CN110358717A - A kind of microbial bacterial agent and its preparation method and application for fomesafen of degrading - Google Patents
A kind of microbial bacterial agent and its preparation method and application for fomesafen of degrading Download PDFInfo
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- CN110358717A CN110358717A CN201910824679.9A CN201910824679A CN110358717A CN 110358717 A CN110358717 A CN 110358717A CN 201910824679 A CN201910824679 A CN 201910824679A CN 110358717 A CN110358717 A CN 110358717A
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- microbial agent
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- fomesafen
- soil
- microcapsule microbial
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Links
- 230000000813 microbial effect Effects 0.000 title claims abstract description 61
- 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 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 230000000593 degrading effect Effects 0.000 title claims abstract description 13
- 230000001580 bacterial effect Effects 0.000 title abstract description 35
- 239000002689 soil Substances 0.000 claims abstract description 98
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 61
- 239000003094 microcapsule Substances 0.000 claims abstract description 50
- 241000894006 Bacteria Species 0.000 claims abstract description 40
- 239000004009 herbicide Substances 0.000 claims abstract description 26
- 230000002363 herbicidal effect Effects 0.000 claims abstract description 25
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 18
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 18
- 241000983364 Stenotrophomonas sp. Species 0.000 claims abstract description 18
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 15
- 239000001301 oxygen Substances 0.000 claims abstract description 15
- 238000004321 preservation Methods 0.000 claims abstract description 14
- 244000005700 microbiome Species 0.000 claims abstract description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 11
- 239000000661 sodium alginate Substances 0.000 claims description 11
- 235000010413 sodium alginate Nutrition 0.000 claims description 11
- 229940005550 sodium alginate Drugs 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 230000002572 peristaltic effect Effects 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000000967 suction filtration Methods 0.000 claims description 4
- 229940058641 actidose Drugs 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 241000726221 Gemma Species 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 abstract description 17
- 238000006731 degradation reaction Methods 0.000 abstract description 17
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract 1
- 238000012545 processing Methods 0.000 description 71
- 238000011282 treatment Methods 0.000 description 52
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 39
- 241000196324 Embryophyta Species 0.000 description 36
- 240000008042 Zea mays Species 0.000 description 34
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 34
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 22
- 235000005822 corn Nutrition 0.000 description 22
- 230000012010 growth Effects 0.000 description 21
- 238000010790 dilution Methods 0.000 description 14
- 239000012895 dilution Substances 0.000 description 14
- 239000002609 medium Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 12
- 230000035784 germination Effects 0.000 description 12
- 235000009973 maize Nutrition 0.000 description 12
- 238000004587 chromatography analysis Methods 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 10
- 239000002068 microbial inoculum Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 108020004465 16S ribosomal RNA Proteins 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 239000003854 herbicide residue Substances 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000009331 sowing Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 4
- 229940124530 sulfonamide Drugs 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000004382 potting Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 238000005527 soil sampling Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- DICDYWOAKAGJLU-UHFFFAOYSA-N S(=O)(C1=CC=C(C=C1)N)(=O)N.[F] Chemical compound S(=O)(C1=CC=C(C=C1)N)(=O)N.[F] DICDYWOAKAGJLU-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Chemical compound C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000299507 Gossypium hirsutum Species 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 241000122971 Stenotrophomonas Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- -1 chloro-trifluoromethyl phenoxy Chemical group 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000003760 hair shine Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Substances CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 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
-
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
Abstract
A kind of microbial bacterial agent and its preparation method and application for fomesafen of degrading, it is related to microorganism field.The problem of the invention solves the microbial bacterial agents for fomesafen of effectively degrading currently without one kind, bacterium used in the microcapsule microbial agent is the Mixed Microbes of Bei Laisi bacillus (Bacillus Velezensis) FB11 and widow oxygen monad (Stenotrophomonas sp.) FB14, deposit number is respectively GMCC No.17732, CGMCC No.17731, preservation date is on May 08th, 2019, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.After two plants of bacterium are mixed, auxiliary material is added, microbial bacterial agent is made.It is used for degrading herbicide fomesafen.Hybrid bacterial strain used in the present invention is significant to herbicide fomesafen degradation effect in soil.
Description
Technical field
The present invention relates to microorganism fields, and in particular to a kind of microbial bacterial agent preparation method for fomesafen of degrading.
Background technique
Entitled 5- (2- the chloro-trifluoromethyl phenoxy)-N- methylsulfonyl -2- nitrobenzamide of fomesafen chemistry,
The herbicide for belonging to diphenyl ether receives more in agriculture weeding since it has high herbicidal active at low concentrations
Carry out more concerns.
When field is using the fomesafen aqua that concentration is 250g/L, dosage is seldom usually 667m2/ 100mL,
But fomesafen is good to the preventive effect of annual broad-leaved shape weeds under this dosage, mainly act on soybean,
The field of corn, cotton and peanut and other crops.But since its residence time in the soil is longer, it generally can reach 6-12
A month, that is to say, that when second year long-term cropping, still having the herbicide residues of a large amount of upper one years, peasant be will continue in the soil
Apply new herbicide fomesafen in the soil, thus in field soil fomesafen amount, can with long-term cropping
The increase of the time limit and constantly accumulate.This this may result in soil degradation, make the forfeiture production capacity of soil temporarily or continuously, shadow
The growth for ringing crop, the nutritional ingredient for reducing crop even result in crop total crop failure, seriously affect the development of China's agricultural economy.It is residual
Herbicide in the soil is stayed not only to will affect crop growth, the habitat that can also destroy terrestrial organism influences the normal of them
Life；The herbicide of residual in the soil can enter rivers,lakes and seas with the water flow on soil surface by the flushing of rainwater, endanger
The growth and breeding of aquatile.These final pesticides all can endanger our strong under the action of food chain, into human body
Health.Therefore it has increasing need for finding environmental-friendly and there is cost-benefit technology to repair the soil polluted by fomesafen
Earth.
