Endophytic fungus Thyronectria cucurbitula YAFEF1102 from Artemisia argyi and its application

Abstract

The invention belongs to the technical field of microbial control, and specifically discloses an endophytic fungus of Artemisia argyi Thyronectria cucurbitula YAFEF1102 and its application, specifically, the endophytic fungus of Artemisia argyi provided by the present invention, the deposited name is: Thyronectria cucurbitula YAFEF1102, deposit number: CCTCC NO: M2024711. Through experiments, it was found that the crude extract of fermentation broth prepared from the fungus YAFEF1102 has good antibacterial activity against Staphylococcus aureus, Bacillus lentus, Vibrio parahaemolyticus, and Escherichia coli, which provides a new way to alleviate the threat posed by pathogenic bacteria resistance to human health and to research and develop new antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control, and specifically relates to an endophytic fungus of Artemisia argyi Thyronectria cucurbitula YAFEF1102 and its applications. Background Art

[0002] Qiai (Artemisia argyi Levl. et Vant. var. argyi. cv. Qiai), a plant of the genus Artemisia in the family Asteraceae, is a Chinese national geographical indication product and a traditional Chinese herbal medicine. Traditional Chinese medicine believes that Qiai leaves have the effects of removing dampness and relieving pain, removing blood stasis and swelling, dredging meridians, warming the meridians and activating blood circulation, and are beneficial for treating cold diseases, gynecological diseases, respiratory diseases, rheumatic diseases and other diseases. Qiai is also considered the king of mugwort. It grows in Qichun County, Hubei Province. It is a high-quality authentic medicinal material. It contains a variety of active ingredients such as volatile oils, organic acids, flavonoids, terpenes, macroelements and trace elements. It has pharmacological effects such as antioxidant, antibacterial, anti-inflammatory, anti-tumor, immunomodulatory, anti-infective, and neuroprotective.

[0003] Endophytic fungi are a type of microorganisms that parasitize healthy plant tissues and organs. In a broad sense, it refers to a large group including mycorrhizal fungi, epiphytic saprophytes, and latent pathogens that are temporarily harmless to the host; in a narrow sense, it refers to a type of fungi that infects and colonizes the host plant at a certain stage or all stages of its life cycle without causing obvious symptoms. Endophytic fungi have established a harmonious and mutually beneficial symbiotic relationship with the host plant. They parasitize in the plant body to absorb nutrients from the host plant to complete their own life cycle. At the same time, the colonization of endophytic fungi provides assistance to the growth and stress tolerance of the host plant. Compared with rhizosphere microorganisms, endophytic bacteria and endophytic actinomycetes, endophytic fungi have the advantages of rich diversity, large number, and the ability to produce active substances with wide application value. Current studies have shown that the active metabolites of endophytic fungi in plants are potential precursors of antibiotics, anticancer drugs or pesticides, which have great research significance and economic value. Summary of the invention

[0004] The main purpose of the present invention is to provide an endophytic fungus of Artemisia argyi Thyronectria cucurbitula YAFEF1102 and a liquid culture method thereof produce bacteria-inhibiting compounds through liquid fermentation.

[0005] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0006] An endophytic fungus of Artemisia argyi, deposited name: Thyronectria cucurbitula YAFEF1102, deposit number: CCTCC NO: M2024711.

[0007] The Qi Artemisia argyi endophytic fungus fermentation liquid.

[0008] The method for preparing the fermentation liquid comprises the following steps:

[0009] (1) Thyronectria cucurbitula YAFEF1102 strain activation;

[0010] (2) Activate Thyronectria cucurbitula YAFEF1102 was inoculated into BPGY liquid medium to obtain fermentation broth;

[0011] The BPGY liquid culture medium is prepared by weighing 0.5 g potassium bromide, 0.1 g peptone, 1.5 g glucose and 0.5 g yeast powder respectively, adding them into a conical flask containing 100 ml water, sterilizing by high pressure, and obtaining the BPGY liquid culture medium after cooling.

