CN114467978A

CN114467978A - Application of rosa roxburghii endophytic fungi and metabolite thereof in broad-spectrum antibacterial agent and preparation

Info

Publication number: CN114467978A
Application number: CN202210249069.2A
Authority: CN
Inventors: 姜于兰; 张洪
Original assignee: GUIZHOU ACADEMY OF TESTING AND ANALYSIS; Guizhou University
Current assignee: GUIZHOU ACADEMY OF TESTING AND ANALYSIS; Guizhou University
Priority date: 2022-03-14
Filing date: 2022-03-14
Publication date: 2022-05-13
Anticipated expiration: 2042-03-14
Also published as: CN114467978B

Abstract

The invention discloses an application and a preparation of roxburgh rose endophytic fungi and metabolites thereof in a broad-spectrum antibacterial agent. The invention provides a Rosa roxburghii endophytic fungus and metabolite thereof, wherein the minimum inhibitory concentration of the Rosa roxburghii endophytic fungus on Lasiodipida theobroma, Borgyphispora dothidea, Colletotrichum capsaici, Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotom, Talaromyces kabodanensis, Pseudomonas syrgae pv.actindiae, Pantoea agglomerans, Staphyloccocus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa is 1.25, 2.50, 10.00, 1.25, 0.31, 1.25, 5.00, 0.62, 2.50 and 2.50mg/mL respectively. The components with broad-spectrum antibacterial activity are obtained by microbial fermentation, and the process is simple, safe and environment-friendly.

Description

Application of rosa roxburghii endophytic fungi and metabolite thereof in broad-spectrum antibacterial agent and preparation

Technical Field

The invention relates to an application of a microorganism, in particular to an application and a preparation of roxburgh rose endophytic fungus and metabolite thereof in a broad-spectrum antibacterial agent.

Background

Endophytic fungi (Endophytic fungi) refer to a class of fungi that live in cells within the plant body or within plant tissues at a certain period of their life history without causing significant disease to the plant tissues. In recent years, many secondary metabolites with potent antipathogenic, insecticidal, antioxidant, cytotoxic and anticancer properties have been discovered from endophytic fungi of plants. Endophytic fungi can not only produce plant hormones to promote the growth of host plants, produce bioactive compounds to increase the resistance of plants to environmental stress, but also promote the accumulation of secondary metabolites, including pharmaceutical ingredients, initially produced by plants. Rosa roxburghii Tratt is known to be rich in vitamin C, superoxide dismutase (SOD) and flavonoids. In the traditional medicine, the rosa roxburghii tratt is mainly used as a medicine by roots, leaves and fruits and is used for treating diseases such as digestion, dysentery, hypertension, vitamin C deficiency and the like, and the pharmacological action of the rosa roxburghii tratt is recorded in the book Ben Cao gang mu Shi Yi (supplement to the compendium of materia Medica). Modern pharmacological studies show that various phytochemicals extracted from rosa roxburghii tratt also have biological activities of resisting fungi, bacteria and oxidation, regulating the immune function of an organism, preventing and treating type 2 diabetes and the like. Since Rosa roxburghii is a plant source of antibacterial compounds, it is also an ideal resource for screening endophytic fungi with antibacterial potential. The endophytic fungi is separated from the rosa roxburghii and the endophytic fungi with potential biological activity is searched for so as to develop a natural product with biological activity.

Disclosure of Invention

The invention aims to provide application of Rosa roxburghii endophytic fungus Epicoccum latenicolulum and metabolite thereof in preparation of broad-spectrum antibacterial agent.

The technical scheme adopted by the invention is as follows:

the application of the rosa roxburghii endophytic fungus Epicoccum latusicolum and metabolite thereof in the broad-spectrum antibacterial agent is characterized in that the rosa roxburghii endophytic fungus Epicoccum latusicolum is preserved to China general microbiological culture collection center (CGMCC), and the biological preservation number is as follows: CGMCC 40110, China academy of sciences microorganism research institute No. 3, Xilu No.1, North Cheng, Chaozhou, the address Beijing, and requests the depositor to specify the name, strain number or symbol of the culture: epicoccum latesulum HGUP191049.

The colony of the Rosa roxburghii endophytic fungus Epicoccum latusoolum HGUP191049 is white, flocculent, round and light gray, and has a light red pigment precipitate near the center.

The use of the endophytic fungi of Rosa roxburghii (Epicococcus lauricum) and its metabolites in broad spectrum antibacterial agents corresponding to the antifungal agents of Erysipelothromyces cacao (Lasiocladia theobroma), Staphylococus botrytis (Botryosphaeria dothidea), Colletotrichum capsici (Colletotrichum capsaicii), Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), Sclerotium sclerotiorum (Sclerotium sclerotiorum) and Fusarium fructicum (Talaromyces kabodanensis), and Pseudomonas syringae (Pseudomonas syringae pv. acta), Pantoea agglomerans (Pantoea agglomerans), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis (Pseudomonas aeruginosa).

