CN103122316B - Phlebia acerina strain and application thereof in degrading metalaxyl pesticide residue - Google Patents
Phlebia acerina strain and application thereof in degrading metalaxyl pesticide residue Download PDFInfo
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- CN103122316B CN103122316B CN201210506098.9A CN201210506098A CN103122316B CN 103122316 B CN103122316 B CN 103122316B CN 201210506098 A CN201210506098 A CN 201210506098A CN 103122316 B CN103122316 B CN 103122316B
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Abstract
The invention relates to phlebia acerina strain and application thereof in degrading metalaxyl pesticide residue, and belongs to the biological technical field of organisms. The strain is utilized for degrading high-concentration metalaxyl pesticide residue. According to the identification, the scientific name of the white-rot fungi is phlebia acerina Y3667 which is stored in the Common Organism Center of the China Committee for Culture Collection of Microorganisms, wherein the storage number is CGMCC 6809; the ITS (Internal Transcribed Spacers) sequences are extracted by the DNA, cloned and measured; compared with the stored sequences in GenBank, the homology of the phlebia acerina strain with the phlebia acerina strain BMC3014 is 99%. The strain is cultivated in liquid medium to obtain microbial inoculum; and the microbial inoculum is added with the high-concentration metalaxyl pesticide for effectively degrading the metalaxyl pesticide residue. The degrading microbial inoculum generated by the technical scheme in the invention can be used for effectively degreasing the metalaxyl pesticide on the surface of water and agricultural products, so that the ecological environment is protected and restored.
Description
Technical field
The invention belongs to environmental microorganism field, is to utilize method of microorganism degraded chemical pesticide residual, relates to a strain maple and penetrates arteries and veins lead fungi bacterial strain Phlebia acerina and its application of application in high density metaxanin chemical residual degradation thereof.
Background technology
That chemical pesticide has is applied widely, controlling object is many, production cost is low, prevention effect is good and economic benefit advantages of higher, and it is applicable development that has greatly promoted modern agriculture extensively, has improved efficiency and the mechanization degree of agriculture production.But inevitably, a large amount of use agricultural chemicals kill and wound Natural Enemies of Insects and beneficial microorganism, make harmful organism produce resistance, cause people, animal poisoning, easily plant is produced to the problems such as poisoning, chemical pesticide in entered environment, a part can be passed through the effect of solar radiation, Soil Microorganism etc., and progressively degraded was lost efficacy; Another part finally can pass through the effect of " food chain ", enters human body, to artificial growth stage harm.All kinds of chemical pesticides extensively enter in the physical environments such as global air and atmospheric fallout, water body, soil, bed mud level organism at present, become immanent pollutent in environment, therefore explore the important research direction that the method that pesticide residue are carried out to effectively degrade becomes researchist's long-term endeavour.And in this area research, the use microorganism of extensively rising in recent years carries out biological degradation to pesticide residue becomes hot fields, by microbial process, agricultural chemicals macromole is resolved into micromolecular compound, and make its loss of activity.Due to features such as microorganism individuality are little, breeding rapid, specific surface is large.They more easily conform than other biology, and can produce novel bacterial by spontaneous mutation, and produce new enzyme system, there is new metabolic function, thereby can participate in degraded and transformation to artificial new synthetic compound, so microorganism has great potential to degrading pesticide residues.White-rot fungi is that a class can be grown on wood substrates, cause a class fungi of timber white rot, except xylogen, Mierocrystalline cellulose and the hemicellulose that can degrade in timber, the multiple environmental pollutant of can also effectively degrading, the synthetic chemical pesticide that comprises current many types, the enzyme systems such as the laccase of secreting in its process of growth, manganese peroxidase and lignin peroxidase can these environmental pollutant of non-specific degraded.White-rot fungi is of a great variety, and its degradation capability to environmental pollutants also varies, and therefore obtains the efficient degrading bacterial strain of chemical pesticide and applies to practice, to reducing pesticide residue, the harm of environment is had to realistic meaning.
This patent is the high-efficiency degradation oomycetes sterilant that separation obtains from the corruption wood of virgin forest---the white-rot fungi maple of metaxanin is penetrated arteries and veins lead fungi Phlebia acerina Y3667, experiment shows that this bacterium can utilize metaxanin to grow as sole carbon source and the energy in water agar, and makes metaxanin degraded; Adding under the liquid culture condition of exogenous nutrition thing, this bacterium 48h can degrade the high density metaxanin of sneaking into 5mg/L in substratum more than 82%, and within the scope of wider pH, all has good degradation effect.Absolutely prove that this bacterium can be for the biological degradation of the metaxanin of water body and remained on surface.There is no at present the bibliographical information that utilizes the research of Phlebia acerina degraded metaxanin pesticide residue.
