CN114467978B - Application of metabolite of Rosa roxburghii endophytic fungi as broad-spectrum antibacterial agent - Google Patents

Abstract

The invention discloses the application and preparation of an endophytic fungus and its metabolites in a broad-spectrum antibacterial agent. The metabolites are fermented by Epicoccum latusicollum, filtered, and the mycelium is discarded. The fermentation liquid adopts ethyl acetate Extract, concentrate under reduced pressure to constant weight, and obtain endophytic fungus metabolites of Rosa roxburghii, which are broad-spectrum antibacterial agents. The present invention provides a thorn pear endophytic fungus and its metabolites against Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Talaromyces kabodanensis, Pseudomonas syringae pv.actinidiae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis , Escherichia coli and Pseudomonas aeruginosa were 1.25, 2.50, 10.00, 1.25, 10.00, 1.25, 1.25, 0.31, 1.25, 5.00, 0.62, 2.50 and 2.50 mg/mL, respectively. The composition with broad-spectrum antibacterial activity is obtained through microbial fermentation, and the process is simple, safe and environmentally friendly.

Description

Application of metabolites of endophytic fungi in Rosa roxburghii as broad-spectrum antimicrobial agents

Technical Field

The invention relates to an application of microorganisms, and in particular to an application of a metabolite of endophytic fungi of roxburghii as a broad-spectrum antibacterial agent.

Background Art

Endophytic fungi refer to a class of fungi that live in the cells of plants or in plant tissues during a certain period of their life history and do not cause obvious diseases to plant tissues. In recent years, many secondary metabolites with strong anti-pathogenic fungi, anti-pathogenic bacteria, insecticide, antioxidant, cytotoxic and anti-cancer properties have been found from endophytic fungi. 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 originally produced by plants, including pharmaceutical ingredients. Rosa roxburghii Tratt. is known for being rich in vitamin C, superoxide dismutase (SOD) and flavonoids. In traditional medicine, the roots, leaves and fruits of Rosa roxburghii are mainly used as medicines to treat diseases such as indigestion, dysentery, hypertension, vitamin C deficiency, etc. Its pharmacological effects are detailed in "Supplement to Compendium of Materia Medica". Modern pharmacological studies have shown that a variety of phytochemical components extracted from roxburghii also have biological activities such as antifungal, antibacterial, antioxidant, regulating the body's immune function, and preventing and treating type 2 diabetes. Since roxburghii is a plant source of antibacterial compounds, it is also an ideal resource for screening endophytic fungi with antibacterial potential. Endophytic fungi are isolated from roxburghii and endophytic fungi with potential biological activity are sought to develop bioactive natural products.

Summary of the invention

The invention aims to provide an application of an endophytic fungus of roxburghii roxburghii Epicoccum latusicollum and its metabolites in preparing a broad-spectrum antibacterial agent.

The technical solution adopted by the present invention is:

The invention discloses an endophytic fungus of roxburghii, Epicoccum latusicollum, and its metabolites in a broad-spectrum antibacterial agent. The endophytic fungus of roxburghii, Epicoccum latusicollum, is deposited in China General Microbiological Culture Collection Center (CGMCC), with biological deposit number: CGMCC NO.40110, and its address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The depositor is requested to designate the name, strain number or symbol of its culture: Epicoccum latusicollum HGUP191049.

The colony of the endophytic fungus Epicoccum latusicollum HGUP191049 of roxburghii is white, flocculent, round, light grey, and has light red pigmentation near the center.

The invention relates to an application of endophytic fungi of roxburghii Epicoccum latusicollum and its metabolites in broad-spectrum antimicrobial agents, wherein the broad-spectrum antimicrobial agents correspond to Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Sclerotiniasclerotiorum and Talaromyces kabodanensis antifungal agents, as well as Pseudomonas syringae pv. actinidiae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

The preparation method of the roxburghii endophytic fungus metabolite is as follows: the roxburghii endophytic fungus Epicoccumlatusicollum is fermented to obtain a culture solution, the culture solution is separated into a fermentation solution and mycelium by suction filtration or high-speed centrifugation, and the mycelium is discarded; the fermentation solution is extracted with ethyl acetate, and the organic phase is concentrated to a constant weight to obtain the Epicoccumlatusicollum metabolite, which is a broad-spectrum antibacterial agent.

