Bacterial strain capable of repairing pinoxaden-polluted soil and application of bacterial strain

Abstract

The invention discloses a bacterial strain capable of repairing pinoxaden-polluted soil and application of the bacterial strain, the bacterial strain is Acinetobacter pittii T4P20-1, the Acinetobacter pittii T4P20-1 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is December 21, 2021, and the preservation number is CGMCC No.24157. The pinoxaden-contaminated soil remediation agent is used for remediation of pinoxaden-contaminated soil. The pinoxaden-contaminated soil remediation agent is used for promoting germination of seeds in pinoxaden-contaminated soil as a remediation agent.

Description

Technical Field

[0001] This invention relates to the agricultural field, specifically to a strain of herbicide that can remediate soil contaminated with clopyralid and its application. Background Technology

[0002] Wheat accounts for approximately 218.5 × 10⁻⁶ kilowatts of the world's total. 6 China's arable land, covering over 100 hectares, makes a significant contribution to the global agricultural economy, exceeding that of any other crop. In 2020, my country's grain output reached 616.74 million tons, a year-on-year increase of 0.5%. With decreasing arable land, sustainable wheat production is crucial for meeting global food security. Weed infestation at all stages of wheat growth is a major obstacle and threat to global wheat production; therefore, herbicides such as clodinafop-propargyl are widely used in wheat fields.

[0003] Clonort herbicides are widely used to control weeds in wheat fields to increase planting intensity and yield. However, herbicide residues in crops may pose risks to humans through consumption. Because herbicides are inherently toxic to organisms, they are likely to affect human health, pollute natural resources, and disrupt the balance of ecosystems.

[0004] Pinoxaden is a phenylpyrazoline herbicide discovered, developed, and manufactured by Syngenta. Its trade names include Axial and others. Its chemical formula is 8-(2,6-diethyl-4-methylphenyl)-1,2,4,5-tetrahydro-7-oxo-7H-pyrazole[1,2-d][1,4,5]oxadiazaphen-9-yl 2,2-dimethylpropionate, and its molecular formula is C0. 23 H 32 N2O4, its chemical structural formula is as follows Figure 1 As shown, the relative molecular mass is 400.5. It is a white, odorless powdery solid. At 25℃, the solubility of clopyralid in water is 200 mg / L, in organic solvents acetone 250 g / L, in dichloromethane >500 g / L, in ethyl acetate 130 g / L, and in methanol 260 g / L. Due to its unique chemical structure, it can inhibit the activity of acetyl-CoA carboxylase (ACCase), hinder the biosynthesis of fatty acids, interfere with cell membrane formation, leading to the cessation of weed growth and eventual death. It has systemic properties and is mainly used for the control of annual grass weeds in barley fields. It has good control efficacy against grass weeds such as wild oats, ryegrass, foxtail grass, hard grass, barnyard grass, and Japanese barnyard grass, especially against noxious grass weeds such as wild oats, ryegrass, hard grass, and barnyard grass, with a control efficacy approaching 100%.

[0005] Clotrimazole is not easily photodegraded or hydrolyzed, and is relatively easy to degrade and adsorb in soil, but not easily leached or volatilized. It is also easily degraded in water-sediment. The median lethal concentrations (LD50) of 5% clotrimazole EC against rainbow trout, daphnia, algae, birds, and silkworms were 24 mg / L, 4 mg / L, 4.17 mg / L, 4780.3 mg / kg, and 4000 mg / L, respectively. It has low biotoxicity in the environment, moderate toxicity to aquatic organisms such as algae, and some impact on the tested plants. Currently, there are no reports on the degradation of clotrimazole, both domestically and internationally. Most literature focuses on the efficacy, safety, analytical methods, and synthetic routes of clotrimazole. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of bacteria capable of remediating soil contaminated with clopyralid and its application.

[0007] The present invention discloses a strain of Acinetobacter pittii T4P20-1, which is capable of remediating soil contaminated with clopyralid. It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 21, 2021, with accession number CGMCC No. 24157.

[0008] The present invention relates to the application of a strain of herbicide that can remediate soil contaminated with clopyralid, which is used as a soil remediation agent to remediate soil contaminated with clopyralid.

[0009] The present invention relates to the application of a strain of herbicide strain for remediating soil contaminated with clodinafop-propargyl, which acts as a remediation agent to promote seed germination in soil contaminated with clodinafop-propargyl.

[0010] This invention screens strains by measuring their growth trends and degradation rates in inorganic salt-added herbicide culture solutions. Strains with high degradation capabilities are selected for morphological observation, physiological and biochemical reactions, and molecular identification to determine their species relationships. The optimal growth conditions for the strains are explored, and finally, the degradation ability of the strains is verified by seed germination tests of sensitive crops in subsequent crops.

