CN107365728A - One plant degraded PAEs endophyte of plant and the application in PAEs contaminated soils are repaired - Google Patents
One plant degraded PAEs endophyte of plant and the application in PAEs contaminated soils are repaired Download PDFInfo
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- CN107365728A CN107365728A CN201710736090.4A CN201710736090A CN107365728A CN 107365728 A CN107365728 A CN 107365728A CN 201710736090 A CN201710736090 A CN 201710736090A CN 107365728 A CN107365728 A CN 107365728A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/11—Bacillus megaterium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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
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- 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
Abstract
The present invention disclose one plant degrade PAEs endophyte of plant and the application in PAEs contaminated soils are repaired.The bacterial strain is bacillus megaterium (Bacillus megaterium) YJB3, on 06 28th, 2017 China typical culture collection centers for being preserved in Wuhan University of Wuhan, China city, deposit number：CCTCC NO：M 2017389.Using YJB3 bacterium solutions by way of rhizosphere soil is inoculated with, spraying treatment or seed soak bacterium, endophyte is colonized in plant rhizosphere or internal, or directly add bacterium to improve soil phthalic acid ester pollution amelioration efficiency into PAEs contaminated soils.The simple to operate, safety of the present invention, economy, do not introduce secondary pollution to soil environment, can evade pollution of agricultural products risk, for efficiently using low concentration pollution soil production safety agricultural product in regional and realize " while production, while repair " it is significant.
Description
Technical field
The invention belongs to organic polluted soil to repair field, and in particular to the plant of one plant of degraded phthalic acid ester (PAEs)
Thing endophyte, and can individually or joint phytoremediation PAEs contaminated soils application process.
Background technology
The activity such as the unreasonable use of the agricultural material products such as agricultural chemicals, chemical fertilizer and agricultural film and Industrial " three Waste " discharge constantly release is big
The PAEs class organic pollutions of amount, cause Farmland Soil Pollution getting worse.PAEs in contaminated soil is absorbed tired by crops
Product, serious threat agricultural product quality and safety.PAEs is a kind of Endocrine Disruptors, has endocrine disruption, reproductive development
Toxicity, hepatotoxicity wind agitation and " three cause " effect.Agricultural product of the ordinary meal intake containing PAEs cause long-term low dose to expose, serious danger
And human health.Soil remediation is to eliminate agricultural land soil PAEs to pollute most direct mode, compared to traditional soil remediation skill
The art such as physics such as heavy metal pollution of soil, chemical leaching, air lift and heat treatment, chemical method, it is biological prosthetic have cost it is low, safety have
The features such as imitating, be environment-friendly.Biological renovation method mainly includes microorganism remediation, phytoremediation, microorganism-plant combined reparation
Deng.Functional microorganism with organic pollutions such as efficient degradation PAEs, obtain in various environment that can be outside plant,
It can separate and obtain out of plant, the latter's research is also seldom, but has many advantages compared with the former in application, such as, work(
Can endophyte of plant individually can also combine the plant organic pollution such as PAEs in pollution degradation soil together, additionally it is possible to
Assist host plant to reduce the accumulation in vivo of the organic pollution such as PAEs and caused toxic effect, while promote host plant
Growth.Because endophyte is entered in plant by approach such as plant leaf blade stomata, stem hole skin and root system wounds, so
The organization internal in each organ of plant and space between cells are colonized afterwards.Endophyte establishes good mutualism with plant host
Relation, important component in plant microecosystem is formed, occupies special ecological niche.
It is worthy of attention that crop rhizosphere or internal can be inoculated in using endophyte of plant, rhizosphere soil is reduced
The organic pollutions such as middle PAEs pollute, and reduce crop PAEs accumulations, effectively evade crop PAEs pollution risks, make special in crop body
It is not that edible position pollutant load is less than food sanitation safe limit standard, and then ensures agricultural product quality and safety.But
As far as we know, there is presently no patent and document report endophyte of plant individually using or with plant combined applied to reparation
PAEs contaminated soils simultaneously reduce crop uptake and accumulation PAEs, efficiently to utilize contaminated soil production safety agricultural product, realize and " give birth on side
Production, side are repaired ".
The content of the invention
The shortcomings that in order to overcome prior art and deficiency, it is an object of the invention to provide in one plant of degraded PAEs plant
Raw bacterium.
Another object of the present invention is to the application for the endophyte of plant for providing above-mentioned degraded PAEs.
