CN116064242A - Lanlight collar mould and its separation method and application - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a blue-light phialides and a separation method and application thereof, and discloses a blue-light phialides YAFEF068 strain which is obtained by separating root parts of lithospermum yunnanense; the preservation name is blue-light collar bottle mould YAFEF068 (Cadophora orchidicola YAFEF 068); the Chinese culture medium is preserved in China center for type culture Collection, and the preservation address is China university of Wuhan; preservation date: 2022, 6, 8; preservation number CCTCC NO: m2022841 has remarkable inhibition effect on various pathogenic bacteria, provides a new way for researching and developing new antibacterial drugs, and relieves the threat of pathogenic bacteria resistance to human health.

Description

Lanlight collar mould and its separation method and application

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a blue-light phialides and a separation method and application thereof.

Background

Endophytic fungi are fungi that can colonize the inside of healthy plant tissue at a certain stage of life history and do not cause significant symptoms in the host plant. Including those that are apparent at a certain stage of their life history, as well as latent pathogenic and mycorrhizal fungi that are temporarily harmless to the host plant. Endophytic fungi are of various types and widely exist in various types of plants, and some of the endophytic fungi can promote plant growth and remarkably improve stress resistance of host plants, including the resistance to biotic stress and abiotic stress. While the plant body provides a place for fungi to grow and inhabit, in return, the fungi synthesize some secondary metabolites for promoting the growth and defense of the plants. Some of these secondary metabolites have great prospects for new drug development. With the acceleration of modern progress, global environmental pollution is also aggravated, and various diseases caused by pathogenic bacteria and fungi are greatly threatened to human health. In order to reduce the toxic and side effects caused by chemical synthesis of drugs, people have been enthusiastic to develop natural drugs from medicinal plants.

Radix Arnebiae (Onosma paniculatum) belongs to perennial herb of Arnebiae of Arnebiaceae, and is mainly distributed in the western part of Sichuan to southwest of China, the northwest of Yunnan to the middle part and the western part of Guizhou, and grows in dry hillside and Quercus salicifolia forest margin and sunny hillside grass at an altitude of 2000-3200 m. The root stem is used as medicine, contains acetyl shikonin, isobutyryl shikonin, beta-dimethyl propylene shikonin, beta-hydroxy isovaleryl shikonin, 3, 4-dimethyl pentene-3-acyl shikonin and other compounds, and has the functions of resisting bacteria, eliminating inflammation, resisting tumor, promoting blood and heart circulation system and the like.

At present, research on endophytic fungi of lithospermum yunnanense (Onosma paniculatum) is not reported.

Disclosure of Invention

The invention mainly aims to provide a strain of phialides lanuginosus with strong antibacterial activity, and in addition, the invention also provides a separation method and application of the strain.

In order to achieve the above purpose, the present invention is realized by the following technical scheme: the strain of the invention, which is the blue-green collar bottle mould YAFEF068 with strong antibacterial activity, is obtained by separating the root of lithospermum yunnanense; the preservation name is blue-light collar bottle mould YAFEF068 (Cadophora orchidicola YAFEF 068); the Chinese culture medium is preserved in China center for type culture Collection, and the preservation address is China university of Wuhan; preservation date: 2022, 6, 8; preservation number: cctccc NO:2022841.

further, the ITS gene sequence of the strain is a nucleotide sequence shown as SEQ ID No. 1.

The invention also provides a microbial inoculum comprising a culture or processed product of the phialides lanuginosus YAFEF068.

Further, the microbial inoculum comprises: crude extract of mycelial of Phycomyces cyrtonema YAFEF068, ultrasonic cleavage supernatant or ultrasonic cleavage precipitate.

Further, the culture medium used for culturing the phialides lanuginosus YAFEF068 is ZMMM solid culture medium.

