CN105400698B - One plant of birch pleat pore fungi and its application in dimethomorph residue degrading - Google Patents
One plant of birch pleat pore fungi and its application in dimethomorph residue degrading Download PDFInfo
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- CN105400698B CN105400698B CN201410420888.4A CN201410420888A CN105400698B CN 105400698 B CN105400698 B CN 105400698B CN 201410420888 A CN201410420888 A CN 201410420888A CN 105400698 B CN105400698 B CN 105400698B
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Abstract
The present invention relates to one plant of birch pleat hole bacteria strain and its applications in dimethomorph chemical residual degradation, are degraded using the bacterial strain to dimethomorph pesticide residue, belong to field of microbial biotechnology.The identified entitled birch pleat pore fungi of the mycology (Lenzites betulina), bacterial strain Y4962, the bacterial strain has been stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC 8783, it is extracted by DNA, ITS sequence expands and measurement sequence, with the comparison of saving sequence in GenBank, find its homology with Lenzites betulina bacterial strain DSH97_071 up to 99%.After the bacterial strain cultivates acquisition microbial inoculum in liquid medium, dimethomorph pesticide is added, it being capable of efficient degradation dimethomorph pesticide residue.It is capable of the dimethomorph pesticide residue of efficient degradation water body and surface of agricultural products by the degradation bacterial agent of technical solution of the present invention production, protection and repairs ecological environment.
Description
Technical field
The invention belongs to field of environment microorganism, it is to be remained using the method degradation chemical pesticide of microorganism, is related to one plant
Birch pleat hole bacteria strain Lenzites betulina Y4962 and its application in dimethomorph chemical residual degradation.
Background technique
Microbial degradation is that the use microorganism risen extensively in recent years remains progress biology to organic pollutants such as pesticides
The hot research field of degradation.The technology is decomposed organic pollutant macromolecular by the degradation and suction-operated of microbial cells
At small molecule compound, and make its loss of biological activity and environmental sound.Since microorganism individual is small, breeding is rapid, compares table
The features such as face is big, they are easier to adapt to environment compared with other biologies, and can generate novel bacterial by natural mutation, generate new enzyme
System obtains new metabolic pathway, to may participate in the degradation and transformation to organic pollutants such as pesticides, therefore microorganism
There is great potential to degrading pesticide residues.
White-rot fungi is that one kind can be grown on wood substrates, causes a kind of Higher basidiomycetes of timber white rot,
Non-specific extracellular enzyme laccase (Laccase), lignin peroxidase (Lignin peroxidase) and the manganese mistake of generation
Oxide enzyme (Manganese peroxidase) other than lignin, cellulose and hemicellulose in the timber that can degrade,
Can also effectively be degraded a variety of environmental contaminants, including number of types of polycyclic aromatic hydrocarbon, artificial synthesized dyestuff and chemical agriculture at present
Medicine.White-rot fungi type is abundant, between type and the different strains of identical type degradation capability to environmental pollutants have it is bright
Significant difference is different, therefore obtains the efficient degrading bacterial strain to chemical pesticide and apply to practice, to the reduction remaining danger of Environmental Pesticide
Evil is of great significance.Dimethomorph is the fungicide of single-minded ovicidal bacterium, can destroy the formation of oomycetes cell envelope and make disease
Bacterium is dead.And the birch pleat pore fungi as Higher basidiomycetes, due to not having the action site of dimethomorph, dimethomorph is to it
Without function and effect.
This patent is isolated high-efficiency degradation oomycetes fungicide --- an alkene acyl from the rotten wood of virgin forest
The white-rot fungi birch pleat pore fungi Lenzites betulina Y4962 of quinoline.It is any that experiment shows that dimethomorph does not generate the bacterium
Inhibition and killing effect, can be grown in water agar using dimethomorph as sole carbon source and the energy instead；
Under the liquid culture condi of addition exogenous nutrition object, which can will be mixed into the high concentration alkene acyl of 10mg/L in culture medium
72% or more morpholine degradation.Absolutely prove that the bacterium can be used for the biology drop of water body and the remaining dimethomorph of surface of agricultural products
Solution.It there is no the document report using Lenzites betulina degradation dimethomorph pesticide residue research at present.
Summary of the invention
The object of the present invention is to provide one plant of birch pleat hole bacteria strain and its application in dimethomorph residue degrading, the bacterium
Strain has the biodegradable industrial applications prospect in residue in water and dimethomorph remained on surface.
