CN109266582B - Thin layer bacterium and application thereof in degrading neonicotinoid insecticide - Google Patents
Thin layer bacterium and application thereof in degrading neonicotinoid insecticide Download PDFInfo
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- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
Abstract
The invention discloses a lamellar bacterium and application thereof in degrading neonicotinoid insecticides, the lamellar bacterium is identified as Hymenobacter lateriticformes with the strain name DG01, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No.16346, with preservation date of 2018, 8 months and 27 days. Compared with the prior pesticide degradation technology, the new strain of TLC 16346 can effectively degrade neonicotinoid insecticides, particularly imidacloprid, can be used as a pesticide degradation microbial inoculum, and has better application prospect, and both growing cells and resting cells of the TLC 16346 can effectively degrade neonicotinoid insecticides.
Description
Technical Field
The invention belongs to the technical field of microbial biology, and particularly relates to a TLC and application thereof in degrading neonicotinoid insecticides.
Background
The neonicotinoid insecticides are nitrogen heterocycle-containing insecticides, and have the action mode of selectively controlling nicotinic acid acetylcholinesterase receptors in insect nervous systems and blocking normal conduction of the insect central nervous systems, so that pests are paralyzed and die. Imidacloprid and acetamiprid are the main representative varieties of the insecticides, and are widely used for various crops such as rice, wheat, vegetables, fruit trees, tea trees, cotton, tobacco and the like. Neonicotinoid insecticides are also one of the main insecticide varieties in China. However, the long-term use of large amounts of neonicotinoid insecticides has serious influence on food safety and ecological environment, such as the problem of ecological chain caused by high toxicity of imidacloprid to powdery insects, bees and birds; imidacloprid and acetamiprid have residues in grains, vegetables and tea leaves, thereby creating food safety problems; 80-98% of the neonicotinoid insecticides finally enter the soil environment, causing pollution to the soil and surface water and threatening aquatic insects. In recent years, the influence of neonicotinoid insecticides on the environmental ecology has attracted global attention, and the environmental return thereof is highly regarded.
Microbial degradation is one of the main routes of soil metabolism of neonicotinoid insecticides and is also one of the most economical and effective methods for eliminating the environmental pollution of the insecticides. Strains which can degrade neonicotinoids pesticides reported at present include Bacillus alkalinitilis, Ensifer meliloti, Ensifer adhaerens CGMCC 6315, Leifsonia sp.PC-21, Klebsiella pneumoniae BCH1, Pseudomonas sp.1G, Pseudomonas aeruginosa CGMCC 6648, Stenotrophoromonas maltophilia CGMCC 1.1788, and Variovorax boronicumCGMCC 4969, but no report that the strain of the genus Hymenobacter degrades neonicotinoids pesticides is reported in the literature.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a thin layer bacterium and application thereof in degrading neonicotinoid insecticides.
The technical scheme is as follows: the invention provides a TLC (TLC) which is classified as TLC (Hymenobacter lateriticoloatus), the strain name is DG01, the TLC is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC No.16346, with preservation date of 2018, 8 months and 27 days.
The culture method of the lamellar bacteria comprises the following steps: inoculating the TLC CGMCC16346 to an R2A agar culture medium, and culturing in an incubator at the temperature of 30 +/-1 ℃; or inoculating into R2A liquid culture medium, and shake culturing at 30 + -1 deg.C in shaker.
The invention also provides application of the TLC bacteria in degrading neonicotinoid insecticides.
The neonicotinoid insecticide is imidacloprid, thiacloprid or acetamiprid.
Further preferably, the application method comprises the following steps: inoculating the TLC CGMCC16346 into a nutrient culture solution containing imidacloprid for shake culture, and degrading the imidacloprid by the TLC CGMCC16346 in the growth process.
Further preferably, the application method comprises the following steps: inoculating the TLC CGMCC16346 into a liquid nutrient medium, and centrifugally collecting and washing the thalli after shaking culture; the thalli is suspended in phosphate buffer solution containing imidacloprid, thiacloprid or acetamiprid, and is subjected to shaking culture, and resting cells of TLC CGMCC16346 degrade the imidacloprid, thiacloprid or acetamiprid.
