CN109604332B

CN109604332B - Application of ochrobactrum intermedium in degradation of triadimefon

Info

Publication number: CN109604332B
Application number: CN201910125508.7A
Authority: CN
Inventors: 张鑫; 杨凯; 徐玮杰; 戴泽元
Original assignee: Shanghai Tobacco Group Co Ltd
Current assignee: Shanghai Tobacco Group Co Ltd
Priority date: 2019-02-20
Filing date: 2019-02-20
Publication date: 2021-06-04
Anticipated expiration: 2039-02-20
Also published as: CN109604332A

Abstract

The invention provides application of ochrobactrum intermedium F1 in degrading triadimefon. Experiments show that the ochrobactrum intermedium F1 can effectively degrade triadimefon and has good application prospects in soil pollution improvement and degradation of residual triadimefon in fruits and vegetables.

Description

Application of ochrobactrum intermedium in degradation of triadimefon

Technical Field

The invention relates to application of ochrobactrum intermedium F1 in degrading triadimefon.

Background

Triadimefon is also called triadimefon and Bailitong and is the first widely used triazole fungicide. Has special effect on powdery mildew, rust disease and smut, thereby obtaining a beautiful name: "Puccining". Triazolone is widely applied in China, more raw pesticide and preparation manufacturers exist, and the developed preparation is of various types, mainly comprising 15 percent of emulsifiable solution, 20 percent of emulsifiable solution, 8 percent of hypertonic emulsifiable solution, 10 percent of hypertonic emulsifiable solution, 12 percent of synergistic emulsifiable solution, 10 percent of wettable powder, 15 percent of wettable powder, 25 percent of hypertonic wettable powder, 15 percent of aerosol, and a plurality of compounded bactericides, bactericidal insecticides and seed coatings containing triazolone. The triazolone can be applied in various modes such as stem leaf spraying, seed treatment, soil treatment and the like.

The data show that the human beings use pesticides to kill insects and weed from the 40 th generation of the 20 th century, and the annual loss of the total agricultural yield is saved by about 15%. However, the use of a large amount of pesticides for a long time greatly increases harmful substances in the environment, harms the ecology and the human beings, and causes pesticide pollution. It is reported that the utilization rate of the agricultural chemicals is generally 10%, and about 90% of the agricultural chemicals remain in the environment, causing pollution to the environment. China is a big agricultural country, the use amount of chemical pesticides is the first worldwide, according to statistics, the use area of pesticides in China is up to 1.8 hundred million hectares every year, 666 which is used since the last 50 th century reaches 400 million tons, more than DDT50 million tons, and 1330 million hectares of polluted farmlands.

At present, although the technology for degrading residual pesticides by using microorganisms is mature, the research on triazolone is blank.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide an application of Ochrobactrum intermedium F1(Ochrobactrum intermedium F1) in degrading triadimefon for residual triadimefon in the environment.

In order to achieve the above objects and other related objects, the present invention provides an application of Ochrobactrum intermedium F1(Ochrobactrum intermedium F1) in degrading triadimefon.

In another aspect, the invention provides an application of Ochrobactrum intermedium F1(Ochrobactrum intermedium F1) in preparing pesticide degradation agents, soil improvement agents or fruit and vegetable detergents.

In another aspect, the invention provides a pesticide degradation agent, which contains an effective dose of ochrobactrum intermedium F1.

The pesticide degradation agent can be a degradation agent specially used for degrading triadimefon, and can also be a degradation agent containing other residual pesticides.

In another aspect, the invention provides a soil improving agent, which contains an effective dose of ochrobactrum intermedium F1.

In another aspect of the invention, the fruit and vegetable detergent contains an effective dose of ochrobactrum intermedium F1.

The pesticide-degrading agent, the soil improving agent, and the fruit and vegetable detergent are not limited to the preparations named "pesticide-degrading agent", "soil improving agent", or "fruit and vegetable detergent", and the preparations having a pesticide-degrading function, a soil-improving function, or a fruit and vegetable-washing function should be the pesticide-degrading agent, the soil improving agent, or the fruit and vegetable detergent referred to in the present application.