The method of herbicide mainly has the modes such as microbial degradation and hydrolysis in degradation soil at present, and microbial bacterial agent is benefit
The microorganism existing for script in soil obtains the micro- life of advantage with our required functions by way of artificially taming
Object, the active bacteria formulation being prepared into conjunction with carrier by immobilization technology, to be carried out to certain substance residual in soil
The technology of degradation.But a kind of microbial bacterial agent effectively degraded for fomesafen how is found, it is that current urgent need solves
The problem of.
Summary of the invention
The purpose of the present invention is to solve a kind of asking for the microbial bacterial agent currently without effectively degradation fomesafen
Topic, and a kind of microbial bacterial agent preparation method of fomesafen of degrading is provided.
A kind of microcapsule microbial agent of the invention, bacterium used in the microcapsule microbial agent are Bei Laisi bacillus
The Mixed Microbes of (Bacillus Velezensis) FB11 and widow oxygen monad (Stenotrophomonas sp.) FB14；Wherein,
Bei Laisi bacillus (Bacillus Velezensis) FB11, is deposited in Chinese microorganism strain preservation management
Committee's common micro-organisms center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is 2019
May 08 day, deposit number was CGMCC No.17732；
Few oxygen monad (Stenotrophomonas sp.) FB14, is deposited in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is in Mays, 2019
08, deposit number was CGMCC No.17731.
A kind of method of preparation microcapsule microbial agent, it is followed the steps below:
One, preparing mass percentage is 2% sodium alginate soln, and mass percentage is 5% Actidose；And it will
The two mixes, and stirs in mixed process when dissolving by heating, obtains mixed solution；
Two, Bei Laisi bacillus (Bacillus Velezensis) FB11 and few oxygen unit cell of logarithmic phase will be grown to
The bacterium solution of bacterium (Stenotrophomonas sp.) FB14, is centrifuged respectively, collects thallus, then mixes the thallus of collection
Afterwards, it is put into spare in refrigerator；
Three, being cooled to temperature to the mixed solution of step 1 is 30 DEG C, and step is added into mixed solution by 1% access amount
Rapid two thallus, and be uniformly mixed；
Four, the mixed liquor of the addition thallus of step 3 is discharged into calcium chloride solution by peristaltic pump, forms microcapsule bacterial
Agent；Wherein, the drain pipe of peristaltic pump is placed at the high 10cm of calcium chloride solution liquid level；
Five, by microcapsule microbial agent drawing liquid, after suction filtration, microcapsule microbial agent is placed in 30 DEG C of baking oven, is dried, is i.e. completion institute
The microcapsule microbial agent preparation stated.
A kind of application of microcapsule microbial agent of the present invention, it is used for degrading herbicide fomesafen.
Bei Laisi bacillus (Bacillus Velezensis) FB11 of the invention, the bacterium colony shape on LB culture medium
State be it is round, edge is irregular, tarnish, milky, opaque, it is in crateriform that surface folding, which has protrusion, concavity,
Colony diameter size is about 2~3mm on nutrient agar, and microscopically observation belongs to gram-positive bacterium to the bacterial strain, there is bud
Spore, somatic cells presentation is rod-shaped, and amphitrichous is movable.
Few oxygen monad (Stenotrophomonas sp.) FB14 of the invention, bacterium colony in it is faint yellow, round, protuberance,
Opaque, neat in edge, has ammonia odor, surface smooth at toughness, on nutrient agar colony diameter size be about 0.5~
1mm, microscopically observation to the bacterial strain belong to gram-negative bacterium, and somatic cells present rod-shaped.
Two plants of bacterium Physiology and biochemistries identification of the invention is as follows:
The biochemical reactions of bacterial strain routine are referring to " common bacteria system identification handbook " and " primary Jie Shi Bacteria Identification hand
Volume " method, carry out the identification of 14 physiological and biochemical indexs respectively to each bacterium bacterial strain, the results are shown in Table 1.
1 degradation bacteria strains physiological and biochemical index of table
Note: "+" indicates reaction as the positive, and "-" indicates reaction for feminine gender.
Two plants of bacterium 16s rDNA identification of the invention is as follows:
Using two plants of bacterium genomic DNAs template, primer is universal primer.Then object in table 2 is sequentially added in PCR pipe
Then matter is reacted in the PCR instrument of setting program.The condition of primer sequence, PCR reaction system and setting respectively as table 2,
Shown in table 3, PCR product is subjected to agarose gel electrophoresis detection, PCR product recycle using DNA QIAquick Gel Extraction Kit pure
Change, be then delivered to the measurement that raw work sequencing company carries out 16S rDNA nucleotide sequence, by the sequence in sequence and Genbank into
According to degree of similarity download sequence after the comparison of row sequence homology, MAGA5.05 software building systematic evolution tree, root are then utilized
According to relationship between the kind of the determination bacterial strain of the visual result of chadogram and with the affiliations of other kinds (result such as Figure 15 and
Shown in 16).
The primer sequence of 2 16S rDNA PCR amplification of table
The pcr amplification reaction system and response procedures of 3 bacterial strain 16S rDNA of table
Product after PCR amplification carries out 1% agarose gel electrophoresis detection, as a result as shown in figure 14, bacterial strain FB11 and
The 16S rDNA genetic fragment size of FB14 is about 1500bp.Identify that bacterial strain FB11 and FB14 are determined respectively by 16S rDNA
For Bacillus velezensis (Bei Laisi bacillus) and Stenotrophomonas sp. (Stenotrophomonas)；Bacterial strain
It is respectively MK828185 and MK828188 that the 16S rDNA sequence of FB11 and FB14, which logs on to Genbank to obtain accession number,.
The present invention include it is following the utility model has the advantages that
Microcapsule microbial agent has finally been made in the present invention, when microbial inoculum is handled soil 30 days, remains fomesafen to rear stubble
The influence of sensitive crop growth of maize is smaller, therefore in practical operation, after selecting microbial inoculum to handle 30 days, is sowed.Examination
It tests result and provides theoretical foundation for microorganism remediation soil from now on.
Carry out plate coating after microcapsule microbial agent produced by the present invention dissolution, measure living bacteria count be 2.936 ×
108cfu/mL。
The residual quantity of herbicide fomesafen in soil, acquired results are measured by high performance liquid chromatography are as follows: the present invention
Hybrid bacterial strain used is significant to herbicide fomesafen degradation effect.
