A strain of microorganism capable of remediating soil contaminated with diflubenzuron and its application

Abstract

A strain of *Klebsiella aerogenes* T2P21-1, capable of remediating soil contaminated with diflubenzuron and its application, is disclosed in this invention. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit date T2P21-1 and accession number CGMCC No. 24156. It is used as a soil remediation agent to remediate soil contaminated with diflubenzuron and to promote seed germination in such soil.

Description

Technical Field

[0001] This invention relates to the agricultural field, specifically to a strain of bacteria capable of remediating soil contaminated with diflubenzuron 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 diflubenzuron are widely used in wheat fields.

[0003] Biflusulfonamide herbicide is 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] Florasulam is the fifth triazole pyrimidine sulfonamide (PTS) product successfully developed by Dow AgroSciences (along with sulfadiazine, pyrimidine sulfadiazine, chlorpyrifos sulfadiazine, and penoxsulam). Its trade names include Maxida, Maxi, and Prunus. Its chemical formula is 2',6'-difluoro-5-ethoxy-8-fluoro[1,2,4]triazole[1,5-C]pyrimidine-2-sulfonylaniline, with a relative molecular mass of 359.3 and a molecular formula of C12H8F3N5O3S. Its chemical structure is shown below. Figure 1 As shown, at 20℃ and pH=7.0, its solubility in water is 6.36 g / L. It is a typical acetolactate synthase (ALS) inhibitor. It mainly inhibits the activity of ALS enzyme in plants to prevent the biosynthesis of branched-chain amino acids, thereby disrupting protein synthesis, which in turn causes the plant to stop growing and die, ultimately leading to plant death. It is a broad-spectrum herbicide for post-emergence foliar application to control broadleaf weeds.

[0005] The adsorption and degradation of herbicides in soil, particularly diflubenzuron, has been a major research focus in recent decades, with the aim of reducing herbicide residue concentrations in the soil. Typically, laboratory studies employ batch equilibrium methods to determine adsorption parameters such as Koc, KF, and 1 / n, followed by incubation over time to determine degradation parameters such as k and t1 / 2. All these parameters contribute to assessing herbicide persistence and mobility, and to improving herbicide fate models, which can provide predicted environmental concentrations or simulate the environmental fate of the pesticide.

[0006] Gerri et al.'s research showed that the selectivity of diflubenzuron for wheat was mainly related to the difference in metabolic rate between wheat with a half-life of 2.4 hours and broadleaf weeds with a half-life of 19-48 hours. The structure of the metabolite was determined by nuclear magnetic resonance and liquid chromatography / mass spectrometry. Wheat plants metabolize diflubenzuron through hydroxylation of nitrogen via the aniline ring and binding with glucose. Broadleaf weeds metabolize very slowly, making it impossible to isolate the metabolites; however, comparative HPLC data indicate that hydroxylation is the primary pathway. Roy et al. investigated the degradation pathway and rate of the triazolopyrimidine sulfonamide herbicide diflubenzuron in six soil types under aerobic conditions at 20°C and 25°C, and separated and identified the degradation products using mass spectrometry. It degrades rapidly under microbial action, with an average half-life of 2.4 days (range 0.7–4.5 days). They also determined the photodegradation rate and pathway of the triazolopyrimidine herbicide diflubenzuron in soil and water. Mark et al. studied the photodegradation rate and pathway in soil and water, estimating a half-life of 14 days in soil and 36 days in sterile buffer water during summer at 40°N latitude. In natural water systems, photodegradation is faster. In summer at 51.5 degrees north latitude, the measured half-life was 3.3 days, indicating that indirect photolysis is an important pathway for the photodegradation of diflubenzuron in aquatic environments. Ma Yunfeng et al. studied the dynamics of diflubenzuron residue dissipation in three wheat fields over two years. The half-life in wheat plants ranged from 1.33 to 5.59 days, and the half-life in soil ranged from 5.66 to 7.84 days. Diflubenzuron was relatively difficult to adsorb in sandy and clay soils. The addition of humic acid and the increase of the initial concentration of diflubenzuron had a certain impact on the leaching capacity. Song Guochun et al. showed that the half-life of diflubenzuron in wheat plants ranged from 2.0 to 5.8 days, and the degradation rate reached more than 82% after 14 days of treatment.