The nutrient medium is preferably R2A medium.
The co-metabolism substrate is glucose, maltose and other saccharides or sodium pyruvate.
The technical effects are as follows: compared with the prior art, the new strain of the TLC bacteria CGMCC16346 can effectively degrade neonicotinoid insecticides, particularly imidacloprid, by both growing cells and resting cells. Therefore, the strain can be used as a pesticide degradation microbial inoculum and has good application prospect.
Drawings
FIG. 1 shows the colony morphology and microscopic morphology of the TLC bacteria CGMCC16346 plate of the invention.
FIG. 2 is an HPLC chart of imidacloprid degradation by the growing cells and resting cells of the TLC 16346 strain of the invention, wherein: a: transforming imidacloprid by resting cells taking maltose as a co-metabolism matrix; b: transforming imidacloprid by resting cells taking glucose as a co-metabolism matrix; c: transforming imidacloprid into resting cells without adding a co-metabolism matrix; d: transforming imidacloprid by resting cells taking sodium pyruvate as a co-metabolism matrix; e: growing cells degrade imidacloprid; f: substrate imidacloprid control (no inoculated cells).
FIG. 3 is an HPLC chart of the resting cells of TLCC 16346 of the invention degrading thiacloprid, wherein: a: thiacloprid substrate control (no inoculated cells); b: the resting cells without the co-metabolism matrix are used for degrading thiacloprid; c: adding resting cells taking maltose as a co-metabolism matrix to degrade thiacloprid; d: and (3) adding resting cells taking sodium pyruvate as a co-metabolism matrix to degrade thiacloprid.
FIG. 4 is an HPLC chart of the resting cells of TLCC 16346 for degrading acetamiprid, wherein: acetamiprid substrate control (no inoculated bacteria); b, degrading acetamiprid by resting cells without the co-metabolism matrix; c: resting cells added with sodium pyruvate as a co-metabolism matrix degrade acetamiprid.
Detailed Description
The TLC of the present invention CGMCC16346 and its application are described in detail with reference to the following specific examples.
Example 1: separation, purification, screening and identification of degradable imidacloprid microorganisms
1. Strain isolation
Collecting soil and water samples from south Jing Xixia region of Jiangsu, adding 2g soil sample or 2mL water sample into 18mL sterile water containing glass beads, oscillating for 2h, standing for 20min, diluting 100 μ L sample to 10-3And 10-4Then, the solid mineral salt culture medium plate containing 50mg/L imidacloprid was coated with the imidacloprid. The composition of the mineral salt culture medium is (g/L): KH (Perkin Elmer)2PO4 1.36，Na2HPO42.13，MgSO4·7H2O0.5 and 10mL of metal ion solution, pH 7.0. The composition of the metal ionic liquid is (g/L): CaCl2·2H2O 0.40,H3BO3 0.30,CuSO4·5H2O 0.04,FeSO4·7H2O 0.20，MnSO4·7H2O 0.40,NaMoO4·2H2O0.20, KI 0.10 and 10.0mL/L concentrated hydrochloric acid. After single bacteria grow on the solid mineral salt culture medium plate containing the imidacloprid, selecting bacterial colonies with different forms, streaking the bacterial colonies to a new imidacloprid mineral salt solid culture medium, culturing the solid plate in an incubator at 30 ℃ for 3 days, and streaking, separating and purifying the grown single bacteria on an R2A culture medium plate again. The formula of the 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，MgSO4.7H2O0.2, glucose 0.5, sodium pyruvate 0.3, peptone 0.25, water 1000mL, pH 7.2. + -. 0.2.
