CN114231422B - Fusarium solani for degrading fomesafen and application thereof - Google Patents
Fusarium solani for degrading fomesafen and application thereof Download PDFInfo
- Publication number
- CN114231422B CN114231422B CN202111490799.3A CN202111490799A CN114231422B CN 114231422 B CN114231422 B CN 114231422B CN 202111490799 A CN202111490799 A CN 202111490799A CN 114231422 B CN114231422 B CN 114231422B
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- CN
- China
- Prior art keywords
- fomesafen
- fusarium solani
- degrading
- fusarium
- strain
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- BGZZWXTVIYUUEY-UHFFFAOYSA-N fomesafen Chemical compound C1=C([N+]([O-])=O)C(C(=O)NS(=O)(=O)C)=CC(OC=2C(=CC(=CC=2)C(F)(F)F)Cl)=C1 BGZZWXTVIYUUEY-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 241000427940 Fusarium solani Species 0.000 title claims abstract description 42
- 230000000593 degrading effect Effects 0.000 title claims abstract description 24
- 238000009629 microbiological culture Methods 0.000 claims abstract description 10
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims abstract description 9
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 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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- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- VWDXGKUTGQJJHJ-UHFFFAOYSA-N Catenarin Natural products C1=C(O)C=C2C(=O)C3=C(O)C(C)=CC(O)=C3C(=O)C2=C1O VWDXGKUTGQJJHJ-UHFFFAOYSA-N 0.000 description 1
- 244000223760 Cinnamomum zeylanicum Species 0.000 description 1
- 239000010282 Emodin Substances 0.000 description 1
- RBLJKYCRSCQLRP-UHFFFAOYSA-N Emodin-dianthron Natural products O=C1C2=CC(C)=CC(O)=C2C(=O)C2=C1CC(=O)C=C2O RBLJKYCRSCQLRP-UHFFFAOYSA-N 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- YOOXNSPYGCZLAX-UHFFFAOYSA-N Helminthosporin Natural products C1=CC(O)=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O YOOXNSPYGCZLAX-UHFFFAOYSA-N 0.000 description 1
- 239000005590 Oxyfluorfen Substances 0.000 description 1
- OQMBBFQZGJFLBU-UHFFFAOYSA-N Oxyfluorfen Chemical compound C1=C([N+]([O-])=O)C(OCC)=CC(OC=2C(=CC(=CC=2)C(F)(F)F)Cl)=C1 OQMBBFQZGJFLBU-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000000103 Potentilla erecta Species 0.000 description 1
- 235000016551 Potentilla erecta Nutrition 0.000 description 1
- NTGIIKCGBNGQAR-UHFFFAOYSA-N Rheoemodin Natural products C1=C(O)C=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1O NTGIIKCGBNGQAR-UHFFFAOYSA-N 0.000 description 1
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- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- KIPLYOUQVMMOHB-MXWBXKMOSA-L [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O Chemical compound [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O KIPLYOUQVMMOHB-MXWBXKMOSA-L 0.000 description 1
- NUFNQYOELLVIPL-UHFFFAOYSA-N acifluorfen Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(OC=2C(=CC(=CC=2)C(F)(F)F)Cl)=C1 NUFNQYOELLVIPL-UHFFFAOYSA-N 0.000 description 1
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- CONWAEURSVPLRM-UHFFFAOYSA-N lactofen Chemical compound C1=C([N+]([O-])=O)C(C(=O)OC(C)C(=O)OCC)=CC(OC=2C(=CC(=CC=2)C(F)(F)F)Cl)=C1 CONWAEURSVPLRM-UHFFFAOYSA-N 0.000 description 1
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- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 1
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- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/40—Organic compounds containing sulfur
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Environmental & Geological Engineering (AREA)
- General Health & Medical Sciences (AREA)
- Soil Sciences (AREA)
- Zoology (AREA)
- Botany (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Materials Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses Fusarium putrescens for degrading fomesafen, which is named as follows: F-F1, classified and named: fusarium solani (CGMCC No. 23288) and the preservation date is: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center. The rDNA-ITS sequence of the strain F-F1 consists of 573 bases, fusarium solani obtained by screening is used as a single strain to co-metabolize herbicide Fusarium solani with a proper amount of nutrient substances, the change of the residual concentration of fomesafen in the continuous 7d culture process is determined, and the result shows that Fusarium solani F-F1 and a proper amount of nutrient substances can well degrade fomesafen.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to Fusarium solani for degrading fomesafen and application thereof.
