Trichoderma fermentation liquor and preparation method thereof

Abstract

The invention relates to trichoderma fermentation liquor and a preparation method thereof. The Trichoderma fermentation broth is a fermentation broth of Trichoderma sulphureum DH4-3, and the preservation number of the Trichoderma sulphureum DH4-3 is CGMCC (China General Microbiological Culture Collection Center) No.40704. The Trichoderma sulphureum DH4-3 can be used for preparing the Trichoderma sulphureum. The preparation method of the trichoderma fermentation liquor comprises the following steps: inoculating a culture medium with the trichoderma thiochromatum DH4-3, and carrying out fermentation culture. The trichoderma fermentation liquor has an inhibition effect on 12 test plant pathogenic bacteria such as poplar skin rot bacteria, nursery rhizoctonia solanacearum, blueberry branch blight bacteria, apple rot bacteria, cucumber sclerotinia sclerotiorum, apple fruit rot bacteria, muskmelon vine cutting bacteria, rice fusarium moniliforme, red adzuki bean colletotrichum gloeosporioides, tobacco alternaria alternata, tobacco target leaf spot bacteria and pythium aphanidermatum; the composition has the advantages of good control effect, wide antibacterial spectrum, wide application range and the like.

Description

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

[0002] The invention belongs to the technical field of microorganisms, and in particular relates to a Trichoderma fermentation liquid and a preparation method thereof. Background Art

[0003] Nursery damping-off is an important soil-borne disease caused by infection by Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Pythium debaryanum and P. aphanidermatum. The disease is widely distributed worldwide. At present, the prevention and control of the disease mainly focuses on chemical control and breeding of disease-resistant varieties, but both have disadvantages. On the one hand, the disease-resistant varieties cultivated have problems such as resistance degradation and low resistance. On the other hand, the large-scale use of chemical pesticides can easily cause environmental pollution and destroy the ecological balance. Therefore, it is imperative to study sustainable control technology based on ecosystem regulation. With the progress of society, people are increasingly aware of the harm of long-term and large-scale use of chemical pesticides to the ecological environment and human health. Biological control can effectively overcome these disadvantages, so biological pesticides are increasingly valued by people, among which the application of antagonistic microorganisms in plant disease biological control has attracted much attention. In biological control, it is necessary to select suitable strains for prevention and control. How to select suitable strains for biological control of pathogenic bacteria of nursery blight is an urgent problem to be solved.

[0004] Trichoderma belongs to Fungi, Deuteromycotina, Hypomycetes, Hypocystales, Hypocystaceae, and is a type of fungus widely used as biological control agents (BCAs). Weindling first discovered in 1932 that Trichoderma lignorum can parasitize on two soil-borne plant pathogens, Pythium sp. and Rhizopus sp., which was only three years later than 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 Trichoderma harzianum can release enzymes to break down the cell wall of Tristeza amylovory, thereby preventing it from infecting cells (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 green Trichoderma 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 antibacterial spectra and control effects, and it is crucial to provide new strains that can effectively control the pathogens of nursery blight. Summary of the invention

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

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

[0007] The present invention provides a strain of Trichoderma, the strain name is Trichoderma sulphureum DH4-3, and the deposit number is CGMCC No. 40704. The strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC) on June 16, 2023, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0008] The present invention isolates and obtains 7 strains of Trichoderma from soil and host plants collected from forest areas in Jilin Province, Liaoning Province and other places, and selects an antagonistic strain that can effectively inhibit nursery damping-off pathogens through primary screening of live bacteria and secondary screening of fermentation liquid, and names it DH4-3. The present invention achieves a breakthrough in the field of nursery damping-off pathogens using Trichoderma, and provides a more efficient, broader antibacterial spectrum, and more favorable application biocontrol strain for biological control (for example, biological control of nursery damping-off disease).

[0009] The invention provides a Trichoderma fermentation liquid, which is the fermentation liquid of Trichoderma sulphureum DH4-3 (Trichoderma sulphureum DH4-3), and the preservation number of Trichoderma sulphureum DH4-3 is CGMCC No.40704.

[0010] The present invention provides a fermentation method of the above-mentioned Trichoderma or a preparation method of the above-mentioned Trichoderma fermentation liquid, comprising the following steps: inoculating the above-mentioned Trichoderma into a culture medium for fermentation and culturing.

[0011] Furthermore, the culture medium may be potato dextrose medium.

[0012] Furthermore, the fermentation culture temperature may be 28°C.

