CN112625938A - Octreobacter xanthipes AVM-2 for degrading abamectin and application thereof in aquaculture - Google Patents
Octreobacter xanthipes AVM-2 for degrading abamectin and application thereof in aquaculture Download PDFInfo
- Publication number
- CN112625938A CN112625938A CN202011234533.8A CN202011234533A CN112625938A CN 112625938 A CN112625938 A CN 112625938A CN 202011234533 A CN202011234533 A CN 202011234533A CN 112625938 A CN112625938 A CN 112625938A
- Authority
- CN
- China
- Prior art keywords
- avm
- abamectin
- strain
- ochrobactrum
- degradation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000005660 Abamectin Substances 0.000 title claims abstract description 117
- IBSREHMXUMOFBB-JFUDTMANSA-N 5u8924t11h Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O3)C=C[C@H](C)[C@@H](C(C)C)O4)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 IBSREHMXUMOFBB-JFUDTMANSA-N 0.000 title claims abstract description 88
- 229950008167 abamectin Drugs 0.000 title claims abstract description 86
- 230000000593 degrading effect Effects 0.000 title claims abstract description 33
- 238000009360 aquaculture Methods 0.000 title claims abstract description 8
- 244000144974 aquaculture Species 0.000 title claims abstract description 8
- 230000015556 catabolic process Effects 0.000 claims abstract description 82
- 238000006731 degradation reaction Methods 0.000 claims abstract description 82
- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 claims abstract description 31
- 241000371997 Eriocheir sinensis Species 0.000 claims abstract description 26
- 210000000514 hepatopancreas Anatomy 0.000 claims abstract description 25
- 241000894006 Bacteria Species 0.000 claims abstract description 24
- 210000003205 muscle Anatomy 0.000 claims abstract description 19
- 239000000575 pesticide Substances 0.000 claims abstract description 18
- 241000588814 Ochrobactrum anthropi Species 0.000 claims abstract description 17
- 230000000877 morphologic effect Effects 0.000 claims abstract description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000002689 soil Substances 0.000 claims abstract description 5
- 241000588843 Ochrobactrum Species 0.000 claims description 34
- 230000000694 effects Effects 0.000 claims description 18
- 238000002474 experimental method Methods 0.000 claims description 17
- 238000011081 inoculation Methods 0.000 claims description 15
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 239000000447 pesticide residue Substances 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 230000007423 decrease Effects 0.000 claims description 7
- 230000003247 decreasing effect Effects 0.000 claims description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 6
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 5
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 claims description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 3
- 238000003794 Gram staining Methods 0.000 claims description 3
- 235000000177 Indigofera tinctoria Nutrition 0.000 claims description 3
- 229910002651 NO3 Inorganic materials 0.000 claims description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 230000003698 anagen phase Effects 0.000 claims description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 230000005540 biological transmission Effects 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 229940097275 indigo Drugs 0.000 claims description 3
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 241000371986 Eriocheir Species 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 6
- 238000000034 method Methods 0.000 description 4
- 241000238557 Decapoda Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000005034 decoration Methods 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- 240000008199 Rhododendron molle Species 0.000 description 1
- 241001233305 Xanthisma Species 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/04—Disinfection
Abstract
The application discloses a caneberry AVM-2 for degrading avermectin and application thereof in aquaculture, wherein a growing strain taking avermectin as a unique carbon source is separated and purified from bottom mud soil of a Chinese mitten crab culture pond polluted by pesticide, the strain takes the avermectin as the carbon source for growing, has degradation capacity and is named as AVM-2, and the caneberry AVM-2 belongs to the caneberry through morphological, physiological and biochemical characteristics and 16SrDNA identification; adding abamectin into Eriocheir sinensis culture water to make its initial concentration reach 5 mg/L, and adding separated waterDegrading bacteria AVM-2 to make the concentration of bacteria liquid in water body be 4.0X 106CFU/mL. In an experimental group added with the degrading strain, the residual quantity of the abamectin in the hepatopancreas and muscle tissues of the Eriocheir sinensis is obviously reduced, and the degradation rate of the ochrobactrum anthropi AVM-2 to the abamectin in the hepatopancreas and the muscle is over 60 percent.
