CN102071163B - Multifunctional engineering strain for degrading chlorpyrifos and construction method thereof - Google Patents
Multifunctional engineering strain for degrading chlorpyrifos and construction method thereof Download PDFInfo
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Abstract
The invention discloses a multifunctional engineering strain for degrading chlorpyrifos and a construction method thereof, and relates to a burkholderia vietnamiensis multifunctional engineering strain P418-M. The collection number of the strain is CGMCC No. 4166. The construction method for the burkholderia vietnamiensis multifunctional engineering strain comprises the following steps that: organic phosphorus degradation enzyme genes from acinetobacter are integrated onto chromosomes of burkholderia vietnamiensis by suicide plasmid through Tn5 transposition by using the burkholderia vietnamiensis, so the function of degrading pesticide residues is increased on the basis of the capacity of removing phosphorus and potassium, fixing nitrogen and promoting plant growth and rhizosphere colonization of a multifunctional microorganism to realize multiple functions of the burkholderia vietnamiensis.
Description
Technical field
The invention belongs to chemical pesticide biodegradation technique field, relate to a strain organophosphorus pesticide Chlorpyrifos 94 tool efficient degradation ability, the multifunctional engineering strain and the construction process thereof that have fixed nitrogen, phosphorus decomposing, potassium decomposing and biological and ecological methods to prevent plant disease, pests, and erosion function simultaneously.
Background technology
Chlorpyrifos 94 (chlorpyrifos) is claimed Le Siben, chlorine pyrazothion etc. again, and chemical name is O; O-diethylammonium-O-(3; 5, the 6-trichloro-2-pyridyl) thiophosphatephosphorothioate, nineteen sixty-five is developed by Dow Chemical company; Tool is efficient, wide spectrum, characteristic such as efficient, is widely used in the control of field crop, fruits and vegetables, flowers and house pest.
China is after completely forbidding high malicious organophosphorus pesticide on January 1st, 2007, and Chlorpyrifos 94 is as low toxicity, the rapid dominate of organophosphorus pesticide substitute efficiently.The frequent a large amount of uses of Chlorpyrifos 94 cause not only that agricultural-food are residual to exceed standard, and have a strong impact on foreign exchange earning and development and national economy; And water body and ecological environment of soil caused serious pollution, especially, not only influence soil fertility to the pollution of soil, and to influences such as soil microorganisms population, microbial diversity, vinelandii activity, soil enzyme activitiess greatly.Therefore, the governing problem of Chlorpyrifos 94 contaminated soil more and more receives people's attention.
In the research work in the past, the various countries scholar has been separated to the chlorpyrifos degrading bacteria of multiple different sources.But the screening of these degradation bacteria strains just obtains from aspect of chlorpyrifos degrading, and is less to the control consideration of soil disease and pest, scarcely has growth-promoting functions, and the peasant is difficult for accepting.Simultaneously, compare with two kinds of original strains while fermentative prodn, the reorganization multifunctional engineering strain can reduce cost, reduce pollution greatly in the production technique link; And, the reorganization multifunctional engineering strain when preparation uses, the restraining effect each other that can avoid 2 kinds or multiple microbial inoculum to use simultaneously being produced.Therefore utilize genetic engineering means to make up multifunctional engineering strain, exploitation had both had degrading pesticide residues, possessed controlling plant diseases and the microbial inoculum that promotes crop growth simultaneously again, was used for the reparation of pesticide contaminated soil, had higher society and ecological benefits.
Vietnam's bulkholderia cepasea (Burkholderia vietnamiensis) P418 separates in the barley rhizosphere soil in Byelorussia Minsk city; This bacterial strain has the rhizosphere colonization ability after deliberation; Phosphorus decomposing, potassium decomposing, fixed nitrogen and promotion plant-growth effect; Be the multi-functional biocontrol strain of a strain, plant root-knot nematode is also had certain control effect.Import through gene, make this biocontrol microorganisms possess the function of degrading organic phosphor pesticides Chlorpyrifos 94 simultaneously, for the preparation research and the exploitation of engineering bacillus strain are laid a good foundation.
Summary of the invention
The objective of the invention is practical problems and demand, Vietnam's bulkholderia cepasea multifunctional engineering strain (Burkholderia vietnamiensis) P418-M and the construction process thereof that provide a strain Chlorpyrifos 94 contaminated soil to repair to China's pesticidal contamination.