Summary of the invention
The object of this invention is to provide that a strain maple is penetrated arteries and veins lead fungi bacterial strain and this bacterial strain of application in high density metaxanin chemical residual degradation has the biodegradable industrial applications prospect at the metaxanin of water body pesticide residue and remained on surface.
One strain maple is penetrated arteries and veins lead fungi bacterial strain, this bacterial strain called after maple is penetrated arteries and veins lead fungi (Phlebia acerina) and is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation date be on November 15th, 2012 deposit number be: CGMCC6809.
2. by maple claimed in claim 1, penetrate arteries and veins lead fungi bacterial strain, it is characterized in that: bacterium colony is flocculence, oyster white; Bacterium colony is comprised of generative hyphae, and generative hyphae thin-walled arrives slightly heavy wall, diameter 1.8-6 micron, and many simple separations, normal branch, mycelia content has in the blue reagent of cotton has a liking for indigo plant reaction, unchanged in potassium iodide reagents; Plate is cultivated 4 centimetres of 3 days colony diameters;
The ITS rDNA sequence of this bacterial strain has been submitted to GenBank database (GenBank accession number: KC218570), find that the gene order homology of this sequence and Phlebia acerina bacterial strain BMC3014 reaches 99%.
The culturing process that described maple is penetrated arteries and veins lead fungi bacterial strain is: described maple is penetrated to arteries and veins lead fungi bacterial strain Phlebia acerina Y3667 mycelium and be inoculated on 60 millimeters of plate substratum of diameter, at 28-30 ℃, 3-5 days grows on plate substratum; Scalper scraping mycelia, in access liquid nutrient medium, the mycelium inoculation of 2 culture dish scrapings is in 100ml nutrient solution, in 28-30 ℃, shaking culture 3-5 days under 140rmp/min speed conditions, the nutrient solution of the outer degrading enzyme of born of the same parents that acquisition comprises its secretion, it is the mix bacterium agent of mycelia and bacterium liquid;
Described plate substratum consists of Fructus Hordei Germinatus and soaks powder 10g, and agar 18g, adds water to 1L, pH nature;
Described liquid nutrient medium consists of Fructus Hordei Germinatus and soaks powder 10g, potassium primary phosphate 0.5g, CuSO
40.1g, CaCl
20.01g, MnSO
40.01g, pH nature, adds water and is settled to 1L.
In the nutrient solution 100ml cultivating through claim 4, add the metaxanin agricultural chemicals of ultimate density 5-10mg/L, in 30 ℃, rotating speed 140rmp/min, shaking table concussion is cultivated, and incubation time is more than 48 hours.
Described maple is penetrated arteries and veins lead fungi bacterial strain in the application of degraded metaxanin pesticide residue.
Carry out as follows:
(1) in the nutrient solution 100-150ml cultivating through claim 3, the metaxanin agricultural chemicals that adds final concentration 5-10mg/L, in 28-30 ℃, rotating speed 140-160rmp/min, shaking table concussion is cultivated, with the metaxanin agricultural chemicals that adds equal concentrations in the nutrient solution without inoculation as a control group;
(2) every 12h sampling once, get 2-3ml sample and be placed in 50mL plastic centrifuge tube, add acetonitrile 10-12mL, vibration 20-30min, centrifugal, take out supernatant liquor, repeat 3 times, merge supernatant liquor, extract the metaxanin pesticide residue in nutrient solution, through Fo Luoli tripoli Solid-Phase Extraction column purification, at 38-40 ℃, Rotary Evaporators is concentrated, lyophilize at-20 ℃, then with a small amount of acetonitrile dissolving, carry out gas chromatographic analysis, calculate degradation rate.
Its degradation rate (%)=(A-B)/A * 100,
Wherein A is control group metaxanin pesticide residue values,
B is the metaxanin pesticide residue value after degradation bacterial agent degraded.
The maple of take is penetrated arteries and veins lead fungi as bacterial classification, take sterilant as supplementary carbon source, degraded metaxanin.