Preferably, the ZMMM solid culture medium comprises 8 g/bottle of corn flour, 15 mL/bottle of MM liquid culture medium, and the mixture is uniformly mixed into 350mL tissue culture bottles, wherein the MM liquid culture medium comprises: 6g/L of sodium nitrate, 0.52g/L of potassium chloride, 0.52g/L of magnesium sulfate heptahydrate and 1.52g/L of monopotassium phosphate.

Furthermore, the invention provides a strain of the phialides lanuginosus YAFEF068 and application of a microbial inoculum prepared from the strain in preparation of medicines for inhibiting pathogenic bacteria, wherein the pathogenic bacteria are bacillus cereus, streptococcus agalactiae, bacillus subtilis or shigella flexneri.

The invention also provides a preparation method of the strain blue-green collar bottle mould YAFEF068, which comprises the following steps:

(1) Cleaning, sterilizing, dicing, re-sterilizing and airing the collected radix arnebiae seu lithospermi root sample;

(2) Respectively taking 8 tissue blocks, equidistantly placing the tissue blocks on a PDA culture medium containing 50ug/mL ampicillin and kanamycin for culture, marking a culture dish, and then placing the culture dish in a 26 ℃ incubator for culture for 8d, and periodically observing during the culture;

(3) After the mycelia grow fully, transferring the mycelia to a PDA culture medium, culturing for 8 days, and carrying out subsequent molecular identification on the selected strain to obtain the lithospermum yunnanense root endophytic fungus YAFEF068;

the PDA medium comprises: potato powder 5g/L, KH ₂ PO ₄ 1g/L、MgSO ₄ 0.5g/L, 20g/L glucose, 5g/L, VB yeast powder ₁ 0.1g/L, agar 16g/L.

Furthermore, the invention also provides a preparation method of the blue-green collar mould YAFEF068 mycelium crude extract, which comprises the following steps:

(1) Scraping the blue-thin collar mould YAFEF068 mycelium, putting into a 2mL centrifuge tube, adding 500uL sterile water, putting into a cell disruption homogenizer for disruption for 2min, inoculating disruption solution into each tissue culture bottle filled with ZMMM solid culture medium, and culturing in a constant temperature incubator at 26 ℃ for 15d;

(2) Culture mycelium extraction: and (3) drying mycelia cultured in the tissue culture bottle in a baking oven at 60 ℃ for 7 hours, removing water in the culture medium, adding 100mL of methanol, carrying out ultrasonic vibration for 40 minutes, standing for 48 hours, separating bacterial liquid by using a funnel, condensing, refluxing and drying an extraction solution by using a rotary evaporator, and thus obtaining the crude extract of the myceliophthora lanuginosus YAFEF068 mycelia.

Compared with the prior art, the invention has the following advantages:

the invention provides a novel strain of phialides (Cadophora orchidicola) YAFEF068, the intermediate mycelium of the strain is black, the edge mycelium is white, the mycelium radially grows to form floccules around, the floccules are dense, the colony is in a regular round shape, and the applicant has verified that the extract of the strain culture has wide antibacterial activity, particularly has remarkable antibacterial effect on bacillus cereus, streptococcus agalactiae, bacillus subtilis or shigella flexneri, and the active substances and antibacterial mechanism of the strain can provide a novel way for developing new medicines.

Drawings

FIG. 1 is a graph showing the growth of mycelium of YAFEF068 strain according to the present invention.

FIG. 2 is a statistical chart of the detection results of the antibacterial activity of the YAFEF068 strain.

FIG. 3 is a graph showing the bacteriostatic effect of YAFEF068 strain cultured in different media according to the present invention (note: 1. JMM solid medium; 2.ZMMM solid medium; 3.OSMM solid medium; 4.PDA solid medium; 5.MY liquid medium; 6.PSA liquid medium; 7.CCMM solid medium; 8.SBMM solid medium; 9.OMAM solid medium).

FIG. 4 is a graph of the bacteriostatic effect of the invention across different species (note: a.Cadophor sp.; b.Cadophor malorum; c.Cadophor fastigiata; d.Cadophor orchidicola YAFEF 068).