Vietnam's bulkholderia cepasea multifunctional engineering strain (Burkholderiavietnamiensis) P418-M of chlorpyrifos degradation provided by the present invention; Submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on September 13rd, 2010; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Biological study institute of the Chinese Academy of Sciences, deposit number: CGMCC No.4166.
The original strain of utilization of the present invention is strain Vietnam bulkholderia cepasea P418, has phosphorus decomposing, potassium decomposing, fixed nitrogen, anti-soil-borne disease and rhizosphere colonization ability.The present invention utilizes genetic engineering means; Through the Tn5 swivel base; To be incorporated on the karyomit(e) of P418 through suicide plasmid from the organic phosphorus degrading enzyme gene of acinetobacter calcoaceticus; Make it on basis, increase the function of degrading pesticide residues, realize the multi-functional of Burkholderia with phosphorus decomposing, potassium decomposing, fixed nitrogen, diseases prevention and rhizosphere colonization ability.
The construction process that falls the Chlorpyrifos 94 multifunctional engineering strain of the present invention may further comprise the steps:
1) structure of recombinant plasmid pUT-M
According to organic phosphorus degrading enzyme gene order design primer mf and mr (introducing Not I restriction enzyme site and SD sequence); Utilize round pcr amplification organic phosphorus degrading enzyme gene; Not I enzyme respectively cuts organic phosphorus degrading enzyme gene and plasmid pUT-Km1; Gene after again enzyme being cut is connected with plasmid pUT-Km1, changes intestinal bacteria S17-1 (λ-pir), obtain recombinant plasmid pUT-M over to;
2) transform Vietnam Burkholderia P418
The intestinal bacteria S17-1 that contains recombinant plasmid mixes to transform with Vietnam Burkholderia P418, through the resistant panel screening, obtain 68 single strains;
3) screening
The degradation capability that reaches Chlorpyrifos 94 through pcr amplification, electrophoresis, Southern hybridization detects; To step 2) in 68 single strains screen; Finally obtain 1 to the highest single strain of chlorpyrifos degrading activity, be Vietnam's bulkholderia cepasea multifunctional engineering (Burkholderia vietnamiensis) P418-M that falls Chlorpyrifos 94.
The above-mentioned screening of falling the Chlorpyrifos 94 multifunctional engineering mainly adopts following three steps to carry out:
(1) pcr amplification and electrophoresis
Utilize mf and mr through pcr amplification and electrophoretic technique to step 2) in 68 single strains screen, have 26 single strains to amplify the purpose band;
(2) Southern hybridization
26 single strains that above-mentioned steps (1) is obtained under equal conditions carry out Southern hybridization respectively, and used probe is an organic phosphorus degrading enzyme full length gene sequence.Through detecting the organic phosphorus degrading enzyme gene that 20 single strains are arranged is that single copy is inserted in the P418 karyomit(e), and it is subsequent use to keep these 20 single strains.
(3) mutant strain is to the detection of organic phosphorus pesticide degradation ability
Through the degradation capability detection 20 bacterial strains in the above-mentioned steps (2) are further screened.Be inoculated under the same terms on the minimal medium flat board that contains organophosphorus pesticide, through the transparent circle screening, referring to Fig. 4, wherein 7 bacterial strains produce transparent circle; Again these 7 bacterial strains are received respectively in the liquid minimal medium that contains organophosphorus pesticide; Cultivated 48 hours; Utilize HPLC to detect the degradation rate of each bacterial strain to organophosphorus pesticide; Obtain the highest bacterial strain of 1 strain degradation rate, be title product of the present invention-fall Vietnam's bulkholderia cepasea multifunctional engineering strain (Burkholderiavietnamiensis) P418-M of Chlorpyrifos 94.
Above-mentioned steps 1) primer mf and mr sequence are following:
mf：5’-GATCGCGGCCGCAGGAGGTTGATATGCCCCTGAAGAAC-3’
mr：5’-GATAGCGGCCGCCTTGGGGTTGACGACCG-3’
The biological characteristic research of Chlorpyrifos 94 multifunctional engineering strain falls in the present invention:
(1) growth curve of multifunctional engineering P418-M and original strain P418 research is referring to Fig. 5
(2) the multifunctional engineering P418-M that obtains is carried out the detection of phosphorus decomposing, potassium decomposing and nitrogen fixing capacity, referring to table 2.