Description
Technical Field
The application relates to the technical field of abamectin degradation in aquaculture, in particular to ochrobactrum anthropi AVM-2 for degrading abamectin and application thereof in aquaculture.
Background
Eriocheir sinensis (A. sinensis)Eriocheir sinemis) Also called river crab, is popular among people because of its delicious taste, rich nutritive value, simple cooking, etc. At present, the breeding quantity and the market demand of river crabs in China are increased at a high speed, and the problems of quality safety and the like are also concerned. In river crab cultivation, abamectin is used to remove wild fish and parasites, however, aquatic animals are sensitive to abamectin, and residual abamectin has a great influence on the growth and quality of river crabs.
The method aims at solving the problem of residual phytotoxicity of abamectin to aquatic crustaceans, takes the abamectin as a research object, enriches and screens out efficient abamectin degrading strains from soil, utilizes the morphological characteristics and physiological and biochemical characteristics of bacteria and combines 16S rDNA gene sequence analysis to identify the strains, researches the optimal culture conditions and the degradation characteristics of the degrading bacteria under pure culture conditions, and then adopts a laboratory culture experimental method to research the influence of the degrading bacteria on the abamectin residue in river crab culture, thereby aiming at reducing or eliminating the problem of the abamectin residue in aquatic economic animals. The experiment aims to reduce the problem of drug residue in the environment by screening the avermectin degrading bacteria and provides scientific basis for bioremediation of the environment.
Content of application
The technical problem to be solved is as follows:
the technical problem to be solved by the application is that residual abamectin in the prior art has great influence on the growth and the quality of river crabs, and the like, and the avibacteroid AVM-2 for degrading abamectin and the application thereof in aquaculture are provided to reduce or eliminate the residual abamectin in aquatic economic animals.
The technical scheme is as follows:
an avermectin-degrading ochrobactrum avium AVM-2 is characterized in that a growing strain taking avermectin as a unique carbon source is separated and purified from bottom mud soil of a Chinese mitten crab culture pond polluted by pesticide, the strain grows by taking the avermectin as the carbon source, has degrading capacity and is named as AVM-2, and the avermectin-degrading AVM-2 belongs to the avermectin avermitilis determined by morphological, physiological and biochemical characteristics and 16SrDNA identification.
The application also discloses an application of the ochrobactrum avium AVM-2 for degrading abamectin in aquaculture, which comprises the following steps:
the first step, the influence of the inoculation amount, the pH value, the temperature and the initial concentration of the pesticide on the degradation capability of the strain: when the bacterial strain of the ochrobactrum anthropi AVM-2 is in low bacterial quantity, the degradation efficiency of the avermectin is increased along with the increase of the inoculation quantity, and when the bacterial inoculation concentration is 4 multiplied by 106 When the concentration of the pesticide is increased, the degradation rate of the avermectin is reduced and tends to be stable, and the degradation rate of the ochrobactrum avium AVM-2 is gradually reduced along with the increase of the initial concentration of the pesticide;
the second step is that: when the concentration of the avermectin is 60 mg/L, the highest degradation rate of the ochrobactrum avium AVM-23 d is 33.59%, the degradation effect is inhibited along with the increase of the substrate concentration, and when the concentration of the avermectin is 240 mg/L, the 3d degradation rate is 25.71%;
thirdly, determining the influence of the pH value on the degradation rate of the ochrobactrum avium AVM-2: under the condition of strong acid or strong base, the degradation effect is poor, when the pH is 5, 6 and 10, the 3d degradation efficiency is respectively 8.64%, 11.23% and 10.06%, when the pH is 7, the 3d degradation rate is 33.15% at most, the degradation efficiency of the strain begins to show a reduction trend along with the environment is alkaline, and when the pH is 8, the degradation efficiency is remarkably reduced to 31.32%;
