CN114958616A - Cinnamomum camphora symbiotic fungus YAFEF008 and separation method thereof - Google Patents
Cinnamomum camphora symbiotic fungus YAFEF008 and separation method thereof Download PDFInfo
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- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 title claims abstract description 70
- 241000723346 Cinnamomum camphora Species 0.000 title claims abstract description 69
- 241000233866 Fungi Species 0.000 title claims abstract description 57
- 238000000926 separation method Methods 0.000 title abstract description 6
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- 229960000723 ampicillin Drugs 0.000 claims description 9
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 9
- 244000061456 Solanum tuberosum Species 0.000 claims description 8
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- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
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- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims 1
- 241000588626 Acinetobacter baumannii Species 0.000 abstract description 7
- 241000193755 Bacillus cereus Species 0.000 abstract description 7
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
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- 241000218195 Lauraceae Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
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- 229940088710 antibiotic agent Drugs 0.000 description 2
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- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
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- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000235349 Ascomycota Species 0.000 description 1
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- 241001061264 Astragalus Species 0.000 description 1
- 241000007126 Codonopsis pilosula Species 0.000 description 1
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- 241001237515 Dothideomycetes sp. Species 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 240000000233 Melia azedarach Species 0.000 description 1
- 206010048723 Multiple-drug resistance Diseases 0.000 description 1
- 244000082946 Tarchonanthus camphoratus Species 0.000 description 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Genetics & Genomics (AREA)
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- Agronomy & Crop Science (AREA)
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Abstract
The invention relates to the technical field of microorganisms, and particularly discloses cinnamomum camphora symbiotic fungus YAFEF008 and a separation method thereof, wherein the ITS gene sequence of the cinnamomum camphora symbiotic fungus YAFEF008 comprises a nucleotide sequence shown in SEQ ID No.1, the nucleotide sequence is preserved in a China center for type culture collection with the preservation number of CCTCC M20211452, the fungus grows well on a PDA culture medium, hyphae grow radially to the periphery in a yellow brown color, the hyphae are dense, the fungus layer is thin, the bacterial colony is in a regular round shape, experiments show that the YAFEF008 mycelium primary extract has good antibacterial activity on bacillus cereus, streptococcus agalactiae, bacillus pumilus, escherichia coli, acinetobacter baumannii, staphylococcus haemolyticus and other bacteria, can effectively relieve crisis caused by drug-resistant strains, provides a new way for exploring new antibacterial drugs, and provides an important guidance basis for developing novel biological pesticides.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to cinnamomum camphora symbiotic fungus YAFEF008 and a separation method thereof.
Background
Plant symbiotic fungi (Plant symptomatology fungi) refer to microorganisms that parasitize within Plant tissues at a specific or complete stage of life history without causing the Plant to produce harmful symptoms, including mycorrhizal fungi as well as endophytic fungi and latent pathogenic bacteria that live on the surface of the Plant. Researches show that in the long-term co-evolution process, symbiotic bacteria and host plants form a special physiological metabolic pathway and generate various active compounds with novel structures, and the diversity of the symbiotic bacteria determines that a plurality of active symbiotic bacteria are not developed, so that the researches on secondary metabolites of the symbiotic bacteria play an important role in developing new antibacterial drugs and can effectively relieve the crisis brought by drug-resistant strains.
At present, the primary metabolism and the secondary metabolite of the endophytic bacteria become research hotspots, and researchers find natural active substances with antibacterial, antioxidant, antifungal and the like in various plant symbiotic bacteria such as chinaberry, Chinese toon, astragalus, codonopsis pilosula and the like. Each part of the plant such as leaves, flowers, barks, roots and the like contains abundant secondary metabolites, and part of the secondary metabolites have excellent antibacterial activity and particularly have stronger antagonistic activity on some drug-resistant bacteria, such as alkaloids, terpenoids, flavonoids, quinones and the like. In order to kill plant germs or inhibit the activity of germs, people invent antibiotics and synthetic antibacterial drugs, pathogenic bacteria generate drug resistance to antibiotics with the wide application of the antibacterial drugs, even multiple drug resistance occurs, the occurrence and spread of the drug resistance of pathogenic bacteria form a serious threat to human health, and the research of new antibacterial drugs becomes an important scientific research proposition.
