CN111378599B

CN111378599B - Degradation strain capable of simultaneously degrading two isomers of chiral herbicide 2, 4-dichlorprop and microbial inoculum produced by same

Info

Publication number: CN111378599B
Application number: CN202010095007.1A
Authority: CN
Inventors: 蒋建东; 张龙; 陈凯; 胡顺利
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-02-17
Filing date: 2020-02-17
Publication date: 2022-02-25
Anticipated expiration: 2040-02-17
Also published as: CN111378599A

Abstract

The invention discloses a degrading strain capable of simultaneously degrading two isomers [ (R, S) -DCPP ] of a chiral herbicide 2, 4-dichlorprop and a microbial inoculum produced by the degrading strain. The strain DCP-6 is identified as Sphingopyxis sp, deposited in China center for type culture Collection in 2019, 12 months and 23 days, and has the strain preservation number of CCTCC M20191084. The degrading strain DCP-6 can degrade phenoxy carboxylic acid chiral herbicides (R, S) -DCPP and 2-methyl-4-chlorophenoxypropionic acid (R, S) -MCPP in a broad spectrum, and phenoxy carboxylic acid achiral herbicides 2, 4-dichlorophenoxyacetic acid (2,4-D), MCPA, 2,4-DB and the like, and the degrading microbial inoculum product can reduce the residual quantity of the (R, S) -DCPP and the like in soil by more than 89%, so that the problems of pollution of the phenoxy carboxylic acid herbicides in soil and water environments, phytotoxicity to crops and the like in the agricultural production process can be effectively solved, and the ecological environment is protected.

Description

Degradation strain capable of simultaneously degrading two isomers of chiral herbicide 2, 4-dichlorprop and microbial inoculum produced by same

Technical Field

The invention relates to a degrading strain capable of simultaneously degrading two isomers of a chiral herbicide 2, 4-dichlorprop and a microbial inoculum produced by the degrading strain, belonging to the field of biological high technology.

Background

Chemical pesticides are important guarantees for rapid development of agriculture and food safety in the world, and play an immeasurable role in the modernization process of agricultural production. Among chemical pesticides currently used in China, pesticides having a chiral structure (chiral pesticides) account for about 40%. Meanwhile, with the continuous development of chemical pesticide varieties, the number of pesticide chiral compounds is remarkably increased. In each large agricultural compound, the amount of herbicide used is the first place. Research shows that the herbicide with chiral structure (chiral herbicide) has considerable enantiomer difference in the aspects of pesticide effect, environment safety, etc. Although enantiomers of chiral compounds have the same physicochemical properties, their isomers may have different biological activities and ecotoxicology. The incomplete recognition of chiral compounds has led to the tradition of tragic pain in humans, as was the case typically with the "reaction stop" (thalidomide) which was a temporary bombing in the medical field. R-thalidomide has sedative effect and can be used for treating vomiting during pregnancy, and S-thalidomide has strong teratogenic effect, which can cause more than 12000 'seal fetus' malformations worldwide in 6 years of use. Since most chiral herbicides have hitherto been produced and applied in the form of racemates (equimolar mixtures of optically active chiral molecules with their enantiomers), it is inevitable that different isomers of chiral herbicides enter into environmental systems such as organisms, soil and water bodies simultaneously. Thus, the depletion of chiral herbicides in the environment has received a great deal of attention.

2, 4-D-propionic acid (DCPP), is one of the typical chiral herbicides widely used throughout the world. DCPP belongs to a medium-toxicity compound, and can release toxic compound gases such as carbon monoxide, hydrogen chloride and the like when being heated and decomposed. The herbicidal activity and the efficacy of growth hormone of DCPP are almost entirely concentrated on the R-type enantiomer [ (R) -DCPP ], the S-type enantiomer [ (S) -DCPP ] has no herbicidal activity and has slight anti-auxin activity. DCPP has potential carcinogenicity and mutagenicity, has weak volatility and large solubility, is difficult to biodegrade and directly photolyze, and has high environmental residual rate. After the DCPP is applied, only a small part of the DCPP can be absorbed by the plant body, and most of other DCPP can directly or indirectly enter the surrounding soil, and easily enters the water environment along with rainwater or irrigation water from the soil due to high water solubility. Therefore, the aquatic environment is greatly damaged. At the same time, the (S) -form enantiomer, although not herbicidally active, may be more toxic to non-target organisms. Therefore, the dynamic extinction of DCPP in the environment is getting more and more attention, and a practical method for effectively removing DCPP residue in the environment is desired.

