CN107649504A - A kind of method of resistance gene contamination in repairing and treating soil - Google Patents
A kind of method of resistance gene contamination in repairing and treating soil Download PDFInfo
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- CN107649504A CN107649504A CN201710983680.7A CN201710983680A CN107649504A CN 107649504 A CN107649504 A CN 107649504A CN 201710983680 A CN201710983680 A CN 201710983680A CN 107649504 A CN107649504 A CN 107649504A
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- soil
- azolla imbricata
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- antibiotic
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- 241000351595 Azolla imbricata Species 0.000 claims abstract description 36
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- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- 238000011068 loading method Methods 0.000 description 1
- ZEKZLJVOYLTDKK-UHFFFAOYSA-N lomefloxacin Chemical compound FC1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNC(C)C1 ZEKZLJVOYLTDKK-UHFFFAOYSA-N 0.000 description 1
- 229960002422 lomefloxacin Drugs 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 238000011880 melting curve analysis Methods 0.000 description 1
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- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 1
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- 238000003908 quality control method Methods 0.000 description 1
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- 238000004445 quantitative analysis Methods 0.000 description 1
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 238000003900 soil pollution Methods 0.000 description 1
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- 238000007619 statistical method Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 229940063650 terramycin Drugs 0.000 description 1
- 101150004433 tetQ gene Proteins 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 101150097091 tnpA gene Proteins 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Soil Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Botany (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of method of resistance gene contamination in repairing and treating soil, pass through Azolla imbricata phytoremediation chronic administration livestock and poultry muck and the soil of sewage irrigation, diversity of soil microorganism is added, promotes the degraded of antibiotic in soil, reduces soil available content of beary metal.Under the conditions of optimal dose Azolla imbricata phytoremediation, the content of antibiotic and available heavy metal declines 28.1% 44.8% and 46.8% 95.9% respectively in soil, and the quantity of resistant gene reduces 27.1%.Azolla imbricata phytoremediation technology can effectively control total resistant gene in soil, the relative abundance of transposase gene.Azolla imbricata phytoremediation technology is a kind of green technology method that can effectively administer heavy metal in soil, antibiotic and resistant gene combined pollution.The advantages that this method is simple to operate, easy to implement, cost is low, green, has broad application prospects.
Description
Technical field
Present invention relates particularly to technical field of soil remediation, and in particular to resistance gene contamination in a kind of repairing and treating soil
Method.
Background technology
Abuse of antibiotics and induce drug resistance pathogenic bacteria and caused what people were spread to antibiotic and resistant gene (ARGs)
Concern.ARGs persistence residual, and environmental hazard of the propagation than antibiotic in itself in varying environment medium are bigger.Contain
Antibiotic irrigation water can cause soil pollution, be the important sources of soil antibiotic as applying the muck containing antibiotic.Poultry
In poultry manure containing high-caliber antibiotic, heavy metal (selection index system is cooperateed between resistant gene), resistant microorganism and
Resistant gene, the influence during its recycling to environment are interesting issue at present.
Soil is the main place of feces of livestock and poultry consumption, is the main source of human pathogen's bacterium resistant gene.Using pig manure
It is that an important factor for influenceing resistance gene abundance in soil, resistance level of the edaphon to antibiotic can be dramatically increased.Heap
Fertile fermentation energy reduces the concentration of residual antibiotic in fresh livestock and poultry muck, and reduces its resistant gene content, has at present
The technology mode for reaching control resistant gene by regulating and controlling compost and propagating.A large amount of administrations of livestock and poultry muck, it is most likely that can cause
The resistant gene pollution of soil.Phytoremediation technology is a kind of inexpensive high effective green environmentally friendly technology, and how to utilize the skill
Art pollutes to administer the resistant gene of soil, not yet solves so far.Azolla imbricata is Filicineae Azolla imbricata section floating plant, in China
It is widely distributed, it is annual herb, the four seasons are in great numbers, and tillering ability is strong, and plant is short and small, network of roots life, and the accumulation of plant nitrogen is obvious, organic
Matter content is high, and purifying water effect is good.
The content of the invention
The defects of in order to overcome above-mentioned prior art to exist, it is dirty that the present invention provides resistant gene in a kind of repairing and treating soil
The method of dye, the degraded of antibiotic in soil is promoted, reduce soil available content of beary metal and resistant gene
Quantity is reduced.
