CN116949102A - Fungus secondary metabolite Terrein and preparation method and application thereof - Google Patents
Fungus secondary metabolite Terrein and preparation method and application thereof Download PDFInfo
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- 241000233866 Fungi Species 0.000 title claims abstract description 47
- MHOOPNKRBMHHEC-UHFFFAOYSA-N Terrein Natural products CC=CC1=CC(=O)C(O)C1O MHOOPNKRBMHHEC-UHFFFAOYSA-N 0.000 title claims abstract description 37
- MHOOPNKRBMHHEC-HZIBQTDNSA-N terrein Chemical compound C\C=C\C1=CC(=O)[C@H](O)[C@H]1O MHOOPNKRBMHHEC-HZIBQTDNSA-N 0.000 title claims abstract description 37
- 229930000044 secondary metabolite Natural products 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 22
- 244000068988 Glycine max Species 0.000 claims abstract description 22
- 201000010099 disease Diseases 0.000 claims abstract description 22
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 22
- 229930000226 fungal secondary metabolite Natural products 0.000 claims abstract description 20
- 241000238631 Hexapoda Species 0.000 claims abstract description 13
- 239000000575 pesticide Substances 0.000 claims abstract description 11
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 108
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 60
- 238000000855 fermentation Methods 0.000 claims description 29
- 230000004151 fermentation Effects 0.000 claims description 29
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000011347 resin Substances 0.000 claims description 18
- 229920005989 resin Polymers 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 14
- 238000010898 silica gel chromatography Methods 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 239000000287 crude extract Substances 0.000 claims description 12
- 239000003208 petroleum Substances 0.000 claims description 11
- 238000001179 sorption measurement Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 8
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 5
- 230000002829 reductive effect Effects 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 239000004570 mortar (masonry) Substances 0.000 claims description 3
- 238000004809 thin layer chromatography Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 241000124058 Plectosphaerella Species 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 230000000968 intestinal effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000000643 oven drying Methods 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 241000948155 Phytophthora sojae Species 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 9
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 7
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 230000002265 prevention Effects 0.000 abstract description 6
- 206010059866 Drug resistance Diseases 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 4
- 239000005730 Azoxystrobin Substances 0.000 description 8
- WFDXOXNFNRHQEC-GHRIWEEISA-N azoxystrobin Chemical compound CO\C=C(\C(=O)OC)C1=CC=CC=C1OC1=CC(OC=2C(=CC=CC=2)C#N)=NC=N1 WFDXOXNFNRHQEC-GHRIWEEISA-N 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 4
- 239000003899 bactericide agent Substances 0.000 description 4
- 239000005807 Metalaxyl Substances 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000009036 growth inhibition Effects 0.000 description 3
- ZQEIXNIJLIKNTD-UHFFFAOYSA-N methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alaninate Chemical compound COCC(=O)N(C(C)C(=O)OC)C1=C(C)C=CC=C1C ZQEIXNIJLIKNTD-UHFFFAOYSA-N 0.000 description 3
- 241000233614 Phytophthora Species 0.000 description 2
- 241000124061 Plectosphaerella cucumerina Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical compound ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002027 dichloromethane extract Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 229940050176 methyl chloride Drugs 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
- C12P7/38—Cyclopentanone- or cyclopentadione-containing products
-
- 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
- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a fungus secondary metabolite Terrein, a preparation method and application thereof, wherein the fungus secondary metabolite Terrein is obtained by separating a secondary metabolite of fungus SN5702 obtained from the intestinal tract of an insect horse, the compound has good antibacterial activity on phytophthora sojae, the inhibition rate reaches 100% at the concentration of 100 mu g/mL, and the compound has an inhibition effect on the zoosporangium growth and development stage of the phytophthora sojae; therefore, the fungal secondary metabolite Terrein prepared by the invention can be used for preventing and treating soybean epidemic diseases or preparing pesticides for preventing and treating soybean epidemic diseases; can effectively reduce the drug resistance problem caused by the prevention of the soybean epidemic disease by using the traditional chemical pesticide, and simultaneously reduce the problems of ecological environment pollution and the like caused by ecological environment pollution.
Description
Technical Field
The invention relates to the field of microorganism secondary metabolites, in particular to a fungus secondary metabolite Terrein, a preparation method and application thereof.