Biodegradation is an important process for the conversion of chemical pesticides in the environment, where microorganisms are the mainstay of biodegradation. The microorganism has the characteristics of large quantity, various varieties, rapid propagation, strong environmental adaptability, difficult generation of secondary pollution and the like, and is widely applied to the removal of herbicide residues in soil and water bodies for controlling herbicide pollution and even agricultural products. The utilization of the metabolism of microorganisms to transform the herbicide which is easy to remain and make the herbicide nontoxic is also a hotspot of the current research.

Disclosure of Invention

Aiming at the practical problems and requirements of environmental remediation, the invention provides a degrading strain capable of simultaneously degrading two isomers of the chiral herbicide 2, 4-dichlorprop.

The invention also aims to provide a microbial inoculum prepared by the degrading strain.

The invention also aims to provide a preparation method and application of the degrading microbial inoculum.

The purpose of the invention can be realized by the following technical scheme:

the invention provides a (R, S) -DCPP high-efficiency degradation strain DCP-6 (deposited in China center for type culture collection in 23.12.2019. month.12. CCTCC NO: M20191084. the strain has negative gram staining reaction, and the morphological characteristics of the strain DCP-6 are that the strain is orange colony on an LB plate, the colony is convex, the surface is moist and smooth, the edge is neat, and the strain is opaque (figure 1A). The main biological characteristics are G^－The thallus is rod-shaped (0.5 μm × 1.4 μm), has no flagellum (FIG. 1B), and is aerobic; the catalase, oxidase and v.p. reactions were positive; indole reaction is negative; can not hydrolyze starch, and can make litmus margaritae solidify lactic acid. Under the condition of laboratory shake flask culture, the strain DCP-6 can completely degrade 30mg/L (R, S) -DCPP in 96h (figure 2). The strain can be produced by fermentation equipment commonly used in the fermentation industry.

The degrading strain DCP-6 is applied to the preparation of a microbial inoculum for degrading two isomers of phenoxy carboxylic acid herbicides; the phenoxy carboxylic acid herbicide is selected from any one or more of (R, S) -DCPP, (R, S) -MCPP, 2,4-D, MCPA or 2,4-DB, preferably any one or more of (R, S) -DCPP, (R, S) -MCPP and 2, 4-D.

The pesticide residue degrading microbial inoculum produced by the degrading strain is prepared by fermenting the degrading strain.

The degrading bacteria agent is preferably produced by the following method:

(1) inoculating test tube species of the (R, S) -DCPP degrading strain DCP-6 into an LB culture medium shake flask, and carrying out shake culture until the logarithmic phase;

(2) inoculating the cultured strain into a seeding tank according to the inoculation amount of 10 percent, culturing to a logarithmic phase, wherein the formula of a culture medium used by the seeding tank is as follows: glucose 8g/L, yeast extract 5g/L, K₂HPO₄ 1g/L，NaCl 5g/L，CaCO₃ 2g/L，MgSO₄ 0.2g/L, 0.1% (v/v) of soybean oil, and the pH value of 7.2-7.5;

(3) inoculating the seed liquid into a production tank according to the inoculation amount of 10% for culture, wherein the culture medium used by the production tank is the same as that of the seed tank;

(4) the ventilation quantity of sterile air is 1:0.6-1.2 in the culture process of a seeding tank and a production tank, the stirring speed is 180-240 rpm, the culture temperature is 30-35 ℃, the whole-process culture time is 96-108 hours, the number of thalli reaches more than 10 hundred million/mL after the fermentation is finished, and the fermentation liquid is directly subpackaged into liquid formulations by a plastic packaging barrel or a packaging bottle or solid microbial inoculum formulations by adopting a packaging bag for peat adsorption after being taken out of the tank.