The preparation method of the endophytic fungus metabolite of roxburghii comprises the following steps: fermenting and culturing endophytic fungus Epicoccum latusicollum to obtain culture solution, separating the culture solution into fermentation solution and mycelium by suction filtration or high-speed centrifugation, and discarding the mycelium; extracting the fermentation solution with ethyl acetate, concentrating the organic phase to constant weight, and obtaining the metabolite of Epicoccum latusicollum HGUP191049; inoculating endophytic fungus Epicoccum latusicollum HGUP191049 in PDB fermentation medium, culturing at 28±1°C and 160-220 r/min for 5-14 days, obtaining culture solution, separating the culture solution into fermentation solution and mycelium by suction filtration or high-speed centrifugation (10000-12000 r/min), and discarding the mycelium. The fermentation broth was extracted with ethyl acetate (volume ratio 1:1-1:2), the extraction was repeated 3 times, and the broth was concentrated under reduced pressure at 35-55°C to a constant weight to obtain the metabolites of Epicoccum latusicollum HGUP191049, which is a broad-spectrum antibacterial agent. The culture medium is potato dextrose broth (PDB): 200 g potato (peeled), 20 g glucose, 1000 mL distilled water, and natural pH.

Furthermore, the endophytic fungus Epicoccum latusicollum of the roxburghii is first activated and cultured before fermentation, and then directly (or after being stored at 4°C for 5 to 30 days) inoculated into the fermentation medium. The activation culture is as follows: Epicoccum latusicollum HGUP191049 is inoculated into a potato dextrose agar (PDA) medium at 28±1°C for 3 to 7 days. The PDA medium is: 200g potato, 20g glucose, 15 to 20g agar, 1000mL distilled water, and natural pH.

Compared with the prior art, the invention provides an endophytic fungus of roxburghii Epicoccum latusicollum, and a broad-spectrum antibacterial active ingredient is obtained by microbial fermentation. The metabolites of Epicoccum latusicollum can be used as a broad-spectrum antibacterial agent. After tests, the broad-spectrum antibacterial agent has an effective antibacterial effect on Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Sclerotiniasclerotiorum, Talaromyces kabodanensis, Pseudomonas syringae pv. actinidiae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, and the like. The minimum inhibitory concentrations of Escherichia subtilis, Escherichia coli and Pseudomonas aeruginosa were 1.25, 2.50, 10.00, 1.25, 10.00, 1.25, 1.25, 0.31, 1.25, 5.00, 0.62, 2.50 and 2.50 mg/mL respectively. Because of its natural ingredients, the broad-spectrum antibacterial agent of natural ingredients is the future market trend, with simple process, safety and environmental protection. The test results are shown in Figures 4 and 5.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the colony morphology of strain HGUP191049 (front and back);

Figure 2 is a phylogenetic tree of strain HGUP191049; Figure 2 is a phylogenetic tree constructed using the maximum likelihood method based on multi-gene (ITS, LSU, TUB and RPB2) sequences. The maximum likelihood bootstrap support rate (ML ≥ 50%) and Bayesian posterior probability (PP ≥ 0.90) are annotated near the nodes (ML/PP). Epicoccum latusicollum HGUP191049.1, Epicoccumlatusicollum HGUP191049.2 and Epicoccum latusicollum HGUP191049.3 represent the results of three independent sequencing of Epicoccum latusicollum HGUP191049. T: model strain.

Figure 3 shows the culture broth of strain HGUP191049 fermented for 5 to 14 days;

FIG4 is a picture showing the antifungal activity of the ethyl acetate extract of Epicoccum latusicollum fermentation product;

In the figure, A is the metabolite of Epicoccum latusicollum HGUP191049 (20 mg/mL), B is the positive control of carbendazim (20 mg/mL), and C is the negative control of dimethyl sulfoxide (DMSO). 1 is the inhibition of Lasiodiplodia theobromae; 2 is the inhibition of Botryosphaeria dothidea; 3 is the inhibition of Colletotrichum capsici; 4 is the inhibition of Rhizoctonia solani; 5 is the inhibition of Fusarium oxysporum; 6 is the inhibition of Sclerotinia sclerotiorum; 7 is the inhibition of Talaromyces kabodanensis.