fourthly, determining the influence of the temperature on the degradation rate of the ochrobactrum anthropi AVM-2: the degradation effect is best under the condition of 30 ℃, and the 3d degradation rate is 33.46 percent at most. When the temperature is 20 ℃ and 25 ℃, the 3d degradation efficiency is 16.67 percent and 22.19 percent respectively, and the degradation efficiency is obviously reduced;
fifthly, the application of the avermectin degrading strain AVM-2 is as follows: by taking a group added with 5.0mg/L of abamectin as a contrast, the content of the abamectin in the hepatopancreas of the eriocheir sinensis shows the trend of increasing firstly and then decreasing secondly, at the 9 th day, the content of the abamectin in the hepatopancreas reaches the maximum value of 98.59 +/-16.21 mg/kg, then the content starts to decrease gradually, after the 18 th day, the residual amount of the abamectin in the hepatopancreas is below the detection limit, the addition amount of the abamectin is 5.0mg/L, and the added bacteria liquid is 4 multiplied by 106 In the CFU/mL experimental group, the content of the abamectin also shows a trend of increasing firstly and then decreasing, at the 9 th day, the content of the abamectin in the hepatopancreas reaches the maximum value of 36.77 +/-4.15 mg/kg, and then gradually decreases, and after 18 days, the residues of the abamectin in the hepatopancreas cannot be detected; the difference analysis shows that the pesticide residue in the experimental group added with the ochrobactrum anthropi AVM-2 is obviously reduced compared with the control group, the degradation rate of the ochrobactrum anthropi AVM-2 to the abamectin is more than 50 percent except the 3 rd, and the degradation rate reaches 65.99 percent at the 15 th day;
and a sixth step: the group with the addition of 5.0mg/L of abamectin is taken as a control group, the content of the abamectin in the muscle tissue of the eriocheir sinensis is detected to show a trend that the abamectin content is increased firstly and then reduced, the trend is consistent with the trend detected by pesticide residue in hepatopancreas, the content of the abamectin in the muscle tissue reaches 24.69 +/-1.02 mg/kg to the maximum at the 9 th day, the content of the abamectin in the muscle tissue starts to be reduced gradually, the minimum value is reached when an experiment is finished, the content of the abamectin is 0.82 +/-0.10 mg/kg, in the experiment group added with the pesticide and the Canalbium fulvidrum AVM-2, the content of the abamectin in the muscle of the eriocheir sinensis also shows a trend that the abamectin content is increased firstly and then reduced, and the difference analysis shows that the pesticide residue in the experiment group is obviously reduced compared with the control group after the degrading bacteria is.
As a preferred technical scheme of the application: the shape of the ochrobactrum AVM-2 strain on an LB solid culture medium is round, slightly convex, milky white and smooth in surface, the strain is rod-shaped and flagellum under the observation of a transmission electron microscope, and a gram-staining strain is gram-negative bacteria. The physiological and biochemical experiments show that the nitrate reduction of the strain is positive, and the indigo substrate, the V.P experiment, the ONPG, the sucrose, the raffinose, the sorbitol, the citrate and the xylose are negative.
As a preferred technical scheme of the application: the growth of the ochrobactrum avium AVM-2 strain is faster, the strain enters a logarithmic growth phase within 3 hours, and the strain enters a plateau phase within 15 hours.
As a preferred technical scheme of the application: the total DNA of AVM-2 strain is used as template, bacteria 16S rDNA universal primer is used for PCR amplification to obtain amplification product with length about 1500 bp, 16SrDNA sequence measured by strain is compared and analyzed in GenBank database by using BLAST, and the sequence is found to be matched with the sequenceOchrobatrumsp. the similarity of the gene sequence of the strain reaches 100%, and the molecular biology combines physiological, biochemical and morphological characteristics to confirm that the strain AVM-2 is the genus Ochrobactrum.