The method for preparing the degrading microbial inoculum comprises the following steps:

(2) inoculating the cultured strain into a seeding tank according to the inoculation amount of 10 percent, culturing to a logarithmic phase, wherein the formula of a culture medium used by the seeding tank is as follows: glucose 8g/L, yeast extract 5g/L, K₂HPO₄ 1g/L，NaCl 5g/L，CaCO₃ 2g/L，MgSO₄0.2g/L, 0.1% (v/v) of soybean oil, and pH value of 7.2-7.5;

The degrading microbial inoculum provided by the invention is applied to degrading phenoxy carboxylic acid herbicides.

The phenoxy carboxylic acid herbicide degrading strain DCP-6 disclosed by the invention is applied to degrading phenoxy carboxylic acid herbicides, and preferably applied to degrading phenoxy carboxylic acid herbicides in soil.

Wherein, the phenoxy carboxylic acid herbicide is preferably chiral herbicide 2, 4-dichlorprop acid [ (R, S) -DCPP ] and 2-methyl-4-dichlorprop acid [ (R, S) -MCPP ] raceme and any one or more of phenoxy carboxylic acid achiral herbicide 2, 4-dichlorphenoxyacetic acid (2,4-D), 2-methyl-4-dichlorphenoxyacetic acid (MCPA) and 2, 4-dichlorprop ester (2,4-DB), and further preferably (R, S) -DCPP and/or (R, S) -MCPP.

Advantageous effects

The invention provides a bacterial DCP-6 capable of efficiently and rapidly degrading phenoxy carboxylic acid chiral herbicide (R, S) -DCPP. The degrading strain DCP-6 has wide degrading spectrum, can degrade racemes of phenoxy carboxylic acid chiral herbicides (R, S) -MCPP, phenoxy carboxylic acid achiral herbicides 2,4-D, MCPA, 2,4-DB and the like, can completely degrade 30mg/L (R, S) -DCPP within 96 hours, and has wide application potential and value. The degrading bacteria agent produced by using the bacteria has the advantages of low production and use cost, convenient use and good removal effect. Is suitable for large-area popularization and use in agricultural production areas or places with green food trademark marks. The invention has important significance for protecting ecological environment, protecting the health of people and improving the added value of agricultural products. The degrading microbial inoculum can be used for normally using chemical pesticide to prevent and control weeds before crop sowing, so that the pesticide residue content in agricultural products meets the requirement of green food.

The invention successfully solves the problem of pesticide residue exceeding standard in agricultural production, not only fully exerts the high-efficiency and quick action of chemical pesticide in the prevention and control of plant diseases and insect pests, but also can successfully treat the soil and water environment polluted by phenoxy carboxylic acid herbicide residue, and protect the ecological environment.

Drawings

FIG. 1 photograph (A) and photograph (B) of colony of Strain DCP-6

FIG. 2 phylogenetic analysis of 16S rRNA genes of Strain DCP-6

FIG. 3 degradation of (R, S) -DCPP by Strain DCP-6

FIG. 4 Effect of temperature on the degradation of (R, S) -DCPP by Strain DCP-6

FIG. 5 Effect of initial pH on the degradation of (R, S) -DCPP by Strain DCP-6

FIG. 6 Effect of inoculum size on the degradation of (R, S) -DCPP by Strain DCP-6

FIG. 7 is a graph of the ultraviolet detection effect of strain DCP-6 substrate spectrum

Biological material preservation information

DCP-6, classified and named as Sphingopyxis sp.DCP-6, is preserved in China center for type culture Collection, the preservation number of the strain is CCTCC NO: M20191084, the preservation date is 2019, 12 and 23 days, the preservation address is Wuhan city, Hubei province, eight paths in flood mountain areas, and the China center for type culture Collection of Wuhan university.

Detailed Description

Example 1 isolation and identification of strains

The invention provides a bacterial strain capable of efficiently degrading two chiral isomers of a chiral herbicide (R, S) -DCPP and a microbial inoculum produced by the bacterial strain, wherein the bacterial strain is a gram-negative bacterial strain DCP-6 which is separated from soil of a certain abandoned farm chemical factory area of Nanjing, Jiangsu.