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

[0014] Furthermore, the method further comprises a step of obtaining a supernatant. For example, the supernatant can be obtained by centrifugation, filtration, etc.

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

[0016] The present invention provides a bacterial agent, comprising the above-mentioned Trichoderma and / or the fermentation product of the above-mentioned Trichoderma. The present invention has no special restrictions on the dosage form of the bacterial agent, for example, the bacterial agent can be a liquid preparation or a solid preparation, or other types of preparations. In addition to Trichoderma, components commonly used in the art for preparing biocontrol preparations can also be added to facilitate its application.

[0017] The Trichoderma and the bacterial agent provided by the invention have the advantages of good prevention and control effect, broad antibacterial spectrum, wide application range, etc.

[0018] The present invention provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: inoculating the above-mentioned Trichoderma into a culture medium for fermentation and culturing.

[0019] The present invention provides application of the above-mentioned Trichoderma and / or the above-mentioned bacterial agent in the prevention and treatment of plant pathogenic bacteria.

[0020] The present invention provides the use of the fermentation liquid of Trichoderma in the prevention and treatment of plant pathogenic bacteria. The fermentation liquid of Trichoderma can be prepared by the following method: inoculating Trichoderma into a culture medium, fermenting and culturing at 28°C to obtain the fermentation liquid.

[0021] Furthermore, the plant pathogenic bacteria can be selected from one or a combination of poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine disease, rice seedling rot, red bean anthracnose, tobacco brown spot, tobacco target spot and citrullus rot.

[0022] The present invention provides application of the above-mentioned Trichoderma and / or the above-mentioned bacterial agent in preventing and treating symptoms caused by the above-mentioned plant pathogenic bacteria.

[0023] The present invention provides the use of the Trichoderma fermentation liquid in preventing and treating the symptoms caused by the above-mentioned plant pathogens. The plant pathogens are selected from one or a combination of poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine slash, rice seedling rot, red bean anthracnose, tobacco brown spot, tobacco target spot and citrus fruit rot.

[0024] The Trichoderma provided by the present invention can be used to prepare a biocontrol agent, which can be used to control the above-mentioned plant pathogens.

[0025] The Trichoderma fermentation liquid provided by the present invention can be used to prepare a biocontrol agent for preventing and controlling the above-mentioned plant pathogens. The biocontrol agent is used to prevent and control diseases caused by plant pathogens; the plant pathogens are selected from one or a combination of poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine slash, rice seedling rot, red bean anthracnose, tobacco brown spot, tobacco target spot and citrus fruit rot.

[0026] The present invention provides application of the above-mentioned Trichoderma and / or the above-mentioned biocontrol agent in preventing and controlling damping-off disease of seedlings.

[0027] The present invention provides application of the fermentation liquid of Trichoderma in preventing and treating damping-off disease of seedlings.

[0028] The invention has the advantages of good prevention and treatment effect, broad antibacterial spectrum, wide application range, etc. when used.

[0029] The present invention provides a biological control method, comprising the following steps: using the above-mentioned Trichoderma and / or the above-mentioned bacterial agent for biological control. The above-mentioned fermentation liquid can also be used for biological control. It is used to prevent and control diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are selected from one or a combination of poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine slash, rice seedling blight, red bean anthracnose, tobacco brown spot, tobacco target spot and citrus fruit rot.

[0030] The method provided by the invention has the advantages of good prevention and treatment effect, broad antibacterial spectrum, wide application range, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the inhibitory effect of DH4-3 live bacteria on nursery blight pathogen, where A is the experimental group and B is the control group.

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

[0033] Figure 3 Results of phylogenetic analysis of strain DH4-3 and related strains.

[0034] Figure 4 It is the inhibitory effect of live DH4-3 bacteria on pathogenic bacteria, among which A is DH4-3 against cucumber sclerotinia disease, B is DH4-3 against tobacco target spot pathogen, and C is DH4-3 against citrus pythium sphaeroides.