In another aspect of the present invention, there is provided a method for degrading triadimefon, which comprises applying ochrobactrum intermedium F1 to a subject having triadimefon remaining thereon. The treatment object is soil or fruits and vegetables, and when the treatment object is soil, the method further comprises the step of spraying ochrobactrum intermedium F1 in the soil; when the treated object is fruits and vegetables, the method comprises the step of soaking the fruits and vegetables in a solvent containing ochrobactrum intermedium F1.

The method or use is carried out in an amount of 0.4 to 1.2X 10 per 1mg of triazolone-O.xanthus F1⁸ CFU。

As described above, the application of ochrobactrum intermedium F1 in degrading triadimefon has the following beneficial effects:

the ochrobactrum intermedium F1 provided by the invention can effectively degrade triazolone residues which exceed the limit requirement of triazolone of grains, cereals, oil plants, vegetables, fruits and tobaccos by tens of times; and the ochrobactrum intermedium F1 is derived from the traditional leavening agent, is safe and reliable, and has good application prospect.

Drawings

FIG. 1 shows an electron micrograph and an under-microscope view of ochrobactrum intermedium.

Figure 2 shows a triadimefon standard chromatogram.

Figure 3 shows the triadimefon standard curve.

FIG. 4 shows the degradation rate of triadimefon by 3 species of example 3.

FIG. 5 shows the growth curves of the 3 groups of bacteria in example 4 and the concentration curves of triadimefon in the experimental group.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art. Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Materials and instruments:

(1) a leavening agent: the traditional food leaven (Fenjiu Daqu) collected in the laboratory contains abundant strain resources such as lactic acid bacteria, bacillus, yeast, mold and actinomycetes, and is provided by Shanxi Xinghua cunfen wine GmbH.

(2) Basal Mineral Salts Medium (MSM): NH (NH)₄NO₃1.5g/L；KH₂PO₄0.5g/L；K₂HPO₄1.5 g/L; NaCl1.0 g/L; MgSO4 & 7H2O 0.2g/L; when MSM solid culture medium is prepared, 15-20g/L agar is added, Beijing Omboxing Biotechnology GmbH.

(3) LB broth culture medium: synthetic dry powder medium, oko star biotechnology limited, beijing.

(4) LB nutrient agar medium: synthetic dry powder medium, oko star biotechnology limited, beijing.

(5) Apparatus and device

Ultraviolet spectrophotometer: 2800UV/VIS SPECTROPHOTOMETER

Superclean bench: TY-CJ-2ND Hardong electronic technology development Co., Ltd

Multi-tube vortex mixer: LE PARD

A vortex mixer: MS2-Minishaker IKA

An electronic balance: SPS202F Mettler-Torledo weighing apparatus, Inc

Constant temperature shake incubator: SKY-2102C Shanghai Sukun industries Ltd

Electric heating constant temperature incubator: DHP-9052 Shanghai-Hengchang scientific instruments Co., Ltd

Freezing a centrifuge: TGL-20M Changsha trivial instruments & instruments Limited

An autoclave: ZDZX-40B1 Shanghai Shenan medical instruments factory

Electric heating constant temperature water bath: s11-2 Beijing Changan scientific instrument factory

Electric heating constant temperature air blast drying cabinet: DHG-9053A Shanghai-Hengchang scientific instruments Co., Ltd

Example 1

(1) Enrichment and domestication of pesticide degrading bacteria

Taking fresh Daqu of Fenjiu factory as a source of a screened strain, weighing 10g of Daqu in a triangular flask of 90ml of normal saline, culturing for 40min at 30 ℃ and 180rpm, taking 1ml of Daqu, transferring into 9ml of MSM culture medium containing 50mg/kg, culturing for 7d at 30 ℃ and 180rpm, sucking 1ml of Daqu, transferring into 9ml of MSM culture medium containing 100mg/kg, culturing for 7d at 30 ℃ and 180rpm, continuously transferring according to the above operation, and gradually increasing the concentration of pesticide to 200-400 mg/kg.

