Cladosporium erythraeum YNF2QT fermentation extract and application thereof
By isolating and fermenting C. red fermenting YNF2QT, its fermented extract was prepared, which solved the problem of difficult inhibiting the pathogen of tobacco red star disease in the prior art, achieved effective inhibition of the pathogen, and had significant agricultural application potential.
Patent Information
- Application Number
- CN202411835592.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to effectively inhibit the pathogens of tobacco red star disease, and there is a lack of effective agricultural application solutions.
The active ingredients that inhibit the pathogens of tobacco Red Arosenis YNF2QT were prepared by isolating and fermenting C. red C. 2QT, and the fermentation extract was prepared, and the active ingredients that inhibited the pathogens of tobacco Red Astrology were obtained through steps such as ethyl acetate extraction and methanol elution.
The fermented extract of C. red C. red has a significant inhibitory effect on the pathogens of tobacco red star disease. The diameter of the antibacterial circle is 35.0mm on average, showing strong antibacterial effect and has potential agricultural application value.
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Figure CN119614385A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial pesticides, and in particular to a Cladosporium rubrum YNF2QT fermentation extract and application thereof. Background Art
[0002] Cladosporium is a type of fungus in the Cladosporiaceae family that can produce conidia and is widely distributed. Cladosporium strains have been reported to produce a variety of active ingredients. For example, Jin Ran screened out a strain of Cladosporium sp. Bio-1 that can produce lignin-degrading enzyme-laccase and can grow with alkaline lignin as the sole carbon source (Hunan University, 2012); Ma Rui isolated two strains of C. tianshanense SL-14 and C. neopsychrotolerans SL-16 with significant lignocellulose degradation ability (China Agricultural University, 2017); Liu Huanhuan et al. isolated multiple secondary metabolites from Cladosporium sp. (Chemical Engineering Design Communications, 2019, 45(3)).
[0003] The research team isolated and obtained multiple strains of Cladosporium from soil samples from many places in Yunnan, and studied the inhibitory effects of their fermentation products on tobacco pathogens such as tobacco brown spot disease, tobacco bacterial wilt, and tobacco black shank disease. A red Cladosporium (Cladosporium rubrum) YNF2QT was obtained, which has a good inhibitory effect on tobacco brown spot disease pathogens and has important agricultural application value and potential. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a red Cladosporium YNF2QT fermentation extract and application thereof, which has a good inhibitory effect on tobacco brown spot disease pathogens and shows certain agricultural application potential.
[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:
[0006] Cladosporium rubrum YNF2QT is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242803.
[0007] A bacterial agent comprises Cladosporium rubrum YNF2QT.
[0008] A Cladosporium rubrum YNF2QT fermentation extract, wherein the fermentation extract is prepared by the following method:
[0009] (1) Solid activation of strains: The strain YNF2QT with the deposit number of CCTCC NO: M 20242803 was inoculated into a solid slant medium in a test tube and cultured at 28°C for 6-9 days to obtain an activated strain;
[0010] (2) Liquid culture of strains: inoculate the activated strain into a liquid culture medium in a triangular flask, and culture at 28°C in a shaker at 180-200 rpm for 4-7 days to obtain a strain seed solution;
[0011] (3) Strain expansion culture: inoculate the strain seed liquid into the liquid fermentation medium in a triangular flask, and culture at 28°C in a shaking incubator at 180-200 rpm for 11-13 days to obtain the strain fermentation liquid;
[0012] (4) Extract preparation: The fermentation broth of the strain was ultrasonicated for 30-45 min, extracted with ethyl acetate for 20-30 h, and concentrated by rotary evaporation. The extract was eluted with methanol to obtain the fermentation extract.
[0013] Preferably, in the above technical solution, in step (1), the solid slant culture medium is PDA culture medium, which is sterilized by high pressure steam at 121° C. for 15 min.
[0014] Preferably, in the above technical solution, in step (2), the liquid culture medium is PDB culture medium, 100 mL of PDB culture medium is placed in each 500 mL Erlenmeyer flask, and sterilized by high pressure steam at 121° C. for 15 min.
[0015] Preferably, in the above technical solution, in step (2), the inoculation amount of the activated bacteria is 1.5-2 cm 2 .
[0016] Preferably, in the above technical solution, in step (3), the liquid culture medium is PDB culture medium, and each 2000 mL Erlenmeyer flask is filled with 250-300 mL PDB culture medium, and the rest of the preparation method is the same as step (2).
[0017] Preferably, in the above technical solution, in step (3), the inoculation amount of the strain seed fermentation liquid is 10-15 mL per Erlenmeyer flask.
[0018] Preferably, in the above technical solution, in step (4), the volume ratio of the strain fermentation broth to ethyl acetate is 1:1.5-1:3; and 2-2.5 mL of methanol is used for elution.