The technical solution that the present invention uses is：The method of resistance gene contamination, described in a kind of repairing and treating soil
Restorative procedure be that, by filling fresh Azolla imbricata plant around tree root, the amount of described Azolla imbricata plant landfill is every
5-15kg Azolla imbricata plants are filled around tree root.
The depth of the described fresh Azolla imbricata plant of landfill is 5-10cm.
The described mode that fresh Azolla imbricata plant is filled around tree root is：Uniformly placed within one week in tree root full
The red plant in river is simultaneously buried.
Region of the described tree root peripheral region for the area centered on tree for 1.4m × 1.4m.
Described trees are Ou Ganshu.
The beneficial effects of the invention are as follows：The invention provides a kind of method of resistance gene contamination in repairing and treating soil,
By Azolla imbricata phytoremediation chronic administration livestock and poultry muck and the soil of sewage irrigation, diversity of soil microorganism is added,
The degraded of antibiotic in soil is promoted, reduces soil available content of beary metal.Repaiied in optimal dose Azolla imbricata plant
Under the conditions of multiple, the content of antibiotic and available heavy metal declines 28.1%-44.8% and 46.8%- respectively in soil
95.9%, and the quantity of resistant gene reduces 27.1%.Azolla imbricata phytoremediation technology can effectively control total resistance in soil
The relative abundance of gene, transposase gene.Azolla imbricata phytoremediation technology is that one kind can effectively administer heavy metal in soil, resist
Raw element and the green technology method of resistant gene combined pollution, this method is simple to operate, easy to implement, cost is low, green
The advantages that, have broad application prospects.
Embodiment
With reference to embodiment, the present invention will be described in detail, and embodiment is only the preferred embodiment of the present invention,
It is not limitation of the invention.
Method
Sample region overview
Sample is in three YangOu Gan gardens of Wenzhou City Ouhai District.Bowl mandarin orange is local characteristic fruit, three YangOu Gan gardens areas
About 1000 mu, a large amount of Xiaohegous are run through in mandarin orange garden, pollution of area source discharge in mandarin orange garden causes river seriously to pollute.Planting household directly utilizes
River recharge Gan Yuan, and chronic administration livestock and poultry muck.Along stream seining cultivation Azolla imbricata plant in mandarin orange garden, plant-micro- is utilized
Bio combined processing river, and salvage Azolla imbricata and be used to repair mandarin orange Orchard Soil.
Sampled point is set and sample collection
In April, 2016,18 Ou Ganshu of the same size are selected in three YangOu Gan gardens.With bowl mandarin orange under every bowl mandarin orange tree crown
Equal area (1.4m × 1.4m) sample is set centered on tree.In sample ground 10cm depths uniformly fill different fresh weights (5kg,
7.5kg, 10kg, 12.5kg, 15kg) Azolla imbricata plant, each amount of landfill repeats with setting 3 pieces of samples, totally 18 pieces, wherein 3
The individual sample for not filling Azolla imbricata, as control.After 45d, 15cm depths is taken by serpentine circuit five-spot in each sample ground
Soil, mixing are fitted into sterile sampling bag.According to plant amount of landfill order from low to high, by soil sample be designated as AS1, AS2, AS3,
AS4, AS5, control sample are designated as CS.The soil of collection removes plant roots and visible organic matter residuum.Each sampling is small by 5
Sample forms, and scene uniformly mixing.Sample loading ice chest is taken back into lab analysis.The fresh samples of 200g carry out physics and chemistry after air-drying sieving
Indexs measure；Progress vacuum freeze drying is stored in -80 DEG C after another 200g samples cross 2mm sieves, in case total genomic dna extracts,
For microorganism and Resistance gene analysis；Remaining sample is stored in -80 DEG C in case heavy metal and antibiotic detection.
Determination of physical and chemical properties
Physiochemical properties of soil determines according to soil agrochemistry conventional method.