The specific separation and screening method of the strain comprises the following steps:

a soil sample (10.0 g) was added to 100mL of a liquid inorganic salt medium (hereinafter abbreviated as MSM) containing 30mg/L of (R, S) -DCPP, shake-cultured at 30 ℃ and 180rpm for 7d, transferred to a fresh medium of the same medium in an inoculum size of 15% (v/v), and continuously subjected to enrichment subculture for four times. And (4) scanning by using an ultraviolet spectrophotometer within the range of 200-350nm to detect the degradation effect of the fifth generation enrichment solution. Diluting and coating the effective fruit enrichment solution on an inorganic salt solid culture medium containing 30mg/L (R, S) -DCPP, culturing for 5 days at 30 ℃, picking a single colony on a plate into 3mL of liquid LB test tube culture medium, then storing and transferring to 20mL of MSM culture medium containing 30mg/L (R, S) -DCPP, and culturing for 5 days at 30 ℃. Then adding 25% hydrochloric acid, adjusting the pH value to be about 3.0, extracting with dichloromethane with the same volume, and detecting the effect by an ultraviolet spectrophotometer to obtain the (R, S) -DCPP degrading strain.

Deposited in China center for type culture Collection in 12 months and 23 days in 2019, the preservation number of the strain is CCTCC NO: M20191084, and the strain is identified to belong to Sphingopyxis sp. The main biological property is G^－The bacteria are rod-shaped, and have a size of about 0.5 μm width and a length of 1.4 μmWithout flagella (fig. 1B), aerobic; the catalase, oxidase and v.p. reactions were positive; indole reaction is negative; can not hydrolyze starch, and can make litmus margaritae solidify lactic acid. The 16S rRNA gene sequence (SEQ ID NO.1) of the strain DCP-6 is compared and analyzed in a database EzBioCloud, and the result shows that the strain DCP-6 has the closest relationship with Sphingopyxis, wherein the genetic relationship with Sphingopyxis taejonnensis JSS54^TThe sequence consistency of the 16S rRNA is as high as 98.72 percent and is as high as Sphingopyxis lindanitolerans WS5A3p^TThe sequence identity of 16S rRNA is 98.65%. And (3) combining the colony morphological characteristics, physiological and biochemical characteristics and 16S rRNA gene comparison analysis of the strains. Finally, the strain DCP-6 was preliminarily identified as belonging to the genus Sphingopyxis (FIG. 2).

Example 2 laboratory degradation experiment

2.1 seed liquid preparation

Inoculating the strain DCP-6 into 100mL LB culture medium, shaking-culturing at 30 deg.C and 180rpm, centrifuging at 6,000rpm after 48h, collecting thallus, washing thallus twice with sterilized MSM, and re-suspending with 10mL sterilized MSM to obtain seed liquid.

2.2 degradation of the Strain DCP-6 on the chiral herbicide (R, S) -DCPP

Inoculating 5% strain DCP-6 into 100mL MSM containing 30mg/L (R, S) -DCPP, shake culturing at 30 deg.C and 180rpm, sampling 3mL every 12 hr, and collecting on day 4. Detecting the residual amount of the (R, S) -DCPP, calculating the degradation rate, and drawing a time-degradation curve of the strain DCP-6 to the chiral herbicide (R, S) -DCPP. As shown in FIG. 3, strain DCP-6 was able to completely degrade 30mg/L of (R, S) -DCPP in 96 h.

Detecting chiral herbicide (R, S) -DCPP by high performance liquid chromatography: taking 3mL of a sample to be detected, adding 25% hydrochloric acid to adjust the pH value of the sample to be detected to be about 3.0, then adding dichloromethane with the same volume for extraction, and standing after vortex oscillation for 2 min. When the aqueous phase and the organic phase appeared to separate clearly, the upper aqueous phase was removed and excess anhydrous sodium sulfate was added to the lower organic phase to remove the residual water completely. Then 2mL of the treated organic phase was aspirated into a centrifuge tube and placed in a fume hood until dichloromethane was completely volatilized, and then 350. mu.L of methanol was added to the centrifuge tube, mixed by shaking, filtered through an organic filter with a diameter of 0.22 μm, and then detected on a liquid chromatograph. Liquid chromatography detection conditions: instrument, Shimadzu LC-20a (Shimadzu corporation); a chiral chromatography column, Superchiral S-AS (Chiralway Biotech co., ltd.),0.46cm i.d. 15cm Length,5 μm; the column temperature was set at 25 ℃; mobile phase, n-hexane: isopropyl alcohol: trifluoroacetic acid 96: 04:0.05(v/v/v), flow rate set to 0.8 mL/min; the ultraviolet detector detects wavelengths of 220nm and 235 nm; sample size, 6 μ L. And calculating the content according to the frontal area of the standard curve.