Cinnamomum camphora (Cinnamomum camphora) is evergreen tree of Lauraceae and Lauraceae, and can be used for extracting camphor and camphor wood oil from wood, root, branch and leaf for pharmaceutical and perfume industries, and has effects of conserving water source, fixing soil, preventing sand and beautifying environment. According to the research, the fungus YAFEF008 with antibacterial activity is obtained by separating and screening the symbiotic bacteria of the cinnamomum camphora, and experiments show that the liquid extract of the YAFEF008 has good drug resistance to bacteria such as bacillus cereus, streptococcus agalactiae, bacillus pumilus, escherichia coli, acinetobacter baumannii, hemolytic staphylococcus and the like, so that a new way is provided for researching and developing new antibacterial drugs.
Disclosure of Invention
The invention aims to solve the technical problem of providing a cinnamomum camphora root symbiotic fungus YAFEF008 with strong antibacterial activity and a separation method thereof.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the cinnamomum camphora symbiotic fungus YAFEF008 disclosed by the invention is cinnamomum camphora root symbiotic fungus YAFEF 008; the deposit name is Dothideomycetes sp. The culture is preserved in China center for type culture Collection, and the preservation address is Wuhan university in China; the preservation date is as follows: 11/19/2021; the preservation number is CCTCCM 20211452.
The cinnamomum camphora symbiotic fungus YAFEF008 comprises an ITS gene sequence of the cinnamomum camphora symbiotic fungus YAFEF008, wherein the ITS gene sequence comprises a nucleotide sequence shown in SEQ ID No. 1.
Further, the present invention provides a microbial agent, wherein the active ingredient of the microbial agent comprises at least one of the following (a), (b) and (c):
(a) primary extraction of fermentation liquor of cinnamomum camphora symbiotic fungus YAFEF 008;
(b) ultrasonically cracking supernatant of cinnamomum camphora symbiotic fungus YAFEF008 cells;
(c) ultrasonic cracking precipitation of cinnamomum camphora symbiotic fungus YAFEF008 cells.
Further, the invention provides a method for separating cinnamomum camphora symbiotic fungus YAFEF008, which comprises the following steps:
(1) washing the collected camphor root sample with tap water, then transferring the camphor root sample to a sterile workbench for sterilization treatment, sucking excess moisture on the surface of the sterilized sample with sterile filter paper, and airing for later use;
(2) cutting roots of cinnamomum camphora into tissue blocks with the size of 0.5cm by using an autoclaved scissors or a dissecting knife in a clean bench, placing the tissue blocks on a PDAKAS antibacterial culture medium (the PDAKAS antibacterial culture medium is 39g/L of potato powder glucose agar culture medium, 50ug/mL of ampicillin and 50ug/mL of kanamycin) at equal intervals for culture, placing 8 tissue blocks on each culture dish, marking the culture dishes, placing the culture dishes in an inverted 26 ℃ culture box for culture for about 8 days, and observing the tissue blocks irregularly;
(3) transferring the hyphae to a solid PDA culture medium, culturing for 15 days, and performing molecular identification on the strain to obtain cinnamomum camphora root symbiotic fungus YAFEF 008.
Further, the invention also provides a preparation method of the primary extract of the fermentation liquor of cinnamomum camphora symbiotic fungus YAFEF008, which comprises the following steps:
(1) adding 1mL of liquid PG culture medium into a 2mL centrifuge tube, scraping a proper amount of grown cinnamomum camphora root symbiotic bacteria YAFEF008 mycelium into the centrifuge tube, crushing for 90S, inoculating 500 mu L of crushed sample liquid into conical flasks filled with the PG liquid culture medium respectively, and placing the conical flasks in a constant-temperature shaking incubator at 150 r.min -1 Culturing at 28 deg.C for 8 days;
(2) extracting mycelium of the culture: after the fermentation culture is finished, separating the mycelium and the bacterial liquid by using a separating funnel, adding ethyl acetate into the bacterial liquid according to the volume ratio of 1:1, carrying out ultrasonic oscillation for 40min, standing for 12h, then loading the bacterial liquid into the separating funnel for layering, recovering the layered lower-layer waste liquid, condensing, refluxing and drying the upper-layer extraction solution by using a rotary evaporator, and obtaining the primary extract of the fermentation liquid cultured by the cinnamomum camphora root symbiotic bacteria YAFEF 008.