The invention provides the endophyte of plant that one plant has efficient degradation PAEs functions, and plant is repaiied alone or in combination
Multiple PAEs contaminated soils, promote PAEs degradeds and reduction crop uptake and accumulation PAEs application in soil.The present invention it is simple to operate,
Safety, cost are low, and secondary pollution is not produced to soil environment, by increasing capacitance it is possible to increase crop biomass, PAEs contaminated soils are effectively repaired,
Uptake and accumulation of the crop to PAEs is reduced, evades crop pollution risk, ensures agricultural product quality and safety.
The purpose of the present invention is achieved through the following technical solutions：
The present invention provides one plant of degraded PAEs endophyte of plant, and strain is named as Bacillus megaterium
YJB3, tamed and be enriched with by gradient, separated out of plant.
Described degraded PAEs endophyte of plant is bacillus megaterium (Bacillus megaterium) YJB3, its
Preservation information：Depositary institution：China typical culture collection center (CCTCC), preservation date：On 06 28th, 2017, preservation
Address：Chinese Wuhan Wuhan Universitys, deposit number：CCTCC NO：M 2017389.
The separation screening of described endophyte of plant comprises the following steps：
(1) plant material surface sterilisation step：After the fresh plant materials sample sterile water wash of collection, use is sterile
Scissors is cut into long 1cm segment, and super-clean bench is interior to carry out surface sterilizing, 1%~3%H according to following procedure2O21~4min is rinsed,
Aseptic water washing 2~4 times, 70% (v/v) alcohol rinse 2~5min, aseptic water washing 2~4 times, the drift of 3~5%NaClO solution
Wash 2~5min, aseptic water washing 2~4 times；
(2) inspection of plant material surface Disinfection Effect：Last time is taken to rinse the μ L of sterilized water 100 of vegetable material respectively
After through the plant sample of surface sterilization in 30 DEG C of culture 24h of LB solid plates, no microorganism grows, and shows plant material surface
Sterilizing is thorough, can carry out endophyte of plant separation screening；
(3) enrichment and domestication of endophyte of plant：By surface sterilization, thoroughly vegetable material is placed in sterile mortar, is added
The sterile quartz sands of 0.1~2g and 5~10mL sterile phosphate buffers (PBS, pH7.0~7.5) solution, then grind to even
Slurry, take 1~5mL supernatants be inoculated in 100~150mL containing PAEs be sole carbon source inorganic salt liquid culture medium in, at 30 DEG C,
Under 120~150rpm speed conditions, lucifuge shaking table culture, every 5~7 days are a cycle, step up PAEs initial concentrations, from
50mg/L to 1000mg/L；
(4) endophyte of plant separation screening：After domestication terminates, by above-mentioned culture bacterium solution gradient dilution to 10-5~10-7Apply
It is distributed on the inorganic salts solid medium flat board containing 500~1000mg/L PAEs, 30 DEG C incubated, treats to grow on flat board
After bacterium colony, with method of scoring purification of target bacterial strain, wherein bacterial strain YJB3 can be using PAEs as sole carbon source, and growing way is vigorous, as mesh
Bacterial strain is marked, i.e.,：Bacillus megaterium (Bacillus megaterium) YJB3.
Plant material surface sterilisation step can also be in the step (2)：The fresh plant materials sample of collection is used
After sterile water wash, long 1cm segment is cut into sterile scissors, by vegetable material be totally submerged with equipped with 150mL~200mL without
In the conical flask with stopper of bacterium water or beaker with cover, ultrasonic sterilization 20min, working frequency 20KHz, power 150W.
Minimal medium (the gL-1)：K2HPO45.8, KH2PO44.5, (NH4)2SO42.0, MgCl20.16,
CaCl20.02, Na2MoO4·2H2O 0.0024, KNO30.0012, FeCl30.0018, MnCl2·2H2O 0.0015, pH
7.0。
Described bacillus megaterium (B.megaterium) YJB3 16S rDNA nucleotide sequences such as SEQ ID NO：1
It is shown.
Described bacillus megaterium (B.megaterium) YJB3 forms and physiological and biochemical property are：Bacterium colony is smooth, light
Yellow, neat in edge, thalline are easily provoked, gram-positive bacteria, thalline elongated rod shape, 1.5~2.0 μm wide, 5.2~7.8 μm long；It is good
Oxygen, catalase reacting positive, sugar fermentation is positive, and Starch Hydrolysis experiment is positive, and methyl red test is positive, and urase experiment is positive, lemon
Hydrochlorate is tested and H2S experiments are negative, and Voges-Proskauer experiments are negative, and indoles experiment is negative, and gelatin hydrolysis experiment is negative.