Sample antibiotic determines
Soil sample crushed the standby survey of 60 mesh sieves in room-dry.Sample pretreating method is with reference to pertinent literature and is improved：
Weigh 1.00g soil samples to be placed in 10mL centrifuge tubes, add 50% magnesium nitrate-ammonia spirit (96/4, volume ratio) 4mL, vibration
5min, ultrasonic extraction 15min, centrifuge (4500rmin-1) 8min, collect supernatant.Residue extracts 2 repeatedly in aforementioned manners again
It is secondary.Merge supernatant, after HLB solid phase extraction columns (successively crossing 6mL methanol and 6mL water) extracting and enriching.Cleaned with 6mL water small
Post, 10min is dried in vacuo, then pillar is eluted with the acetic acidacetonitriles of 3mL 1%.Eluent is blown to closely under 40 DEG C of water-baths with nitrogen
It is dry, 1mL is settled to acetonitrile-water (20/80, volume ratio), solution is crossed 0.22 μm of filter membrane and is collected in sample bottle, protected at 4 DEG C
Deposit for LC-MS/MS analysis measure.
Sample determining heavy metals
Soil sample by pre-treatment, air-dry, after the levigate mesh nylon mesh of mistake 100 it is stand-by.Soil sample heavy metal (Pb, Zn, Cu, Cd)
Three steps that form (exchangeable species, reducible state, oxidable state, residual form) analysis is proposed using reference material administration of the European Community are continuous
Grading extraction method.National standard material is added during analysis and carries out quality control.Pb, Zn, Cu and Cd are used after sample treatment
Atomic absorption spectrometry.
The biological community structure of sample
Equivalent sample is respectively taken from 3 repetition soil samples of vacuum freeze drying, is mixed, then therefrom takes sample 0.1g, is used
DNA extraction kit PowerMicroorganism total DNA in DNA Isolation Kit extraction samples.Using based on
The biological community structure of Illumina HiSeq 2000 high throughput sequencing technologies analysis sample.Goal in research fragment is thin
Bacterium 16S rDNA V6 hypervariable regions, PCR amplification using primers F 985 (5 '-CNACGCGAAGAACCTTANC-3 ')/R1046 (5 '-
CGACAGCCATGCANCACCT-3 '), amplification program and conditioned reference pertinent literature.The sequence measured is by splicing and screening
Afterwards, it is that standard obtains activity classification unit (OTU) with similitude 97%, OTU passes through RDP databases (http：//
Rdp.cme.msu.edu/ Classifer programs progress searching classification in), analysis microbiologic population Shannon diversity indices,
Species composition, relative abundance, contrast the biological community structure of each sample.
The high throughput fluorescence quantitative analysis of resistant gene
Equivalent sample is respectively taken from 3 repetition soil samples of vacuum freeze drying, is mixed, then therefrom weighs soil sample 0.5g, is adopted
Soil sample STb gene is extracted with Fast DNA Spin Kit for Soil kits, then carries out electrophoresis with 1% Ago-Gel
Checking.The DNA sample extracted determines concentration and corresponding purity with trace dna protein analyzer, extracts DNA sample
A260/A280Value is between 1.8~2.0.
Using the quantitative reaction platform of Smart Chip Real-time PCR Systems high throughput fluorescences.Fluorescent quantitation
Reagent is the SYBR Green I Master of Light Cycler 480.Quantifying system volume 100n L, wherein each reagent is dense eventually
Spend and be：The SYBR Green I Master Mix 1 of Light Cycler 480 ×, Nuclease-free PCR-Grade
The μ g μ L of water, BSA 1-1, DNA concentration 5ng μ L-1, 1 μm of olL of primer-1.Quantitative PCR response procedures：95 DEG C of pre-degenerations
10min；95 DEG C of denaturation 30s, 60 DEG C of annealing extension 30s, 40 circulate；Program automatic heating carries out melting curve analysis.Resistance
Gene primer has used 334 pairs of primers, according to Smart Chip Real-Time PCR System instruments with reference to pertinent literature
Setting have selected 295 antagonism gene primers and the general 16S rRNA gene primers of a pair of bacteriums.