[0012] In the preparation method, in step (2), the culture conditions are: 27°C, 170 rpm constant temperature shaker, culture for 15 days, and obtain the fermentation liquid.

[0013] The crude extract of the fermentation liquid of endophytic fungi of Artemisia argyi.

[0014] The preparation method of the crude extract of fermentation broth comprises the following steps: filtering the mycelium in the fermentation broth to obtain a bacterial broth, adding ethyl acetate to the bacterial broth, ultrasonically treating the bacterial broth, standing it to separate layers, and then extracting the supernatant; condensing, refluxing, and drying the mixture using a rotary evaporator to obtain the crude extract of the fungal fermentation broth.

[0015] In the preparation method, ethyl acetate is added to the bacterial solution at a volume ratio of 1:1.5.

[0016] A bacterial agent, which contains the endophytic fungus of Artemisia argyi, the fermentation liquid or the crude extract of the fermentation liquid.

[0017] A liquid culture medium for endophytic fungi of Artemisia argyi, wherein each 100 ml of the liquid culture medium contains 0.5 g potassium bromide, 0.1 g peptone, 1.5 g glucose and 0.5 g yeast powder.

[0018] The use of the Qi Ai endophytic fungus, the fermentation broth, the crude extract of the fermentation broth or the bacterial agent in the preparation of antibacterial and / or bactericidal products.

[0019] The bacteria include Staphylococcus aureus, Bacillus lentus, Vibrio parahaemolyticus or Escherichia coli.

[0020] The present invention has the following beneficial effects:

[0021] The present invention obtains a fungus with antibacterial activity by separating and screening endophytic fungi from Artemisia argyi, and the deposited name is: Thyronectria cucurbitula YAFEF1102, deposit number: CCTCC NO: M 2024711, classification name: Thyronectria cucurbitula Through experiments, it was found that the crude extract of fermentation broth prepared by the fungus YAFEF1102 has good antibacterial activity against Staphylococcus aureus, Bacillus lentus, Vibrio parahaemolyticus or Escherichia coli, which provides a new way to alleviate the threat posed by pathogenic bacteria resistance to human health and to research and develop new antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The endophytic fungus of Artemisia argyi described in the present invention Thyronectria cucurbitula YAFEF1102 mycelium growth diagram;

[0024] Figure 2 The endophytic fungus of Artemisia argyi described in the present invention Thyronectria cucurbitula Micrograph of mycelium of YAFEF1102;

[0025] Figure 3 The endophytic fungus of Artemisia argyi of the present invention Thyronectria cucurbitula The inhibitory effect of YAFEF1102 on Staphylococcus aureus (1: DSMO; 2: BPGY liquid culture medium crude extract; 3: Thyronectria cucurbitula YAFEF1102 fermentation broth crude extract; 4: Thyronectria cucurbitula YAFEF1102 mycelium crude extract)

[0026] Figure 4 The endophytic fungus of Artemisia argyi of the present invention Thyronectria cucurbitula The inhibitory effect of YAFEF1102 on Bacillus lentus (1: DSMO; 2: BPGY liquid culture medium crude extract; 3: Thyronectria cucurbitula YAFEF1102 fermentation broth crude extract; 4: Thyronectria cucurbitula YAFEF1102 mycelium crude extract)

[0027] Figure 5 The endophytic fungus of Artemisia argyi of the present invention Thyronectria cucurbitulaThe inhibitory effect of YAFEF1102 on Vibrio parahaemolyticus (1: DSMO; 2: BPGY liquid culture medium crude extract; 3: Thyronectria cucurbitula YAFEF1102 fermentation broth crude extract; 4: Thyronectria cucurbitula YAFEF1102 mycelium crude extract)

[0028] Figure 6 The endophytic fungus of Artemisia argyi of the present invention Thyronectria cucurbitula The inhibitory effect of YAFEF1102 on Escherichia coli (1: DSMO; 2: BPGY liquid culture medium crude extract; 3: Thyronectria cucurbitula YAFEF1102 fermentation broth crude extract; 4: Thyronectria cucurbitula YAFEF1102 mycelium crude extract)

