CN108410759B - Rhodococcus rhodochrous for efficiently degrading cyhalothrin and application thereof - Google Patents

Abstract

The invention discloses rhodococcus rhodochrous for efficiently degrading cyhalothrin and application thereof. The invention takes microbial resources in the traditional fermentation process as research objects, and obtains the degradation bacteria of the cyhalothrin which has more usage, large residual quantity and long degradation period in the planting process of crops such as tobacco and the like by screening, wherein the degradation bacteria is Rhodococcus rhodochrous L5 strain which is from Fenjiu Daqu, has high safety, can efficiently degrade the cyhalothrin with the concentration of 5mg/kg, has certain application prospect for degrading the low-concentration cyhalothrin pesticide residue of the crops such as tobacco leaves and the like, and provides a safe and effective biodegradation way for improving the quality and safety of tobacco products and solving the pesticide residue problem of the crops such as the tobacco leaves and the like.

Description

Rhodococcus rhodochrous for efficiently degrading cyhalothrin and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a function and characteristics of a strain for efficiently degrading cyhalothrin rhodococcus rhodochrous.

Background

With the centralized development of agriculture, the pesticide is widely used in the world, and on one hand, the use of the pesticide can improve the yield of crops; on the other hand, pesticide residues in the product pose potential health hazards and cause environmental pollution. In recent years, studies on the degradation of pesticide residues by microorganisms have been greatly advanced. However, currently, pesticide degrading bacteria are mainly screened from soil and water body sludge with serious pesticide pollution and mainly used for solving the pesticide pollution in the soil and the water body, most of screened strains are pseudomonas, klebsiella and the like, the safety of the strains is questioned, and the application of the strains is limited to a certain extent. Therefore, the search for safe and reliable sources of strains becomes the key to the degradation of pesticide residues.

Research reports that some strains in the food fermentation process have strong pesticide degradation capability. The traditional Chinese fermented food has a long history and a plurality of varieties, and contains rich food microbial resources. Some microorganisms can grow pesticides as carbon sources and nitrogen sources in the fermentation process, so that the effect of degrading pesticides is achieved, and for example, yeast and other microorganisms can degrade pesticides such as pyrethroid, organochlorine insecticide, organophosphorus insecticide and the like.

Disclosure of Invention

An object of the present invention is to provide a strain of Rhodococcus rhodochrous L5.

The preservation number of the Rhodococcus rhodochrous L5 provided by the invention is CGMCC No. 15232.

The Rhodococcus rhodochrous L5 is classified and named as Rhodococcus rhodochrous, and the strain has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 of West Lu 1 of Beijing Ind. area, Ministry of microbiology of China academy of sciences, postal code 100101) in 2018, 17.01.8.D.and the preservation number is CGMCC No. 15232.

Another object of the present invention is to provide a novel use of Rhodococcus rhodochrous or a bacterial suspension thereof or a bacterial agent thereof or a fermentation broth thereof or a metabolic broth thereof or a culture thereof.

The invention provides application of Rhodococcus rhodochrous or bacterial suspension thereof or microbial inoculum thereof or fermentation liquor thereof or metabolic liquid thereof or culture solution thereof in degrading pyrethroid pesticides.

The invention also provides the application of Rhodococcus rhodochrous or its bacterial suspension or its bacterial agent or its fermentation liquor or its metabolic liquor or its culture liquor in preparing the product for degrading pyrethroid pesticide.

In the application, the pyrethroid pesticide is cyhalothrin.

In the above application, the Rhodococcus rhodochrous is Rhodococcus rhodochrous L5CGMCC No. 15232.

It is a further object of the invention to provide a product for degrading pyrethroid insecticides.

The active ingredient of the product for degrading the pyrethroid pesticide provided by the invention is Rhodococcus rhodochrous or bacterial suspension thereof or microbial inoculum thereof or fermentation liquor thereof or metabolic liquid thereof or culture solution thereof.

In the product, the pyrethroid pesticide is cyhalothrin.

In the above product, said Rhodococcus rhodochrous is Rhodococcus rhodochrous L5CGMCC No. 15232.