Advantage of the present invention is: this patent is the high-efficiency degradation oomycetes sterilant that separation obtains from the corruption wood of virgin forest---the white-rot fungi bacterial strain maple of metaxanin is penetrated arteries and veins lead fungi Phlebiaacerina Y3667.It is that a strain has high vigor that this maple is penetrated arteries and veins lead fungi, and its cultural method is simple, and fast growth is difficult for variation, and experiment shows that this bacterium can utilize metaxanin to grow as sole carbon source and the energy in water agar, and makes metaxanin degraded; Adding under the liquid culture condition of exogenous nutrition thing, this bacterium 48h can be by the metaxanin degraded of sneaking into 5mg/L in substratum more than 82%, and within the scope of wider pH, all there is good degradation effect, absolutely prove that this bacterium can be for the biological degradation of the metaxanin of water body and remained on surface, there is the biodegradable industrial applications prospect of pesticide residue, also can be used as the type strain of research white-rot fungi to metaxanin chemical residual degradation mechanism.
Embodiment
The separation from the rotten wood of virgin forest of the bacterial strain of degraded metaxanin pesticide residue provided by the present invention obtains.This bacterium separation method is as follows:
By specific examples, describe enforcement of the present invention in detail below, object is to help reader to understand better spirit of the present invention, but not as the restriction to the scope of the present invention.
1. substratum preparation:
(1) culture presevation substratum (solid, 1L): Fructus Hordei Germinatus soaks powder 10g, glucose 10g, agar 18g, adds water to 1L, pH value nature;
(2) strain activation and culture base (solid, 1L): Fructus Hordei Germinatus soaks powder 10g, agar 18g, adds water to 1L, pH value nature;
(3) liquid nutrient medium (liquid, 1L): Fructus Hordei Germinatus soaks powder 10g, potassium primary phosphate 0.5g, CuSO
40.1g, CaCl
20.01g, MnSO
40.01g, pH nature, adds water and is settled to 1L.
The all 121 ℃ of sterilizings 25 minutes in pressure kettle of above substratum.
2. actication of culture: the mycelia of choosing degradation bacteria, be inoculated in the 60mm culture dish that is added with culture presevation substratum, in 28 ℃, cultivate 4 days, then use scalpel scraping media surface mycelia, connect bacterium and enter in liquid nutrient medium, in 30 ℃, shaking culture 4-5 days under 140 revs/min of speed conditions, the nutrient solution of the outer degrading enzyme of born of the same parents that acquisition comprises its secretion; The microbial inoculum of being produced by the technical solution of the present invention metaxanin pesticide residue of can effectively degrading.
The cultivation of embodiment 1 thalline: penetrating the mycelium inoculation of arteries and veins lead fungi Phlebia acerinaY3667 with inoculating needle picking maple in above-mentioned strain activation and culture base plate, is to cultivate 4 days in 28 ℃ of incubators in temperature, bacterium colony is loose, snowy white, flocculence.With the loose mycelia of scalpel scraping, be inoculated in the Erlenmeyer flask that 500ml nutrient solution of autoclaving 20min under 121 ℃, 0.1MPa is housed.Be placed in again 30 ℃, rotating speed 140rmp/min, shaking table concussion is cultivated 4 days, obtains mycelia and bacterium liquid mix bacterium agent.
The degraded of metalaxyl residue in embodiment 2 supplementary carbon source solid mediums: when the sterilizing of strain activation and culture base cools to 40 ℃, sneak into through the ultraviolet sterilization metaxanin agricultural chemicals of 30 minutes, concentration is 5mg/L, after fully mixing, pour in the culture dish of diameter 60mm, every culture dish 3mL substratum, this substratum pinkiness.Cultured mycelia in embodiment 1 is broken into small pieces along colony edge with the punch tool of 6mm diameter.After being mixed with the substratum condensation of metaxanin agricultural chemicals, access mycelia small pieces, mycelia is close to media surface downwards.Be placed in 30 ℃ of incubators and cultivate 10 days, the pink in substratum is taken off, and the molecular structure of metaxanin agricultural chemicals is degraded, and it is cream-colored translucent that substratum is.
Embodiment 3: the degraded of degradation bacterium preparation to high density metaxanin pesticide residue
(1) in the nutrient solution 100ml cultivating through claim 3, add the metaxanin agricultural chemicals of 5mg/L, in 30 ℃, rotating speed 140rmp/min, shaking table concussion is cultivated, with the metaxanin agricultural chemicals that adds equal concentrations in the nutrient solution without inoculation as a control group;
(2) every 12h sampling once, get 3ml sample and be placed in 50mL plastic centrifuge tube, add acetonitrile 10mL, 30min vibrates on vibrator, centrifugal under 3000rmp/min, take out supernatant liquor, repeat 3 times, merge supernatant liquor, extract after the metaxanin pesticide residue in nutrient solution, in solid-phase extraction device through Fo Luoli tripoli solid-phase extraction column (specification: 500mg3ml) purify, Rotary Evaporators concentrated (40 ℃ following), lyophilize at-20 ℃, then with a small amount of acetonitrile, dissolve the pesticide residue of extracting, it is carried out to gas chromatographic analysis.Calculate degradation rate: degradation rate (%)=(A-B)/A * 100, wherein A is control group metaxanin pesticide residue value (after 48 hours, residual value is 4.7mg/L), and B is the metaxanin pesticide residue value (after 48 hours, residual value is 0.8mg/L) after degradation bacterial agent degraded.Under this culture condition, maple is penetrated arteries and veins lead fungi Phlebia acerina Y3667 to 48 hours Nei Keda of degradation rate of high density metaxanin pesticide residue more than 82%.