FIG5 is a picture showing the antibacterial activity of the ethyl acetate extract of Epicoccum latusicollum fermentation product;

In the figure, A is the metabolite of Epicoccum latusicollum HGUP191049 (20 mg/mL), B is the positive control of streptomycin sulfate or penicillin sodium (20 mg/mL), and C is the negative control of dimethyl sulfoxide (DMSO). 1 is Pseudomonas syringae pv.actinidiae; 2 is Pantoea agglomerans; 3 is Staphylococcus aureus; 4 is Bacillus subtilis; 5 is Escherichia coli; 6 is Pseudomonas aeruginosa.

The endophytic fungus of sea buckthorn, Epicoccum latusicollum, was mailed to the China General Microbiological Culture Collection (CGMCC) on March 6, 2022, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, requesting the depositor to assign the name, strain number or symbol to its culture: Epicoccum latusicollum HGUP191049.

The endophytic fungus of roxburghii, Epicoccum latusicollum, is deposited in China General Microbiological Culture Collection Center (CGMCC), with biological deposit number: CGMCC NO.40110, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The depositor is requested to assign the name, strain number or symbol to its culture: Epicoccum latusicollum HGUP191049

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

The roxburghii roxburghii (scientific name: Rosa roxburghii Tratt.) of the present invention is a deciduous shrub of the genus Rosa in the family Rosaceae, and its fruit is known as the "king of vitamin C" among fruits.

Embodiment 1:

The material double distilled water used refers to water with a resistivity of 18MΩ*cm (25°C). In addition to water molecules, there are almost no impurities in the water, no bacteria, viruses, organic matter such as chlorinated dioxins, and no mineral trace elements required by the human body. The sterile double distilled water is obtained by sterilizing double distilled water in a high-pressure steam sterilizer at 121°C for 15 to 30 minutes.

1. Plant sample collection: Fresh and healthy samples of roxburghii tissue (roots, stems, leaves, flowers, fruits and seeds) were collected in Xiuwen County, Guiyang City, Guizhou Province and Panzhou City, Liupanshui City, Guizhou Province. All samples were immediately sent to the laboratory and stored in a 4°C refrigerator. Endophytic fungi were isolated within 48 hours after sample tissue collection.

2. Isolation of endophytic fungi: All tissues were rinsed under tap water for 30 minutes, rinsed with double distilled water for 10 minutes, and dried under natural conditions. The tissues were then cut into small pieces and transferred to a clean bench for surface disinfection. All samples were surface disinfected with 75% ethanol (1 minute) and rinsed 3 times with sterile double distilled water. Subsequently, the surface was disinfected with an aqueous solution of sodium hypochlorite (1% effective chlorine) (roots and fruits, 2 minutes; stems and seeds, 3 minutes; leaves and flowers, 1 minute), and then rinsed 3 times with sterile double distilled water. After rinsing, the surface water of the tissue was blotted with sterile filter paper and transferred to potato dextrose agar (PDA) medium. To avoid bacterial contamination, 30 μg/mL streptomycin was added to the PDA medium. Each culture plate contained 3 to 5 tissue fragments. After culturing at 28±1°C for 3 to 7 days, the mycelium was subcultured to a fresh PDA plate to obtain a pure culture of endophytic fungi. Three methods were used to detect the surface disinfection effect to ensure that all the isolated strains were endophytic fungi of Rosa roxburghii: (1) In the clean bench, a sterile plate of PDA culture medium was placed as blank control 1 to check the cleanliness of the clean bench; (2) 100 μL of the final rinse liquid was inoculated on a sterile plate of PDA culture medium as blank control 2 for the inspection of the rinse liquid; (3) The Rosa roxburghii tissue after surface disinfection was placed in a sterile plate of PDA culture medium and rolled once, and then taken out after 20 minutes as blank control 3, which was a sterile tissue block screened by plant tissue blotting method. PDA culture medium composition: 200 g potato (peeled), 20 g glucose, 15-20 g agar, 1000 mL distilled water, natural pH.