(3) Vietnam's Burkholderia P418 and fall Chlorpyrifos 94 multifunctional engineering P418-M and detect at the colonization ability of wheat root is referring to Fig. 6.
(4) Vietnam's Burkholderia P418 and fall Chlorpyrifos 94 multifunctional engineering P418-M the inhibition ability of soil-borne pathogen is detected is referring to table 3.
(5) multifunctional engineering P418-M and original organic phosphorus degrading bacterial strain N16 are carried out the detection of the dead tick degradation capability of soil poisoning, referring to table 4
The application of Chlorpyrifos 94 multifunctional engineering P418-M falls in the present invention:
The biological degradation that Chlorpyrifos 94 multifunctional engineering strain P418-M can be used for soil organic phosphorus agricultural chemicals chlopyrifos residue falls in the present invention; The control and short the giving birth to that also can be used for the frequently seen plants disease simultaneously, like miliary damping-off germ (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), cereal rhizoctonia (Rhizoctonia cerealis), plant root-knot nematode etc.Therefore, this multifunctional engineering strain can be used as the preparation that main effective constituent is used for microbial fertilizer.
Advantage of the present invention is: bring problems such as serious environmental pollution to present chemical pesticide especially organophosphorus pesticide; The using gene engineering means through the Tn5 swivel base, are incorporated into the organic phosphorus degrading enzyme coding gene on Vietnam Burkholderia P418 karyomit(e); Make multifunctional microbial have diseases prevention, phosphorus decomposing, potassium decomposing, fixed nitrogen; Promoting increases it and falls agricultural residual function on the basis of plant-growth and rhizosphere colonization ability, realizes falling that farming is residual, diseases prevention and short multi-functional such as living.In dull and stereotyped antagonistic experiment, engineering strain is compared with original strain Vietnam Burkholderia P418, and its bacteriostasis is not affected; Degradation capability detects, and 5 days degradation rates to the lab simulation contaminated soil of the engineering bacteria of structure reach 82%.The growth curve of engineering bacteria P418-M and original bacterium P418 is close, utilize the substratum of original strain that engineering bacteria is fermented after, be that carrier is processed pulvis with the turfy soil, said preparation reached about 70% the degradation rate of cell simulations contaminated soil in 7 days.
The present invention will be incorporated on the chromosomal DNA of Vietnam Burkholderia P418 through suicide plasmid from the organic phosphorus degrading enzyme gene of acinetobacter calcoaceticus, and what obtained stability and high efficiency falls Chlorpyrifos 94 multifunctional engineering P418-M.The pulvis of processing after the substratum fermentation with original strain has good repairing effect to the Chlorpyrifos 94 contaminated soil.Description of drawings
Fig. 1 is the restriction enzyme mapping of recombinant plasmid pUT-M, and wherein swimming lane 1 is the standard DNA molecular weight Marker of 1kb, swimming lane 23 positive plasmid enzyme restriction results.
Fig. 2 is the PCR electrophoretogram of engineering strain, and wherein swimming lane 1 is the standard DNA molecular weight Marker of 1kb, and other is the PCR result of part engineering strain.
Fig. 3 is a Southern hybridization photo, and as dna probe, 26 single bacterium colonies that the step of (1) in the step 3) is obtained carry out Southern hybridization with organic phosphorus degrading enzyme gene order total length.Through detecting the organic phosphorus degrading enzyme gene that 20 single strains are arranged in these 26 single strains is that single copy is inserted in the karyomit(e) of Vietnam Burkholderia P418, and Fig. 3 is the Southern hybridization photo of part single strain.
Fig. 4 is the dull and stereotyped photo of Chlorpyrifos 94; 20 single strains that in the step 3) (2) step is obtained are inoculated on the inorganic salt flat board that contains organophosphorus; Through transparent circle screening bacterial strain, 7 single strains can normal growths and produce transparent circle among the figure, have the organic phosphorus degrading enzymic activity.
Fig. 5 is engineering strain P418-M and original strain P418 growth curve.
Fig. 6 be fall Chlorpyrifos 94 multifunctional engineering strain P418-M and original strain P418 the wheat tip of a root decide to grow quantity, wherein, A figure is that multifunctional engineering strain P418-M and original strain P418 decide to grow quantitative relation figure at the wheat tip of a root in the sterilization soil; B figure is that the two decides to grow quantitative relation figure at the wheat tip of a root in the nature soil.