Further, the PG liquid culture medium comprises the following components: 5g/L of potato extract powder, 5g/L of yeast powder, 20g/L of glucose, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of monopotassium phosphate and 10.1g/L of vitamin B.
Further, the PDAKAS antibacterial medium comprises: potato powder glucose agar culture medium 39g/L + ampicillin 50ug/mL + kanamycin 50ug/mL, PDA solid culture medium includes: potato powder glucose agar culture medium 39g/L + agar 15 g/L.
Compared with the prior art, the invention has the following advantages:
the invention obtains a new YaFEF008 strain of cinnamomum camphora (dothieomycetes sp.) by separating and screening symbiotic bacteria at the root of cinnamomum camphora, the fungi grow well on PDA culture medium, the hypha is tawny and grows radially to the periphery, the hypha is dense, the bacterial layer is thin, the bacterial colony is in a regular round shape, experiments show that the primary extract of the YAFEF008 strain has better antibacterial activity to bacillus cereus, streptococcus agalactiae, bacillus pumilus, escherichia coli, acinetobacter baumannii, hemolytic staphylococcus and other bacteria, can effectively relieve the crisis caused by drug-resistant strains, and provides a new approach for exploring new antibacterial drugs.
Drawings
FIG. 1 is a graph showing the growth of YAFEF008 hyphae according to the present invention;
FIG. 2 is a photograph of mycelia under a YAFEF008 fluorescence microscope of the present invention;
FIG. 3 is a graph showing the inhibitory effect of YAFEF008 according to the present invention on Bacillus cereus, wherein a, b and d represent control groups and c represents an experimental group;
FIG. 4 is a graph showing the inhibitory effect of YAFEF008 according to the present invention on Streptococcus agalactiae, a, b, d represent control groups, and c represents experimental groups;
FIG. 5 is a graph showing the inhibitory effect of YAFEF008 according to the present invention on Bacillus pumilus, wherein a, b and d represent control groups and c represents experimental group;
FIG. 6 is a graph showing the inhibitory effect of YAFEF008 according to the present invention on Escherichia coli, wherein a, b, and d represent control groups, and c represents an experimental group;
FIG. 7 is a graph showing the inhibitory effect of YAFEF008 according to the present invention on Acinetobacter baumannii, wherein a, b and d represent control groups and c represents an experimental group;
FIG. 8 is a graph showing the inhibitory effect of YAFEF008 according to the present invention on Staphylococcus hemolyticus, wherein a, b, and d represent control groups, and c represents an experimental group;
FIG. 9 shows a phylogenetic tree display diagram of the cinnamomum camphora symbiotic fungus YAFEF008 constructed based on ITS in the present invention.
Detailed Description
The present invention is further illustrated by the following figures and examples, wherein the chemicals used in the present invention are of analytical grade and are commercially available.
Example 1
YAFEF008, the strain is Cinnamomum camphora root symbiotic fungus YAFEF 008; the deposit name is dothideomyces sp. The culture is preserved in China center for type culture Collection, and the preservation address is Wuhan university in China; the preservation date is as follows: 11/19/2021; the preservation number is CCTCCM 20211452. The strain is in a form shown in figures 1 and 2, fungi grow well on a PDA culture medium, hyphae grow radially to the periphery in a yellow brown color, the hyphae are dense, the bacterial layer is thin, bacterial colonies are in a regular round shape, the effect is tested to be shown in figures 3-7, primary extracts of fermentation liquor obtained after the strain is cultured have good antibacterial activity on pathogenic bacteria such as bacillus cereus, streptococcus agalactiae, bacillus pumilus, escherichia coli, acinetobacter baumannii and hemolytic staphylococcus, and the pathogenic bacteria are purchased from Guangdong province bacteria drug resistance monitoring and quality control centers. 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.