3. DNA extraction: Use a sterilized scalpel to scrape 50 mg of pure culture mycelium from 1 to 3 weeks old, and use the Fungal gDNA Kit GD2416 (produced by Biomiga) to extract fungal DNA according to its instructions. The DNA product is stored in a -20°C refrigerator for later use.

4. PCR amplification: The polynucleotide chain reaction (PCR) method was used to amplify the internal transcribed spacer (ITS) (ITS4/ITS5), large ribosomal subunit rDNA (LSU) (LR5/LR0R), β-tubulin gene (TUB) (Bt2a/Bt2b) and RNA polymerase II second largest subunit gene (RPB2) (fRPB2-5F/fRPB2-7cR), a total of 4 gene fragments.

The primers for ITS amplification were ITS4 (5′-TCCTCCgCTTATTgATATgC-3′) and

ITS55′-ggAAgTAAAAgTCgTAACAAgg-3′); LSU amplification primer is LR5

(5′-TCCTGAGGGAAACTTCG-3′) and LR0R (5′-ACCCGCTGAACTTAAGC-3′); TUB amplification primers are Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3′) and Bt2b

(5′-ACCCTCAGTGTAGTGACCCTTGGC-3′); RPB2 amplification primer is fRPB2-5F

(5′-GAYGAYMGWGATCAYTTYGG-3′), fRPB2-7cR (5′-CCCATRGCTTGYTTRCCCAT-3′), where M=A/C, R=A/G, W=A/T, Y=C/T.

The reaction system was: DNA template 1 μL, upstream primer 1 μL, downstream primer 1 μL, PCRMix 12.5 μL, ddH2O 9.5 μL.

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

5. Confirmation of PCR reaction products: Mix 5 μL of PCR product with 1 μL of DAN Green dye and spot on 1.2% agarose gel. Perform electrophoresis at 110V for 15 minutes and observe the bands in the gel imaging system. If the bands are clear, it is preliminarily judged that the amplification is successful.

6. PCR reaction product sequencing: The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

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

7. Data analysis: The sequence of strain HGUP191049 was compared with the sequence in GenBank by Blast. The BLAST search showed that the ITS sequence of strain HGUP191049 might belong to the genus Epicoccum. By consulting the latest references, a multi-gene phylogenetic tree was drawn as shown in Figure 2. As can be seen from Figure 2, according to the comparison of genetic affinity, the strain HGUP191049 is determined to be Epicoccum latusicollum, which is deposited in the Plant Pathology Laboratory, College of Agriculture, Guizhou University, with a deposit number of GUCC 191049, which is equivalent to HGUP191049 in the present invention, and the deposit date is April 25, 2020, and the address is Guizhou University, Guiyang, China, with a zip code of 550025; at the same time, the endophytic fungus of Rosa roxburghii, Epicoccum latusicollum, is deposited in the China General Microbiological Culture Collection Center (CGMCC), with a biological deposit number of CGMCC NO.40110, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the depositor is requested to specify the name, strain number or symbol for its culture: Epicoccum latusicollum HGUP191049.

Example 2: Isolation of Metabolites of Endophytic Bacteria in Rosa roxburghii

1. Strain resuscitation and activation: Inoculate Epicoccum latusicollum HGUP191049 stored in a 4°C refrigerator into PDA medium and culture it in a constant temperature incubator at 28±1°C for 3 to 7 days.

2. Preparation of metabolites of endophytic bacteria of roxburghii: In a clean bench, use a sterile puncher to punch a 6mm diameter bacterial cake along the edge of the colony of the HGUP191049 strain in step 1, inoculate it into a 250mL conical flask containing 100mL fermentation medium, and culture it at 28±1℃ and 220r/min for 5-14d. Take the culture broth after fermentation, separate the culture broth into fermentation broth and mycelium by suction filtration or high-speed centrifugation (10000-12000r/min), and discard the mycelium. The fermentation broth is extracted with ethyl acetate (volume ratio 1:1-1:2), and the extraction is repeated 3 times. It is concentrated under reduced pressure at 35-55℃ to constant weight to obtain the metabolites of Epicoccumlatusicollum HGUP191049, and stored at -20℃.