Fig. 7 is the degradation capability of engineering bacteria preparation to land for growing field crops simulating pollution soil, and wherein, left figure death by poisoning tick is pressed recommended dose (0.5kg.a.i.ha
-1), right figure death by poisoning tick is doubling dose (1kg.a.i.ha
-1).Embodiment
Below in conjunction with specific embodiment the present invention is described further.
Embodiment 1: the structure that falls the Chlorpyrifos 94 multifunctional engineering strain
1) structure of recombinant plasmid pUT-M
According to organic phosphorus degrading enzyme gene design primer:
mf：5’GATCGCGGCCGCAGGAGGTTGATATGCCCCTGAAGAAC-3’
mr：5’-GATAGCGGCCGCCTTGGGGTTGACGACCG-3’
Introducing Not I restriction enzyme site and SD sequence at these gene two ends, is template with pGM-T-M, carries out pcr amplification.Reaction conditions is: 94 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 30min, 55 ℃ of annealing 50min, 72 ℃ are extended 1min, totally 30 circulations, last 72 ℃ are extended 10min.
Utilize Not I single endonuclease digestion organic phosphorus degrading enzyme gene and plasmid pUT-Km1, connect, (λ-pir), transformant utilizes above-mentioned primer to carry out PCR and detects, and reaction conditions is the same to be transformed into E.coli S17-1.
Detect the positive colony that obtains through PCR, extract plasmid, utilize Not I further to carry out enzyme and cut evaluation, referring to Fig. 1, positive colony is recombinant plasmid pUT-M
2) transform
Recombinant plasmid pUT-M is transformed into E.coli S17-1, and (in the competent cell of λ-pir), as the donor bacterium, P418 does recipient bacterium with bacterial strain with it, and the two mixing makes the organic phosphorus degrading enzyme gene integration to original bacterium P418 karyomit(e).The donor bacteria suspension, the recipient bacterium suspension that are about to incubated overnight mix in the 1.5ml centrifuge tube with 1: 1 (volume ratio), collect thalline, and aseptic water washing once; Collect once more in the sterilized water that thalline is suspended in 200 μ l, bacterium liquid point be connected on the P418 bacterium flat board, 28 ℃ cultivate 6-8h after; Collect thalline with sterilized water; Be applied to after the dilution on the P418 flat board that contains Kan, cultivate 48h, obtain 68 single strains for 28 ℃.The single bacterium colony smear for microscopic examination of picking part, the bacterial strain thalline after the conversion is very little, is little rod-short, and is consistent with the original strain form.
3) screening of Chlorpyrifos 94 multifunctional engineering P418M is fallen
The main screening of adopting following three steps to fall agricultural residual multifunctional engineering strain:
(1) pcr amplification and electrophoretic technique
Utilize mf and mr through pcr amplification and electrophoretic technique to step 2) in 68 single strains carry out the muton screening, the PCR reaction conditions is referring to the PCR reaction conditions among the embodiment 1.Obtain 26 single strains through pcr amplification and electrophoretic technique detection, the result sees Fig. 2.
(2) Southern hybridization
Through Southern hybridization 26 single bacterial classifications in (1) are verified that method is with reference to molecular cloning test guide (third edition).As probe, indicate dna probe with organic phosphorus degrading enzyme gene order total length with DIG HIGH PRIME LABELING MIX (Roche).Through detecting among the embodiment 1 3) in the organic phosphorus degrading enzyme gene of 20 single strains to be arranged in 26 single strains obtaining of (1) step be that single copy is inserted in the karyomit(e) of P418, the result sees Fig. 3.
(3) the organic phosphorus degrading ability detects
20 single strains that (2) Southern in step 3) hybridization back is obtained are inoculated on the inorganic salt flat board that contains organophosphorus (Chlorpyrifos 94) simultaneously; Cultivate after 3-5 days for 28 ℃; Screen 7 single strains through the transparent circle detection and have the organic phosphorus degrading enzymic activity, referring to Fig. 4.These 7 single strains are inoculated in the inorganic salt liquid substratum (contain Chlorpyrifos 94 100mgL
-1), shaking table utilizes performance liquid chromatography (HPLC) to detect the degradation capability of each bacterial strain to organophosphorus (Chlorpyrifos 94) after cultivating 48h.
Can find out that by table 1 to be numbered 43 engineering strain degrading activity higher, be 70%, and it is confirmed as purpose product-fall Chlorpyrifos 94 multifunctional engineering strain (Burkholderia vietnamiensis) P418-M.