Claims (7)
1. a strain maple is penetrated arteries and veins lead fungi bacterial strain, it is characterized in that: this bacterial strain called after maple is penetrated arteries and veins lead fungi (Phlebia acerina) and is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date be on November 15th, 2012 deposit number be: CGMCC6809.
2. a maple claimed in claim 1 is penetrated arteries and veins lead fungi bacterial strain in the application of degraded metaxanin pesticide residue.
3. by maple claimed in claim 2, penetrate arteries and veins lead fungi bacterial strain in the application of degraded metaxanin pesticide residue, it is characterized in that:
The culturing process that described maple is penetrated arteries and veins lead fungi bacterial strain is: described maple is penetrated to arteries and veins lead fungi bacterial strain Phlebia acerina mycelium and be inoculated on 60 millimeters of plate substratum of diameter, at 28-30 ℃, 3-5 days grows on plate substratum; Scalper scraping mycelia, in access liquid nutrient medium, the mycelium inoculation of 2 culture dish scrapings is in 100ml nutrient solution, in 28-30 ℃, shaking culture 3-5 days under 140rpm speed conditions, the nutrient solution of the outer degrading enzyme of born of the same parents that acquisition comprises its secretion, it is the mix bacterium agent of mycelia and bacterium liquid;
Described plate substratum consists of Fructus Hordei Germinatus and soaks powder 10g, and agar 18g, adds water to 1L, pH nature;
Described liquid nutrient medium consists of Fructus Hordei Germinatus and soaks powder 10g, potassium primary phosphate 0.5g, CuSO
40.1g, CaCl
20.01g, MnSO
40.01g, pH nature, adds water and is settled to 1L.
4. by maple claimed in claim 3, penetrate arteries and veins lead fungi bacterial strain in the application of degraded metaxanin pesticide residue, it is characterized in that:
In the nutrient solution 100ml cultivating through claim 3, add the metaxanin agricultural chemicals of ultimate density 5-10mg/L, in 30 ℃, rotating speed 140rpm, shaking table concussion is cultivated, and incubation time is more than 48 hours.
5. by maple claimed in claim 3, penetrate arteries and veins lead fungi bacterial strain in the application of degraded metaxanin pesticide residue, it is characterized in that:
Described application is carried out as follows:
(1) in the nutrient solution 100ml cultivating through claim 3, the metaxanin agricultural chemicals that adds final concentration 5-10mg/L, in 28-30 ℃, rotating speed 140rpm, shaking table concussion is cultivated, with the metaxanin agricultural chemicals that adds equal concentrations in the nutrient solution without inoculation as a control group;
(2) every 12h sampling once, get 2-3ml sample and be placed in 50mL plastic centrifuge tube, add acetonitrile 10-12mL, vibration 20-30min, centrifugal, take out supernatant liquor, repeat 3 times, merge supernatant liquor, extract the metaxanin pesticide residue in nutrient solution, through Fo Luoli tripoli Solid-Phase Extraction column purification, at 38-40 ℃, Rotary Evaporators is concentrated, lyophilize at-20 ℃, then with a small amount of acetonitrile dissolving, carry out gas chromatographic analysis, calculate degradation rate.
6. by maple claimed in claim 5, penetrate arteries and veins lead fungi bacterial strain in the application of degraded metaxanin pesticide residue, it is characterized in that: its degradation rate (%)=(A-B)/A * 100,
Wherein A is control group metaxanin pesticide residue values,
B is the metaxanin pesticide residue value after degradation bacterial agent degraded.
7. by the maple described in claim 2 or 3, penetrate arteries and veins lead fungi bacterial strain in the application of degraded metaxanin pesticide residue, it is characterized in that: take metaxanin as supplementary carbon source, utilize maple to penetrate arteries and veins lead fungi for bacterial classification, degraded metaxanin.
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