2. Determination of imidacloprid degradation ability of screened strains
The purified strain was streaked on R2A solid medium and cultured in an incubator at 30 ℃ until a single colony grew. A single colony was picked with a sterile toothpick, inoculated into a 100mL Erlenmeyer flask containing 20mL of R2A liquid medium, and shake-cultured at 30 ℃ and 220rpm for 16 hours to serve as a seed solution. Then, the cells were inoculated at an inoculum size of 1% into 500mL Erlenmeyer flasks containing 100mL of R2A liquid medium, and cultured at 30 ℃ and 220rpm for 14 hours with shaking. The bacterial suspension was centrifuged at 8000rpm for 6min at 4 ℃ to collect the bacterial cells, which were then washed twice with 30mL of phosphate buffer (pH 7.0), and the bacterial cell pellet was resuspended in the above phosphate buffer (containing 120mg/L imidacloprid) and OD was adjusted600Is 5. 2mL of the above resting cell transformation solution was put into a 50mL centrifuge tube, which was sealed with a gas-permeable membrane, and the centrifuge tube was placed at 30 ℃ and shaking-cultured for 96 hours at 220 rpm. And analyzing and detecting the imidacloprid content change by using an Agilent 1200 type HPLC instrument. HPLC conditions: the mobile phase is 25% acetonitrile and 75% deionized water (containing 0.01% acetic acid), and the flow rate is 1 mL/min; the HPLC column is Agilent HC-C18 reversed phase column (4.6 × 250mm, 5 μm), and the column temperature is 30 deg.C; the detection wavelength was 269 nm. The content change of imidacloprid in the zero time sample and the 96h sample is compared.
3. Identification of the DG01 Strain
Identifying strains of which the HPLC peak area of the imidacloprid is obviously reduced, wherein firstly, observing the microscopic morphology of the strains by using an optical microscope after dyeing; secondly, extracting genome DNA of the strain, amplifying 16S rRNA gene by PCR, and performing blastn analysis on the amplified gene sequence. The DG01 single colony on the solid R2A medium plate is characterized by red, convex, smooth, round, sticky bacteria. The colony morphology of DG01 and the shape of the optical microscope of the crystal violet single stain are shown in FIG. 1. The methods for extracting genomic DNA and PCR amplification of 16S rRNA gene were as follows: the Takara MiniBEST bacterial Genomic DNA Extraction Kit Ver 3.0 genome Extraction Kit was used, and Gram-negative bacterial lysis was used to extract Genomic DNA. The primers are 16S rRNA gene PCR amplification universal primers K1 and K2. The K1 primer sequence is: 5'-AACTGAAGAGTTTGATCC-3' (SEQ ID No: 1), the K2 primer sequence is: 5'-TAGGTTACCTTGTTGTTACGACTT-3' (SEQ ID No: 2). The primers were synthesized by Biotechnology engineering (Shanghai) Co., Ltd. Reaction system: 10 × LA buffer 2.0 μ L, MgCl22.0 μ L, dNTP (2.5mmol/L)2.0 μ L, K1(50mmol/L)0.4 μ L, K2(50mmol/L)0.4 μ L deionized water 11.0 μ L LA Taq enzyme (2U/. mu.L) 0.2 μ L, DNA template 2.0 μ L for a total reaction volume of 20 μ L. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 50s, annealing at 58 ℃ for 40s, extension at 72 ℃ for 2min for 30 cycles, and extension at 72 ℃ for 10 min. The obtained sequence was sequenced by Biotechnology engineering, Inc., and the obtained 16S rRNA gene sequence (SEQ ID No: 3) was analyzed by nucleic acid alignment in the Genbank database of the national center for Biotechnology information. The alignment showed that the strain DG01 had 99% similarity to the 16S rRNA gene of the TLC bacterium Hymenobacter lateriticcola. The results of morphological observation and 16S rRNA gene sequence analysis showed that the strain DG01 is a TLC bacterium, Hymenobacter lateriticcola.
The strain DG01 was deposited in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms (No. 3, institute of microbiology, China academy of sciences, West Lu 1, North Chen of the rising-oriented region, Beijing) on 27.8.2018, and was classified and named as TLC, Hymenobacter lateriticalis, with the deposit number of CGMCC 16346.