The fermentation medium consists of 200 g of potatoes (peeled), 20 g of glucose, 1000 mL of distilled water, and a natural pH. The medium is sterilized in a high-pressure steam autoclave at 121°C for 15 min.

3. Antifungal test is as follows:

Activation of test fungi: Plant pathogenic fungi Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Sclerotiniasclerotiorum and Talaromyces kabodanensis were inoculated into PDA medium in a clean bench and cultured in a constant temperature incubator at 28±1℃ for 3 to 7 days as test fungi.

Weigh 20 mg of the metabolite of Epicoccum latusicollum HGUP191049 obtained in step 2, dissolve it in 1 mL DMSO, prepare a 20 mg/mL stock solution, filter it through a microporous filter membrane (0.22 μm) and set it aside. The antifungal test was carried out by the filter paper diffusion method: the filter paper was made into a circular filter paper with a diameter of 6 mm using a puncher, and sterilized at 121°C for 30 min before use. On the clean bench, the bacterial cake (diameter: 6 mm) of the test bacteria was inoculated on a PDA plate (diameter: 9 cm) at a distance of 2 cm from the edge. And a sterile filter paper was placed at an equal distance on the other edge of the plate, and then the filter paper was impregnated with 10 μL of the metabolite (20 mg/mL). DMSO and carbendazim aqueous solution (20 mg/mL) were used as negative and positive controls, respectively. All plates were cultured at 28°C for 2-7 days, and the radial growth radius of mycelium on the negative control plate R1 and the radial growth radius of mycelium on the experimental plate containing metabolites (R2) were measured. The inhibition rate (%) = (R1-R2)/R1×100%. The greater the inhibition rate, the better the antibacterial effect on the test bacteria.

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

4. Antibacterial test is as follows:

Activation of test bacteria: In a clean bench, Pseudomonas syringae pv.actinidiae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were activated. aeruginosa) was inoculated on nutrient agar (NA) medium by streaking, Pseudomonas syringae pv. kiwifruit and Pantoea agglomerans were cultured in a constant temperature incubator at 25°C for 48 hours, and then single colonies were picked and inoculated in LB medium, cultured at 220r/min, 25°C for 12-24 hours as test bacteria; Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were cultured in a constant temperature incubator at 37°C for 48 hours, and then single colonies were picked and inoculated in LB medium, cultured at 220r/min, 37°C for 12-24 hours as test bacteria.

Weigh 20 mg of the metabolite of Epicoccum latusicollum HGUP191049 obtained in step 2, dissolve it in 1 mL of DMSO, prepare a 20 mg/mL stock solution, filter it through a microporous filter membrane (0.22 μm) and set it aside. Use the filter paper diffusion method to carry out the antibacterial test: use a puncher to make a circular filter paper with a diameter of 6 mm, sterilize it at 121°C for 30 min and set it aside. On the clean bench, quickly add the test bacterial suspension to the unsolidified NA medium (45-55°C), add 10 mL of the test bacterial suspension to every 100 mL of NA medium, shake well and quickly pour it into the plate, and cool it to obtain the culture medium of different test bacteria. Place a sterile filter paper (diameter: 6 mm) in the center of the NA plate of the test bacteria, and then impregnate the filter paper with 10 μL of metabolite (20 mg/mL). DMSO was used as a negative control, and streptomycin sulfate aqueous solution or penicillin sodium solution (both 20 mg/mL) was used as a positive control. Plates containing Pseudomonas syringae pv. kiwifruit and Pantoea agglomerans were placed in a 25°C constant temperature incubator, and plates containing Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were placed in a 37°C constant temperature incubator. After 24 to 48 hours of incubation, the inhibitory effect of Epicoccum latusicollum HGUP191049 metabolites on the test bacteria was observed. The diameters of the inhibition zones of each treatment group are shown in Table 1 and Figure 4. When the diameter of the inhibition zone is greater than 8 mm, it indicates that the fermentation broth of the strain contains antibacterial active ingredients. The larger the diameter of the inhibition zone, the better the antibacterial effect on the test bacteria.

