CN102296041B - Bacterium for efficiently degrading residual pesticide carbendazim and application thereof - Google Patents
Bacterium for efficiently degrading residual pesticide carbendazim and application thereof Download PDFInfo
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- CN102296041B CN102296041B CN 201110247634 CN201110247634A CN102296041B CN 102296041 B CN102296041 B CN 102296041B CN 201110247634 CN201110247634 CN 201110247634 CN 201110247634 A CN201110247634 A CN 201110247634A CN 102296041 B CN102296041 B CN 102296041B
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- derosal
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- zymomonas mobilis
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Abstract
本发明公开了一株高效降解残留农药多菌灵的细菌及其用途。所述降解残留农药多菌灵的细菌是寡养单胞菌(Stenotrophomonas sp.)DJL1614，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为CGMCC No.5151。本发明提供的寡养单胞菌(Stenotrophomonas sp.)DJL1614 CGMCC No.5151在无机盐培养基中10d对500mg/L多菌灵的降解率达到73.41％。这表明该菌株能够高效降解多菌灵，在修复土壤多菌灵污染方面具有广阔的应用前景。The invention discloses a bacterium for efficiently degrading residual pesticide carbendazim and its application. The bacterium that degrades the residual pesticide carbendazim is Stenotrophomonas sp. DJL1614, and its preservation number in the General Microorganism Center of China Committee for Microbial Culture Collection is CGMCC No.5151. The degradation rate of Stenotrophomonas sp. DJL1614 CGMCC No.5151 provided by the present invention to 500 mg/L carbendazim in inorganic salt medium for 10 days reaches 73.41%. This shows that the strain can efficiently degrade carbendazim, and has broad application prospects in remediating soil carbendazim pollution.
Description
Technical field
The present invention relates to bacterium of a high-efficiency degradation remains of pesticide derosal and uses thereof.
Background technology
Derosal (carbendazim) chemical name is N-(2-benzimidazolyl base)-Urethylane, and English chemical name is methyl benzimidazol-2-ylcarbamate.Molecular formula is C
9H
9N
3O
2, structural formula is as follows.
Derosal can interfere RNA synthesize and the cell mitogen process, fungal disease to various crop has prevention effect, it is the agriculture at present main sterilant of going up controlling plant diseases, use range is very wide, mainly act on paddy rice, peanut, cotton, tobacco, beet and various vegetables, can prevent and treat cucurbits powdery mildew, tomato early blight, beans anthrax, eqpidemic disease, sclerotinia rot of colza, shallot, leek gray mold, eggplant, cucumber timberrot, melon, bean anthracnose, powdery mildew of pea etc. also have certain effect to the prosperous length of control peanut.But the residual period of derosal in soil, is long, can cause people's hepatopathy, causes the effect of chromosome aberration, has that potential is carcinogenic, a teratogenesis, mutagenesis, to the toxic effect of Mammals, so the degraded of research derosal just seems particularly important.
Less relatively to the research report of derosal degradation bacteria both at home and abroad at present.As, external, Holtman etc. have been separated to the gram-positive red city of 6 strains rhodococcus (Rhodococcus erythropolis), the derosal of 16mg/L in the inorganic salt nutrient solution of wherein having degraded behind the 5 strain bacterium 15d, and average degradation rate is 1.07mg/ (L.d).Domestic, it is 19.16% to the degradation rate of 500mg/L derosal that the Rolls that Zhang Guishan was separated in 2004 leads to Salmonella (Ralstonia sp.) 24d in minimal medium.If can find to make derosal obtain more effective degraded to the higher bacterial strain of derosal degradation capability.
Summary of the invention
The purpose of this invention is to provide the bacterium that a strain can efficient degradation remains of pesticide derosal.
Bacterium provided by the present invention is oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 17th, 2011 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.5151.
Another object of the present invention provides a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is described oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151.
Described microbial inoculum is following 1) or 2) described microbial inoculum:
1) be used to the to degrade microbial inoculum of derosal;
2) be used for the microbial inoculum that the rehabilitating soil derosal pollutes.
Described oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 or be that the application of microbial inoculum in the degraded derosal of activeconstituents also belongs to protection scope of the present invention with oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151.
Described oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 or be that the application of microbial inoculum in the rehabilitating soil derosal pollutes of activeconstituents also belongs to protection scope of the present invention with oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151.
A further object of the present invention provides the method for a kind of cultivation oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151, and this method comprises the step that oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 is cultivated at the substratum that is used for cultivating the oligotrophy Zymomonas mobilis.
It is the preparation method of the microbial inoculum of activeconstituents with oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 that another purpose of the present invention provides a kind of, comprise the steps: described oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 is obtained described microbial inoculum as activeconstituents.
The present invention is that soil sample is gathered in places such as vegetable plot, farmland, pond that derosal pollutes are half congealed, insecticide factory from being subjected to for a long time, and therefrom separates, screens the derosal degradation bacteria DJL1614 that obtains.Experiment showed, that this bacterial strain 10d in minimal medium reaches 73.41% to the degradation rate of 500mg/L derosal.This shows that this bacterial strain energy efficient degradation derosal is having broad application prospects aspect the pollution of rehabilitating soil remains of pesticide derosal.
