CN104498389B - Bacterium for degrading herbicide namely Pursuit (imazethapyr-ammonium) and acetochlor and application of bacterium - Google Patents

Bacterium for degrading herbicide namely Pursuit (imazethapyr-ammonium) and acetochlor and application of bacterium Download PDF

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CN104498389B
CN104498389B CN201410705124.XA CN201410705124A CN104498389B CN 104498389 B CN104498389 B CN 104498389B CN 201410705124 A CN201410705124 A CN 201410705124A CN 104498389 B CN104498389 B CN 104498389B
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acetochlor
rhizobium
shite
root nodule
bacterium
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高淼
孙建光
罗琼
张燕春
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Institute of Agricultural Resources and Regional Planning of CAAS
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Abstract

The invention discloses a bacterium for degrading herbicide namely Pursuit (imazethapyr-ammonium) and acetochlor and an application of the bacterium. The bacterium for degrading Pursuit and acetochlor is (Rhizobium sp.) 198-R-46, and has a collection number of CGMCC (China General Microbiological Culture Collection Center) No.9866 in the CGMCC. The degradation rate of the (Rhizobium sp.) 198-R-46 CGMCC No. 9866 provided by the invention to 100mg/L Pursuit reaches 92% after the bacterium is put into an inorganic salt culture medium for 7 days, and the degradation rate to 50mg/L acetochlor reaches 88.11%, which shows that the strain namely (Rhizobium sp.) 198-R-46 can be used for degrading Pursuit or acetochlor and has a wide application prospect in repair of the herbicide namely Pursuit and/or acetochlor pollutions of soil.

Description

Antibacterial of one plant of degrading herbicide Pu Shite and Acetochlor and application thereof
Technical field
The present invention relates to antibacterial of one plant of degrading herbicide Pu Shite and Acetochlor and application thereof in microorganism field.
Background technology
Pu Shite, also known as Imazethapyr, English name pursuit (imazethapyr-ammonium), chemical name (rs)- 5- ethyl -2- (4- isopropyl-4-methyl -5- oxo -2- imidazoline -2- base) nicotinic acid.Chemical constitution is as shown in Equation 1:
Pu Shite is imidazolinone herbicide, is side chain amino acid synthetic inhibitor, all can apply before bud or after bud, right The grassy weed in Semen sojae atricolor and other leguminous plant farmlands and some broad leaved weeds for example Amaranthus mangostanus L., knotweed, Herba chenopodii, Herba Solani Nigri, Herba Xanthii, barnyard grass, Herba Setariae Viridis, Herba Digitariae, broomcorn millet etc. have excellent prevention effect.
Acetochlor, english common name acetochlor, chemical name is 2- ethyl -6 methyl -- n- ethoxyl methyl-α - Chloro acetanilide, structural formula is as shown in Equation 2:
Acetochlor is absorbability acetamide-group herbicides, is selective preemergence herbicide.Can be by the plumelet of weeds and young root Absorb, suppress weeds protein synthesis, and make weeds dead.It is mainly used in the crops such as Semen sojae atricolor, Semen arachidis hypogaeae, Semen Maydiss, Cotton Gossypii and prevent and kill off one Year raw grassy weed and part broad leaved weed, have good preventive effect to Soybean Dodder, invalid to perennial weeds.