[0019] The invention discloses an application of Cladosporium rubrum YNF2QT or a bacterial agent or a fermented extract in preparing a medicine for inhibiting pathogenic bacteria of tobacco brown spot disease.
[0020] The above technical solution of the present invention has the following beneficial effects:
[0021] The present application provides a method for preparing a red Cladosporium YNF2QT fermentation extract, and the fermentation extract is used to inhibit tobacco brown spot disease. The results of the plate antibacterial test show that the average diameter of the antibacterial zone of the red Cladosporium YNF2QT fermentation extract prepared by the method of the present invention against the tobacco brown spot disease pathogen is 35.0 mm, indicating that the fermentation extract has strong inhibitory activity against the tobacco brown spot disease pathogen, and the red Cladosporium YNF2QT fermentation extract provided by the present invention can be effectively used to inhibit tobacco brown spot disease pathogen. The present invention has the advantages of simple preparation process and strong antibacterial effect, and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
[0023] Figure 1 This is the phylogenetic tree of Cladosporium rubrum YNF2QT;
[0024] Figure 2 The figure is a plate culture picture of Cladosporium rubrum YNF2QT, the left picture is the front picture of the culture medium, and the right picture is the back picture of the culture medium;
[0025] Figure 3 This is a graph showing the inhibition of the fermentation extract of Cladosporium rubrum YNF2QT on tobacco brown spot disease pathogens. The left figure shows the inhibition zone diagram of methanol on tobacco brown spot disease pathogens (CK), and the right figure shows the inhibition zone diagram of the fermentation extract on tobacco brown spot disease pathogens (YNF2QT). DETAILED DESCRIPTION
[0026] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.
[0027] Unless otherwise specified, the reagents used in this application are all commercially available or obtained through commercial channels, or can be prepared by referring to existing chemical methods.
[0028] Example 1
[0029] Identification of Cladosporium rubrum YNF2QT:
[0030] A fungus strain numbered YNF2QT was screened from the soil of Yunnan Stone Forest. The strain DNA was extracted using the conventional CTAB method. The ITS sequence was amplified using fungal universal primers ITS1 and ITS4. The PCR product was sent to Beijing Qingke (Kunming) Xinye Biotechnology Co., Ltd. for sequencing. The sequenced DNA sequence was compared by BLAST search in the NCBI database. The ITS sequence of the strain had a similarity of 90.54% with the effectively published species Cladosporium rubrum (GenBank No: MN053018). Therefore, the strain was identified as Cladosporium rubrum.
[0031] Cladosporium rubrum YNF2QT was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on December 12, 2024, with the accession number CCTCCNO: M 20242803.
[0032] Example 2
[0033] The preparation method of the Cladosporium rubrum YNF2QT fermentation extract comprises the following steps:
[0034] (1) Solid activation of strains: strain YNF2QT was inoculated into a test tube solid slant medium and cultured at 28°C for 6-9 days to obtain activated strains; the solid slant medium was PDA medium, sterilized by high pressure steam at 121°C for 15 min;
[0035] (2) Liquid culture of strains: The activated strains were inoculated into the liquid culture medium of the triangular flask. The inoculation amount of the activated strains was 1.5-2.0 cm per triangular flask. 2 , cultured at 28°C in a shaker at 180-200 rpm for 4-7 days to obtain a strain seed solution; the liquid culture medium is PDB culture medium, 100 mL of PDB culture medium is placed in each 500 mL conical flask, and sterilized by high-pressure steam at 121°C for 15 min;
[0036] (3) Strain expansion culture: inoculate the strain seed liquid into a liquid fermentation medium in a triangular flask, the inoculation amount of the strain seed fermentation liquid is 10-15 mL per triangular flask, and culture at 28°C in a shaker at 180-200 rpm for 11-13 days to obtain the strain fermentation liquid; the liquid medium is PDB medium, and each 2000 mL triangular flask is filled with 250-300 mL PDB medium, and the rest of the preparation method is the same as step (2);
[0037] (4) Extract preparation: The fermentation broth of the strain was sonicated for 20-30 min, extracted with 1:1.5-1:3 ethyl acetate for 20-30 h, and eluted with 2-2.5 mL of methanol to obtain a fermentation extract.
[0038] Example 3
[0039] The plate antibacterial test of the fermentation extract against the pathogen of tobacco brown spot disease includes the following steps:
[0040] (1) Preparation of the fermentation extract of Cladosporium rubrum YNF2QT for inhibiting tobacco brown spot disease pathogen: The fermentation extract of the present invention for inhibiting tobacco brown spot disease pathogen by the microorganism is prepared from strain YNF2QT by a liquid fermentation method, and the specific preparation method is the same as that in Example 2.
[0041] (2) Cultivation of tobacco brown spot pathogen: Inoculate tobacco brown spot pathogen onto test tube slant medium and culture at 28°C for 4-5 days until the bacteria grow well. The test tube slant medium is PDA medium.