[0011] There are few reports on the degradation mechanisms of three types of wheat field herbicides (including physical, chemical, and biological degradation). Microbial degradation is the best way to degrade herbicides. Therefore, the main purpose of this experiment is to isolate strains that grow with clopyralid as the sole carbon and nitrogen source from different soils, study the growth characteristics and degradation patterns of the isolated strains, and explore the degradation mechanism of the isolated strains. The aim is to provide microbial resources and technical support for the remediation of soils contaminated by these herbicides, and to lay the foundation for the research on the microbial degradation and bioremediation of soils contaminated by these herbicides. Attached Figure Description

[0012] Figure 1 This is the chemical structure diagram of cyclophosphamide;

[0013] Figure 2 Growth curve of a bacterial strain that is highly efficient at degrading cyclohexane after 72 hours;

[0014] Figure 3 Standard curves for three herbicide standards;

[0015] Figure 4 The graph shows the degradation rate of herbicides by the degrading strains.

[0016] Figure 5 Morphological identification diagram of highly efficient degrading bacterial strains;

[0017] Figure 6 Agarose gel electrophoresis image of 16S rDNA from a highly efficient bacterial degrading strain; Note: M: Marker DL2000; 1: PCR product of strain T4P20-116S.

[0018] Figure 7 This is a graph showing the comparison results of the strains using BLAST on NCBI.

[0019] Figure 8 Figure 1 shows the effect of different pH values ​​and inoculum amounts on the growth of bacteria strains that are highly efficient at degrading clopyralid; Figure 2a shows the effect of different pH values ​​on the growth of strain T4P20-1, and Figure 3b shows the effect of different inoculum amounts on the growth of strain T4P20-1.

[0020] Figure 9 Figure 1 shows the effect of different carbon and nitrogen sources on the growth of bacteria strains that are highly efficient at degrading clopyralid; Figure 2a shows the effect of different carbon sources on the growth of strain T4P20-1, and Figure 3b shows the effect of different nitrogen sources on the growth of strain T4P20-1.

[0021] Figure 10 Figure showing the effect of culture time on the growth and degradation of clopyralid by strain T4P20-1.

[0022] Figure 11 The graph shows the linear regression equations of the sensitive concentrations of herbicides on the germination of seeds of subsequent sensitive crops; curve A represents linearity (germination rate (%)) and curve B represents linearity (stem length (cm)). Detailed Implementation

[0023] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0025] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0026] Example 1

[0027] 1. Soil sample for testing

[0028] The soil samples were collected from soils grown in experimental fields of Heilongjiang University (growing corn, wheat, potatoes, and peanuts), field soils grown in Dujia Town, Wuchang City, Harbin (growing rice, potatoes, corn, and wheat), forest soils from Minyi Township, and soils grown in Binzhou City, Shandong Province (growing corn, wheat, potatoes, cotton, peanuts, and apples). The collected soil samples were numbered as shown in Table 1. After sieving, the soil samples were stored in a refrigerator at 4°C.

[0029] Table 1. Soil sample collection locations, sample numbers, and crop varieties.

[0030]

[0031] 2. Reagents

[0032] Phosphatidylcholine (95%) Syngenta Crop Protection Ltd., Switzerland; Chromatographic grade methanol (Shandong Yuwang, chromatographic purity); Dichloromethane (Tianjin Guangfu, analytical purity).

[0033] 3. Culture medium

[0034] (1) Basic Inorganic Salt Medium (MSM): K₂HPO₄ 1.79g, KH₂PO₄ 0.45g, MgSO₄·7H₂O 0.2g, NaCl 0.4g, distilled water 1000mL, pH=7.0. Solid medium: add 1.5-2.0% agar powder.

[0035] (2) LB (Luria-Bertani) medium: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl, 1000mL distilled water, pH 7.0, autoclaved at 121℃ for 15min.

[0036] 4. Preparation of standard stock solution

[0037] Preparation of stock solution: Weigh 0.5 g of the standard and place it in a 10 mL volumetric flask. Dilute to volume with chromatographic grade methanol, filter through a 0.45 μL organic filter membrane, and store for later use.

[0038] 5. Preparation of bacterial suspension

[0039] Pick a purified single colony and inoculate it into a test tube containing 5 mL of LB liquid medium. Incubate at 30 °C and 160 r / min for 12 h to activate the bacterial culture. Take 100 μL of the activated strain and place it in a 96-well plate to measure the OD value. Based on the OD600 value, take a sample and place it in a 1.5 mL centrifuge tube. Centrifuge at 5000 r / min for 5 min, discard the supernatant, add sterile 0.9% physiological saline, vortex for 1 min, centrifuge again, discard the supernatant, and add sterile 0.9% physiological saline to adjust the OD600 to 1.0 to prepare a bacterial suspension.