One plant of birch pleat hole bacteria strain, the Strain Designation are birch pleat pore fungi (Lenzites betulina), and bacterial strain Y4962 is protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica)；Preservation date is deposit number on January 24th, 2014 are as follows:
CGMCC No.8783。
Mycelium growth vigor is vigorous, and bacterium colony is just villiform, rear at finer and close felt shape, snowy white, not chromogenesis；Bacterium
It falling and is made of generative hyphae and skeletal hyphae, generative hyphae thin-walled to slightly heavy wall, there is clamp connection to separate by 2-3 microns of diameter,
Branch once in a while；Skeletal hyphae slightly heavy wall is to heavy wall, and mycelia wall has thermophilic blue reaction in cotton orchid reagent, without change in potassium iodide reagents
Change, 1-2.5 microns of diameter, common right angle branch；4.5 centimetres of colony diameter of plate culture 3 days.The ITS rDNA sequence of the bacterial strain
It has been submitted to GenBank database, GenBank accession number KJ845635, the sequence and Lenzites betulina bacterial strain
The gene homology of DSH97_071 is up to 99%.
The incubation of birch pleat hole bacteria strain are as follows: by the birch pleat hole bacteria strain Lenzites betulina
The mycelium of Y4962 bacterial strain is inoculated on 60 millimeters of plating mediums of diameter, and 4-5 is grown on 28-30 DEG C, plating medium
It；Scalpel scrapes mycelia, accesses in fluid nutrient medium, and the mycelium inoculation of 4 culture dishes scraping is to being equipped with 100ml culture solution
In 250ml conical flask, shaken cultivation 6-7 days under 28-30 DEG C, 140rpm speed conditions obtains the extracellular drop comprising its secretion
The culture solution of enzyme is solved, is the mix bacterium agent of mycelia and bacterium solution；
The plating medium group becomes yeast extract 10g, and agar 17g adds water to 1L, and pH is naturally, in 121 DEG C of high pressures
Sterilizing 20 minutes；
The fluid nutrient medium group becomes yeast extract 8-12g, KH2PO40.4-0.6g,CuSO40.05-0.1g,
CaCl20.01-0.02g,MnSO40.01-0.02g, Vitamin B10.01-0.02g, Tween 800.4-0.6g, pH naturally,
Add water to be settled to 1L, in 121 DEG C high pressure sterilization 20-25 minutes.
The culture solution for being greater than 0 to 200mg/L above-mentioned culture containing dimethomorph pesticide ultimate density shakes in 28-30 DEG C
Bed shake culture, incubation time 96-120 hour, progress dimethomorph pesticide can degradation.
Birch pleat hole is inoculated in the culture solution for the above-mentioned culture for being greater than 0 to 200mg/L containing dimethomorph pesticide ultimate density
Bacterium, using birch pleat pore fungi as strain, using fungicide as supplementary carbon source, dimethomorph of degrading.
The birch pleat hole bacteria strain and its application in dimethomorph residue degrading are tested as follows:
(1) in birch pleat pore fungi strain cultured solution 90-120ml, the dimethomorph pesticide of final concentration 8-10mg/L is added, in
28-30 DEG C, revolving speed 140-160rpm, shaking table shake culture, the alkene acyl of equal concentrations to be added in the culture solution without inoculation
Quinoline pesticide is as a control group；
(2) primary every sampling for 24 hours, it takes 2-3ml sample to be placed in osculum 100ml conical flask, acetonitrile 8-12mL is added, surpass
Sound extract 15min, after pour into the plastic centrifuge tube equipped with 8g Nacl, be mixed by inversion repeatedly, 3500rpm be centrifuged 5min,
50min is stood, takes upper layer acetonitrile in 100ml round-bottomed flask, 33-37 DEG C of rotary evaporation is done to close, and 2ml n-hexane adds 0.5ml
Acetone cleans round-bottomed flask, pours into sample presentation bottle, and be repeated once, is settled to 5ml, takes 1ml for GC-ECD gas-chromatography
Analysis calculates degradation rate.
Its degradation rate (%)=(A-B)/A × 100, in which:
A is control group dimethomorph pesticide residue value,
B is the dimethomorph pesticide residue value after degradation bacterial agent is degraded.
Using birch pleat pore fungi as strain, using fungicide as supplementary carbon source, dimethomorph of degrading.
