Sagonosporopsis pogostemonis YNF4655 fermentation extract and application thereof

Abstract

The invention discloses a Stagnosporopsis pogostemonis YNF4655 fermentation extract and application of the Stagnosporopsis pogostemonis YNF4655 fermentation extract, the Stagnosporopsis pogostemonis YNF4655 is preserved in the China Center for Type Culture Collection, and the preservation number of the Stagnosporopsis pogostemonis YNF4655 is CCTCC NO: M 20242804. The strain is fermented to prepare a Stagonosporopsis pogostemonis YNF4655 extracting solution, and the average diameter of an inhibition zone of the Stagonosporopsis pogostemonis YNF4655 extracting solution on pathogenic bacteria of the tobacco brown spot is 32.5 mm, so that the fermented extract has strong inhibition activity on the pathogenic bacteria of the tobacco brown spot and can be applied to inhibition of the pathogenic bacteria of the tobacco brown spot. Meanwhile, the invention has the advantages of simple preparation process flow and low raw material cost, and has better potential application value in the technical field of crop pesticides.

Description

Technical Field

[0001] The invention relates to the technical field of microbial pesticides, and in particular to a Stagonosporopsis pogostemonis YNF4655 fermentation extract and application thereof. Background Art

[0002] Tobacco brown spot disease (TBSD) is a fungal disease caused by Alternaria alternata, which mainly harms tobacco leaves at maturity, seriously affecting the yield and quality of tobacco leaves. It has become one of the major fungal diseases affecting tobacco production worldwide. For many years, chemical agents have been mainly used to prevent and control the disease. Although they can effectively control the disease, tobacco is a leaf-producing plant, and chemical agents are prone to produce pesticide residues or environmental pollution, and even produce resistance, which has affected the quality and safety of tobacco leaves and the ecological environment. Biocontrol bacteria have the advantages of being green, highly effective and leaving no residue when used to control crop diseases. Currently, there have been studies on microbial agents for controlling tobacco brown spot disease, such as Trichoderma harzianum Rifai (CN114467979B), Bacillus paralicheniformis (CN117264834A), Streptomyces globisporus (CN101422170B) and other agents. In addition, Yang Shuiying et al. screened and obtained the chitinase-producing bacterium FEC-1, which has an inhibitory effect on the hyphae growth of tobacco brown spot disease bacteria, with an inhibition bandwidth of 12.6 mm (Journal of Northwest Agricultural University, 2007, 16(4):5).

[0003] Therefore, screening and developing microbial agents that antagonize tobacco brown spot disease pathogens is one of the key contents of biological control of tobacco brown spot disease and has important agricultural application value. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a Stagonosporopsis pogostemonis YNF4655 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] Stagonosporopsis pogostemonis YNF4655, which is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20242804.

[0007] A Stagonosporopsis pogostemonis YNF4655 fermentation extract, wherein the fermentation extract is prepared by the following method:

[0008] (1) Solid activation of strains: The strain YNF4655 with the deposit number of CCTCC NO: M 20242804 was inoculated into a solid slant medium in a test tube and cultured at 28°C for 4-5 days to obtain an activated strain;

[0009] (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 3-4 days to obtain a strain seed solution;

[0010] (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 shaker at 180-200 rpm for 10-12 days to obtain the strain fermentation liquid;

[0011] (4) Extract preparation: The fermentation broth of the strain was sonicated for 30-45 min, extracted with ethyl acetate for 15-25 h, and concentrated by rotary evaporation to obtain a crude extract, which was eluted with methanol to obtain a fermentation extract.

[0012] 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.

[0013] 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.

[0014] Preferably, in the above technical solution, in step (2), the inoculation amount of the activated bacteria is 1-1.5 cm 2 .

[0015] 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).

[0016] 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.

[0017] Preferably, in the above technical solution, in step (4), the volume ratio of the strain fermentation broth to ethyl acetate is 1:2-1:3; and 1.5-2 mL of methanol is used for elution.

[0018] A bacterial agent comprising Stagonosporopsis pogostemonis YNF4655.

[0019] The invention discloses an application of Stagonosporopsis pogostemonis YNF4655 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 fermentation extract of Stagonosporopsis pogostemonis YNF4655, 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 inhibition zone of the Stagonosporopsis pogostemonis YNF4655 fermentation extract prepared by the method of the present invention against tobacco brown spot disease pathogens is 40.0 mm, indicating that the fermentation extract has strong inhibitory activity against tobacco brown spot disease pathogens, and the Stagonosporopsis pogostemonis YNF4655 fermentation extract provided by the present invention can be effectively used to inhibit tobacco brown spot disease pathogens. At the same time, the present invention has the advantages of simple preparation process and low raw material cost, and has good potential application value in the field of crop pesticide technology. 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 Phylogenetic tree for Stagonosporopsis pogostemonis YNF4655;