Detailed description of the invention
Fig. 1 is the bacterial strain acclimating test photo of screening；
Fig. 2 is inorganic salts fomesafen culture medium bacterial strain screening photo；Wherein, left hand view is blank control in figure；It is right
Side figure is 1000 times of coating results of dilution；
Fig. 3 is colonial morphology of the bacterial strain FB11 and FB14 on LB culture medium；
Fig. 4 is strain growth curve graph；
Fig. 5 is microcapsule microbial agent morphologies observation figure, wherein left hand view is that undried microcapsule microbial agent shines in figure
Piece；Right part of flg is dry microcapsule microbial agent photo；
Fig. 6 is corn potted plant experiment result figure；Wherein, growth of maize situation is shone after left hand view is processing 30 days in figure
Piece；Right part of flg is growth of maize situation photo after processing 60 days in figure；
Fig. 7 is soil treatment 30 days and 60 days corn seedling processing groups and control group emergence rate, wherein ■ goes out for control group
Seedling rate, ■ are processing group emergence rate；
Fig. 8 is soil treatment 30 days and 60 days corn seedling processing groups and control group plant height, wherein ■ is control group strain
Height, ■ are processing group plant height；
Fig. 9 is soil treatment 30 days and 60 days corn seedling processing groups and control group strain fresh weight, wherein ■ is control group strain
Fresh weight, ■ are processing group strain fresh weight；
Figure 10 is soil treatment 30 days and 60 days corn seedling processing groups and control group plant dry weight, wherein ■ is control group
Plant dry weight, ■ are processing group plant dry weight；
Figure 11 is soil treatment 30 days and 60 days corn seedling processing groups and control group root fresh weight, wherein ■ is control group
Root fresh weight, ■ are processing group root fresh weight；
Figure 12 is soil treatment 30 days and 60 days corn seedling processing groups and control group root dry weight, wherein ■ is control group
Root dry weight, ■ are processing group root dry weight；
Figure 13 is fomesafen canonical plotting；
Figure 14 is that microbial inoculum handles different time fomesafen residue change figure；Wherein, ■ is control group, and ■ is processing
Group；
Figure 15 is 16S rDNA segment PCR amplification electrophoresis detection figure；Wherein, 1:FB11；2:FB14；M:Marker
DL10000；
Figure 16 is fomesafen degradation bacteria strains FB11 phylogenetic tree；
Figure 17 is fomesafen degradation bacteria strains FB14 phylogenetic tree.
Specific embodiment
Specific embodiment 1: a kind of microcapsule microbial agent of present embodiment, used in the microcapsule microbial agent
Bacterium is Bei Laisi bacillus (Bacillus Velezensis) FB11 and few oxygen monad (Stenotrophomonas sp.)
The Mixed Microbes of FB14；Wherein,
Bei Laisi bacillus (Bacillus Velezensis) FB11, is deposited in Chinese microorganism strain preservation management
Committee's common micro-organisms center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is 2019
May 08 day, deposit number was CGMCC No.17732；
Few oxygen monad (Stenotrophomonas sp.) FB14, is deposited in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is in Mays, 2019
08, deposit number was CGMCC No.17731.
Specific embodiment 2: present embodiment is with one difference of specific embodiment: the microcapsule microbial agent
It is by Bei Laisi bacillus (Bacillus Velezensis) FB11 and few oxygen monad (Stenotrophomonas sp.)
Mixed Microbes, sodium alginate, active carbon and the calcium chloride of FB14 is prepared；Wherein, Bei Laisi bacillus (Bacillus
Velezensis) mass ratio of FB11 and widow oxygen monad (Stenotrophomonas sp.) FB14 is 1:1.
It is other same as the specific embodiment one.
Specific embodiment 3: present embodiment is with one difference of specific embodiment: being mixed in microcapsule microbial agent
The mass percentage of bacterium is 92%, and the mass percentage of sodium alginate is 2%, and the mass percentage of active carbon is 5%,
The mass percentage of calcium chloride is 1%.
It is other same as the specific embodiment one.
Specific embodiment 4: a kind of method of microcapsule microbial agent of present embodiment, it is followed the steps below:
One, preparing mass percentage is 2% sodium alginate soln, and mass percentage is 5% Actidose；And it will
The two mixes, and stirs in mixed process when dissolving by heating, obtains mixed solution；
Two, Bei Laisi bacillus (Bacillus Velezensis) FB11 and few oxygen unit cell of logarithmic phase will be grown to
The bacterium solution of bacterium (Stenotrophomonas sp.) FB14, is centrifuged respectively, collects thallus, then mixes the thallus of collection
Afterwards, it is put into spare in refrigerator；
Three, being cooled to temperature to the mixed solution of step 1 is 30 DEG C, and step is added into mixed solution by 1% access amount
Rapid two thallus, and be uniformly mixed；
Four, the mixed liquor of the addition thallus of step 3 is discharged into calcium chloride solution by peristaltic pump, forms microcapsule bacterial
Agent；Wherein, the drain pipe of peristaltic pump is placed at the high 10cm of calcium chloride solution liquid level；
Five, by microcapsule microbial agent drawing liquid, after suction filtration, microcapsule microbial agent is placed in 30 DEG C of baking oven, is dried, is i.e. completion institute
The microcapsule microbial agent preparation stated.
Specific embodiment 5: present embodiment is with four difference of specific embodiment: the quality hundred of sodium alginate
Dividing content is 2%；The mass percentage of active carbon is 5%；The mass percentage of calcium chloride is 1%.
It is other identical as specific embodiment four.
Specific embodiment 6: a kind of application of microcapsule microbial agent of present embodiment, it is used for degrading herbicide fluorine sulfanilamide (SN)
Careless ether.
The content of present invention is not limited only to the content of the respective embodiments described above, the group of one of them or several specific embodiments
The purpose of invention also may be implemented in contract sample.