Described phthalic acid ester (PAEs) is diethyl phthalate (DEP) or/and repefral
Or/and dibutyl phthalate (DBP) and/or di-n-octyl phthalate (DnOP) and/or phthalic acid two (DMP)
(2- ethylhexyls) ester (DEHP) and/or butyl benzyl phthalate (BBP).
Application of the described degraded PAEs endophyte of plant in phytoremediation PAEs contaminated soils alone or in combination.
Described bacillus megaterium (B.megaterium) YJB3 bacterium solution preparation methods are：Take slant preservation
B.megaterium YJB3 strain transfers are in 1000mL LB fluid nutrient mediums, 30 DEG C, after 7~9h of 120rpm shaking table cultures,
Thalline is collected by centrifugation in 3000r/min, is then suspended in PBS solution, is diluted to concentration as 106~109CFU/mL bacteria suspension is simultaneously
The sodium acetate for supplementing 0.5~1.5g/L is starting carbon source.
By described bacillus megaterium (B.megaterium) YJB3 according to inoculum density be 106~109CFU/mL bacterium
Suspension is directly added into by PAEs contaminated soil media, after stirring and is ventilated.
By described bacillus megaterium (B.megaterium) YJB3 according to concentration be 106~109CFU/mL inoculum concentrations are
1~10mL is inoculated in plant rhizosphere soil or is inoculated in plant and plant combined reparation PAEs contaminated soils.
The method being inoculated in plant of described endophyte of plant can be：The vegetable seeds of full grains is taken, it is first
First soaked seed 15~20min of sterilization with 5% sodium hypochlorite, then rinsed 2~3 times with aseptic deionized water, immerse and be containing concentration
106~10930~180min in CFU/mL bacillus megaterium YJB3 bacteria suspensions.
The method being inoculated in plant of described endophyte of plant can be：Using spraying or mode is smeared by concentration
For 106~109CFU/mL bacillus megaterium YJB3 bacterial suspension inoculations are in plant leaf surface.
The method being inoculated in plant of described endophyte of plant can be：It is by plant seedlings root system immersion concentration
106~10930~90min in CFU/mL bacillus megaterium YJB3 bacteria suspensions.
Described plant can be for the Sedum alfredii Hance of phytoremediation for soil, clover, rye grass, black nightshade etc. or water
The crops such as rice, corn, cabbage heart, tomato.The seed of above-mentioned plant is commercially available to be obtained.
The present invention is had the following advantages and effect relative to prior art：
The present invention provides through gradient domestication and enrichment, in the plant that one plant of degraded PAEs is separated out of plant
Raw bacterium Bacillus megaterium YJB3.Pass through rhizosphere soil using endophyte of plant B.megaterium YJB3 bacterium solutions
The mode of inoculation, spraying treatment or seed leaching bacterium, makes endophyte colonize in plant rhizosphere or internal, or directly adds bacterium dirty to PAEs
Contaminate in soil, can improve soil phthalic acid ester pollution amelioration efficiency, and reduce crop uptake and accumulation PAEs.Present invention behaviour
Make simple, safety, economy, secondary pollution is not introduced to soil environment, pollution of agricultural products risk can be evaded, for efficiently utilizing
In regionality low concentration pollution soil produce Safe Agricultural Product and realize " while production, while repair " it is significant.
Brief description of the drawings
Fig. 1 is bacillus megaterium (B.megaterium) YJB3 bacterium colonies and cell morphological characteristic.
Fig. 2 is bacillus megaterium (B.megaterium) YJB3 16S rDNA PCR results.
Fig. 3 is bacillus megaterium (B.megaterium) YJB3 phylogenetic trees.
Fig. 4 is that bacillus megaterium (B.megaterium) YJB3 cuts down to PAEs in cabbage heart rhizosphere micro-region in earth culture environment
The influence of behavior；Wherein, S0 refers to root growth area, and S0-1 spans are from the rhizosphere area with a distance from root surface 1mm；S1-3 spans are from root table
Face 1-3mm rhizosphere area；Rhizosphere area of the S3-5 spans from root surface 3-5mm.
