CN106520613B - The bacterium that one plant of degrading herbicide Acetochlor and weeds are burnt - Google Patents

The bacterium that one plant of degrading herbicide Acetochlor and weeds are burnt Download PDF

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CN106520613B
CN106520613B CN201611006095.3A CN201611006095A CN106520613B CN 106520613 B CN106520613 B CN 106520613B CN 201611006095 A CN201611006095 A CN 201611006095A CN 106520613 B CN106520613 B CN 106520613B
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高淼
孙建光
孙颖飞
张磊
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Institute of Agricultural Resources and Regional Planning of CAAS
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Abstract

The invention discloses the bacteriums that one plant of degrading herbicide Acetochlor and weeds are burnt.The bacterium that the degradation Acetochlor and weeds are burnt is rhizobium (Rhizobium sp.) 198-R-47, is CGMCC No.9867 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 provided by the invention reaches 73.65% to the degradation rate of 50mg/L Acetochlor in 7 days in minimal medium；The degradation rate burnt to 100mg/L weeds reaches 9.04%.This shows that the bacterial strain can degrade Acetochlor and/or weeds are burnt, and has broad application prospects in terms of rehabilitating soil Determination of Herbicide Acetochlor and/or weeds burn pollution.

Description

The bacterium that one plant of degrading herbicide Acetochlor and weeds are burnt
It is that November 26, invention and created name in 2014 are that the application, which is application No. is the 201410705138.1, applying date, The divisional application of " bacterium and application thereof that one plant of degrading herbicide Acetochlor and weeds are burnt ".
Technical field
The present invention relates to the bacteriums that one plant of degrading herbicide Acetochlor and weeds are burnt in microorganism field.
Background technique
Acetochlor, english common name acetochlor, chemical name are -6 methyl-N- ethoxyl methyl-α of 2- ethyl - Chloro acetanilide.
Acetochlor is absorbability acetamide-group herbicides, is selective preemergence herbicide.It can be by the young shoot and young root of weeds It absorbs, inhibits the synthesis of weeds protein, and keep weeds dead.It is mainly used for the crops such as soybean, peanut, corn, cotton and prevents and kill off one Year raw gramineae weed and part broadleaf weeds, have good preventive effect to Soybean Dodder, invalid to perennial weeds.
Weeds burn also known as acifluorfen, English name acifluorfen, chemical name 5- (2- chloro- α, α, α-trifluoro To toloxyl) 2- nitrobenzoic acid.
It is diphenyl ether herbicide that weeds, which are burnt, belongs to contact herbicide, is protoporphyrin oxidase inhibitor.It generallys use 25% aqua carries out surface soil processing and Miao Houjing foliar spray mist before big bean seedlings, can effectively prevent and kill off Soybean Field broadleaf weeds such as Siberian cocklebur, piemarker Fiber crops black nightshade, amaranth, Chenopodiaceae, knotweed, are led a cow, artemisiifolia, datura, purslane, acalypha copperleaf, beggar-ticks, weasel hemp nettle, dayflower etc., Miao Houzao Phase processing can also prevent and kill off the annual gramineous weed including herba setariae viridis.After spray, soybean meeting damage to different degrees, performance Restore normal for yellowing leaf, shrinkage, brown stain etc., but after 6~7d.
Acetochlor and weeds burn that half-life period is shorter, have always been considered as be a kind of low toxicity environmentally friendly agricultural chemical, but in recent years Come frequent, excess or improper use, different degrees of pollution is caused to agricultural land soil and underground water etc., to the danger of succession crop Evil is also increasingly severe.Studies have shown that largely application Acetochlor and weeds are burnt and cause certain residual toxicity in the environment, The several years can be remained in underground water, surface water, entered in environment and be constantly enriched in vivo by food chain, had to fish 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 U.S.EPA.
The degradation 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, microorganism renovation agent is developed, Herbicidal agent is accelerated by artificial infection It is a very necessary job and practicable approach that farmland herbicide herbicide carryover is eliminated in remaining degradation.
Summary of the invention
The technical problem to be solved by the present invention is to how degrading herbicide Acetochlor and weeds are burnt.
In order to solve the above technical problems, the present invention provides one plant can be burnt with degrading herbicide Acetochlor and weeds it is thin Bacterium.