It is a final object of the invention to provide a method for degrading pyrethroid insecticides.

The method for degrading pyrethroid pesticide provided by the invention comprises the step of treating the substance containing pyrethroid pesticide with Rhodococcus rhodochrous or its bacterial suspension or its microbial inoculum or its fermentation liquor or its metabolic liquid or its culture solution.

In the method, the pyrethroid pesticide is cyhalothrin. The substance containing the pyrethroid pesticide can be crops such as tobacco leaves with residual cyhalothrin and the like.

In the above method, said Rhodococcus rhodochrous is Rhodococcus rhodochrous L5CGMCC No. 15232.

The invention takes microbial resources in the traditional fermentation process as research objects, and obtains the degradation bacteria of the cyhalothrin which has more usage, large residual quantity and long degradation period in the planting process of crops such as tobacco and the like by screening, wherein the degradation bacteria is Rhodococcus rhodochrous L5 strain which is from Fenjiu Daqu, has high safety, can efficiently degrade the cyhalothrin with the concentration of 5mg/kg, has certain application prospect for degrading the low-concentration cyhalothrin pesticide residue of the crops such as tobacco leaves and the like, and provides a safe and effective biodegradation way for improving the quality and safety of tobacco products and solving the problem of pesticide residue of the crops such as the tobacco leaves and the like.

Drawings

FIG. 1 is a chromatogram of cyhalothrin standard.

FIG. 2 is a standard curve of cyhalothrin.

FIG. 3 shows the result of measuring the degradation rate of cyhalothrin.

FIG. 4 is a graph showing the growth curve of Rhodococcus rhodochrous and the degradation kinetics curve of cyhalothrin. Note: tangle-solidup, change in cyhalothrin concentration in the group not inoculated with rhodococcus rhodochrous; ■, inoculation of a change in concentration of cyhalothrin in the Rhodococcus rhodochrous group; o, growth change of Rhodococcus rhodochrous in the group without addition of cyhalothrin; □, growth changes of Rhodococcus rhodochrous in the group of addition of cyhalothrin.

Deposit description

The strain name is as follows: rhodococcus rhodochrous

Latin name: rhodococcus rhodochrous

The strain number is as follows: l5

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: year 2018, month 01, day 17

Registration number of the preservation center: CGMCC No.15232

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The leavening agent in the following examples is a product of cunninghami fenjiu ltd, west, which is a traditional food leavening agent (fen jiu daqu) containing abundant strain resources such as lactic acid bacteria, bacillus, yeast, mold and actinomycetes.

The pesticide standard cyhalothrin in the following examples is the product of carbofuran J & K.

The basic inorganic salt medium (MSM) in the following examples is composed of solvent and solute, the solvent is water, and the solute and its concentration in the medium are as follows: NH (NH)₄NO₃，1.5g/L；KH₂PO₄，0.5g/L；K₂HPO₄，1.5g/L；NaCl，1.0g/L；MgSO₄·7H₂O, 0.2 g/L; when MSM solid culture medium is prepared, 15-20g/L agar is added.

The LB broth medium (synthetic dry powder medium) and LB nutrient agar medium (synthetic dry powder medium) in the following examples are both products of Oboxing Biotechnology Co., Ltd, Beijing.

The solvent used for diluting the pesticide in the following examples is acetonitrile, and the solvent is a product of Shirong chemical Co., Ltd.

Example 1 isolation, identification and preservation of L5 Strain

Enrichment, domestication, separation and purification of pesticide degrading bacteria

1. Enrichment and domestication of pesticide degrading bacteria

The strain is screened by taking fresh yeast of Fenjiu factory as a source. The method comprises the following specific steps: weighing 10g Fenjiu Daqu in a triangular flask of 90mL physiological saline, culturing at 30 ℃ and 180rpm for 40min, taking 1mL of Fenjiu Daqu, transferring into 9mL of MSM culture medium containing 50mg/kg of cyhalothrin, culturing at 30 ℃ and 180rpm for 7d, sucking 1mL of Fenjiu Daqu, transferring into 9mL of MSM culture medium containing 100mg/kg of cyhalothrin, culturing at 30 ℃ and 180rpm for 7d, continuously transferring according to the above operation, and gradually increasing the concentration of the cyhalothrin to 400mg/kg of 200-.

