CN116574657B

CN116574657B - Pseudomonas friedel and application thereof

Info

Publication number: CN116574657B
Application number: CN202310630227.3A
Authority: CN
Inventors: 刘晓辉; 于淼; 李杨; 敖静; 高晓梅; 孙玉禄; 宋立群; 朱正威; 池景良
Original assignee: LIAONING SCIENTIFIC ACADEMY OF MICROBIOLOGY
Current assignee: LIAONING SCIENTIFIC ACADEMY OF MICROBIOLOGY
Priority date: 2023-05-30
Filing date: 2023-05-30
Publication date: 2024-05-07
Anticipated expiration: 2043-05-30
Also published as: CN116574657A

Abstract

The invention discloses a Pseudomonas friedel and application thereof, belonging to the field of microorganisms. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 3 and 9 days of 2023, and has a preservation address of CGMCC No.26782, which is China academy of sciences of China, no. 3, north Star, kogyo, beijing, city. Experiments show that the strain is cultured for 48 hours in a basic salt culture medium added with 100mL/L metolachlor, the degradation rate reaches 93.9%, and the strain has high-efficiency degradation capability on herbicide metolachlor; the strain fermentation liquor is applied to treat the soil containing the metolachlor for 35d, the degradation rate reaches 82.1%, and the strain can be fully proved to repair the soil polluted by the metolachlor.

Description

Pseudomonas friedel and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to a Pseudomonas fradonepsis and application thereof.

Background

Metolachlor is an amide herbicide, and the English name is: METOLACHLO, molecular formula: c ₁₅H₂₂ClNO₂, molecular weight: 283.8, metolachlor is a broad-spectrum post-emergence herbicide, which is primarily absorbed by the shoots and conducted upward to inhibit the growth of the shoots and roots. The action mechanism mainly inhibits the protein synthesis of germinated seeds, and secondly inhibits the penetration of choline into phospholipid to interfere with the formation of lecithin. Because the capability of the gramineous weed buds to absorb the metolachlor is stronger than that of broadleaf weeds, the effect of the herbicide on preventing and killing gramineous weeds is far better than that of broadleaf weeds. The metolachlor is applied to dry crops, vegetable crops, orchards and nursery gardens, and is used for various crops such as: the herbicide is used for crops such as soybean, corn, cotton, peanut, potato, cabbage, spinach, garlic, sunflower sesame, rape, radish, sugarcane and the like, can also be used for crops such as orchards and other leguminous, cruciferous, solanaceae, asteraceae and umbrella-type families, can prevent and remove annual gramineous weeds such as eleusine indica, crabgrass, green bristlegrass, cotton grass and the like, broadleaf weeds such as amaranth, purslane and the like, and crushed rice sedge and cyperus esculentus, and has good control effect on gramineous weeds and broadleaf weeds. Thus, metolachlor may also be used as a non-selective herbicide against fallow lands. The metolachlor has long retention and half-life in soil and is easy to generate chemical injury to rice, wheat, soybean and other aftercrop sensitive organisms; the pollution of groundwater and surface water threatens the health and ecological environment of human beings and animals. Because serious environmental pollution is caused at present by long-term and large-scale use, the presence of the metolachlor in soil and water body for crop growth is detected, and the metolachlor not only damages the ecological environment, but also seriously endangers the health of human beings.

The mechanism of microbial degradation of pesticides is mainly classified into 2 types, one is that microorganisms directly act on pesticides and then perform enzymatic reaction so as to degrade the pesticides, and most microbial degradation of pesticides is realized through the mechanism; the other is that microorganisms indirectly affect pesticides by changing the surrounding environment, and the common main effects are mineralization, accumulation and co-metabolism. The mode of degrading pesticides by the microorganism through enzymatic reaction mainly comprises dehydrogenation, reduction, hydrolysis, oxidation and other reaction types.