The preservation explanation
Strain name: oligotrophy Zymomonas mobilis
Latin name: (Stenotrophomonas sp.)
Strain number: DJL1614
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on August 17th, 2011
The preservation center numbering of registering on the books: CGMCC No.5151
Description of drawings
Fig. 1 is a derosal bioassay standard curve.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Enrichment medium: K
2HPO
45.71g/L, KH
2PO
41.70g/L, (NH
4)
2SO
42.63g/L, MgSO
47H
2O 0.095g/L, MnSO
40.05g/L, FeSO
40.05g/L, CaCl
20.003g/L, pH value 7.0.
Isolation medium: enrichment medium adds 2% agar.
Minimal medium: NH
4CL 1.0g/L, KH
2PO
40.5g/L, K
2HPO
41.5g/L, MgSO
40.2g/L, NaCl 0.5g/L, pH7.0.
Beef-protein medium: peptone 10.0g/L, extractum carnis 5.0g/L, NaCl 5.0g/L, pH7.2.
Separation and the evaluation of embodiment 1, derosal degradation bacteria DJL1614
One, the separation of derosal degradation bacteria DJL1614
At the 100ml carbendazim concentration is to add 10g soil sample (pick up from the BeiJing, China and be subjected to the agricultural land soil that derosal pollutes) in the enrichment culture liquid of 100mg/L, 25 ℃, 200r/min shaking culture 3d, it is in the 200mg/L enrichment medium that absorption 5ml nutrient solution is forwarded to carbendazim concentration, cultivate 3d, enrichment continuously successively, transfer 5 times, make carbendazim concentration be followed successively by 100,200,400,500,800 and 1000mg/L.After enrichment finishes, getting nutrient solution evenly coats on the derosal isolation medium and minimal medium of same concentrations, 25 ℃ of constant temperature culture, after treating the single bacterium colony of dull and stereotyped upward appearance, the bigger single bacterium colony of picking is forwarded on the beef-protein medium, 25 ℃ of constant temperature culture are purified to after the pure culture standby repeatedly.
Found that bacterium colony is chosen bacterium colony separation and purification on the minimal medium in that to contain on the derosal minimal medium of same concentrations score growing way on substratum good.To separate and the resulting strain bacterium called after derosal degradation bacteria DJL1614 of purifying.
Two, the evaluation of derosal degradation bacteria DJL1614
From the derosal degradation bacteria DJL1614 that the following aspects authentication step one is separated and purifying obtains:
1, morphology is identified
To be in logarithmic phase, and the bacterium colony size is stable, the derosal degradation bacteria DJL1614 that above-mentioned steps one is separated and purifying obtains carries out single bacterium colony state description, mainly comprises size, color, transparency, wettability, bacterium colony condition of surface (whether smooth, projection, fold, depression etc.), the colony edge state (whether neat, irregular, radial etc.) of bacterium colony.
For the described derosal degradation bacteria DJL1614 that is in logarithmic phase, behind smear staining, adopt the form of observation by light microscope thalline.
The result shows, the derosal degradation bacteria DJL1614 elliptical shaped lobes that above-mentioned steps one is separated and purifying obtains, and faint yellow, translucent, smooth surface is moistening, neat in edge; The thalline rod-short, 0.3 * 2.5 μ m, Gram-negative.
2, analysis of physio biochemical characteristics
With reference to " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: Science Press, 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 derosal degradation bacteria DJL1614.
The physiological and biochemical property measurement result of described derosal degradation bacteria DJL1614 is as shown in table 1:
The physiological and biochemical property of table 1 derosal degradation bacteria DJL1614
Annotate: "+" expression is positive, and "-" expression is negative.
3,16s rDNA sequence homology analysis
Ordinary method is cultivated the derosal degradation bacteria DJL1614 that above-mentioned steps one separation and purification obtains, the total DNA that extracts bacterial strain is as the gene amplification template, with bacterium 16s rDNA universal primer, 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACCTTGTTACGACTT-3 ' carry out the PCR reaction.Reaction system adopts Shanghai biotechnology company limited pcr amplification test kit.Response procedures is: 95 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ of extensions 2min, totally 30 circulations.Dna sequencing is finished by Beijing three rich polygala root biotech companies, sequence assembly and similarity analysis use DNAStar software to finish, and gene is compared by online the finishing of American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov).
The sequence of derosal degradation bacteria DJL161416s rDNA sees sequence 1 in the sequence table for details.
4, growth characteristics analysis
Bacterial strain optimum temperuture and optimal pH growth experiment have been carried out.Adopt minimal medium, at the thermal adaptability of 4 ℃, 28 ℃, 37 ℃, 60 ℃ cultivations, observation, record bacterial strain, each is handled 3 times and repeats respectively.Adjust acidity and be respectively pH3, pH4, pH5, pH6, pH7, pH8, pH9, pH10, pH11, each is handled 3 times and repeats, the optimal pH of cultivation, observation, record strain growth.