Background
Phytophthora sojae root rot (Phytophthora root rot), also known as soybean epidemic disease, is a worldwide devastating disease of soybean caused by Phytophthora sojae (Phytophthora sojae). The disease is not a single occurrence, is typically a large area hazard, and the earlier the occurrence the more serious the hazard to the crop. The most convenient and efficient method for preventing and treating soybean epidemic diseases is to use a systemic bactericide, wherein metalaxyl is a special-effect bactericide for preventing and treating soybean epidemic diseases, but the metalaxyl has single action site, so that pathogenic bacteria generate drug resistance mutation after long-term administration, and the metalaxyl cannot be continuously and stably used in actual production. With the frequent use of chemical pesticides, soybean phytophthora generates the problem of drug resistance of different degrees, and the use effect and the service life of the existing chemical pesticides are seriously reduced. The emergence of drug resistant strains has raised concerns about bactericides, and the development of new bactericides to address this problem is highly desirable. The microorganism has the advantages of rich yield, easy acquisition, rich secondary metabolites, higher biological activity and the like, and becomes an important resource for developing and screening novel natural product pesticides, and the development of the secondary metabolites with the prevention and treatment effects on soybean epidemic diseases is particularly important.
Disclosure of Invention
In order to solve the technical problems, the invention provides a fungus secondary metabolite Terrein, and a preparation method and application thereof.
In order to achieve the above purpose, the invention is implemented according to the following technical scheme:
the first object of the present invention is to provide a method for preparing a fungal secondary metabolite Terrein, comprising the steps of:
s1, separating a fungus, namely, a spherical shell fungus PLectosphaerella sp, from an insect horse intestinal flora, and storing the isolated fungus in a refrigerator at the temperature of minus 80 ℃; the strain is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and the preservation number is: CGMCC No.40710, the preservation date is 2023.6.25, and the preservation address is Hospital No. 1 and No. 3 in Beijing Chaoyang district;
s2, carrying out 24L liquid fermentation on the fungi SN5702, and obtaining a fermentation crude extract of the fungi SN5702 by adopting macroporous resin adsorption, methanol leaching and dichloromethane extraction;
s3, separating and purifying the fermentation crude extract of the fungi SN5702 to obtain a fungus secondary metabolite-Terrein.
Further, the step S1 specifically includes:
the insects Ma Liuyong% alcohol were soaked on an ultra clean bench for 3min, followed by multiple washes of the insect body surface with sterile water. Placing the treated insect in a sterilized culture dish, taking its intestinal tract with a sterilized scalpel, placing in a sterilized mortar, adding appropriate amount of sterile water, sufficiently grinding, and diluting to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 The doubling liquid is evenly coated on PDA culture medium by using a liquid-transferring gun to respectively take 100 mu L of grinding liquid, and is cultivated in the environment of 25 ℃; single colonies of different forms were picked using an inoculating needle and cultured repeatedly to obtain fungus SN5702.
Further, the step S2 specifically includes:
s21, transferring fungus SN5702 stored in a refrigerator at the temperature of minus 80 ℃ into a culture dish containing PDA culture medium, and culturing for 6 days at the constant temperature of 25 ℃; 5mL of PDB culture solution is added into a 25mL test tube, and sterilization is carried out for 30min at 121 ℃; collecting bacterial cakes with the diameter of 5mm at the edge of an SN5702 bacterial colony by using a puncher in a sterile operation table, inoculating the bacterial cakes into a test tube, and carrying out shake culture at 25 ℃ and 180r/min for 48 hours to obtain primary seed fermentation liquor;
s22, adding 50mL of PDB culture solution into a 250mL triangular flask, sterilizing for 30min at 121 ℃, transferring the primary seed fermentation solution into the triangular flask in a sterile operation table, and carrying out shake culture for 48h at 25 ℃ and 180r/min to obtain a secondary seed fermentation solution;
s23, adding 400mL of PDB culture solution and 4% of XAD-16 macroporous adsorption resin into a 2L triangular flask, and sterilizing for 30min at 121 ℃; transferring the secondary seed fermentation liquid into a 2L triangular flask in a sterile operation table, and carrying out shake culture at 25 ℃ and 180r/min for 7d to carry out total fermentation for 24L; filtering to remove mycelium and fermentation liquid after fermentation, cleaning resin with clear water, mixing macroporous adsorption resin, and oven drying at 28deg.C;
s24, placing the dried macroporous adsorption resin in a 2L separating funnel, adding methanol until the macroporous adsorption resin is not passed through, oscillating, standing and soaking for 3 hours;
s25, repeating the step S24 for four times, collecting methanol eluent, concentrating to obtain a methanol crude extract, and weighing for later use; four extractions of the methanol crude extract with a methylene chloride-distilled water-methanol system were performed, wherein V Dichloromethane (dichloromethane) :V Distilled water :V Methanol ＝2:1:1，V Total (S) =1200 mL, combining dichloromethane eluates, concentrating under reduced pressure to obtain crude fermentation extract of fungus SN5702.