FIG. 5 is a diagram showing mycelia of YAFEF068 strain under a fluorescence microscope according to the present invention.

FIG. 6 shows the inhibition of Bacillus cereus by YAFEF068 strain of the present invention (note a. Pure medium (ZMMM solid medium), b. ZMMM solid medium (Cadophor sp.); c. ZMMM solid medium (Cadophora orchidicola YAFEF 068); d. Dimethyl sulfoxide).

FIG. 7 shows the inhibition of Streptococcus agalactiae by YAFEF068 strain of the present invention, (note a. Pure medium (ZMMM solid medium), b. ZMMM solid medium (Cadophor sp.); c. ZMMM solid medium (Cadophora orchidicola YAFEF 068); d. Dimethyl sulfoxide).

FIG. 8 is a graph showing the inhibition of Bacillus subtilis by YAFEF068 strain of the present invention, (note a. Pure medium (ZMMM solid medium), b. ZMMM solid medium (Cadophor sp.); c. ZMMM solid medium (Cadophora orchidicola YAFEF 068); d. Dimethyl sulfoxide).

FIG. 9 shows the inhibition of Shigella flexneri by YAFEF068 strain of the present invention, (note a. Pure medium (ZMMM solid medium), b. ZMMM solid medium (Cadophor sp.); c. ZMMM solid medium (Cadophora orchidicola YAFEF 068); d. Dimethyl sulfoxide).

FIG. 10 is a phylogenetic tree of YAFEF068 strains of the present invention.

Detailed Description

The invention is further described with reference to the drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

As shown in figures 6 to 9, the invention screens out the Phycomyces lividans YAFEF068, and the obtained strain is preserved in China center for type culture collection, and is separated and purified from the root of lithospermum yunnanense. The strain is deposited with China center for type culture collection (China university) under the name of blue-thin collar bottle mould YAFEF068 (Cadophora orchidicola YAFEF 068); preservation number: cctccc NO: m2022841. The cultures were received by the collection at 2022, month 06 and 08, and were registered for storage for thirty years from that day, and for further five years after receiving a request to provide a sample of the cultures before expiration, the viability of the cultures was checked by the collection at 2022, month 06 and 15, and the result was survival.

YAFEF068 strain grows well on PDA culture medium, the middle mycelium is black, the color of edge mycelium is white, the mycelium grows radially to the periphery and is flocculent, and is denser, and the colony is in a regular round shape. The mycelium crude extract cultivated by the strain has better antibacterial activity on pathogenic bacteria such as bacillus cereus, streptococcus agalactiae, bacillus subtilis, shigella flexneri and the like. Provides a new way for researching and developing new antibacterial drugs in order to relieve the threat of pathogenic bacteria drug resistance to human health.

Isolation and identification of YAFEF068 Strain

1. Isolation of YAFEF068 Strain

The root of the lithospermum yunnanense is firstly washed for 24 hours by tap water, then is transferred into an ultra-clean workbench, is firstly washed by 1L of sterile water, and is then cut into tissue blocks of 0.5cm by a knife. Placing the cut tissue blocks into a 50mL centrifuge tube, pouring ethanol with different proportions for full sterilization, washing with sterilized water, placing into sterile filter paper for absorbing water, and airing for later use. The tissue blocks were placed equidistantly on PDA medium containing 50ug/mL ampicillin and kanamycin, 8 tissue blocks were placed on each dish, the dishes were marked, the dishes were placed upside down in a 27℃incubator for cultivation for about 8d, and the periodic observations were made during the cultivation. After hypha grows fully in the culture medium, transferring the hypha to PDA culture medium, culturing for 8d, and carrying out subsequent molecular identification on the selected strain to obtain the phialides lanuginosus YAFEF068.

PDA medium included: potato powder 5g/L, KH ₂ PO ₄ 1g/L、MgSO ₄ 0.5g/L, 20g/L glucose, 5g/L, VB yeast powder ₁ 0.1g/L, agar 16g/L.