The result shows that the optimum growth temperature of described derosal degradation bacteria DJL1614 is 25 ℃, and the suitableeest growth pH is pH7～8.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis result, the derosal degradation bacteria DJL1614 that the step 1 separation and purification is obtained is accredited as the yellow monospore Cordycepps oligotrophy zygosaccharomyces (Stenotrophomonas sp.) of modification bacterium γ subgroup.This derosal degradation bacteria DJL1614 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 17th, 2011 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.5151.
The quantitative assay of embodiment 2, oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 degraded derosal ability
One, derosal bioassay standard curve plotting
With carbendazim standard product (available from Sigma company) preparation series standard suspension, according to derosal these characteristics of maximum absorption band are arranged at the 281nm place, by determined by ultraviolet spectrophotometry OD281, and then draw derosal bioassay standard curve, the OD281 measurement result of different concns derosal is as shown in table 2:
The OD281 value of table 2 different concns derosal
According to table 1 data, obtaining derosal bioassay standard curvilinear equation is y=0.0769x+0.0064, R
2=0.999.Derosal bioassay standard curve as shown in Figure 1.
Two, oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 degraded derosal ability quantitative assay
Ability according to following scheme quantitative assay oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 degraded derosal:
In above-mentioned minimal medium, go into the derosal of 500mg/L, insert oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151, simultaneously only containing derosal, but the substratum that does not meet described oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 is as blank.In 25 ℃, the 200r/min shaking table is cultivated, and takes a sample respectively behind 3d, 5d, 8d, 10d, 15d and measures the derosal residual quantity according to the derosal bioassay standard curve of step 1 drafting.
The result shows that the derosal starting point concentration is 500mg/L, and described oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 reaches 73.41% (as shown in table 3) to the degradation rate of derosal during 10d.This result shows, oligotrophy Zymomonas mobilis provided by the present invention (Stenotrophomonas sp.) but DJL1614CGMCC No.5151 efficient degradation derosal.
Table 3 oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 degraded derosal effect (starting point concentration: 500mg/L)
Claims (7)
1. oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614, it is numbered CGMCC No.5151 in the Tibetan at China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. microbial inoculum, it is characterized in that: the activeconstituents of described microbial inoculum is the described oligotrophy Zymomonas mobilis of claim 1 (Stenotrophomonas sp.) DJL1614CGMCC No.5151.
3. microbial inoculum according to claim 2 is characterized in that: described microbial inoculum is following 1) or 2) described microbial inoculum:
1) be used to the to degrade microbial inoculum of derosal;
2) be used for the microbial inoculum that the rehabilitating soil derosal pollutes.
4. the described oligotrophy Zymomonas mobilis of claim 1 (Stenotrophomonas sp.) DJL1614CGMCC No.5151 or claim 2 or the 3 described microbial inoculums application in the degraded derosal.
5. the described oligotrophy Zymomonas mobilis of claim 1 (Stenotrophomonas sp.) DJL1614CGMCC No.5151 or claim 2 or the 3 described microbial inoculums application in the rehabilitating soil derosal pollutes.
6. cultivate the method for the described oligotrophy Zymomonas mobilis of claim 1 (Stenotrophomonas sp.) DJL1614CGMCC No.5151, comprise the step that described oligotrophy Zymomonas mobilis (Stenotrophomonas sp.) DJL1614CGMCC No.5151 is cultivated at the substratum that is used for cultivating the oligotrophy Zymomonas mobilis.
7. the preparation method of claim 2 or 3 described microbial inoculums comprises the steps: the described oligotrophy Zymomonas mobilis of claim 1 (Stenotrophomonas sp.) DJL1614CGMCC No.5151 is obtained described microbial inoculum as activeconstituents.
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| CN102296041B (en) * | 2011-08-26 | 2013-07-31 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for efficiently degrading residual pesticide carbendazim and application thereof |
| CN103305439B (en) * | 2013-05-27 | 2015-06-03 | 上海阿斯沃特企业发展有限公司 | Stenotrophomonassp. H-4 and preparation method of degrading enzyme preparation thereof as well as application |
| CN103333833B (en) * | 2013-07-01 | 2014-08-13 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for degrading herbicide chlorimuron-ethyl and application of bacterium |
| CN103343100B (en) * | 2013-07-01 | 2014-08-13 | 中国农业科学院农业资源与农业区划研究所 | Bacterium capable of degrading pesticides chlorimuron-ethyl and carbendazim and application thereof |
| CN103333835B (en) * | 2013-07-01 | 2015-03-11 | 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 |
| CN104862245B (en) * | 2015-04-16 | 2017-09-19 | 西北农林科技大学 | A carbendazim-degrading bacterium MBC‑6F and its application |
| CN106119114A (en) * | 2016-06-07 | 2016-11-16 | 南通市产品质量监督检验所 | The separating screening method of a kind of chlorpyrifos degrading bacteria and application thereof |
| CN110305814B (en) * | 2019-07-14 | 2020-08-28 | 甘肃省科学院生物研究所 | Stenotrophomonas and application thereof in codonopsis pilosula |
| CN115739973B (en) * | 2022-11-29 | 2024-04-12 | 湖南农业大学 | Method for treating carbendazim-polluted soil by combining fungus dreg with nitrification inhibitor |
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