CN109266582A - A kind of application of thin layer bacterium and its anabasine insecticide of degrading - Google Patents
A kind of application of thin layer bacterium and its anabasine insecticide of degrading Download PDFInfo
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Abstract
The invention discloses a kind of thin layer bacterium and its applications in degradation anabasine insecticide, the thin layer bacterium is identified as Hymenobacter latericoloratus, the entitled DG01 of bacterial strain, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC No.16346, the deposit date is on August 27th, 2018.Compared with existing degradation of pesticide technology, new strains thin layer bacterium CGMCC 16346 of the invention, growing cell and resting cell can effectively degrade anabasine insecticide, especially imidacloprid, can be used as degradation of pesticide microbial inoculum, with good application prospect.
Description
Technical field
The invention belongs to field of microbial biotechnology, and in particular to a kind of thin layer bacterium and its in degradation anabasine desinsection
Application in agent.
Background technique
Anabasine insecticide is a kind of nitrogenous heterocyclic insecticide, and the mode of action is selectivity control insect nerveous system
Nicotinic acetylcholine esterase receptor in system, blocks the normal conduction of insect CNS, benumbs so as to cause pest
And then it is dead.Imidacloprid and Acetamiprid are the main representative kinds of the insecticides, are widely used in rice, wheat, vegetables, fruit
In the multiple kinds of crops such as tree, tea tree, cotton and tobacco.Anabasine insecticide is also one of main insecticide variety in China.
However largely seriously affected for a long time using food-safe produced with ecological environment of anabasine insecticide, if imidacloprid is to biography
Powder insect honey bee and birds toxicity are high, thus the ecological chain problem caused；Imidacloprid and Acetamiprid are in cereal, vegetables and tealeaves
There is residual, to generate food-safety problem；80~98% anabasine insecticide enters eventually into soil environment, causes
Contaminated soil and surface water simultaneously generate threat to aquatic insect.In recent years, influence of the anabasine insecticide to environmental ecology
Caused the whole world extensive concern, in the environment return the great attention for becoming and being subject to.
Microbial degradation is one of the main path of anabasine insecticide soil metabolism, and eliminates the insecticides ring
One of the most economical effective method of border pollution.The bacterial strain of degradable anabasine insecticide reported at present has Bacillus
alkalinitrilicus、Ensifer meliloti、Ensifer adhaerens CGMCC 6315、Leifsonia
sp.PC-21、Klebsiella pneumoniae BCH1、Pseudomonas sp.1G、Pseudoxanthomonas
Indica CGMCC 6648, Stenotrophomonas maltophilia CGMCC 1.1788 and Variovorax
Boronicumulans CGMCC 4969 etc., but there are no thin layer Pseudomonas Hymenobacter strains for degrading anabasine desinsection
The document report of agent.
Summary of the invention
Goal of the invention: the present invention provides a kind of thin layer bacterium and its applications in degradation anabasine insecticide.
Technical solution: the present invention provides a kind of thin layer bacterium, categorized is accredited as thin layer bacterium (Hymenobacter
Latericoloratus), the entitled DG01 of bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, deposit number are as follows: CGMCC No.16346, the deposit date is on August 27th, 2018.
The cultural method of the thin layer bacterium are as follows: the thin layer bacterium CGMCC 16346 is inoculated on R2A agar medium,
It is cultivated in the incubator that temperature is 30 ± 1 DEG C；Or be inoculated in R2A fluid nutrient medium, training is vibrated in 30 ± 1 DEG C of shaking table
It supports.
The present invention also provides application of the thin layer bacterium in degradation anabasine insecticide.
The anabasine insecticide is imidacloprid, thiacloprid or Acetamiprid.
Further preferably, application method are as follows: the thin layer bacterium CGMCC 16346 is inoculated in the training of the nutrition containing imidacloprid
Shaken cultivation in nutrient solution, thin layer bacterium CGMCC 16346 degrade imidacloprid during the growth process.