Quantitative PCR data processing is carried out according to relevant document.According to Smart Chip Real-Time System detection
Limit and sensitivity, determine CTBe worth for 31 when detection threshold value as instrument.Each sample carries out 3 technologies and repeats to test, 3 skills
Art, which repeats all to amplify, is considered positive when coming；3 sampling repetitions at least two with a sampled point amplify, it is believed that sample
The target gene of product is effectively detected；Work as CTWhen value exceeds 31, corresponding CTValue is replaced by 31.The statistical method of relative quantification
Calculated with formula (1), (2) and (3)：
ΔCT=CT(ARGs)-CT(16S) (1)
ΔΔCT=Δ CT(test)-ΔCT(control) (2)
FC=2[-(ΔΔCT)] (3)
In formula, ARGs is 295 kinds of antibiotics resistance genes to be determined, and 16S refers to the 16S rRNA bases of testing sample
Cause, CTIt is that high throughput fluorescence quantifies determined cycle threshold, Δ CTThe target gene C of certain sampleTValue and 16S rRNA gene CsT
The difference of value, Δ Δ CTRefer to the target gene Δ C of (test) sample to be measuredTIt is worth the Δ C with compareing (control) sampleTDifference
Value, FC values refer to that the target gene of (test) sample to be measured is the enrichment times for compareing (control) sample.
Data analysis
Data analysis is carried out using Excel 2010, the significance of difference of each achievement data is examined using student t.Meter
Calculate FC lower limits FC=2[-(ΔΔCT+s)], s is the parallel functional gene C of 3 samplingsTStandard variance is the same as 16S rRNA gene CsTStandard
The root of quadratic sum between variance.S=(S1 2+S2 2)1/2.In formula, s1For the parallel functional gene C of 3 samplingsTThe standard side of value
Difference, s2For the 16S rRNA gene Cs in same sampling siteTThe standard variance of value.Work as FC=2[-(ΔΔCT+s)]＞ 1, and student
T is examined thinks testing sample relative to reference sample significant enrichment when having conspicuousness.
Testing result
The physicochemical property of the soil of table 1
Explanation：Each data are the average value repeated three times；In same column data, same letter represents
Difference is not notable (P ＞ 0.05), and different alphabets show significant difference (P ＜ 0.05)；Following table is same
The pH value of each treatment group soil, validity nitrogen, available phosphorus, organic carbon, NH4 +-N、NO3 -- N content is above compareing
Soil.
Antibiotic content (μ gkg in the soil of table 2-1)
Norfloxacin, Ciprofloxacin, Lomefloxacin, Enrofloxacin, tetracycline, terramycin and gold in each treatment group soil
Mycin content is below compareing soil.Compared with CS, the antibiotic concentration in AS3 is remarkably decreased (P ＜ 0.05), have dropped
37.6%-70.2%, minimum is reached.
Each form content of beary metal (μ gg in the soil of table 3-1)
Explanation：Available state=exchangeable species+reducible state+oxidable state
Available state Pb, Zn, Cu, Cd content in each treatment group soil is less than control soil, and residual form heavy metal contains
Amount is significantly higher than control soil (P ＜ 0.05).Under the conditions of optimal dose (the fresh Azolla imbricata/Ou Ganshu of 15kg), an available state huge sum of money
The content of category have dropped 46.8%-95.9%.
The relative abundance of resistant gene in the soil of table 4
Resistant gene recall rate in the soil of table 5
Explanation：MLSB=macrolide lipase, lincolamycin, streptavidin B resistant
Gene,
FCA=quinolone chloramphenicol resistance gene
Detect 220 kinds of ARGs in pedotheque altogether, soil bacteria show antibiotic (sulfamido, beta-lactam, FCA,
MLSB, Tetracyclines and vancomycin) resistance, the accountings of these antibiotic resistances is respectively 1.4%, 24.1%, 19.6%,
26.3%th, 19.1% and 9.5%.
9 kinds of transposase genes (tnpA-01, tnpA-02, tnpA-03, tnpA-04, tnpA- are detected in pedotheque altogether
05th, tnpA-06, tnpA-07, Tp614 and IS613).The detection of transposase gene (tnpA, IS613 and Tp614) in AS1-3
Rate is 4.1%-5.1%.Compared with AS1-3, the diversity of transposase gene have dropped respectively with relative abundance in AS4-5
66.7%-87.5% and 69.3%-89.4%, illustrate that suitable Azolla imbricata dosage can effectively control transposase gene in soil
Propagation.