2. Separation and purification of pesticide degrading bacteria

The enrichment and domestication experiment of the degrading bacteria lasts for 28 days at the early stage, and the bacteria cultured for 28 days by the cyhalothrin with the concentration of 400mg/kg for the fourth transfer are subjected to stock solution and 10^-1、10^-2、10^-3Spread on MSM solid medium containing 100mg/kg cyhalothrin, and cultured in a constant temperature incubator at 37 deg.C for 2-5 days. Colonies of different forms with fast growth and good growth condition are picked and continuously purified and cultured for 3 times on MSM solid culture medium containing 100mg/kg cyhalothrin. After the culture is finished, selecting a single bacterial colony which grows vigorously and is large in bacterial colony after streaking to be placed on an LB solid culture medium flat plate, and finally obtaining a pesticide-tolerant strain which is named as an L5 strain.

II, identification of L5 Strain

1. Physicochemical characterization of L5 Strain

The physicochemical properties of the L5 strain are shown in Table 1.

TABLE 1 physicochemical Properties of L5 Strain

Strain name	Name of the genus	Color of colony	Cell shape	Gram stain results	Mode of dissimilarity
						Rhodococcus rhodochrous	Rhodococcus genus	Reddish colour	Spherical shape	Positive (+)	Aerobic treatment

2. Molecular characterization of L5 Strain

Colony PCR was performed on the isolated and purified L5 strain. The PCR reaction system is 25 mu L, and the PCR reaction conditions are as follows: keeping at 94 deg.C for 5 min; the main circulation is at 94 ℃ for 30 s; 30s at 54 ℃; circulating for 35 times at 72 deg.C for 1 min; final extension at 72 ℃ for 7 min; storing at 4 ℃. After colony PCR, detecting the PCR product by using 1% agarose gel electrophoresis, then sending the PCR product to Huada gene (Beijing) Limited for purification and sequencing, applying Vector NTI 11.5 software to process the bidirectional sequencing result, and then logging in NCBI for BLAST comparison to determine the strain species. The 16S rDNA sequence of the strain L5 is shown in SEQ ID No. 1.

By combining the above identification results, it was determined that the L5 strain was named Rhodococcus rhodochrous, which was classified and named Rhodococcus rhodochrous, and was deposited in the China general microbiological culture Collection center (CGMCC for short, address: No. 3 of the institute for microbiology, China academy of sciences, zip code 100101) on 17.01.2018 in the Ministry of culture Collection of microorganisms, and the deposition number was CGMCC No. 15232.

Example 2 use of Rhodococcus rhodochrous L5 for degrading Cyhalothrin

Firstly, preparation of bacterial suspension

Picking Rhodococcus rhodochrous L5 from LB solid medium, transferring into a vial containing LB broth, and culturing at 30 deg.C in shaker (150rpm) until the colony OD600 is 1.0 + -0.1; centrifuging the bacterial liquid under 5000 Xg for 5min, discarding supernatant, washing thallus precipitate with MSM liquid culture medium, mixing, washing for 2 times, adding MSM liquid culture medium, and oscillating with vortex oscillator for resuspension to obtain thallus suspension with bacterial concentration of 10⁷cfu/mL。

Second, degradation of cyhalothrin

1. According to the tobacco leaf pesticide residue standard YQ 50-2014 of the Chinese tobacco general company, the maximum limit standard of cyhalothrin pesticide in tobacco is regulated to be 1mg/kg, 2 times of pesticide limit value in the tobacco leaf pesticide residue standard YQ 50-2014 is selected for carrying out experiments by referring to 2 times of pesticide limit value mentioned in the feedback information of Shanghai tobacco company.

