CN103243060B - Quinclorac degrading bacteria and application thereof - Google Patents

Quinclorac degrading bacteria and application thereof Download PDF

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CN103243060B
CN103243060B CN201310201538.4A CN201310201538A CN103243060B CN 103243060 B CN103243060 B CN 103243060B CN 201310201538 A CN201310201538 A CN 201310201538A CN 103243060 B CN103243060 B CN 103243060B
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quinclorac
degradation
tobacco
application
degrading bacteria
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CN103243060A (en
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柏连阳
罗坤
董俊宇
周小毛
曾爱平
刘祥英
胡利锋
刘开林
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Hunan Agricultural University
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Abstract

The invention discloses a quinclorac degrading bacteria and application thereof. The quinclorac degrading bacteria J3 belongs to Alcaligenes sp. and is collected in CGMCC (China General Microbiological Culture Collection Center); the collection date is March 28, 2013, the collection number is CGMCC No.7384, and the GenBank accession number of a bacterial strain 16S rDNA is KC693648. The quinclorac degrading bacteria is a Gram negative bacterium, and a bacterial colony has a smooth surface and a raised center, is in a faint yellow color and a circular shape and is regular on the edge and non-transparent. The quinclorac degrading bacteria disclosed by the invention has a quinclorac degradation function and can prevent the tobaccos from being injured by quinclorac.

Description

A kind of quinclorac degradation bacteria and application thereof
Technical field
The invention belongs to environment pollutant biological treatment technical field, specifically, relate to a kind of bacterium of quinclorac of degrading, and be subject to the application in quinclorac poisoning at degraded quinclorac and control tobacco.
Background technology
Quinclorac (quinclorac), chemical name: 3,7-dichloro-8-quinoline carboxylic acid, it is Novel rice paddy weedicide before the bud of BASF Aktiengesellschaft's exploitation, after bud, because it has, consumption is few, the lasting period long, be widely used in agriculture production to advantages such as barnyard grass special efficacys, is mainly used in the anti-barnyard grass in rice field.Its structural formula is as follows:
Quinclorac is in acid; in acid soil, degraded slowly; easily cause serious harm to late stubble sensitive crop; particularly in paddy rice and tobacco crop rotation district; quinclorac residual in soil easily produces harm to solanaceous crops tobacco growing; have a strong impact on the seed output and quality of tobacco leaf, there is no method or the medicament of the poisoning that available protecting tobacco remains by quinclorac at present.
The main path alleviating dichloroquinoline acid pollution in soil is at present photodegradation and microbiological deterioration.Large quantity research proves, the multiple-microorganism that occurring in nature exists is the important factor of remains of pesticide in degraded environment.It is reported, Lv Zhenmei etc. (Chinese Journal of Applied Ecology, 2004,15 (4): 605-609) were separated in 2004 the efficient degrading bacteria (WZ1) obtaining a strain quinclorac, through identifying that it belongs to onion pseudomonas sp, can by the dichloroquinoline acid degradation 90% of 1000mg/L in 11d; Liu Huashan etc. (safety and environment journal, 2012,2:45-49) were separated in 2012 the efficient degrading bacteria (HN36) obtaining a strain quinclorac, and this Pseudomonas, can by the dichloroquinoline acid degradation 96% of 400mg/L in 48h in Bordetella.But, tobacco is subject to the poisoning of quinclorac, there is no provide protection.And there is not yet the research report of relevant Alcaligenes degraded quinclorac at present both at home and abroad.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide one and can to degrade quinclorac, and the Alcaligenes of tobacco by quinclorac poisoning can be prevented and treated.