Bacterium provided by the invention is rhizobium (Rhizobium sp.) 198-R-47, and the bacterial strain is in October, 2014 It is preserved within 28th China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: 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 constituent of the microbial inoculum is the 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 high-molecular compound；The mineral material can be clay, talcum, kaolin, montmorillonite, white At least one of carbon, zeolite, silica and diatomite；The vegetable material can be at least one in corn flour, bean powder and starch Kind；The high-molecular compound can be polyvinyl alcohol and/or polyglycols.The liquid-carrier can be organic solvent, vegetable oil, mine Object oil or water；The organic solvent can be decane and/or dodecane.In the microbial inoculum, the active constituent can be to be cultured Living cells, the fermentation liquid of living cells, the filtrate of cell culture or cell and the mixture of filtrate form exist.Described group The dosage form for closing object can be a variety of dosage forms, such as liquor, emulsion, suspending agent, pulvis, granule, wettable powder or water dispersible granules.
As needed, surfactant (such as polysorbas20, Tween 80), adhesive, stabilization can be also added in the microbial inoculum Agent (such as antioxidant), pH adjusting agent.
Rhizobium (Rhizobium sp.) the 198-R-47 CGMCC No.9867 or with rhizobium (Rhizobium Sp.) 198-R-47 CGMCC No.9867 is microbial inoculum the answering in degrading herbicide Acetochlor and/or weeds are burnt of active constituent With also belonging to protection scope of the present invention.
Rhizobium (Rhizobium sp.) the 198-R-47 CGMCC No.9867 or with rhizobium (Rhizobium Sp.) 198-R-47 CGMCC No.9867 is that the microbial inoculum of active constituent burns dirt in rehabilitating soil Determination of Herbicide Acetochlor and/or weeds Application in dye also belongs to 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 rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 is trained in the culture medium for cultivating rhizobium Feeding step.
A further object of the present invention is to provide one kind with rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 is the preparation method of the microbial inoculum of active constituent.
The preparation method of above-mentioned microbial inoculum provided by the present invention includes the following steps: the rhizobium (Rhizobium Sp.) 198-R-47 CGMCC No.9867 obtains the microbial inoculum as active constituent.
The present invention is to be acquired soil sample, and the herbicide that therefrom separation screening obtains by the farmland of pollution of herbicide from for a long time 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 rate in 7 days in minimal medium；The drop that 100mg/L weeds are burnt 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 burn pollution aspect and have broad application prospects.
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 abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on October 28th, 2014
Collection is registered on the books number: CGMCC No.9867
Detailed description of the invention
Fig. 1 is Acetochlor standard curve.
Fig. 2 is that weeds burn standard curve.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Culture medium as used in the following examples 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: add agar to the culture that its content is that 15-20g/L is obtained into nitrogen-free fluid nutrient medium 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: into inorganic salt liquid culture medium plus agar to its content is 2% obtained culture medium.
Rhizobium (Rhizobium sp.) LD1616 CGMCC No.7775 (abbreviation rhizobium in following embodiments (Rhizobium sp.) LD1616) it is disclosed in 103343100 B of Chinese invention patent document CN.
The separation and identification of embodiment 1, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867
One, Acetochlor/weeds burn the separation of degradation bacteria 198-R-47
1,10g pedotheque is added in 100ml sterile water and (picks up from Chinese Heihe In The Heilongjiang River by the agriculture of pollution of herbicide Field), dirty solution is made in shaken cultivation 30min.The above-mentioned dirty solution of 1ml is drawn to be added in the test tube filled in 9ml sterile water sufficiently (dilution is denoted as 10 at this time for mixing-1), then it is added in another test tube for filling 9ml sterile water from absorption 1ml in this test tube Be uniformly mixed, and so on be made 10-2、10-3、10-4、10-5The bacteria suspension of different dilutions.Take 0.1ml uniform each dilution Be coated in nitrogen-free solid medium tablets, 28 DEG C constant temperature stationary culture 2-3 days.After bacterium colony is formed, in nitrogen-free solid culture Purifying agaric is carried out on base plate.