Azolla imbricata phytoremediation technology is greatly reduced the relative abundance of ARGs in soil, including aac (6 ')-Ib-02,
AadE, acrA-05, vanXD, blaCTX-M-04, blaCMY2-01, blaCMY tetR-01, tetQ, tetPA, tetM-02 with
tet(34).Compared with compareing soil, bla TEM relative abundance have dropped 95.4%, 81.0% respectively in AS1, AS2 and AS3
With 99.8%.In AS3, in addition to aadA2-03, tetG-02 and mexF, other ARGs relative abundance substantially reduces.With
Control soil compares, when plant dosage increases to 10kg (AS3), erm B, erm F, ermT-01 and erm C relative abundance
Drop to 0.02%, 0.002%, 0.001% and 0.01% respectively.
When plant dosage increases to the fresh Azolla imbricatas of 15kg/bowl mandarin orange tree, soil ARGs quantity have dropped 27.1%.With compareing
Soil compares, and in addition to AS2, total ARGs of each treatment group soil relative abundance has decline, wherein AS3 total ARGs's
Relative abundance have dropped 44.5%, illustrate that suitable plant dosage can effectively suppress the relative abundance of total ARGs in soil.
Compared with compareing soil, the ermT-01 quantity in AS1-3 significantly reduces, and the ermT-01 quantity in AS4 and AS5
Slight rebound.It can be seen that suitable Azolla imbricata dosage can effectively control the ermT-01 in soil.
In treatment group soil, there is obvious Succession in biological community structure.Compared with compareing soil, everywhere
Microbial diversity in reason group soil adds, and wherein AS3 (the fresh Azolla imbricata/Ou Ganshu of 10kg) microbial diversity is most rich
Richness, and AS4-5 diversity of soil microorganism slightly falls after rise.Therefore, the Azolla imbricata plant of different amounts is more to edaphon
Sample generates different influences.
4 action principles
Contain substantial amounts of nutrient and organic matter in Azolla imbricata plant, be applied to energy improved soil structure after soil,
Improve soil physico-chemical property.Organic matter can strengthen retentivity of the heavy metal in soil surface after entering soil.With exchangeable species,
Heavy metal existing for reducible state and oxidable state has bioactivity for environment, belongs to biologically effective state；Residual form category is former
Rawore thing reference state, inanimate object validity.
The Spreading and diffusion of resistant gene includes vertical and horizontal and shifts two important channels, and vertical transfer refers to by resistance base
Host's bacterium colony succession of cause is propagated, and horizontal transfer is situated between by Mobile genetic elements (transposons, integron, plasmid etc.)
Lead and propagated between bacterium.Antibiotic and the selection pressure that heavy metal is resistant gene in environment, can co-induction resistant gene.One
Aspect, Azolla imbricata phytoremediation make antibiotic in soil be decreased obviously with available heavy metal content, meanwhile, also make in soil
The propagation of transposase gene has obtained effective suppression, so as to effectively control the propagation and diffusion of resistant gene in soil；It is another
Aspect, structure of soil microbial community and composition cause to carry ARGs there occurs obvious succession under Azolla imbricata repairing condition
Flora change, ARGs vertical transmission is have impact on, so as to have impact on ARGs distribution and return.Therefore, Azolla imbricata is planted
Thing recovery technique can effectively control the resistant gene of soil to pollute.
Described above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
Those of ordinary skill for, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (5)
1. a kind of method of resistance gene contamination in repairing and treating soil, it is characterised in that described restorative procedure is, by
Fresh Azolla imbricata plant is filled around tree root, the amount of described Azolla imbricata plant landfill is to fill 5- around each tree wood root system
15kg Azolla imbricata plants.
2. the method for resistance gene contamination in a kind of repairing and treating soil according to claim 1, it is characterised in that described
The depth of the fresh Azolla imbricata plant of landfill be 5-10cm.
3. the method for resistance gene contamination in a kind of repairing and treating soil according to claim 1, it is characterised in that described
The mode that fresh Azolla imbricata plant is filled around tree root be：Azolla imbricata plant is uniformly placed within one week in tree root and is covered
Bury.
4. the method for resistance gene contamination in a kind of repairing and treating soil according to claim 1, it is characterised in that described
Tree root peripheral region for centered on tree area for 1.4m × 1.4m region.
5. the method for resistance gene contamination in a kind of repairing and treating soil according to claim 1, it is characterised in that described
Trees be Ou Ganshu.
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