General apply specially for long residual herbicides, advantage is that herbicidal effect is good, herbicide spectrum width, dosage are few, easy to use, medication Low cost, but their residence times in soil are long, typically up to 2-3, using easily causing in continuous cropping or crop rotation farmland Succession crop poisoning, the underproduction, even have no harvest, and had a strong impact on the adjustment of agricultural planting industry structure.After long residual herbicides make Stubble crop is happened occasionally by the poisoning underproduction, total crop failure event.It is mainly shown as: one is that some are local in Heilungkiang, Jilin, the Inner Mongol Soybean Field is changed to paddy field, rice cultivation is aggrieved serious, or even total crop failure.Two is that herbicide is applied rear 3-4 and still had poisoning, leads to Beet plant industry glides.Three is that industrial crops development is impacted, and Semen Cucurbitae, Helianthi, Rhizoma Solani tuber osi, Caulis et Folium Lini and vegetable etc. are hindered Evil, severe patient base is ruined.Northeastern Inner Mongolia is just wanted to adjust crop mix, because Semen Tritici aestivi, Soybean Field use for many years several years ago Long residual herbicides make the industrial crops such as plantation Rhizoma Solani tuber osi, Caulis et Folium Lini, Semen Benincasae, Semen Phaseoli Vulgariss not enable, and seriously constrain local warp Ji development.Four is the crop damage such as Semen Maydiss, Sorghum vulgare Pers., millet, downgrades the underproduction or total crop failure.
The Acetochlor half-life is shorter, is considered as a kind of environmentally friendly agricultural chemical of low toxicity always, but frequent in recent years, mistake Amount or improper use, cause different degrees of pollution to agricultural land soil and subsoil water etc., the harm to succession crop is also more next More serious.Research shows, a large amount of Acetochlors of applying cause certain residual toxicity in the environment, and it enters into and passes through food in environment Thing chain is constantly enriched with vivo, can cause the endocrine regulation of organism, causes sterile, abnormal sex difference very To carcinogenic, carcinogen is set to by EPA.
The degraded of nature herbicide residue relies primarily on Soil Microorganism to complete, but natural degradation is very slow. Therefore, targetedly screen high-effective microorganism bacterial strain, develop microorganism renovation agent, Herbicidal agent is accelerated by artificial vaccination It is a very necessary job and practicable approach that the degraded of residual eliminates farmland herbicide herbicide carryover.
Content of the invention
The technical problem to be solved is how degrading herbicide Pu Shite and Acetochlor.
For solving above-mentioned technical problem, the invention provides one plant can be thin with degrading herbicide Pu Shite and Acetochlor Bacterium.
The antibacterial that the present invention provides is root nodule bacteria (rhizobium sp.) 198-r-46, and this bacterial strain is in November 6 in 2014 Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and (abbreviation cgmcc, address is: court of Beijing Positive area North Star West Road 1 institute 3), deposit number is cgmcc no.9866.
It is a further object to provide a kind of microbial inoculum, the active component of this microbial inoculum is described root nodule bacteria (rhizobium sp.)198-r-46cgmcc no.9866.
Above-mentioned microbial inoculum can be following 1) or 2) described microbial inoculum:
1) it is used for the microbial inoculum of degrading herbicide Pu Shite and/or Acetochlor；
2) it is used for rehabilitating soil herbicide Pu Shite and/or the microbial inoculum of Acetochlor pollution.
Described microbial inoculum can also include carrier.Described carrier can be solid carrier or liquid-carrier.Described solid carrier can For mineral material, vegetable material or macromolecular compound；Described mineral material can be clay, Talcum, Kaolin, montmorillonite, white At least one in carbon, zeolite, Silicon stone and kieselguhr；Described vegetable material can be at least in Semen Maydis powder, Semen Glycines powder and starch Kind；Described macromolecular compound can be polyvinyl alcohol and/or polyglycols.Described liquid-carrier can be organic solvent, vegetable oil, ore deposit Thing oil or water；Described organic solvent can be decane and/or dodecane.In described microbial inoculum, described active component can be to be cultured Living cells, presented in the mixture of the fermentation liquid of living cells, the filtrate of cell culture or cell and filtrate.Described group The dosage form of compound can be multiple dosage forms, such as liquor, Emulsion, suspending agent, powder, granule, wettable powder or water dispersible granules.
As needed, also can add surfactant (as polysorbas20, Tween 80 etc.), binding agent in described microbial inoculum, stablize Agent (as antioxidant), ph regulator etc..
Described root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 or with root nodule bacteria (rhizobium Sp.) 198-r-46cgmcc no.9866 is microbial inoculum the answering in degrading herbicide Pu Shite and/or Acetochlor of active component With falling within protection scope of the present invention.