Further, the step S3 specifically includes:
s31, performing primary silica gel column chromatography by using a dichloromethane-methanol system as an eluent, wherein the elution proportion is that dichloromethane is sequentially used for eluting 2L and V (dichloromethane) :V (methanol) Elution of 4l, v=100:1, 100:2, 100:4, 100:8, 100:16, respectively (dichloromethane) :V (methanol) 2L eluted with 1:1, 2L eluted with methanol, 26L co-eluted; combining similar components according to a thin layer chromatography result after the first-stage silica gel column chromatography to obtain a component A and a component B;
s32, carrying out petroleum ether-ethyl acetate system gradient elution on the component A, wherein the elution proportion is V in sequence (Petroleum ether) :V (Ethyl acetate) After combining the components with similar polarities, respectively obtaining five components A1, A2, A3, A4 and A5, wherein the components are respectively shown in the specification of (9:1, 8:2, 7:3 and 6:4);
s33, performing silica gel column chromatography on the A4 component by taking a methylene dichloride-methanol system as an eluent, wherein the elution proportion is V in sequence (II)Methyl chloride) :V (methanol) Two gradients of 100:1.5 and 100:2 were eluted and similar fractions were pooled and concentrated to give three components A4A, A4B, A C;
s34, using petroleum ether-ethyl acetate system as eluent, performing silica gel column chromatography on the A4C component, V (Petroleum ether) :V (Ethyl acetate) =7:3, yielding three components A4C1, A4C2, A4C 3; performing silica gel column chromatography on the A4C2 component by using a methylene dichloride-methanol system as an eluent, V (dichloromethane) :V (methanol) =100:2.5, yielding A4C2A component; and (3) performing methanol system gel column chromatography on the A4C2A component, and repeatedly purifying to obtain a fungus secondary metabolite Terrein.
A second object of the present invention is to provide a fungal secondary metabolite Terrein prepared by the above method.
The third object of the invention is to provide an application of a fungus secondary metabolite Terrein in preventing and treating soybean epidemic diseases.
The fourth object of the invention is to provide an application of a fungus secondary metabolite Terrein in preparing pesticides for preventing and treating soybean epidemic diseases.
Compared with the prior art, the fungus secondary metabolite Terrein is obtained by separating the fungus SN5702 secondary metabolite obtained from the intestinal tracts of insects, the compound has good antibacterial activity on phytophthora sojae, the inhibition rate reaches 100% at the concentration of 100 mug/mL, and the compound has an inhibition effect on the zoosporangium growth and development stage of phytophthora sojae; therefore, the fungal secondary metabolite Terrein prepared by the invention can be used for preventing and treating soybean epidemic diseases or preparing pesticides for preventing and treating soybean epidemic diseases; can effectively reduce the drug resistance problem caused by the prevention of the soybean epidemic disease by using the traditional chemical pesticide, and reduce the problems of ecological environment pollution and the like caused by ecological environment pollution.
Drawings
FIG. 1 shows colony morphology of strain SN5702 on PDA.
FIG. 2 is a mass spectrum of the fungal secondary metabolite Terrein.
FIG. 3 shows nuclear magnetic resonance hydrogen spectrum of the fungal secondary metabolite Terrein.
FIG. 4 is a nuclear magnetic carbon spectrum of the fungal secondary metabolite Terrein.
FIG. 5 is a scanning electron microscope image of the fungus secondary metabolite Terrein before and after treatment of Phytophthora sojae filaments.