In recent years, immobilized microorganism technology is rapidly developed, and is gradually applied to pesticide degradation and sewage treatment, so that the immobilized microorganism technology becomes a new research field. Because the immobilized cells can hinder the diffusion of the substrate and oxygen, thereby reducing the enzymatic activity of the cells. The metolachlor has stable self structure, is difficult to degrade and is insoluble in water. The mobility of the material in the environment is high, and the degradation rate in the natural environment is low. The traditional method for removing the metolachlor generally adopts physical and chemical methods such as adsorption, catalysis and oxidation reduction, but the methods have the problems of high repair cost, complex operation process, uncertain degradation intermediate products, larger pollution byproducts generated in the degradation process are diffused to water environment, soil environment and atmospheric environment, secondary pollution is caused, and the like. Based on these problems, a biodegradation technology which does not cause secondary pollution is sought. The biodegradation method has simple operation, low cost, no secondary pollution and the like, and is widely studied in recent years. The biodegradation of metolachlor at present mainly comprises plant degradation and microbial degradation. Microbial degradation refers to a process for converting metolachlor in the presence of microorganisms in the environment into simple, non-toxic inorganic substances. Therefore, the work of screening the microorganism strain for efficiently degrading the metolachlor is highly valued by scientific researchers, and the microorganism types which are separated at present mainly comprise bacteria, fungi, actinomycetes, algae and the like. Bacteria in microbial degradation are the main group for degrading metolachlor. Since the 80 s of the last century, bacteria have been widely used in the field of repair of microbial contamination due to simple cultivation and strong adaptability, and have an extremely important role in degrading metolachlor. Many bacteria have been isolated that can partially and even completely degrade metolachlor. The Pseudomonas sp has better adaptability to soil environment, stronger alkali resistance and drought resistance, better growth in soil and extremely important significance for degrading the metolachlor in the environment.

Disclosure of Invention

The invention aims to provide a Pseudomonas fraden-Sail and application thereof, so as to solve the problems in the prior art, and the strain can efficiently degrade metolachlor and soil polluted by metolachlor and has important significance for soil restoration.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a Pseudomonas (Pseudomonas fredenkscergensis) of Fredenlcer root, which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 3 months and 9 days in 2023, and has a preservation address of CGMCC No.26782, which is China academy of sciences of China, no. 3, north Star, korean area, beijing.

The invention also provides application of the Francisco root pseudomonas in preparation of a microbial preparation for degrading metolachlor.

The invention also provides a microbial preparation which comprises the pseudomonas friedel.

The invention also provides an application of the Francisco root pseudomonas in preparing a soil restoration agent.

The invention also provides a soil restoration agent, which comprises the pseudomonas friedel.

The invention also provides a method for fermenting and culturing the pseudomonas freudenreichii, which comprises the steps of inoculating the pseudomonas freudenreichii with an LB culture medium and obtaining fermentation liquor through fermentation culture.

Preferably, the temperature of the fermentation culture is 26-36 ℃, the fermentation time is 12-84 hours, the rotating speed is 120-220rpm, and the initial pH value is 5-7.

Preferably, the temperature of the fermentation culture is 30 ℃, the fermentation time is 48 hours, the rotating speed is 180rpm, and the initial pH value is 6.

The invention also provides application of the pseudomonas friedel, the microbial preparation or the soil restoration agent in soil restoration.

Preferably, the application is in the remediation of metolachlor contaminated soil.

The invention discloses the following technical effects:

According to the invention, a strain of Pseudomonas fradontocarvae is separated from soil, experiments show that the strain is cultured for 48 hours in a basic salt culture medium added with 100mL/L metolachlor, the degradation rate reaches 93.9%, and the strain has high-efficiency degradation capability on herbicide metolachlor; the strain fermentation liquor is applied to treat the soil containing the metolachlor for 35d, the degradation rate reaches 82.1%, and the strain can be fully proved to repair the soil polluted by the metolachlor. The invention provides a new bacterial source for the restoration of the soil polluted by the metolachlor, so as to provide a new direction for the restoration of the soil by biodegradation of the metolachlor in the soil.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows colony morphology of strain A ₂₁;

FIG. 2 shows the microscopic morphology of strain A ₂₁;

FIG. 3 is a phylogenetic tree constructed from 16S rDNA of strain A ₂₁; the number 2021-16-2 in the figure is the strain A21;

FIG. 4 shows the effect of fermentation temperature on the number of strains A ₂₁ and the degradation rate;

FIG. 5 shows the effect of fermentation time on the number of strains A ₂₁ and the degradation rate;

FIG. 6 shows the effect of shaking table rotation speed on the bacterial count and degradation rate of strain A ₂₁;

FIG. 7 shows the effect of initial pH on the number of strains A ₂₁ and degradation rate;

FIG. 8 shows the effect of bottling amount on bacterial count and degradation rate of strain A ₂₁;

FIG. 9 is a graph of the 3D response surface for the initial pH of the fermentation temperature vs;

FIG. 10 is a graph of the 3D response surface of fermentation temperature vs. shaker speed;

FIG. 11 is a graph of 3D response surface for fermentation temperature vs. bottled amount;

FIG. 12 is a graph of the 3D response surface for the initial pH vs. shaker speed;

FIG. 13 is a 3D response surface plot of initial pH vs. bottle volume;

FIG. 14 is a graph of 3D response surface for shaker speed vs. bottled amount;

FIG. 15 shows the degradation rate of metolachlor in soil by strain A ₂₁.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Example 1

1. Test soil

The soil is collected from karst county of Liaoning province, is corn field surface soil (0-20 cm) for long-term application of metolachlor, and is subjected to air drying, impurity removal, grinding, sieving with a 2mm sieve, and packaging into a sealing bag for storage in a refrigerator at 4 ℃.