[0040] 6. Enrichment, domestication, culture, isolation, and purification of highly efficient degrading bacteria

[0041] Herbicide-degrading strains were isolated and acclimatized using a shake-flask enrichment culture method. 5g of soil sample, 25mL of inorganic salt liquid culture medium, and an appropriate amount of herbicide were added to 50mL Erlenmeyer flasks to achieve a herbicide concentration of 200mg / L. The flasks were then incubated in a constant-temperature shaker at 30℃ and 180r / min. Every 5 days, 2.5mL of the culture was inoculated into fresh inorganic salt liquid culture medium, with the herbicide concentration increased by 200mg / L. This acclimatization culture was continued for 5 weeks until the herbicide concentration reached 1000mg / L. The enriched cultures were then serially diluted with sterile water (10⁻⁶ ppm). -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 The bacteria were spread separately on inorganic salt solid medium containing the corresponding herbicide and incubated at 30°C for 3 days. Colonies with different colors and shapes were selected for purification. The purified strains were added to 1 mL of LB medium and cultured on a shaker. When the OD value reached 1.0, the culture was centrifuged at 5000 r / min for 5 min, the supernatant was discarded, and 70% glycerol was added and mixed by aspiration. The culture was then stored at -20°C.

[0042] 7. Determination of growth curves of highly efficient degrading bacterial strains

[0043] 20 μL of bacterial suspension was added to 180 μL of herbicide-inorganic salt medium (herbicide concentrations of 25 mg / L, 50 mg / L, and 75 mg / L) and LB liquid medium, respectively, with 4 replicates. The bacterial growth was measured every 12 hours and analyzed using Bioscreen for 72 hours. The bacterial growth curve was plotted.

[0044] 8. Determination of recovery and degradation rate of highly efficient degrading bacterial strains

[0045] 8.1 Sample Pretreatment Method

[0046] (1) Add 1 mL of culture medium, 0.25 g of NaCl and 5 mL of CH2Cl2 to a 10 mL centrifuge tube respectively, and shake on a Vortex for 1 min;

[0047] (2) Take out the upper layer and place it in a new centrifuge tube. Add 1 mL of CH2Cl2 and shake with Vortex for 1 min. Take out the lower organic phase and place it in the first corresponding centrifuge tube. Add 1 mL of CH2Cl2 to the second centrifuge tube and shake for 1 min. Take out the organic phase and place it in the first centrifuge tube.

[0048] (3) Add 0.2g of anhydrous Na2SO4 to the first centrifuge tube after merging and shake. Put the upper liquid into a 10mL volumetric flask and place the volumetric flask in a 50℃ water bath to evaporate CH2Cl2.

[0049] (4) Dilute to 10 mL with chromatographic grade methanol, sonicate for 30 min, filter through a 0.45 μm organic filter membrane, place in a 2 mL centrifuge tube, store at 4 °C for later testing.

[0050] 8.2 HPLC Chromatographic Detection Conditions

[0051] Column: INERTSIL ODS-35UM (5μm, 4.6×250mm)

[0052] Injection volume: 20 μL

[0053] Liquid Chromatographic Conditions for Cyclophosphamide

[0054] Column temperature: 40℃; flow rate: 1.0 mL / min; mobile phase: acetonitrile:water (0.05% phosphoric acid aqueous solution) = 65:35 (v:v); detection wavelength: 260 nm.

[0055] 8.3 Construction of herbicide standard curve

[0056] The mother liquor of clopyralid was diluted to 12.5 mg / L, 7.5 mg / L, 5 mg / L, 2.5 mg / L, and 1 mg / L, respectively, and analyzed by high performance liquid chromatography according to the chromatographic conditions.

[0057] 8.4 Determination of degradation rate of highly efficient degrading bacterial strains

[0058] Add 5 mL of inorganic salt and herbicide liquid culture medium (25 mg / L, 50 mg / L, 75 mg / L) and 50 μL of bacterial suspension to test tubes, respectively. Mark the liquid level before shaking, and incubate at 30℃ and 200 rpm for 5 days. A blank control group (CG) is cultured together with the inoculated test tubes. Before taking samples, replenish the liquid level according to the records. Extract the culture medium and perform high-performance liquid chromatography (HPLC) analysis to determine its degradation capacity.

[0059] 8.5 Determination of recovery rate and calculation of degradation rate

[0060] Recovery rate (%) = (Measured concentration / Added concentration) × 100% (Formula 2-1)

[0061] Degradation rate (%) = [1 - (actual concentration of the treatment / actual concentration of the control)] × 100% (Formula 2-2)

[0062] 9. Identification of highly efficient degrading bacterial strains

[0063] 9.1 Morphological identification of highly efficient degrading bacterial strains

[0064] 9.1.1 Gram staining

[0065] Gram staining was performed on the bacterial strain, and then the characteristics of the bacterial cells were determined using an oil immersion optical microscope. Referring to the three-step Gram staining method, the specific steps are as follows:

[0066] (1) Add half a drop of physiological saline to the center of a clean, unused glass slide. Under aseptic conditions, pick a small number of single colonies from the culture medium, dissolve them in the physiological saline, and spread them evenly into a thin film.