Described root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 or with root nodule bacteria (rhizobium Sp.) 198-r-46cgmcc no.9866 is the microbial inoculum of active component in rehabilitating soil herbicide Pu Shite and/or Acetochlor dirt 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 root nodule bacteria (rhizobium sp.) 198-r-46cgmcc The method of no.9866.
The method of culture root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 provided by the present invention, bag Include and root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 is cultivated in the culture medium for cultivating root nodule bacteria Step.
A further object of the present invention is to provide one kind with root nodule bacteria (rhizobium sp.) 198-r-46cgmcc No.9866 is the preparation method of the microbial inoculum of active component.
The preparation method of above-mentioned microbial inoculum provided by the present invention, comprises the steps: described root nodule bacteria (rhizobium Sp.) 198-r-46cgmcc no.9866, as active component, obtains described microbial inoculum.
The present invention is from the farmland collection soil sample being polluted by herbicide Pu Shite and Acetochlor for a long time, and therefrom bolter Select the herbicide Pu Shite obtaining and/or Acetochlor degradation bacteria root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866.It is demonstrated experimentally that this bacterial strain 7 days degradation rates to 100mg/l Pu Shite in minimal medium reach 92%；Right 50mg/l Acetochlor degradation rate reaches 88.11%.This shows this bacterial strain energy degrading herbicide Pu Shite and/or Acetochlor, repairing Earth backing earth herbicide Pu Shite and Acetochlor pollution aspect have broad application prospects.
Preservation explanation
Strain name: root nodule bacteria
Latin name: (rhizobium sp.)
Strain number: 198-r-46
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 November 6th, 2014
Collection is registered on the books numbering: cgmcc no.9866
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
In following embodiments, culture medium used is as follows:
Nitrogen-free fluid 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 the culture that agar obtains to its content for 15-20g/l in nitrogen-free fluid medium Base.
Beef extract-peptone fluid medium: solute and its concentration are Carnis Bovis seu Bubali cream 5g/l, peptone 10g/l, nacl 5g/ l；Solvent is distilled water；ph7.2.
Beef extract-peptone solid medium: solute and its concentration are Carnis Bovis seu Bubali cream 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, k2hpo41.5g/ L, mgso40.2g/l, nacl 0.5g/l；Solvent is distilled water；ph7.0.
Inorganic salt solid medium: adding agar to its content in inorganic salt liquid culture medium is 2% culture medium obtaining.
Root nodule bacteria (rhizobium sp.) ld1616cgmcc no.7775 (abbreviation root nodule bacteria in following embodiments (rhizobium sp.) ld1616) disclosed in Chinese invention patent document cn 103343100b.
Embodiment 1, the separation of root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 and identification
First, the separation of Pu Shite/Acetochlor degradation bacteria 198-r-46
1st, 10g pedotheque is added (to pick up from the agriculture that Chinese Heihe In The Heilongjiang River is subject to pollution of herbicide in 100ml sterilized water Field), shaken cultivation 30min makes dirty solution.Draw the above-mentioned dirty solution of 1ml to add in the test tube filling in 9ml sterilized water fully (now dilution factor is designated as 10 for mixing-1), from this test tube, then draw 1ml be added in another test tube filling 9ml sterilized water Mix homogeneously, makes 10 by that analogy-2、10-3、10-4、10-5Different dilution bacteria suspensions.Each dilution factor is taken 0.1ml uniform It is coated on nitrogen-free solid medium flat board, 28 DEG C of constant temperature quiescent culture 23 days.After bacterium colony is formed, in nitrogen-free solid culture Purifying agaric is carried out on base flat board.