Table 1: the degrading activity of 7 single strains detects after the dull and stereotyped detection of inorganic salt
Embodiment 2: the biological characteristics that falls Chlorpyrifos 94 multifunctional engineering strain P418-M
(1) growth curve of multifunctional engineering strain P418-M and original strain P418 is measured
The speed of growth of 2 bacterial strains is determined in the P418 liquid nutrient medium and detects.The result shows (Fig. 5), has inserted foreign gene on the engineering strain karyomit(e), but its growth curve compares with original strain P418, and evident difference is not arranged, and shows that the importing of foreign gene has just increased the function of original strain, to not influence of its growth.The culture medium condition of this explanation original strain can be used for the cultivation of engineering strain fully.
Phosphorus decomposing, potassium decomposing, the nitrogen fixing capacity of (2) falling Chlorpyrifos 94 multifunctional engineering strain P418-M and original strain P418 compare
Utilize following method multifunctional engineering strain P418-M and original strain P418 to be carried out the mensuration of relevant fixed nitrogen, phosphorus decomposing, ability of dissolving potassium respectively.
Nitrogenase activity is measured (full nitrogen colourimetry)
In the 250mL triangular flask, add 50mL Ah Xu Bei Shi (Ashby) liquid nutrient medium, 121 ℃ of sterilization 30min.The bacteria suspension of 2mL inclined-plane flushing is gone in aseptic technique, every bottle graft, does the blank that does not connect bacterium simultaneously, all after 30 ℃, 160r/min are cultivated 5d, takes out and surveys nitrogen.
Draw vinelandii liquid 1.0mL in the 30mL alimentary canal, add 3mL sulfuric acid, add 0.1g catalyzer and 5 ydrogen peroxide 50 and disappear and boil to limpid, unclear if reaction solution boils for a long time, add 1-2 after can cooling off and drip ydrogen peroxide 50, continue to boil to water white transparency, take off cooling.Add a little zero(ppm) water, shake up, dropping sodium (400g/L) is to verditer deposition (about 11-12mL) occurring; Add 20 Seignette salts; Shelter calcium magnesium to remove precipitation of hydroxide, test tube liquid all is transferred in the 100mL volumetric flask, the alimentary canal washings is poured volumetric flask in the lump into; Be diluted to scale, shake up.Filter, filtrating 10.00mL adds 1.0mL sodium hydroxide in tube comparison tubes, the 1.0mL Seignette salt, and the 3.0mL Nessler's reagent shakes up, and behind the colour developing 2-3min, promptly pours in the cuvette, and tintometer is measured in the 420nm place.
The phosphorus decomposing determination of activity
The bacterial strain to be measured that activation is good is cultivated 48h for 28 ℃, and cell concentration reaches 10
8Subsequent use behind the cfu/ml.If connect bacterium, connect inactivated bacteria, (contrast) 3 processing that connect substratum, each handles 3 repetitions.Respectively above-mentioned seed liquor is seeded in the 50ml fermention medium by 5% inoculum size, cultivates 10d for 28 ℃.Add 6%H behind 121 ℃ of deactivation 40min
2 O
22 in triangular flask, 60 ℃ of water-bath 48h so that microbial biomass phosphorus discharge.To clarification, constant volume is surveyed the filtrating available phosphorus content with molybdenum antimony resistance colorimetric method to fermented liquid with without phosphorus filter paper filtering.
The potassium decomposing determination of activity
Add potassium felspar sand 0.1g and scarce K substratum 50ml in every 250ml triangular flask, at 121 ℃ of sterilization 20min, insert bacteria suspension 3ml then, contrast connects the equivalent inactivated bacterial liquid and does not add bacterium liquid, and 28 ℃ of shaking culture 5 days are got the rare H of fermented liquid with 60g/L
2O
2Disappear boil, filtration, constant volume, sodium tetraphenylborate turbidimetry survey K.
The phosphorus decomposing of table 2 multi-functional bacterial strain and original strain, potassium decomposing determination of activity
The result is as shown in table 2, and the reorganization multifunctional engineering is compared with original strain P418, and its phosphorus decomposing, potassium decomposing and nitrogenase activity do not have notable difference, explain that the importing of organic phosphorus degrading enzyme gene does not have influence on original phosphorus decomposing of engineering strain, potassium decomposing and fixed nitrogen function.