Fig. 5 be in earth culture environment bacillus megaterium (B.megaterium) YJB3 to cabbage heart uptake and accumulation PAEs shadow
Ring.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
Embodiment 1
From artificial swamp herborization sample, endophyte separation screening experiment is carried out immediately.Using the endophyte point of routine
From method.After the fresh plant materials sample sterile water wash of collection, long 1cm segment is cut into sterile scissors, it is ultra-clean
In platform surface sterilizing, 3%H are carried out according to following procedure2O22.5min, aseptic water washing 4 times are rinsed, 70% alcohol rinses 3 times,
Aseptic water washing 3 times, 3%NaClO solution rinsing 5min, aseptic water washing 3 times, takes last time to rinse vegetable material respectively
The μ L of sterilized water 100 and the plant sample through surface sterilization, immigration LB solid mediums (peptone 10g, yeast extract 5g,
NaCl 10g, agar powder 1.8%, the sterilizing of 1000mL, pH7.0,121 DEG C of constant volume) 30 DEG C of culture 24h of flat board, no microorganism grows,
Plant material surface sterilizing is thorough, and the vegetable material is placed in sterile mortar, adds the sterile quartz sands of 0.5g and 10mL is sterile
Phosphate buffer (pH7.2), grinds to homogenate, takes supernatant 1mL, is inoculated in 100mL and contains the nothing that DBP is sole carbon source
In machine salt fluid nutrient medium, 30 DEG C, 150rpm, lucifuge shaking table culture, every 5 days are a cycle, step up the initial dense of DBP
Degree, from 50mg/ to 1000mg/L, gradient is tamed；After domestication terminates, by above-mentioned nutrient solution gradient dilution to 10-5Be coated on containing
On 500mg/L DBP inorganic salts solid medium flat boards, 30 DEG C incubated, and after flat board grows bacterium colony, method of scoring purifying obtains
Aimed strain YJB3 is obtained, transfers and is preserved in LB test tube slants, 4 DEG C of refrigerators.
Embodiment 2
From artificial swamp herborization sample, endophyte separation screening experiment is carried out immediately.By the fresh plant material of collection
After expecting sample sterile water wash, long 1cm segment is cut into sterile scissors, vegetable material is totally submerged in equipped with 150mL
In the conical flask with stopper or beaker with cover of~200mL sterilized waters, ultrasonic sterilization 25min, working frequency 20KHz, power
For 150W；The vegetable material is placed in sterile mortar, adds the sterile quartz sands of 2g and 10mL sterile phosphate buffers
(pH7.2), grind to homogenate, take supernatant 0.5mL, be inoculated in 100mL and contain the inorganic salt liquid that DBP is sole carbon source
In culture medium, 30 DEG C, 150rpm, lucifuge shaking table culture, every 7 days are a cycle, step up DBP initial concentration,
100mg/L to 800mg/L gradients tame；After domestication terminates, by above-mentioned nutrient solution gradient dilution to 10-6Be coated on containing
On 1000mg/L DBP inorganic salts solid medium flat boards, 30 DEG C are incubated, after flat board grows bacterium colony, method of scoring purifying
Aimed strain YJB3 is obtained, transfers and is preserved in LB test tube slants, 4 DEG C of refrigerators.
Embodiment 3
(1) strain morphology characterization：By bacterial strain YJB3 streak inoculations in LB solid mediums, flat board is inverted constant temperature training
Case is supported, 30 DEG C of incubated 12~18h, observes colonial morphology, bacterium colony is smooth, faint yellow, neat in edge, easily provokes；To bacterial strain
Gram's staining is carried out, gram-positive bacteria, is observed using SEM, the bacterial strain is long bacillus, 1.5~2.0 μm
Width, it is 5.2~7.8 μm long, see Fig. 1.
(2) bacterial strain relevant physiological biochemical test
Methyl red experiment, urase experiment, catalase experiment, sugared fermenting experiment, starch are carried out to bacterial strain YJB3 cultures respectively
Hydrolysising experiment, citrate experiment, H2One systems such as S experiments, Voges-Proskauer experiments, indoles experiment, gelatin hydrolysis experiment
Row relevant physiological biochemical test, detailed results are shown in Table 1.