[0067] (2) After drying under natural conditions, fix it over an alcohol lamp flame. The time should not be too long, and it is best to keep the slide warm to the touch to prevent the bacteria from dying due to excessive temperature.

[0068] (3) Add a drop of crystal violet to the bacterial cells, cover the bacterial film, stain for 1-2 minutes, and rinse with running water.

[0069] (4) Rinse off the residual water on the bacterial film with iodine solution, cover the bacterial film, stain for 1 minute, and rinse with running water.

[0070] (5) Remove residual water, rinse with 95% ethanol until colorless, and rinse with running water.

[0071] (6) Add safranin staining solution, cover the bacterial film, stain for 2 minutes, and rinse with running water.

[0072] (7) After drying, observe under an oil immersion microscope. Gram-positive bacteria appear blue-purple, and Gram-negative bacteria appear red.

[0073] 9.1.2 Observation of colony morphology

[0074] Prepare LB solid medium, dilute and spread the purified strains on the medium, and incubate at 30°C for 72 hours. Observe the size, edge, transparency, color, shape and other characteristics of the single colonies produced.

[0075] 9.2 Biochemical identification of highly efficient degrading bacterial strains

[0076] Identification was performed using biochemical identification tubes. The purified strain was inoculated into nutrient broth medium and incubated at 37°C for 18-24 hours. 50 μL of the broth culture was then added to a micro-volume vial and incubated at 35-37°C. Physiological and biochemical characteristics were identified with reference to Bergey's Manual of Bacterial Identification (9th Edition) and the Handbook of Common Bacteria.

[0077] 9.3 Identification of 16S rDNA sequences of highly efficient degrading bacterial strains

[0078] 9.3.1 Extraction of bacterial genomic DNA

[0079] (1) Sample processing

[0080] Gram-negative bacteria: Take 1 mL of overnight cultured bacterial suspension, add it to a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacterial cells. Add 180 μL of buffer digestion, then add 20 μL of proteinase K solution, and vortex to mix. Incubate at 56°C for 1 h until the cells are completely lysed.

[0081] Gram-positive bacteria: Take 1 mL of overnight bacterial culture and add it to a 1.5 mL centrifuge tube. Centrifuge at 8000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial culture in 180 μL of lysozyme solution (prepare a 20 mg / mL lysozyme solution by adding the corresponding lysozyme to Enzymatic lysis buffer before use) and incubate at 37°C for 30–60 min. Then add 20 μL of Proteinase K solution and vortex to mix. Incubate at 56°C for 30 min until the cells are completely lysed.

[0082] (2) Add 200 μL of Buffer BD and mix thoroughly by inverting.

[0083] (3) Add 200L of anhydrous ethanol and mix thoroughly by inverting.

[0084] (4) Place the adsorption column into the collection tube, use a pipette to add all the solution and translucent fibrous suspension into the adsorption column, let it stand for 2 minutes, then centrifuge at 12000 r / min at room temperature for 1 minute, and discard the waste liquid in the collection tube.

[0085] (5) Place the adsorption column back into the collection tube, add 500 μL of PW Solution, centrifuge at 10000 r / min for 30 s and discard the filtrate.

[0086] (6) Place the adsorption column back into the collection tube, add 500 μL Wash Solution, centrifuge at 10000 r / min for 30 s and discard the filtrate.

[0087] (7) Place the adsorption column back into the collection tube and centrifuge at 12000r / min at room temperature for 2min to remove the residual WashSolution.

[0088] (8) Remove the adsorption column and place it in a new 1.5 mL centrifuge tube. Add 50-100 μL of CE Buffer and let stand for 3 min. Centrifuge at 12000 r / min at room temperature for 2 min and collect the DNA solution. The extracted DNA can be used immediately for the next experiment or stored at -20℃.

[0089] 9.3.2 PCR amplification of 16S rDNA

[0090] Using 16S rDNA universal primers and the extracted bacterial DNA as a template, amplification was performed according to the following reaction system and amplification conditions. The universal primers, reaction system, and cycling conditions for bacterial identification are shown in Tables 2 and 3, respectively. PCR products were detected by 1% agarose gel electrophoresis.

[0091] Table 216S rDNA PCR amplification primer sequences

[0092]

[0093] Table 3.16S rDNA PCR amplification reaction system and cycling procedure

[0094]

[0095] 9.3.3 Recovery and purification of PCR products

[0096] The PCR product electrophoresis bands are cleaved to obtain the desired DNA target band, and the purification method is as follows:

[0097] (1) Cut a piece of gel containing the target fragment from the agarose gel and weigh it.