The preparation method of the metabolite of the rosa roxburghii endophytic fungi comprises the steps of fermenting and culturing the rosa roxburghii endophytic fungi Epicoccum latusicollum to obtain a culture solution, separating the culture solution into a fermentation solution and a mycelium through suction filtration or high-speed centrifugation, and discarding the mycelium; the fermentation liquor is extracted by ethyl acetate, and the organic phase is concentrated to constant weight to obtain Epicoccum lateucinolum metabolite, namely the broad-spectrum antibacterial agent.

The preparation method of the metabolite of the rosa roxburghii endophytic fungi comprises the following steps: fermenting and culturing the Rosa roxburghii endophytic fungus Epicoccum latusicolum to obtain a culture solution, separating the culture solution into a fermentation solution and a mycelium through suction filtration or high-speed centrifugation, and discarding the mycelium; extracting the fermentation liquor by ethyl acetate, concentrating the organic phase to constant weight, and obtaining Epicoccum lateuciolum HGUP191049 metabolite: inoculating Rosa roxburghii endophytic fungus Epicoccum latusollum HGUP191049 into a PDB fermentation culture medium, culturing at the temperature of 28 +/-1 ℃ at 160-220 r/min for 5-14 d to obtain a culture solution, separating the culture solution into a fermentation solution and a mycelium through suction filtration or high-speed centrifugation (10000-12000 r/min), and discarding the mycelium. Extracting the fermentation liquor by ethyl acetate (the volume ratio is 1: 1-1: 2), repeatedly extracting for 3 times, and concentrating under reduced pressure at 35-55 ℃ to constant weight to obtain Epicoccum latusollum HGUP191049 metabolite, namely the broad-spectrum antibacterial agent. The culture medium is Potato Dextrose Broth (PDB): 200g of potato (peeled), 20g of glucose and 1000mL of distilled water, and the pH value is natural.

Furthermore, before the fermentation of the roxburgh rose endophytic fungus Epicoccum latusicollum, activation culture is carried out, and then the roxburgh rose endophytic fungus Epicoccum latusicollum is directly inoculated into a fermentation medium (or is stored at the low temperature of 4 ℃ for 5-30 days). The activation culture comprises the following steps: epicoccum latenicolum HGUP191049 is inoculated into a Potato Dextrose Agar (PDA) culture medium and cultured for 3-7 days at the temperature of 28 +/-1 ℃. The PDA culture medium is as follows: 200g of potato, 20g of glucose, 15-20 g of agar, 1000mL of distilled water and natural pH.

Compared with the prior art, the invention provides the roxburgh rose endophytic fungus Epicoccum latusicollum, broad-spectrum antibacterial active ingredients are obtained through microbial fermentation, and Epicoccum laticolelum metabolite can be used as a broad-spectrum antibacterial agent, for the strains of Erysiphe cacao (Lasiodipia theobromae), Staphylococus viticola (Botryosphaeria dothidea), Colletotrichum capsici (Colletotrichum capsaici), Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Phycomyces carotovora (Talaromyces kabodanensis), Pseudomonadactylus syringii (Pseudomonas syringica pv. acidianus), Pantoea agglomerans (Pantoea agglomerans), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa (Pseudomonas aeruginosa) the concentrations of the strains are respectively, 25.00, 25.0.25.0, 25.0, 25.0.5, 25.0.0, 25.0.0.5, 25.0.0, 25.0.0.0, 25.0.0, and/or more. Because the components are natural, the broad-spectrum antibacterial agent with natural components is a future market trend, the process is simple, safe and environment-friendly, and the test results are shown in fig. 4 and fig. 5.

Drawings

FIG. 1 shows the colony morphology (front and back) of the strain HGUP 191049;

FIG. 2 is a phylogenetic tree of strain HGUP 191049; FIG. 2 is a phylogenetic tree constructed using the maximum likelihood method based on multiple gene (ITS, LSU, TUB and RPB2) sequences. The maximum likelihood method self-expansion support rate (ML is more than or equal to 50 percent) and the Bayes posterior probability (PP is more than or equal to 0.90) are marked near the node (ML/PP). Epicoccum latusicolum HGUP191049.1, Epicoccum latusicolum HGUP191049.2 and Epicoccum latusicolum HGUP191049.3 represent the results of 3 independent sequencing of Epicoccum latusicolum HGUP191049, respectively. T: a model strain.

FIG. 3 is a culture solution of strain HGUP191049 fermented for 5-14 d;

FIG. 4 is a photograph showing the antifungal activity of an ethyl acetate extract phase of Epicoccum latusicollum fermentation broth;

in the figure, A is Epicoccum latenicolumm HGUP191049 metabolite (20mg/mL), B is carbendazim positive control (20mg/mL), and C is dimethyl sulfoxide (DMSO) negative control. 1 is inhibiting Erysiphe cacao (Lasiodipia theobromae); 2 inhibiting Staphylococus (Botryosphaeria dothidea); 3 is Colletotrichum capsici (Colletotrichum capsicii); 4, inhibiting Rhizoctonia solani (Rhizoctonia solani); 5 is the inhibition of Fusarium oxysporum (Fusarium oxysporum); sclerotinia sclerotiorum (sclerotiorum) is inhibited at 6, and Phycomyces kabodanensis (Talaromyces kabodanensis) is inhibited at 7.