FIG. 6 shows the effect of the fungal secondary metabolite Terrein on the prevention of soybean epidemic disease
FIG. 7 shows the protective effect of the fungal secondary metabolite Terrein on soybean epidemic disease.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.
Example 1
The insects Ma Liuyong% alcohol were soaked on an ultra clean bench for 3min, followed by multiple washes of the insect body surface with sterile water. Placing the treated insect in a sterilized culture dish, taking its intestinal tract with a sterilized scalpel, placing in a sterilized mortar, adding appropriate amount of sterile water, sufficiently grinding, and diluting to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 The doubling liquid is respectively and evenly coated on a PDA culture medium by taking 100 mu L of grinding liquid by a liquid-transferring gun, and is placed in a dark condition at 25 ℃ for inversion culture for 3-7 d; picking single colonies with different forms by using an inoculating needle, and repeatedly culturing to obtain fungi SN5702; the molecular biological analysis initially proves that the strain is a phylogenetic fungus (plectophaerella), and the result of phylogenetic comparison analysis is carried out by amplifying rDNA-ITS sequences, so that the ITS-rDNA sequences of the strain are gathered on the same branch with Plectosphaerella cucumerina (the GenBank accession number is OP 847797.1), and the similarity is more than 99%. SN5702 has the following features: the hyphae are white and paste-like, as shown in figure one.
Phylogenetic characteristics: by amplifying rDNA-ITS sequence and performing phylogenetic comparison analysis, the result shows that the ITS-rDNA sequence of the strain is gathered on the same branch with Plectosphaerella cucumerina (GenBank accession number is OP 847797.1) and the similarity is more than 99%. The results of gene sequencing were as follows: TGCGGAGGGATCATTACTGAGTACTACACTCTCTACCCTTTGTGAACTATTATACCTGTTGCTTCGGCGGCGCCCGCGAGGGTGCCCGCCGGTCTCATCAGAATCTCTGTTTTCGAACCCGACGATACTTCTGAGTGTTCTTAGCGAACTGTCAAAACTTTTAACAACGGATCTCTTGGCTCCAGCATCGATGAAGAACGCAGCGAAACGCGATATGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATGGCGCCTTCCAGTATCCTGGGAGGCATGCCTGTCCGAGCGTCGTTTCAACCCTCGAGCCCCCGTGGCCCGGCGTTGGGGATCTGCCACGGCAGGCCCCTAAAACCAGTGGCGGACCCGAAGGCCCTCTCCTTTGCGCAGTAGCATCAGCCTCGCATTGGGATCCCTCGGCGTCCTGCCTCTAAACCCCCCACAAGTCCGCTCCGGCGGCACCAAGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATAT.
The spherical shell fungus plectophaerella sp is preserved in China general microbiological culture Collection center, and has the preservation number: CGMCC No.40710, the preservation date is 2023.6.25, and the preservation address is Hospital No. 1 and No. 3 of Beichen Xiyun in the Chaoyang area of Beijing city.
Example 2
Performing 24L fermentation treatment by using the fungus SN5702 obtained in the example 1, and obtaining a fermentation crude extract of the fungus SN5702 by macroporous resin adsorption, methanol leaching and dichloromethane extraction; separating and purifying the fermentation crude extract of the fungi SN5702 to obtain a fungi secondary metabolite-Terrein; the specific process is as follows:
strain SN5702, stored in a-80℃refrigerator, was transferred to a petri dish containing PDA medium and incubated at 25℃for 6d, the colony morphology of which is shown in FIG. 1.5 mL of PDB medium was added to a 25mL tube and sterilized at 121℃for 30min. Collecting bacterial cakes with the diameter of 5mm at the edge of a SN5702 bacterial colony by using a puncher in a sterile operation table, inoculating the bacterial cakes into a test tube, and carrying out shake culture at 25 ℃ and 180r/min for 48 hours;
culturing a secondary seed solution: 50mL of PDB culture medium was added to a 250mL Erlenmeyer flask, and the flask was sterilized at 121℃for 30min. In a sterile operation table, transferring the first-stage seed liquid into a triangular flask, and culturing for 48h at 25 ℃ under 180r/min in a shaking way.