2.3 Effect of temperature on the degradation of (R, S) -DCPP by the Strain DCP-6

Inoculating seed liquid of strain DCP-6 into MSM culture medium added with (R, S) -DCP with final concentration of 30mg/L, respectively culturing at 4, 16, 25, 30, 35, 37 and 45 ℃ by shaking at 180rpm, sampling after 3d, detecting residual amount of (R, S) -DCP, calculating degradation rate, and determining influence of temperature on degradation of (R, S) -DCP by strain DCP-6. As shown in FIG. 4, the strain DCP-6 has the highest degradation rate on (R, S) -DCPP at 30 ℃.

2.4 Effect of the initial pH on the degradation of (R, S) -DCPP by the Strain DCP-6

Adding 30mg/L (R, S) -DCPP into MSM culture medium with initial pH of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, inoculating seed liquid of strain DCP-6 according to 5% inoculation amount, shake culturing at 30 deg.C and 180rpm, sampling after 3 days, detecting residual amount of (R, S) -DCPP, calculating degradation rate, and determining influence of pH on degradation of (R, S) -DCPP by strain DCP-6. Control was performed without inoculation of the degrading strain. As shown in FIG. 5, strain DCP-6 showed the best degradation effect on (R, S) -DCPP at pH 7.0; the (R, S) -DCPP can be well degraded in the pH range of 6.0-8.0; and when the pH is less than 5.0 and the pH is more than 9.0, the degradation ability is remarkably reduced.

2.5 Effect of inoculum size on the degradation of (R, S) -DCPP by the Strain DCP-6

Inoculating to MSM culture medium containing 30mg/L (R, S) -DCPP in an amount of 1%, 3%, 5%, 8%, 10% and 12%, respectively, shake culturing at 30 deg.C and 180rpm, and measuring the content of (R, S) -DCPP once in 48 hr. As shown in FIG. 6, the size of the inoculum amount has a direct relationship with the degradation efficiency of (R, S) -DCPP, and the larger the inoculum amount, the higher the degradation efficiency of (R, S) -DCPP.

2.6 substrate spectra of Strain DCP-6

Inoculating the mixture to MSM culture medium containing 30mg/L different substrates [ (R, S) -DCPP, (R, S) -MCPP, 2,4-D, MCPA, 2,4-DB and 2,4,5-T ] according to 5%, shaking culturing at 30 ℃ and 180rpm for 5 days, and detecting the degradation of different substrates by an ultraviolet spectrophotometer (FIG. 7). The results show that strain DCP-6 can degrade (R, S) -DCPP, (R, S) -MCPP, 2,4-D, MCPA and 2, 4-DB.

Example 3 preparation of microbial inoculum

The original seed of the chiral herbicide (R, S) -DCPP degrading strain DCP-6 is activated on a test tube inclined plane, the degradation performance is measured, and then the original seed is inoculated on the test tube inclined plane for standby. The test tube seed was inoculated into a 1000mL shake flask containing 200mL LB medium (LB medium formulation: peptone 10g, yeast powder 5g, sodium chloride 5g, water 1L, pH 7.4), and shake-cultured at constant temperature until logarithmic phase, to prepare for inoculation of the first-stage seed tank. 50L of first-level seed tank, 40L of batch size and the formula of culture medium as follows: glucose 8g/L, yeast extract 5g/L, K₂HPO₄ 1g/L，NaCl 5g/L，CaCO₃ 2g/L，MgSO₄0.2g/L, 0.1% (v/v) of soybean oil, and pH value of 7.2-7.5.