(3) Burkholderia P418 with fall Chlorpyrifos 94 multifunctional engineering strain P418-M wheat root decide grow
Wheat seed with 70% ethanol disinfection after 28～30 ℃ of vernalization, seed show money or valuables one carries unintentionally the back with 2 * 10
8Vietnam Burkholderia P418 of cfu/ml Rifampin mark and the seed soaking of the Burkholderia multifunctional engineering bacterium liquid of Rifampin and kantlex mark are sowed respectively in sterilization soil and natural soil.Get the tip of a root (the above 2cm of the tip of a root) after 21 days, add aqua sterilisa, gradient dilution behind the mixing.Coated plate statistics colony count.
The result is as shown in Figure 6, no matter in sterilization soil, still be that multifunctional engineering strain is compared with original strain P418 in natural soil, colonization ability is suitable.Explain that the insertion of foreign gene does not have influence on the colonization ability of original bacterial strain.
(4) bacteriostatic activity of Burkholderia P418 and multifunctional engineering strain P418-M is measured
Adopt dull and stereotyped face-off method; Respectively dry thread Pyrenomycetes (Rhizoctoniasolani), cereal rhizoctonia (Rhizoctonia cerealis), Fusarium oxysporum (Fusarium oxysporum) pathogenic fungi that flat board is cultivated broken into the bacterium sheet that diameter is 5mm with aseptic punch tool, insert the dull and stereotyped central authorities of PDA; Point meets Burkholderia P418 and falls Chlorpyrifos 94 multifunctional engineering strain P418-M on the circumference of pathogenic bacteria lawn 2cm, 28 ℃ of width of cultivating the antibacterial band of record after 5～7 days.
From table 3, can find out; Engineering strain and original strain the P418 inhibiting rate to cereal rhizoctonia (Rhizoctoniacerealis) are the highest; Engineering strain is compared with original strain P418; Antagonistic ability to pathogenic bacteria does not have notable difference, explains after original strain P418 inserts foreign gene and does not lose bacteriostasis.
Table 3 multifunctional engineering strain and original strain P418 compare the antagonistic ability of pathogenic bacteria
Annotate: CK is the pathogenic bacteria expansion radius at the fungi-proofing place of not delivering a child; Data are three multiple MVs in the table.
(5) the existing degradation capability detection of falling Chlorpyrifos 94 bacterial strain N16 to lab simulation Chlorpyrifos 94 contaminated soil in Chlorpyrifos 94 multifunctional engineering strain P418-M and laboratory is fallen.
Get field soil (pH 6.7), cross 20 mesh sieves behind the natural air drying, sterilization claims that 50g in the 250ml triangular flask, adds the Chlorpyrifos 94 ethanolic soln, and mixing and making final concentration is 50mgkg
-1Dry ground, absorption 24h after as mimic Chlorpyrifos 94 contaminated soil.Test is established and not being connect the bacterium control group and connecing the bacterium treatment group, and treatment group adds multifunctional engineering strain P418-M and the existing bacterium liquid that falls Chlorpyrifos 94 bacterial strain N16 respectively, makes that bacteria concentration is 10 in the soil
8CFUg
-1Dry ground, it is 50% of soil water retaining capacity that soil water content is respectively organized in adjustment, cultivate the dark place.Inoculate sampling after 5 days, measure the content of the dead tick of soil poisoning.
Can be found out that by table 4 engineering strain P418-M is also higher slightly than N16 to the degradation rate of organophosphorus in the soil (Chlorpyrifos 94), possibly be that the colonization ability of engineering strain in soil is better than N16, can better play a role.
Table 4 multifunctional engineering strain and existing degradation bacteria strains N16 are to the comparison of Chlorpyrifos 94 polluted soil degrading ability
Annotate: each data is three multiple MVs in the table
Degradation rate (%)=(contrast death by poisoning tick content-sample death by poisoning tick content)/contrast Chlorpyrifos 94 content * 100%
(6) the multifunctional engineering strain formulation preparation reaches the degradation capability to land for growing field crops simulating pollution soil
Behind the culture media shaking vase fermentation 36h with original strain; Fermenation raw liquid (bacteria containing amount at every gram more than 9,000,000,000) is regulated the pH value to 7.0-7.3; Add in the aseptic turfy soil in 10% ratio; Add XG 550 (1.0%), SODLUM FULVATE (0.1%), SDS (2.5%) simultaneously, fully mixing packs.