Has the advantages that:
compared with the prior art, the ochrobactrum avium AVM-2 for degrading abamectin and the application thereof adopt the technical scheme, and have the following technical effects:
1. the degrading bacteria can be used for the abamectin content in the aquatic products such as Eriocheir sinensis and the like, so that the food safety is improved;
2. separating and screening candidate bacterium AVM-2 for degrading abamectin from bottom mud of a Chinese mitten crab culture pond using abamectin, and considering that the strain belongs to the genus Ochrobactrum (Ochrobactrum) (III) through morphological observation, physiological and biochemical identification and 16S rDNA gene sequence analysisOchrobatrumsp.). The degradation characteristics of the strain are analyzed, the degradation characteristics of the strain are researched by a method of controlling single-factor variables, and the influence of different inoculation amounts, pH values, temperatures and initial concentrations of pesticides on the degradation capability of the strain is determined. The results show that the strain degradation effect is good when the inoculation pH is 7, the temperature is 30 ℃, the substrate concentration is low, and the inoculation amount is large;
3. the degradation effect of the strain AVM-2 on the abamectin is verified under the laboratory culture condition. Adding abamectin into the water body for cultivating Chinese mitten crab to make its initial concentration reach 5 mg/L, then adding separated degradation bacteria AVM-2 to make the concentration of bacteria liquid in water body be 4.0×106CFU/mL. The result shows that in the experimental group added with the degrading strain, the residual quantity of the avermectin in the hepatopancreas and muscle tissues of the eriocheir sinensis is obviously reduced, and the degrading rate of the ochrobactrum anthropi AVM-2 to the avermectin in the hepatopancreas and the muscle reaches more than 60 percent. The bacterial strain is proved to be capable of effectively reducing the residue of the abamectin in the eriocheir sinensis body.
Description of the drawings:
FIG. 1 is a graph showing the effect of the inoculum size of the present application on the degradation rate of the strain.
FIG. 2 is a graph showing the effect of temperature on the degradation rate of a strain according to the present invention.
FIG. 3 is a graph showing the effect of avermectin concentration on the degradation rate of a strain.
FIG. 4 is a graph showing the effect of the amount of inoculated ochrobactrum on the degradation rate of the strain.
FIG. 5 is a graph of the residual amount of avermectin in the hepatopancreas of Eriocheir sinensis of the present application.
FIG. 6 is a graph showing the residual amount of avermectin in the muscle of Eriocheir sinensis.
Detailed Description
The technical scheme of the invention is further explained in detail by combining the attached drawings:
it will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Example 1:
separating and purifying a growing strain taking abamectin as a unique carbon source from bottom mud soil of a Chinese mitten crab culture pond polluted by pesticide, wherein the strain grows by taking the abamectin as the carbon source, has the degradation capability and is named as AVM-2, and is determined by morphological, physiological and biochemical characteristics and 16SrDNA identificationIt belongs to ochrobactrum. Is preserved in China center for type culture Collection, Wuhan university, China, with the preservation number of CCTCC NO: m2019720, the preservation date is 2019, 9, 12 and the Latin classification of the biological material is namedOchrobatrum sp.AVM。
The application of the ochrobactrum avium AVM-2 for degrading the abamectin comprises the following steps:
the first step, the influence of the inoculation amount, the pH value, the temperature and the initial concentration of the pesticide on the degradation capability of the strain: when the bacterial strain of the ochrobactrum anthropi AVM-2 is in low bacterial quantity, the degradation efficiency of the avermectin is increased along with the increase of the inoculation quantity, and when the bacterial inoculation concentration is 4 multiplied by 106 When the concentration of the pesticide is increased, the degradation rate of the avermectin is reduced and tends to be stable, and the degradation rate of the ochrobactrum avium AVM-2 is gradually reduced along with the increase of the initial concentration of the pesticide;
the second step is that: when the concentration of the avermectin is 60 mg/L, the highest degradation rate of the ochrobactrum avium AVM-23 d is 33.59%, the degradation effect is inhibited along with the increase of the substrate concentration, and when the concentration of the avermectin is 240 mg/L, the 3d degradation rate is 25.71%;