2. Identification of endophytic fungi at root of lithospermum yunnanense

(1) Identification of morphology

Fungi grow well on PDA culture medium, the white of hypha earlier stage is radial to the surrounding growth, and the fungus silk is denser, and the fungus layer is thicker, and the bacterial colony is regular circular shape, grows to later stage and is black.

(2) DNA extraction

Before the experiment, the water bath kettle is opened for preheating CTAB; separating and purifying cultured mycelium from radix Arnebiae root endophytic fungi, placing in 2mL centrifuge tube containing steel ball, soaking in stainless steel tank containing liquid nitrogen for 6min, and crushing with cell homogenizing crusher for 1min until mycelium is broken; transferring the centrifuge tube until the centrifuge tube is ventilated, adding 1mL of CTAB, 200uL of PVP and 20uL of beta-mercaptoethanol, shaking uniformly (15 s), placing into a water bath kettle for water bath for 1.5h, and shaking once every 10min during the water bath; after the water bath is finished, putting the mixture into a low-temperature centrifuge for centrifugation (4 ℃,10min and 13000 r/min); 1mL of the supernatant was aspirated, 500uL of a mixture of phenol and chloroform-isoamyl alcohol was added, and the mixture was shaken for 10min, followed by centrifugation (4 ℃,10min,13000 r/min); taking 900uL of supernatant to a 2mL new centrifuge tube, adding 500uL of phenol and chloroform-isoamyl alcohol mixture for rewashing once, shaking uniformly and continuing low-temperature centrifugation; taking 700uL of supernatant to a 2mL new centrifuge tube, adding 700uL of chloroform-isoamyl alcohol mixture, shaking for 10min, and centrifuging again at low temperature; taking 500uL of supernatant to a 1.5mL centrifuge tube, adding 50uL of sodium acetate and 1mL of absolute ethyl alcohol placed at the temperature of minus 20 ℃, and slowly shaking up and down for 4-6 times; placing in a refrigerator at-20deg.C for standing for 1 hr, pouring out supernatant, adding 500uL ethanol into a centrifuge tube containing precipitate, slightly shaking for 10 times, and standing for 3min; centrifuging at normal temperature for 3min after repeating again, and performing 12000r; sucking residual ethanol by using a 100uL pipetting gun, centrifuging at normal temperature again, then replacing the 10uL pipetting gun to suck residual ethanol, and uncovering and standing for 15min; adding 60uLTE buffer, standing for 2min, flicking with finger until the precipitate is dissolved, centrifuging at normal temperature for 30s, and detecting by gel electrophoresis.

(3) ITS analysis and identification

The fungal rDNA spacer sequence (containing ITS1 region, 5.8S region, ITS2 region) was amplified using fungal universal primers ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and the resulting ITS sequencing sequence was shown in SEQ ID No. 1.

(4) Construction of developmental trees

Based on phylogenetic tree (figure 10) of ITS radix arnebiae root endophytic fungus YAFEF068, comprehensive strain morphology identification and molecular biology identification results, the fungus has the closest relationship with Phycomyces laneanus (Cadophora orchidicola), so radix arnebiae root endophytic fungus YAFEF068 is identified as Cadophora orchidicola.

Example 2

Extraction of active substance of fungi YAFEF068

1. Solid culture

The ZMMM medium comprises the following components: corn flour 8 g/bottle, MM liquid culture medium 15 mL/bottle (sodium nitrate 6g/L, potassium chloride 0.52g/L, magnesium sulfate heptahydrate 0.52g/L, potassium dihydrogen phosphate 1.52 g/L), and mixing into 350mL tissue culture bottle.

Scraping a proper amount of grown blue-and-light collar mould YAFEF068 mycelium, putting the mycelium into a 2mL sterilizing centrifugal tube, adding 1mL sterile water, crushing for 2min, taking 500 mu L of crushed sample liquid, respectively inoculating into each tissue culture bottle filled with ZMMM culture medium, and culturing for 15d at the temperature of 26 ℃ in a constant-temperature incubator.