Beneficial effects of the present invention are verified by following embodiment:
Embodiment 1
Prepared by the degrading herbicide fomesafen microcapsule microbial agent of the present embodiment and degradation experiment is specific as follows:
1, culture medium
LB liquid medium: tryptone 10g/L；Yeast extract 5g/L；NaCl:10g/L；Distilled water (uses 5mol/
LNaOH is adjusted to pH7.0).
Minimal medium: ammonium nitrate 1g/L；Potassium dihydrogen phosphate 0.5g/L；Disodium hydrogen phosphate 1.5g/L；NaCl:1g/L；
Epsom salt 0.2g/L；Agar 15g/L (is adjusted to pH7.2 with 5mol/L NaOH).
2, the screening of bacterial strain
The domestication of 2.1 soil enrichments
The fomesafen aqua that 20 μ L specifications are 250g/L is drawn, the sterilized inorganic salt liquid culture of 50mL is added to
In base；It then weighs 0.5g fresh soil samples to be added in the minimal medium containing herbicide, seal, in 30 DEG C shake
To be cultivated for 24 hours under the revolving speed of 180rpm in bed.The enrichment culture object generated in the absorption above-mentioned bottle of 5mL afterwards for 24 hours is transferred to fresh
It is in the sterile inorganic salt liquid culture medium of 50mL of 250g/L fomesafen aqua containing 20 μ L specifications, culture is for 24 hours.In repetition
Process is stated, until the concentration of fomesafen aqua in bottle reaches 500mg/L.It can be observed that concentration is the nothing of 500mg/L
Muddiness is generated in machine salt culture medium, grows the bacterial strain that can be survived in fomesafen in bottle.
The isolation and purification of 2.2 bacterial strains
200mL inorganic salts solid medium is prepared, is sterilized 20 minutes at 121 DEG C, to the reduction of solid medium temperature but not
The fomesafen aqua of 80 μ L 250g/L is added when solidification, is poured into clean and sterile plate after mixing, it is cooling solidifying
Gu after, it is inverted spare.
Drawing the fomesafen aqua concentration that acclimating is turned out is the bacterium solution 1mL in 500mg/L triangular flask, is added
Into the centrifuge tube containing 9mL sterile water, mixing is played in suction, and diluted concentration is 10 times；Then draw diluted concentration be 10 times from
Liquid 1ml in heart pipe is added in the new centrifuge tube containing 9mL sterile water, and mixing is played in suction, and diluted concentration is initial
100 times；An aforesaid operations are repeated, gradient dilution is carried out, 1000 times that multiple is initial are released in last centrifuge tube.
The 100 μ L of bacterium solution for drawing these three extension rates respectively, is added drop-wise to ready containing fomesafen herbicide
Inorganic salts solid medium in, be coated with uniformly with spreading rod, each dilution applies three plates, as Duplicate Samples；One flat
Bacterium is not applied on plate, as blank control.Whole plates are placed upside down in 30 DEG C of bacteriological incubator and are inverted culture, taken out after 36h
It is observed.The preferable single colonie of growing way carries out scribing line purifying on picking fomesafen inorganic salts solid medium.
The measurement of 2.3 strain growth curves
The LB liquid medium of certain volume is prepared in triangular flask, sterilize 20min at 121 DEG C, cold after the completion of sterilizing
But culture medium is chosen raw on fomesafen inorganic salts solid medium after culture medium temperature reaches room temperature with sterile pipette tips
Long single colonie is linked into sterilized LB culture medium.It puts it in the 30 DEG C of shaking tables adjusted, with the revolving speed of 180rpm
Culture, carries out spreading cultivation for bacterial strain, was measured every 2 hours to absorbance value of the bacterium solution at 600nm in incubation, measures
Period of delay, logarithmic growth phase, stationary phase and the decline phase of hybrid bacterial strain growth, draw growth curve.
3, the preparation of capsule microbial inocula
The preparation process of 3.1 microcapsule microbial agents
Be up to logarithmic growth latter stage bacterium solution carry out thalline were collected by centrifugation, the thallus being collected into is placed on to 4 DEG C of ice
It is spare in case.It is equipped with the mixed solution of 2% sodium alginate, 5% active carbon, water is met due to sodium alginate and becomes thick, so
Outfit process is dissolved by heating, cooling after dissolution completely, and when temperature is cooled to 30 DEG C, the thallus after centrifugation is connect by 1%
Enter amount to be linked into mixed liquor.The suction pipe of peristaltic pump is put into mixed liquor, drain pipe is placed on 1% calcium chloride solution liquid of distance
At the high 10cm in face, after mixed liquor is added drop-wise to calcium chloride solution by pipette sucking, microcapsule microbial agent is formed.Microcapsule microbial agent drop
After complete, the microcapsule microbial agent formed in calcium chloride solution is poured into the drawing liquid funnel for being placed with filter paper, connects bottle,suction and pumping
Filter pump is filtered.After suction filtration, the microcapsule microbial agent in funnel is poured into the plate for being placed with filter paper, being put into temperature is 30
DEG C baking oven in, drying, be stored in dry local back up.
3.2 microcapsule microbial agent determination of quality index
The physical behavior of microcapsule microbial agent after drying is observed and recorded, after record by microcapsule microbial agent be dissolved in
In the sodium citrate solution of original bacteria liquid volume identical 2%, dilution spread is on LB solid medium, calculating its effective viable bacteria
Number.
4, pot experiment
4.1 soil pre-treatments
Soil preparation process is arranged two groups altogether, and every group is designed with three repetitions, and processing method is as shown in table 4.
4 soil preparation method of table
Prepare the identical big flowerpot of 6 sizes, respectively marked as control group 1, control group 2, control group 3, processing group 1, place
Reason group 2, processing group 3 are put into the identical fresh soil sample of 4kg in each basin, take 20g soil sample to be fitted into sack, marked the time, put
Enter in refrigerator and refrigerates.Wherein control group and every group of processing group are designed with three repetitions.80 μ L are all separately added into each basin
250g/L fomesafen aqua makes the final concentration of 5mg/kg soil of fomesafen in soil, soil is stirred evenly, dry
Soil sampling after one night, is put into refrigerator cold-storage；100mL water is separately added into control group 1, control group 2, control group 3, stirring is equal
It is even, micro- glue that 10g is dissolved with the sodium citrate solution of 100mL 2% is separately added into processing group 1, processing group 2, processing group 3
Capsule microbial inoculum, stirs evenly.After one month, soil sampling, is placed in refrigerator and refrigerates respectively.
