spot bacterium Alternaria alternata Tobacco target spot pathogen Rhizoctonia solani and Pythium spp. Pythium aphanidermatum One or more combinations of them.

9. The application of the Trichoderma fermentation broth according to claim 1 in the prevention and control of damping-off disease in seedlings.

10. A biological control method, characterized in that, The procedure includes the following steps: using the Trichoderma fermentation broth described in claim 1 for biological control, to prevent and control diseases caused by plant pathogens; wherein the plant pathogen is selected from *Poplar Bark Rot*. Valsa sordida Nursery damping-off pathogen Fusarium oxysporum Apple rot bacteria Valsa mali Cucumber sclerotinia stem rot Sclerotinia scleroriorum Melon vine pruning pathogens Fusarium oxysporium Rice seedling blight pathogen Fusarium moniliforme Red bean anthracnose bacteria Colletotrichum sp. Tobacco red spot bacterium Alternaria alternata Tobacco target spot pathogen Rhizoctonia solani and Pythium spp. Pythium aphanidermatum One or more combinations of them.

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