After the feeding is finished, carrying out high-pressure moist heat sterilization at 121 ℃, cooling to 30 ℃, inoculating the cultured shake flask strain into a 50L first-class seed tank according to the inoculation amount of 10%, culturing to logarithmic phase (about 98 hours), wherein the stirring speed is 220rpm, and the introduction amount of sterile air is 1: 0.8. inoculating the seed liquid reaching logarithmic phase into a secondary seed tank according to the inoculation amount of 10%. 500L of secondary seed tank, 400L of material feeding amount, and the formula and culture conditions of the culture medium are consistent with those of the primary seed tank. Inoculating the seed liquid reaching logarithmic phase into a production tank according to the inoculation amount of 10% for culture, wherein the culture medium composition of the production tank is the same as that of a seed tank. The capacity of the production tank is 5 tons, and the feeding amount is 4.5 tons. 1.1kg/cm production tank after feeding²Sterilizing at 121 deg.C, cooling to 30 deg.C, and introducing sterile air to maintain sterile state. The temperature of the production tank after inoculation is controlled to be 30-35 ℃, and the ventilation of sterile air in the culture process of the production tank is 1: 1.0, the stirring speed is 240rpm, and the culture time of the whole process flow is 100 hours. The number of thalli reaches after the fermentation is finishedMore than 10 hundred million/mL.

After fermentation, the culture solution is directly taken out of the tank and is subpackaged into liquid dosage forms by using a plastic packaging barrel or a packaging bottle or into solid microbial inoculum dosage forms by adopting a packaging bag for peat adsorption.

Example 4 soil degradation experiment

Vegetable garden soil was taken as the soil sample to be tested. The soil sample is sieved by a 2mm sieve, a certain amount of (R, S) -DCPP, (R, S) -MCPP and 2,4-D powder are respectively taken and dissolved in 10mL of methanol, and then diatomite is soaked to enable the pesticide to be completely adsorbed. And drying the soaked diatomite in a fume hood, and mixing the diatomite into the soil to ensure that the concentration of the pesticide in the soil is about 50 mg/kg. 500 g of each soil sample is cultured in a constant temperature incubator at 30 ℃, the DCP-6 seed solution is inoculated according to the inoculation amount of 10 percent, and the water holding capacity of the soil is kept at 60 percent by taking the non-inoculated one as a control. After 7 days of incubation, 3 samples each weighing 20g were taken for each treatment. The sample was shaken with an equal volume of the extract (methanol: water: acetic acid ═ 49: 49: 2) in a mechanical shaker for 2h (30 ℃,200 rpm). After shaking, the extract was transferred to a 50mL centrifuge tube and centrifuged for 10min (6,000rpm) and then filtered. The above procedure was repeated twice with the extract reduced to 15 mL. The filtrates were combined and adjusted to pH 3.0 with concentrated HCl. The mixture was extracted 3 times with dichloromethane as the extractant (the lower layer liquid was removed), and the volumes of the extracts were 30mL, 15mL and 15mL, respectively. Combining the extracts, concentrating in a rotary evaporation concentrator, and blowing nitrogen to dry. The volume is determined by using l mL of methanol, the solution is filtered through an organic phase microporous filter membrane with the diameter of 0.22 mu m, and then HPLC detection is carried out to determine the residual quantity of the three compounds.

As can be seen from Table 1, the degradation rates of (R, S) -DCPP, (R, S) -MCPP and 2,4-D by strain DCP-6 reached 89.6%, 87.5% and 85.6% respectively after 7 days of culture. The results show that the bacterial strain DCP-6 has no phenomena of no degradation or sharp degradation efficiency reduction after being applied to soil, and has stable degradation performance, thereby providing scientific basis for the bacterial strain DCP-6 to repair the (R, S) -DCPP, (R, S) -MCPP and 2,4-D polluted soil.

TABLE 1 degradation of related pesticides in soil by strain DCP-6

Sequence listing

<110> Nanjing university of agriculture

<120> a degrading strain capable of simultaneously degrading two isomers of chiral herbicide 2, 4-dichlorprop and microbial inoculum produced by the same

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Sphingopyxis sp.