Example 2
Isolation and identification of Cinnamomum camphora symbiotic fungus YAFEF008 strain
1. Isolation of Cinnamomum camphora symbiotic bacteria
Washing the collected camphor root sample with tap water for 12 hours, removing fungi or other microorganisms on the surface of the sample, then transferring the sample to a sterile workbench for disinfection treatment, washing the sample with 1L of sterile water, putting the sample in a culture dish, cutting the sample into small sections with an inoculating knife, and putting the small sections in a 50mL sterile centrifuge tube; soaking in 75% ethanol for 1min (shaking), pouring off ethanol, and washing with sterile water for 3 times; after being washed by sterile water, 10mL of prepared 5% H is poured 2 O 2 Soaking for 3min (shaking continuously during the soaking period), and pouring off H 2 O 2 Then, washing with sterile water for 3 times; and (4) absorbing the redundant water on the surface of the sterilized sample by using sterile filter paper, and airing for later use.
Then, the roots of cinnamomum camphora are cut into tissue blocks with the size of 0.5cm by using an autoclaved scissors or a dissecting knife in a clean bench, the tissue blocks are equidistantly placed on a PDAKAS antibacterial culture medium (the PDAKAS antibacterial culture medium is 39g/L of potato powder glucose agar culture medium, 50ug/mL of ampicillin and 50ug/mL of kanamycin) for culture, 8 tissue blocks are placed on each culture dish, the culture dishes are marked, the culture dishes are placed in an incubator at 26 ℃ upside down for culture for about 8 days, and the culture period is observed irregularly.
And finally, transferring the hyphae growing in the culture medium to a PDA (potato starch glucose agar) culture medium (39 g/L) for culture under a microscope, continuously observing for 8-10 days, and performing subsequent molecular identification on the picked strains to obtain the cinnamomum camphora symbiotic fungus YAFEF 008.
2. Identification of Cinnamomum camphora symbiotic bacteria
(1) Identification of morphology
The fungi grow well on the PDA culture medium, hyphae grow radially to the periphery in a yellow brown color, the hyphae are dense, the fungus layer is thin, and the bacterial colony is in a regular round shape.
(2) DNA extraction
Before the test, CTAB water bath is carried out for more than 3 ℃ at 65 ℃; 0mi taking mycelium obtained by separating, purifying and culturing n cinnamomum camphora symbiotic bacteria in a 2mL centrifuge tube, putting the centrifuge tube into a stainless steel tank filled with liquid nitrogen, soaking for 6min, crushing for 1.5min by using a crusher, and crushing the mycelium; adding 1mL of CTAB and 20uL of beta-mercaptoethanol into a centrifugal tube, shaking up (10min), and putting into a water bath kettle for 1 h; after the water bath is finished, putting the mixture into a centrifugal machine for centrifugation for 10min & 4; 0 r/liquid m of 12 supernatant 00 is taken, 1mL (phenol: chloroform: isoamyl alcohol 25: 24: 1) is added in, the mixture is shaken for 10min and centrifuged for 10min (the two times are repeated); taking the supernatant, adding 50uLNaAc and 1mL absolute ethyl alcohol (-20, DEG C), shaking uniformly, and putting into a refrigerator at the temperature of-20 ℃ for precipitation for 1 h; centrifuging for 10min after precipitation, adding 500uL 75% ethanol for eluting for 2 times (3 min/each time), and adding 500uL anhydrous ethanol for eluting for 1 time; centrifuging for 3min, removing ethanol, and standing to dry; adding 40uL elution buffer, and performing instantaneous centrifugation to obtain the genomic DNA of the fungus YAFEF 008.