And uniformly mixing the cyhalothrin and the MSM liquid culture medium to ensure that the concentrations of the cyhalothrin in the MSM liquid culture medium are respectively 2mg/kg, 5mg/kg and 10mg/kg, thereby obtaining the MSM liquid culture medium containing the cyhalothrin with different concentrations.

2. And (3) adding the bacterial suspension prepared in the first step into the MSM liquid culture medium containing cyhalothrin with different concentrations according to the addition amount of 5% by volume and 1% by mass of glucose to obtain a rhodococcus rhodochrous-cyhalothrin system containing cyhalothrin with different concentrations, wherein the volume of the rhodococcus rhodochrous-cyhalothrin system is 10 mL.

3. Subjecting the rose red ball prepared in step twoThe bacterial-cyhalothrin system is cultured for 7d in a shaking way at 30 ℃ and 180rpm and taken as a treatment group, meanwhile, MSM culture medium which does not contain bacterial suspension and contains cyhalothrin with different concentrations is taken as a control group, and the control group and the treatment group are respectively carried out in 3 parallels. The concentration of cyhalothrin in the rhodococcus rhodochrous-cyhalothrin system containing cyhalothrin with different concentrations after 7 days is measured by a gas chromatography-mass spectrometry combined technology (GC-MS), and the degradation rate is calculated. The manufacturing method of the standard curve of cyhalothrin is as follows: preparing a standard curve working solution of cyhalothrin with the following concentration: 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg, 20 mg/kg. And (3) carrying out quantitative detection on the working solution of the standard curve with different concentrations by adopting GC-MS, taking the concentration of the cyhalothrin as an abscissa, taking the peak area of the characteristic peak as an ordinate, and carrying out linear regression according to the relation between the concentration and the peak area to draw the standard curve. Wherein, the GC-MS quantitative detection method comprises the following steps: sample pretreatment: 2mL of the sample was aspirated and centrifuged at 12000 Xg for 5 min. Collecting 1.5mL of supernatant, adding acetonitrile with the same volume, violently shaking for 1min, adding 1.5g of sodium chloride, shaking for 1min, centrifuging at a rotating speed of 3800rpm for 5min, taking 1mL of supernatant into a 2mL centrifuge tube filled with 150mg of anhydrous magnesium sulfate, shaking for 1min, centrifuging at a rotating speed of 10000rpm for 1min, taking the supernatant, passing the supernatant through a 0.22 mu m organic filter membrane into a sample vial, and waiting for GC-MS detection. Chromatographic conditions are as follows: carrier gas: helium, column flow rate 1.0mL min^-1(ii) a The sample inlet temperature is 280 ℃, the non-split sample injection mode is adopted, the sample injection time is 1.0min, and the sample injection amount is 1 mu L; column temperature program: initial temperature 100 ℃ at 30 ℃ min^-1Heating to 250 deg.C, maintaining for 0min, and heating to 20 deg.C/min^-1The temperature is raised to 310 ℃ and kept for 10 min. Mass spectrum conditions: carrier gas: helium gas; a collision device: argon gas; the transmission line temperature is 290 ℃; an ion source E1 source; the ion source temperature is 250 ℃; electron energy 70 eV; the solvent delay time is 2.5 min; the scanning mode is as follows: multiple selection ion storage assay (SIS).

The three characteristic ions of cyhalothrin are 152, 181 and 208, respectively. According to the characteristic ion, a characteristic peak can be separated from the chromatogram, the characteristic ion peak with the highest responsivity is used for quantification, the other two characteristic ion peaks are used for qualification, the peak area representing the characteristic peak of the pesticide is calculated, and the degradation rate is calculated. The degradation rate calculation formula is as follows:

third, experimental results

1. Rate of degradation

The chromatogram of cyhalothrin standard and the standard curve of cyhalothrin are shown in FIG. 1 and FIG. 2. And (3) measuring each group of samples according to a standard GC-MS (gas chromatography-mass spectrometry) method and a mass spectrometry method, wherein peak areas of characteristic ions of 152, 181 and 208 are basically matched, and the peak represents cyhalothrin. The 181 characteristic ion peak with the highest responsiveness is used for quantification, the 152 and 208 characteristic ion peaks are used for qualification, and the SPSS software is used for carrying out differential analysis on the degradation rates of the cyhalothrin with different concentrations to obtain the degradation rate results of Rhodococcus rhodochrous (Rhodococcus rhodochrous) on the cyhalothrin with the concentrations of 2mg/kg, 5mg/kg and 10 mg/kg.