[0029] Figure 7 The present invention shows a phylogenetic tree of endophytic fungi constructed based on ITS. Specific implementation plan

[0030] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The endophytic fungus of Artemisia argyi provided by the present invention is deposited under the following name: Thyronectria cucurbitula YAFEF1102, deposit number: CCTCC NO: M 2024711, classification name: Thyronectria cucurbitula ; Depository: China Center for Type Culture Collection, deposit address: Wuhan University, China; The culture was received by the Collection on April 18, 2024 and registered. According to the request, it was preserved for thirty years from that date. The viability of the culture was tested by the Collection on April 25, 2024, and the result was viable.

[0032] Example 1 Isolation and identification of endophytic fungi from Artemisia argyi

[0033] 1. Isolation of endophytic fungi from Artemisia argyi

[0034] After digging the roots of Qi Ai from the soil, they were immediately placed in a ziplock bag and the samples were stored in dry ice until the next step. The samples were then cleaned of surface debris with tap water and cut into 2 cm small pieces with a scalpel. The pieces were rinsed with 1 L of distilled water and then soaked in 15% hydrogen peroxide solution for 1 min. The hydrogen peroxide solution was discarded and rinsed 6 times with 50 mL of sterilized water.

[0035] Cut the 2cm small pieces into tissue blocks with a length and width of 0.5cm, put them into a 2ml centrifuge tube, add 3 steel balls and 0.7mL sterilized water, and break them for 2 minutes (180rpm). After diluting 1000 times, evenly spread them on 10 KASMPA solid culture medium plates (KASMPA: 15g / L maltose + 5g / L potato powder + 1g / L Qi Ai powder + 200μg / mL karanomycin + 200μg / mL ampicillin + 200μg / mL streptomycin + 15g / L agar powder), culture for 15 days, grow colonies, select colonies, and identify single colonies.

[0036] The grown hyphae were transferred to MPA solid medium and cultured for 15 days. The strain was molecularly identified and obtained. Thyronectria cucurbitula The MPA solid culture medium comprises the following components: 15 g / L maltose+5 g / L potato flour+1 g / L Artemisia argyi powder+15 g / L agar powder.

[0037] 2. Identification of endophytic fungi in Artemisia argyi

[0038] (1) Identification of morphology

[0039] The strain morphology is Figure 1 , 2 As shown, the fungus grew well on MPA solid culture medium, the surface of the colony was wrinkled and raised, the fungal layer was thick, and the colony was irregular in shape.

[0040] (2) DNA extraction

[0041] ① Before the experiment, heat CTAB (hexadecyltrimethylammonium bromide) in a 65℃ water bath for 30 minutes; ② Take 50 mg of fresh Thyronectria cucurbitulaYAFEF1102 mycelium was placed in a 2mL centrifuge tube, 3 small steel balls were added, the centrifuge tube was placed in liquid nitrogen for 3 minutes, and immediately crushed with a crusher for 2 minutes, 1mL of preheated CTAB solution was added, and the mixture was blown and mixed with a pipette, and then all were transferred to a centrifuge tube containing 200μL PVP (polyvinyl pyrrolidone), 20μL β-mercaptoethanol was added in the air in a fume hood, and the mixture was shaken for 15s to fully grind, and then placed in a 65°C water bath for 1.5h, flipped up and down 5-6 times every 10min, and centrifuged at 12000r / min, 4°C for 10min after the water bath ended; ③ Take 1mL of supernatant into a new centrifuge tube, add 500μL of DNA phenol reagent and chloroform-isoamyl alcohol mixture, flipped up and down for 10min, and centrifuged (4℃, 12000r / min) for 10min (③ steps were repeated twice); ④ Take 900μL of supernatant and put it into a new centrifuge tube, add 50μL 3mol sodium acetate solution and 900μL 95% anhydrous ice ethanol (-20℃), shake well and put into -20℃ refrigerator for precipitation for 3h; ⑤ After precipitation, centrifuge (4℃·12000r / min) for 10min, discard the supernatant, add 500μL 75% alcohol, turn upside down 2-3 times, let stand for 3min, and discard the supernatant; ⑥ Add 500μL 95% alcohol, turn upside down 2-3 times, let stand for 3min, centrifuge at room temperature (13000rpm) for 3min, discard ethanol, and let dry; ⑦ Add 40μL elution buffer EB, centrifuge at room temperature (13000rpm) for 1.5min to obtain fungi Thyronectria cucurbitula Genomic DNA of YAFEF1102.

