CN106520613A - Bacterium for degrading herbicide acetochlor and acifluorfen sodium - Google Patents
Bacterium for degrading herbicide acetochlor and acifluorfen sodium Download PDFInfo
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Abstract
The invention discloses a bacterium for degrading herbicide acetochlor and acifluorfen sodium. The bacterium for degrading the herbicide acetochlor and the acifluorfen sodium is rhizobium (Rhizobium sp.) 198-R-47, and has a preservation number being CGMCC No. 9867 in China General Microbiological Culture Collection Center. After the rhizobium (Rhizobium sp.) 198-R-47 CGMCC No. 9867 provided by the invention is cultured in an inorganic salt culture medium for 7 days, the degradation rate on 50mg/L of acetochlor achieves 73.65 percent, and the degradation rate on 100mg/L of acifluorfen sodium achieves 9.04 percent. The experiment shows that the bacterial strain can degrade the acetochlor and/or the acifluorfen sodium, and has a wide application prospect on repairing pollution of the soil herbicide acetochlor and/or the acifluorfen sodium.
Description
The application is that the Application No. 201410705138.1, applying date is for November 26, invention and created name in 2014
The divisional application of " bacterium that one plant of degrading herbicide Acetochlor and weeds are burnt and application thereof ".
Technical field
The present invention relates to the bacterium that one plant of degrading herbicide Acetochlor and weeds are burnt in microorganism field.
Background technology
Acetochlor, english common name acetochlor, chemical name be -6 methyl-N- ethoxyl methyl-α of 2- ethyls -
Chloro acetanilide.
Acetochlor is absorbability acetamide-group herbicides, is selective preemergence herbicide.Can be by the young shoot of weeds and young root
Absorb, suppress the synthesis of weeds protein, and make weeds dead.It is mainly used in the crops such as soybean, peanut, corn, cotton and prevents and kill off one
Year raw grassy weed and part broad leaved weed, have good preventive effect to Soybean Dodder, invalid to perennial weeds.
Weeds are burnt, also known as acifluorfen, English name acifluorfen, chemical name 5- (the chloro- α of 2-, α, α-trifluoro
To toloxyl) 2- nitrobenzoic acids.
It is diphenyl ether herbicide that weeds are burnt, and belongs to contact herbicide, is protoporphyrin oxidase inhibitor.Generally adopt
25% aqua carries out soybean before seedling table soil and processes and after seedling Jing foliar spray mist, can effectively prevent and kill off Soybean Field broad leaved weed such as Siberian cocklebur, piemarker
Fiber crops, black nightshade, amaranth, lamb's-quarters, knotweed, lead a cow, artemisiifolia, datura, purslane, acalypha copperleaf, beggar-ticks, weasel hemp nettle, dayflower etc., after seedling morning
Phase process can be also prevented and kill off including the annual gramineous weed including green bristlegrass.After spray, soybean meeting damage to different degrees, performance
For yellowing leaf, shrinkage, brown stain etc., but recover after 6～7d normal.
Acetochlor and weeds burn that the half-life is shorter, are considered as a kind of environmentally friendly agricultural chemical of low toxicity always, but in recent years
Come frequent, excess or improper use, different degrees of pollution is caused to agricultural land soil and underground water etc., the danger to succession crop
Evil is also increasingly severe.Research shows, applies Acetochlor in a large number in the environment and weeds are burnt and cause certain residual toxicity, its
The several years can be remained in underground water, surface water, is entered in environment and is constantly enriched with by food chain in vivo, fish are had
Stronger toxicity, environmental pollution are very big.Especially Acetochlor can cause the endocrine disturbance of organism, cause infertility, not just
Normal gender differences are even carcinogenic, are set to carcinogenic substance by EPA.
The degraded of nature herbicide residue relies primarily on Soil Microorganism to complete, but natural degradation is very slow.
Therefore, high-effective microorganism bacterial strain is targetedly screened, develops microorganism renovation agent, Herbicidal agent is accelerated by artificial infection
It is a very necessary job and practicable approach that the degraded of residual eliminates farmland herbicide herbicide carryover.