[0042] (3) Plate antibacterial test method: Oxford cup method was used.
[0043] Add an appropriate amount of sterile water to the cultured test tube of tobacco brown spot pathogen, blow with a sterile pipette tip, collect spores and prepare a spore suspension with a concentration of 10 6 / mL, take 1mL spore suspension and mix it with 20mL PDA medium, pour it into a sterile culture dish with an inner diameter of 9cm to make a bacterial plate. Take a piece of sterile filter paper, put it in an Oxford cup, make it close to the cup wall, place the Oxford cup on the bacterial plate, and let it stand for 3-4h. Add 0.2mL of the prepared red Cladosporium (Cladosporium rubrum) YNF2QT fermentation extract to the Oxford cup, use methanol as a blank control, and repeat 3 times for each treatment. After culturing in a 28℃ constant temperature box for 48h, the diameter of the inhibition zone is measured by the cross method. The results of the inhibition zone diameters of the test samples of the red Cladosporium (Cladosporiumrubrum) YNF2QT fermentation extract were 32.0mm, 37.0mm, 35.0mm, and 36.0mm, respectively, with an average of 35.0mm, and the diameter of the methanol control inhibition zone was 0mm.
[0044] The experimental results showed that the average diameter of the inhibition zone of the fermentation extract of Cladosporium rubrum YNF2QT against tobacco brown spot disease pathogens was 35.0 mm.
[0045] Example 4
[0046] Preparation and application of Cladosporium rubrum YNF2QT bacterial agent:
[0047] The preparation method of the bacterial agent is the same as steps (1) to (3) of Example 2. The prepared bacterial agent is sprayed onto the surface of tobacco leaves by spraying, which has a preventive effect on tobacco brown spot disease.
[0048] The Cladosporium rubrum YNF2QT fermentation extract of the present application has strong inhibitory activity against tobacco brown spot pathogens, has the advantages of simple preparation process and strong antibacterial effect, and has potential application value.
[0049] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.
Claims
1. Cladosporium rubrum YNF2QT, characterized in that It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20242803.
2. A Cladosporium rubrum YNF2QT fermentation extract, characterized in that: The fermentation extract is prepared by the following method: (1) Solid activation of strains: The strain YNF2QT with the deposit number of CCTCC NO: M20242803 as claimed in claim 1 is inoculated into a test tube solid slant medium, and statically cultured at 28° C. for 6-9 days to obtain an activated strain; (2) Liquid culture of strains: inoculate the activated strain into a liquid culture medium in a triangular flask, and culture at 28°C in a shaker at 180-200 rpm for 4-7 days to obtain a strain seed solution; (3) Strain expansion culture: inoculate the strain seed liquid into the liquid fermentation medium in a triangular flask, and culture at 28°C in a shaking incubator at 180-200 rpm for 11-13 days to obtain the strain fermentation liquid; (4) Extract preparation: The fermentation broth of the strain was ultrasonicated for 30-45 min, extracted with ethyl acetate for 20-30 h, and concentrated by rotary evaporation. The extract was eluted with methanol to obtain the fermentation extract.
3. The Cladosporium rubrum YNF2QT fermentation extract according to claim 2, characterized in that In step (1), the solid slant culture medium is PDA culture medium, which is sterilized by high pressure steam at 121° C. for 15 min.
4. The Cladosporium rubrum YNF2QT fermentation extract according to claim 2, characterized in that In step (2), the liquid culture medium is PDB culture medium, and each 500 mL Erlenmeyer flask is filled with 100 mL PDB culture medium, and sterilized by high pressure steam at 121° C. for 15 min.
5. The Cladosporium rubrum YNF2QT fermentation extract according to claim 4, characterized in that In step (2), the inoculation amount of the activated bacteria is 1.5-2cm per triangular flask. 2 .
6. The Cladosporium rubrum YNF2QT fermentation extract according to claim 2, characterized in that In step (3), the liquid culture medium is PDB culture medium, and each 2000 mL Erlenmeyer flask is filled with 250-300 mL PDB culture medium. The rest of the preparation method is the same as step (2).
7. The Cladosporium rubrum YNF2QT fermentation extract according to claim 6, characterized in that In step (3), the inoculation amount of the strain seed fermentation liquid is 10-15 mL per Erlenmeyer flask.
8. The Cladosporium rubrum YNF2QT fermentation extract according to claim 2, characterized in that In step (4), the volume ratio of the fermentation broth of the strain to ethyl acetate is 1:1.5-1:3; and 2-2.5 mL of methanol is used for elution.
9. A bacterial agent, characterized in that The bacterial agent comprises the Cladosporium rubrum YNF2QT described in claim 1.
10. Use of the Cladosporium rubrum YNF2QT according to claim 1, the fermentation extract according to any one of claims 2 to 8, or the bacterial agent according to claim 9 in the preparation of a medicament for inhibiting the pathogen of tobacco brown spot disease.