The results are shown in FIG. 3. As can be seen from FIG. 3, Rhodococcus rhodochrous L5 has a certain degradation rate to cyhalothrin at concentrations of 2mg/kg, 5mg/kg and 10 mg/kg. Wherein, the degradation rate of Rhodococcus rhodochrous L5 to cyhalothrin with the concentration of 5mg/kg is 71.44 percent, the peak value of the degradation rate to cyhalothrin with different concentrations is reached, the degradation rate to cyhalothrin with the concentration of 2mg/kg is 28.87 percent, and the degradation rate to cyhalothrin with the concentration of 10mg/kg is 40.85 percent. The inter-group data difference analysis of the Rhodococcus rhodochrous-cyhalothrin shows that the Rhodococcus rhodochrous L5 has the highest degradation rate on the cyhalothrin with the concentration of 5mg/kg, the second highest degradation rate on the cyhalothrin with the concentration of 10mg/kg, the lowest degradation rate on the cyhalothrin with the concentration of 2mg/kg, and the degradation rates of the groups with the concentrations of 2mg/kg and 10mg/kg are significantly different from the degradation rate of the group with the concentration of 5 mg/kg. It was demonstrated that Rhodococcus rhodochrous L5 is an effective degrading bacterium of cyhalothrin.

2. Dynamic change curve of pesticide concentration and dynamic change curve of strain growth

In order to explore the interaction between cyhalothrin and degradation bacteria and the degradation speed change of cyhalothrin in 0-7d, the concentration change of cyhalothrin in the presence of degradation bacteria in 0-7d and the growth change curve of degradation bacteria in the presence of cyhalothrin in 0-7d were determined. The method comprises the following specific steps:

(1) uniformly mixing cyhalothrin with an MSM liquid culture medium to ensure that the concentration of the cyhalothrin in the MSM liquid culture medium is 5mg/kg, thereby obtaining the MSM liquid culture medium containing the cyhalothrin;

(2) adding the bacterial suspension prepared in the first step into the MSM liquid culture medium containing cyhalothrin prepared in the step (1) according to the addition amount of 5% by volume and the addition amount of 1% by mass of glucose to obtain a rhodococcus rhodochrous-cyhalothrin system; simultaneously, taking the MSM liquid culture medium containing cyhalothrin and the MSM liquid culture medium containing no cyhalothrin as controls;

(3) the concentration of cyhalothrin and the OD of the bacterial solution were measured at 0 th, 1 th, 2 th, 3 th, 4 th, 5 th, 6 th and 7 th days of culture, respectively_600nm。

The results are shown in FIG. 4. It can be seen from the figure that: in the control group not inoculated with Rhodococcus rhodochrous, the amount of change in the concentration of cyhalothrin in the MSM liquid medium containing the concentration of 5mg/kg was small, and the concentration of cyhalothrin was decreased in the experimental group of cyhalothrin inoculated with Rhodococcus rhodochrous. In addition, the growth of Rhodococcus rhodochrous strain was significantly promoted in the experimental group to which cyhalothrin was added at a concentration of 5mg/kg, as compared with the control group to which cyhalothrin was not added. The degradation rate of the cyhalothrin is related to the growth density of the strain. Cyhalothrin can promote the growth of Rhodococcus rhodochrous, and the degradation rate of cyhalothrin is improved by Rhodococcus rhodochrous.