2. Culture medium

Inorganic salt culture medium: k ₂HPO₄ 1.6g,KH₂PO₄ 0.4g,MgSO₄·7H₂ O0.4 g, naCl 0.1g, glucose 3g, metolachlor 0.1g, constant volume to 1000 mL, pH to neutral (7.0), and autoclaving at 121deg.C for 30min.

LB medium: 10g of tryptone, 10g of NaCl, 5g of yeast extract powder, 1000mL of distilled water, pH 7.2 and sterilization at 121 ℃ for 30min.

LBA medium: tryptone 10g, naCl 10g, yeast extract 5g, distilled water 1000mL, pH 7.2, agar 15g, and sterilization at 121 ℃ for 30min.

3. Isolation and screening of strains

5G of the treated soil sample was weighed and added to 100mL of an inorganic salt medium containing 100mL/L of metolachlor, and the mixture was subjected to shaking culture at 32℃and 180r/min for 3d. 5mL of the fermentation broth after 3d is absorbed and inoculated into 100mL of new inorganic salt culture medium, the concentration of metolachlor in the culture medium is 200mL/L, the concentration of metolachlor (300, 400 and 500 mL/L) is sequentially increased according to the same culture method, and the culture is finished after continuous enrichment for 15 d. The finally enriched culture solution is diluted by 10 ^-2、10^-3、10^-4、10^-5 times and 10 times ^-6 times respectively, coated on an inorganic salt solid culture medium containing 100mL/L metolachlor, placed in a constant temperature incubator at 30 ℃ for culture, and colonies with good growth vigor are picked from a flat plate for purification after 3d. The bacterial strain A ₂₁ with the best degradation effect is selected as the test target strain.

4. Identification results of strains

4.1 Colony morphology and microscopic morphology

Colony morphology: the colony edge is neat, milky white, slightly convex and smooth in surface, as shown in fig. 1.

Microscope morphology: the two ends of the crude bacillus are flat, as shown in figure 2.

4.2 Physiological biochemistry

Purified strain A21 was identified by reference to "Berger's Manual of bacteria identification (8 th edition) and" Manual of common bacteria System identification ". The physiological and biochemical results are shown in Table 1.

TABLE 1 physiological and Biochemical results

4.3 Molecular biological characterization results

16S rDNA sequence alignment and phylogenetic analysis: with the primer of bacterial 16S rDNA, namely the upstream primer F (SEQ ID NO: 1): 5' -CAGAGTTTGATCCTGGCT-3, downstream primer R (SEQ ID NO: 2): 5' -AGGAGGTGATCCAGCCGCA-3, and amplification of 16SrDNA was performed.

The amplification system is as follows: rTaq enzyme (5U/. Mu.L) 12.5. Mu.L, each of the upstream primer and the downstream primer (2.5. Mu. Mol/L) 1. Mu.L, template DNA 2. Mu.L, and finally double distilled water to 50. Mu.L were added, mixed and then centrifuged instantaneously, and the mixture was placed on a PCR apparatus to denature at 98℃for 3min, then denature at 98℃for 25s, denature at 55℃for 25s, extend at 72℃for 1min for 30 cycles, and finally extend at 72℃for 10 min. The PCR products were electrophoresed through a 1% agarose gel, and the results were recorded by observation and photographing under a gel imaging system. Sequencing was delegated to Mejie biosome. Inputting a sequencing result into GenBank for homology analysis, and comparing by using software such as Mega, clustal and the like to construct a evolutionary tree, wherein the repetition number is 1000. The evolutionary tree is shown in fig. 3, and the result of the combined physiological and biochemical experiment shows that A ₂₁ is pseudomonas (Pseudomonas fredenkscergensis).