Further preferably, application method are as follows: the thin layer bacterium CGMCC 16346 is inoculated in liquid nutrient media,
It is collected by centrifugation after shaken cultivation and washing thalline；Thallus is suspended in the phosphate containing imidacloprid, thiacloprid or Acetamiprid to delay
It rushes in solution, shaken cultivation, resting cell degradation imidacloprid, thiacloprid or the Acetamiprid of thin layer bacterium CGMCC 16346.
The nutrient medium is preferably R2A culture medium.
The co-metabolic substance is glucides or the Sodium Pyruvates such as glucose, maltose.
Technical effect: compared with the existing technology, the new strains thin layer bacterium CGMCC 16346 of invention grows cell and quiet
Breath cell can effectively degrade anabasine insecticide, especially imidacloprid.Therefore the bacterial strain can be used as degradation of pesticide microbial inoculum, have
There is good application prospect.
Detailed description of the invention
Fig. 1 is 16346 flat-plate bacterial colony form of thin layer bacterium CGMCC of the present invention and microscopic morphology.
The HPLC figure of growth cell and resting cell degradation imidacloprid that Fig. 2 is thin layer bacterium CGMCC 16346 of the present invention,
In: A: using maltose as the conversion of resting cells imidacloprid of co-metabolic substance；B: thin as the tranquillization of co-metabolic substance using glucose
Dysuria with lower abdominal colic imidacloprid；C: it is not added with the conversion of resting cells imidacloprid of co-metabolic substance；D: using Sodium Pyruvate as co-metabolic substance
Conversion of resting cells imidacloprid；E: growth cell degradation imidacloprid；F: substrate imidacloprid compares (not being inoculated with thallus).
The HPLC figure for the resting cell degradation thiacloprid that Fig. 3 is thin layer bacterium CGMCC 16346 of the present invention, in which: A: thiophene worm
Quinoline Substrate controls (are not inoculated with thallus)；B: it is not added with the resting cell degradation thiacloprid of co-metabolic substance；C: adding maltose is
The resting cell degradation thiacloprid of co-metabolic substance；D: addition Sodium Pyruvate is the resting cell degradation thiophene worm of co-metabolic substance
Quinoline.
The HPLC figure for the resting cell degradation Acetamiprid that Fig. 4 is thin layer bacterium CGMCC 16346 of the present invention, in which: A: pyridine worm
Amidine Substrate controls (are not inoculated with thallus)；B: it is not added with the resting cell degradation Acetamiprid of co-metabolic substance；C: addition Sodium Pyruvate
For the resting cell degradation Acetamiprid of co-metabolic substance.
Specific embodiment
Thin layer bacterium CGMCC 16346 of the present invention and its application are elaborated in conjunction with following specific embodiment.
Embodiment 1: degradable imidacloprid microorganism isolates and purifies, screens and identifies
1, strain isolation
Soil and water body sample are acquired from Nanjing Qixia District, 2g soil sample or 2ml water sample is taken to be added containing bead
In 18mL sterile water, 2h is vibrated, after standing 20min, 100 μ L samples is taken to be diluted to 10-3With 10-4Afterwards, it is coated on containing 50mg/L
On the solid mineral salt culture medium plate of imidacloprid.The group of mineral salts medium becomes (g/L): KH2PO41.36 Na2HPO4
2.13 MgSO4·7H2O 0.5 and 10mL metal ion liquid, pH 7.0.Metal ion liquid group becomes (g/L): CaCl2·2H2O
0.40,H3BO3 0.30,CuSO4·5H2O 0.04,FeSO4·7H2O 0.20, MnSO4·7H2O 0.40,NaMoO4·2H2O
0.20, KI 0.10 and 10.0mL/L concentrated hydrochloric acid.After growing single colonie on the solid mineral salt culture medium plate containing imidacloprid,
The bacterium colony of picking different shape is crossed to new imidacloprid mineral salt solid medium, and solid plate is cultivated in 30 DEG C of incubators
3d, the single colonie grown carry out scribing line again on R2A culture medium flat plate and isolate and purify.The formula of R2A culture medium is (g/L):
Tryptone 0.25, acid hydrolyzed casein 0.5, yeast powder 0.5, soluble starch 0.5, K2HPO4.3H2O 0.3, MgSO4.7H2O
0.2, glucose 0.5, Sodium Pyruvate 0.3, peptone 0.25, water 1000mL, pH 7.2 ± 0.2.