(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 at the pesticide concentration of 400mg/kg and transferred for the fourth time are subjected to stock solution and 10^-1、10^-2、10^-3Spreading on MSM solid culture medium containing 100mg/kg pesticide, and culturing in 37 deg.C incubator 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 pesticide. After the culture is finished, selecting the single colony with better growth condition after streaking on an LB solid culture medium flat plate. And obtaining single bacterial colonies of the pesticide-resistant bacteria.

(3) Identification of colonies

Carrying out colony PCR on the separated and purified strains, adopting a 25 mu L PCR reaction system, and carrying out PCR reaction conditions: 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 in the laboratory, PCR products were detected by electrophoresis on a 1% agarose gel. And (3) delivering the PCR product to Huada Gene (Beijing) Co Ltd for purification and sequencing, applying Vector NTI 11.5 software to process the bidirectional sequencing result, and logging in NCBI for BLAST comparison to determine the strain species.

TABLE 1 identification of 16s rRNA sequence of Strain

The physicochemical properties of each pesticide-degrading strain are shown in table 2:

TABLE 2 physicochemical Properties of the strains

Example 2

Adding the bacterial liquid after the heavy suspension of each strain into MSM liquid culture media containing pesticides with different concentrations according to the addition of 5% and the addition of 1% of glucose, wherein the pesticide concentrations are 2, 5 and 10 times of the pesticide residue limit standard of each tobacco leaf in sequence. The total volume is 10 ml. After culturing for 7 days at 30 ℃ in a shaking table (150rpm), the degradation rate of each strain for the pesticide in MSM medium with 2, 5, and 10 times of pesticide concentration after 7 days was measured. In the control test, no bacterial suspension is added, the change of pesticide concentration is measured after the test piece is placed for 7 days under the same condition, and 3 control groups and 3 treatment groups are respectively carried out in parallel.

Sampling 3ml each day, wherein 1ml is measured by ultraviolet spectrophotometer to characterize bacterial liquid OD of strain growth_600nmValue, another 2ml were pretreated and then tested for triadimefon concentration by gas chromatography-mass spectrometry (GC-MS):

gas mass chromatograph (GC-MS): agilent Technologies 5977B MSD; a chromatographic column: HP-5 capillary column (30m × 0.25mm × 0.25 μm)

Sample pretreatment: 2ml of the selectively cultured bacterial suspension 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, placing the 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 temperature of a sample inlet is 280 ℃, the sample injection mode is not divided, the sample injection time is 1.0min, and the sample injection amount is 1 mu L; column temperature program: initial temperature 100 ℃ at 20 ℃ min^-1Heating to 225 deg.C, maintaining for 0min, and heating to 10 deg.C/min^-1Heating to 265 deg.C, maintaining for 0min, and heating at 30 deg.C for min^-1The temperature is raised to 290 ℃ and kept for 4 min.

Mass spectrum conditions: carrier gas: helium gas; a collision device: argon gas; transmission line temperature: 290 ℃; an ion source: a source of E1; ion source temperature: 250 ℃; electron energy: 70 eV; solvent delay time: 2.5 min; the scanning mode is as follows: multiple selection ion storage assay (SIS).

The triadimefon standard chromatogram and triadimefon standard curve are shown in fig. 2 and fig. 3.

The degradation rate of the three strains on the triadimefon is shown in figure 4, and the efficiency of degrading the triadimefon by the ochrobactrum intermedium F1 is obviously higher than that of the other two strains.

Example 3

The amplification medium contained 1.5g/L NH₄NO₃、0.5g/L KH₂PO₄、1.5g/L K₂HPO₄、1.0g/L NaCl、0.2g/L MgSO₄·7H₂O MSM medium. Sterilizing and reserving for later use.

Triazolone was purchased from carbofuran J & K with a purity of 98.5%.