The original sequence SEQ ID NO:3 is:

TGGTAACCGTCCTCCCGAAGGTTAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCGATCCATCTCTGGAAAGTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAGAGCTCAAGGCTCCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCATACTCTAGCTTGTCAGTTTTGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCAACTTAACAAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAGACACCAACGTATTAGGTTAATGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCGTCCGTTTCCGAGCGTTATCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTCTCAAGAGAAGCAAGCTTCTCTCTACCGCTCGACTGCA.

the strain A ₂₁ obtained by separation, namely the Pseudomonas fradenciclovir (Pseudomonas fredenkscergensis) is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 3 months and 9 days in 2023, and the preservation address is CGMCC No.26782, which is the China national academy of sciences of China, no. 3, north Star, chaoyang area, beijing.

Example 2 fermentation conditions optimization of Strain A ₂₁ for degradation of metolachlor

1. Influence of fermentation temperature on bacterial count and degradation rate of bacterial strain A ₂₁

The strain A21 is inoculated in an inorganic salt culture medium by an inoculating loop, the culture temperature is set to 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃ and 36 ℃ respectively, and after 48 hours of shaking culture at 180rpm, the number of the A ₂₁ bacteria and the degradation rate of the metolachlor in the fermentation broth are measured.

As shown in FIG. 4, the cell count and degradation rate of strain A ₂₁ reached the highest at 30℃and were 90.9X10- ⁸ CFU/mL and 92.5%, respectively.

2. Influence of fermentation time on degradation rate of bacterial count of strain A ₂₁

The strain A21 is inoculated into a culture medium, and after being respectively cultured for 24 hours, 36 hours, 48 hours, 60 hours, 72 hours and 84 hours at the temperature of 32 ℃ and the speed of 180rpm, the bacterial count (plate colony counting method) of the strain A ₂₁ and the degradation rate of the metolachlor in the fermentation liquid are measured.

As shown in FIG. 5, the number of bacteria and the degradation rate of the strain A ₂₁ reached the highest at 48 hours, which were 90.1X10. 10 ⁸ CFU/mL and 92.8%, respectively.

3. Influence of rotation speed of shaking table on degradation rate of bacterial count of strain A ₂₁

The rotation speed of the shaking table is set to be 140rpm, 160rpm, 180rpm, 200rpm and 220rpm respectively, and after culturing for 48 hours at 32 ℃, the bacterial count of the bacterial strain A ₂₁ and the degradation rate of the metolachlor in the fermentation liquid are measured.

As a result, as shown in FIG. 6, the number of bacteria and the degradation rate of the strain A ₂₁ reached the highest at a rotation speed of 180rpm, which were 90.3X10. 10 ⁸ CFU/mL and 92.1%, respectively.

4. Effect of initial pH on the number of bacteria of Strain A21 and degradation Rate

The initial pH of the culture medium was set to be 5.0, 5.5, 6.0, 6.5 and 7.0, and the number of A ₂₁ bacteria and the degradation rate of metolachlor in the fermentation broth were measured after culturing at 32 ℃ and 180rpm for 48 hours.

As shown in FIG. 7, the cell number of A ₂₁ and the degradation rate of metolachlor reached the highest at pH 6, which were 87.1X10. 10 ⁸ CFU/mL and 92.2%, respectively.

5. Influence of bottling amount on bacterial count and degradation rate of strain A ₂₁

Bottling according to 60mL, 80mL, 100mL, 120mL, 140mL and 160mL, culturing at 32deg.C and 180rpm for 48h, and measuring bacterial count of strain A ₂₁ and degradation rate of metolachlor in fermentation broth.

As shown in FIG. 8, the number of strains A ₂₁ and the degradation rate of metolachlor reached the highest at the bottling rate of 100mL, which were 88.5X10. 10 ⁸ CFU/mL and 91.6%, respectively.

6. Box-Behnken response surface test

According to the single-factor test result, selecting a factor level which has obvious influence on the degradation of the metolachlor by the strain A ₂₁, designing a Box-Behnken response surface test with a factor 3 level by using Design-Expert 8.0.6 software, wherein the specific scheme is shown in table 2, using the degradation rate of the metolachlor as a response value, applying a Box-Benhnken Design 29 group test, and designing and obtaining the Box-Benhnken test results shown in table 2.

TABLE 2Box-Benhnken test design and results

And (3) fitting a quadratic multiple regression equation of the degradation rate of the metolachlor to obtain a regression equation of the functional relation between each factor and the response value, and determining the optimal fermentation condition according to the generated response surface diagram.

And performing secondary multiple regression fitting on the metolachlor degradation rate result in the table 2 by using Design-Expert 8.0.6 software to obtain a regression equation of the functional relation between each factor and the response value, and determining the optimal fermentation condition according to the generated response surface diagram.