In order to realize the object of the invention, the invention provides a strain quinclorac degradation bacteria, through being accredited as Alcaligenes (Alcaligenes sp.), numbering J3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), preservation date is on March 28th, 2013, deposit number is CGMCC No.7384, it is characterized in that: this bacterium is Gram-negative bacteria, bacterium colony smooth surface, central protuberance, in faint yellow, circular, neat in edge, opaque, thalline is shaft-like, (0.5 ~ 1.2) μm × (0.5 ~ 2.6) μm, this bacterium obligate aerobic, oxidase test and nitrate reduction test are positive, and the test of urea test, nitrate aerogenesis, hydrogen sulfide production test and DNA hydrolysis experiment are negative, and can not utilize dextrose plus saccharose.
Described degradation bacteria obtains through enrichment culture-screening purifying, and concrete steps are as follows:
1, enrichment culture: get the pedotheque containing quinclorac, add in the minimal medium containing 100mg/L quinclorac, 30 DEG C, shaking culture after a week in the shaking table of 180r/min, inoculum size with 4% is linked in the minimal medium after fresh sterilizing, transfer 1 time weekly, dichloroquinoline acid concentration increases progressively by 100mg/L gradient, and enrichment culture 5 times is 500mg/L to quinclorac final concentration;
Wherein, described minimal medium is SODIUMNITRATE 4g, potassium primary phosphate 1.5g, Sodium phosphate dibasic 0.5g, iron trichloride 0.01g, calcium chloride 0.01g, yeast powder 0.05g, micro-mother liquor (EDTA-disodium 0.01g/L, Manganous chloride tetrahydrate 39mg/L, zinc sulfate 43mg/L, Sodium orthomolybdate 36mg/L) 10mL.
2, screening purifying: described degradation bacteria, after enrichment culture, adopts method of scoring purifying bacterial strain, being seeded to being separated the degradation bacteria obtained on LB inclined-plane, at being stored in 4 DEG C.
Present invention also offers the application of described degradation bacteria in degraded quinclorac.
The application of described degraded quinclorac, by measuring the degradation rate of single bacterium, concrete steps are: be forwarded in the inorganic salt liquid substratum that dichloroquinoline acidity is 50mg/L by the inoculum size with 4% after bacterial strain J3 LB liquid medium activation (24h) after purifying, 30 DEG C, cultivate 1 week under dark condition in 150r/min shaking table, detection bacterial strain J3 is to the degradation rate of quinclorac.
Present invention also offers described degradation bacteria control tobacco by the application in quinclorac poisoning.
Quinclorac is added soil with mixed local method, and dosage is respectively 25%, 50% and 100% of its field recommendation consumption, loads diameter 18cm, the basin alms bowl of high 20cm.To cultivate to the cigarette transplantation of seedlings of 4 ~ 5 leaf phases in basin alms bowl, the strain of every alms bowl 1, carries out root irrigation with J3 bacterium liquid after transplanting, connecing bacterium amount is that 4%(degradation bacteria J3 is through liquid LB overnight incubation, OD600=0.42, adds 8mlJ3 bacterium liquid in 200ml sterilized water, finally to join in basin alms bowl near cigarette strain root).Establish CK group (not adding bacterium not dosing), biological and ecological methods to prevent plant disease, pests, and erosion group (add bacterium dosing, quinclorac consumption is that consumption is recommended in field) and poisoning group (dosing does not add bacterium, and quinclorac consumption is respectively field and recommends consumption) respectively, each process 5 repetition.
Respectively at 20d, 40d and 60d " Invest, Then Investigate " cigarette strain leaf wide (instituting an inquiry the 4th leaf from top), data acquisition Microsoft Excel2000 software processes, and analyze with DPS software package, draw the mean blade width of each process, calculate inhibiting rate and recovery rate respectively according to formula.
Present invention also offers a kind of comprise above-mentioned degradation bacteria microbial inoculum and described microbial inoculum degraded quinclorac and control tobacco by the application in quinclorac poisoning.
Degradation bacteria of the present invention is Alcaligenes, compensate for both at home and abroad to the blank of Alcaligenes degraded quinclorac.And degradation bacteria of the present invention not only has the function of degraded quinclorac, can also effectively prevent and treat quinclorac and cause poisoning to tobacco.