[0035] Figure 5 The results are as follows: A is the antibacterial activity of DH4-3 fermentation liquid against pathogenic bacteria, B is the control group nursery blight pathogen, and C is the DH4-3 fermentation liquid against poplar bark rot pathogen. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0037] The present invention isolates 30 kinds of fungi from the soil and branches under the forest in various protection areas in Jilin Province and Dalian City, screens the target fungi with the nursery damping-off pathogen as the target fungi, and finally selects a fungus DH4-3 collected from the healthy larch branches in the Mudangang Forest Farm in Dunhua City, Jilin Province. Through morphological observation and ITS-DNA sequence analysis method, the classification status of the strain DH4-3 is determined to belong to Trichoderma, which is Trichoderma sulphureum. The antibacterial activity of the live bacteria and the fermentation liquid of DH4-3 is further determined by the plate confrontation method and the cup-disc method, respectively, and the biocontrol effect of the strain is clarified. The strain has an inhibitory effect on 12 kinds of tested plant pathogens including the nursery damping-off pathogen, and the antibacterial spectrum is wide. Among them, the strain has a significant inhibitory effect on the nursery damping-off pathogen, the diameter of the pathogen confronted by the live bacteria is only 24.66mm, and the diameter of the antibacterial circle of the fermentation liquid is 22.41mm. The strain has good application and development prospects and can be used as or prepared as a biocontrol agent. The present invention is the first to report the strain and the fungicidal activity of the strain.

[0038] In the embodiments, the plant pathogens tested: poplar bark rot Valsa sordida, nursery blight Fusarium oxysporum, apple rot Valsa mali, apple fruit rot Monilinia polystroma were isolated and preserved by the Forest Pathology Laboratory of Jilin Academy of Forestry Sciences; cucumber sclerotinia sclerotiorum, melon vine blight Fusarium oxysporium, rice seedling blight Fusarium moniliforme, red bean anthracnose Colletotrichum sp., tobacco brown spot pathogen Alternaria alternata, tobacco target spot pathogen Rhizoctonia solani, citrus fruit rot pathogen Pythium aphanidermatum were donated by the Plant Virus Research Laboratory of Shenyang Agricultural University; blueberry branch blight Neofusicoccum parvum was donated by Associate Professor Xu Chengnan of Yan'an University; the public can obtain the embodiments recorded in the present invention for non-commercial purposes only.

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

[0040] Test medium: Potato dextrose agar (PDA) medium formula includes: glucose 20g, agar 20g, potato flour 200g, distilled water 1L, pH natural. Potato dextrose liquid fermentation medium is based on potato dextrose agar medium without adding agar.

[0041] Test reagents: Ezup column fungal genomic DNA extraction kit was purchased from Shanghai Biotech Co., Ltd.

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

[0043] When data analysis is involved in the embodiments, SPSS 23.0 software was used for statistical analysis, and the new multiple range method was used for difference significance analysis.

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

[0045] The following is an introduction through specific embodiments.

[0046] Example 1 Screening of Trichoderma strains

[0047] From July to September 2022, healthy larch branches were collected from Mudangang Forest Farm in Dunhua City, Jilin Province, healthy Korean pine branches in Wangqing County, Yanji City, Jilin Province, soil from Chixi Nature Reserve in Changbai Mountain, Jilin Province, under the white birch forest in Hongshipizhou, Jilin City, Jilin Province, and under the Mongolian oak forest in Jinhu Mountain, Dalian City, Liaoning Province.

[0048] The strain screening was carried out by the stepwise dilution and spreading separation method. 10 g of branches / soil were baked at 60 °C for 1 h, then added to 100 mL of sterile water and shaken for 30 min. 1 mL of the suspension was drawn and diluted with sterile water from 10 to 10. -3 , 10 -4 and 10 -5 Pipette 0.1 mL of the dilution tube onto the PDA plate and spread evenly. Then invert the culture dish in a 28°C incubator and culture for 3-10 days. Observe and select strains with different colony morphology and transfer them to the PDA slant for culture in time. Re-purify 3-5 times using the dilution separation method, and store them in a 4°C refrigerator with numbers for later use.

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

[0050] The activated Rhizoctonia solani was inoculated into the PDA plate and cultured at 28°C for 7 days before use. All isolated strains were transferred to the PDA plate for culture. After each strain grew vigorously, the Rhizoctonia solani was used as the target bacteria, and the antibacterial activity of the Trichoderma isolated above was determined by the plate confrontation culture method. Experimental group: Trichoderma and Rhizoctonia solani were made into 5mm cakes, respectively, and placed on both sides of the center of the PDA plate (90mm in diameter), with the distance between Trichoderma and the center of the PDA plate being 2.5cm, and the distance between Rhizoctonia solani and the center of the PDA plate being 2.5cm. Control group: Rhizoctonia solani was made into 5mm cakes and placed 2.5cm away from the center of the PDA plate.

[0051] The experimental group and the control group were cultured at 28℃ for 72h, and the diameter of the pathogen of Rhizoctonia solani was measured by the cross measurement method. Each treatment was repeated 3 times, and the inhibition rate was calculated.