Example 2: imidacloprid degradation by resting cells of maltose-promoted lamellar bacteria CGMCC16346
The TLC CGMCC16346 preserved at-80 ℃ is streaked on an R2A solid culture medium and cultured in an incubator at 30 ℃ for 24h until a single colony grows out. The imidacloprid degradation assay was performed as described in example 1 "assay for screening strains for their ability to degrade imidacloprid". One group of the experimental groups was supplemented with co-metabolism substrate (co-substrate) maltose. After the resting cell transformation liquid is oscillated and transformed for 96 hours, evaporated water is replenished, then a sample is taken, and after being filtered by a microporous filter membrane with the aperture of 0.22 mu m, the sample is subjected to HPLC analysis. The result shows that the residual imidacloprid amount in the experimental group (containing co-metabolism matrix maltose) inoculated with the TLCC 16346 is 33.15mg/L (the HPLC analysis result is shown in figure 2A), while the imidacloprid content in the control group without the co-metabolism matrix maltose is 94.42mg/L (the HPLC analysis result is shown in figure 2C); the imidacloprid concentration of the experimental group is reduced by 81.88 mg/L. The results show that the resting cells of the TLC CGMCC16346 can degrade 71.50% of imidacloprid within 4 days, and the addition of the co-metabolism substrate maltose promotes the degradation of the imidacloprid.
Example 3: imidacloprid degradation by resting cells of glucose-promoted lamellar bacteria CGMCC16346
Essentially the same as example 2, except that the co-metabolic substrate was glucose. The result shows that the residual imidacloprid amount in the experimental group (containing co-metabolism matrix glucose) inoculated with the TLCC 16346 is 42.72mg/L (the HPLC analysis result is shown in figure 2B), and the imidacloprid concentration is reduced by 65.95 mg/L. The result shows that the resting cells of the TLC CGMCC16346 can degrade 60.98 percent of imidacloprid in 96 hours.
Example 4: sodium pyruvate promotes resting cells of TLC CGMCC16346 to degrade imidacloprid
Essentially the same as example 2, except that the co-metabolic substrate was sodium pyruvate. As shown in figure 2D, the imidacloprid residue in the experimental group inoculated with the TLC CGMCC16346 (containing co-metabolism substrate sodium pyruvate) is 32.03 mg/L; the imidacloprid concentration of the experimental group is reduced by 83.63 mg/L. The result shows that the resting cells of the TLC CGMCC16346 can degrade 72.63 percent of imidacloprid in 96 hours.
Example 5: imidacloprid degraded by growing cells of TLC (microorganism culture medium CGMCC 16346)
The TLC CGMCC16346 preserved at-80 ℃ is streaked on an R2A solid culture medium and cultured in an incubator at 30 ℃ for 24h until a single colony grows out. A single colony was picked with a toothpick, inoculated into a 100mL Erlenmeyer flask containing 20mL of R2A liquid medium, and shake-cultured at 30 ℃ and 220rpm for 16 hours to serve as a seed solution. Then, the cells were inoculated at a concentration of 1% into a 500mL Erlenmeyer flask containing 100mL of R2A (containing 120mg/L imidacloprid) liquid medium, cultured with shaking at 30 ℃ and 220rpm for 48 hours, and subjected to HPLC analysis by sampling with water. The result shows that the imidacloprid concentration of the inoculated TLC (CGMCC 16346) is reduced by 31.93mg/L (the HPLC analysis result is shown in figure 2E), and the growing cells of the TLC (CGMCC 16346) can degrade 22.33 percent of imidacloprid in 2 d.