The content of the invention
The technical problem to be solved is how degrading herbicide Acetochlor and weeds are burnt.
For solve above-mentioned technical problem, the invention provides one plant can with degrading herbicide Acetochlor and weeds burn it is thin
Bacterium.
The bacterium that the present invention is provided is rhizobium (Rhizobium sp.) 198-R-47, and the bacterial strain is in October, 2014
(abbreviation CGMCC, address is to be preserved within 28th China Committee for Culture Collection of Microorganisms's common micro-organisms center：Beijing
The institute 3 of Chaoyang District North Star West Road 1), deposit number is CGMCC No.9867.
It is a further object to provide a kind of microbial inoculum, the active component of the microbial inoculum is described rhizobium
(Rhizobium sp.)198-R-47 CGMCC No.9867。
1) or 2) above-mentioned microbial inoculum can be following microbial inoculums：
1) microbial inoculum burnt for degrading herbicide Acetochlor and/or weeds；
2) microbial inoculum of pollution is burnt for rehabilitating soil Determination of Herbicide Acetochlor and/or weeds.
The microbial inoculum can also include carrier.The carrier can be solid carrier or liquid-carrier.The solid carrier can
For mineral material, vegetable material or macromolecular compound；The mineral material can be clay, talcum, kaolin, montmorillonite, white
At least one in carbon, zeolite, silica and diatomite；The vegetable material can be at least in corn flour, bean powder and starch
Kind；The macromolecular compound can be polyvinyl alcohol and/or polyglycols.The liquid-carrier can be organic solvent, vegetable oil, ore deposit
Thing oil or water；The organic solvent can be decane and/or dodecane.In the microbial inoculum, the active component can be being cultured
Living cells, the zymotic fluid of living cells, in the form of the mixture of the filtrate of cell culture or cell and filtrate.Described group
The formulation of compound can be various formulations, such as liquor, emulsion, suspending agent, pulvis, granule, wettable powder or water dispersible granules.
As needed, can also add surfactant (such as polysorbas20, Tween 80 etc.), adhesive in the microbial inoculum, stablize
Agent (such as antioxidant), pH adjusting agent etc..
Described rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 or with rhizobium (Rhizobium
Sp.) microbial inoculum answering in degrading herbicide Acetochlor and/or weeds burn of the 198-R-47 CGMCC No.9867 for active component
With falling within protection scope of the present invention.
Described rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 or with rhizobium (Rhizobium
Sp.) 198-R-47 CGMCC No.9867 burn dirt in rehabilitating soil Determination of Herbicide Acetochlor and/or weeds for the microbial inoculum of active component
Application in dye falls within protection scope of the present invention.
It is also another object of the present invention to provide a kind of culture rhizobium (Rhizobium sp.) 198-R-47 CGMCC
The method of No.9867.
The method of culture rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 provided by the present invention,
Including the training in the culture medium for cultivating rhizobium by rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867
Foster step.
A further object of the present invention is to provide one kind with rhizobium (Rhizobium sp.) 198-R-47 CGMCC
Preparation methods of the No.9867 for the microbial inoculum of active component.
The preparation method of above-mentioned microbial inoculum provided by the present invention, comprises the steps：By described rhizobium (Rhizobium
Sp.) 198-R-47 CGMCC No.9867 obtain the microbial inoculum as active component.
The present invention is to gather soil sample, and the herbicide that therefrom separation screening is obtained from the long-term farmland for being subject to pollution of herbicide
Acetochlor and/or weeds burn degradation bacteria rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867.It is demonstrated experimentally that
The bacterial strain reaches 73.65% to 50mg/L Acetochlor degradation rates in 7 days in minimal medium；The drop burnt by 100mg/L weeds
Solution rate reaches 9.04%.This shows that the bacterial strain energy degrading herbicide Acetochlor and/or weeds are burnt, in rehabilitating soil herbicide second grass
Amine and weeds have broad application prospects in terms of burning pollution.