[0052] Antibacterial rate (%) = (colony 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 confrontation culture with Trichoderma, there are 7 strains with a diameter of less than 50 mm for the pathogenic bacteria of nursery blight, and 4 strains with a diameter of less than 35 mm. Among them, the strain with the best antibacterial effect is named DH4-3. The diameter of the pathogen of this strain is 24.66 mm, and the antibacterial rate is 72.60%. Figure 1 The figure shows the inhibitory effect of DH4-3 live bacteria on the seedling wilt pathogen. A is the experimental group and B is the control group. It can be seen that DH4-3 has a significant inhibitory effect on the seedling wilt pathogen. Therefore, DH4-3 was selected as the strain for the next test.

[0054] Table 1 Antibacterial activity of Trichoderma against Rhizoctonia solani

[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] The morphological observation results of strain DH4-3 are as follows Figure 2 shown.

[0059] On the PDA plate, the colony is initially white and velvety, and later there are round hyphae and dense conidial areas, which are green or dark green in color. Conidiophores are arranged in a ring, and the branches are curved or wavy. Conidia are spherical. Strain DH4-3 grows vigorously on PDA medium.

[0060] The DH4-3 strain was inoculated onto a PDA plate, and a sterilized cover slip was inserted into the culture medium at a 45° angle. After culturing at 28°C, the cover slip was removed and observed under a BX53 Olympus optical microscope. The results of observation under the microscope showed that no spores were generated when cultured at 28°C for 1-3 days, hyphae were generated in 1-3 days, spores appeared after 5 days, and spores spread in a radial manner after 7 days.

[0061] After extracting the DNA of strain DH4-3 according to the instructions of the Ezup column fungal genomic DNA extraction kit, the ITS-DNA gene fragment was amplified, and the recovered product of the amplified fragment was sent to Treasure Biotechnology (Dalian) Engineering Co., Ltd. for bidirectional sequencing. After successful sequencing and splicing, the full length of the ITS-DNA gene amplified fragment of strain DH4-3 was 573bp. After the sequence was analyzed by software such as BioEdit 7.0.1 and manually corrected, the BLAST program of NCBI was used to compare the measured sequence with the sequence of known similar model species downloaded from GenBank for homology analysis, and the neighbor-joining method (NJ) of MEGA 6.0 software was used to align the sequence and draw a phylogenetic tree. Figure 3 A phylogenetic tree was constructed for the sequences with high homology to the DH4-3 strain, and it can be seen that the strain DH4-3 is a species of the genus Trichoderma, which is Trichoderma sulphurochromis.

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

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

[0064] According to the results of detecting the diameter of pathogens in Example 1, the strain DH4-3 with the best antagonistic effect was selected to measure the antibacterial spectrum.

[0065] Poplar bark rot pathogen, nursery blight pathogen, blueberry branch blight pathogen, apple rot pathogen, cucumber sclerotinia pathogen, apple fruit rot pathogen, melon vine disease pathogen, rice seedling pathogen, red bean anthracnose pathogen, tobacco brown spot pathogen, tobacco target spot pathogen, and ictaluri pathogen were selected as test strains, and the antibacterial spectrum of DH4-3 against the above test strains was determined by the flat plate confrontation culture method. The detection method refers to Example 1.

[0066] The experimental results are shown in Table 2. It can be seen from Table 2 that the antagonistic bacteria DH4-3 has an inhibitory effect on all 12 test strains; among them, the inhibitory effect on the nursery blight pathogen is the strongest. The diameter of the pathogen after confrontation culture is only 24.66mm, which is significantly different from the other test pathogens; Figure 4 C) Cucumber Sclerotinia sclerotiorum ( Figure 4 A) and tobacco brown spot pathogens, the growth of pathogens was inhibited. When the diameter of the control pathogen reached 90mm, the diameters of the pathogens cultured with DH4-3 were 25.78mm (Pythium aphanidermatum), 25.08mm (Cucumber Sclerotinia sclerotiorum), and 26.35mm (Tobacco brown spot pathogens). Compared with the above test strains, DH4-3 had a strong inhibitory effect on tobacco target spot pathogens ( Figure 4 B) The inhibitory effect of poplar bark rot bacteria 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 Determination of the preparation of fermentation broth and its antibacterial spectrum

[0070] Prepare 40 mL of potato glucose liquid fermentation medium and put it into a 200 mL conical flask. Add 6 DH4-3 bacterial cakes with a diameter of 5 mm to each flask. After constant temperature shaking culture at 28°C and 150 r / min for 6 days, place it in a centrifuge at 7800 rpm and 4°C for 10 min. Discard the precipitate and absorb the supernatant.