2. Culture mycelium extraction

And (3) drying mycelia cultured in the tissue culture bottle in a baking oven at 60 ℃ for 7 hours, removing water in the culture medium, adding 100mL of methanol, carrying out ultrasonic vibration for 40 minutes, standing for 48 hours, separating bacterial liquid by a funnel, condensing, refluxing and drying an extraction solution by a rotary evaporator, and thus obtaining the crude extract of mycelia cultured by the Phycomyces cyrtophyllus YAFEF068.

Test example 1

Antibacterial Activity detection of mycelium crude extract of Phycomyces cyrtonema YAFEF068 culture

1. Activation of pathogenic bacteria

Pathogenic bacteria such as bacillus cereus, streptococcus agalactiae, bacillus subtilis, shigella flexneri and the like purchased from the Guangdong province bacteria drug resistance monitoring and quality control center are respectively taken and added into a 2mL centrifuge tube, then LB liquid culture medium is added, and then the centrifuge tube is placed into a constant temperature shaking table at 37 ℃ for culturing for 12 hours, so that pathogenic bacteria bacterial liquid is obtained.

2. Filter paper sheet method for detecting antibacterial activity

Taking a proper amount of crude extract of the blue-light phialides YAFEF068 mycelium, weighing, adding a proper amount of 20mg/uLDMSO (dimethyl sulfoxide) solution, and diluting to 50mg/mL. Marking the reverse side of an LB solid culture medium, uniformly smearing pathogenic bacteria liquid obtained by activation on the LB solid culture medium respectively, airing, sucking a small amount of dissolved blue-and-light collar bottle mould YAFEF068 mycelium crude extract by using 5mm filter paper, placing the blue-and-light collar bottle mould YAFEF068 mycelium crude extract at a position corresponding to the mark of the culture medium, dipping d (DMSO solution) by using a 5mm filter paper wafer as negative control, and designing a. Pure culture medium (ZMMM solid culture medium); and b, culturing the crude extract of Cadophora sp with the ZMMM solid culture medium as a control, wherein C is the crude extract of YAFEF068 cultured with the ZMMM solid culture medium, placing the culture medium in a constant temperature incubator at 37 ℃ for culturing for 12 hours, observing whether a bacteriostasis ring appears, and measuring the diameter of the bacteriostasis ring by using a crisscross method.

As shown in the results of figures 2 and 6-9, the fermentation broth primary extract of the strain culture of the phialides lanuginosus YAFEF068 obtained by screening has good antibacterial activity on pathogenic bacteria such as bacillus cereus, streptococcus agalactiae, bacillus subtilis and shigella flexneri, wherein the antibacterial activity difference between bacillus cereus and shigella flexneri is obvious (P < 0.05), and the antibacterial activity difference between streptococcus agalactiae and bacillus subtilis is not obvious. In order to relieve the threat of pathogenic bacteria drug resistance to human health, a new way is provided for researching and developing new antibacterial drugs.

Comparative example

To further determine the bacteriostatic effect of the YAFEF068 strain, antibacterial tests were performed and the bacteriostatic properties of different species were compared and the results are shown below.

1. The YAFEF068 strain is cultured by different culture mediums, and the antibacterial activity of the crude extract is detected, and the pathogenic bacteria are bacillus cereus, so that the bacterial strain is best in growth effect and most obvious in antibacterial activity when cultured in the ZMMM solid culture medium (figure 3).