[0098] (2) Add Buffer B2 at 3-6 times the weight of the gel block and solute in a 50℃ water bath for 5-10 minutes.

[0099] (3)(Optional) For fragments <500bp, add 1 / 3 volume of isopropanol to Buffer B2.

[0100] (4) Transfer the sol solution into the adsorption column and centrifuge at 8000×g for 30s. Discard the liquid in the collection tube.

[0101] (5) Add 500 μL of Wash Solution, centrifuge at 9000 × g for 30 s, and discard the liquid in the collection tube. Repeat once.

[0102] (6) Centrifuge the empty adsorption column at 9000×g for 1 min.

[0103] (7) Place the adsorption column into a clean 1.5 mL centrifuge tube, add 15-40 μL of solution buffer to the center of the adsorption membrane, let it stand at room temperature for 1 min, and then centrifuge for 1 min. Save the DNA solution in the tube.

[0104] 9.3.416S rDNA sequence analysis

[0105] The 16S rDNA sequence was aligned to the ribosome database at http: / / rdp.cme.msu.edu / index.jsp, and the accession number was submitted.

[0106] 10. Determination of growth characteristics of highly efficient degrading bacterial strains

[0107] 10.1 Growth determination of highly efficient degrading bacterial strains at different pH values

[0108] Pick a purified single colony and inoculate it into a test tube containing 5 mL of LB liquid medium. Incubate at 30 °C and 160 rpm for 12 h on a shaker to activate the culture. Take 100 μL of the activated strain and place it in a 96-well plate to measure the OD. 600 According to OD 600 Take a sample and place it in a 1.5 mL centrifuge tube. Centrifuge at 5000 r / min for 5 min, discard the supernatant, add sterile 0.9% physiological saline, vortex for 1 min, centrifuge again, discard the supernatant, add sterile 0.9% physiological saline to adjust OD600 to 1.0 to prepare a bacterial suspension.

[0109] 10% bacterial suspension was added to liquid culture medium containing 75 mg / L cyclohexane inorganic salts at pH 6.0, 7.0, and 8.0, respectively, with three replicates. One control group was set up without the bacterial strain. The bacterial growth curve was plotted every 12 hours using Bioscreen for 72 hours.

[0110] 10.2 Growth determination of highly efficient degrading bacterial strains at different inoculum sizes

[0111] Pick a purified single colony and inoculate it into a test tube containing 5 mL of LB liquid medium. Incubate at 30 °C and 160 rpm for 12 h on a shaker to activate the culture. Take 100 μL of the activated strain and place it in a 96-well plate to measure the OD. 600 According to OD 600 Samples were taken and placed in 1.5 mL centrifuge tubes, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and sterile 0.9% physiological saline was added. The mixture was vortexed in votex for 1 min, centrifuged again, the supernatant was discarded, and sterile 0.9% physiological saline was added to adjust the OD600 to 1.0 to prepare a bacterial suspension. 1%, 5%, and 10% of the bacterial suspensions were added to liquid culture medium containing 75 mg / L of clopyralid inorganic salts, with three replicates. A control group without the bacterial strain was included. Detection was performed every 12 h, and the bacterial growth curves were plotted using Bioscreen after 72 h.

[0112] 10.3 Growth assay of highly efficient degrading bacterial strains with added carbon source

[0113] Pick a purified single colony and inoculate it into a test tube containing 5 mL of LB liquid medium. Incubate at 30 °C and 160 rpm for 12 h on a shaker to activate the culture. Take 100 μL of the activated strain and place it in a 96-well plate to measure the OD. 600 According to OD 600 Samples were taken and placed in 1.5 mL centrifuge tubes, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and sterile 0.9% physiological saline was added. The mixture was vortexed in votex for 1 min, centrifuged again, the supernatant was discarded, and sterile 0.9% physiological saline was added to adjust the OD600 to 1.0 to prepare a bacterial suspension. 10% of the bacterial suspension was added to inorganic salt liquid culture medium containing 75 mg / L oxychloride, glucose, sucrose, and lactose, with three replicates. One control group without the bacterial strain was included. The samples were measured every 12 h and analyzed using Bioscreen for 72 h to plot the bacterial growth curve.

[0114] 10.4 Growth determination of highly efficient degrading bacterial strains by external nitrogen source

[0115] Pick a purified single colony and inoculate it into a test tube containing 5 mL of LB liquid medium. Incubate at 30 °C and 160 rpm for 12 h on a shaker to activate the culture. Take 100 μL of the activated strain and place it in a 96-well plate to measure the OD. 600 According to OD 600Samples were taken and placed in 1.5 mL centrifuge tubes, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and sterile 0.9% physiological saline was added. The mixture was vortexed in votex for 1 min, centrifuged again, the supernatant was discarded, and sterile 0.9% physiological saline was added to adjust the OD600 to 1.0 to prepare a bacterial suspension. 10% of the bacterial suspension was added to an inorganic salt liquid culture medium containing 75 mg / L oxychloride, urea, potassium nitrate, and ammonium sulfate, with three replicates. One control group without the bacterial strain was included. The samples were measured every 12 h and analyzed using Bioscreen for 72 h to plot the bacterial growth curve.