FIG. 5 is a photograph showing the antibacterial activity of an ethyl acetate extract of Epicoccum latusicollum fermentation broth;

in the figure, A is Epicoccum latusoolum HGUP191049 metabolite (20mg/mL), B is streptomycin sulfate or penicillin sodium (20mg/mL) positive control, and C is dimethyl sulfoxide (DMSO) negative control. 1 is clove Pseudomonas kiwi fruit pathogenic variety (Pseudomonas syringae pv. actiniae); 2 is Pantoea agglomerans (Pantoea agglomerans); 3 Staphylococcus aureus (Staphylococcus aureus); 4 is Bacillus subtilis; escherichia coli (Escherichia coli) 5; 6 is Pseudomonas aeruginosa (Pseudomonas aeruginosa).

The Rosa roxburghii endophytic fungus Epicoccum latusicollum is sent to China general microbiological culture Collection center (CGMCC) at 3 months and 6 days in 2022, and the microbial research institute of China academy of sciences, No. 3 of Xilu No.1 of Beijing area facing Yang requires the name, strain number or symbol of the culture appointed by the depositor: epicoccum latesulum HGUP191049.

The rosa roxburghii endophytic fungus Epicoccum latusicollum is preserved to China general microbiological culture Collection center (CGMCC), and the biological preservation number is as follows: CGMCC 40110, China academy of sciences microorganism research institute No. 3, Xilu No.1, North Cheng, Chaozhou, the address Beijing, and requests the depositor to specify the name, strain number or symbol of the culture: epicoccum latusicollum HGUP191049

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the roxburghii Tratt is deciduous shrub of Rosa in Rosaceae. The fruit is known as the 'Vc king' in fruit.

Example 1:

the material used, double distilled water, refers to water having a resistivity of up to 18M Ω cm (25 ℃). Besides water molecules, the water has almost no impurities, no organic matters such as bacteria, viruses and chlorine-containing dioxin, and no mineral trace elements required by human bodies. The sterile double distilled water is obtained by sterilizing double distilled water through a high-pressure steam sterilizing pot under the conditions of 121 ℃ and 15-30 minutes.

1. Collecting a plant sample: fresh and healthy tissue samples (roots, stems, leaves, flowers, fruits and seeds) of Rosa roxburghii are collected from Xiuwen county, Guiyang, Guizhou province and Diazhou, Liudianshui, Guizhou province. All samples were immediately sent to the laboratory and stored in a 4 ℃ freezer. The separation of endophytic fungi from the sample tissue is completed within 48 hours after collection.

2. And (3) separating endophytic fungi: all tissues were rinsed under tap water for 30min, with double distilled water for 10min, and allowed to air dry under natural conditions. The tissue is then cut into small pieces and transferred to a clean bench for surface sterilization. All samples were surface sterilized with 75% ethanol (1 min) and rinsed 3 times with sterile double distilled water. Followed by surface disinfection with aqueous sodium hypochlorite (1% available chlorine) (roots and fruits, 2 min; stems and seeds, 3 min; leaves and flowers, 1 min) and rinsing 3 times with sterile double distilled water. After rinsing, the surface water of the tissue was blotted dry with sterile filter paper and transferred to Potato Dextrose Agar (PDA) medium. To avoid bacterial contamination, 30. mu.g/mL streptomycin was added to PDA medium. Each culture plate contains 3-5 tissue segments, and after 3-7 days of culture at 28 +/-1 ℃, hyphae are subcultured on a fresh PDA plate to obtain a pure endophytic fungus culture. The surface disinfection effect was tested by 3 methods to ensure that all isolated strains were rosa roxburghii endophytic fungi: (1) placing a sterile flat plate of a PDA culture medium in a clean bench as a blank control 1 for checking the cleanliness of the clean bench; (2) inoculating 100 μ L of the final rinse solution to a sterile plate of PDA culture medium as a blank control 2 for examination of the rinse solution; (3) placing the roxburgh rose tissue with the sterilized surface in a sterile flat plate of a PDA culture medium, rolling for a circle, placing for 20 minutes, and taking out to serve as a blank control 3, wherein the blank control is a sterile tissue block screened by a plant tissue blotting method. PDA culture medium composition: 200g of potato (peeled), 20g of glucose, 15-20 g of agar, 1000mL of distilled water and natural pH.

DNA extraction: 50mg of pure culture mycelia were scraped off for 1 to 3 weeks with a sterilized scalpel, and Fungal DNA was extracted using Fungal gDNA Kit GD2416 (manufactured by Biomiga) according to the instruction. The DNA product was stored in a freezer at-20 ℃ for further use.

PCR amplification: the method of Polynucleotide Chain Reaction (PCR) is used to amplify an in vivo transcribed spacer (ITS) (ITS4/ITS5), ribosomal large subunit rDNA (LSU) (LR5/LR0R), beta-tubulin gene (TUB) (Bt2a/Bt2b) and RNA polymerase II second large subunit gene (RPB2) (fRPB2-5F/fRPB2-7cR), which are 4 gene segments in total.