2nd, the preliminary screening that herbicide Pu Shite, Acetochlor, chlorimuronethyl and weeds are burnt with degradation capability adopts flat board saturating Bright circle method.It is 1000mg/l that Pu Shite standard substance (fluka Products) addition inorganic salt solid medium is made its content, obtains Apply dead falt sheet to general；Acetochlor standard substance (fluka Products) addition inorganic salt solid medium is made its content be 1000mg/l, obtains Acetochlor flat board；Chlorimuron ethyl (fluka Products) addition inorganic salt solid medium is made Its content is 1000mg/l, obtains chlorimuronethyl flat board；Weeds are burnt standard substance (fluka Products) and adds inorganic salt solid It is 1000mg/l that culture medium makes its content, obtains weeds and burns flat board.Apply dead falt sheet, Acetochlor flat board, chlorimuronethyl flat board by general Burn flat board with weeds and carry out subregion, the bacteria suspension 10 μ l of every plant of bacterium that aspiration step 1 purification obtains and the root nodule bacteria of this laboratory The bacteria suspension 10 μ l (the thalline content of various bacterial strain bacteria suspensions is identical) of (rhizobium sp.) ld1616 dibbling 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.Screen one plant and apply dead falt sheet and second grass general The bacterial strain of larger transparent circle all can be formed on amine flat board, be named as Pu Shite/Acetochlor degradation bacteria 198-r-46.General apply Spy/Acetochlor degradation bacteria 198-r-46 is burnt in chlorimuronethyl flat board and weeds and all can not be formed transparent circle on flat board, and general applying is described Spy/Acetochlor degradation bacteria 198-r-46 can not degrade chlorimuronethyl and weeds are burnt.Root nodule bacteria (rhizobium sp.) ld1616 is only Larger transparent circle is formed on chlorimuronethyl flat board, general apply dead falt sheet, Acetochlor flat board and weeds and burn shape is all unable on flat board Become transparent circle.Illustrate that Pu Shite/Acetochlor degradation bacteria 198-r-46 can degrade Pu Shite and Acetochlor it is impossible to degraded chlorimuronethyl Burn with weeds；Root nodule bacteria (rhizobium sp.) ld1616 can degrade chlorimuronethyl it is impossible to degraded Pu Shite, Acetochlor and miscellaneous Grass burns.
Table 1. Pu Shite/Acetochlor degradation bacteria 198-r-46 and root nodule bacteria (rhizobium sp.) ld1616 remove to four kinds Careless agent degradation capability primary dcreening operation result
Strain number Pu Shite Acetochlor Chlorimuronethyl Weeds are burnt
Pu Shite/Acetochlor degradation bacteria 198-r-46 + + - -
Root nodule bacteria (rhizobium sp.) ld1616 - - + -
Note: "+" representing the larger transparent circle of formation, "-" indicates that no transparent circle is formed.
2nd, the identification of Pu Shite/Acetochlor degradation bacteria 198-r-46
Pu Shite/Acetochlor degradation bacteria 198-r-46 from the following aspects authentication step one separates and purification obtains:
1st, Morphological Identification
Exponential phase will be in and bacterium colony size will be stable, the Pu Shite that above-mentioned steps one separate and purification obtains/second grass Amine degradation bacterium 198-r-46 carries out single bacterium colony state observation, the main size including bacterium colony, color, transparency, wettability, bacterium colony Apparent condition (whether flat, projection, fold, depression etc.), colony edge state (whether neat, irregular, radial etc.).
Result shows, Pu Shite/Acetochlor degradation bacteria 198-r-46 bacterium colony table that above-mentioned steps one separate and purification obtains Face is raised, and sticky, edge is irregular；Faint yellow, bacterium colony is larger, diameter 2.3-3.1mm.
2nd, analysis of physio biochemical characteristics
Reference " common bacteria system identification handbook " (east show pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: section Publishing house, 2011.) and " Microbiology Experiment " (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (third edition). Beijing: Higher Education Publishing House, 1999.) measure the physiological and biochemical property of above-mentioned Pu Shite/Acetochlor degradation bacteria 198-r-46.
The physiological and biochemical property measurement result of described Pu Shite/Acetochlor degradation bacteria 198-r-46 is as shown in table 2:
The physiological and biochemical property of table 2. Pu Shite/Acetochlor degradation bacteria 198-r-46
Note: "+" representing positive, "-" represents negative.