The invention has the advantages that this patent is that an isolated high-efficiency degradation oomycetes from the rotten wood of virgin forest kills
The white-rot fungi bacterial strain birch pleat pore fungi (Lenzites betulina) of microbial inoculum --- dimethomorph, bacterial strain Y4962.The birch pleat hole
Bacterium, which is one plant, has higher vigor, and cultural method is simple, and the speed of growth is fast, is not easy the bacterial strain to make a variation, and experiment shows that the bacterium can
It is grown in water agar using dimethomorph as sole carbon source and the energy, and dimethomorph is made to degrade；It is adding
Under the liquid culture condi of exogenous nutrition object, which can will be mixed into the dimethomorph degradation 72% of 10mg/L in culture medium
More than, it absolutely proves that the bacterium can be used for the biodegrade of water body and the remaining dimethomorph of surface of agricultural products, has pesticide residual
Biodegradable industrial applications prospect is stayed, can also be used as research white-rot fungi to dimethomorph chemical residual degradation mechanism
Type strain.
Specific embodiment
The bacterial strain of degradation dimethomorph pesticide residue provided by the present invention is isolated from virgin forest rotten wood.
The bacterium separation method is as follows:
Below by way of the specific example implementation that the present invention will be described in detail, it is therefore intended that reader is helped to more fully understand the present invention
Spirit Essence, but not as the restriction to the scope of the present invention.
1. culture medium is prepared:
(1) culture presevation culture medium (solid, 1L): yeast extract 10g, agar 17g adds water to 1L, and pH is naturally, in 121
DEG C high pressure sterilization 20 minutes；
(2) strain activation and culture base (solid, 1L): yeast extract 10g, agar 17g adds water to 1L, and pH is naturally, in 121
DEG C high pressure sterilization 20 minutes；
(3) fluid nutrient medium (liquid, 1L): yeast extract 10g, KH2PO40.5g,CuSO40.1g,CaCl20.01g,
MnSO40.01g, vitamin B10.01g, Tween 800.5g, pH naturally, plus water be settled to 1L, in 121 DEG C of high pressure sterilizations 20
Minute.
2. actication of culture: the mycelia of picking birch pleat pore fungi is inoculated in the 60mm culture dish added with culture presevation culture medium,
In 28 DEG C, cultivate 5 days.
3. prepared by microbial inoculum: with mycelia of the scalpel scraping media surface after overactivation, connects bacterium and enter in fluid nutrient medium,
In 30 DEG C, shaken cultivation 6-7 days under the conditions of 140rmp obtain the culture solution of the extracellular degrading enzyme comprising its secretion；By the present invention
The microbial inoculum of technical solution production can effectively degrade dimethomorph pesticide residue.
The culture of 1 thallus of embodiment: it is connect with the mycelia of transfer needle picking birch pleat pore fungi Lenzites betulina Y4962
Kind is to cultivate 5 days in 28 DEG C of incubators in temperature, bacterium colony is loose, snowy white, cotton-wool into above-mentioned strain activation and culture base plate
Shape.Mycelia is cut with scalpel, is inoculated into the cone equipped with 500ml through the culture solution of high pressure sterilization 20min under 121 DEG C, 0.1MPa
In shape bottle.It is placed in 30 DEG C, revolving speed 140rmp, shaking table shake culture 96h again, obtains mycelia and bacterium solution mix bacterium agent.
Embodiment 2 is using dimethomorph as the remaining degradation of dimethomorph in the activation medium of supplementary carbon source: in strain
When activation medium sterilizing cools to 40 DEG C, it is mixed into the dimethomorph pesticide by ultraviolet sterilization 30 minutes, final concentration of 10mg/
L after mixing well, is poured into the culture dish of diameter 60mm, every culture dish 3mL culture medium, and the culture medium is colourless.By embodiment 1
In cultured mycelia along colony edge break into round bacterium piece with the punch of 6mm diameter.The training of dimethomorph pesticide to be mixed with
After supporting base condensation, mycelia small pieces are accessed, mycelia is close to downwards media surface.It is placed in 30 DEG C of incubators and cultivates 12 days, cultivate
Mycelia grows vigorous base in ware, and culture medium is translucent in cream color, and the chemical structure of dimethomorph pesticide is degraded at this time, and
It is absorbed and utilized by birch pleat pore fungi mycelia as carbon source.