400mL of PDB culture medium and 4% XAD-16 macroporous adsorbent resin were placed in a 2L flask, and sterilized at 121℃for 30min. In a sterile operating table, transferring the secondary seed fermentation liquid into a 2L triangular flask, and carrying out shaking culture at 25 ℃ and 180r/min for 7d to carry out total fermentation for 24L. After the strain fermentation is completed, filtering out hypha and fermentation liquor, cleaning resin with clear water, combining the resin, and drying at a constant temperature of 28 ℃.
Placing the dried resin in a 2L separating funnel, adding methanol until the resin is over, oscillating, standing and soaking for 3h, repeating the steps four times, collecting methanol eluent, concentrating to obtain a methanol crude extract, and weighing for later use. Four extractions of the methanol crude extract with a dichloromethane-distilled water-methanol system (V Dichloromethane (dichloromethane) :V Distilled water :V Methanol ＝2:1:1，V Total (S) =1200 mL), and the dichloromethane eluates were combined and concentrated under reduced pressure to give 21.8g of a crude dichloromethane extract.
120g of silica gel column and 22g of silica gel (100-200 meshes) are selected and stirred, and a dichloromethane-methanol system is used as an eluent to carry out primary silica gel column chromatography. Elution ratio dichloromethane 2L, V (dichloromethane) :V (methanol) =100:1, 100:2, 100:4, 100:8, 100:16, eluting 4l, v respectively (dichloromethane) :V (methanol) 2L eluted with 1:1, 2L eluted with methanol, 26L co-eluted. According to the thin layer chromatography results, the similar fractions were combined to obtain 2 fractions, 15.1. 15.1g A fraction and 1.2. 1.2g B fraction, respectively.
The component A is subjected to petroleum ether-ethyl acetate system gradient elution, and the elution proportion is V in sequence (Petroleum ether) :V (Ethyl acetate) After combining the components of similar polarity, they were divided into 5 components of 1.37g A1, 67.5mg A2, 4.36g A3, 7.06g A4 and 250.4mg A5, =9:1, 8:2, 7:3, 6:4.
Carrying out silica gel column chromatography on the A4 component, and selecting dichloromethane: two gradients of methanol=100:1.5 and 100:2 were eluted and similar fractions were pooled and concentrated to give three fractions of 515.2mg a4a, 3.2g a4b, 575.8mg a4c, respectively.
Carrying out silica gel column chromatography on the A4C component, wherein an eluent system is petroleum ether: ethyl acetate=7:3, 130mg of a4c1, 388.3mg of a4c2, 32.3mg of a4c3 component. The A4C2 component is subjected to silica gel column chromatography, and a system is selected from dichloromethane: methanol=100:2.5, giving A4C2A component. And (3) performing methanol system gel column chromatography on the A4C2A component, and repeatedly purifying to obtain 128mg of fungus secondary metabolite.
The structural identification of the fungal secondary metabolite was performed according to the low resolution ESI-MS M/z153[ M-H ] shown in FIG. 2] - Calculated value C 8 H 10 O 3 Determining the molecular formula of C 8 H 10 O 3 The unsaturation was 4. According to FIGS. 3, 4 and Table 1 1 H NMR spectrum, 13 C NMR spectrum analysis, the structure is identified as terrein, and the structural formula is:
TABLE 1
Example 3
Determining antibacterial activity of the fungus secondary metabolite terrein obtained in example 2 on phytophthora sojae by using a hypha growth rate method, using azoxystrobin treatment as a positive control, and further calculating EC of the fungus secondary metabolite terrein and azoxystrobin for inhibiting growth of phytophthora sojae silk by using a hypha growth inhibition test 50 The final concentrations of the fungus secondary metabolite tertein and azoxystrobin are respectively set to be 6.25 mug/mL, 12.5 mug/mL, 25 mug/mL, 50 mug/mL and 100 mug/mL which are 5 gradient concentrations, and the EC of the fungus secondary metabolite tertein and azoxystrobin for inhibiting the growth of phytophthora sojae wire is further calculated through a hypha growth inhibition test 50 The values and results are shown in Table 2.
TABLE 2
As is clear from Table 2, the growth inhibition rate of tertein against phytophthora sojae reached 100% and EC at a concentration of 100. Mu.g/mL 50 EC of azoxystrobin with a value of 23.79 μg/mL 50 The value was 9.62. Mu.g/mL.