Sequence listing

<110> university of agriculture in China

<120> rhodococcus rhodochrous for efficiently degrading cyhalothrin and application thereof

<160>1

<170>PatentIn version 3.5

<210>1

<211>1400

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gtcgaacgat gaagcccagc ttgctgggtg gattagtggc gaacgggtga gtaacacgtg 60

ggtgatctgc cctgcactct gggataagcc tgggaaactg ggtctaatac cggatatgac 120

ctcttgctgc atggcgaggg gtggaaagtt tttcggtgca ggatgagccc gcggcctatc 180

agcttgttgg tggggtaatg gcctaccaag gcgacgacgg gtagccggcc tgagagggcg 240

accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 300

attgcacaat gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg 360

ttgtaaacct ctttcagcag ggacgaagcg aaagtgacgg tacctgcaga agaagcaccg 420

gccaactacg tgccagcagc cgcggtaata cgtagggtgc gagcgttgtc cggaattact 480

gggcgtaaag agctcgtagg cggtttgtcg cgtcgtctgt gaaatcccgc agctcaactg 540

cgggcttgca ggcgatacgg gcagactcga gtactgcagg ggagactgga attcctggtg 600

tagcggtgaa atgcgcagat atcaggagga acaccggtgg cgaaggcggg tctctgggca 660

gtaactgacg ctgaggagcg aaagcgtggg tagcgaacag gattagatac cctggtagtc 720

cacgccgtaa acggtgggcg ctaggtgtgg gtttccttcc acgggatccg tgccgtagcc 780

aacgcattaa gcgccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga 840

cgggggcccg cacaagcggc ggagcatgtg gattaattcg atgcaacgcg aagaacctta 900

cctgggtttg acatgtaccg gacgactgca gagatgtggt ttcccttgtg gccggtagac 960

aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020

gcgcaaccct tgtcctgtgt tgccagcacg tgatggtggg gactcgcagg agactgccgg 1080

ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat gtccagggct 1140

tcacacatgc tacaatggtc ggtacagagg gctgcgatac cgtgaggtgg agcgaatccc 1200

ttaaagccgg tctcagttcg gatcggggtc tgcaactcga ccccgtgaag tcggagtcgc 1260

tagtaatcgc agatcagcaa cgctgcggtg aatacgttcc cgggccttgt acacaccgcc 1320

cgtcacgtca tgaaagtcgg taacacccga agccggtggc ctaacccctt gtgggaggga 1380

gccgtcgaag gtgggatcgg 1400

Claims (7)

1. A strain of Rhodococcus rhodochrous L5 with preservation number of CGMCC No. 15232.

2. The use of Rhodococcus rhodochrous (Rhodococcus rhodochrous) or a bacterial suspension thereof or a bacterial preparation thereof for degrading pyrethroid pesticides;

or, the use of Rhodococcus rhodochrous (Rhodococcus rhodochrous) or a bacterial suspension thereof or a bacterial preparation thereof for the preparation of a product for degrading pyrethroid insecticides;

the Rhodococcus rhodochrous (Rhodococcus rhodochrous) is Rhodococcus rhodochrous (Rhodococcus rhodochrous) with a accession number of CGMCC No.15232 as set forth in claim 1.

3. Use according to claim 2, characterized in that: the pyrethroid pesticide is cyhalothrin.

4. A product for degrading pyrethroid pesticide comprises Rhodococcus rhodochrous (Rhodococcus rhodochrous) or its suspension or its inoculum as active ingredient;

the Rhodococcus rhodochrous (Rhodococcus rhodochrous) is Rhodococcus rhodochrous (Rhodococcus rhodochrous) with a accession number of CGMCC No.15232 as set forth in claim 1.

5. The product of claim 4, wherein: the pyrethroid pesticide is cyhalothrin.

6. A method for degrading a pyrethroid pesticide comprises treating a substance containing the pyrethroid pesticide with Rhodococcus rhodochrous (Rhodococcus rhodochrous) or a bacterial suspension thereof or a bacterial preparation thereof;

the Rhodococcus rhodochrous (Rhodococcus rhodochrous) is Rhodococcus rhodochrous (Rhodococcus rhodochrous) with a accession number of CGMCC No.15232 as set forth in claim 1.

7. The method of claim 6, wherein: the pyrethroid pesticide is cyhalothrin.

Images