Embodiment 3: degradation of the degradation bacterium preparation to dimethomorph pesticide residue in liquid
(1) in the culture solution 100ml that embodiment 1 is cultivated, the dimethomorph pesticide of 10mg/L is added, in 30 DEG C, revolving speed
140rmp, shaking table shake culture, the dimethomorph pesticide of equal concentrations to be added in the culture solution without inoculation as a control group；
(2) primary every sampling for 24 hours, it takes 2-3ml sample to be placed in osculum 100ml conical flask, acetonitrile 10mL, ultrasound is added
Extract 15min, after pour into the plastic centrifuge tube equipped with 8gNacl, be mixed by inversion repeatedly, 3500rpm be centrifuged 5min, it is quiet
50min is set, takes upper layer acetonitrile in 100ml round-bottomed flask, 35 DEG C of rotary evaporations are done to close, and 2ml n-hexane adds 0.5ml acetone clear
Round-bottomed flask is washed, pours into sample presentation bottle, and be repeated once, is settled to 5ml, takes 1ml for GC-ECD gas chromatographic analysis, meter
Calculate degradation rate.Its degradation rate (%)=(A-B)/A × 100, wherein A is control group dimethomorph pesticide residue value (after 96 hours
Residue is 8.7mg/L), B is the dimethomorph pesticide residue value after degradation bacterial agent is degraded, and (residue is after 96 hours
1.5mg/L)。
Under the condition of culture, degradation rate is calculated, birch pleat pore fungi Lenzites betulina Y4962 is to high concentration alkene
Up to 72% or more in degradation rate 96 hours of morpholide pesticide residue.
Claims (4)
1. one plant of birch pleat hole bacteria strain or its culture solution are in the application of dimethomorph pesticide residue of degrading, it is characterised in that: the bacterium
Strain is named as birch pleat pore fungi (Lenzites betulina) bacterial strain Y4962；It is preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preservation date is deposit number on January 24th, 2014 are as follows: CGMCC No.8783.
2. application according to claim 1, it is characterised in that:
The acquisition process of the birch pleat pore fungi strain cultured solution are as follows: be inoculated with the mycelium of the birch pleat hole bacteria strain Y4962
Onto plating medium, grown 4-5 days on 28-30 DEG C, plating medium；Mycelia is scraped, is inoculated into and is trained equipped with 100mL liquid
In the conical flask for supporting base, shaken cultivation 6-7 days under 28-30 DEG C, 140rpm speed conditions obtains the extracellular drop comprising its secretion
The culture solution of enzyme is solved, the culture solution is the mix bacterium agent of mycelia and bacterium solution；
The plating medium group becomes yeast extract 10g, and agar 17g adds water to 1L, and pH is naturally, in 121 DEG C of high pressure sterilizations
20 minutes；
The fluid nutrient medium group becomes yeast extract 8-12g, KH2PO4 0.4-0.6g, CuSO4 0.05-0.1 g,
CaCl2 0.01-0.02 g, MnSO40.01-0.02 g, Vitamin B1 0.01-0.02g, 80 0.4-0.6 of Tween
G, pH naturally, plus water be settled to 1L, in 121 DEG C high pressure sterilization 20-25 minutes.
3. application as described in claim 2, it is characterised in that:
Ultimate density is added in culture solution as stated in claim 2 to be greater than 0 and be less than or equal to the dimethomorph of 10mg/L
Pesticide, in 28-30 DEG C, shaking table shake culture incubation time 96-120 hours, carries out the degradation of dimethomorph pesticide.
4. application according to claim 3, it is characterised in that:
It is inoculated with birch pleat pore fungi in the culture solution of addition dimethomorph pesticide described in claim 3, using birch pleat pore fungi as bacterium
Kind, dimethomorph of degrading.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003035561A2 (en) * | 2001-09-10 | 2003-05-01 | Universite Catholique De Louvain | Sustainable process for the treatment and detoxification of liquid waste |
| CN103122316A (en) * | 2012-11-30 | 2013-05-29 | 中国科学院沈阳应用生态研究所 | Phlebia acerina strain and application thereof in degrading metalaxyl pesticide residue |
-
2014
- 2014-08-22 CN CN201410420888.4A patent/CN105400698B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003035561A2 (en) * | 2001-09-10 | 2003-05-01 | Universite Catholique De Louvain | Sustainable process for the treatment and detoxification of liquid waste |
| CN103122316A (en) * | 2012-11-30 | 2013-05-29 | 中国科学院沈阳应用生态研究所 | Phlebia acerina strain and application thereof in degrading metalaxyl pesticide residue |
Non-Patent Citations (2)
| Title |
|---|
| Yuan H.S. and Cao W.J..KJ845635.1.《Genbank》.2014,LOCUS、DEFINITION、SOURCE、REFERENCE、FEATURES、ORIGIN. * |
| 余欣欣等.微生物和酶在降解环境有机污染物中的应用.《环境工程》.2012,97-100. * |
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