Background
At present, the quality condition of farmland soil in China is worry, and the unreasonable use of chemical fertilizers and pesticides is one of the main reasons. Diphenyl ether herbicides (diphenyl ether herbicides, DEHs) are widely used worldwide due to their high efficiency, low toxicity, high selectivity and the like. Most of the existing fluorine-containing varieties with high activity mainly comprise fomesafen, oxyfluorfen, acifluorfen, lactofen and the like. The fomesafen is applied to 80% -90% of soybean fields in China, and in order to promote agricultural structure optimization, the national planting industry structure in 2020 is adjusted, so that the soybean area is increased to 1.4 hundred million mu. With the succession of soybean farmland weed groups, the number of weeds insensitive to pesticides is increased, and the use amount and application area of fomesafen are increased in recent years. Because fomesafen accumulates in the soil for a long period of residual effect, the residual quantity in the field is high due to continuous application for many years, and the yield reduction and even the harvest failure of aftercrop sensitive crops such as cabbages, beet, corn, wheat and the like are caused, the adjustment of the agricultural planting structure and the safety of agricultural production in the main production area of Chinese grains are seriously threatened, and the foggy-side agricultural fertilizer also becomes an important factor of agricultural non-point source pollution in China. Therefore, how to clean and efficiently remove the residual toxicity of fomesafen in soil is a current urgent problem to be solved. Although DEHs may also be digested by photodegradation, leaching, etc., microbial degradation is a major mode of degradation due to the presence of enzymes in many microorganisms that readily convert nitro groups. The pollution of DEHs in farmland soil is cooperatively repaired by utilizing the main biological groups of the soil, and the method has important significance for exploring a new environment-friendly pollution repair technology and realizing green sustainable development of agriculture.
The current research considers that Fusarium solani is soil-dwelling bacteria, which can cause systemic or local infection of plants and cause plant wilting and rot. However, at the same time, the scholars find that the fusarium bran has an inhibiting effect on various pathogenic bacteria. Also, research reports that Fusarium solani can produce rhein and emodin as pharmaceutical monomer components and degrade functions of agricultural antibiotics of terramycin, norfloxacin and sulfadimidine.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides Fusarium solani for degrading fomesafen and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
fusarium putrescens for degrading fomesafen, wherein the name of the strain is: F-F1, classified and named: fusarium solani (CGMCC No. 23288) and the preservation date is: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
Further, rDNA-ITS sequence of the strain F-F1 is SEQ ID NO.1.
Further, the Fusarium solani F-F1 can degrade the herbicide fomesafen under the co-metabolism effect of the additional nutrient substances, and the degradation rate of the fomesafen can reach 96.2%.
Further, the nutrient is basic salt culture medium plus 2 percent (mass fraction) glucose;
wherein, the basic salt culture medium is: 1.0g/L of ammonium sulfate, 1.5g/L of dipotassium hydrogen phosphate, 0.5g/L of monopotassium phosphate, 1.0g/L of sodium chloride, 0.4g/L of magnesium sulfate heptahydrate, and constant volume of distilled water, and adjusting pH to be neutral by hydrochloric acid; the mixture was autoclaved at 121℃for 20 minutes before use.
A fomesafen degrading agent comprising a fomesafen degrading fusarium solani as described above.
Further, the reagent further comprises an additional carbon source and/or nitrogen source.
Further, the reagent further comprises glucose as an additional carbon source.
The use of fusarium solani as described above as a medicament for degrading fomesafen.
The application of Fusarium solani as soil remediation agent.
The application of Fusarium solani as the water body restoration agent is provided.
The beneficial effects obtained by the invention are as follows:
1. the rDNA-ITS sequence of the strain F-F1 consists of 573 bases, fusarium solani obtained by screening is used as a single strain to co-metabolize herbicide Fusarium solani with a proper amount of nutrient substances, the change of the residual concentration of fomesafen in the continuous 7d culture process is determined, and the result shows that Fusarium solani F-F1 and a proper amount of nutrient substances can well degrade fomesafen.
2. According to the invention, degradation and repair researches of Shi Chucao doses of fomesafen in farmland through the combination of Fusarium solani and nutrient glucose in an inorganic salt culture medium are carried out, and the change of fomesafen in the culture medium in the process of culturing for 168h (7 d) is determined. Preliminary proof that the fusarium can be used as a degrading strain to degrade fomesafen and can be used as a candidate strain for restoring pollution of farmland soil herbicide fomesafen.