[0007] Currently, there are no reports in domestic or international literature on the degradation of diflubenzuron. Summary of the Invention

[0008] The purpose of this invention is to provide a strain of bacteria capable of remediating soil contaminated with diflubenzuron and its application.

[0009] The present invention discloses a strain of Klebsiella aerogenes T2P21-1, which is capable of remediating soil contaminated with diflubenzuron. 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. 24156.

[0010] The present invention relates to the application of a strain of bacteria capable of remediating soil contaminated with diflubenzuron, which is used as a soil remediation agent for soil contaminated with diflubenzuron.

[0011] The present invention relates to the application of a strain of bacteria that can remediate soil contaminated with diflubenzuron, which acts as a remediation agent to promote seed germination in soil contaminated with diflubenzuron.

[0012] 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.

[0013] There are few reports on the degradation mechanisms (including physical, chemical, and biological degradation) of three types of wheat field herbicides. Microbial degradation is the best way to degrade herbicides. Therefore, the main purpose of this experiment is to isolate and cultivate strains that grow with diflubenzuron 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

[0014] Figure 1 Chemical structure diagram of diflubenzuron;

[0015] Figure 2 The growth curve of a bacterium strain that is highly efficient at degrading diflubenzuron after 72 hours;

[0016] Figure 3 Standard curves for three herbicide standards;

[0017] Figure 4 The graph shows the degradation rate of herbicides by the degrading strains.

[0018] Figure 5 Morphological identification diagram of highly efficient degrading bacterial strains;

[0019] Figure 6 Agarose gel electrophoresis image of 16S rDNA from a highly efficient bacterial degrading strain; Note: M: Marker DL2000; 1: 16S PCR product of strain T2P21-1;

[0020] Figure 7 This is a graph showing the comparison results of the strains using BLAST on NCBI.

[0021] Figure 8Figure 1 shows the effect of different pH values ​​and inoculum amounts on the growth of the bacterium strain T2P21-1 that is highly efficient at degrading diflubenzuron; Figure 2a shows the effect of different pH values ​​on the growth of strain T2P21-1, and Figure 2b shows the effect of different inoculum amounts on the growth of strain T2P21-1.

[0022] Figure 9 Figure 1 shows the effect of different carbon and nitrogen sources on the growth of bacterium strain T2P21-1 that is highly efficient at degrading diflubenzuron; Figure 2a shows the effect of different carbon sources on the growth of strain T2P21-1, and Figure 2b shows the effect of different nitrogen sources on the growth of strain T2P21-1.

[0023] Figure 10 Figure showing the effect of culture time on the growth and degradation of diflubenzuron by strain T2P21-1;

[0024] Figure 11 The graph shows the linear regression equation for the effect of herbicides on the seed germination of subsequent sensitive crops based on their concentration. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Example 1

[0029] 1. Soil sample for testing

[0030] 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.

[0031] Table 1. Soil sample collection locations, sample numbers, and crop varieties.

[0032]

[0033]

[0034] 2. Reagents

[0035] Diflubenzuron (99%), Dr. Ehrenstorfer Standards GmbH, Germany; Chromatographic grade methanol (Shandong Yuwang, chromatographic purity); Dichloromethane (Tianjin Guangfu, analytical purity).

[0036] 3. Culture medium

[0037] (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.

[0038] (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.

[0039] 4. Preparation of standard stock solution

[0040] 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.

[0041] 4. Preparation of bacterial suspension

[0042] 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.

[0043] 5. Enrichment, domestication, culture, isolation, and purification of highly efficient degrading bacteria

[0044] 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.