Accompanying drawing explanation
Fig. 1 is the electromicroscopic photograph of bacterial strain J3;
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The screening of embodiment 1 degradation bacteria J3 and Activity determination
This experiment adopts the method for enrichment culture and coating line to be separated from three soil samples of the long-term application quinclorac collected and obtains a strain has better degradation capability degradation bacteria to quinclorac, by its called after J3.The electromicroscopic photograph of bacterial strain J3 as shown in Figure 1.
Near Agricultural University Of Hunan, to have collected three pedotheque naming numbers as follows for rice terrace, vega and Hunan insecticide factory water port: water paddy soil (Q) near Agricultural University Of Hunan; Water port place of Hunan insecticide factory soil sample (T); Tobacco Farm soil (J) near Agricultural University Of Hunan.
The enrichment of dichloroquinoline acidolysis bacterium: every part of pedotheque gets 5g, joins 50mL respectively containing in the minimal medium of 100mg/L quinclorac, in 30 DEG C, shaking culture one week under 180r/min.Afterwards with 4%(volume fraction) inoculum size be linked in the minimal medium after fresh sterilizing, transfer 1 time weekly, dichloroquinoline acid concentration increases progressively by 100mg/L gradient, and enrichment culture 5 times is 500mg/L to quinclorac final concentration.After screening purifying, being seeded to being separated the degradation bacteria strains obtained on LB inclined-plane, being stored in 4 DEG C of refrigerators.
Liquid chromatographic detection: joined by 1mL testing sample in 2mL centrifuge tube, 3 repetitions are established in each process.Add 0.25mL chloroform-propyl carbinol mixing solutions [V (chloroform): V (propyl carbinol)=4:1] respectively, after shake well 30min, centrifugal 2min under 12000r/min.By upper water phase transition in new centrifuge tube, repeat top-operation and extract 2 times.The centrifugal 5min of sample 12000r/min will handled well more afterwards, uses liquid chromatogram measuring after crossing 0.22 μm of filter membrane.Liquid chromatographic detection condition: adopt Athana C18 chromatographic column, moving phase V (methyl alcohol): V (0.2% acetic acid aqueous solution)=60:40, column temperature 30 DEG C, flow velocity 0.8mL/min, determined wavelength (UV) 240nm, sample size 20 μ L.
The mensuration of single bacterium degradation rate: the inoculum size with 4% after bacterial strain J3 LB liquid medium activation (24h) after purifying is forwarded in the inorganic salt liquid substratum that dichloroquinoline acidity is 50mg/L, 30 DEG C, cultivate 1 week under dark condition in 150r/min shaking table, detection bacterial strain J3 is to the degradation efficiency of quinclorac.
By be separated from soil sample J the single bacterium J3 obtained activate after with 4%(volume fraction, OD600=0.42) it is in the triangular flask of 50mg/L that inoculum size is inoculated in dichloroquinoline acid concentration respectively, cultivate and within 7 days, adopt high-performance liquid chromatogram determination list bacterium to the degradation rate of quinclorac afterwards, its degradation rate is 71.5%.
The qualification of example 2 bacterial strain J3
The Morphology and physiology biochemical characteristic of J3: by microscope and the bacterium colony of scanning electron microscopic observation bacterial strain J3, single bacterium form.The Physiology and biochemistry qualification of J3 adopts test kit (single box biochemical identification pipe, Huankai Microbes Tech Co., Ltd., Guangdong produces).
The 16S rDNA sequencing of J3: adopt test kit to extract degradation bacteria DNA(DNA and extract test kit, Beijing Quanshijin Biotechnology Co., Ltd produces), amplimer used is synthesized by Hua Da genome company:
Upstream primer is 5 '-AGAGTTTGATCMTGGCTCAG-3 ';
Downstream primer is 5 '-TACGGCTACCTTGTTACGACTT-3 '.