3rd, 16s rdna sequence homology analysis
Conventional method culture above-mentioned steps one isolate and purify Pu Shite/Acetochlor degradation bacteria 198-r-46 obtaining, and extract Total dna of bacterial strain as gene amplification template, with antibacterial 16s rdna universal primer, 27f:5 '- Agagtttgatcctggctcag-3 ', 1492r:5 '-taccttgttacgactt-3 ' carries out pcr reaction.Reaction system adopts Shanghai biological engineering company limited pcr amplification kit.Response procedures are: 95 DEG C of degeneration 30s, 55 DEG C of annealing 1min, 72 DEG C prolong Stretch 2min, 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 American National Biotechnology Information center ncbi data base (http: // Www.ncbi.nlm.nih.gov) complete online with eztaxon.
Pcr gene amplification obtains the 16s rdna genetic fragment about 1.3kb of Pu Shite/Acetochlor degradation bacteria 198-r-46, Carry out online sequence analysis, result with 16s rdna sequence published in ncbi and eztaxon data base after measuring sequence Display Pu Shite/Acetochlor degradation bacteria 198-r-46 and rhizobium rhizobium nepotum (genbank accession number Fr870231) homology highest, reaches 99.85%.
The 16s rdna sequence of Pu Shite/Acetochlor degradation bacteria 198-r-46 refers to sequence 1.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rdna sequence homology analysis result, by step one point The Pu Shite obtaining from purification/Acetochlor degradation bacteria 198-r-46 is accredited as rhizobium (rhizobium sp.).This is general to apply It is general that spy/Acetochlor degradation bacteria 198-r-46 is preserved in China Committee for Culture Collection of Microorganisms on November 06th, 2014 Logical microorganism center (abbreviation cgmcc, address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is cgmcc no.9866.The full name of Pu Shite/Acetochlor degradation bacteria 198-r-46 is root nodule bacteria (rhizobium sp.) 198-r-46cgmcc No.9866, referred to as root nodule bacteria (rhizobium sp.) 198-r-46.
Embodiment 2, root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 degraded Pu Shite ability are quantitative Measure
First, the drafting of Pu Shite bioassay standard curve
Accurately weigh Pu Shite standard substance (fluka Products) 10.0mg in 10ml volumetric flask, molten with a small amount of methanol Solution, volumetric flask is placed on vibration 10min in ultrasound wave bath, then with methanol constant volume to 10ml, shakes up, be made into 1000mg/l general Apply special mother solution.Then proceed to methanol dilution obtain concentration be respectively 20,40,60,80, the standard solution of 100mg/l.Using height Effect liquid phase chromatogram (hplc) measures the peak area of variable concentrations Pu Shite standard substance, 3 repetitions.With Pu Shite concentration for horizontal seat Mark, peak area is vertical coordinate, draws Pu Shite standard 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: 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 Pu Shite bioassay standard curve: y=24.482x-6.6979 (r2=0.9998).Wherein, y is peak area, x For Pu Shite concentration.
2nd, root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 degraded Pu Shite ability quantitative determination
Root nodule bacteria (rhizobium sp.) 198-r-46 process: by rhizobial nodule bacterium (rhizobium sp.) 198-r- 46 cgmcc no.9866 are seeded in culture 24h on beef extract-peptone solid medium, and picking 1 ring is seeded in 5ml Carnis Bovis seu Bubali cream In peptone fluid medium, 180r/min cultivates 12h, and centrifugation is removed culture medium, is adjusted to inorganic salt liquid culture medium od600It is worth for 1.0.Draw 0.2ml bacteria suspension (1 × 109Cfu/ml) it is inoculated into the inorganic salt liquid that 5ml contains Pu Shite 100mg/l Culture medium (adds Pu Shite (fluka Products) so that the concentration of Pu Shite is obtained for 100mg/l in inorganic salt liquid culture medium The culture medium arriving) test tube in, 28 DEG C, 180r/min cultivate 7 days, obtain degradation solution.Take 4ml degradation solution to 50ml centrifuge tube In, add 8ml dichloromethane, vibrate 2min, stand 10min, add a little anhydrous sodium sulfate.Accurately draw 800 μ l organic faciess It is transferred in 1.5mlep centrifuge tube, Nitrogen evaporator dries up, add 400 μ l methanol (chromatographic grade), molten on ultrasonic washing instrument Solution 10min, is collected by filtration to sample bottle with liquid spectrum filter, measures Pu Shite according to above-mentioned hplc method.