The processing of 4.2 corn seed germinations
Corn fresh seeds 400 of surface sterilization will be carried out, and 12h was impregnated in 30 DEG C of warm water, then by corn
Seed is spread wet gauze above and below and is put into basin, cultivates in 30 DEG C of environment.Until being calculated after germination
The germinating energy and germination percentage of corn seed, it is unusable if germination percentage and germinating energy are too low；If germination percentage germinating energy reaches
When normal seed, can the seed of picking bud similar length sowed.
The calculation formula of germination percentage and germinating energy is as follows:
Sub- sum × 100% is planted experimentally in the normal chitting piece number/confession in germinating energy (%)=4th day；
Sub- sum × 100% is planted experimentally in the normal chitting piece number/confession in germination percentage (%)=7th day；
4.3 sensitive crop growth of seedling index determinings
Every basin sows the corn seed of 14 grain germinations, totally 6 basin, select maize bud growing way it is good sow, needed before sowing
It loosens the soil, and sowing is not required to too deep, otherwise influence corn seed emergence.By the basin of sowing be placed in illumination cultivation room into
Row culture, keeps each basin local environment identical, using Na lamp as the light source of corn growth in culturing room, carries out daily to maize seedling
Illumination 10h helps maize seedling to carry out photosynthesis, and maize seedling carries out normal respiration after turning off the light.When soil surface is dry
When, a small amount of water can be sprayed in soil surface, but the water volume sprayed in 6 basins should be kept identical.Keep potting room temperature and wet
Degree.After sowing one month, soil sampling, emergence rate, plant height, strain fresh weight, plant dry weight, root fresh weight and the root that note calculates corn seed is done
Weight, after weighing, is dried in 30 DEG C of baking oven, after dry, weighs again, and record data are counted.
4.4 potting soil residues detectons
4.4.1 the pre-treatment of soil sample
N-hexane and acetone are configured to extracting solution according to the ratio of 1:1 first；By chromatography methanol and water in the ratio of 3:1
It is mixed with as chromatogram flow phase, and is filtered using filter.
Weigh each 8g of treated pedotheque, be respectively put into the centrifuge tube of clean 50mL, then to it is each from
It is separately added into 15mL extracting solution in heart pipe, carries out ultrasound assisted extraction 5min using ultrasonic washing instrument, promotes dissolution.After ultrasound
Room temperature is centrifuged 5min under the revolving speed of 5000rpm, and after centrifugation, Aspirate supernatant arrives nitrogen evaporator adjusting into clean test tube
50 DEG C, supernatant is dried up using nitrogen evaporator in draught cupboard.The extracting solution of 15mL is added into centrifuge tube again, repeats the above step
Suddenly, until joined the total extract of 60mL in each centrifuge tube, all after drying, the color of 2mL is separately added into test tube
Methanol is composed, invisible spectro residue is completely dissolved, using the Ultrasound Instrument ultrasonic several seconds after dissolution, is filled into centrifuge tube using filter
In.Spare analysis under the conditions of being stored in -20 DEG C.
4.4.2 the preparation of standard curve
Chromatography methanol and pure water are mixed according to the ratio of 3:1, after mixing, with phosphorus acid for adjusting pH to 3.0, then
It is filtered, all after the completion of filtering, ultrasonic 30min seals the In Shade preservation of bottle.
0.01g fomesafen standard specimen is dissolved in 1mL chromatography methanol, uniformly mixes, is formulated as the fluorine of 10000mg/L
Sulfanilamide (SN) grass ether mother liquor；10 μ L fomesafen mother liquors are drawn, 990 μ L chromatography methanol are subsequently added into, diluted concentration is at this time
100mg/L；The dilution that 100 μ L concentration are 100mg/L is drawn, is subsequently added into the chromatography methanol of 100 μ L, at this time diluent concentration
For 50mg/L；The dilution for drawing 80 μ L, 100mg/L, is subsequently added into the chromatography methanol of 120 μ L, and the concentration of dilution is at this time
40mg/L；The dilution that 160 μ L concentration are 100mg/L is drawn, is subsequently added into the chromatography methanol of 140 μ L, at this time diluent concentration
For 30mg/L；The dilution that 40 μ L concentration are 100mg/L is drawn, is subsequently added into the chromatography methanol of 160 μ L, at this time diluent concentration
For 20mg/L；The dilution that 20 μ L concentration are 100mg/L is drawn, is subsequently added into the chromatography methanol of 180 μ L, at this time diluent concentration
For 10mg/L；The dilution that 10 μ L concentration are 190mg/L is drawn, is subsequently added into the chromatography methanol of 100 μ L, at this time diluent concentration
For 5mg/L；The dilution that 2 μ L concentration are 100mg/L is drawn, is subsequently added into the chromatography methanol of 198 μ L, diluent concentration is at this time
1mg/L.It is measured using high performance liquid chromatograph and corresponds to peak area, draws standard curve.
4.4.3 chromatographic test strip part
Chromatographic test strip part: mobile phase: methanol: water (3:1) (phosphorus acid for adjusting pH to 3.0)；Column temperature: 40 DEG C；Sample volume: 20
μL；Wavelength: 230nm；Flow velocity: 0.8mL/min；Appearance time: 5.045min.
4.4.4 sample measures
By extracting solution by sample injector, it is injected separately into high performance liquid chromatography detection device, after sample has all been surveyed, record
Peak area and retention time calculate the residual quantity of fomesafen in soil according to peak area and standard curve.
Analysis of experimental results
5, the screening of degradation bacteria strains
The suspension (Fig. 1) that soil enrichment is tamed is coated on containing herbicide fomesafen after gradient dilution
Inorganic salts solid medium in, cultivated in 30 DEG C of incubator for 24 hours afterwards observation as a result, result as shown in Fig. 2, in blank pair
There is any bacterium colony according to no on culture medium, illustrates no living contaminants；It is coated under the conditions of 1000 times of dilution, fluorine sulfanilamide (SN) grass
Ether minimal medium grows the hybrid bacterial strain that can decompose herbicide fomesafen.Purified, degradation rate measurement and identification,
Two plants of fomesafen efficient degrading bacterias are obtained, as shown in Figure 3.