ITS amplification primers are ITS4 (5'-TCCTCCgCTTATTgATATgC-3') and ITS55 '-ggAAgTAAAAgTCgTAACAAgg-3'); the LSU amplification primers were LR5 (5'-TCCTGAGGGAAACTTCG-3') and LR0R (5'-ACCCGCTGAACTTAAGC-3'); TUB amplification primers are Bt2a (5'-GGTAACCAAATCGGTGCTGCTTTC-3') and Bt2b (5'-ACCCTCAGTGTAGTGACCCTTGGC-3'); the amplification primer of the RPB2 is fRPB2-5F (5 '-GAYGAYMGWGATCAYTTYGG-3'), fRPB2-7cR (5 '-CCCATRGCTTGYTTRCCCAT-3'), wherein M is A/C, R is A/G, W is A/T, and Y is C/T.

The reaction system is as follows: DNA template 1. mu.L, forward primer 1. mu.L, reverse primer 1. mu. L, PCRMix 12.5.5. mu. L, ddH2O 9.5.5. mu.L.

PCR amplification procedure: ITS and LSU are pre-denatured at 94 deg.C for 3min, denatured at 94 deg.C for 30s, annealed at 55 deg.C for 30s, extended at 72 deg.C for 45s, and stored at 4 deg.C for 35 cycles, extended at 72 deg.C for 10 min. TUB is pre-denatured at 95 deg.C for 5min, denatured at 94 deg.C for 1min, annealed at 55 deg.C for 1min, extended at 72 deg.C for 2min, and stored at 4 deg.C for 35 cycles, extended at 72 deg.C for 10 min. RPB2 is pre-denatured at 94 deg.C for 3min, denatured at 94 deg.C for 45s, annealed at 55 deg.C for 45s, extended at 72 deg.C for 65s, and stored at 4 deg.C for 35 cycles, extended at 72 deg.C for 10 min.

Confirmation of PCR reaction product: mixing 5 mu L of PCR product with 1 mu L of DAN Green dye, then spotting the mixture on 1.2% agarose gel, carrying out electrophoresis for 15min under the condition of 110V, and observing a band in a gel imaging system, wherein if the band is clear, the success of amplification is preliminarily judged.

Sequencing PCR reaction products: the PCR product was sent to Biotechnology engineering (Shanghai) Co., Ltd for measurement

The ITS, LSU, TUB and RPB2 sequences of the strain HGUP191049 are shown in SEQ ID NO. 1.

7. And (3) data analysis: the sequence of the strain HGUP191049 was subjected to homology alignment with the sequence in GenBank by Blast alignment, and BLAST search showed that the ITS sequence of the strain HGUP191049 may belong to the genus Epicoccum, and by referring to the latest reference, a multigene phylogenetic tree was constructed as shown in FIG. 2. As can be seen from FIG. 2, the strain HGUP191049 is determined to be Epicoccum latusicollum according to genetic affinity comparison, is preserved in the plant pathology laboratory of the college of agriculture of Guizhou university, has the preservation number GUCC 191049, is equivalent to the strain HGUP191049 in the invention, has the preservation date of 2020, 4 and 25 days, and has the address of Guiyang in China, Guizhou university, zip code 550025; meanwhile, the rosa roxburghii endophytic fungus Epicoccum latusicollum is preserved to China general microbiological culture Collection center (CGMCC), and the biological preservation number is as follows: CGMCC 40110, China academy of sciences microorganism research institute No. 3, Xilu No.1, North Cheng, Chaozhou, the address Beijing, and requests the depositor to specify the name, strain number or symbol of the culture: epicoccum latesulum HGUP191049.

Example 2: separation of metabolite of rosa roxburghii endophyte

1. And (3) recovering and activating the strain: epicoccum latucidolum HGUP191049 stored in a refrigerator at 4 ℃ is inoculated into a PDA culture medium and is placed in a constant temperature incubator at 28 +/-1 ℃ for culture for 3-7 days.

2. Preparing a roxburgh rose endophyte metabolite: and (3) beating the HGUP191049 strain obtained in the step (1) into a bacterial cake with the diameter of 6mm along the edge of a bacterial colony by using a sterile puncher in a super clean bench, inoculating the bacterial cake into a 250mL conical flask containing 100mL of fermentation medium, and culturing the bacterial cake at the temperature of 28 +/-1 ℃ and at 220r/min for 5-14 d. Taking the culture solution after fermentation, separating the culture solution into a fermentation solution and mycelium through suction filtration or high-speed centrifugation (10000-12000 r/min), and discarding the mycelium. Extracting the fermentation liquor by ethyl acetate (the volume ratio is 1: 1-1: 2), repeatedly extracting for 3 times, concentrating under reduced pressure at 35-55 ℃ to constant weight to obtain Epicoccum latusollum HGUP191049 metabolite, and storing at-20 ℃.