[0024] Figure 2 The plate culture pictures of Stagonosporopsis pogostemonis YNF4655, 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 the inhibition chart of the fermentation extract of Stagonosporopsis pogostemonis YNF4655 on the pathogen of tobacco brown spot disease. The left picture is the inhibition circle chart of methanol on the pathogen of tobacco brown spot disease (CK), and the right picture is the inhibition circle chart of the fermentation extract on the pathogen of tobacco brown spot disease (YNF4655). 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 Stagonosporopsis pogostemonis YNF4655:

[0030] A fungus strain numbered YNF4655 was screened from the soil of Lincang, Yunnan. 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 similarity between the ITS sequence of the strain and the effectively published species Stagonosporopsis pogostemonis (GenBank No: PP349771) was 93.80%. Combined with the colony morphology and microscopic characteristics of the strain, the strain was identified as Stagonosporopsis pogostemonis.

[0031] Stagonosporopsis pogostemonis YNF4655 was deposited on December 12, 2024 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the accession number CCTCC NO: M20242804.

[0032] Example 2

[0033] The preparation method of the fermentation extract of Stagonosporopsis pogostemonis YNF4655 comprises the following steps:

[0034] (1) Solid activation of strains: strain YNF4655 was inoculated into a test tube solid slant medium and cultured at 28°C for 4-5 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 a liquid culture medium in a triangular flask, with an inoculation amount of 1-1.5 cm2 of the activated strains per triangular flask, and cultured at 28°C in a shaker at 180-200 rpm for 3-4 days to obtain a strain seed solution; the liquid culture medium was PDB culture medium, and 100 mL of PDB culture medium was placed in each 500 mL triangular 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 and 180-200 rpm for 10-12 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 30-45 min, extracted with 1:2-1:3 ethyl acetate for 15-25 h, and concentrated by rotary evaporation to obtain a crude extract, which was eluted with 1.5-2.0 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 Stagonosporopsis pogostemonis YNF4655 for inhibiting tobacco brown spot disease pathogens: The fermentation extract of the microorganisms for inhibiting tobacco brown spot disease pathogens of the present invention is prepared from strain YNF4655 by a liquid fermentation method. The specific preparation method is the same as that of 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 of spore suspension and mix it with 20mL of 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 Stagonosporopsis pogostemonis YNF4655 fermentation extract to the Oxford cup, use methanol as a blank control, and repeat 3 times for each treatment. After culturing in a 28℃ incubator 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 Stagonosporopsis pogostemonis YNF4655 fermentation extract were 33.0mm, 35.0mm, 26.0mm, and 36.0mm, respectively, with an average of 32.5mm, and the diameter of the methanol control inhibition zone was 0mm.

[0044] The experimental results show that the average diameter of the inhibition zone of the fermentation extract of Stagonosporopsis pogostemonis YNF4655 against tobacco brown spot pathogen is 32.5 mm.

[0045] Example 4

[0046] Preparation and application of Stagonosporopsis pogostemonis YNF4655 inoculant:

[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 Stagonosporopsis pogostemonis YNF4655 fermentation extract of the present application has strong inhibitory activity against tobacco brown spot disease pathogens, and can be used in the prevention and treatment of tobacco brown spot disease to prevent the pathogens from developing drug resistance.

[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. Stagonosporopsis pogostemonis YNF4655, characterized in that It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242804.

2. A Stagonosporopsis pogostemonis YNF4655 fermentation extract, characterized in that The fermentation extract is prepared by the following method: (1) Solid activation of strains: The strain YNF4655 with the deposit number of CCTCC NO: M20242804 as claimed in claim 1 is inoculated into a solid slant medium in a test tube, and cultured at 28° C. for 4-5 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 3-4 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 shaker at 180-200 rpm for 10-12 days to obtain the strain fermentation liquid; (4) Extract preparation: The fermentation broth of the strain was sonicated for 30-45 min, extracted with ethyl acetate for 15-25 h, and concentrated by rotary evaporation to obtain a crude extract, which was eluted with methanol to obtain a fermentation extract.

3. The Stagonosporopsis pogostemonis YNF4655 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 Stagonosporopsis pogostemonis YNF4655 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 Stagonosporopsis pogostemonis YNF4655 fermentation extract according to claim 4, characterized in that In step (2), the inoculation amount of the activated bacteria is 1-1.5 cm per triangular flask. 2 .

6. The Stagonosporopsis pogostemonis YNF4655 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 Stagonosporopsis pogostemonis YNF4655 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 Stagonosporopsis pogostemonis YNF4655 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 1:2-1:3; and 1.5-2 mL of methanol is used for elution.

9. A bacterial agent, characterized in that The bacterial agent comprises the Stagonosporopsispogostemonis YNF4655 described in claim 1.

10. Use of Stagonosporopsis pogostemonis YNF4655 according to claim 1, the fermented extract according to any one of claims 2 to 8, or the bacterial agent according to claim 9 in the preparation of a drug for inhibiting pathogenic bacteria of tobacco brown spot disease.

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