The bacterial strain relevant physiological biochemical results of table 1
| Bio-chemical characteristics | As a result |
| Methyl red is tested | + |
| Aerobic growth | + |
| Catalase reacts | + |
| Sugared fermenting experiment | + |
| Starch Hydrolysis is tested | + |
| Urase is tested | + |
| Citrate is tested | - |
| H2S is tested | - |
| Voges-Proskauer is tested | - |
| Indoles is tested | - |
| Gelatin hydrolysis is tested | - |
(3) bacterial strain 16S rDNA are analyzed
Using bacterial genomes DNA extraction kit, strain gene group DNA is extracted, using strain gene group DNA as template,
Using the 16S rDNA fragments of bacterial 16 S rDNA universal primers (27F, 1492R) amplification bacterial strain, Shanghai life work sequencing is delivered to, is led to
Cross 1% agarose gel electrophoresis and find that a specific band is occurring in product about at 1100bp, as a result as shown in Fig. 2 sequence
Such as SEQ ID NO：Shown in 1.Obtained bacterial 16 S rDNA sequences and ncbi database are subjected to Blast
(www.ncbi.nlm.nih.gov/Blast) known bacterium Bacillus megaterium in sequence analysis, with database
The similitudes of 16S rDNA sequences reached 99%, the phylogenetic tree of structure is as shown in figure 3, combining form and Physiology and biochemistry
Feature, the bacterial strain is accredited as Bacillus megaterium.Strain is named as bacillus megaterium (B.megaterium)
YJB3。
Described bacillus megaterium (Bacillus megaterium) YJB3 preservation information：Depositary institution：Chinese allusion quotation
Type culture collection (CCTCC), preservation date：On 06 28th, 2017, preservation address：Chinese Wuhan Wuhan Universitys,
Deposit number：CCTCC NO：M 2017389.
Embodiment 4
DBP concentration is set in soil as 100mgkg–1, take part soil (about 5kg) to add DBP acetone solns, mixed
It is even, parent-soil is as polluted, is then mixed into remaining soil, stirs, treats to load root case after acetone solvent volatilization, slowly
Drip irrigation adds running water, keeps the 60% of field capacity, and shading aging 14d.Take the B.megaterium of slant preservation
YJB3 strain transfers are in 100mL LB fluid nutrient mediums, 30 DEG C, after 120rpm shaking table cultures 7h, 3000rpm centrifugation 3min, receive
Collect thalline, after being rinsed 2~3 times with PBS, resuspension obtains bacteria suspension, and adjustment cell concentration is 108CFU/mL, and supplement 0.5g/L
Sodium acetate be starting carbon source.It is stable after cabbage heart seedling growth and after thering is young leaves to grow, with 10mL inoculum concentrations by bacteria suspension
Cabbage heart rhizosphere is inoculated in using root-pouring method.As a result show, DBP cut rates averagely improve in green precious (LB) rhizosphere soil of cabbage heart
54.2% (Fig. 4), its overground part DBP contents reduce 45.2%, and root DBP contents reduce 40.6% (Fig. 5), cabbage heart Hua Guan
(HG) DBP cut rates averagely improve 50.6% (Fig. 4) in rhizosphere soil, and its overground part DBP contents reduce 43.6%, root
DBP contents reduce 32.7% (Fig. 5).
Embodiment 5
DEHP concentration is set in soil as 100mgkg–1, not add the control of DEHP soil.Take part soil (about
DEHP acetone solns 5kg) are added, is mixed thoroughly, as pollutes parent-soil, be then mixed into remaining soil, stir, treat acetone
Solvent loads root case after volatilizing, slow drip irrigation adds running water, keeps the 60% of field capacity, and shading aging 14d.
The B.megaterium YJB3 strain transfers of slant preservation are taken in 100mL LB fluid nutrient mediums, 30 DEG C, the training of 120rpm shaking tables
After supporting 7h, 3000rpm centrifugation 3min, thalline is collected, after being rinsed 2~3 times with PBS, resuspension obtains bacteria suspension, adjusts cell concentration
For 107CFU/mL, and the sodium acetate for supplementing 1.0g/L is starting carbon source.Treat Growth of Tomato Seedling balanced condition and there is young leaves to grow
Afterwards, spraying treatment is used with 10mL inoculum concentrations, is inoculated in tomato overground part.Handled relative to bacterium is not connect, inoculating strain
B.megateriumYJB3 makes DEHP cut rates in tomato rhizosphere soil averagely improve 24.1%.