The method for obtaining culture by solid culture medium culture is the same as the method for obtaining culture by solid culture, and the method for obtaining culture by liquid culture medium is to scrape appropriate amount of YAFEF068 strain mycelium, put it into 2mL off-tube, add 500uL of sterile water, put it into cell disruption homogenizer for disruption for 1min, take disruption solution and inoculate into each conical flask filled with liquid culture medium, put it into constant temperature shaking incubator for culturing for 8d (150 r.min ^-1 28 deg.c). After fermentation culture is completed, separating mycelium from bacterial liquid by using a separating funnel, adding ethyl acetate 1:1 into the bacterial liquid, shaking uniformly, standing for 12 hours, taking supernatant solution after full extraction, condensing, refluxing and drying by using a rotary evaporator to obtain the fermentation broth extract cultivated by the YAFEF068 strain, wherein the activity detection method is consistent with the test method, observing whether a bacteriostasis ring appears by using a filter paper disc method, and measuring the diameter of the bacteriostasis ring by using a crisscross method.

The formula of the culture medium for comparison experiments:

JRMM solid medium: 8 g/bottle of walnut residue and 15 mL/bottle of MM culture medium (6 g/L of sodium nitrate, 0.52g/L of potassium chloride, 0.52g/L of magnesium sulfate heptahydrate and 1.52g/L of potassium dihydrogen phosphate), wherein the walnut residue is the residual oil residue after the dry walnut seed oil is extracted;

ZMMM solid medium: corn flour 8 g/bottle + MM medium 15 mL/bottle (sodium nitrate 6g/L, potassium chloride 0.52g/L, magnesium sulfate heptahydrate 0.52g/L, potassium dihydrogen phosphate 1.52 g/L);

OSMM solid medium: 10 g/bottle of rice and 15 mL/bottle of MM medium (6 g/L of sodium nitrate, 0.52g/L of potassium chloride, 0.52g/L of magnesium sulfate heptahydrate, 1.52g/L of potassium dihydrogen phosphate);

PDA solid medium: 5g/L of potato soaked powder, 5g/L of yeast powder, 20g/L of glucose, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of potassium dihydrogen phosphate, 0.1g/L of vitamin B and 16g/L of agar;

MY liquid medium: 21g/L of yeast malt extract broth;

PSA liquid medium: 5g/L of potato soaked powder, 5g/L of yeast powder, 20g/L of sucrose, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of potassium dihydrogen phosphate and 0.1g/L of vitamin B;

CCMM medium comprises 8 g/bottle of hickory dreg and 15 mL/bottle of MM medium (sodium nitrate 6g/L, potassium chloride 0.52g/L, magnesium sulfate heptahydrate 0.52g/L, potassium dihydrogen phosphate 1.52 g/L), wherein the hickory dreg is the residue of dried hickory dreg after oil extraction;

SBMM medium, sorghum flour 7 g/bottle+MM medium 15 mL/bottle (sodium nitrate 6g/L, potassium chloride 0.52g/L, magnesium sulfate heptahydrate 0.52g/L, potassium dihydrogen phosphate 1.52 g/L);

OMAM solid culture medium comprises soybean peptone 20g/L, glucose 20g/L, yeast powder 1g/L, and sucrose 20g/L.

2. The strain phialides lanuginosus YAFEF068 (Cadophora orchidicola) and a strain a.cadophora sp; cadophora malorum; cadophor fastigiata; the bacteriostasis contrast effect (figure 4) is that the pathogenic bacteria are bacillus cereus, the culture modes are solid fermentation culture, the culture mediums are ZMMM solid culture mediums, the treatment mode is the same as the method for obtaining the culture through the solid culture, and the activity detection method is the same as the test method. The different species of bacteria (Cadophora sp., cadophora malorum and Cadophora fastigiata) are isolated from cypripedium ricenoki plants by methods consistent with the methods of the present patent. As can be seen from FIG. 4, the strain of the invention, YAFEF068, has remarkable antibacterial effect on inhibiting Bacillus cereus, compared with similar strains of different species.