Preservation explanation
Strain name：Rhizobium
Latin name：(Rhizobium sp.)
Strain number：198-R-47
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On October 28th, 2014
Collection is registered on the books numbering：CGMCC No.9867
Description of the drawings
Fig. 1 is Acetochlor calibration curve.
Fig. 2 burns calibration curve for weeds.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
In following embodiments, culture medium used is as follows：
Nitrogen-free fluid nutrient medium：Solute and its concentration are sucrose 10g/L, NaCl 0.12g/L, K2HPO4·3H2O 0.5g/
L, CaCO31g/L, MgSO4·7H2O 0.2g/L；Solvent is distilled water；pH7.2.
Nitrogen-free solid medium：To in nitrogen-free fluid nutrient medium, add agar to obtain to its content for 15-20g/L culture
Base.
Beef extract-peptone fluid nutrient medium：Solute and its concentration are beef extract 5g/L, peptone 10g/L, NaCl 5g/
L；Solvent is distilled water；pH7.2.
Beef extract-peptone solid medium：Solute and its concentration are beef extract 5g/L, peptone 10g/L, NaCl 5g/
L, agar 15-20g/L；Solvent is distilled water；pH7.2.
Inorganic salt liquid culture medium：Solute and its concentration are NH4Cl 1.0g/L, KH2PO40.5g/L, K2HPO4 1.5g/
L, MgSO40.2g/L, NaCl 0.5g/L；Solvent is distilled water；pH7.0.
Inorganic salts solid medium：To in inorganic salt liquid culture medium, add agar to its content to be 2% culture medium for obtaining.
Rhizobium (Rhizobium sp.) LD1616 CGMCC No.7775 (abbreviation rhizobium in following embodiments
(Rhizobium sp.) LD1616) disclosed in 103343100 B of Chinese invention patent document CN.
The separation of embodiment 1, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 and identification
First, Acetochlor/weeds burn the separation of degradation bacteria 198-R-47
1st, in 100ml sterilized waters 10g pedotheques are added (to pick up from agriculture of the Chinese Heihe In The Heilongjiang River by pollution of herbicide
Field), shaken cultivation 30min makes dirty solution.Draw the above-mentioned dirty solutions of 1ml to add in the test tube filled in 9ml sterilized waters fully
(now dilution factor is designated as 10 for mixing-1), then 1ml is drawn from this test tube be added in another test tube for filling 9ml sterilized waters
It is well mixed, makes 10 by that analogy-2、10-3、10-4、10-5Different dilution bacteria suspensions.Each dilution factor is taken into 0.1ml uniform
It is coated on nitrogen-free solid medium flat board, 28 DEG C of constant temperature quiescent cultures 23 days.After bacterium colony is formed, in nitrogen-free solid culture
Purifying agaric is carried out on base flat board.
2nd, Determination of Herbicide Acetochlor, weeds are burnt, the preliminary screening of Pu Shite and chlorimuronethyl degradation capability it is saturating using flat board
Bright circle method.It is 1000mg/L that Acetochlor standard items (Fluka Products) addition inorganic salts solid medium is made its content, is obtained
To Acetochlor flat board；Weeds are burnt standard items (Fluka Products) adds inorganic salts solid medium to make its content be
1000mg/L, obtains weeds and burns flat board；Pu Shite standard items (Fluka Products) addition inorganic salts solid medium is made into which
Content is 1000mg/L, obtains general applying dead falt sheet；Chlorimuron ethyl (Fluka Products) is added into the training of inorganic salts solid
It is 1000mg/L that foster base makes its content, obtains chlorimuronethyl flat board.By Acetochlor flat board, weeds burn flat board, it is general apply dead falt sheet and
Chlorimuronethyl flat board carries out subregion, the 10 μ L of bacteria suspension of every plant of bacterium that the purifying of aspiration step 1 is obtained and the rhizobium in this laboratory
The 10 μ L of bacteria suspension (the thalline content of various bacterial strain bacteria suspensions is identical) of (Rhizobium sp.) LD1616 dibblings to four kinds respectively
On flat board (every plant of bacterium is repeated 3 times on a flat board), 28 DEG C of cultures, observations.One plant is screened in Acetochlor flat board and weeds
Burn on flat board and can form the bacterial strain of larger transparent circle, which is named as Acetochlor/weeds and burns degradation bacteria 198-R-47 by the bacterial strain.