[0042] (3) ITS analysis and identification

[0043] The fungal universal primers ITS1-F (5'-CTTGGTCATTTAGAGGAAGTAA -3') and LR3 (5'-CCGTGTTTCAAGACGGG -3') were used to amplify the intergenic sequence (including ITS1 region, 5.8S region, and ITS4 region) of fungal rDNA. The resulting ITS sequencing sequence is shown in SEQ ID No.1.

[0044] SEQ ID No.1

[0045]

[0046] (4) Construction of developmental tree

[0047] Phylogenetic tree of plant endophytic fungi was constructed using MEGA software based on ITS ( Figure 4 ), based on the results of strain morphological and molecular biological identification, strain YAFEF1102 was identified as Thyronectria cucurbitula , and then deposited with the deposit number: CCTCC NO: M 2024711.

[0048] Example 2 Liquid fermentation crude extract of endophytic fungi of Artemisia argyi

[0049] 1. Liquid culture

[0050] Endophytic fungi of Artemisia argyi Thyronectria cucurbitula The mycelium of YAFEF1102 was inoculated into a 250 mL conical flask containing 100 mL BPGY liquid culture medium, and placed in a constant temperature shaker at 27 °C and 170 rpm for 15 days. The preparation method of 100 mL BPGY liquid culture medium was as follows: 0.5 g potassium bromide, 0.1 g peptone, 1.5 g glucose and 0.5 g yeast powder were weighed separately and added into a conical flask containing 100 mL water. The bottle mouth was sealed with a sealing film and then sterilized by high pressure. After sterilization, it was set aside for use.

[0051] 2. Preparation of crude extracts from liquid culture fermentation broth and antibacterial testing

[0052] The liquid cultured mycelium was filtered with gauze to obtain a bacterial solution, and ethyl acetate was added to the bacterial solution at a volume ratio of 1:1.5, mixed evenly, and ultrasonicated for 45 minutes. Then it was poured into a separatory funnel, left to stand for 12 hours to allow stratification, and then the supernatant was extracted. The extracted solution was condensed and refluxed on a rotary evaporator to obtain the endophytic fungi of Qi Ai. Thyronectria cucurbitula YAFEF1102 liquid fermentation crude extract.

[0053] 3. Preparation of crude mycelial extract

[0054] After filtering the liquid fermented mycelium with gauze, liquid fermented mycelium was obtained. The mycelium was placed in a triangular flask, and 95% ethanol was added to submerge the mycelium. After standing for 12 hours, the mycelium extract was obtained by gauze filtration. The mycelium extract was condensed and refluxed and dried by a rotary evaporator to obtain the endophytic fungus of Qi Ai. Thyronectria cucurbitula Crude extract of YAFEF1102 mycelium.

[0055] 4. Preparation of crude extract from BPGY liquid medium

[0056] Place a 250mL conical flask containing 100mL of BPGY liquid medium in a constant temperature shaker at 27°C and 170rpm for 15 days. Add ethyl acetate to the liquid medium at a volume ratio of 1:1.5, mix well, and then ultrasonicate for 45 minutes. Then pour it into a separatory funnel, let it stand for 12 hours to allow stratification, and then extract the supernatant. The extract solution is condensed and refluxed on a rotary evaporator to dry to obtain a crude extract of BPGY liquid medium.

[0057] 5. Activation of pathogenic bacteria

[0058] Take 5 μL of the pathogenic bacteria Staphylococcus aureus, Bacillus lentus, Vibrio parahaemolyticus, and Escherichia coli stored in the laboratory and add them to a 2 mL centrifuge tube, then add 750 μL of LB liquid culture medium (10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract), and then culture the centrifuge tube in a constant temperature shaker at 37°C and 180 r / min for 12 hours to obtain the pathogenic bacteria solution, which is placed in a 4°C refrigerator for use. Take it out before use and dilute the activated bacteria to 1 / 10.