3. The invention is proved by experiments that: the culture medium of the strain F-F1 is: co-metabolism of 50mg L in the presence of 2% glucose -1 When the fomesafen is cultured for 7d, the degradation rate of the fomesafen in F-F1+2% Glucose (GFM) treatment can reach 96.2% respectively, and the degradation rate of the fomesafen in blank (CK) treatment is less than 4.4%. Whereas the degradation rate of fomesafen in the treatment (FM) with only fungi is only 19.5%.
4. The invention analyzes and tests the degradation effect of the pure strain F-F1 on the herbicide fomesafen under the co-metabolism effect of the additional nutrient substances, and the strain has the prospect of being used as a candidate strain for degrading and repairing the farmland herbicide fomesafen.
Drawings
FIG. 1 is a diagram showing colony morphology and microscopic features of Fusarium solani on potato dextrose agar medium;
FIG. 2 is a diagram showing the growth of Fusarium solani in pure culture and with an added carbon/nitrogen source according to the present invention;
FIG. 3 is a graph showing the degradation of fomesafen in a blank basal salt medium and a nutrient-added control according to the present invention;
FIG. 4 is a graph showing the degradation rate of fomesafen by Fusarium solani under the co-metabolism of the blank basal salt medium and the nutrient substances.
Detailed Description
The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are conventional commercial products unless otherwise specified, the methods used in the invention are conventional methods in the art unless otherwise specified, and the mass of each substance used in the invention is conventional.
Fusarium putrescens for degrading fomesafen, wherein the name of the strain is: F-F1, classified and named: fusarium solani (CGMCC No. 23288) and the preservation date is: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
Preferably, the rDNA-ITS sequence of the strain F-F1 is SEQ ID NO.1.
Preferably, the fusarium solani F-F1 can degrade herbicide fomesafen under the co-metabolism effect of additional nutrient substances, and the degradation rate of fomesafen can reach 96.2%.
Preferably, the nutrient is basal salt medium+2% (mass fraction) glucose;
wherein, the basic salt culture medium is: 1.0g/L of ammonium sulfate, 1.5g/L of dipotassium hydrogen phosphate, 0.5g/L of monopotassium phosphate, 1.0g/L of sodium chloride, 0.4g/L of magnesium sulfate heptahydrate, and constant volume of distilled water, and adjusting pH to be neutral by hydrochloric acid; the mixture was autoclaved at 121℃for 20 minutes before use.
A fomesafen degrading agent comprising a fomesafen degrading fusarium solani as described above.
Preferably, the reagent further comprises an additional carbon source and/or nitrogen source.
Preferably, the reagent further comprises an additional carbon source, glucose.
The use of fusarium solani as described above as a medicament for degrading fomesafen.
The application of Fusarium solani as soil remediation agent.
The application of Fusarium solani as the water body restoration agent is provided.
Specifically, the preparation and detection of the correlation are as follows:
the invention separates a strain of fusarium solani from the soybean soil of a farm in Harbin city, and respectively carries out morphological and molecular biological method identification. Morphological analysis including culture methods, pellet staining and mini-culture identification, molecular biology methods selected ITS (the internal transcribed spacer region ofthe rRNA gene, the gene transcription spacer) and β -tubulin gene (TUB), and by sequencing by amplification, the results showed high homology with fusarium solani Fusarium solani rDNA ITS gene. The classification is named: fusarium solani, deposit unit: china general microbiological culture Collection center (CGMCC) with a collection number of CGMCC No.23288 and a collection date of: 2021, 10 and 19, the deposit address is: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
According to the invention, the degradation condition of fomesafen under the action of the Fusarium solani strain F-F1 and glucose co-metabolism of nutrient substances is researched in a laboratory by utilizing an inorganic salt culture medium, and a solid scientific basis is provided for future application of the fungus in farmland polluted soil and water.
The screening and physiological property test process of the strain is as follows:
1. materials and methods
1.1 samples, instruments, reagents and Medium
Sample source: the soil sample of the isolated and screened strain is collected from soybean soil of a farm in Harbin city.
Instrument: thermostatic shaker (gold jar, huate, changzhou), centrifuge (Centrifuge 5810R, eppendorf Germany), microfiltration membrane (0.22 μm, pall, U.S.A.), gene Analyzer (3730xl DNAanalyzer,ABI, U.S.A.), capillary (50 cm,96 lanes, 4331246, ABI, U.S.A.), PCR apparatus (2720, ABI, U.S.A.), electrophoresis apparatus (DYY-8C, six instrument works, beijing), ultraviolet analyzer (UV-IV, new technology application institute, beijing), VORTEX apparatus (VORTEX-5, manufactured by Linbell instruments Co., jiangsu sea door), ultra-high Performance liquid chromatograph (H-Class, waters, U.S.A.).