Root nodule bacteria (rhizobium sp.) ld1616 process: except by above-mentioned root nodule bacteria (rhizobium sp.) 198-r-46 Root nodule bacteria (rhizobium sp.) 198-r-46 in process replaces with root nodule bacteria (rhizobium sp.) ld1616 cgmcc No.7775, remaining all same.
Blank is processed: meanwhile, with non-Rhizobium Inoculation (rhizobium sp.) 198-r-46cgmcc no.9866 The above-mentioned inorganic salt liquid culture medium containing Pu Shite 100mg/l as blank, measure Pu Shite's according to the method described above Concentration.
Pu Shite degradation rate: x=(1-a/b) × 100%, in formula, x is degradation rate (%), and a is root nodule bacteria (rhizobium Sp.) remain in the Pu Shite concentration of residual or root nodule bacteria (rhizobium sp.) ld1616 treatment fluid in 198-r-46 treatment fluid Pu Shite concentration, b is the Pu Shite concentration not connecing in bacterium blank treatment fluid residual.Experiment sets 3 repetitions, repeats every time Each processes 10 test tubes of inoculation.Measurement result is as shown in table 3.
Table 3. root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 degraded Pu Shite effect
Result shows, Pu Shite initial concentration is 100mg/l, described root nodule bacteria (rhizobium sp.) 198-r- after 7 days 46cgmcc no.9866 reaches 92% (as shown in table 3) to the degradation rate of Pu Shite；Root nodule bacteria (rhizobium sp.) Ld1616cgmcc no.7775 reaches 0.02% to the degradation rate of Pu Shite.This result shows, root nodule provided by the present invention Bacterium (rhizobium sp.) 198-r-46cgmcc no.9866 degradable Pu Shite.
Embodiment 3, root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 degraded Acetochlor ability are quantitative Measure
First, Acetochlor standard curve making
Accurately weigh Acetochlor standard substance (fluka Products) 10.0mg in 10ml volumetric flask, molten with a small amount of methanol Solution, volumetric flask is placed on vibration 10min in ultrasound wave bath, then with methanol constant volume to 10ml, shakes up, be made into 1000mg/l second Careless amine mother solution.Then with methanol dilution obtain concentration be respectively 10,20,40,60,80,100mg/l standard solution, using efficient Liquid chromatography (hplc) measures the peak area of variable concentrations Acetochlor standard substance, 3 repetitions.With the concentration of Acetochlor for horizontal seat Mark, peak area is vertical coordinate, draws Acetochlor standard 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: methanol: water=80:20 (v/v), water glacial acetic acid is 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, root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 degraded Acetochlor ability quantitative determination
Root nodule bacteria (rhizobium sp.) 198-r-46 process: by root nodule bacteria (rhizobium sp.) 198-r- 46cgmccno.9866 is seeded in culture 24h on beef extract-peptone solid medium, and picking 1 ring is seeded in 5ml Carnis Bovis seu Bubali cream egg In white peptone fluid medium, 180r/min cultivates 12h, and centrifugation is removed culture medium, is adjusted to od with inorganic salt liquid culture medium600 It is worth for 1.0.Draw 0.2ml bacteria suspension (1 × 109Cfu/ml) it is inoculated into the inorganic salt liquid culture that 5ml contains Acetochlor 50mg/l Base (adds Acetochlor (fluka Products) so that the concentration of Acetochlor is obtained for 50mg/l in inorganic salt liquid culture medium Culture medium) test tube in, 28 DEG C, 180r/min cultivate 7 days, obtain degradation solution, as follows measure Residual Determination of Acetochlor: Take 3ml degradation solution to 50ml centrifuge tube, 8000r/min centrifugation 5min collects supernatant, adds 3ml dichloromethane, acutely shakes Swing 5min, stand 10min, treat aqueous phase and organic faciess layering, add a small amount of anhydrous sodium sulfate that organic faciess are dehydrated, accurately inhale Take 800 μ l organic faciess in the centrifuge tube of 1.5ml, dried up with Nitrogen evaporator, add 400 μ l methanol, super in ultrasonic cleaner Sound 10min, is filtered to liquid chromatograph sample bottle with 0.22 μm of organic faciess acupuncture type filter, measures second grass according to above-mentioned hplc method Amine.