2, the measurement of bacterium degradation imidacloprid ability
It takes the bacterial strain of above-mentioned purifying to line on R2A solid medium, is cultivated in 30 DEG C of incubators to growing single colonie.
With sterile toothpick picking individual colonies, it is inoculated in the 100mL conical flask of the fluid nutrient medium of R2A containing 20mL, 30 DEG C, under 220rpm
Shaken cultivation 16h, as seed liquor.Then, according to 1% inoculum concentration, transferred in the fluid nutrient medium of R2A containing 100mL
In 500mL conical flask, 30 DEG C, shaken cultivation 14h under 220rpm.For bacterium solution in 4 DEG C, 8000rpm is centrifuged 6min, collects thallus, with
It is washed twice afterwards with 30mL phosphate buffer (pH=7.0), bacterial sediment is resuspended in above-mentioned phosphate buffer and (contains
The imidacloprid of 120mg/L) and adjust OD600It is 5.Take the above-mentioned conversion of resting cells liquid of 2mL in the centrifuge tube of 50mL, with ventilative
After film sealing, centrifuge tube is placed in 30 DEG C, 220rpm shaking table shaken cultivation 96h.Using 1200 type HPLC instrument analysis detection of Agilent
Imidacloprid changes of contents.HPLC condition: mobile phase is 25% acetonitrile and 75% deionized water (containing 0.01% acetic acid), and flow velocity is
1mL/min；HPLC column is Agilent HC-C18 reversed-phase column (4.6 × 250mm, 5 μm), and column temperature is 30 DEG C；Detection wavelength is
269nm.The changes of contents of sample and the imidacloprid in 96h sample when comparing zero.
3, the identification of DG01 bacterial strain
There is the bacterial strain significantly reduced to carry out strain idenfication the HPLC peak area of above-mentioned imidacloprid, first is that using light after dyeing
Learn micro- sem observation thallus microscopic morphology；Second is that extracting the genomic DNA of bacterial strain, its 16S rRNA gene of PCR amplification, to amplification
Gene order carry out blastn analysis.DG01 single colonie is red, raised, smooth, round on solid R2A culture medium flat plate
The characteristic of bacteria of shape, toughness.DG01 colonial morphology and the monochromatic optical microscopy shape of crystal violet are as shown in Fig. 1.It mentions
It takes the method for genomic DNA and PCR amplification 16S rRNA gene as follows: using TakaRa MiniBEST Bacteria
3.0 genome extraction kit of Genomic DNA Extraction Kit Ver, and use Gram-negative
Genomic DNA is extracted in Bacteria cracking.Primer is that 16S rRNA gene PCR expands universal primer K1 and K2.K1 primer sequence
Are as follows: 5 '-AACTGAAGAGTTTGATCC-3 ' (SEQ ID No:1), K2 primer sequence are as follows: 5 '-
TAGGTTACCTTGTTGTTACGACTT-3 ' (SEQ ID No:2).Primer is closed by Sangon Biotech (Shanghai) Co., Ltd.
At.Reaction system: 10 × LA buffer, 2.0 μ L, MgCl2(25mmol/L)2.0μL、dNTP(2.5mmol/L)2.0μL、K1
(50mmol/L) 0.4 μ L, 0.4 μ L of K2 (50mmol/L), 11.0 μ L of deionized water, LA Taq enzyme (2U/ μ L) 0.2 μ L, DNA profiling
2.0 μ L, totally 20 μ L total reaction volume.PCR reaction condition are as follows: after 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 50s, 58 DEG C of annealing
40s, 72 DEG C of extension 2min, totally 30 recycle, then 72 DEG C of extension 10min.Gained sequence is by Sheng Gong bioengineering Co., Ltd
After sequencing, obtained 16S rRNA gene order (SEQ ID No:3) is in the Genbank of National Center for Biotechnology Information
Nucleic acid is carried out in database compares analysis.Comparison result shows, DG01 bacterial strain and thin layer bacterium Hymenobacter
The similitude of the 16S rRNA gene of latericoloratus reaches 99%.Morphologic observation and 16S rRNA gene sequencing
The result shows that DG01 bacterial strain is thin layer bacterium Hymenobacter latericoloratus.