(3) ITS analysis and identification
The sequences of the fungal rDNA spacer sequence (containing ITS1 region, 5.8S region and ITS4 region) are amplified by using fungal universal primers ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and the obtained ITS sequencing sequence is shown in SEQ ID No. 1.
(4) Construction of developmental trees
A phylogenetic tree of cinnamomum camphora symbiotic fungus YAFEF008 is constructed based on ITS (fig. 8), and the fungus has a closest relationship with the ascomycete sp (dothideomyces sp.) by integrating the results of strain morphology identification and molecular biology identification, so that cinnamomum camphora symbiotic fungus YAFEF008 is identified as the ascomycete sp.
Example 3
Extraction of active substance of cinnamomum camphora symbiotic bacteria YAFEF008
1. Liquid fermentation culture
Liquid fermentation medium PG: 15g/L of lactose, 6g/L of sodium nitrate, 0.52g/L of potassium chloride, 0.52g/L of magnesium sulfate heptahydrate, 1.52g/L of monopotassium phosphate, 1mL/L of trace elements and 1L of purified water. And (5) subpackaging the culture medium.
Adding 1mL of liquid PG culture medium into a 2mL centrifuge tube, scraping a proper amount of grown and formed cinnamomum camphora root symbiotic bacteria YAFEF008 mycelium, putting the mycelium into the centrifuge tube, crushing for 90S, inoculating 500 mu L of crushed sample liquid into conical flasks filled with the PG liquid culture medium respectively, putting the conical flasks in a constant-temperature shaking incubator for 150 r.min < -1 >, and culturing for 10d at 28 ℃;
2. culture mycelium extraction
After the fermentation culture is finished, separating the mycelium and the bacterial liquid by using a separating funnel, adding ethyl acetate (1:1) into the bacterial liquid, carrying out ultrasonic oscillation for 40min, standing for 12h, then loading the bacterial liquid into the separating funnel for layering, recovering the layered lower-layer waste liquid, condensing, refluxing and drying the upper-layer extraction solution by using a rotary evaporator, and obtaining the primary extract of the fermentation liquid cultured by the cinnamomum camphora root symbiotic bacteria YAFEF 008.
Test example 1
Antibacterial activity detection of primary extract of fermentation liquor of cinnamomum camphora symbiotic bacteria YAFEF008 culture
1. Activation of pathogenic bacteria
Pathogenic bacteria such as bacillus cereus, streptococcus agalactiae, bacillus pumilus, escherichia coli, acinetobacter baumannii, staphylococcus hemolyticus and the like which are stored in a laboratory are respectively added into a 2mL centrifugal tube in a small amount, 700ul of LB liquid culture medium is added, then the centrifugal tube is put into a constant temperature shaking table with the rotation speed of 180 r.min < -1 > at 37 ℃ for culture for 12h to obtain a pathogenic bacteria liquid, the pathogenic bacteria liquid is put into a refrigerator with the temperature of 4 ℃ for standby, and the activated bacteria are taken out and diluted by 1/10 times.
2. Antibiotic for positive control and its preparation
Ampicillin (Ampicillin) (batch No. 0339, purity: 95%) was purchased from Kunming Shuoyang technologies, Inc. 50mg of ampicillin is precisely weighed and respectively placed in a centrifuge tube, and 1mL of DMSO solution is added to prepare 50mg/mL of antibiotic DMSO solution for later use.