The reagent consumables are shown in Table 1.
Table 1 Table of reagent consumables list for experiments
Enrichment medium: 5g/L peptone, 3g/L beef extract, 5g/L sodium chloride, distilled water to constant volume, adjusting pH to 7.0, and autoclaving at 121deg.C for 20min before use.
Basic salt medium: 1.0g/L of ammonium sulfate, 1.5g/L of dipotassium hydrogen phosphate, 0.5g/L of monopotassium phosphate, 1.0g/L of sodium chloride, 0.4g/L of magnesium sulfate heptahydrate, and distilled water for constant volume, and adjusting the pH value to be neutral by hydrochloric acid. The mixture was autoclaved at 121℃for 20 minutes before use.
Purification medium (potato dextrose agar medium): potato extract powder 5.0g/L, glucose 20.0g/L, agar 14.0g/L, pH (25 ℃) 5.6+ -0.2. Distilled water is used for constant volume, and the distilled water is sterilized for 20min at the temperature of 121 ℃ after subpackaging, and is uniformly shaken for standby.
Medium for degradation experiments: with basal salt medium.
2 method
2.1 Strain screening
Taking 10g of a soil sample from soybean soil in a farm in Harbin city, adding the soil sample into a 100mL triangular flask containing fomesafen with the concentration of 50mg/L and culturing for 5 days at the oscillation frequency of 150r/min at 30 ℃; then, the mixture was transferred to a medium containing fomesafen at a concentration of 100mg/L at an inoculum size of 10%Culturing in 100mL triangular flask with enriched culture medium for 5 days; transferring the strain into a triangular flask containing 100mL of enrichment medium with the concentration of fomesafen of 200mg/L according to the inoculation amount of 10%, and continuously culturing for 5 days; then 1mL of the enrichment culture medium bacterial liquid is taken and added into a 10mL centrifuge tube, distilled water is used for constant volume, and the enrichment culture medium bacterial liquid is diluted step by step for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Transferring 0.1mL of diluent to a purification culture medium plate with the concentration of fomesafen of 100mg/L, coating the plates, placing the plates in a constant temperature incubator at 30 ℃ for 3 days, selecting single colonies with different forms of characteristics, inoculating the single colonies to the purification culture medium with the concentration of fomesafen of 100mg/L, and respectively carrying out 3 times of separation and purification by adopting a plate streaking method. After purification, single colony strains with better growth vigor on a flat plate are selected and stored in an inclined plane test tube with potato dextrose agar culture medium.
2.2 screening of Fomesafen function degrading bacteria
Culturing the purified strain in an enrichment culture medium until the logarithmic period (the logarithmic period is determined by measuring an OD600 value, the OD600 value is 0.9, namely the logarithmic period), and then inoculating the strain into a basic inorganic salt culture solution with the concentration of fomesafen of 50mg/L at 5% of inoculation amount (in the experiment, "5% of inoculation amount" refers to the volume ratio of inoculation solution to inoculated culture medium), wherein the inorganic salt culture solution is treated by the following steps: 2% glucose (mass fraction) was added separately before sterilization, and a control of the non-inoculated broth was set, and each sample was treated 3 times. After the completion of the culture, 2mL of the supernatant was collected, 10mL of acetonitrile, 2g of sodium chloride, vortexing for 1min, and centrifugation at 8000r/min for 5min were added, 1mL of the supernatant was collected, and the residual amount of fomesafen was measured by passing through a 0.22 μm filter membrane using an ultra performance liquid chromatograph. Through screening, a fungus strain with excellent co-metabolism function of fomesafen is obtained and named as F-F1. The specific settings and results are shown in tables 2 and 3.
TABLE 2 interpretation of the meaning of the different numbering treatments
TABLE 3 residual and degradation Rate of Fomesafen under Co-metabolism of the strain and nutrient of the invention
As shown in fig. 2, the results indicate that: after F-F1 inoculation, the degradation effect of fomesafen in the treatment (FM) without adding nutrient substances is higher than that of CK (the degradation rate of the fomesafen in 168h of culture is 4.4%), but the degradation rate of fomesafen in 168h of culture is only 19.5%. After 2% Glucose (GFM) nutrient substances are added, the degradation of fomesafen in a basic salt culture medium is greatly accelerated, and after 168 hours of culture, the residual fomesafen is degraded to 1.7mg/L, and the degradation rate reaches 96.2%.