NA medium composition: peptone 10.0 g, beef powder 3.0 g, sodium chloride 5.0 g, agar 15.0 g, distilled water 1000 mL, pH 7.3±0.1.

LB medium composition: peptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, pH 7.0.

5. Antifungal test results: The antifungal activities of metabolites of Epicoccum latusicollum HGUP191049 are shown in Figure 4 and Table 1.

Table 1

6. Antibacterial test results: The antibacterial activities of the metabolites of Epicoccum latusicollum HGUP191049 are shown in Figure 5 and Table 2.

Table 2

Example 3: Determination of the inhibitory ability of endophytic fungi metabolites of Rosa roxburghii against Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum and Talaromyces kabodanensis.

Determination of the minimum inhibitory concentrations of metabolites of the endophytic fungus Epicoccum latusicollum HGUP191049 from Rosa roxburghii against Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum and Talaromyces kabodanensis.

Activation of test bacteria: Plant pathogenic fungi Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Sclerotiniasclerotiorum and Talaromyces kabodanensis were inoculated into PDA medium in a clean bench and cultured in a constant temperature incubator at 28±1℃ for 3-7 days as test bacteria.

Weigh 20 mg of the metabolite of Epicoccum latusicollum HGUP191049 obtained in step 2, dissolve it in 1 mL DMSO, prepare a 20 mg/mL stock solution, filter it through a microporous filter membrane (0.22 μm), and dilute it with DMSO to different concentrations of metabolites (10.00, 5.00, 2.50, 1.25, 0.62, 0.31 mg/mL). Use a punch to make a circular filter paper with a diameter of 6 mm, sterilize it at 121°C for 30 minutes, and then set it aside. On the clean bench, inoculate the test bacteria cake (diameter: 6 mm) on a PDA plate (diameter: 9 cm), 2 cm away from the edge. Place a sterile filter paper at an equal distance on the other edge of the plate, and then impregnate the filter paper with 10 μL of metabolites of different concentrations. DMSO is used as a negative control. All plates were cultured at 28°C for 2-7 days, and the radial growth radius of mycelium on the negative control plate R1 and the radial growth radius of mycelium on the experimental plate containing metabolites (R2) were measured. The inhibition rate (%) = (R1-R2)/R1×100%. When the inhibition rate is close to 0, it means that there is no antibacterial effect, which is the minimum inhibitory concentration. Repeat three times. The test results are shown in Table 3.

Table 3

Example 4: Determination of the inhibitory ability of endophytic fungal metabolites of Rosa roxburghii against Pseudomonas syringae pv. actinidiae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

Determination of the minimum inhibitory concentrations of metabolites of the endophytic fungus Epicoccum latusicollum HGUP191049 from Rosa roxburghii against Pseudomonas syringae pv.actinidiae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

Activation of test bacteria: In an ultra-clean workbench, Pseudomonas syringae pv. kiwifruit, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were inoculated on nutrient agar (NA) medium by streaking. Pseudomonas syringae pv. kiwifruit and Pantoea agglomerans were cultured in a 25°C constant temperature incubator for 48 hours, and then single colonies were picked and inoculated in LB medium, cultured at 220 r/min, 25°C for 12 to 24 hours as test bacteria; Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were cultured in a 37°C constant temperature incubator for 48 hours, and then single colonies were picked and inoculated in LB medium, cultured at 220 r/min, 37°C for 12 to 24 hours as test bacteria.