Further observing EC by scanning electron microscope 50 The phytophthora sojae silk form with the value of 23.79 mug/mL is found to have obvious difference between the control mycelium form and the mycelium form of the treatment group. The control Phytophthora sojae silks (FIG. 5, A-B) were uniform in size, smooth in surface, relatively regular cylindrical in shape and intact in hyphae ends. The mycelium of the treatment group has obvious shrinkage and shrinkage in the form of (FIG. 5, C-D), the volume is reduced, the tail end of the mycelium is obviously deformed, and the structure is incomplete.
Example 4
The inhibition effect of the fungal secondary metabolite terrein on zoosporangium formation, zoosporangium germination and zoosporangium germination of Phytophthora sojae was tested using spore germination inhibition test, the final concentration of the fungal secondary metabolite terrein was set to 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL and 10. Mu.g/mL for 5 gradient concentrations, and the EC thereof was calculated 50 The values and results are shown in Table 3.
TABLE 3 Table 3
As is clear from Table 3, as the concentration of the fungal secondary metabolite terrein increases, the inhibition rate against zoosporangium and spores of Phytophthora sojae increases, and EC 50 Values of 4.70, 1.25 and 0.92. Mu.g/mL, respectively, control azoxystrobin EC 50 The values were 0.18, 0.05 and 0.017. Mu.g/mL, respectively.
Example 5
The prevention and treatment effects of the fungus secondary metabolite terrein on soybean epidemic diseases are determined by adopting an in-vitro leaf-blade method and a root irrigation method, and specific experimental data are shown in tables 4 and 5.
TABLE 4 Table 4
TABLE 5
As can be seen from tables 4 and 5, the effect of preventing phytophthora sojae was 61.26% and the effect of treating phytophthora sojae was 61.40% when the concentration of terrein as a secondary metabolite of eubacteria was 300. Mu.g/mL, which is comparable to that of azoxystrobin as a control agent. The results of the potting root method are shown in fig. 6 and 7. From FIGS. 6 and 7, it is clear that the concentration of the fungal secondary metabolite tertein is 800 μg/mL, and the preventive effect and the therapeutic effect are 63.00% and 74.00% respectively, which are lower than the positive control azoxystrobin, but have smaller differences; it can be seen that the fungus secondary metabolite terrein of the embodiment can be used for preventing and treating soybean epidemic diseases or preparing pesticides for preventing and treating soybean epidemic diseases; can effectively reduce the drug resistance problem caused by the prevention and treatment of soybean epidemic diseases by using traditional chemical pesticides in China.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Claims (7)
1. A method for preparing a fungus secondary metabolite Terrein, which is characterized by comprising the following steps:
s1, separating a fungus, namely, a spherical shell fungus PLectosphaerella sp, from an insect horse intestinal flora, and storing the fungus in a refrigerator at the temperature of minus 80 ℃; the strain is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and the preservation number is: CGMCCNO.40710 with a preservation date of 2023.6.25 and a preservation address of Beijing Chaoyang area North Chenxi No. 1 and 3;
s2, carrying out 24L liquid fermentation on the fungi SN5702, and obtaining a fermentation crude extract of the fungi SN5702 by adopting macroporous resin adsorption, methanol leaching and dichloromethane extraction;
s3, separating and purifying the fermentation crude extract of the fungi SN5702 to obtain a fungus secondary metabolite-Terrein.
2. The method for preparing a fungal secondary metabolite Terrein according to claim 1, wherein said step S1 specifically comprises:
soaking insect Ma Liuyong% alcohol on ultra-clean bench for 3min, and then repeatedly soaking with sterile waterWashing the surface of the insect body; placing the treated insect in a sterilized culture dish, taking its intestinal tract with a sterilized scalpel, placing in a sterilized mortar, adding appropriate amount of sterile water, sufficiently grinding, and diluting to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 The doubling liquid is evenly coated on PDA culture medium by using a liquid-transferring gun to respectively take 100 mu L of grinding liquid, and is cultivated in the environment of 25 ℃; single colonies of different forms were picked using an inoculating needle and cultured repeatedly to obtain fungus SN5702.