2, Determination of Herbicide Acetochlor, weeds are burnt, the preliminary screening of Pu Shite and chlorimuronethyl degradation capability it is saturating using plate Bright circle method.Acetochlor standard items (Fluka Products) addition inorganic salts solid medium is made into its content 1000mg/L, is obtained To Acetochlor plate；Weeds, which are burnt standard items (Fluka Products) addition inorganic salts solid medium, makes its content 1000mg/L obtains weeds and burns plate；Pu Shite standard items (Fluka Products) addition inorganic salts solid medium is made it Content is 1000mg/L, obtains general applying dead falt sheet；The training of inorganic salts solid is added in chlorimuron ethyl (Fluka Products) Feeding base makes its content 1000mg/L, obtains chlorimuronethyl plate.By Acetochlor plate, weeds burn plate, it is general apply dead falt sheet and Chlorimuronethyl plate carries out subregion, and aspiration step 1 purifies the rhizobium of bacteria suspension 10 the μ L and this laboratory of every plant of obtained bacterium 10 μ L of bacteria suspension (the thallus content of various bacterial strain bacteria suspensions is identical) the difference dibbling of (Rhizobium sp.) LD1616 is to four kinds On plate (every plant of bacterium is repeated 3 times on a plate), 28 DEG C of cultures, observation.One plant is screened in Acetochlor plate and weeds The bacterial strain that can form larger transparent circle is burnt on plate, it is named as Acetochlor/weeds and burns degradation bacteria 198-R-47 by the bacterial strain. Acetochlor/weeds, which burn degradation bacteria 198-R-47, cannot form transparent circle in the general dead falt sheet and chlorimuronethyl plate applied.Rhizobium (Rhizobium sp.) LD1616 only forms larger transparent circle on chlorimuronethyl plate, applies dead falt sheet, Acetochlor plate general Transparent circle cannot be formed by burning with weeds on plate.Illustrate Acetochlor/weeds burn degradation bacteria 198-R-47 can degrade Acetochlor and Weeds are burnt, and cannot 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 removes 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: "+" indicates to form larger transparent circle, and "-" indicates that no transparent circle is formed.
Two, Acetochlor/weeds burn the identification of degradation bacteria 198-R-47
Acetochlor/the weeds for separating and purifying from the following aspects authentication step one burn degradation bacteria 198-R-47:
1, Morphological Identification
Logarithmic growth phase will be in and bacterium colony size is stablized, Acetochlor/weeds that above-mentioned steps one are separated and purified It burns degradation bacteria 198-R-47 and carries out single colonie state observation, main includes size, color, transparency, the wettability, bacterium colony of bacterium colony Surface state (whether flat, protrusion, fold, recess etc.), colony edge state (whether neat, irregular, radial etc.).
The result shows that above-mentioned steps one separate and the Acetochlor/weeds purified burn degradation bacteria 198-R-47 bacterium colony circle Shape protrusion, milky moisten unclarity, regular edges, diameter 0.8-1.5mm.
2, analysis of physio biochemical characteristics
With reference to " common bacteria system identification handbook " (east show pearl, Beijing Cai Miaoying common bacteria system identification handbook: section Publishing house, 2011.) and " Microbiology Experiment " (Beijing Shen Ping, Fan Xiurong, Li Guangwu Microbiology Experiment (third edition): Higher Education Publishing House, 1999.) measure 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: "+" indicates positive, and "-" indicates negative.
3,16S rDNA sequence homology analysis
Acetochlor/weeds that conventional method culture above-mentioned steps one isolate and purify burn degradation bacteria 198-R-47, extract The total DNA of bacterial strain is as gene magnification template, with bacterial 16 S rDNA universal primer, 27f:5 '- AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACCTTGTTACGACTT-3 ' carry out PCR reaction.Reaction system uses Shanghai bioengineering Co., Ltd PCR amplification kit.Response procedures are as follows: 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 Radix Polygalae biotech company by Beijing three and is completed, sequence assembly and similarity analysis Completed using DNAStar software, gene compare by National Center for Biotechnology Information ncbi database (http: // Www.ncbi.nlm.nih.gov it) is completed online with EzTaxon.
Pcr gene expands to obtain the 16S rDNA genetic fragment about 1.3kb that Acetochlor/weeds burn degradation bacteria 198-R-47, Online sequence analysis is carried out with 16S rDNA sequence published in NCBI and EzTaxon database after measurement sequence, as a result Display Acetochlor/weeds burn degradation bacteria 198-R-47 and rhizobium Rhizobium pusense NRCPB10THomology is most Height reaches 99.31%.