DG01 bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on August 27th, 2018
The heart (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), classification naming are thin layer bacterium
Hymenobacter latericoloratus, deposit number are CGMCC 16346.
Embodiment 2: maltose promotes the resting cell degradation imidacloprid of thin layer bacterium CGMCC 16346
It takes the thin layer bacterium CGMCC 16346 of -80 DEG C of preservations to line on R2A solid medium, is trained in 30 DEG C of incubators
It supports for 24 hours to growing single colonie.According to " measurement of bacterium degradation imidacloprid ability " the method in embodiment 1, pyrrole is carried out
The Degrading experiment of worm quinoline.Co-metabolic substance (co-substrate) maltose is wherein added in one group of experimental group.Above-mentioned tranquillization is thin
After born of the same parents' conversion fluid transition 96h, sampled after supplying the moisture content of evaporation, after the filtering with microporous membrane in 0.22 μm of aperture, sample
Carry out HPLC analysis.The result shows that pyrrole in the experimental group (containing co-metabolic substance maltose) of inoculation thin layer bacterium CGMCC 16346
Worm quinoline residual quantity is 33.15mg/L (HPLC analyzes result as shown in fig. 2), and does not contain pair of co-metabolic substance maltose
It is 94.42mg/L according to imidacloprid content in group (HPLC analyzes result as shown in attached drawing 2C)；Experimental group imidacloprid concentration is reduced
81.88mg/L.The above results show the resting cell of thin layer bacterium CGMCC 16346 degradable 71.50% imidacloprid in 4d, and
Add the degradation that co-metabolic substance maltose promotes imidacloprid.
Embodiment 3: glucose promotes the resting cell degradation imidacloprid of thin layer bacterium CGMCC16346
Essentially identical with example 2, only co-metabolic substance is glucose.The result shows that inoculation thin layer bacterium CGMCC 16346
Experimental group (containing co-metabolic substance glucose) in Determination of Imidacloprid Residue amount be 42.72mg/L (HPLC analyze result such as attached drawing 2B
It is shown), imidacloprid concentration reduces 65.95mg/L.The result shows that the resting cell of thin layer bacterium CGMCC 16346 can in 96h
It degrades 60.98% imidacloprid.
Embodiment 4: Sodium Pyruvate promotes the resting cell degradation imidacloprid of thin layer bacterium CGMCC 16346
Essentially identical with example 2, only co-metabolic substance is Sodium Pyruvate.As shown in attached drawing 2D, it is inoculated with thin layer bacterium CGMCC
Determination of Imidacloprid Residue amount is 32.03mg/L in 16346 experimental group (containing co-metabolic substance Sodium Pyruvate)；Experimental group imidacloprid is dense
Degree reduces 83.63mg/L.The result shows that the resting cell of thin layer bacterium CGMCC 16346 is in degradable 72.63% imidacloprid of 96h.
Embodiment 5: the growth cell degradation imidacloprid of thin layer bacterium CGMCC 16346
It takes the thin layer bacterium CGMCC 16346 of -80 DEG C of preservations to line on R2A solid medium, is trained in 30 DEG C of incubators
It supports for 24 hours to growing single colonie.With toothpick picking individual colonies, it is inoculated in the 100mL conical flask of the fluid nutrient medium of R2A containing 20mL,
30 DEG C, shaken cultivation 16h under 220rpm, as seed liquor.Then, according to 1% inoculum concentration, transferred in containing 100mL
In the 500mL conical flask of R2A (imidacloprid containing 120mg/L) fluid nutrient medium, in 30 DEG C, shaken cultivation 48h under 220rpm, mend
Water sampling carries out HPLC analysis.The result shows that inoculation 16346 imidacloprid concentration of thin layer bacterium CGMCC reduces 31.93mg/L (HPLC
Result is analyzed as shown in attached drawing 2E), the growth cell of thin layer bacterium CGMCC 16346 is in degradable 22.33% imidacloprid of 2d.