6, the growth curve of bacterial strain
Two plants of degradation bacteria 1:1 are mixed in LB liquid medium of transferring, measure bacterium solution absorbance at 600nm every 2h
It is worth the growth curve drawn as shown in figure 4,0-12h is growth lag phase, 12-24h is logarithmic growth phase, and 24-36h is that growth declines
Move back the phase.It follows that bacterial strain when cultivating 12-24h thallus present logarithmic growth state, it is most vibrant, for 24 hours when be logarithmic growth
Latter stage viable bacteria body is most.
7, microcapsule microbial agent determination of quality index result
It tests and passes through microcapsule microbial agent such as Fig. 5 that wriggling pumping action is added dropwise out in the process, outside the microcapsule microbial agent after drying
Seeing is that black is spherical, and no special odor, diameter is about 2.8mm, and every microcapsule microbial agent quality is about 0.0149g, and volume is
2.305g/cm3, it is not soluble in water, it is placed in the sodium citrate solution that concentration is 2% and is dissolved after 3h.Pass through solid medium culture
It is 2.936 × 10 that counting, which measures its living bacteria count,8cfu/mL。
8, sensitive crop growth of seedling index determining result
The corn seed quantity that germination treatment is carried out in test is 400, the germinating energy of seed after carrying out germination treatment 4 days
It is 90.25%, illustrates that the vigor for testing selected corn seed is very high；After germination treatment 7 days, the germination percentage of seed is 95.75%,
After the completion of corn germination processing, sowed, as shown in Figure 6.
The corn seedling that processing is sowed after 30 days, 3 basin control groups are grown without maize seedling, and 3 basin processing groups grow 6 respectively
Strain, 5 plants, 6 plants of maize seedlings, the corn seedling that processing is sowed after 60 days, 3 basin control groups grow 3 plants, 2 plants, 1 plant of maize seedling respectively,
3 basin processing groups grow 8 plants, 11 plants, 9 plants of maize seedlings respectively, calculate emergence rate, the corn seedling measurement plant height of extraction, strain fresh weight,
Plant dry weight, root fresh weight and root dry weight, as shown in Fig. 7-12.
According to Fig. 7,30 days processing group emergence rates of soil treatment are higher than control group, and difference is extremely significant；At soil treatment 60 days
Reason group emergence rate is higher than control group, and difference is extremely significant；60 days control group emergence rates of soil treatment, which are higher than soil treatment 30 days, to be compareed
Group, significant difference, but be not extremely significant；60 days processing group emergence rates of soil treatment are higher than 30 days processing groups of soil treatment, difference
It is extremely significant.
According to Fig. 8,30 days processing group plant heights of soil treatment are higher than control group, and difference is extremely significant；It handles within soil treatment 60 days
Group plant height is higher than control group, and difference is extremely significant；60 days control group plant heights of soil treatment are higher than 30 days control groups of soil treatment, difference
Significantly, but it is not extremely significant；60 days processing group plant heights of soil treatment are higher than 30 days processing groups of soil treatment, and difference is not significant.
According to Fig. 9,30 days processing group strain fresh weights of soil treatment are higher than control group, and difference is extremely significant；At soil treatment 60 days
Reason group strain fresh weight is higher than control group, and difference is extremely significant；60 days control group strain fresh weights of soil treatment, which are higher than soil treatment 30 days, to be compareed
Group, difference is not significant；60 days processing group strain fresh weights of soil treatment are higher than 30 days processing groups of soil treatment, and difference is not significant.
According to Figure 10,30 days processing group plant dry weights of soil treatment are higher than control group, and difference is extremely significant；At soil treatment 60 days
Reason group plant dry weight is higher than control group, significant difference, but is not extremely significant；60 days control group plant dry weights of soil treatment are higher than at soil
30 days control groups are managed, difference is not significant；60 days processing group plant dry weights of soil treatment are higher than 30 days processing groups of soil treatment, and difference is not
Significantly.
According to Figure 11,30 days processing group root fresh weights of soil treatment are higher than control group, and difference is extremely significant；At soil treatment 60 days
Reason group root fresh weight is higher than control group, and difference is not significant；60 days control group root fresh weights of soil treatment, which are higher than soil treatment 30 days, to be compareed
Group, difference are extremely significant；60 days processing group root fresh weights of soil treatment are higher than 30 days processing groups of soil treatment, and difference is not significant.
According to Figure 12,30 days processing group root dry weights of soil treatment are higher than control group, significant difference, but not extremely significant；Soil
Earth handles 60 days processing group root dry weights and is higher than control group, and difference is not significant；60 days control group root dry weights of soil treatment are higher than soil
30 days control groups are handled, difference is not significant；60 days processing group root fresh weights of soil treatment are higher than 30 days processing groups of soil treatment, difference
Significantly, but it is not extremely significant.
According to the analysis to growth of maize situation, it can be found that in emergence rate, plant height, strain fresh weight and plant dry weight
Processing group is substantially better than control group, significant difference.30 days processing groups of soil treatment are significantly higher than in root fresh weight and root dry weight
Control group, 60 days processing groups of soil treatment are higher than control group, but difference is not significant.30 days processing groups of soil treatment and soil
60 days processing groups are handled, difference is extremely significant in emergence rate reparation, the difference in plant height, strain fresh weight, plant dry weight, root fresh weight reparation
It is not significant, the significant difference but be not extremely significant on root dry weight.From the above results, in Field information, the microbial inoculum soil
It, being capable of fomesafen phytotoxicity effect in rehabilitating soil well when handling 30 days.
9, potting soil retention analysis result
Measured respectively using high performance liquid chromatograph concentration be 1mg/L, 5mg/L, 10mg/L, 20mg/L, 30mg/L,
The fomesafen titer of 40mg/L and 50mg/L measures peak area.Using concentration of standard solution as abscissa, peak area is vertical sits
Plotting standard curve, linear equation are as follows: y=37165x+7807.3, R2=0.9995.As a result as shown in figure 13.