The 16S rDNA sequence that Acetochlor/weeds burn degradation bacteria 198-R-47 is detailed in sequence 1.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16S rDNA sequence homology analysis as a result, by step 1 point Degradation bacteria 198-R-47, which is burnt, from the obtained Acetochlor/weeds of purifying is accredited as rhizobium (Rhizobium sp.).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 Logical microorganism center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number CGMCC No.9867.The full name that Acetochlor/weeds burn degradation bacteria 198-R-47 is rhizobium (Rhizobium sp.) 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 degradation Acetochlor ability are quantitative Measurement
One, Acetochlor measures standard curve making
It is molten with a small amount of methanol in 10mL volumetric flask to accurately weigh Acetochlor standard items (Fluka Products) 10.0mg Solution, volumetric flask is placed in ultrasonic wave bath and vibrates 10min, then with methanol constant volume to 10mL, shakes up, is made into 1000mg/L second Careless amine mother liquor.Then obtaining concentration with methanol dilution is respectively 10,20,40,60,80,100mg/L standard solution, using efficient Liquid chromatography (HPLC) measures the peak area of various concentration Acetochlor standard items, 3 repetitions.With the concentration of Acetochlor for horizontal seat Mark, peak area is ordinate, draws Acetochlor standard curve, as shown in Figure 1.
Testing conditions are as follows:
Detection system: Agilent 1100Series.Chromatographic column: C18DiamosilTMReversed-phase column, 250mm × 4.6mm, grain 5 μm of diameter.Chromatographic condition: mobile phase: methanol: water=80:20 (v/v), water is adjusted to pH=3 with glacial acetic acid；Detection wavelength, 215nm； Flow velocity, 1.0mL/min；Sampling volume, 20 μ L；30 DEG C of column temperature.
Gained Acetochlor measures standard curve: y=55.363x+144.5 (R2=0.9999).Wherein, y is peak area, x For Acetochlor concentration.
Two, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 degradation Acetochlor ability quantitative determination
Rhizobium (Rhizobium sp.) 198-R-47 processing: by rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 is seeded on beef extract-peptone solid medium and cultivates for 24 hours, and 1 ring of picking is seeded in 5mL beef extract albumen In peptone fluid nutrient medium, 180r/min cultivates 12h, and culture medium is removed in centrifugation, is adjusted with inorganic salt liquid culture medium to OD600Value It is 1.0.Draw 0.2mL bacteria suspension (1 × 109Cfu/ml) it is inoculated into the inorganic salt liquid culture medium of 5mL 50mg/L containing Acetochlor (training that Acetochlor (Fluka Products) obtain the concentration 50mg/L of Acetochlor is added into inorganic salt liquid culture medium Support base) test tube in, 28 DEG C, 180r/min cultivate 7 days, obtain degradation solution, measure Residual Determination of Acetochlor as follows: taking Into 50mL centrifuge tube, 8000r/min is centrifuged 5min and collects supernatant 3mL degradation solution, and 3mL methylene chloride is added, acutely vibrates 5min stands 10min, is layered to water phase and organic phase, a small amount of anhydrous sodium sulfate is added and is dehydrated to organic phase, accurate to draw 800 μ L organic phases are dried up in the centrifuge tube of 1.5mL with nitrogen evaporator, and 400 μ L methanol are added, ultrasonic in ultrasonic cleaner 10min is filtered with 0.22 μm of organic phase aculeus type filter to liquid chromatogram sample bottle, according to above-mentioned HPLC method measurement second grass Amine.
Rhizobium (Rhizobium sp.) LD1616 processing: it removes above-mentioned rhizobium (Rhizobium sp.) 198-R-47 Rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 in processing replaces with rhizobium (Rhizobium Sp.) LD1616 CGMCC No.7775, remaining is all the same.
Blank control processing: meanwhile, with non-Rhizobium Inoculation (Rhizobium sp.) 198-R-47 CGMCC No.9867 The above-mentioned 50mg/L containing Acetochlor minimal medium as blank control, measure the concentration of Acetochlor according to the method described above.
Acetochlor degradation rate: X=(1-A/B) × 100%, X is degradation rate (%) in formula, and A is rhizobium (Rhizobium Sp. it) is remained in remaining Acetochlor concentration or rhizobium (Rhizobium sp.) LD1616 treatment fluid in 198-R-47 treatment fluid Acetochlor concentration, B be do not connect remaining Acetochlor concentration in bacterium blank control treatment fluid.Experiment sets 3 repetitions, repeats every time Each processing is inoculated with 10 test tubes.
3. rhizobium of table (Rhizobium sp.) 198-R-47 CGMCC No.9867 degradation Acetochlor effect
The results show that 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 that, rhizobium (Rhizobium provided by the present invention Sp.) the degradable Acetochlor of 198-R-47 CGMCC No.9867.