Embodiment 6
DEP concentration is set in soil as 100mgkg–1, not add the control of DEP soil.Take part soil (about 5kg)
DEP acetone solns are added, is mixed thoroughly, as pollutes parent-soil, be then mixed into remaining soil, stir, treat acetone solvent
Load root case after volatilization, slow drip irrigation adds running water, keeps the 60% of field capacity, and shading aging 14d.Take tiltedly
The B.megaterium YJB3 strain transfers of face preservation are in 100mL LB fluid nutrient mediums, 30 DEG C, 120rpm shaking table cultures 8h
Afterwards, 3000rpm centrifuges 3min, collects thalline, and after being rinsed 2~3 times with PBS, resuspension obtains bacteria suspension, and adjustment cell concentration is
108CFU/mL, and the sodium acetate for supplementing 1.5g/L is starting carbon source.The vegetable seeds of full grains is taken, first with 5% hypochlorous acid
Sodium seed soaking sterilization 20min, is then rinsed 3 times with aseptic deionized water, and it is 10 to immerse containing the concentration8CFU/mL endophyte
180min in nutrient solution.Warm indoor growing 55 days, relative to bacterium processing is not connect, inoculating strain B.megateriumYJB3 makes rye
DEP cut rates averagely improve 50.4% in careless rhizosphere soil.
Embodiment 7
DMP concentration is set in soil as 100mgkg–1, using the soil for not adding DMP as control, take part soil (about
DMP acetone solns 5kg) are added, is mixed thoroughly, as pollutes parent-soil, be then mixed into remaining soil, stir, treat acetone
Solvent loads root case after volatilizing, slow drip irrigation adds running water, keeps the 60% of field capacity, and shading aging 14d.
The B.megaterium YJB3 strain transfers of slant preservation are taken in 100mL LB fluid nutrient mediums, 30 DEG C, the training of 120rpm shaking tables
After supporting 8h, 3000rpm centrifugation 3min, thalline is collected, after being rinsed 2~3 times with PBS, resuspension obtains bacteria suspension, adjusts cell concentration
For 106CFU/mL, and the sodium acetate for supplementing 0.8g/L is starting carbon source.Rice seedling root system is immersed into bacterium solution suspension 60min, so
Transplant afterwards in DMP contaminated soils, warm indoor growing 50d, relative to bacterium processing is not connect, inoculating strain B.megateriumYJB3 makes
DMP degradation rates averagely improve 43.3% in rhizosphere soil.
Embodiment 8
DBP concentration is set in soil as 100mgkg–1, using the soil for not adding DBP as control, take part soil (about
DBP acetone solns 5kg) are added, is mixed thoroughly, as pollutes parent-soil, be then mixed into remaining soil, stir, treat acetone
Solvent slow drip irrigation addition running water after volatilizing, keeps the 60% of field capacity, and shading aging 14d.Inclined-plane is taken to protect
The B.megaterium YJB3 strain transfers of Tibetan are in 100mL LB fluid nutrient mediums, 30 DEG C, after 120rpm shaking table cultures 8h,
3000rpm centrifuges 3min, collects thalline, and after being rinsed 3 times with PBS, resuspension obtains bacteria suspension, and adjustment cell concentration is 108CFU/
ML, and the sodium acetate for supplementing 0.8g/L is starting carbon source, and the bacteria suspension is directly admixed in soil, stirs and ventilates.Soil
DBP degradation rates are 35.2% in earth, and plus in soil bacteria DBP degradation rates are not only 11.3%.Bacterial strain is directly added into soil
B.megaterium YJB3, it can effectively remove DBP in soil and pollute.
Embodiment 9
DnOP concentration is set in soil as 100mgkg–1, using the soil for not adding DnOP as control, take part soil (about
DnOP acetone solns 5kg) are added, is mixed thoroughly, as pollutes parent-soil, be then mixed into remaining soil, stir, treat acetone
Solvent loads root case after volatilizing, slow drip irrigation adds running water, keeps the 60% of field capacity, and shading aging 14d.
The B.megateriumYJB3 strain transfers of slant preservation are taken in 100mL LB fluid nutrient mediums, 30 DEG C, the training of 120rpm shaking tables
After supporting 8h, 3000rpm centrifugation 3min, thalline is collected, after being rinsed 2~3 times with PBS, resuspension obtains bacteria suspension, adjusts cell concentration
For 108CFU/mL, and the sodium acetate for supplementing 0.8g/L is starting carbon source.The bacteria suspension is directly accessed with 3~4 true leaves
In rye grass rhizosphere soil, warm indoor culture 55 days, mutually relative to bacterium processing is not connect, inoculating strain B.megateriumYJB3 makes
DnOP degradation rates improve 23.5% in plant rhizosphere soil.