thirdly, determining the influence of the pH value on the degradation rate of the ochrobactrum avium AVM-2: under the condition of strong acid or strong base, the degradation effect is poor, when the pH is 5, 6 and 10, the 3d degradation efficiency is respectively 8.64%, 11.23% and 10.06%, when the pH is 7, the 3d degradation rate is 33.15% at most, the degradation efficiency of the strain begins to show a reduction trend along with the environment is alkaline, and when the pH is 8, the degradation efficiency is remarkably reduced to 31.32%;
fourthly, determining the influence of the temperature on the degradation rate of the ochrobactrum anthropi AVM-2: the degradation effect is best under the condition of 30 ℃, and the 3d degradation rate is 33.46 percent at most. When the temperature is 20 ℃ and 25 ℃, the 3d degradation efficiency is 16.67 percent and 22.19 percent respectively, and the degradation efficiency is obviously reduced;
fifthly, the application of the avermectin degrading strain AVM-2 is as follows: taking the group added with 5.0mg/L of abamectin as a contrast, the content of the abamectin in the hepatopancreas of the Eriocheir sinensis shows the trend of increasing firstly and then decreasing, at the 9 th day, the content of the abamectin in the hepatopancreas reaches the maximum value of 98.59 +/-16.21 mg/kg, and then the content of the abamectin in the hepatopancreas starts to gradually increaseGradually decreases until 18 d, the residual amount of abamectin in the liver and pancreas is below the detection limit, the addition amount of abamectin is 5.0mg/L, and the added bacteria liquid is 4 multiplied by 106 In the CFU/mL experimental group, the content of the abamectin also shows a trend of increasing firstly and then decreasing, at the 9 th day, the content of the abamectin in the hepatopancreas reaches the maximum value of 36.77 +/-4.15 mg/kg, and then gradually decreases, and after 18 days, the residues of the abamectin in the hepatopancreas cannot be detected; the difference analysis shows that the pesticide residue in the experimental group added with the ochrobactrum anthropi AVM-2 is obviously reduced compared with the control group, the degradation rate of the ochrobactrum anthropi AVM-2 to the abamectin is over 50 percent except for 3 days, and the degradation rate reaches 65.99 percent at 15 days;
and a sixth step: the group with the addition of 5.0mg/L of abamectin is taken as a control group, the content of the abamectin in the muscle tissue of the eriocheir sinensis is detected to show a trend of increasing firstly and then decreasing, which is consistent with the trend detected by pesticide residue in hepatopancreas, the content of the abamectin in the muscle tissue reaches 24.69 +/-1.02 mg/kg to the maximum at the 9 th day, the content of the abamectin in the muscle tissue starts to decrease gradually, reaches the minimum value at the end of an experiment, and is 0.82 +/-0.10 mg/kg, in the experiment group with the pesticide and the ochrobactrum xanthium AVM-2, the content of the abamectin in the muscle of the eriocheir sinensis also shows a trend of increasing firstly and then decreasing, and the difference analysis shows that the pesticide residue in the experiment group is obviously reduced compared with the control group after the degrading bacteria is added, and the degradation efficiency is highest and reaches 66.78% at the 15 th day
The shape of the ochrobactrum AVM-2 strain on an LB solid culture medium is round, slightly convex, milky white and smooth in surface, the strain is rod-shaped and flagellum under the observation of a transmission electron microscope, and a gram-staining strain is gram-negative bacteria. The physiological and biochemical experiments show that the nitrate reduction of the strain is positive, and the indigo substrate, the V.P experiment, the ONPG, the sucrose, the raffinose, the sorbitol, the citrate and the xylose are negative.
The growth of the ochrobactrum avium AVM-2 strain is faster, the strain enters a logarithmic growth phase within 3 hours, and the strain enters a plateau phase within 15 hours.
The method comprises the steps of taking the total DNA of the AVM-2 strain as a template, carrying out PCR amplification by using a bacterial 16S rDNA universal primer to obtain an amplification product with the length of about 1500 bp, carrying out comparison analysis on a 16SrDNA sequence measured by the strain in a GenBank database by using BLAST, finding that the similarity of the sequence and a gene sequence of an Ochrobatrum sp strain reaches 100%, and confirming that the AVM-2 strain is Ochrobactrum by combining the molecular biology with the physiological biochemistry and morphological characteristics.