Embodiment 6: maltose promotes the resting cell degradation thiacloprid of thin layer bacterium CGMCC16346
Essentially identical with the condition of example 2, substrate is thiacloprid, concentration 120mg/L, degradation time 4d, HPLC item
Mobile phase is the deionized water containing 0.01% acetic acid of 35% acetonitrile and 65% in part, and Detection wavelength 242nm, other conditions are same
The HPLC condition of imidacloprid.The result shows that inoculation 16346 experimental group of thin layer bacterium CGMCC (containing co-metabolic substance maltose) thiophene
Worm quinoline concentration reduces 41.19mg/L (it is as shown in Figure 3 C that HPLC analyzes result).The substrate thiacloprid control for not being inoculated with thallus has no
(HPLC analyzes result figure as illustrated in figure 3) is reduced, (HPLC analyzes result figure such as to the control group without containing co-metabolic substance maltose
Shown in 3B) degradation rate is only 7.27%, and the degradation rate of experimental group thiacloprid can reach 33.30%.
Embodiment 7: Sodium Pyruvate promotes the resting cell degradation thiacloprid of thin layer bacterium CGMCC16346
Essentially identical with the condition of example 6, substrate is thiacloprid, concentration 120mg/L, degradation time 4d, Co metabolism
Matrix is Sodium Pyruvate.As a result (it is as shown in Figure 3D that HPLC analyzes result) display thiacloprid reduces 27.54mg/L, the drop of thiacloprid
Solution rate is 22.22%.
Embodiment 8: the resting cell degradation Acetamiprid of thin layer bacterium CGMCC16346
Essentially identical with the condition of example 4, substrate is Acetamiprid, concentration 120mg/L, using propionic acid acid sodium as Co metabolism
Matrix.Mobile phase is the deionized water containing 0.01% acetic acid of 30% acetonitrile and 70% in HPLC condition, and Detection wavelength is
235nm, other conditions are the same as imidacloprid HPLC condition.As a result (it is as shown in Figure 4 C that HPLC analyzes result) shows that Acetamiprid subtracts after 4d
Few 20.42mg/L, the degradation rate of Acetamiprid are 15.76%, and not plus the Acetamiprid degradation rate of the experimental group of co-metabolic substance is only
4.92% (it is as shown in Figure 4 B that HPLC analyzes result) is not inoculated with the Substrate controls of thallus (HPLC analyzes result as shown in figure Fig. 4 A)
Acetamiprid degradation is not generated.The above results show the degradable Acetamiprid of resting cell of thin layer bacterium CGMCC 16346, and pyruvic acid
Sodium promotes its Acetamiprid of degrading.
Sequence table
<110>Ministry of Environmental Protection, Nanjing Environment Science Institute
Nanjing Normal University
<120>application of a kind of thin layer bacterium and its anabasine insecticide of degrading
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aactgaagag tttgatcc 18
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
taggttacct tgttgttacg actt 24
<210> 3
<211> 1421
<212> DNA
<213>thin layer bacterium (Hymenobacter latericoloratus)
<400> 3
gggtaagcgg cagctataca tgcaagtcga acgggtggtc gcaagaccac tagtggcgca 60
cgggtgcgta acgcgtaacc aacctgcccc gatctggggg atagcccgcc gaaaggcgga 120
ttaataccgc atatacccgc agcctggcat caggcaacgg ggaaagattt attggatcgg 180
gatggggttg cgtgacatta gctagttggc ggggtaacgg cccaccaagg cgacgatgtc 240
taggggacct gagagggtga tcccccacac tggcactgag atacgggcca gactcctacg 300
ggaggcagca gtagggaata ttgggcaatg ggcgagagcc tgacccagcc atgccgcgtg 360
ccggatgaag gccttctggg ttgtaaacgg cttttctcag ggaagaaaaa gaccttgcga 420
ggtacactga cggtacctga ggaataagca ccggctaact ccgtgccagc agccgcggta 480
atacggaggg tgcaagcgtt gtccggattt attgggttta aagggtgcgt aggcggcttg 540