[0116] 10.5 Determination of the effect of incubation time on the growth and degradation ability of highly efficient degrading bacterial strains

[0117] Pick a purified single colony and inoculate it into a test tube containing 5 mL of LB liquid medium. Incubate at 30 °C and 160 rpm for 12 h on a shaker to activate the culture. Take 100 μL of the activated strain and place it in a 96-well plate to measure the OD. 600 According to OD 600 Samples were taken and placed in 1.5 mL centrifuge tubes, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and sterile 0.9% physiological saline was added. The mixture was vortexed in votex for 1 min, centrifuged again, the supernatant was discarded, and sterile 0.9% physiological saline was added to adjust the OD600 to 1.0 to prepare a bacterial suspension. 10% of the bacterial suspension was added to liquid culture medium containing 75 mg / L of clopyralid inorganic salts, with three replicates. One control group without the bacterial strain was included. The samples were tested every 12 h, and Bioscreen was used to detect the residues for 72 h. The degradation rate was calculated, and the growth curve and degradation curve of the bacterial strain were plotted.

[0118] 11. Determination of the degradation ability of highly efficient degrading bacterial strains by bioassay method

[0119] The method for surface disinfection of seeds is as follows: First, soak the seeds in 75% ethanol for 2 minutes, then discard the ethanol and rinse them with sterile water. After discarding the water, soak the seeds in 3% NaClO solution for 20 minutes. After discarding the sodium hypochlorite solution, rinse the seeds repeatedly with sterile water until the odor of sodium hypochlorite disappears. Then, soak the surface-disinfected seeds in warm water at 30℃ for 12 hours.

[0120] The sensitivity concentration screening test method is as follows: First, prepare a medium containing gradient concentrations (5, 25, 50, 75, 100 mg / L) of clopyralid inorganic salts. Cut sterile gauze into appropriate sizes and place them in sterile plates. Add 40 mL of culture medium to each plate, and then evenly place 40 seeds in each plate. Each treatment has 3 replicates, and sterile water is set as a blank control. Incubate in a 25℃ light incubator for 6 days, and pay attention to replenishing the same amount of water every day. Observe and record the germination rate, shoot length and root length, etc., and statistically analyze to determine the more sensitive concentration of seeds.

[0121] The bioassay method for the degradation capacity of the strain is as follows: The strain with a high degradation rate was inoculated into the medium of sensitive concentration of herbicide inorganic salt, and shaken at 30℃. The strain was then applied to sensitive crop seeds according to the above method. This was the treatment group. In addition, sterile water was set as the blank group, and the medium of uninoculated herbicide inorganic salt was set as the control group. After each batch of treated seeds had grown for 6 days, the germination rate, shoot length and root length were observed and recorded.

[0122] 12. Enrichment, domestication, culture, isolation, and purification of highly efficient degrading bacteria

[0123] Enrichment culture was carried out on inorganic salt medium with added clodinafop-propargyl, and strains with clear zones in the medium with added inorganic salt were screened. The strains were further purified on LB solid medium, and 45 highly efficient degrading strains were isolated from the tested soil samples, as shown in Table 4. Among them, 14 strains were able to degrade clodinafop-propargyl.

[0124] 13. Determination of growth curves of highly efficient degrading bacterial strains

[0125] The growth curve of the bacterium strain that efficiently degrades clopyralid after 72 hours is shown in the figure. Figure 2 As shown, strains T4P20-1, T4P20-3, and T8P20-2 showed a growth trend under the condition of an initial concentration of 25 mg / L of clopyralid, while strains T1P20-3 and T3P20-1 showed a growth trend under the condition of an initial concentration of 50 mg / L of clopyralid.

[0126] Table 4. Number of Highly Degrading Bacterial Strains

[0127]

[0128] 14. Determination of the recovery rate of highly efficient degrading bacterial strains

[0129] 14.1 Construction of the standard curve for highly efficient degrading bacterial strains

[0130] Plot the standard curve of the chlorpyrifos herbicide standard as follows: Figure 3As shown. The linear regression equation for clodinafop-propargyl is: y = 342906x + 41167, and its correlation coefficient is: R. 2 =0.9993.

[0131] 14.2 Determination of the recovery rate of highly efficient degrading bacterial strains

[0132] The recovery rates of cyclophosphamide addition are shown in Table 5, ranging from 92.42% to 108.26%, which meets the requirements of the national standard (80-120%), proving that the above-mentioned herbicide extraction and high-performance liquid chromatography detection methods are feasible.