3. The method for preparing a fungal secondary metabolite Terrein according to claim 1, wherein said step S2 comprises:
s21, transferring fungus SN5702 stored in a refrigerator at the temperature of minus 80 ℃ into a culture dish containing PDA culture medium, and culturing for 6 days at the constant temperature of 25 ℃; 5mL LPDB culture solution is added into a 25mL test tube, and sterilization is carried out for 30min at 121 ℃; collecting bacterial cakes with the diameter of 5mm at the edge of an SN5702 bacterial colony by using a puncher in a sterile operation table, inoculating the bacterial cakes into a test tube, and carrying out shake culture at 25 ℃ and 180r/min for 48 hours to obtain primary seed fermentation liquor;
s22, adding 50mL of PDB culture solution into a 250mL triangular flask, sterilizing for 30min at 121 ℃, transferring the primary seed fermentation solution into the triangular flask in a sterile operation table, and carrying out shake culture for 48h at 25 ℃ and 180r/min to obtain a secondary seed fermentation solution;
s23, adding 400mLPDB culture solution and 4% XAD-16 macroporous adsorption resin into a 2L triangular flask, and sterilizing for 30min at 121 ℃; transferring the secondary seed fermentation liquid into a 2L triangular flask in a sterile operation table, and carrying out shake culture at 25 ℃ and 180r/min for 7d to carry out total fermentation for 24L; filtering to remove mycelium and fermentation liquid after fermentation, cleaning resin with clear water, mixing macroporous adsorption resin, and oven drying at 28deg.C;
s24, placing the dried macroporous adsorption resin in a 2L separating funnel, adding methanol until the macroporous adsorption resin is not passed through, oscillating, standing and soaking for 3 hours;
s25, repeating the step S24 for four times, collecting methanol eluent, concentrating to obtain a methanol crude extract, and weighing for later use; four extractions of the methanol crude extract with a methylene chloride-distilled water-methanol system were performed, whereinV Dichloromethane (dichloromethane) :V Distilled water :V Methanol ＝2:1:1，V Total (S) =1200 mL, combining dichloromethane eluates, concentrating under reduced pressure to obtain crude fermentation extract of fungus SN5702.
4. The method for preparing a fungal secondary metabolite Terrein according to claim 1, wherein said step S3 comprises:
s31, performing primary silica gel column chromatography by using a dichloromethane-methanol system as an eluent, wherein the elution proportion is that dichloromethane is sequentially used for eluting 2L and V (dichloromethane) :V (methanol) Elution of 4l, v=100:1, 100:2, 100:4, 100:8, 100:16, respectively (dichloromethane) :V (methanol) 2L eluted with 1:1, 2L eluted with methanol, 26L co-eluted; combining similar components according to a thin layer chromatography result after the first-stage silica gel column chromatography to obtain a component A and a component B;
s32, carrying out petroleum ether-ethyl acetate system gradient elution on the component A, wherein the elution proportion is V in sequence (Petroleum ether) :V (Ethyl acetate) After combining the components with similar polarities, respectively obtaining five components A1, A2, A3, A4 and A5, wherein the components are respectively shown in the specification of (9:1, 8:2, 7:3 and 6:4);
s33, performing silica gel column chromatography on the A4 component by taking a methylene dichloride-methanol system as an eluent, wherein the elution proportion is V in sequence (dichloromethane) :V (methanol) Two gradients of 100:1.5 and 100:2 were eluted and similar fractions were pooled and concentrated to give three components A4A, A4B, A C;
s34, using petroleum ether-ethyl acetate system as eluent, performing silica gel column chromatography on the A4C component, V (Petroleum ether) :V (Ethyl acetate) =7:3, yielding three components A4C1, A4C2, A4C 3; performing silica gel column chromatography on the A4C2 component by using a methylene dichloride-methanol system as an eluent, V (dichloromethane) :V (methanol) =100:2.5, yielding A4C2A component; and (3) performing methanol system gel column chromatography on the A4C2A component, and repeatedly purifying to obtain a fungus secondary metabolite Terrein.
5. A fungal secondary metabolite Terrein prepared by the method of any one of claims 1-4.
6. Use of the fungal secondary metabolite Terrein according to claim 5 for controlling soybean epidemic disease.
7. Use of the fungal secondary metabolite Terrein according to claim 5 in the manufacture of a pesticide for controlling soybean epidemic disease.
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