Root nodule bacteria (rhizobium sp.) ld1616 process: except by above-mentioned root nodule bacteria (rhizobium sp.) 198-r-46 Root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 in process replaces with root nodule bacteria (rhizobium sp.) Ld1616cgmcc no.7775, remaining all same.
Blank is processed: meanwhile, with non-Rhizobium Inoculation (rhizobium sp.) 198-r-46cgmcc no.9866 The above-mentioned minimal medium containing Acetochlor 50mg/l as blank, measure 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 root nodule bacteria (rhizobiumsp.) at the Acetochlor concentration remaining in 198-r-46 treatment fluid or root nodule bacteria (rhizobium sp.) ld1616 The Acetochlor concentration of residual in reason liquid, b is the Acetochlor concentration not connecing residual in bacterium blank treatment fluid.Experiment sets 3 weights Multiple, repeat each every time and process 10 triangular flasks of inoculation.
Result shows, Acetochlor initial concentration is 50mg/l, described root nodule bacteria (rhizobium sp.) 198-r- after 7 days 46 cgmcc no.9866 reach 88.11% to the degradation rate of Acetochlor；Root nodule bacteria (rhizobium sp.) ld1616cgmcc No.7775 is 0% (as shown in table 4) to the degradation rate of Acetochlor.This result shows, root nodule bacteria provided by the present invention (rhizobium sp.) 198-r-46cgmcc no.9866 degradable Acetochlor.
Table 4. root nodule bacteria (rhizobium sp.) 198-r-46cgmcc no.9866 degraded Acetochlor effect

Claims (7)

1. root nodule bacteria (rhizobium sp.) 198-r-46, it is in China Committee for Culture Collection of Microorganisms's commonly micro- life The deposit number at thing center is cgmcc no.9866.
2. a kind of microbial inoculum it is characterised in that: the active component of described microbial inoculum be claim 1 described in root nodule bacteria (rhizobium sp.)198-r-46.
3. microbial inoculum according to claim 2 it is characterised in that: described microbial inoculum be following 1) or 2) described microbial inoculum:
1) it is used for the microbial inoculum of Pu Shite and/or Acetochlor of degrading；
2) it is used for rehabilitating soil Pu Shite and/or the microbial inoculum of Acetochlor pollution.
4. (rhizobium sp.) 198-r-46 of the root nodule bacteria described in claim 1 or the microbial inoculum described in Claims 2 or 3 exist Application in degraded Pu Shite and/or Acetochlor.
5. (rhizobium sp.) 198-r-46 of the root nodule bacteria described in claim 1 or the microbial inoculum described in Claims 2 or 3 exist Application in rehabilitating soil Pu Shite and/or Acetochlor pollution.
6. the method for root nodule bacteria (rhizobium sp.) 198-r-46 described in culture claim 1, including by described root nodule bacteria The step that (rhizobium sp.) 198-r-46 cultivates in the culture medium for cultivating root nodule bacteria.
7. the preparation method of microbial inoculum described in Claims 2 or 3, comprises the steps: the root nodule bacteria described in claim 1 (rhizobium sp.) 198-r-46, as active component, obtains described microbial inoculum.
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