The fermentation medium comprises the following components: 200g of potato (peeled), 20g of glucose and 1000mL of distilled water, and the pH value is natural. Sterilizing with high pressure steam sterilizing kettle at 121 deg.C for 15min.

3. The antifungal tests were as follows:

activation of test fungi: the plant pathogenic fungi including Erysipelothromyces cacao (Lasiodipia theobromae), Staphylaxis botrytis (Botryosphaeria dothidea), Colletotrichum capsici (Colletotrichum capsicii), Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), Sclerotium sclerotiorum (Sclerotium sclerotiorum) and Phycomyces kabodanensis (Talaromyces kabodanensis) are inoculated in a PDA culture medium in an ultraclean workbench and cultured in a constant temperature incubator at 28 +/-1 ℃ for 3-7 days to serve as test fungi.

20mg of Epicoccum latusollum HGUP191049 metabolite obtained in step 2 is weighed, dissolved in 1mL of DMSO to prepare a stock solution of 20mg/mL, and filtered through a microporous filter membrane (0.22 μm) for later use. The antifungal test is carried out by adopting a filter paper diffusion method: making filter paper into circular filter paper sheet with diameter of 6mm with a puncher, and sterilizing at 121 deg.C for 30 min. On a clean bench, a cake of test bacteria (diameter: 6mm) was inoculated onto a PDA plate (diameter: 9cm) 2cm from the edge. And a sterile filter paper sheet was placed at the other edge of the plate at equal distances, and then the filter paper sheet was impregnated with 10. mu.L of metabolite (20 mg/mL). DMSO and aqueous carbendazim solution (20mg/mL) were used as negative and positive controls, respectively. All plates were incubated at 28 ℃ for 2-7 days, and the negative control plate hypha radial growth radius R1 and the experimental plate hypha radial growth radius containing metabolites (R2) were measured for inhibition (%) (R1-R2)/R1 × 100%. The higher the inhibition rate is, the better the inhibition effect on the test bacteria is.

PDA culture medium composition: 200g of potato (peeled), 20g of glucose, 15-20 g of agar, 1000mL of distilled water and natural pH.

4. The antibacterial test is as follows:

activation of test bacteria: inoculating Pseudomonas syringae pathogenic variety of kiwifruit (Pseudomonas syringae pv. actindiae), Pantoea agglomerans (Pantoea agglomerans), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis, Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa (Pseudomonas aeruginosa) to a Nutrient Agar (NA) culture medium by streaking in an ultraclean workbench, culturing the Pseudomonas syringae pathogenic variety and the Pantoea agglomerans in a constant temperature incubator at 25 ℃ for 48 hours, picking out a single colony, inoculating the single colony in the culture medium, culturing at 220r/min at 25 ℃ for 12-24 hours, and taking the single colony as a test bacterium; after culturing staphylococcus aureus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa in a 37 ℃ constant temperature incubator for 48 hours, selecting a single colony, inoculating the single colony in an LB (LB) culture medium, culturing at 220r/min at 37 ℃ for 12-24 hours, and taking the single colony as a test bacterium.

20mg of Epicoccum latusollum HGUP191049 metabolite obtained in step 2 is weighed, dissolved in 1mL of DMSO to prepare a stock solution of 20mg/mL, and filtered through a microporous filter membrane (0.22 μm) for later use. The antibacterial test is carried out by adopting a filter paper diffusion method: making filter paper into circular filter paper sheet with diameter of 6mm with a puncher, and sterilizing at 121 deg.C for 30 min. Quickly adding the bacterial suspension to be tested into an unset NA culture medium (45-55 ℃) on a clean bench, adding 10mL of bacterial suspension to be tested into every 100mL of NA culture medium, quickly pouring the bacterial suspension to a flat plate after shaking up, and cooling to obtain different culture media of the bacteria to be tested. A sterile filter paper sheet (diameter: 6mm) was placed in the center of the NA plate of the test bacteria, and then the filter paper sheet was impregnated with 10. mu.L of the metabolite (20 mg/mL). DMSO was used as a negative control, and an aqueous solution of streptomycin sulfate or a solution of penicillin sodium (both 20mg/mL) was used as a positive control. The plate containing the clove pseudomonas kiwi fruit pathogenic variety and pantoea agglomerans is placed in a constant temperature incubator at 25 ℃, the plate containing staphylococcus aureus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa is placed in a constant temperature incubator at 37 ℃, after culturing for 24-48 hours, the inhibition effect of Epicoccum latisulum HGUP191049 metabolites on test bacteria is observed, and the diameter of the inhibition zone of each treatment group is shown in table 1 and fig. 4. When the diameter of the inhibition zone is larger than 8mm, the strain fermentation liquor contains antibacterial active ingredients, and the larger the diameter of the inhibition zone is, the better the inhibition effect on the tested bacteria is.

The NA culture medium comprises: 10.0g of peptone, 3.0g of beef powder, 5.0g of sodium chloride, 15.0g of agar and 1000mL of distilled water, wherein the pH value is 7.3 +/-0.1.