2.3 method for detecting residual quantity of pesticide fomesafen
2.3.1 external standard method: the standard fomesafen solution is diluted to 0.02, 0.1, 0.2, 0.5, 1, 2 and 5mg/L concentration by using an equal volume of acetonitrile and deionized water (1:1), then the concentration is measured by using an ultra-high performance liquid chromatograph, 1 mu L of each sample is injected, each sample is treated for 3 times, and the average value of peak areas is obtained. And (3) taking the concentration as an abscissa and the peak area as an ordinate to make a standard curve.
2.3.2 liquid chromatography conditions of fomesafen: watersaCQUITYBEH C18 column, 1.7 μm x 2.1mm x 100mm; column oven temperature: 40 ℃; mobile phase: acetonitrile: ultrapure water: formic acid (80:20:0.1, v/v/v); wavelength: 275nm; sample injection amount: 5. Mu.L.
The residual quantity of the fomesafen in the culture solution is obtained through a standard curve, and then the degradation rate of the fomesafen is calculated through a formula 1, and the results are shown in figures 3 and 4.
Equation 1: degradation rate (%) = (concentration of fomesafen in initial culture solution-residual concentration of fomesafen in treatment culture solution)/concentration of fomesafen in initial culture solution x 100%
2.4 identification of Fomesafen functional degradation Strain F-F1
Extraction of strain genome DNA: isolated strains were grown on potato dextrose agar based on 30 ℃ incubator for 5 days using RAidlab DN14 fungus extraction kit according to the instructions for the following procedure: scraping 0.2-0.5mg mycelium, and placing into a 2.0ml centrifuge tube; adding 2 glass beads with the diameter of 3-4MM and 100 mu l of CTAB lysate, and grinding twice for 2min on a Retsch MM400 refiner; adding 400 μl of the CTAB lysate preheated in 65 deg.C water bath, mixing, and standing in 65 deg.C water bath for 0.5-1hr; adding equal volume chloroform and isoamyl alcohol (24:1), and gently reversing and mixing for 50 times; centrifuging at 12000rpm at room temperature for 5min, and collecting supernatant to a new centrifuge tube; adding 1.5 times volume of binding solution into the supernatant, uniformly mixing, transferring to an adsorption column, centrifuging at 12000rpm at room temperature for 30sec, and discarding the waste liquid; adding 500 μl of inhibitor removing solution, centrifuging at 12000rpm at room temperature for 30sec, and discarding the waste liquid; adding 600 μl of the rinse solution, centrifuging at 12000rpm at room temperature for 30sec, and discarding the waste liquid; placing the adsorption column back into an empty collecting tube, and centrifuging at 12000rpm at room temperature for 2min; transferring the adsorption column into a sterilized 1.5ml centrifuge tube, and drying the adsorption column at room temperature for 5min; adding 50-100 μl of elution buffer, standing at room temperature for 3-5min, centrifuging at 12000rpm for 30sec to collect genomic DNA solution, and immediately using or storing at-20deg.C for use.
Ribosomal gene (rDNA) sequence amplification sequencing: the PCR MIX buffer and primer (upstream primer ITS1:5'-TCCGTAGGTGAACCTGCGG-3' and downstream primer ITS4:5 '-TCCTCCGCTT-ATTGATATGC-3') solutions were removed and thawed on ice at 4℃for 30 minutes.
PCR amplification System (50 μl): dd H2O 19 μl; 30. Mu.l of PCR MIX enzyme buffer; 1 μl of the upstream primer; 1 μl of the downstream primer; template 1.5. Mu.l (first 4 components were mixed and the PCR tube was packed, and then DNA template was added).
PCR amplification reaction procedure: denaturation at 94℃for 5min, melting at 94℃for 0.5min, renaturation at 50℃for 30s, extension at 72℃for 1min, extension at 72℃for 7min after 35 cycles of amplification, and preservation of the product at 4℃after completion.
And (3) purifying a PCR product: preparing 2% agarose gel: 2g of agarose was added to 100mL of 1 XTBE and dissolved by heating; electrophoresis voltage is 100V, electrophoresis time is 30min; and (3) purifying the PCR product according to the operation of the kit instruction after tapping.