Amplification reaction system is: 10 × Buffer (Mg 2+) 4 μ L, dNTPs3 μ L, each 1 μ L of primer, thallus DNA 2 μ L, Taq archaeal dna polymerase 1 μ L, adds deionized water to 50 μ L.
PCR reaction conditions: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C of annealing 40s, 72 DEG C extend 60s; 40 circulations; Finally extend 10min in 72 DEG C.
PCR primer adopts sepharose DNA recovery test kit (sepharose DNA reclaims test kit, and TIANGEN company produces) to carry out the recovery of object fragment, reclaims product examining order and is completed by Hua Da genome company.Sequencing result enters GenBank database and compares.
Result shows, J3 bacterial strain Gram-negative, obligate aerobic, and oxidase test and nitrate reduction test are positive, and the test of urea test, nitrate aerogenesis, hydrogen sulfide production test and DNA hydrolysis experiment are negative, and can not utilize dextrose plus saccharose.Identical with the biological characteristics of Alcaligenes faecalis (Alcaligenes faecalis).
By the 16S rDNA sequence of J3 bacterial strain 1888bp altogether, sign in (accession number is KC693648) in GenBank.Be 99% with the homology of Alcaligenes faecalis (Alcaligenes faecalis) (FN667794).Result and Identification of Biological Characteristics come to the same thing.Through being accredited as Alcaligenes (Alcaligenes sp.), numbering J3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), preservation date is on March 28th, 2013, and deposit number is CGMCC No.7384.
The degradation characteristic research of example 3 degradation bacteria J3
Incubation time is on degradation bacteria J3 growth and the impact of degradation rate: preparation 600ml minimal medium is sub-packed in (every bottle of 150ml) in the Erlenmeyer flask of 4 300ml, if an Erlenmeyer flask is CK, other three is three repetitions, regulates pH to be 7.0 ~ 7.2, for subsequent use after sterilising treatment.Quinclorac is added in the Erlenmeyer flask after sterilizing, its concentration is made to be 50mg/L, its excess-three Erlenmeyer flask except CK accesses with the inoculum size (OD600=0.42) of 4% the degradation bacteria J3 activated, and puts into 30 DEG C, the constant-temperature table shaking culture of 150rmin-1 afterwards.Every its OD600 value of 24h sampling and measuring and degradation rate change.
Quinclorac starting point concentration is on degradation bacteria J3 growth and the impact of degradation rate: preparation 2700ml minimal medium is sub-packed in 15 Erlenmeyer flasks respectively, regulate pH to be 7.0 ~ 7.2, add quinclorac after sterilising treatment and make its concentration be respectively each process of 10mg/L, 50mg/L, 100mg/L, 200mg/L, 300mg/L and 400mg/L(3 repetitions).Again with 4%(volume fraction) inoculum size (OD600=0.42) access the degradation bacteria J3 bacterium liquid activated.During 0h, every bottle of sampling is once as CK.Put into 30 DEG C, the constant-temperature table shaking culture of 150rmin-1 afterwards.Its OD600 value and degradation rate of sampling and measuring after 7d.
Inoculum size is on degradation bacteria J3 growth and the impact of degradation rate: preparation 2700ml minimal medium is sub-packed in 15 Erlenmeyer flasks respectively, pH is regulated to be 7.0 ~ 7.2, quinclorac is added after sterilising treatment, the concentration of quinclorac in each Erlenmeyer flask is made to be 50mg/L, afterwards respectively with 2%, 4%, 10%, 15%, 18%, 20%(volume fraction) inoculum size the degradation bacteria J3 bacterium liquid activated is inoculated into (each process 3 repetition) in each Erlenmeyer flask, during 0h, every bottle of sampling is once as CK.Put into 30 DEG C, the constant-temperature table shaking culture of 150rmin-1 afterwards.Its OD600 value and degradation rate of sampling and measuring after 7d.