[0133] Table 5. Recovery Rate of Highly Degradative Bacterial Strains Added

[0134]

[0135] 14.3 Determination of Degradation Rate of Highly Efficient Degrading Bacterial Strains

[0136] By assessing the degradation ability of degrading bacteria on clopyralid in inorganic salt culture media, strains with degradation effects were screened. The results are as follows: Figure 4 As shown, strains T4P20-1, T4P20-3, and T6P20-1 showed the best degradation effect on the initial concentration of 75 mg / L of cyclohexane within 5 days, with degradation rates of 93.87%, 93.89%, and 97.40%, respectively.

[0137] 15. Identification of Highly Efficient Degrading Bacterial Strains

[0138] 15.1 Morphological identification of highly efficient degrading bacterial strains

[0139] The colony morphology of strain T4P20-1, such as Figure 5 As shown, the colony morphology of strain T4P20-1 on LB medium was observed. The colonies were milky white, opaque, round, with neat edges, smooth surface, and a raised center. They stained red with Gram stain and were short rod-shaped, indicating that the strain was a Gram-negative bacterium.

[0140] 15.2 Biochemical identification of highly efficient degrading bacterial strains

[0141] The biochemical reaction results of the highly efficient degrading bacterial strains are shown in Table 6. Compared with Dong Xiuzhu's "Handbook for the Identification of Common Bacteria" and "Bergey's Handbook for the Identification of Bacteria (Ninth Edition)", the experimental results are basically the same.

[0142] Table 6 Biochemical characteristics of highly efficient degrading bacterial strains

[0143]

[0144] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0145] 15.3 Identification of 16S rDNA sequences of highly efficient degrading bacterial strains

[0146] 15.3.1 Agarose gel electrophoresis detection of 16S rDNA from highly efficient degrading strains

[0147] The results of 1.7% agarose gel electrophoresis analysis of the amplified 16S rDNA products of the highly efficient degrading strain are as follows: Figure 6 As shown, the gene fragment size is approximately 1400 bp.

[0148] 15.3.216S rDNA nucleotide sequence determination results

[0149] Blast comparison results of strain T4P20-1 on NCBI Figure 7 As shown, the results indicate that the 16S rDNA sequence of strain T4P20-1 has high homology with Acinetobacter. Based on the morphological and biochemical identification results, the strain was identified as Acinetobacter pittii and submitted to GenBank to obtain accession number MW350066.

[0150] 16. Determination of growth characteristics of highly efficient degrading bacterial strains

[0151] 16.1 Determination of the growth of bacterial strains that are highly efficient at degrading clodinafop-propargyl at different pH values ​​and inoculum amounts

[0152] The results of growth assays on bacterial strains that are highly efficient at degrading clodinafop-propargyl at different pH values ​​and inoculum sizes are as follows: Figure 8 As shown, the strain was cultured in a shaker at 30℃ and 160r / min for 72h. Samples were taken every 12h to measure the absorbance of the strain at pH=6, pH=7 and pH=8. The optimal pH for strain T4P20-1 was 7. Samples were taken every 12h to measure the absorbance of the strain at inoculum sizes of 1%, 5% and 10%. The optimal inoculum size for strain T4P20-1 was 10%.

[0153] 16.2 Growth determination of bacterial strains that efficiently degrade clodinafop-propargyl from different carbon and nitrogen sources

[0154] The results of growth assays on bacterial strains that efficiently degrade clodinafop-propargyl with different carbon and nitrogen sources are as follows: Figure 9 As shown, the strain was cultured in a shaker at 30℃ and 160r / min for 72h. Samples were taken every 12h to measure the absorbance of the strain under the conditions of added glucose, sucrose and lactose. The optimal carbon source for strain T4P20-1 was lactose. Samples were taken every 12h to measure the absorbance of the strain under the conditions of added urea, ammonium nitrate and potassium sulfate. The optimal nitrogen source for strain T4P20-1 was ammonium sulfate.

[0155] 16.3 Determination of the effect of incubation time on the growth and degradation ability of bacterial strains that are highly efficient at degrading clodinafop-propargyl.

[0156] The results of the determination of the effect of incubation time on the growth and degradation ability of bacterial strains that are highly efficient at degrading clodinafop-propargyl are as follows: Figure 10 As shown, strain T4P20-1 was in the logarithmic growth phase after 12-36 hours of cultivation on a medium containing inorganic salts and clopyralid, with an absorbance value of 0.35. After 72 hours, the concentration of clopyralid in strain T4P20-1 decreased from 75 mg / L to 5.11 mg / L, with a degradation rate of 92.23%.