Surveying peak area and linear equation according to sample can be obtained control group and handles 30 days herbicide residue amounts, be after calculating
1.795mg/kg soil, it is 1.399mg/kg soil that control group, which handles 60 days herbicide residue amounts, and it is residual that control group handles 90 days herbicides
Allowance is 1.229mg/kg soil, and it is 0.835mg/kg soil that processing group, which handles 30 days herbicide residue amounts, and processing group is handled 60 days and removed
Careless agent residual quantity is 0.431mg/kg soil, and it is 0.208mg/kg soil that processing group, which handles 90 days herbicide residue amounts,.Being computed can obtain
It is 53.48% that microbial inoculum, which handles 30 days herbicide degradation rates, and it is 69.19% that microbial inoculum, which handles 60 days herbicide degradation rates, microbial inoculum processing
90 days herbicide degradation rates are 83.08%.Statistical chart is drawn to herbicide residue amount in soil, as a result as shown in figure 14.
According to Figure 14 observable herbicide treatment 1 day, control group and processing group residual quantity difference was not significant；Herbicide treatment
30,60 and 90 days, processing group residual quantity is extremely significant to be lower than control group；Control group handle 1 day, 30 days, 60 days, 90 days herbicides it is residual
Stay presentation downward trend, and significant difference between each group；Processing 1 day, processing 30 days with handle 60 days three group differences it is extremely significant,
Processing 1 day, processing 30 days with processing 90 days three group differences it is extremely significant, but handle 60 days with handle 90 days difference be not extremely aobvious
It writes.Processing group handle 1 day, 30 days, 60 days, the presentation of 90 days herbicide residues be decreased obviously trend, and difference is aobvious between each group
It writes；Processing 1 day, processing 30 days with processing 60 days three group differences it is extremely significant, processing 1 day, processing 30 days with handle 90 days three groups
Between difference it is extremely significant, but handle 60 days with handle 90 days difference be not extremely significant.
Find out that at soil treatment 30,60 and 90 days, processing group herbicide residue amount was remarkably decreased by the above results,
And it is extremely significant with control group difference.Difference is extremely significant between 60 days processing groups of 30 days processing groups of soil treatment and soil treatment, soil
Earth handles significant difference but not extremely significant between 60 days and soil treatment 90 days, therefore when using microbial inoculum to soil treatment 30 days,
Fomesafen residual quantity has been in reduced levels in soil, has good repairing effect and application potential.
Sequence table
<110>Heilongjiang University
<120>a kind of microbial bacterial agent and its preparation method and application for fomesafen of degrading
<160> 4
<210> 1
<211> 1487
<212> 16srDNA
<213>Bei Laisi bacillus (Bacillus velezensis).
<220>
<223>Bei Laisi bacillus (Bacillus Velezensis) FB11
<400>1
tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcggacag atgggagctt 60
gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg 120
ggataactcc gggaaaccgg ggctaatacc ggatggttgt ttgaaccgca tggttcagac 180
ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg 240
aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact 300
gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360
acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420
gttgttaggg aagaacaagt gccgttcaaa tagggcggca ccttgacggt acctaaccag 480
aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 540
ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg 600
gctcaaccgg ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa 660
ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact 720
ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc 780
ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg 840
ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900
ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 960
gaaccttacc aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg 1020
gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080
cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag 1260
ccaatcccac aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct 1320
ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380
caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta acctttatgg 1440
agccagccgc cgaaggtggg acagatgatt ggggtgaagt cgtaaca 1487
<210> 2
<211> 1489
<212> 16srDNA
<213>few oxygen monad (Stenotrophomonas sp.).
<220>
<223>widow oxygen monad (Stenotrophomonas sp.) FB14
<400>1
tggctcagag tgaacgctgg cggtaggcct aacacatgca agtcgaacgg cagcacagta 60
agagcttgct cttatgggtg gcgagtggcg gacgggtgag gaatacatcg gaatctacct 120
tttcgtgggg gataacgtag ggaaacttac gctaataccg catacgacct tcgggtgaaa 180
gcaggggacc ttcgggcctt gcgcggatag atgagccgat gtcggattag ctagttggcg 240
gggtaaaggc ccaccaaggc gacgatccgt agctggtctg agaggatgat cagccacact 300
ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat tggacaatgg 360
gcgcaagcct gatccagcca taccgcgtgg gtgaagaagg ccttcgggtt gtaaagccct 420
tttgttggga aagaaaagca gtcggctaat acccggttgt tctgacggta cccaaagaat 480
aagcaccggc taacttcgtg ccagcagccg cggtaatacg aagggtgcaa gcgttactcg 540
gaattactgg gcgtaaagcg tgcgtaggtg gttgtttaag tctgttgtga aagccctggg 600
ctcaacctgg gaattgcagt ggatactggg cgactagagt gtggtagagg gtagtggaat 660
tcccggtgta gcagtgaaat gcgtagagat cgggaggaac atccatggcg aaggcagcta 720
cctggaccaa cactgacact gaggcacgaa agcgtgggga gcaaacagga ttagataccc 780
tggtagtcca cgccctaaac gatgcgaact ggatgttggg tgcaatttgg cacgcagtat 840
cgaagctaac gcgttaagtt cgccgcctgg ggagtacggt cgcaagactg aaactcaaag 900
gaattgacgg gggcccgcac aagcggtgga gtatgtggtt taattcgatg caacgcgaag 960
aaccttacct ggtcttgaca tgtcgagaac tttccagaga tggattggtg ccttcgggaa 1020
ctcgaacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080
ccgcaacgag cgcaaccctt gtccttagtt gccagcacgt aatggtggga actctaagga 1140
gaccgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcatcat ggcccttacg 1200
accagggcta cacacgtact acaatggtag ggacagaggg ctgcaaaccc gcgagggcaa 1260
gccaatccca gaaaccctat ctcagtccgg attggagtct gcaactcgac tccatgaagt 1320
cggaatcgct agtaatcgca gatcagcatt gctgcggtga atacgttccc gggccttgta 1380
cacaccgccc gtcacaccat gggagtttgt tgcaccagaa gcaggtagct taaccttcgg 1440
gagggcgctt gccacggtgt ggccgatgac tggggtgaag tcgtaacag 1489
<210> 3
<211>17
<212> DNA
<213>artificial sequence
<220>
<223>upstream primer P1.