Medium pH is on degradation bacteria J3 growth and the impact of degradation rate: preparation 2700ml minimal medium is sub-packed in 15 Erlenmeyer flasks respectively, regulate Medium's PH Value to be respectively 4,5,6,7,8, each process of 9(3 repetitions), quinclorac is added after sterilising treatment, make the concentration of quinclorac in each Erlenmeyer flask be 50mg/L, then with 4%(volume fraction) inoculum size (OD600=0.42) access the degradation bacteria J3 bacterium liquid activated.During 0h, every bottle of sampling is once as CK.Put into 30 DEG C, the constant-temperature table shaking culture of 150rmin-1 afterwards.Its OD600 value and degradation rate of sampling and measuring after 7d.
Culture temperature is on degradation bacteria J3 growth and the impact of degradation rate: preparation 2700ml minimal medium is sub-packed in 15 Erlenmeyer flasks respectively, pH is regulated to be 7.0 ~ 7.2, quinclorac is added after sterilising treatment, make the concentration of quinclorac in each Erlenmeyer flask be 50mg/L, with 4%(volume fraction) inoculum size (OD600=0.42) access the degradation bacteria J3 bacterium liquid activated.During 0h, every bottle of sampling is once as CK.Be positioned over dark shaking culture in the constant-temperature table of 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C and 45 DEG C (each process 3 repetition) afterwards respectively.Its OD600 value and degradation rate of sampling and measuring after 7d.
Result shows, quinclorac degradation bacteria J3 cultivate after 6d 30 DEG C, pH value 7, inoculum size 4%, quinclorac initial mass concentration shows best degradation effect under being the condition of 100mg/L, degradation rate is more than 70%.
Example 4 bacterial strain J3 is to the prevention effect of tobacco poisoning
With cloud and mist 87 for supplying examination tobacco.The degradation bacteria J3 of preservation is transferred in sterilized LB substratum, in 30 DEG C, the dark shaking culture of 150r/min constant-temperature table spends the night for examination.
Quinclorac is added soil with mixed local method, and dosage is respectively 25%, 50% and 100% of its field recommendation consumption, loads diameter 18cm, the basin alms bowl of high 20cm.To cultivate to the cigarette transplantation of seedlings of 4 ~ 5 leaf phases in basin alms bowl, the strain of every alms bowl 1, carries out root irrigation with J3 bacterium liquid after transplanting, connecing bacterium amount is that 4%(degradation bacteria J3 is through liquid LB overnight incubation, OD600=0.42, adds 8mlJ3 bacterium liquid in 200ml sterilized water, finally to join in basin alms bowl near cigarette strain root).Establish CK group (not adding bacterium not dosing), biological and ecological methods to prevent plant disease, pests, and erosion group (add bacterium dosing, quinclorac consumption is that consumption is recommended in field) and poisoning group (dosing does not add bacterium, and quinclorac consumption is respectively field and recommends consumption) respectively, each process 5 repetition.
Respectively at 20d, 40d and 60d " Invest, Then Investigate " cigarette strain leaf wide (instituting an inquiry the 4th leaf from top), data acquisition Microsoft Excel2000 software processes, and analyze with DPS software package, draw the mean blade width of each process, calculate inhibiting rate and recovery rate respectively according to formula.
Investigation result is: the tobacco mean blade width 2.5cm of poisoning group, the tobacco mean blade width 4.5cm of biological and ecological methods to prevent plant disease, pests, and erosion group, the wide 7.0cm of tobacco leaf of CK group.
The inhibiting rate going out quinclorac according to formulae discovery is 64.3%, and the recovery rate of bacterial strain J3 is 44.4%.
Calculation result illustrates, bacterial strain J3 can prevent and treat the poisoning of tobacco by quinclorac.
The preparation of embodiment 5 Tiny ecosystem degradation bacterial agent
1, the cultivation of bacterial strain
Preparation bacterium slant medium, inoculation, puts into 30 DEG C of incubators and cultivates 48 hours, save backup.