As shown in figures 1-4, a candidate bacterium AVM-2 for degrading abamectin is separated and screened from bottom mud of a eriocheir sinensis culture pond using abamectin, and the strain is considered to belong to the genus Ochrobactrum (Ochrobactrum species) (through morphological observation, physiological and biochemical identification and 16S rDNA gene sequence analysis)Ochrobatrumsp.). The degradation characteristics of the strain are analyzed, the degradation characteristics of the strain are researched by a method of controlling single-factor variables, and the influence of different inoculation amounts, pH values, temperatures and initial concentrations of pesticides on the degradation capability of the strain is determined. The results show that the strain degradation effect is good when the inoculation pH is 7, the temperature is 30 ℃, the substrate concentration is low, and the inoculation amount is large.
As shown in figures 5 and 6, the effect of the strain AVM-2 on the degradation of abamectin is verified under the condition of laboratory culture. Adding abamectin into Eriocheir sinensis culture water to make its initial concentration reach 5 mg/L, adding separated degrading bacteria AVM-2 to make the bacteria liquid concentration in water 4.0 × 106CFU/mL. The result shows that in the experimental group added with the degrading strain, the residual quantity of the avermectin in the hepatopancreas and muscle tissues of the eriocheir sinensis is obviously reduced, and the degrading rate of the ochrobactrum anthropi AVM-2 to the avermectin in the hepatopancreas and the muscle reaches more than 60 percent. The bacterial strain is proved to be capable of effectively reducing the residue of the abamectin in the eriocheir sinensis body.
The foregoing is only a partial embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. An ochrobactrum avium AVM-2 for degrading abamectin is characterized in that: a strain which takes avermectin as a unique carbon source and grows is separated and purified from bottom mud soil of a Chinese mitten crab culture pond polluted by pesticide, the strain grows by taking the avermectin as the carbon source, has the degradation capability and is named as AVM-2, and the strain is determined to belong to the ochrobactrum through morphological, physiological and biochemical characteristics and 16SrDNA identification.
2. The application of the ochrobactrum avium AVM-2 for degrading abamectin in aquaculture, which is characterized by comprising the following steps:
the first step, the influence of the inoculation amount, the pH value, the temperature and the initial concentration of the pesticide on the degradation capability of the strain: when the bacterial strain of the ochrobactrum anthropi AVM-2 is in low bacterial quantity, the degradation efficiency of the avermectin is increased along with the increase of the inoculation quantity, and when the bacterial inoculation concentration is 4 multiplied by 106 When the concentration of the pesticide is increased, the degradation rate of the avermectin is reduced and tends to be stable, and the degradation rate of the ochrobactrum avium AVM-2 is gradually reduced along with the increase of the initial concentration of the pesticide;
the second step is that: when the concentration of the avermectin is 60 mg/L, the highest degradation rate of the ochrobactrum avium AVM-23 d is 33.59%, the degradation effect is inhibited along with the increase of the substrate concentration, and when the concentration of the avermectin is 240 mg/L, the 3d degradation rate is 25.71%;
thirdly, determining the influence of the pH value on the degradation rate of the ochrobactrum avium AVM-2: under the condition of strong acid or strong base, the degradation effect is poor, when the pH is 5, 6 and 10, the 3d degradation efficiency is respectively 8.64%, 11.23% and 10.06%, when the pH is 7, the 3d degradation rate is 33.15% at most, the degradation efficiency of the strain begins to show a reduction trend along with the environment is alkaline, and when the pH is 8, the degradation efficiency is remarkably reduced to 31.32%;
fourthly, determining the influence of the temperature on the degradation rate of the ochrobactrum anthropi AVM-2: the degradation effect is best under the condition of 30 ℃, the 3d degradation rate is 33.46 percent at most, when the temperature is 20 ℃ and 25 ℃, the 3d degradation efficiency is 16.67 percent and 22.19 percent respectively, and the degradation efficiency is obviously reduced;