Composition of LB medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 7.0 of pH value.

5. Results of antifungal tests: antifungal activity of Epicoccum latenicolulum HGUP191049 metabolite is shown in FIG. 4 and Table 1.

TABLE 1

6. Results of antibacterial tests: the antibacterial activity of Epicoccum latenicolulum HGUP191049 metabolite is shown in FIG. 5 and Table 2.

TABLE 2

Example 3: determination of inhibitory Activity of metabolite of the endophytic fungi of Rosa roxburghii on Diospora cacao (Lasiodipia theobroma), Staphylaxis botrytis (Botryosphaeria dothidea), Colletotrichum capsici (Colletotrichcapsicii), Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), Sclerotium sclerotiorum (Sclerotinia sclerotiorum) and Pectinophora fructicola (Talaromyces kabodanensis).

Determination of minimum inhibitory concentrations of metabolites of the endophytic fungus Epicoccum latusiolum HGUP191049 against Lasiodipidia theobromae (Lasiodipia theobromae), Staphyloccocus botrytis (Botryosphaeria dothiea), Colletotrichum capsici (Colletotrichum capsicii), Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), Sclerotium sclerotiorum (Sclerotinia sclerotiorum) and Stachybotrys canus (Talaromyces kabodanensis).

Activation of test bacteria: the plant pathogenic fungi of the brown color diplodia theobromae (Lasiodipodia theobromae), the botrytis cinerea (Botryosphaeria dothidea), the Colletotrichum capsici (Colletotrichum capsicium), the Rhizoctonia solani (Rhizoctonia solani), the Fusarium oxysporum (Fusarium oxysporum), the Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) and the fruit rot fungi (Talaromyces kabodanensis) are inoculated into a PDA culture medium in an ultra-clean workbench and cultured in a constant temperature incubator at 28 +/-1 ℃ for 3-7 days to serve as test bacteria.

20mg of Epicoccum latusollum HGUP191049 metabolite obtained in step 2 is weighed, dissolved in 1mL of DMSO to prepare a stock solution of 20mg/mL, and after filtering through a microporous membrane (0.22 μm), the stock solution is sequentially diluted with DMSO to obtain metabolites with different concentrations (10.00, 5.00, 2.50, 1.25, 0.62, 0.31 mg/mL). Making filter paper into circular filter paper sheet with diameter of 6mm with a puncher, and sterilizing at 121 deg.C for 30 min. On a clean bench, a cake of test bacteria (diameter: 6mm) was inoculated onto a PDA plate (diameter: 9cm) 2cm from the edge. And a sterile filter paper sheet was placed at the other edge of the plate at equal distances, and then the filter paper sheet was impregnated with 10. mu.L of various concentrations of metabolites. DMSO served as a negative control. All plates were incubated at 28 ℃ for 2-7 days, and the negative control plate hypha radial growth radius R1 and the experimental plate hypha radial growth radius containing metabolites (R2) were measured, with the inhibition (%) (R1-R2)/R1 × 100%. If the inhibition rate is close to 0, no bacteriostatic effect is shown, namely the minimum bacteriostatic concentration is obtained, and the steps are repeated for three times. The test results are shown in Table 3.

TABLE 3

Example 4: determination of inhibitory capacity of metabolite of endophytic fungi of Rosa roxburghii on pathogenic variants of Syzygium aromaticum Kiwi (Pseudomonas syringae pv. actiniae), Pantoea agglomerans (Pantoea agglomerans), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis, Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa.

Determination of minimum inhibitory concentration of metabolite of endophytic fungus Epicoccum latuciolum HGUP191049 on pathogenic variants of Syzygium aromaticum Kiwi (Pseudomonas syringica pv. actindiae), Pantoea agglomerans (Pantoea agglomerans), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis, Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa (Pseudomonas aeruginosa).

Activation of test bacteria: in an ultra-clean workbench, inoculating pathogenic variants of the clove pseudomonas kiwi fruit, the pantoea agglomerans, the staphylococcus aureus, the bacillus subtilis, the escherichia coli and the pseudomonas aeruginosa in a Nutrient Agar (NA) culture medium by streaking, culturing the pathogenic variants of the clove pseudomonas kiwi fruit and the pantoea agglomerans in a constant-temperature incubator at 25 ℃ for 48 hours, selecting a single colony, inoculating the single colony in an LB culture medium, culturing at 220r/min at 25 ℃ for 12-24 hours, and taking the single colony as a test bacterium; after culturing staphylococcus aureus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa in a 37 ℃ constant temperature incubator for 48 hours, selecting a single colony, inoculating the single colony in an LB (LB) culture medium, culturing at 220r/min at 37 ℃ for 12-24 hours, and taking the single colony as a test bacterium.