Embodiment 10
BBP concentration is set in soil as 100mgkg–1, not add the control of BBP soil.Take part soil (about 5kg)
BBP acetone solns are added, is mixed thoroughly, as pollutes parent-soil, be then mixed into remaining soil, stir, treat acetone solvent
Load root case after volatilization, slow drip irrigation adds running water, keeps the 60% of field capacity, and shading aging 14d.Take tiltedly
The B.megaterium YJB3 strain transfers of face preservation are in 100mL LB fluid nutrient mediums, 30 DEG C, 120rpm shaking table cultures 7h
Afterwards, 3000rpm centrifuges 3min, collects thalline, and after being rinsed 2~3 times with PBS, resuspension obtains bacteria suspension, and adjustment cell concentration is
109CFU/mL, and the sodium acetate for supplementing 1.0g/L is starting carbon source.Treat growth of maize balanced condition and there are 3~4 leaf length
After going out, spraying treatment is used with 10mL inoculum concentrations, is inoculated in blade.Warm indoor growing 45 days, compared with not connecing bacterium processing, connect bacterium
B.megaterium YJB3 make DEHP cut rates in Rhizosphere of Crops soil averagely improve 14.1%.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Ji'nan University
<120>One plant degraded PAEs endophyte of plant and the application in PAEs contaminated soils are repaired
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1096
<212> DNA
<213> Artificial Sequence
<220>
<223>Bacillus megaterium（B.megaterium）YJB3 16S rDNA nucleotide sequences
<400> 1
gggcatgtgc gcggcgtgct atacatgcca agtcgagcga tactgataga agcttgcaac 60
tatgacgtta gcggcggacg ggtgagtaac acgtccgcaa cgctgcctgt aagactggga 120
taacttcggg ataccgaagc taataccgga taggatcttc tccttcatgg gagatgattg 180
aaagatggaa tcggctatca cttacagatc gcgccgcggt gcattagcta gttggtgagg 240
ttacggctca cctaggcaac gatgcatagc cgacctgaga gggtgatcgg cgacactggg 300
actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg 360
aaagtctgac ggagcaacgc cgcgtgagtg atgaaggctt tcgcgtcgta taactctgtt 420
gttagggaag aacaagtacg agagtaactg cttgtacctt gacggtacct aaccagaaag 480
ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggaa 540
ttattgggcg taaagcgcgc gcaggcggtt tcataagtct gatgtgaaag cgcacggctc 600
aaccgtggag ggtcaatgga aactggggaa cttgagtgct gaagagaaaa gcggaattcc 660
acgtgtagcg gtgaattgcg tagagatgtg gaggaacacc agtggcgaag gcggctttta 720
ggtctgtaac tgacgctgag gcgcgtaagc gtggggagca aacacgatta gataccctgg 780
tagtccacgc cgttaacgat gagtgctaag tgttagaggg tttccgccct atagtgctgc 840
agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900
ttgacggggg ccggcacaag cggtggagca tgtggattaa ttcgaagcaa cgcgaagaac 960
cataccaggt cttgacatcc tctgacactc tagagataga gcgttcggct tcgggggaca 1020
gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt ggagatgaag ggttaagtcc 1080
cgctacgagc cccgaa 1096
Claims (9)
1. one plant of degraded PAEs endophyte of plant, it is characterised in that：Entitled bacillus megaterium (Bacillus
Megaterium) YJB3, on 06 28th, 2017 China typical culture collections for being preserved in Wuhan University of Wuhan, China city
Center, deposit number：CCTCC NO：M 2017389.
2. the endophyte of plant of the degraded PAEs described in claim 1 is in phytoremediation PAEs contaminated soils alone or in combination
Using.
3. application according to claim 2, it is characterised in that：
Described PAEs be diethyl phthalate or/and repefral or/and dibutyl phthalate and/
Or di-n-octyl phthalate and/or (2- ethylhexyls) ester of phthalic acid two and/or butyl benzyl phthalate.