Weigh 20 mg of the metabolite of Epicoccum latusicollum HGUP191049 obtained in step 2, dissolve it in 1 mL DMSO, prepare a 20 mg/mL stock solution, filter it through a microporous filter membrane (0.22 μm), and dilute it with DMSO to different concentrations of metabolites (10.00, 5.00, 2.50, 1.25, 0.62, 0.31 mg/mL). Use a punch to make a circular filter paper with a diameter of 6 mm, sterilize it at 121°C for 30 min, and then set it aside. On the clean bench, quickly add the test bacterial suspension to the unsolidified NA medium (45-55°C), add 10 mL of the test bacterial suspension to every 100 mL of NA medium, shake well and quickly pour it into the plate, and cool it to obtain the culture medium of different test bacteria. A sterile filter paper (diameter: 6 mm) was placed in the center of the NA plate of the test bacteria, and then the filter paper was impregnated with 10 μL of metabolites (20 mg/mL). DMSO was used as a negative control, and streptomycin sulfate aqueous solution or penicillin sodium solution (both 20 mg/mL) were used as positive controls. The plates containing Pseudomonas syringae pv. kiwifruit and Pantoea agglomerans were placed in a 25°C constant temperature incubator, and the plates containing Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were placed in a 37°C constant temperature incubator. After 24 to 48 hours of incubation, the inhibitory effect of Epicoccum latusicollum HGUP191049 metabolites on the test bacteria was observed. If the diameter of the inhibition zone is less than or equal to 8 mm, it means that there is no antibacterial effect, and the experiment was repeated three times. The test results are shown in Table 4.

Table 4

Sequence Listing

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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 (5)

1. The application of a metabolite of the endophytic fungus of Rosa roxburghii as a broad-spectrum antibacterial agent. The broad-spectrum antibacterial agent corresponds to Lasiodiplodia theobromae , Botryosphaeria dothidea , Antifungal agents for Colletotrichum capsici , Rhizoctonia solani , Fusarium oxysporum , and Sclerotinia sclerotiorum , and Pseudomonas syringae syringae pv. actinidiae ), Pantoea agglomerans , Staphylococcus aureus , Bacillus subtilis , Escherichia coli and Pseudomonas aeruginosa Bacterial agent; the preparation method of the endophytic fungus metabolites of Rosa roxburghii is to ferment the endophytic fungus ( Epicoccum latusicollum ) HGUP191049 to obtain the culture liquid, and separate the culture liquid into fermentation liquid and bacteria by suction filtration or high-speed centrifugation. The mycelium was discarded; the fermentation liquid was extracted with ethyl acetate, and the organic phase was concentrated to constant weight to obtain the metabolite of Epicoccum latusicollum HGUP191049. The biological deposit number of the endophytic fungus is : CGMCC NO.40110. 2. The application of the metabolites of the endophytic fungus of Rosa roxburghii as a broad-spectrum antibacterial agent according to claim 1, characterized in that: the fungus Epicoccum latusicollum ( Epicoccum latusicollum ) HGUP191049 is inoculated in the fermentation medium, 28 ±1°C, 160-220 r/min culture for 5-14 days, obtain the culture medium, separate the culture medium into fermentation broth and mycelium by suction filtration or high-speed centrifugation at 10000-12000 r/min, discard the mycelium, The fermentation broth was extracted with ethyl acetate, the volume ratio was 1:1-1:2, the extraction was repeated three times, and concentrated under reduced pressure at 35-55°C to constant weight to obtain the metabolite of Epicoccum latusicollum HGUP191049. 3. the metabolite of Rosa roxburghii endophytic fungus according to claim 2 is used as broad-spectrum antibacterial agent, it is characterized in that: described substratum is potato glucose broth medium: peeled potato 200 g, glucose 20 g, distilled water 1000 mL, pH natural. 4. The application of the metabolites of the endophytic fungus Rosa roxburghii according to claim 3 as a broad-spectrum antibacterial agent, characterized in that: the endophytic fungus Epicoccum latusicollum ( Epicoccum latusicollum ) HGUP191049 is activated and cultivated before fermentation, and then Store at 4°C for 5-30 days or directly into the fermentation medium; the activation culture is: inoculate the endophytic fungus ( Epicoccum latusicollum ) HGUP191049 in potato dextrose agar PDA medium, culture at 28±1°C 3 to 7 days. 5. the metabolite of Rosa roxburghii endophyte according to claim 4 is in the application as broad-spectrum antibacterial agent, it is characterized in that: described potato dextrose agar PDA medium is: potato 200 g, glucose 20 g, agar 15-20 g, 1000 mL distilled water, natural pH.

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