Selecting a single colony of ochrobactrum intermedium F1 from an LB solid medium plate of a preserved strain, inoculating the single colony into an LB broth medium bottle at the temperature of 30℃,Culturing in 150rpm shaking table to bacterial liquid OD₆₀₀Centrifuging the bacterial solution at 5000 × g 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 mix thallus completely to obtain degraded bacteria seed solution, wherein the viable count of intermediate ochrobactrum F1 is 80 × 10⁸CFU/ml。

3 sets of experiments as shown in Table 3, 5% by volume (about 4X 10 colonies) of MSM broth supplemented with 1% glucose carbon source was added⁸CFU) of ochrobactrum intermedium F1 degraded bacterial seed fluid and/or triadimefon (final concentration 10mg/kg), and 3 replicates were set up for each set of experiments. The cells were incubated at 30 ℃ for 7d on a shaker at 150 rpm.

TABLE 3 analysis set up for ability of ochrobactrum intermedium to degrade triazolone

Additive substance	Test group	Control group A	Control group B
				5% Ochrobactrum intermedium suspension	+	+	-
10mg/kg triazolone	+	-	+

The detection conditions and method were the same as in example 2.

It can be seen that: 3 days before the test process, the ochrobactrum intermedium F1 is in a logarithmic growth phase, the bacterial liquid concentrations in the test group and the control group A both rapidly rise, and the concentration of the test group is higher than that of the control group A; meanwhile, the concentration of the triazolone in the test group is reduced to 5.1mg/kg, and the concentration of the triazolone in the control group B is reduced to 9.6mg/kg only. The 4 th to 7 th days in the test process are in a stable growth period, the bacterial liquid concentrations of the test group and the control group A are both slightly reduced, and the concentration of the test group is still higher than that of the control group A; the degradation rate of triadimefon in the test group was slowed to a final concentration of 4.0mg/kg, and 9.0mg/kg for control group B.

The result shows that the provided ochrobactrum intermedium F1 can effectively decompose triadimefon, the degradation rate reaches 60%, and the triadimefon can be utilized to promote the growth of the ochrobactrum intermedium F1.

Example 4

The seed liquid of the degrading bacteria of example 1 was mixed at 5% by volume (about 4X 10 colonies contained)⁸CFU) were transferred to MSM liquid medium (containing 1% glucose carbon source) containing 10mg/kg, 25mg/kg, 50mg/kg triazolone, respectively, and 3 replicates were set for each set of experiments. The cells were incubated at 30 ℃ for 7d on a shaker at 150 rpm.

The concentration of triazolone in the culture broth after 7d was determined for the different test groups using gas chromatography-mass spectrometry (GC-MS).

After being treated by the ochrobactrum intermedium F1 for 7 days, the concentration of the triazolone in the triazolone solution with the original concentration of 10mg/kg, 25mg/kg and 50mg/kg is respectively reduced by 5.75mg/kg, 10.55mg/kg and 11.64 mg/kg.

In GB2763 maximum limit of pesticide residues in food, the limit of triazolone in cereals, oil plants, vegetables and fruits is required to be more than 0.2-1.0mg/kg, and the results show that the ochrobactrum intermedium F1 can effectively degrade the triazolone residues which exceed the standard by tens of times.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. The application of ochrobactrum intermedium in degrading triadimefon has the similarity of the identification result of 16s rRNA sequence of the ochrobactrum intermedium strain with GenBank accession number KT808883.1 of 100%.

2. Use according to claim 1, characterized in that the triadimefon is a triadimefon remaining on the soil or fruits and vegetables.

3. The use according to claim 1, wherein 0.4 to 1.2 x 10 of ochrobactrum intermedium is used per 1mg of triazolone⁸CFU。

4. A method for degrading triadimefon, which comprises applying ochrobactrum intermedium to a treated object with residual triadimefon, wherein the similarity of the 16s rRNA sequence identification of the ochrobactrum intermedium strain with the GenBank number KT808883.1 is 100%.

5. The method according to claim 4, wherein the amount of the ochrobactrum intermedium used is 0.4 to 1.2 x 10 per 1mg of triazolone⁸CFU。

6. The method according to claim 5, wherein the treatment object is soil or fruit and vegetable, and when the treatment object is soil, the method further comprises spraying ochrobactrum intermedium into the soil; when the treated object is fruits and vegetables, the method comprises the step of soaking the fruits and vegetables in a solvent containing ochrobactrum intermedium.