3. Method for detecting antibacterial activity by using filter paper sheet method
Weighing a proper amount of primary extract of cinnamomum camphora symbiotic bacteria YAFEF008 fermentation liquor, adding a proper amount of DMSO (dimethyl sulfoxide) solution, and diluting to 50 mg/mL. Respectively and uniformly coating the pathogenic bacteria liquid obtained by activation on an LB solid culture medium, airing, placing a 5mm filter paper wafer which absorbs the dissolved primary extract of the cinnamomum camphora symbiotic bacteria YAFEF008 fermentation liquid at a corresponding mark position of the culture medium, dipping the 5mm filter paper wafer into a DMSO solution and ampicillin to serve as negative control, placing the culture medium in a constant-temperature incubator at 37 ℃ for culturing for 12 hours, observing whether a bacteriostatic circle appears, and measuring the diameter of the bacteriostatic circle by using a cross method. Results the antibacterial activity of a primary extract (50mg/mL) of the fermentation broth of the cinnamomum camphora symbiotic YAFEF008 culture is shown in Table 1:
TABLE 1 comparison of antibacterial Activity of the first extract of the symbiotic fungus YAFEF008 of Cinnamomum camphora with the Positive control
As shown in fig. 1 to fig. 7 and table 1, a cinnamomum camphora symbiotic fungus YAFEF008 (dothideomyces sp.) is obtained by screening, and a primary extract of a fermentation broth of a strain culture of the cinnamomum camphora symbiotic fungus has good antibacterial activity on pathogenic bacteria such as bacillus cereus, streptococcus agalactiae, bacillus pumilus, escherichia coli, acinetobacter baumannii, staphylococcus haemolyticus and the like, and the pathogenic bacteria are purchased from the centers for monitoring drug resistance and controlling quality of the bacteria in Guangdong province. 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.
In addition to fermentation broth extraction by cultured strains, other culturing or processing methods, such as ultrasonic lysis supernatant of cinnamomum camphora symbiotic fungus YAFEF008 cells; ultrasonic cracking precipitation of cinnamomum camphora symbiotic fungus YAFEF008 cells, and based on the antibacterial effect of fungus YAFEF008, processed products or cultures of cinnamomum camphora symbiotic fungus YAFEF008 cells have corresponding antibacterial activity, and drugs, microbial agents and the like with related antibacterial activity prepared by using the cinnamomum camphora symbiotic fungus YAFEF008 cells are within the protection range of the invention.
The foregoing shows and describes the general principles, essential 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 embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
SEQUENCE LISTING
<110> Wang Yizheng Yuan
<120> cinnamomum camphora symbiotic fungus YAFEF008 and separation method thereof
<130> 2021
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 575
<212> DNA
<213> Artificial sequence (ITS Gene sequence)
<400> 1
cgtagtgacc gtgcggaagg atcattaccc tttcaatgca caaggatcga gcgggtgtag 60
gcaactatac cctctctcct tgctgtatta cgcccttgtt tttcaaattc tatttgtttc 120
ctcggcgggg tttcccgccg gttggataaa ctataacctt tttaattttc aatcagcgtc 180
tgaaataaat taataattac aactttcaac aacggatctc ttggttctgg catcgatgaa 240
gaacgcagcg aaatgcgata agtagtgtga attgcagaat tcagtgaatc atcgaatctt 300
tgaacgcaca ttgcgcccct tggtattcca tggggcatgc ctgttcgagc gtcatttgta 360
ccttcaagct ctgcttggtg ttgggtgttt gtcctcgctc ctctggggta ggactcgcct 420
taaagtaatt ggcagccagt gttttggttt gaagcgcagc acaagtcgcg attcaagctt 480
aatcagtagc tttccataag acatctatca cttttgacct cggatcaggt agggataccc 540
gctgaactta agcatatcaa aagggggggg aaaaa 575
Claims (10)
1. The cinnamomum camphora symbiotic fungus YAFEF008 is characterized in that the ITS gene sequence of the cinnamomum camphora symbiotic fungus YAFEF008 comprises a nucleotide sequence shown in SEQ ID No. 1.
2. The cinnamomum camphora symbiotic fungus YAFEF008 according to claim 1, wherein the symbiotic fungus YAFEF008 has a preservation number of CCTCC M20211452.
3. The culture of the cinnamomum camphora symbiotic fungus YAFEF008 according to claim 1 or 2, or a processed product thereof.
4. Use of the symbiotic fungus of cinnamomum camphora YAFEF008 according to claim 1 or 2 for preparing an antibacterial drug.