fifthly, the application of the avermectin degrading strain AVM-2 is as follows: using the group added with 5.0mg/L of abamectin as a reference, the abamectin in the hepatopancreas of the Eriocheir sinensisThe content of abamectin in the hepatopancreas reaches the maximum value of 98.59 +/-16.21 mg/kg at the 9 th day, and then gradually decreases until the 18 th day, the residual amount of abamectin in the hepatopancreas is below the detection limit, the addition amount of abamectin is 5.0mg/L, and the addition amount of bacteria liquid is 4 multiplied by 106 In the CFU/mL experimental group, the content of the abamectin also shows a trend of increasing firstly and then decreasing, at the 9 th day, the content of the abamectin in the hepatopancreas reaches the maximum value of 36.77 +/-4.15 mg/kg, and then gradually decreases, and after 18 days, the residues of the abamectin in the hepatopancreas cannot be detected; the difference analysis shows that the pesticide residue in the experimental group added with the ochrobactrum anthropi AVM-2 is obviously reduced compared with the control group, the degradation rate of the ochrobactrum anthropi AVM-2 to the abamectin is more than 50 percent except the 3 rd, and the degradation rate reaches 65.99 percent at the 15 th day;
and a sixth step: the group with the addition of 5.0mg/L of abamectin is taken as a control group, the content of the abamectin in the muscle tissue of the eriocheir sinensis is detected to show a trend that the abamectin content is increased firstly and then reduced, the trend is consistent with the trend detected by pesticide residue in hepatopancreas, the content of the abamectin in the muscle tissue reaches 24.69 +/-1.02 mg/kg to the maximum at the 9 th day, the content of the abamectin in the muscle tissue starts to be reduced gradually, the minimum value is reached when an experiment is finished, the content of the abamectin is 0.82 +/-0.10 mg/kg, in the experiment group added with the pesticide and the Canalbium fulvidrum AVM-2, the content of the abamectin in the muscle of the eriocheir sinensis also shows a trend that the abamectin content is increased firstly and then reduced, and the difference analysis shows that the pesticide residue in the experiment group is obviously reduced compared with the control group after the degrading bacteria is.
3. The ochrobactrum avium AVM-2 for degrading abamectin according to claim 1, wherein the avermectin-degrading AVM-2 is prepared from the following raw materials in percentage by weight: the shape of the strain of the ochrobactrum anthropi AVM-2 on an LB solid culture medium is round, slightly convex, milky white and smooth in surface, the strain is rod-shaped and flagellated when observed under a transmission electron microscope, and a gram-staining strain is gram-negative bacteria;
the physiological and biochemical experiments show that the nitrate reduction of the strain is positive, and the indigo substrate, the V.P experiment, the ONPG, the sucrose, the raffinose, the sorbitol, the citrate and the xylose are negative.
4. The ochrobactrum avium AVM-2 for degrading abamectin according to claim 1, wherein the avermectin-degrading AVM-2 is prepared from the following raw materials in percentage by weight: the growth of the ochrobactrum avium AVM-2 strain is faster, the strain enters a logarithmic growth phase within 3 hours, and the strain enters a plateau phase within 15 hours.
5. The ochrobactrum avium AVM-2 for degrading abamectin according to claim 1, wherein the avermectin-degrading AVM-2 is prepared from the following raw materials in percentage by weight: the total DNA of AVM-2 strain is used as template, bacteria 16S rDNA universal primer is used for PCR amplification to obtain amplification product with length about 1500 bp, 16SrDNA sequence measured by strain is compared and analyzed in GenBank database by using BLAST, and the sequence is found to be matched with the sequenceOchrobatrumsp. the similarity of the gene sequence of the strain reaches 100%, and the molecular biology combines physiological, biochemical and morphological characteristics to confirm that the strain AVM-2 is the genus Ochrobactrum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011234533.8A CN112625938A (en) | 2020-11-07 | 2020-11-07 | Octreobacter xanthipes AVM-2 for degrading abamectin and application thereof in aquaculture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011234533.8A CN112625938A (en) | 2020-11-07 | 2020-11-07 | Octreobacter xanthipes AVM-2 for degrading abamectin and application thereof in aquaculture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112625938A true CN112625938A (en) | 2021-04-09 |