Acetochlor/weeds are burnt degradation bacteria 198-R-47 and apply dead falt sheet and chlorimuronethyl flat board can not form transparent circle general.Rhizobium
(Rhizobium sp.) LD1616 only forms larger transparent circle on chlorimuronethyl flat board, applies dead falt sheet, Acetochlor flat board general
Burn with weeds and can not be formed on flat board transparent circle.Illustrate Acetochlor/weeds burn degradation bacteria 198-R-47 can degrade Acetochlor and
Weeds are burnt, it is impossible to which degrade chlorimuronethyl and Pu Shite；Rhizobium (Rhizobium sp.) LD1616 can degrade chlorimuronethyl, no
Can degrade Pu Shite, Acetochlor and weeds are burnt.
1. Acetochlors of table/weeds burn degradation bacteria 198-R-47 and rhizobium (Rhizobium sp.) LD1616 is removed to four kinds
Careless agent degradation capability primary dcreening operation result
| Strain number | Pu Shite | Acetochlor | Chlorimuronethyl | Weeds are burnt |
| Acetochlor/weeds burn degradation bacteria 198-R-47 | - | + | - | + |
| Rhizobium (Rhizobium sp.) LD1616 | - | - | + | - |
Note："+" represents to form larger transparent circle that "-" is indicated and formed without transparent circle.
2nd, Acetochlor/weeds burn the identification of degradation bacteria 198-R-47
Acetochlor/the weeds for obtaining are separated and are purified from the following aspects authentication step one burns degradation bacteria 198-R-47：
1st, Morphological Identification
Will be in exponential phase and bacterium colony size is stable, above-mentioned steps one separate and purify Acetochlor/weeds for obtaining
Burning degradation bacteria 198-R-47 carries out single bacterium colony state observation, the main size for including bacterium colony, color, transparency, wettability, bacterium colony
Surface state (whether flat, projection, fold, depression etc.), colony edge state (whether neat, irregular, radial etc.).
As a result show, above-mentioned steps one are separated and purify Acetochlor/weeds for obtaining burns degradation bacteria 198-R-47 bacterium colony circle
Shape is raised, milky, moistens unclarity, regular edges, diameter 0.8-1.5mm.
2nd, analysis of physio biochemical characteristics
With reference to《Common bacteria system identification handbook》(east show pearl, Cai Miaoying. common bacteria system identification handbook. Beijing:Section
Learn publishing house, 2011.) and《Microbiology Experiment》(Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (third edition). Beijing:
Higher Education Publishing House, 1999.) determines the physiological and biochemical property that above-mentioned Acetochlor/weeds burn degradation bacteria 198-R-47.
The physiological and biochemical property measurement result that the Acetochlor/weeds burn degradation bacteria 198-R-47 is as shown in table 2：
2. Acetochlors of table/weeds burn the physiological and biochemical property of degradation bacteria 198-R-47
Note："+" represents positive, and "-" represents negative.
3rd, 16S rDNA sequence homology analysis
Conventional method culture above-mentioned steps one isolate and purify the Acetochlor/weeds for obtaining and burn degradation bacteria 198-R-47, extract
The STb gene of bacterial strain as gene magnification template, with bacterial 16 S rDNA universal primers, 27f：5′-
AGAGTTTGATCCTGGCTCAG-3 ', 1492r：5 '-TACCTTGTTACGACTT-3 ' enter performing PCR reaction.Reaction system is adopted
Shanghai bioengineering Co., Ltd PCR amplification kits.Response procedures are：95 DEG C of denaturation 30s, 55 DEG C of annealing 1min, 72 DEG C prolong
2min is stretched, totally 30 circulations.DNA sequencing is won polygala root biotech company by Beijing three and is completed, sequence assembly and similarity analysis
Completed using DNAStar softwares, gene is compared by American National Biotechnology Information center ncbi database (http://
Www.ncbi.nlm.nih.gov) completed with EzTaxon online.