[0059] 6. Filter paper method to detect antibacterial activity

[0060] The activated pathogenic bacteria solution was evenly spread on LB solid medium (tryptone 10g / L + sodium chloride 10g / L + agar 15g / L + yeast extract 5g / L) and allowed to stand for use. Take 25 mg Thyronectria cucurbitula YAFEF1102 fermentation broth crude extract, mycelium crude extract and BPGY liquid culture medium crude extract were placed in a 1.5 mL centrifuge tube, and 500 μL DMSO (dimethyl sulfoxide) solution was added to obtain a 50 mg / mL crude extract solution. 10 ul of the above crude extract solution was added to a 5 mm sterilized filter paper disc. When the filter paper disc fully absorbed the crude extract solution, it was placed on the corresponding mark of the culture medium. The culture medium was placed in a constant temperature incubator at 37°C and cultured for 12 hours. Observe whether an antibacterial zone appears and measure the diameter of the antibacterial zone by the cross method. The antibacterial activity results of the crude extract showed that Thyronectria cucurbitula The inhibition zones of the crude extract of YAFEF1102 fermentation broth against Staphylococcus aureus, Bacillus lentus, Vibrio parahaemolyticus, and Escherichia coli were 16.5±0.4 mm, 15.3±0.3 mm, 14.5±0.2 mm, and 14.2±0.1 mm, respectively. Figure 3 , Figure 4 , Figure 5 , Figure 6 ),illustrate Thyronectria cucurbitula The crude extract of YAFEF1102 fermentation broth has good antibacterial activity against Staphylococcus aureus, Bacillus lentus, Vibrio parahaemolyticus and Escherichia coli.

Claims

1. An endophytic fungus of Artemisia argyi, characterized in that: Deposit name: Thyronectria cucurbitula YAFEF1102, deposit number: CCTCC NO: M2024711.

2. The fermentation liquid of endophytic fungi of Artemisia argyi according to claim 1.

3. The method for preparing the fermentation broth according to claim 2, characterized in that: The following steps are involved: (1) Activation of Thyronectria cucurbitula YAFEF1102 strain; (2) inoculating the activated Thyronectria cucurbitula YAFEF1102 into BPGY liquid culture medium to obtain fermentation broth; The BPGY liquid culture medium is prepared by weighing 0.5 g potassium bromide, 0.1 g peptone, 1.5 g glucose and 0.5 g yeast powder respectively, adding them into a conical flask containing 100 ml water, sterilizing by high pressure, and obtaining the BPGY liquid culture medium after cooling.

4. The preparation method according to claim 3, characterized in that: In step (2), the culture conditions are: 27° C., 170 rpm in a constant temperature shaker, culture for 15 days, and obtain a fermentation liquid.

5. The crude extract of endophytic fungus fermentation broth of Artemisia argyi according to claim 2, characterized in that: The preparation method of the crude extract of the fermentation broth is as follows: filtering the mycelium in the fermentation broth of claim 2 to obtain a bacterial liquid, adding ethyl acetate to the bacterial liquid, ultrasonically treating it, standing it to separate layers, and then extracting the supernatant; condensing and refluxing it with a rotary evaporator and drying it to obtain the crude extract of the fungal fermentation broth.

6. A bacterial agent, characterized in that: The bacterial agent contains the endophytic fungus of Qi Ai described in claim 1 or the fermentation liquid described in claim 2.

7. Use of the endophytic fungus of Artemisia argyi according to claim 1, the fermentation broth according to claim 2, the crude extract of the fermentation broth according to claim 5 or the microbial agent according to claim 6 in the preparation of antibacterial and / or bactericidal products, characterized in that: The bacteria are Staphylococcus aureus, Bacillus lentus, Vibrio parahaemolyticus or Escherichia coli.

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