<400> 1

agagtttgat cctggctcag aacgaacgct ggcggcatgc ctaacacatg caagtcgaac 60

gaagtcttcg gacttagtgg cgcacgggtg cgtaacgcgt gggaatctgc ccttgggtac 120

ggaataactc agagaaattt gtgctaatac cgtataatga cttcggtcca aagatttatc 180

gcccaaggat gagcccgcgt aagattagct agttggtggg gtaaaagcct accaaggcga 240

cgatctttag ctggtctgag aggatgatca gccacactgg gactgagaca cggcccagac 300

tcctacggga ggcagcagtg gggaatattg gacaatgggc gaaagcctga tccagcaatg 360

ccgcgtgagt gatgaaggcc ctagggttgt aaagctcttt tacccgggat gataatgaca 420

gtaccgggag aataagctcc ggctaactcc gtgccagcag ccgcggtaat acggagggag 480

ctagcgttgt tcggaattac tgggcgtaaa gcgcgcgtag gcggcttttt aagtcagagg 540

tgaaagcccg gggctcaacc ccggaattgc ctttgaaact ggaaagctag aatcttggag 600

aggtcagtgg aattccgagt gtagaggtga aattcgtaga tattcggaag aacaccagtg 660

gcgaaggcga ctgactggac aagtattgac gctgaggtgc gaaagcgtgg ggagcaaaca 720

ggattagata ccctggtagt ccacgccgta aacgatgata actagctgtc cgggtgcatg 780

gcacttgggt ggcgcagcta acgcattaag ttatccgcct ggggagtacg gtcgcaagat 840

taaaactcaa aggaattgac gggggcctgc acaagcggtg gagcatgtgg tttaattcga 900

agcaacgcgc agaaccttac cagcgtttga catcctgatc gcggttacca gagatggttt 960

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gatgttgggt taagtcccgc aacgagcgca accctcatcc ctagttgcca tcattcagtt 1080

gggcactcta aggaaactgc cggtgataag ccggaggaag gtggggatga cgtcaagtcc 1140

tcatggccct tacgcgctgg gctacacacg tgctacaatg gcggtgacag tgggcagcaa 1200

ccgggcgacc ggtagctaat ctccaaaagc cgtctcagtt cggattgttc tctgcaactc 1260

gagagcatga aggcggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1320

ccaggccttg tacacaccgc ccgtcacacc atgggagttg gtttcacccg aaggcagtgc 1380

tctaacccgc aagggaggaa gctgaccacg gtgggatcag cgactggggt gaagtcgtaa 1440

caaggtaacc 1450

Claims

1. A bacterial strain DCP-6 capable of degrading two isomers of 2, 4-dichlorprop-p of chiral herbicide simultaneously is characterized by being classified and named as Sphingopyxis sp. DCP-6, and deposited in China center for type culture collection in 2019, 12 months and 23 days, wherein the preservation number of the bacterial strain is CCTCC NO: M20191084.

2. The application of the strain DCP-6 in the claim 1 in preparing fungicide for degrading two isomers of phenoxy carboxylic acid herbicides; the phenoxy carboxylic acid herbicide is selected from any one or two of (R, S) -DCPP and (R, S) -MCPP.

3. The application of the strain DCP-6 of claim 1 in preparing a fungicide for degrading phenoxy carboxylic acid herbicides; the phenoxy carboxylic acid herbicide is selected from any one or more of 2,4-D, MCPA or 2, 4-DB.

4. A microbial inoculum produced by the strain DCP-6 of claim 1, which is prepared by fermentation of the strain of claim 1.

5. The microbial preparation according to claim 4, which is produced by the following method:

(1) inoculating the test tube strain of the strain DCP-6 of claim 1 into an LB culture medium shake flask, and culturing with shaking to logarithmic phase;

(2) inoculating the cultured strain into a seeding tank according to the inoculation amount of 10 percent, culturing to a logarithmic phase, wherein the formula of a culture medium used by the seeding tank is as follows: glucose 8g/L, yeast extract 5g/L, K₂HPO₄1 g/L，NaCl 5 g/L，CaCO₃ 2 g/L，MgSO₄0.2g/L, 0.1 percent of soybean oil volume ratio and 7.2 to 7.5 of pH value;

6. A method for producing the microbial agent according to claim 4, characterized by comprising the steps of:

7. The use of the fungicide of claim 4 for degrading phenoxy carboxylic acid herbicides; the phenoxy carboxylic acid herbicide is selected from any one or more of (R, S) -DCPP, (R, S) -MCPP, 2,4-D, MCPA or 2, 4-DB.

8. The use of claim 7, wherein the phenoxy carboxylic acid herbicide is selected from any one or more of (R, S) -DCPP, (R, S) -MCPP, and 2, 4-D.