<400> 3
GTTTGATCCTGGCTCAG 17
<210> 4
<211>18
<212> DNA
<213>artificial sequence
<220>
<223>downstream primer P2.
<400> 4
CTAGCGATTCCGACTTCA 18
Claims (6)
1. a kind of microcapsule microbial agent, it is characterised in that bacterium used in the microcapsule microbial agent is Bei Laisi bacillus
The Mixed Microbes of (Bacillus Velezensis) FB11 and widow oxygen monad (Stenotrophomonas sp.) FB14；Wherein,
Bei Laisi bacillus (Bacillus Velezensis) FB11, is deposited in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is in Mays, 2019
08, deposit number was CGMCC No.17732；
Few oxygen monad (Stenotrophomonas sp.) FB14, it is general to be deposited in China Committee for Culture Collection of Microorganisms
Logical microorganism center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the deposit date is on May 08th, 2019,
Deposit number is CGMCC No.17731.
2. a kind of microcapsule microbial agent according to claim 1, it is characterised in that the microcapsule microbial agent is by Bei Laisi gemma
The Mixed Microbes of bacillus (Bacillus Velezensis) FB11 and widow oxygen monad (Stenotrophomonas sp.) FB14,
Sodium alginate, active carbon and calcium chloride are prepared；Wherein, Bei Laisi bacillus (Bacillus Velezensis) FB11
Mass ratio with few oxygen monad (Stenotrophomonas sp.) FB14 is 1:1.
3. a kind of microcapsule microbial agent stated according to claim 2, it is characterised in that the quality percentage of Mixed Microbes in microcapsule microbial agent
Content is 92%, and the mass percentage of sodium alginate is 2%, and the mass percentage of active carbon is 5%, the quality of calcium chloride
Percentage composition is 1%.
4. the method for preparing a kind of microcapsule microbial agent described in claim 1, it is characterised in that it is to follow the steps below
:
One, preparing mass percentage is 2% sodium alginate soln, and mass percentage is 5% Actidose；And by the two
It mixes, is stirred in mixed process when dissolving by heating, obtain mixed solution；
Two, Bei Laisi bacillus (Bacillus Velezensis) FB11 and few oxygen monad of logarithmic phase will be grown to
The bacterium solution of (Stenotrophomonas sp.) FB14, is centrifuged respectively, is collected thallus, is then mixed the thallus 1:1 of collection
After conjunction, it is put into spare in refrigerator；
Three, being cooled to temperature to the mixed solution of step 1 is 30 DEG C, and step 2 is added into mixed solution by 1% access amount
Thallus, and be uniformly mixed；
Four, the mixed liquor of the addition thallus of step 3 is discharged into calcium chloride solution by peristaltic pump, forms microcapsule microbial agent；Its
In, the drain pipe of peristaltic pump is placed at the high 10cm of calcium chloride solution liquid level；
Five, by microcapsule microbial agent drawing liquid, after suction filtration, microcapsule microbial agent is placed in 30 DEG C of baking oven, is dried, that is, completed described
Microcapsule microbial agent preparation.
5. a kind of preparation method of microcapsule microbial agent according to claim 4, it is characterised in that the quality hundred of sodium alginate
Dividing content is 2%；The mass percentage of active carbon is 5%；The mass percentage of calcium chloride is 1%.
6. a kind of application of microcapsule microbial agent as described in claim 1, it is characterised in that it is for herbicide in soil of degrading
Fomesafen.
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| CN111069275A (en) * | 2019-12-31 | 2020-04-28 | 南京农业大学 | Method for reinforcing reduction of sulfonamide antibiotics in soil by earthworm intestinal contents |
| CN111662839A (en) * | 2020-06-01 | 2020-09-15 | 华南农业大学 | Bacillus belgii for degrading tetracycline, magnetic immobilized microorganism composite material and application |
| CN113265254A (en) * | 2021-07-01 | 2021-08-17 | 山西大地生态环境技术研究院有限公司 | Microcapsule type soil conditioner containing microorganisms and preparation method and application thereof |
| CN114503992A (en) * | 2022-02-23 | 2022-05-17 | 江苏仁信作物保护技术有限公司 | Herbicide containing imazamox and imazapyr and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111069276A (en) * | 2019-12-31 | 2020-04-28 | 南京农业大学 | Method for enhancing tetracycline antibiotic reduction in soil by earthworm intestinal content |
| CN111069275A (en) * | 2019-12-31 | 2020-04-28 | 南京农业大学 | Method for reinforcing reduction of sulfonamide antibiotics in soil by earthworm intestinal contents |
| CN111069276B (en) * | 2019-12-31 | 2021-12-07 | 南京农业大学 | Method for enhancing tetracycline antibiotic reduction in soil by earthworm intestinal content |
| CN111069275B (en) * | 2019-12-31 | 2021-12-07 | 南京农业大学 | Method for reinforcing reduction of sulfonamide antibiotics in soil by earthworm intestinal contents |
| CN111662839A (en) * | 2020-06-01 | 2020-09-15 | 华南农业大学 | Bacillus belgii for degrading tetracycline, magnetic immobilized microorganism composite material and application |
| CN111662839B (en) * | 2020-06-01 | 2022-03-25 | 华南农业大学 | Bacillus belgii for degrading tetracycline, magnetic immobilized microorganism composite material and application |
| CN113265254A (en) * | 2021-07-01 | 2021-08-17 | 山西大地生态环境技术研究院有限公司 | Microcapsule type soil conditioner containing microorganisms and preparation method and application thereof |
| CN114503992A (en) * | 2022-02-23 | 2022-05-17 | 江苏仁信作物保护技术有限公司 | Herbicide containing imazamox and imazapyr and preparation method thereof |
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