Family
ID=75304361
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011234533.8A Pending CN112625938A (en) | 2020-11-07 | 2020-11-07 | Octreobacter xanthipes AVM-2 for degrading abamectin and application thereof in aquaculture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112625938A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117736938A (en) * | 2024-02-07 | 2024-03-22 | 南京万瑞环境科技有限公司 | Microorganism composite flora and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928688A (en) * | 2010-06-21 | 2010-12-29 | 南京农业大学 | Abamectin pesticide residue degrading bacteria and bactericide thereof |
| CN102732455A (en) * | 2012-06-05 | 2012-10-17 | 山东省科学院生物研究所 | Avermectin pesticide residual degrading bacterium, and microbial inoculum produced thereby |
-
2020
- 2020-11-07 CN CN202011234533.8A patent/CN112625938A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928688A (en) * | 2010-06-21 | 2010-12-29 | 南京农业大学 | Abamectin pesticide residue degrading bacteria and bactericide thereof |
| CN102732455A (en) * | 2012-06-05 | 2012-10-17 | 山东省科学院生物研究所 | Avermectin pesticide residual degrading bacterium, and microbial inoculum produced thereby |
Non-Patent Citations (2)
| Title |
|---|
| 张卫卫: "阿维菌素在模拟水产养殖生态系统中的蓄积与消除规律", 《中国水产科学》 * |
| 胡秀虹: "阿维菌素降解菌AW1-12的筛选与分类鉴定", 《西南农业学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117736938A (en) * | 2024-02-07 | 2024-03-22 | 南京万瑞环境科技有限公司 | Microorganism composite flora and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676756B (en) | Bei Laisi bacillus and its application as aquatic pathogenic bacterium inhibitor | |
| CN109897803B (en) | Aquatic probiotic and preparation method and application thereof | |
| CN111394272B (en) | Brevibacillus laterosporus and application thereof | |
| Pang et al. | Dynamic changes of total bacteria and Vibrio in an integrated seaweed–abalone culture system | |
| CN104328075B (en) | Bacillus subtilis strain capable of killing algae and application thereof | |
| CN108004182A (en) | One plant of lactobacillus paracasei and its application in aquaculture | |
| CN106967647B (en) | Pseudoalteromonas foli and application method thereof | |
| CN112625938A (en) | Octreobacter xanthipes AVM-2 for degrading abamectin and application thereof in aquaculture | |
| CN107964524A (en) | A kind of Lactococcus lactis HKS2 and its separating screening method and application with lactic acid activity | |
| CN108676734B (en) | Entomopathogenic fungi and application thereof | |
| CN107937301B (en) | Bacillus amyloliquefaciens and application thereof in aquaculture | |
| CN108048412A (en) | Fragility Vibriophage ValLY-4 and the bactericidal composition comprising the bacteriophage and its application | |
| CN108070570A (en) | Fragility Vibriophage ValDsh-1 and the bactericidal composition comprising the bacteriophage and its application | |
| CN107779422A (en) | One plant height effect suppresses the Leclercia adecarboxylata biocontrol bacterial strain of aspergillus flavus synthesis aflatoxin | |
| CN105907664A (en) | Bacillus amyloliquefaciens PSBHY-3 used for organic phosphorus decomposition in aquaculture pond and application thereof | |
| CN114717149B (en) | South-sea deep-sea fish-source heterologous alkane-eating bacterium AXMZ1 and application thereof | |
| CN114717140A (en) | Bacillus licheniformis and application thereof | |
| JP2006158396A (en) | Novel lactic acid bacterium strain for producing cactus lactic acid fermentation product and method for producing cactus fermentation product by using the same | |
| CN110713955A (en) | Lactic acid bacteria and application thereof in aquaculture | |
| CN108048340B (en) | Bacillus cereus and application thereof | |
| CN113249272B (en) | Novel marine-source halotolerant zoogloea forming strain and application thereof | |
| CN117070418B (en) | Acetylmicrobacterium strain with high salt tolerance, microbial agent and application thereof | |
| JP4469941B2 (en) | Methods and structures for the control of pathogenic microorganisms and organic matter degradation in fish farming | |
| CN116240123B (en) | Paenibacillus polymyxa, microecological preparation and preparation method thereof | |
| CN108048409A (en) | Fragility Vibriophage ValLY-3 and the bactericidal composition comprising the bacteriophage and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210409 |
|
| RJ01 | Rejection of invention patent application after publication |