[0071] The following strains were used as test strains: poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine disease, rice seedling blight, red bean anthracnose, tobacco brown spot, tobacco target spot and citrus fruit rot.

[0072] The cup-and-disc method was used to detect the antibacterial spectrum of the DH4-3 fermentation broth, comprising the following steps: placing an Oxford cup in the center of a PDA culture dish, adding 200 μL of fermentation broth (i.e., the supernatant prepared by the above method) into the Oxford cup, placing four pathogenic bacteria cakes with a diameter of 5 mm at 1.5 cm above, below, left, and right of the Oxford cup, and measuring the diameter of the inhibition zone by the cross method after constant temperature culture at 28°C for 72 hours.

[0073] The antibacterial activity of the fermentation liquid of the antagonistic strain DH4-3 was determined by the cup-plate method. The experimental results are shown in Table 3. The results show that the fermentation liquid of DH4-3 still maintains good antibacterial activity. Among them, the fermentation liquid of this strain has the strongest antibacterial effect on the fungus of nursery blight ( Figure 5 A and B), the diameter of the inhibition zone reached 22.41mm, and it also had a strong antagonistic effect on blueberry branch blight pathogen, with the diameter of the inhibition zone reaching 20.19mm, and on poplar bark rot pathogen ( Figure 5 C), tobacco brown spot pathogen and tobacco target spot pathogen also had good inhibitory effects, with the diameters of the inhibition zones being 18.36mm, 18.53mm and 17.83mm respectively. The fermentation broth of the antagonistic bacteria had obvious inhibitory effects on all the tested pathogens, with the minimum inhibition zone diameter reaching 10.77mm.

[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 P < 0.01 and P < 0.05 levels tested by Duncan's new multiple range method.

[0078] The DH4-3 provided by the present invention has strong fungicidal activity. Since the strain produces antifungal substances during physiological metabolism, its live bacteria and fermentation liquid show strong inhibitory effects on nursery blight pathogens, and can be used as a biological control agent for the prevention and control of pathogenic bacteria.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Trichoderma fermentation broth, characterized in that: It is the fermentation liquid of Trichoderma sulphureum DH4-3, and the preservation number of Trichoderma sulphureum DH4-3 is CGMCC No.40704.

2. The method for preparing the Trichoderma fermentation liquid according to claim 1, wherein The following steps are involved: The culture medium was inoculated with Trichoderma sulphuroides DH4-3 for fermentation.

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

4. The preparation method according to claim 2, characterized in that: The fermentation temperature is 28°C and the fermentation time is 5-7 days.

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

6. Use of the Trichoderma fermentation liquid of claim 1 in the prevention and treatment of plant pathogenic bacteria; the plant pathogenic bacteria are selected from one or a combination of poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine disease, rice seedling rot, red bean anthracnose, tobacco brown spot, tobacco target spot and citrus fruit rot.

7. Use of the Trichoderma fermentation liquid of claim 1 in preventing and treating symptoms caused by plant pathogenic bacteria; the plant pathogenic bacteria are selected from one or a combination of poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine disease, rice seedling blight, red bean anthracnose, tobacco brown spot, tobacco target spot and citrus fruit rot.

8. Use of the Trichoderma fermentation liquid of claim 1 in the preparation of a biocontrol agent; the biocontrol agent is used to control diseases caused by plant pathogens; the plant pathogens are selected from one or a combination of poplar bark rot, nursery blight, blueberry branch blight, apple rot, cucumber sclerotinia, apple fruit rot, melon vine disease, rice seedling blight, red bean anthracnose, tobacco brown spot, tobacco target spot and citrus fruit rot.

9. Use of the Trichoderma fermentation liquid according to claim 1 in preventing and treating damping-off disease of seedlings.

10. A biological control method, characterized in that: The method comprises the following steps: using the Trichoderma fermentation liquid described in claim 1 for biological control to prevent and control diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are selected from one or a combination of poplar bark rot pathogen, nursery blight pathogen, blueberry branch blight pathogen, apple rot pathogen, cucumber sclerotinia pathogen, apple fruit rot pathogen, melon vine disease pathogen, rice seedling rot pathogen, red bean anthracnose pathogen, tobacco brown spot pathogen, tobacco target spot pathogen and citrullus rot pathogen.

Images