[0045] 6. Determination of growth curves of highly efficient degrading bacterial strains

[0046] Take 20 μL of bacterial suspension and add it to 180 μL of herbicide inorganic salt medium (herbicide concentrations of 25 mg / L, 50 mg / L, and 75 mg / L, respectively) and LB liquid medium, respectively. Four replicates were made, and the bacterial growth was measured every 12 h. Bioscreen was used to detect the growth for 72 h and the bacterial growth curve was plotted.

[0047] 7. Determination of recovery and degradation rate of highly efficient degrading bacterial strains

[0048] 7.1 Sample Pretreatment Method

[0049] (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;

[0050] (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.

[0051] (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.

[0052] (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.

[0053] 7.2 HPLC Chromatographic Detection Conditions

[0054] Column: INERTSIL ODS-35UM (5μm, 4.6×250mm)

[0055] Injection volume: 20 μL.

[0056] 7.3 Drawing up the standard curve for herbicides

[0057] The mother liquor of diflubenzuron 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.

[0058] 7.4 Determination of Degradation Rate of Highly Efficient Degrading Bacterial Strains

[0059] 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.

[0060] 7.5 Determination of recovery rate and calculation of degradation rate

[0061] Recovery rate (%) = (Measured concentration / Added concentration) × 100% (Formula 2-1)

[0062] Degradation rate (%) = [1 - (actual concentration of the treatment / actual concentration of the control)] × 100% (Formula 2-2)

[0063] 8. Identification of highly efficient degrading bacterial strains

[0064] 8.1 Morphological identification of highly efficient degrading bacterial strains

[0065] 8.1.1 Gram staining

[0066] 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:

[0067] (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.

[0068] (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.

[0069] (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.

[0070] (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.

[0071] (5) Remove residual water, rinse with 95% ethanol until colorless, and rinse with running water.

[0072] (6) Add safranin staining solution, cover the bacterial film, stain for 2 minutes, and rinse with running water.

[0073] (7) After drying, observe under an oil immersion microscope. Gram-positive bacteria appear blue-purple, and Gram-negative bacteria appear red.

[0074] 8.1.2 Observation of colony morphology

[0075] 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.

[0076] 8.2 Biochemical identification of highly efficient degrading bacterial strains

[0077] 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.

[0078] 8.3 Identification of 16S rDNA sequences of highly efficient degrading bacterial strains

[0079] 8.3.1 Extraction of bacterial genomic DNA

[0080] (1) Sample processing

[0081] Gram-negative 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. 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.

[0082] Gram-positive bacteria: Take 1 mL of overnight cultured bacterial suspension 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. Add 180 μL of lysozyme solution (prepare a 20 mg / mL lysozyme solution by adding the corresponding lysozyme to Enzymatic lysis buffer before use) to resuspend the bacterial suspension 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.

[0083] (2) Add 200 μL of Buffer BD and mix thoroughly by inverting.

[0084] (3) Add 200L of anhydrous ethanol and mix thoroughly by inverting.

[0085] (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 rpm at room temperature for 1 minute, and discard the waste liquid in the collection tube.

[0086] (5) Place the adsorption column back into the collection tube, add 500 μL of PW Solution, centrifuge at 10,000 rpm for 30 seconds and discard the filtrate.

[0087] (6) Place the adsorption column back into the collection tube, add 500 μL Wash Solution, centrifuge at 10000 rpm for 30 s and discard the filtrate.

[0088] (7) Place the adsorption column back into the collection tube and centrifuge at 12000 rpm at room temperature for 2 min to remove the residual WashSolution.

[0089] (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 rpm 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℃.

[0090] 8.3.2 PCR amplification of 16S rDNA

[0091] 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.

[0092] Table 216S rDNA PCR amplification primer sequences

[0093]

[0094] Table 3.16S rDNA PCR amplification reaction system and cycling procedure

[0095]

[0096]

[0097] 8.3.3 Recovery and purification of PCR products

[0098] The PCR product electrophoresis bands are cleaved to obtain the desired DNA target band, and the purification method is as follows:

[0099] (1) Cut a piece of gel containing the target fragment from the agarose gel and weigh it.