4. application according to claim 2, it is characterised in that：
By the bacillus megaterium YJB3 described in claim 1 according to inoculum density be 106~109CFU/mL bacteria suspension is direct
Add in by PAEs contaminated soil media, after stirring and ventilate.
5. application according to claim 2, it is characterised in that：
By the bacillus megaterium YJB3 described in claim 1 according to concentration be 106~109CFU/mL inoculum concentrations are that 1~10mL connects
Kind in plant rhizosphere soil or be inoculated in plant and plant combined reparations PAEs pollution.
6. application according to claim 5, it is characterised in that：
The described method being inoculated in plant is：The vegetable seeds of full grains is taken, is disappeared first with the seed soaking of 5% sodium hypochlorite
15~20min of poison, then rinsed 2~3 times with aseptic deionized water, it is 10 to immerse containing concentration6~109CFU/mL huge bud
30~180min in spore bacillus YJB3 bacteria suspensions.
7. application according to claim 5, it is characterised in that：
The described method being inoculated in plant is：Use spraying or smear mode by concentration as 106~109CFU/mL's is huge
Bacterium anthracoides YJB3 bacterial suspension inoculations are in plant leaf surface.
8. application according to claim 5, it is characterised in that：
The described method being inoculated in plant is：It is 10 that plant seedlings root system is immersed into concentration6~109CFU/mL's is huge
30~90min in bacillus YJB3 bacteria suspensions.
9. according to the application described in any one of claim 2 or 5~8, it is characterised in that：
Described plant is Sedum alfredii Hance, clover, rye grass, black nightshade, rice, corn, cabbage heart, tomato.
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| CN107593769A (en) * | 2017-08-24 | 2018-01-19 | 暨南大学 | Applications of the bacillus megaterium YJB3 in plant growth and biological and ecological methods to prevent plant disease, pests, and erosion is promoted |
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| CN109365520A (en) * | 2018-12-04 | 2019-02-22 | 华南农业大学 | A kind of method of the remediating heavy metal cadmium pollution soil in production |
| CN109456129A (en) * | 2018-12-07 | 2019-03-12 | 佛山科学技术学院 | The charcoal processing method discharged for reducing PAE in plastics micro- in soil |
| CN110883084A (en) * | 2019-12-02 | 2020-03-17 | 淮阴工学院 | Method for in-situ remediation of soil phthalate pollution by using mushroom dregs and alfalfa |
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| CN115058252A (en) * | 2022-01-17 | 2022-09-16 | 江苏省农业科学院 | Microbial soil conditioner for phthalate ester contaminated soil remediation and application thereof |
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| CN107593769A (en) * | 2017-08-24 | 2018-01-19 | 暨南大学 | Applications of the bacillus megaterium YJB3 in plant growth and biological and ecological methods to prevent plant disease, pests, and erosion is promoted |
| CN107593769B (en) * | 2017-08-24 | 2020-04-14 | 暨南大学 | Application of bacillus megaterium YJB3 in promoting plant growth and biocontrol |
| CN108906866A (en) * | 2018-06-27 | 2018-11-30 | 北京高能时代环境技术股份有限公司 | The restorative procedure of petroleum hydrocarbon contaminated soil |
| CN109365520A (en) * | 2018-12-04 | 2019-02-22 | 华南农业大学 | A kind of method of the remediating heavy metal cadmium pollution soil in production |
| CN109456129A (en) * | 2018-12-07 | 2019-03-12 | 佛山科学技术学院 | The charcoal processing method discharged for reducing PAE in plastics micro- in soil |
| CN110883084A (en) * | 2019-12-02 | 2020-03-17 | 淮阴工学院 | Method for in-situ remediation of soil phthalate pollution by using mushroom dregs and alfalfa |
| CN110883084B (en) * | 2019-12-02 | 2021-08-17 | 淮阴工学院 | Method for in-situ remediation of soil phthalate pollution by using mushroom dregs and alfalfa |
| CN111849800A (en) * | 2020-06-09 | 2020-10-30 | 暨南大学 | Application of bacillus megatherium YJB3 in relieving plant oxidative stress |
| CN115058252A (en) * | 2022-01-17 | 2022-09-16 | 江苏省农业科学院 | Microbial soil conditioner for phthalate ester contaminated soil remediation and application thereof |
| CN115058252B (en) * | 2022-01-17 | 2023-08-08 | 江苏省农业科学院 | Microbial soil conditioner for restoring phthalate polluted soil and application thereof |
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