Pcr gene amplification obtains the 16S rDNA genetic fragment about 1.3kb that Acetochlor/weeds burn degradation bacteria 198-R-47,
Online sequence analysis are carried out with published 16S rDNA sequences in NCBI and EzTaxon databases after determining sequence, as a result
Show that Acetochlor/weeds burn degradation bacteria 198-R-47 and rhizobium Rhizobium pusense NRCPB10THomology is most
Height, reaches 99.31%.
Acetochlor/weeds are burnt the 16S rDNA sequences of degradation bacteria 198-R-47 and refer to sequence 1.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16S rDNA sequence homology analysis results, by step one point
Acetochlor/the weeds obtained from purifying are burnt degradation bacteria 198-R-47 and are accredited as rhizobium (Rhizobium sp.).The second grass
Amine/weeds burn degradation bacteria 198-R-47, and to be preserved in China Committee for Culture Collection of Microorganisms on October 28th, 2014 general
(abbreviation CGMCC, address is at logical microorganism center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC
No.9867.It is rhizobium (Rhizobium sp.) 198-R-47 that Acetochlor/weeds burn the full name of degradation bacteria 198-R-47
CGMCC No.9867, referred to as rhizobium (Rhizobium sp.) 198-R-47.
Embodiment 2, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 degradeds Acetochlor ability is quantitative
Determine
First, Acetochlor bioassay standard curve plotting
Acetochlor standard items (Fluka Products) 10.0mg is weighed accurately in 10mL volumetric flasks, molten with a small amount of methyl alcohol
Solution, volumetric flask is placed in ultrasonic wave bath and vibrates 10min, then shaken up to 10mL with methanol constant volume, be made into 1000mg/L second
Careless amine mother liquor.Then concentration respectively 10,20,40,60,80,100mg/L standard liquids is obtained with methanol dilution, using efficient
Liquid chromatography (HPLC) determines the peak area of variable concentrations Acetochlor standard items, 3 repetitions.With the concentration of Acetochlor as horizontal seat
Mark, peak area is ordinate, draws Acetochlor calibration curve, as shown in Figure 1.
Testing conditions are as follows：
Detecting system：Agilent 1100Series.Chromatographic column：C18DiamosilTMReversed-phase column, 250mm × 4.6mm, grain
5 μm of footpath.Chromatographic condition：Mobile phase：Methyl alcohol：Water=80：20 (v/v), water glacial acetic acid are adjusted to pH=3；Detection wavelength, 215nm；
Flow velocity, 1.0mL/min；Sampling volume, 20 μ L；30 DEG C of column temperature.
Gained Acetochlor bioassay standard curve：Y=55.363x+144.5 (R2=0.9999).Wherein, y is peak area, x
For Acetochlor concentration.