Regression equation is ：Y＝+93.66+2.85*A+7.18*B+3.38*C+4.68*D-6.45*A*B-3.75*A*C-3.05*A*D-0.85*B*C-1.33*B*D+0.30*C*D-7.40*A²-6.24*B²-1.53*C²-10.26*D².

TABLE 3 analysis of variance

The determination coefficient R ² = 0.9719 of the model and the correction determination coefficient a _djR ² = 0.9438 show that the fitting degree of the actual value and the predicted value of the test is good. From table 3, it can be seen that, from the effect on degradation rate, the effect of the primary term A, B, C, D on degradation rate reaches a very significant level, and the effect sequence is B > D > C > a, namely: the initial pH value is larger than the bottling amount, the rotating speed of the shaking table is larger than the fermentation temperature.

7. Response surface interaction analysis

The 3D response surface graphs of the interaction between the two factors were made by Design-experert 8.0.6 software, see fig. 9-14. The greater the slope of the curved surface in fig. 9, 11 and 14, and the darker the color closer to the top of the curved surface, the more abrupt the change, the more pronounced the effect, indicating significant interaction between the two factors involved. Whereas the curves in fig. 10, 12 and 13 vary relatively smoothly, the interaction between the two factors involved is not apparent.

Example 3 determination of degradation Rate of metolachlor in soil

1. Determination of the content of metolachlor

The method is carried out according to the steps of the metolachlor detection kit.

The soil sample treatment method comprises the following steps:

(1) In a 60mL plastic bottle, 10g of soil sample (dried in shade at room temperature) was weighed;

(2) 30mL of a sample extract (the sample extract is an aqueous solution containing 75% methanol, for example, 30mL of methanol+10 mL of water) is added, and the cap is screwed down;

(3) Shaking vigorously and centrifuging at low speed for 30min;

(4) Standing overnight at room temperature so that the metolachlor in the sample is fully dissolved in the extracting solution;

(5) Shaking vigorously for 30min, and standing for 10min;

(6) Taking 1mL of supernatant in a test tube;

(7) The supernatant was diluted with sample diluent (50-fold dilution), i.e., 1mL supernatant+49 mL sample diluent.

2. Soil remediation test of degradation Strain A ₂₁

The air-dried soil is screened to prepare the polluted soil with the concentration of the metolachlor of 50 mL/kg. The test was run on a single treatment and a single control, each with 500g of soil and 3 replicates. 50mL of strain A ₂₁ fermentation broth (fermentation broth prepared under the optimal conditions described above) with a concentration of 1X 10 ⁸ CFU/mL was inoculated into the contaminated soil, and 50mL of the culture medium without bacteria was inoculated into the control group. Culturing at room temperature, periodically supplementing sterile water according to constant weight method, sampling at 7d, 14d, 21d, 28d and 35d, and measuring the content of metolachlor in soil to calculate degradation rate.

As can be seen from FIG. 15, the degradation rate of metolachlor in the soil is continuously increased along with the continuous extension of the culture time, and the degradation rate reaches 82.1% on the 35 th day. Also, a portion of the metolachlor in the control group to which the medium was added was degraded, which may be caused by the action of indigenous microorganisms in the soil, and the action of photolysis or hydrolysis. This result is a full indication that strain A ₂₁ can restore soil contaminated with metolachlor.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. Pseudomonas (Pseudomonas fredenkscergensis) for Francisco root, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 26782 in 2023, 3 and 9.

2. Use of the pseudomonas freudenreichii according to claim 1 for the preparation of a microbial preparation for degrading metolachlor.

3. A microbial preparation comprising pseudomonas fredenciclovir according to claim 1.

4. Use of the pseudomonas freudenreichii according to claim 1 for the preparation of a metolachlor contaminated soil remediation agent.

5. A soil restoration agent comprising the pseudomonas fredenskyi of claim 1.

6. A method for the fermentative cultivation of pseudomonas freudenreichii according to claim 1, comprising the steps of inoculating the pseudomonas freudenreichii with LB medium and obtaining a fermentation broth through fermentative cultivation.

7. The method according to claim 6, wherein the fermentation culture is carried out at a temperature of 26-36 ℃, a fermentation time of 12-84 hours, a rotation speed of 120-220rpm, and an initial pH of 5-7.

8. The method of claim 7, wherein the fermentation culture is performed at a temperature of 30℃for 48 hours at 180rpm and an initial pH of 6.

9. Use of the pseudomonas freudenreichii according to claim 1, the microbial preparation according to claim 3 or the soil remediation agent according to claim 5 in the remediation of metolachlor contaminated soil.