Embodiment 3, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 degradation weeds burn ability and quantify Measurement
One, weeds burn the drafting of measurement standard curve
Accurately weighing weeds, to burn standard items (Fluka Products) 10.0mg molten with a small amount of methanol in 10mL volumetric flask Solution, volumetric flask is placed in ultrasonic wave bath and vibrates 10min, then with methanol constant volume to 10mL, shakes up, it is miscellaneous to be made into 1000mg/L Grass burns mother liquor.Then obtaining concentration with methanol dilution is respectively 10,20,40,60,80,100mg/L standard solution.Using efficient Liquid chromatogram (HPLC) measurement various concentration weeds burn the peak area of standard items, 3 repetitions.Concentration is burnt as abscissa using weeds, Peak area is ordinate, draws weeds and burns standard curve, as shown in Figure 2.
Testing conditions are as follows:
Detection system: Agilent 1100Series.Chromatographic column: C18DiamosilTMReversed-phase column, 250mm × 4.6mm, grain 5 μm of diameter.Chromatographic condition: mobile phase: acetonitrile: water (glacial acetic acid tune 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 measurement standard curve: y=7.3196x+2.1212 (R2=0.9875).Wherein, y is peak area, x Concentration is burnt for weeds.
Two, rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 degradation weeds burn ability quantitative determination
Rhizobium (Rhizobium sp.) 198-R-47 processing: 5mL is accurately added in test tube containing weeds and burns 100mg/L Minimal medium (into inorganic salt liquid culture medium be added weeds burn the training that the concentration 100mg/L for burning weeds is obtained Support base), add the OD of 0.2ml600Rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 bacterium that value is 1.0 Liquid (1 × 109Cfu/ml), 28 DEG C, 200r/min culture 7 days, obtain degradation solution.It takes 4mL degradation solution into 50mL centrifuge tube, adds Enter 4mL acetonitrile, vibrate 2min, stands 10min, a little anhydrous sodium sulfate is added.Accurate 800 μ L organic phases of drawing are transferred to It in 1.5mL EP centrifuge tube, is dried up on nitrogen evaporator, 400 μ L methanol (chromatographic grade) is added, burn weeds under ultrasonic wave effect It is completely dissolved, is collected by filtration with liquid spectrum filter into sample bottle, burnt according to above-mentioned HPLC method measurement weeds.
Rhizobium (Rhizobium sp.) LD1616 processing: it removes above-mentioned rhizobium (Rhizobium sp.) 198-R-47 Rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 in processing replaces with rhizobium (Rhizobium Sp.) LD1616 CGMCC No.7775, remaining is all the same.
Blank control processing: 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 control containing weeds, measure what weeds were burnt according to the method described above Concentration.
Weeds burn degradation rate: X=(1-A/B) × 100%, and X is degradation rate (%) in formula, and A is rhizobium (Rhizobium Sp.) remaining weeds are burnt in concentration or rhizobium (Rhizobium sp.) LD1616 treatment fluid and remain in 198-R-47 treatment fluid Weeds burn concentration, B is not connect remaining weeds in bacterium blank control treatment fluid to burn concentration.Experiment sets 3 repetitions, repeats every time Each processing is inoculated with 10 test tubes.Measurement result is as shown in table 4.
The results show that it is 100mg/L, rhizobium (Rhizobium sp.) 198-R- after 7 days that weeds, which burn initial concentration, The degradation rate 9.04% that 47 CGMCC No.9867 burn weeds；Rhizobium (Rhizobium sp.) LD1616 burns weeds Degradation rate is 0.02% (as shown in table 4).This result shows that, 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 degradation weeds burn effect
<110>INST OF AGRICULTURAL RESOURCES
The bacterium that<120>one plants of degrading herbicide Acetochlors 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, in China Committee for Culture Collection of Microorganisms's commonly micro- life The deposit number at object center is CGMCC No.9867.
2. a kind of microbial inoculum, it is characterised in that: the active constituent of the microbial inoculum be rhizobium described in claim 1 (Rhizobium sp.)198-R-47。
1) or 2) 3. microbial inoculum according to claim 2, it is characterised in that: the microbial inoculum is following microbial inoculums:
1) microbial inoculum for degrading Acetochlor and/or weeds are burnt；
2) microbial inoculum of pollution is burnt for rehabilitating soil Acetochlor and/or weeds.
4. rhizobium described in culture claim 1 (Rhizobium sp.) 198-R-47 method, including by the rhizobium (Rhizobium sp.) 198-R-47 CGMCC No.9867 is for cultivating the step of cultivating in the culture medium of rhizobium.
5. the preparation method of microbial inoculum described in Claims 2 or 3 includes the following steps: rhizobium described in claim 1 (Rhizobium sp.) 198-R-47 as active constituent, obtains the microbial inoculum.
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