5. A microbial preparation comprising the culture according to claim 3 or a processed product thereof.
6. A bacterial agent according to claim 5, wherein the active ingredient comprises at least one of the following (a), (b) and (c):
(a) primary extraction of fermentation liquor of cinnamomum camphora symbiotic fungus YAFEF 008;
(b) ultrasonically cracking supernatant of cinnamomum camphora symbiotic fungus YAFEF008 cells;
(c) ultrasonic cracking precipitation of cinnamomum camphora symbiotic fungus YAFEF008 cells.
7. A method of isolating the cinnamomum camphora symbiotic fungus YAFEF008 of claim 1 or 2, comprising the steps of:
(1) washing the collected camphor root sample with tap water, then transferring the camphor root sample to a sterile workbench for sterilization treatment, sucking excess moisture on the surface of the sterilized sample with sterile filter paper, and airing for later use;
(2) the roots of cinnamomum camphora are cut into tissue blocks with the size of 0.5cm by using an autoclaved scissors or a dissecting knife in a clean bench, the tissue blocks are equidistantly placed on a PDAKAS antibacterial culture medium (the PDAKAS antibacterial culture medium is 39g/L of potato powder glucose agar culture medium, 50ug/mL of ampicillin and 50ug/mL of kanamycin) for culture, 8 tissue blocks are placed on each culture dish, the culture dishes are marked, and the culture dishes are placed in an inverted 26 ℃ culture box for culture for about 8 days and are observed irregularly.
(3) Transferring the mycelium to a solid PDA culture medium (the PDA culture medium is 39g/L of potato powder glucose agar culture medium), culturing for 15d, and performing molecular identification on the strain to obtain cinnamomum camphora root symbiotic fungus YAFEF 008.
8. The microbial inoculum according to claim 6, wherein the preparation method of the primary extract of the fermentation broth of the cinnamomum camphora symbiotic fungus YAFEF008 comprises the following steps:
(1) adding 1mL of liquid PDA culture medium into a 2mL centrifuge tube, scraping a proper amount of grown Cinnamomum camphora root symbiotic bacteria YAFEF008 mycelium, placing into the centrifuge tube, crushing for 90S, inoculating 500 μ L of crushed sample liquid into conical flasks containing PG liquid culture medium, placing in a constant temperature shaking incubator at 150r min -1 Culturing at 28 deg.C for 8 days;
(2) extracting mycelium of the culture: after the fermentation culture is finished, separating the mycelium and the bacterial liquid by using a separating funnel, adding ethyl acetate into the bacterial liquid according to the volume ratio of 1:1, carrying out ultrasonic oscillation for 40min, standing for 12h, then loading the bacterial liquid into the separating funnel for layering, recovering the layered lower-layer waste liquid, condensing, refluxing and drying the upper-layer extraction solution by using a rotary evaporator, and obtaining the primary extract of the fermentation liquid cultured by the cinnamomum camphora root symbiotic bacteria YAFEF 008.
9. The method of claim 8, wherein the PG liquid medium comprises the following components: 5g/L of potato extract powder, 5g/L of yeast powder, 20g/L of glucose, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of monopotassium phosphate and vitamin B 1 0.1 g/L; the liquid PDA culture medium comprises: 39g/L of potato starch glucose agar culture medium.
10. The method of claim 7, wherein the PDAKAS antibacterial medium comprises: potato starch glucose agar medium 39g/L + ampicillin 50ug/mL + kanamycin 50ug/mL, the PDA medium includes: 39g/L of potato starch glucose agar culture medium.
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| CN116355763A (en) * | 2023-05-26 | 2023-06-30 | 云南省林业和草原科学院 | Symbiotic fungus for oil wheat and spruce and application thereof |
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| CN118126845A (en) * | 2023-05-05 | 2024-06-04 | 云南省林业和草原科学院 | Inula longissima endophytic fungus YAFEF048 strain and separation method and application thereof |
| CN116355763A (en) * | 2023-05-26 | 2023-06-30 | 云南省林业和草原科学院 | Symbiotic fungus for oil wheat and spruce and application thereof |
| CN116355763B (en) * | 2023-05-26 | 2023-08-29 | 云南省林业和草原科学院 | Symbiotic fungus for oil wheat and spruce and application thereof |
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