20mg of Epicoccum latusollum HGUP191049 metabolite obtained in step 2 is weighed, dissolved in 1mL of DMSO to prepare a stock solution of 20mg/mL, and after filtering through a microporous membrane (0.22 μm), the stock solution is sequentially diluted with DMSO to obtain metabolites with different concentrations (10.00, 5.00, 2.50, 1.25, 0.62, 0.31 mg/mL). Making filter paper into circular filter paper sheet with diameter of 6mm with a puncher, and sterilizing at 121 deg.C for 30 min. Quickly adding the bacterial suspension to be tested into an unset NA culture medium (45-55 ℃) on a clean bench, adding 10mL of bacterial suspension to be tested into every 100mL of NA culture medium, quickly pouring the bacterial suspension to a flat plate after shaking up, and cooling to obtain different culture media of the bacteria to be tested. A sterile filter paper sheet (diameter: 6mm) was placed in the center of the NA plate of the test bacteria, and then the filter paper sheet was impregnated with 10. mu.L of the metabolite (20 mg/mL). DMSO was used as a negative control, and an aqueous solution of streptomycin sulfate or a solution of penicillin sodium (both 20mg/mL) was used as a positive control. The plate containing the pathogenic variant and the pantoea agglomerans of the clove pseudomonas kiwi fruit is placed in a constant temperature incubator at 25 ℃, the plate containing staphylococcus aureus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa is placed in a constant temperature incubator at 37 ℃, and after culturing for 24-48 hours, the inhibition effect of the Epicoccum latisulum HGUP191049 metabolite on the test bacteria is observed. If the diameter of the inhibition zone is less than or equal to 8mm, no inhibition effect is shown, and the steps are repeated for three times. The test results are shown in Table 4.

TABLE 4

Sequence listing

<110> Guizhou university, Guizhou province analysis and test research institute

<120> Rosa roxburghii endophytic fungus Epicocculatucinolum and application thereof in preparation of broad-spectrum antifungal agent