Example 6: thiacloprid degradation by resting cells of maltose-promoted TLC (microorganism culture medium) CGMCC16346
Basically the same as the conditions of example 2, the substrate was thiacloprid, the concentration was 120mg/L, the degradation time was 4d, the mobile phase in HPLC conditions was 35% acetonitrile and 65% deionized water containing 0.01% acetic acid, the detection wavelength was 242nm, and other conditions were the same as those of imidacloprid HPLC. The result shows that the concentration of thiacloprid inoculated with the TLC CGMCC16346 experimental group (containing the co-metabolism substrate maltose) is reduced by 41.19mg/L (the HPLC analysis result is shown in figure 3C). The control of the substrate thiacloprid without inoculated thalli is not reduced (HPLC analysis result is shown in a figure of 3A), the degradation rate of the control group without co-metabolism matrix maltose (HPLC analysis result is shown in a figure of 3B) is only 7.27%, and the degradation rate of the thiacloprid in the experimental group can reach 33.30%.
Example 7 sodium pyruvate promotes the resting cells of TLC CGMCC16346 to degrade thiacloprid
In essentially the same conditions as in example 6, the substrate was thiacloprid, the concentration was 120mg/L, the degradation time was 4d, and the co-metabolic matrix was sodium pyruvate. The results (HPLC analysis is shown in FIG. 3D) show that thiacloprid is reduced by 27.54mg/L, and the degradation rate of thiacloprid is 22.22%.
Example 8 degradation of acetamiprid by resting cells of TLC CGMCC16346
In essentially the same conditions as in example 4, the substrate was acetamiprid at a concentration of 120mg/L with sodium propionate as the co-metabolism substrate. The mobile phase in HPLC condition is 30% acetonitrile and 70% deionized water containing 0.01% acetic acid, the detection wavelength is 235nm, and other conditions are the same as those of imidacloprid HPLC. The results (HPLC analysis results are shown in FIG. 4C) show that the acetamiprid is reduced by 20.42mg/L after 4 days, the degradation rate of the acetamiprid is 15.76%, the degradation rate of the acetamiprid in the experimental group without the co-metabolism matrix is only 4.92% (HPLC analysis results are shown in FIG. 4B), and the acetamiprid degradation is not generated in the substrate control without inoculated bacteria (HPLC analysis results are shown in FIG. 4A). The results show that the resting cells of the TLC CGMCC16346 can degrade the acetamiprid, and the sodium pyruvate can promote the degradation of the acetamiprid.
Sequence listing
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NANJING NORMAL University
<120> thin-layer bacterium and application thereof in degrading neonicotinoid insecticide
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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
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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
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Claims (7)
1. The TLC (Hymenobacter lateriticcola) DG01 is preserved in the China general microbiological culture Collection center with the preservation number as follows: CGMCC number 16346 with preservation date of 2018, 8 months and 27 days.
2. The method for culturing a TLC bacterium as set forth in claim 1, characterized in that the TLC bacterium CGMCC number 16346 is inoculated on an R2A agar medium and cultured in an incubator at a temperature of 30 ± 1 ℃; or inoculating into R2A liquid culture medium, and shake culturing in shaking table at 30 + -1 deg.C.
3. The use of a TLC bacterium of claim 1 to degrade a neonicotinoid insecticide, wherein the neonicotinoid insecticide is imidacloprid, thiacloprid or acetamiprid.
4. The application of claim 3, wherein the application method comprises: the thin-layer bacterium CGMCC number 16346 is inoculated into a nutrient culture solution containing the neonicotinoid insecticide and is subjected to shake culture, and the neonicotinoid insecticide is degraded by the thin-layer bacterium CGMCC number 16346 in the growth process.
5. The application of claim 3, wherein the application method comprises: inoculating the thallus of the TLC (thin-layer bacterium CGMCC number 16346) into a nutrient culture solution, and collecting thallus cells after shaking culture; the bacterial cells are suspended in phosphate buffer solution containing the neonicotinoid insecticide and the co-metabolism matrix, and are subjected to shaking culture, and the neonicotinoid insecticide is degraded by resting cells of the thin layer bacterium CGMCC number 16346.
6. The use according to claim 4 or 5, wherein the nutrient medium is R2A liquid medium.
7. Use according to claim 5, wherein the co-metabolic substrate is glucose, maltose or sodium pyruvate.
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