2nd, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 degraded Acetochlors ability quantitative determination
Rhizobium (Rhizobium sp.) 198-R-47 process：By rhizobium (Rhizobium sp.) 198-R-47
CGMCC No.9867 are seeded on beef extract-peptone solid medium and cultivate 24h, and 1 ring of picking is seeded in 5mL beef extract albumen
In peptone fluid nutrient medium, 180r/min culture 12h, centrifugation remove culture medium, are adjusted to OD with inorganic salt liquid culture medium600Value
For 1.0.Draw 0.2mL bacteria suspensions (1 × 109Cfu/ml) it is inoculated into inorganic salt liquid culture mediums of the 5mL containing Acetochlor 50mg/L
(in inorganic salt liquid culture medium Acetochlor (Fluka Products) is added to make the training that the concentration of Acetochlor is obtained for 50mg/L
Foster base) test tube in, 28 DEG C, 180r/min cultivate 7 days, obtain degradation solution, determine Residual Determination of Acetochlor as follows：Take
Into 50mL centrifuge tubes, 8000r/min centrifugation 5min collect supernatant to 3mL degradation solutions, add 3mL dichloromethane, acutely vibrate
5min, stands 10min, treats water phase and organic phase layering, adds a small amount of anhydrous sodium sulfate to be dehydrated organic phase, accurately draw
800 μ L organic phases are dried up with Nitrogen evaporator in the centrifuge tube of 1.5mL, add 400 μ L methyl alcohol, the ultrasound in ultrasonic cleaner
10min, is filtered to liquid chromatogram sample bottle with 0.22 μm of organic phase aculeus type filter, determines second grass according to above-mentioned HPLC methods
Amine.
Rhizobium (Rhizobium sp.) LD1616 process：Except by above-mentioned rhizobium (Rhizobium sp.) 198-R-47
Rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 in process replace with rhizobium (Rhizobium
Sp.) LD1616 CGMCC No.7775, remaining all same.
Blank process：Meanwhile, with non-Rhizobium Inoculation (Rhizobium sp.) 198-R-47 CGMCC No.9867
The above-mentioned minimal medium containing Acetochlor 50mg/L as blank, determine the concentration of Acetochlor according to the method described above.
Acetochlor degradation rate：X=(1-A/B) × 100%, in formula, X is degradation rate (%), and A is rhizobium (Rhizobium
Sp. remain in the Acetochlor concentration for) remaining in 198-R-47 treatment fluids or rhizobium (Rhizobium sp.) LD1616 treatment fluids
Acetochlor concentration, B be do not connect bacterium blank treatment fluid in the Acetochlor concentration that remains.Experiment sets 3 repetitions, repeats every time
Each processes 10 test tubes of inoculation.
3. rhizobium of table (Rhizobium sp.) 198-R-47 CGMCC No.9867 degraded Acetochlor effects
As a result show, Acetochlor initial concentration is 50mg/L, rhizobium (Rhizobium sp.) 198-R- after 7 days
47 CGMCC No.9867 reach 73.65% (as shown in table 3) to the degradation rate of Acetochlor；Rhizobium (Rhizobium sp.)
LD1616 reaches 0% to the degradation rate of Acetochlor.This result shows, rhizobium (Rhizobium provided by the present invention
Sp.) the degradable Acetochlors of 198-R-47 CGMCC No.9867.
It is quantitative that embodiment 3, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 degraded weeds burn ability
Determine
First, weeds burn the drafting of bioassay standard curve
Accurately weighing weeds, to burn standard items (Fluka Products) 10.0mg in 10mL volumetric flasks, molten with a small amount of methyl alcohol
Solution, volumetric flask is placed in ultrasonic wave bath and vibrates 10min, then shaken up to 10mL with methanol constant volume, be made into 1000mg/L miscellaneous
Grass burns mother liquor.Then concentration respectively 10,20,40,60,80,100mg/L standard liquids is obtained with methanol dilution.Using efficient
Liquid chromatogram (HPLC) determines the peak area that variable concentrations weeds burn standard items, 3 repetitions.Concentration is burnt as abscissa with weeds,
Peak area is ordinate, draws weeds and burns calibration curve, as shown in Figure 2.
Testing conditions are as follows：
Detecting system：Agilent 1100Series.Chromatographic column：C18DiamosilTMReversed-phase column, 250mm × 4.6mm, grain
5 μm of footpath.Chromatographic condition：Mobile phase：Acetonitrile：Water (glacial acetic acid adjusts pH3.0)=40:60(v/v)；Detection wavelength, 258nm；Flow velocity,
1.0mL/min；Sampling volume, 10 μ L；30 DEG C of column temperature.
Gained weeds burn bioassay standard curve：Y=7.3196x+2.1212 (R2=0.9875).Wherein, y is peak area, x
Concentration is burnt for weeds.