2, degradation bacterial agent is prepared
Wherein prepare different formulations according to different application aspect:
(1) liquid preparation: with 500mL triangle bottled 100ml LB substratum, conventional sterilant, after cooling again on Bechtop, directly washes lower inclined plane thalline with sterilized water and inoculates in fermentation triangular flask, shake-flask culture 30 DEG C, 200r/min, cultivate 24h.Proceed to ferment tank, the preparation of fermentation using bacteria tank substratum, sterilizing, inoculation and fermentation after cooling, 30 DEG C, 200r/min, cultivates 48h stuck fermentation at once.Adopt Plastic Bottle or plastic bag packaging fermented liquid.
(2) solid powder: with reference to the preparation of bacterium Shake flask medium, with 500mL triangle bottled 100ml LB substratum, conventional sterilant, after cooling again on Bechtop, directly wash lower inclined plane thalline with sterilized water and inoculate in fermentation triangular flask, shake-flask culture 30 DEG C, 200r/min, cultivates 24h.Proceed to ferment tank, the preparation of fermentation using bacteria tank substratum, sterilizing, inoculation and fermentation after cooling, 30 DEG C, 200r/min, cultivate 48h stuck fermentation, centrifugal fermented liquid, obtains thalline, adds wood charcoal powder absorption thalline wherein, packs with plastics bag at once.
Embodiment 6 Tiny ecosystem degradation bacterial agent is to the prevention effect of tobacco poisoning
Tiny ecosystem degradation bacterial agent (liquid preparation) is adopted to fill with root when tobacco transplant.The mixing of every 1 kilogram of degradation bacterial agent 600-800 times of clear water, after tobacco transplant, drench tobacco rhizosphere and around.In tobacco group's phase, then carry out dispenser 1 time.The effect of this insecticide-applying way and the quinclorac be characterized as around degrading tobacco rhizosphere, prevent quinclorac from invading tobacco root and absorbing, improve tobacco production 20%.
Adopt Tiny ecosystem degradation bacterial agent (solid preparation) to mix with tobacco base manure to use.Mix by every 1 kilogram of degradation bacterial agent and the tobacco base manure for 1 mu of vega, execute to field.The effect of this insecticide-applying way and the quinclorac be characterized as around degrading tobacco rhizosphere, microbial inoculum will invade tobacco, surely grow in tobacco rhizosphere and stem stalk, improve tobacco production 20%.Test proves, illustrates that described microbial inoculum can prevent and treat the poisoning of tobacco by quinclorac.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (6)

1. a strain quinclorac degradation bacteria, through being accredited as Alcaligenes (Alcaligenes sp.), numbering J3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on March 28th, 2013, and deposit number is CGMCC No.7384.
2. the application of degradation bacteria according to claim 1 in degraded quinclorac.
3. degradation bacteria according to claim 1 is subject to the application in quinclorac poisoning control tobacco.
4. one kind comprises the microbial inoculum of degradation bacteria according to claim 1.
5. the application of microbial inoculum according to claim 4 in degraded quinclorac.
6. microbial inoculum according to claim 4 is subject to the application in quinclorac poisoning control tobacco.
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CN103820372B (en) * 2014-03-17 2015-09-30 中国农业科学院烟草研究所 One strain quinclorac efficient degrading bacteria and uses thereof and using method
CN103834599A (en) * 2014-03-17 2014-06-04 中国农业科学院烟草研究所 Quinclorac effective degradation bacteria, and application and use method thereof
CN105861352B (en) * 2015-01-22 2019-08-16 北京禾和润生科技有限公司 With the Ludwig enterobacteria of dichloro quinolinic acid degradation function and its application
CN106497816B (en) * 2015-09-07 2019-09-03 粮华生物科技(北京)有限公司 Pseuomonas denitrifican and its microbial inoculum and they degradation dichloro quinolinic acid in application
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