[0157] 17. Verification of the degradation ability of highly efficient degrading bacterial strains using bioassay methods

[0158] 17.1 Determination of Sensitive Concentrations for the Effects of Herbicides on Seed Germination of Subsequent Sensitive Crops

[0159] The results of the sensitivity concentration test of the effect of clopyralid (P20) on soybean seed germination are shown in Table 7. Compared with the group with 0 added herbicide, the sensitive concentrations at which clopyralid had a significant difference in the effect of clopyralid on soybean seed germination rate and stem length at the 5% level were 75 mg / L and 100 mg / L. Therefore, a concentration of 75 mg / L of clopyralid was selected for subsequent tests.

[0160] A linear regression equation was established based on the experimental results, as follows: Figure 11 As shown, the sensitive concentration equations for the effects of clodinafop-propargyl on soybean seed germination rate, stem length, and root length are: y1 = -0.279x + 95.884, with correlation coefficients R0. 2 =0.9768; y2 = -0.0041x + 1.8167, correlation coefficient R 2 =0.9114; y3 = -0.003x + 1.3356, correlation coefficient R 2 =0.94, where y1, y2, y3, and x represent the germination rate of soybean seeds, stem length, root length, and concentration of oxychloride, respectively.

[0161] 17.2 Validation of the degradation ability of highly efficient degrading bacterial strains by bioassay method

[0162] The changes in germination rate, stem length, and shoot length of seeds treated with clopyralid (P20) by its degrading strain T4P20-1 are shown in Table 8. Among them, the stem length of soybean seeds in the initial concentration of 75 mg / L clopyralid culture solution was significantly lower than that of the blank group. After treatment with its degrading strain T4P20-1, the germination rate and stem length of soybean seeds were significantly higher than those of the control group. Compared with the control group, the stem length and root length showed an increasing trend, increasing by 16.77% and 59.26%, respectively.

[0163] Table 7. Results of bioassay on the sensitivity of subsequent crop seeds to herbicide concentrations.

[0164]

[0165] Note: The significance analysis used Duncan's method for pairwise comparisons. Different letters in each column indicate significant differences, which are at the 0.05 significance level.

[0166] Table 8 Seed germination test results after herbicide treatment with bacterial strains.

[0167]

[0168] Note: The significance analysis used Duncan's method for pairwise comparisons. Different letters in each column indicate significant differences, which are at the 0.05 significance level.

[0169] In summary, current research on the degradation of clodinafop-propargyl is still in its infancy both domestically and internationally. This invention screened Acinetobacter pittii strains from soil long-term contaminated with clodinafop-propargyl to identify strains capable of degrading it. Strain T4P20-1 achieved a 93.87% degradation rate of clodinafop-propargyl at an initial concentration of 75 mg / L within 5 days, a first-time report. Most existing literature reports on the remediation of petroleum pollution using Acinetobacter, such as Peng Sudi et al.'s finding that Acinetobacter sp. AC achieved an 80% degradation rate after 7 days of culture. There are also reports on Acinetobacter degrading phthalate plasticizers, deltamethrin, cellulose, nitrites, and phenolic compounds, but reports on Acinetobacter degrading herbicides in soil are scarce.

[0170] This invention domesticated and isolated strain T4P20-1, which uses clopyralid as the sole carbon and nitrogen source, from soil samples tested in Heilongjiang Province and Shandong Province. Based on morphological observation, physiological and biochemical identification, and 16S rDNA sequence isotype comparison, the strain was identified as Acinetobacter pittii, with the GenBank accession number MW350066. The degradation rate of 75 mg / L clopyralid by strain T4P20-1 reached 93.87% within 5 days. The optimal growth conditions for strain T4P20-1 were: pH 7, inoculum size of 10%, and optimal external carbon and nitrogen sources of lactose and urea, respectively. At 72 h, the clopyralid concentration decreased from 75 mg / L to 5.11 mg / L, with a degradation rate of 92.23%. The addition of the degrading strain had a restorative effect on seed germination. Soybean seeds in the initial 75 mg / L clopyralid culture medium had significantly shorter stem lengths than the control group. After treatment with the degrading strain T4P20-1, the germination rate and stem length of soybean seeds were significantly higher than those of the control group. Compared with the control group, the stem length and root length showed an increasing trend, increasing by 16.77% and 59.26%, respectively.

Claims

1. A strain of bacteria capable of remediating soil contaminated with clopyralid, characterized in that... It is Acinetobacter pittii T4P20-1, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 21, 2021, with accession number CGMCC No. 24157.

2. The application of the strain of herbicide as described in claim 1, which is characterized in that... It is used as a soil remediation agent to remediate soil contaminated with clopyralid.

3. The application of the strain of herbicide-containing soil remediation technology as described in claim 1, characterized in that... It is used as a remediation agent to promote seed germination in soil contaminated with clodinafop-propargyl.

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