2nd, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 degradeds weeds burn ability quantitative determination
Rhizobium (Rhizobium sp.) 198-R-47 process：In test tube, accurately add 5mL to burn 100mg/L containing weeds
Minimal medium (in inorganic salt liquid culture medium add weeds burn the training that the concentration for burning weeds is obtained for 100mg/L
Foster base), add the OD of 0.2ml600It is worth rhizobium (Rhizobium sp.) the 198-R-47 CGMCC No.9867 bacterium for 1.0
Liquid (1 × 109Cfu/ml), 28 DEG C, 200r/min cultivate 7 days, obtain degradation solution.4mL degradation solutions are taken into 50mL centrifuge tubes, plus
Enter 4mL acetonitriles, vibrate 2min, stand 10min, add a little anhydrous sodium sulfate.Accurately draw 800 μ L organic phases to be transferred to
In 1.5mL EP centrifuge tubes, dry up on Nitrogen evaporator, add 400 μ L methyl alcohol (chromatographic grade), under ul-trasonic irradiation burn weeds
Be completely dissolved, be collected by filtration into sample bottle with liquid spectrum filter, weeds determined according to above-mentioned HPLC methods and burnt.
Rhizobium (Rhizobium sp.) LD1616 process：Except by above-mentioned rhizobium (Rhizobium sp.) 198-R-47
Rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 in process replace with rhizobium (Rhizobium
Sp.) LD1616 CGMCC No.7775, remaining all same.
Blank process：Meanwhile, with non-Rhizobium Inoculation (Rhizobium sp.) 198-R-47 CGMCC No.9867
It is above-mentioned burn the inorganic salt liquid culture medium of 100mg/L as blank containing weeds, determine what weeds were burnt according to the method described above
Concentration.
Weeds burn degradation rate：X=(1-A/B) × 100%, in formula, X is degradation rate (%), and A is rhizobium (Rhizobium
Sp.) weeds remained in 198-R-47 treatment fluids are burnt in concentration or rhizobium (Rhizobium sp.) LD1616 treatment fluids and remain
Weeds burn concentration, B be do not connect bacterium blank treatment fluid in the weeds that remain burn concentration.Experiment sets 3 repetitions, repeats every time
Each processes 10 test tubes of inoculation.Measurement result is as shown in table 4.
As a result show, weeds burn initial concentration for 100mg/L, rhizobium (Rhizobium sp.) 198-R- after 7 days
The degradation rate 9.04% that 47 CGMCC No.9867 are burnt to weeds；Rhizobium (Rhizobium sp.) LD1616 is burnt to weeds
Degradation rate is 0.02% (as shown in table 4).This result shows, rhizobium (Rhizobium sp.) provided by the present invention
The degradable weeds of 198-R-47 CGMCC No.9867 are burnt.
4. rhizobium of table (Rhizobium sp.) 198-R-47 CGMCC No.9867 degraded weeds burn effect
<110>INST OF AGRICULTURAL RESOURCES
<120>The bacterium that one plant of degrading herbicide Acetochlor and weeds are burnt
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1300
<212> DNA
<213>Rhizobium (Rhizobium sp.)