<130> 2022

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 530

<212> DNA

<213> Epicoccum latusicollum

<400> 1

tatggggtcg tactgatcga ggtcagagtg taaaaatgta cttttggatg tcgtcgtcat 60

gagtgcaaag cgcgagatgt actgcgctcc gaaatcaata cgccggctgc caattgtttt 120

aaggcgagtc tacaggagac aaacacccaa caccaagcag agcttgaagg tacaaatgac 180

gctcgaacag gcatgcccca tggaatacca aggggcgcaa tgtgcgttca aagattcgat 240

gattcactga attctgcaat tcacactact tatcgcattt cgctgcgttc ttcatcgatg 300

ccagaaccaa gagatccgtt gttgaaagtt gtaactatta agttttttca gacgctgatt 360

tcaactgcaa agggtttaaa tttgtccaat cggtgggcga acccaccgag gaaacgtaag 420

gtactcaaaa gacatgggta agagatagca ggcaaagcct acaactctag gtaatgatcc 480

ttccgcaggt tcacctacgg aaaccttgtt acgattttta cttccactcg 530

<210> 2

<211> 844

<212> DNA

<213> Epicoccum latusicollum

<400> 2

cgattagtct ttcgccccta tgcccaaatt tgacgatcga tttgcacgtc agaaccgctg 60

cgagcctcca ccagagtttc ctctggcttc accctattca agcatagttc accatctttc 120

gggtcccaac agctatgctc ttactcaaat ccatccgaag acatcaggat cggtcgatgg 180

tgcaccccga aaggttccca cctccgttca ctttcattac gcgctcgggc ttgacaccca 240

aacactcgca tagatgttag actccttggt ccgtgtttca agacgggccg cttacagcca 300

ttacgccagc atcctagcag atgcgcggac ctcagtccgg gctggttgca tgtcgtctcc 360

cctataagtt ctccccgaga ggaggtacat gacagagacc tttatccaac cgcccaaact 420

gatgctggcc tgcccgtaga agagtgcacc gggtaaaaac ccggatgagc aactacaggc 480

aagtctggct gcaagcgctt ccctttcaac aatttcacgt gctttttaac tctctttcca 540

aagtgctttt catctttcga tcactctact tgtgcgctat cggtctctgg ccagtattta 600

gctttagaag aaatttacct cccatttaga gctgcattcc caaacaactc gactcgtcga 660

aggggcttta cacggtagag gctagcgacc acgtacggga ttctcaccct ctgtgacgtc 720

ctgttccaag gaacttggac cgctgccaat gccaaagcgc cctctgcaaa ttacaactcg 780

gacgccaaag acgccagatt tcaaatttga gctgttgccg cttcactcgc cgttactagg 840

gcaa 844

<210> 3

<211> 334

<212> DNA

<213> Epicoccum latusicollum

<400> 3

cctcagtgta gtgacccttg gcccagttgt taccagcacc agactggccg aagacgaagt 60

tgtcgggacg gaagaagctg accgaaggga ccagcgcgga cagcgtccat tgtgccgggc 120

tccaaatcga cgaggacggc acggggaacg aacttgttgc cagaggcctg tggaaggtca 180

gcactcgcag tccgtctatg ggaagagtgt catttctagt acctcgttga agtagacgtt 240

catgcgctcg agctggaggt ccgaggtgcc gttgtagaca ccggagccgt cgaggccatg 300

ctcgccggag atggtctgcc agaaagcagc accg 334

<210> 4

<211> 866

<212> DNA

<213> Epicoccum latusicollum

<400> 4

cagtgttgac agatacactt acgcatccac attatcgcat ttgcgccgta cgaatacacc 60

agtcggccgt gacggcaagc tcgcaaagcc ccgccaattg cacaacagtc attggggtct 120

cgtgtgcccc gctgagacgc ctgaagggca agcctgtggt cttgtcaaga acttgtctct 180

gatgtgctac gtcagtgtcg gcagtgatgc cgggcccatt tctgatttca tgagccagcg 240

aaacatgcag ctactcgagg agtacgatca aaatcagaat cccgatgcta ccaaggtttt 300

cgtcaacggt gtttgggtcg gtgtgcattc caacgcacag cagcttgtct ccacagtgca 360

ggaattgcgt cgtaatggaa cactgtccta tgagatgagt ttgatccgtg acatccgtga 420

ccgagagttc aagatcttca cggacgctgg acgtgtcatg agaccacttt tcgtggtgga 480

gagcgatgtt cgcaagccaa accgcaacca tctcgtcttc agccaagagc actacaacaa 540

gctggttgaa gagcagcagg cgatggcaca agcaggcata ggcgaggagg agaagacaga 600

actctcttat ggctggaagg gtctaattca agacggtgtc attgaatatc ttgacgccga 660

agaagaggag actgccatga ttgtcatgtc acccgaggac ctcggtgagt ggcgcgacat 720

gaagatgggt atccctcagg acgatcgcaa ccctcaagga aaggaccgtc ttgcacgcat 780

caagcctaag cctgaccctc gcatccatgc ctacactcat tgcgaaattc atcctgctat 840

gattcttggt atctgtgcta gtatca 866

Claims

1. Application of Rosa roxburghii endophytic fungi and metabolite thereof in broad-spectrum antibacterial agent is provided.

2. The use of the endophytic fungi of Rosa roxburghii and metabolites thereof according to claim 1 in broad spectrum antibacterial agents, wherein: the broad-spectrum antibacterial agent corresponds to antifungal agents of two-color theobroma cacao (Lasiodipia theobroma), botrytis (Botryosphaeria dothidea), Colletotrichum capsici (Colletotrichum capsicii), Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), Sclerotinia sclerotiorum (sclerotiorum) and pythium fructicola (Talaromyces kabodanensis); and antibacterial agents of Pseudomonas syringae kiwifruit pathogenic variety (Pseudomonas syringae pv. actinoidea), Pantoea agglomerans (Pantoea agglomerans), Staphylococcus aureus (Staphylococcus aureus) ATCC 6538, Bacillus subtilis CMCC (B) 63501, Escherichia coli (Escherichia coli) CMCC (B) 44102 and Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC 27853.

3. A method of producing a metabolite of rosa roxburghii endophytic fungi according to claim 1 or 2, characterized in that: fermenting and culturing the Rosa roxburghii endophytic fungus Epicoccum latusicolum to obtain a culture solution, separating the culture solution into a fermentation solution and a mycelium through suction filtration or high-speed centrifugation, and discarding the mycelium; and extracting the fermentation liquor by using ethyl acetate, and concentrating an organic phase to constant weight to obtain a metabolite of the roxburgh rose endophytic fungus Epicoccum latusicolum.

4. The method for preparing a metabolite of Rosa roxburghii endophytic fungi according to claim 3, wherein: inoculating roxburgh rose endophytic fungus Epicoccum latusoolum into a fermentation culture medium, culturing at the temperature of 28 +/-1 ℃ and 160-220 r/min for 5-14 d to obtain a culture solution, separating the culture solution into a fermentation solution and a mycelium through suction filtration or high-speed centrifugation at the speed of 10000-12000 r/min, discarding the mycelium, extracting the fermentation solution by ethyl acetate at the volume ratio of 1: 1-1: 2, repeatedly extracting for 3 times, and concentrating at the temperature of 35-55 ℃ under reduced pressure to constant weight to obtain a metabolite of Epicoccum latusoolum UP191049.

5. The method for preparing a metabolite of Rosa roxburghii endophytic fungi according to claim 4, wherein: the culture medium is Potato Dextrose Broth (PDB): peeled potato 200g, glucose 20g, distilled water 1000mL, pH nature.

6. A method of producing a metabolite of Rosa roxburghii endophytic fungi according to any one of claims 3-5, wherein: the roxburgh rose endophytic fungus Epicoccum latusicolum is subjected to activation culture before fermentation, and then is stored at a low temperature of 4 ℃ for 5-30 d or is directly inoculated into a fermentation culture medium; the activation culture comprises the following steps: inoculating the Rosa roxburghii endophytic fungus Epicoccum latusicolum into a potato glucose agar PDA culture medium, and culturing at 28 +/-1 ℃ for 3-7 days.

7. The method of claim 6, wherein the method comprises: the potato glucose agar PDA culture medium comprises: 200g of potato, 20g of glucose, 15-20 g of agar, 1000mL of distilled water and natural pH.