Other culture or processing methods, such as ultrasonic cleavage supernatant of endophytic fungus YAFEF068 cells in radix Arnebiae; the ultrasonic pyrolysis precipitation of the endophytic fungus YAFEF068 cells at the root of the lithospermum yunnanense is based on the antibacterial effect of the YAFEF068 strain, and the processed product or the culture of the lithospermum yunnanense has corresponding antibacterial activity, and the preparation of related antibacterial active medicaments, bacterial agents and the like by using the lithospermum yunnanense is within the protection scope of the invention.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.

Claims (10)

1. A strain is characterized in that the strain is named as a phialides lanuginosus YAFEF068 (Cadophora orchidicola YAFEF 068) and has a preservation number of CCTCC NO: m2022841.

2. A strain according to claim 1, characterized in that: the ITS gene sequence of the strain is a nucleotide sequence shown as SEQ ID No. 1.

3. A microbial inoculum comprising a culture or processed product of the phialides lanuginosus YAFEF068 of claim 1 or 2.

4. A microbial agent according to claim 3, comprising: crude extract of mycelial of Phycomyces cyrtonema YAFEF068, ultrasonic cleavage supernatant or ultrasonic cleavage precipitate.

5. A microbial inoculum according to claim 3 wherein the medium used for the culture of the phialides lanuginosus YAFEF068 is ZMMM solid medium.

6. The microbial inoculum of claim 5, wherein the ZMMM solid medium comprises corn flour 8 g/bottle, MM liquid medium 15 mL/bottle, and is mixed into a 350mL tissue culture bottle, the MM liquid medium comprising: 6g/L of sodium nitrate, 0.52g/L of potassium chloride, 0.52g/L of magnesium sulfate heptahydrate and 1.52g/L of monopotassium phosphate.

7. Use of a strain of phialides lanuginosus YAFEF068 according to any one of claims 1-2 or of a microbial inoculum according to any one of claims 4-5 for the preparation of a medicament for inhibiting pathogenic bacteria.

8. The use according to claim 6, wherein the pathogenic bacteria is bacillus cereus, streptococcus agalactiae, bacillus subtilis or shigella flexneri.

9. The method for preparing the strain phialides lanuginosus YAFEF068 according to any one of claims 1 to 2, comprising the following steps:

(1) Cleaning, sterilizing, dicing, re-sterilizing and airing the collected radix arnebiae seu lithospermi root sample;

(2) Respectively taking 8 tissue blocks, equidistantly placing the tissue blocks on a PDA culture medium containing 50ug/mL ampicillin and kanamycin for culture, marking a culture dish, and then pouring the culture dish into a 26 ℃ incubator for culture for 8d, and periodically observing during the culture;

(3) After the mycelia grow fully, transferring the mycelia to a PDA culture medium, culturing for 8 days, and carrying out subsequent molecular identification on the selected strain to obtain the lithospermum yunnanense root endophytic fungus YAFEF068;

the PDA medium comprises: potato powder 5g/L, KH ₂ PO ₄ 1g/L、MgSO ₄ 0.5g/L, 20g/L glucose, 5g/L, VB yeast powder ₁ 0.1g/L, agar 16g/L.

10. The microbial inoculum according to claim 4, wherein the preparation of the crude extract of the mycelial mat of the phialides YAFEF068 comprises the following steps:

(1) Scraping the blue-thin collar mould YAFEF068 mycelium, putting into a 2mL centrifuge tube, adding 500uL sterile water, putting into a cell disruption homogenizer for disruption for 2min, inoculating disruption solution into each tissue culture bottle filled with ZMMM solid culture medium, and culturing in a constant temperature incubator at 26 ℃ for 15d;

(2) Culture mycelium extraction: and (3) drying mycelia cultured in the tissue culture bottle in a baking oven at 60 ℃ for 7 hours, removing water in the culture medium, adding 100mL of methanol, carrying out ultrasonic vibration for 40 minutes, standing for 48 hours, separating bacterial liquid by using a funnel, condensing, refluxing and drying an extraction solution by using a rotary evaporator, and thus obtaining the crude extract of the myceliophthora lanuginosus YAFEF068 mycelia.

Images