<400> 1
cagacgggtg agtaacgcgt gggaatctac ccatctctgc ggaatagctc tgggaaactg 60
gaattaatac cgcatacgcc ctacggggga aagatttatc ggggatggat gagcccgcgt 120
tggattagct agttggtggg gtaaaggcct accaaggcga cgatccatag ctggtctgag 180
aggatgatca gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagtg 240
gggaatattg gacaatgggc gcaagcctga tccagccatg ccgcgtgagt gatgaaggcc 300
ttagggttgt aaagctcttt caccgatgaa gataatgacg gtagtcggag aagaagcccc 360
ggctaacttc gtgccagcag ccgcggtaat acgaaggggg ctagcgttgt tcggaattac 420
tgggcgtaaa gcgcacgtag gcggatattt aagtcagggg tgaaatcccg cagctcaact 480
gcggaactgc ctttgatact gggtatcttg agtatggaag aggtaagtgg aattccgagt 540
gtagaggtga aattcgtaga tattcggagg aacaccagtg gcgaaggcgg cttactggtc 600
cattactgac gctgaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 660
ccacgccgta aacgatgaat gttagccgtc gggcagtata ctgttcggtg gcgcagctaa 720
cgcattaaac attccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg 780
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc 840
agctcttgac attcggggta tgggcattgg agacgatgtc cttcagttag gctggcccca 900
gaacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 960
acgagcgcaa ccctcgccct tagttgccag catttagttg ggcactctaa ggggactgcc 1020
ggtgataagc cgagaggaag gtggggatga cgtcaagtcc tcatggccct tacgggctgg 1080
gctacacacg tgctacaatg gtggtgacag tgggcagcga gacagcgatg tcgagctaat 1140
ctccaaaagc catctcagtt cggattgcac tctgcaactc gagtgcatga agttggaatc 1200
gctagtaatc gcagatcagc atgctgcggt gaatacgttc ccgggccttg tacacaccgc 1260
ccgtcacacc atgggagttg gttttacccg aaggtagtgc 1300
Claims (5)
1. rhizobium (Rhizobium sp.) 198-R-47, which is in the common micro- life of China Committee for Culture Collection of Microorganisms
The deposit number at thing center is CGMCC No.9867.
2. a kind of microbial inoculum, it is characterised in that：The active component of the microbial inoculum is the rhizobium (Rhizobium described in claim 1
sp.)198-R-47CGMCC No.9867。
3. microbial inoculum according to claim 2, it is characterised in that：1) or 2) microbial inoculum is following microbial inoculums：
1) microbial inoculum burnt for degrade Acetochlor and/or weeds；
2) microbial inoculum of pollution is burnt for rehabilitating soil Acetochlor and/or weeds.
4. the method for cultivating rhizobium (Rhizobium sp.) 198-R-47CGMCC No.9867 described in claim 1, including
The rhizobium (Rhizobium sp.) 198-R-47CGMCC No.9867 are trained in the culture medium for cultivating rhizobium
Foster step.
5. the preparation method of microbial inoculum described in Claims 2 or 3, comprises the steps：By the rhizobium described in claim 1
(Rhizobium sp.) 198-R-47 CGMCC No.9867, as active component, obtain the microbial inoculum.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381690A (en) * | 2008-10-22 | 2009-03-11 | 东北农业大学 | Method for preparing anti-acetochlor soybean rhizobium inoculant |
| CN103031261A (en) * | 2012-11-21 | 2013-04-10 | 浙江工业大学 | Achromobacter sp. D-12 and application thereof in microbial degradation of acetochlor |
| CN103333836A (en) * | 2013-07-01 | 2013-10-02 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for degrading herbicides chlorimuron-ethyl and acetochlor and application of bacterium |
| CN103333835A (en) * | 2013-07-01 | 2013-10-02 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for degrading herbicide acifluorfen-sodium and application of bacterium |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381690A (en) * | 2008-10-22 | 2009-03-11 | 东北农业大学 | Method for preparing anti-acetochlor soybean rhizobium inoculant |
| CN103031261A (en) * | 2012-11-21 | 2013-04-10 | 浙江工业大学 | Achromobacter sp. D-12 and application thereof in microbial degradation of acetochlor |
| CN103333836A (en) * | 2013-07-01 | 2013-10-02 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for degrading herbicides chlorimuron-ethyl and acetochlor and application of bacterium |
| CN103333835A (en) * | 2013-07-01 | 2013-10-02 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for degrading herbicide acifluorfen-sodium and application of bacterium |
Non-Patent Citations (2)
| Title |
|---|
| CORINNE BOUQUARD ET AL.: "Dechlorination of Atrazine by a Rhizobium sp. Isolate", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 闫春秀等: "微生物降解长残效除草剂的研究进展", 《东北农业大学学报》 * |
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