Ethanol precipitation and purification are carried out on the PCR product, then the PCR product conforming to the size of the band is delivered to Tianjin Jing Yang biotechnology Co., ltd for analysis and sequencing by using ABI 3730XL sequencer electrophoresis on a BigDyeTrv 3.1 Cycle Seq Kit (4336921) Kit, and rDNA-ITS sequence composition of the strain is obtained:
the rDNA-ITS sequence consists of 573 bases (bp).
2.5 colony morphology characterization
F-F1 strain grows fast on potato glucose culture medium, and grows luxuriantly when cultured at 28 ℃ for 5 days with colony diameter of 50-90 mm and single colony hypha is aerial hypha, and the single colony hypha is low-level cotton-like and paste-based. The strain has white edge, light purple cinnamon in the middle and pale yellow back. The absence of diaphragm in hypha was observed under microscope and was long tubular single cell. The small conidium is grown in a pseudo-head mode, and the spore is elliptic, oblong, short sausage-shaped or comma-shaped, has no partition, is transparent and smooth. The large conidium is sickle-shaped, spindle-shaped, needle-shaped or cylindrical, and the like, is slightly bent, has short top cells, is slightly narrow and thin or becomes blunt, has a wall thickness and is free from partition; no foot cell. The colony morphology and microscopic morphology of strain F-F1 are shown in FIG. 1.
2.5 identification of Strain F-F1 as a novel functional Strain
Blast alignment is performed on the sequencing result and GenBank in NCBI, so that the homology of F-F1 and ITS sequence of Fusarium solani is 99%. Based on morphological features of the strain F-F1 and rDNA-ITS sequence analysis results, the strain was identified as Fusarium solani and designated Fusarium solani F-F1 (Fusarium solani F-F1). At present, no report of the function of degrading fomesafen of Fusarium solani (Fusarium solani) is found by researchers at home and abroad. Therefore, the strain F-F1 belongs to a new functional strain, and has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23288 in the year 10 and 19 of 2021.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Sequence listing
<110> agricultural rural environmental protection scientific research monitoring institute
<120> Fusarium putrescens for degrading fomesafen and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 573
<212> DNA
<213> rDNA-ITS sequence of Strain F-F1 (Unknown)
<400> 1
cctccgtagg gggtgactgc ggagggatca ttaccgagtt atacaactca tcaaccctgt 60
gaacatacct aaaacgttgc ttcggcggga acagacggcc ctgtaacaac gggccgcccc 120
cgccagagga cccctaactc tgtttttata atgtttttct gagtaaacaa gcaaataaat 180
taaaactttc aacaacggat ctcttggctc tggcatcgat gaagaacgca gcgaaatgcg 240
ataagtaatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc acattgcgcc 300
cgccagtatt ctggcgggca tgcctgttcg agcgtcatta caaccctcag gcccccgggc 360
ctggcgttgg ggatcggcgg aagccccctg tgggcacacg ccgtccctca aatacagtgg 420
cggtcccgcc gcagcttcca ttgcgtagta gctaacacct cgcaactgga gagcggcgcg 480
gccatgccgt aaaacaccca acttctgaat gttgacctcg aatcaggtag gaatacccgc 540
tgaacttaag catatcaaaa ggcggaggaa ctt 573
<210> 2
<211> 19
<212> DNA
<213> upstream primer ITS1 (Unknown)
<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 20
<212> DNA
<213> downstream primer ITS4 (Unknown)
<400> 3
tcctccgctt attgatatgc 20
Claims (7)
1. Fusarium putrescens for degrading fomesafen is characterized in that: the name of the strain is: F-F1, classified and named: fusarium putrescensFusarium solaniThe preservation number is CGMCC No.23288, and the preservation date is: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
2. A fomesafen degrading agent comprising fomesafen degrading fusarium putrescens of claim 1.
3. The fomesafen degrading reagent according to claim 2, characterized in that: the reagent further comprises an additional carbon source and/or nitrogen source.
4. The fomesafen degrading reagent according to claim 2, characterized in that: the reagent further comprises glucose as an additional carbon source.
5. Use of fusarium solani as claimed in claim 1 as a medicament for degrading fomesafen.
6. Use of fusarium solani as claimed in claim 1 as a soil remediation agent.
7. Use of fusarium solani as claimed in claim 1 as a water remediation agent.
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| CN109136097A (en) * | 2018-07-12 | 2019-01-04 | 农业部环境保护科研监测所 | The penicillium oxalicum of degradation isopropyl methoxalamine and its application |
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