The detection of cell simulations polluted soil degrading ability is carried out in unvegetated vegetable plot, and following 5 processing are established in test: 1) contrast; 2) Chlorpyrifos 94 recommended dose; 3) Chlorpyrifos 94 recommended dose+degradation bacterial agent; 4) Chlorpyrifos 94 doubling dose; 5) Chlorpyrifos 94 doubling dose+degradation bacterial agent.3 sub-districts are established in each processing, and each sub-district area is 5m
2Chlorpyrifos 94 is pressed recommended dose (0.5kg.a.i.ha respectively
-1), doubling dose (1kg.a.i.ha
-1) convert water and spray.
Behind the 24h, (effectively the bacterium number is 30 * 10 with the degradation bacteria preparation after the above-mentioned processing
8Cfug
-1) press 15Kgha
-1Dosage convert water and spray, the aqua sterilisa of the same dosage of sprinkling in the sub-district of contrast and Chlorpyrifos 94 individual curing simultaneously.0d behind the sprinkling degradation bacterial agent, 1d, 7d, 14d, the 21d soil sampling is got 10 grams excessively and is detected the chlopyrifos residue amounts behind 20 mesh sieves.
Can find out that by Fig. 7 multi-functional degradation bacterial agent is higher than the degradation rate that doubling dose is handled to the degradation rate of Chlorpyrifos 94 recommended dose, but the absolute clearance that doubling dose is handled is high.Compare in indoor; Though degradation bacterial agent is slow to the degradation rate of the dead tick of soil poisoning under the field condition; But do not compare with inoculating soil, the adding of the degradation bacterial agent chlopyrifos residue in the soil of fast and effeciently degrading, this has great importance concerning agricultural chemicals soil is pushed up the reparation of pollution.
Claims (3)
1. Vietnam's bulkholderia cepasea multifunctional engineering strain (Burkholderia vietnamiensis) P418-M of a strain chlorpyrifos degradation; China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, deposit number: CGMCC No.4166 have been submitted on 09 13rd, 2010.
2. the purposes of the multifunctional engineering strain of chlorpyrifos degradation as claimed in claim 1; It is characterized in that to be used for the repairing and treating of Chlorpyrifos 94 contaminated soil; Can prevent and treat miliary damping-off germ (Rhizoctonia solani) again; Fusarium oxysporum (Fusarium oxysporum), cereal rhizoctonia (Rhizoctonia cerealis) also has phosphorus decomposing, potassium decomposing, fixed nitrogen growth-promoting functions.
3. the purposes of the multifunctional engineering strain of chlorpyrifos degradation as claimed in claim 2 is characterized in that being used for the preparation of microbial fertilizer.
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| CN101144066A (en) * | 2007-08-21 | 2008-03-19 | 山东省科学院中日友好生物技术研究中心 | Burkholderia multifunctional engineering strain and construction method thereof |
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| KR101122276B1 (en) * | 2009-05-14 | 2012-03-16 | 부산대학교 산학협력단 | Microorganism capable of solubilizing insoluble phosphoric acid and growing in high salinity and broad pH range of soil and method for increasing ginseng saponin using the microorganism |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101144066A (en) * | 2007-08-21 | 2008-03-19 | 山东省科学院中日友好生物技术研究中心 | Burkholderia multifunctional engineering strain and construction method thereof |
Non-Patent Citations (3)
| Title |
|---|
| Amit,Kumar et al..Bacterial dynamics of biofilm development during toluene degradation by burkholderia vietnamiensis G4 in a gas phase membrane bioreactor.《Journal of microbiology and biotechnology》.2009,第19卷(第9期),1028-1033. * |
| 陈凯等.越南伯克霍尔德氏工程菌株B418 - 37的温室防病增产效果.《山东农业科学》.2008,(第7期),58-60. |
| 陈凯等.越南伯克霍尔德氏工程菌株B418- 37的温室防病增产效果.《山东农业科学》.2008,(第7期),58-60. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988189A (en) * | 2017-11-29 | 2018-05-04 | 华南农业大学 | Applications of the enzyme FadD1 in degraded DSF families signaling molecule a kind of in Pseudomonas aeruginosa |
| CN107988189B (en) * | 2017-11-29 | 2020-08-07 | 华南农业大学 | Application of enzyme FadD1 in pseudomonas aeruginosa in degradation of DSF family signal molecules |
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