ttaagtccgg ggtgaaagcc cactgctcaa cagtggaact gccctggata ctggcaagct 600
tgagtccaga cgaggttggc ggaatggatg gtgtagcggt gaaatgcata gataccatcc 660
agaaccccga ttgcgaaggc agctgactag gctggtactg acgctgaggc acgaaagcgt 720
ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatgg atactcgctg 780
gtggcgatag acagtcactg gcttagggaa accggtaagt atcccacctg gggagtacgc 840
tcgcaagagt gaaactcaaa ggaattgacg ggggcccgca caagtggtgg agcatgtggt 900
ttaattcgat gatacgcgag gaaccttacc taggctagaa tgcgcgtgac cggctcagag 960
atgagccttt ccttcgggac acaaagcaag gtgctgcatg gccgtcgtca gctcgtgccg 1020
tgaggtgttg ggttaagtcc cgcaacgagc gcaaccccta tgtttagttg ccatcaggtg 1080
atgctgggga ctctaaacag actgcctgcg caagcagtga ggaaggcggg gacgacgtca 1140
ggtcatcatg gcccttacgc ctagggctac acacgtgcta caatgggcgg tacagagggt 1200
cgctacctgg tgacaggatg ccaatctcaa aaaaccgttc tcagttcgga ttgaagtctg 1260
caactcgact tcatgaagct ggaatcacta gtaatcgcgt atcagccatg acgcggtgaa 1320
tacgttcccg ggccttgtac acaccgcccg tcaagccatg gaagtttggt agacctgaag 1380
ctggtgctcg tcacagaagc cagtaggtga gcaaatggcc t 1421
Claims (8)
1. a kind of thin layer bacterium, which is characterized in that be identified as thin layer bacterium Hymenobacter latericoloratus, bacterial strain name
For DG01, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC
No.16346, the deposit date is on August 27th, 2018.
2. the cultural method of thin layer bacterium described in claim 1, which is characterized in that the thin layer bacterium CGMCC 16346 to be inoculated with
In on R2A agar medium, cultivated in the incubator that temperature is 30 ± 1 DEG C；Or it is inoculated in R2A fluid nutrient medium, Yu Wen
Degree is shaken cultivation in 30 ± 1 DEG C of shaking table.
3. application of the thin layer bacterium described in claim 1 in degradation anabasine insecticide.
4. application of the thin layer bacterium according to claim 3 in degradation anabasine insecticide, which is characterized in that described new
Nicotinic insecticide is imidacloprid, thiacloprid or Acetamiprid.
5. application according to claim 3, which is characterized in that application method are as follows: meet the thin layer bacterium CGMCC 16346
Kind shaken cultivation, thin layer bacterium CGMCC 16346 in the nutrient medium containing anabasine insecticide degrade during the growth process
Anabasine insecticide.
6. application according to claim 3, which is characterized in that application method are as follows: by the thin layer bacterium CGMCC's 16346
Thallus is inoculated in liquid nutrient media, and somatic cells are collected after shaken cultivation；Somatic cells are suspended in containing anabasine
In the phosphate buffer solution of insecticide and co-metabolic substance, shaken cultivation, the resting cell degradation of thin layer bacterium CGMCC 16346
Anabasine insecticide.
7. application according to claim 5 or 6, which is characterized in that the nutrient medium is R2A fluid nutrient medium.
8. application according to claim 6, which is characterized in that the co-metabolic substance is glucose, maltose or acetone
Sour sodium.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811201349.6A CN109266582B (en) | 2018-10-16 | 2018-10-16 | Thin layer bacterium and application thereof in degrading neonicotinoid insecticide |
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| CN201811201349.6A CN109266582B (en) | 2018-10-16 | 2018-10-16 | Thin layer bacterium and application thereof in degrading neonicotinoid insecticide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110835618A (en) * | 2019-11-27 | 2020-02-25 | 南京国环环境研究院有限公司 | Rhodococcus ruber and application thereof in degrading nicotine pesticide |
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