[0100] (2) Add Buffer B2 at 3-6 times the weight of the gel block and saturate in a 50°C water bath for 5-10 minutes.

[0101] (3)(Optional) For fragments <500bp, add 1 / 3 volume of isopropanol to Buffer B2.

[0102] (4) Transfer the sol solution into the adsorption column and centrifuge at 8000×g for 30 seconds. Discard the liquid in the collection tube.

[0103] (5) Add 500 μl of Wash Solution, centrifuge at 9000×g for 30 seconds, and discard the liquid in the collection tube. Repeat once.

[0104] (6) Centrifuge the empty adsorption column at 9000×g for 1 minute.

[0105] (7) Place the adsorption column into a clean 1.5 ml centrifuge tube, add 15-40 μl of Elution Buffer to the center of the adsorption membrane, let stand at room temperature for 1 minute, and then centrifuge for 1 minute. Save the DNA solution in the tube.

[0106] 8.3.416S rDNA sequence analysis

[0107] The 16S rDNA sequence was aligned to the ribosome database at http: / / rdp.cme.msu.edu / index.jsp, and the accession number was submitted.

[0108] 9. Determination of growth characteristics of highly efficient degrading bacterial strains

[0109] 9.1 Growth determination of highly efficient degrading bacterial strains at different pH values

[0110] 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.

[0111] 10% bacterial suspension was added to liquid culture medium containing 50 mg / L diflubenzuron 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 was measured every 12 hours and analyzed using Bioscreen for 72 hours to plot the bacterial growth curve.

[0112] 9.2 Growth determination of highly efficient degrading bacterial strains at different inoculum sizes

[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. 1%, 5%, and 10% of the bacterial suspensions were added to liquid culture medium containing 50 mg / L diflubenzuron 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.

[0114] 9.3 Growth assay of highly efficient degrading bacterial strains with added carbon 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 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 a vortex 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 50 mg / L diflubenzuron, glucose, sucrose, and lactose, with three replicates. A control group without the bacterial strain was included. Detection was performed every 12 h, and the bacterial growth curve was plotted using Bioscreen after 72 h.

[0116] 9.4 Growth determination of highly efficient degrading bacterial strains by external nitrogen source

[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. Centrifugation was performed at 5000 rpm for 5 min. The supernatant was discarded, and sterile 0.9% physiological saline was added. The mixture was vortexed in a vortex for 1 min, centrifuged again, and the supernatant was discarded. 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 50 mg / L diflubenzuron, urea, potassium nitrate, and ammonium sulfate. Three replicates were performed, with one control group without the bacterial strain. Detection was performed every 12 h, and the bacterial growth curve was plotted using Bioscreen after 72 h.

[0118] 9.5 Determination of the effect of incubation time on the growth and degradation ability of highly efficient degrading bacterial strains

[0119] 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 liquid culture medium containing 50 mg / L diflubenzuron 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.

[0120] 10. Determination of the degradation ability of highly efficient degrading bacterial strains by bioassay method

[0121] 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.

[0122] The sensitivity concentration screening test method is as follows: First, prepare a medium containing diflubenzuron inorganic salts with gradient concentrations (5, 25, 50, 75, 100 mg / L). Cut sterile gauze into appropriate sizes and place it in a sterile plate. Add 40 mL of culture medium to each plate, and then evenly place 40 seeds in each plate. Each treatment is repeated three times, and sterile water is set as a blank control. The plates are placed in a 25℃ light incubator and cultured for 6 days, with equal amounts of water added daily. Observe and record indicators such as germination rate, shoot length, and root length. Statistical analysis is performed to determine the more sensitive concentration of the seeds.

[0123] The bioassay method for the degradation ability of the strain is as follows: The strain with a high degradation rate is inoculated into the medium of sensitive concentration of herbicide inorganic salt, shaken at 30℃, and applied to sensitive crop seeds according to the above method. This is the treatment group. In addition, sterile water is set as the blank group, and the medium of uninoculated herbicide inorganic salt is set as the control group. After each batch of treated seeds grows for 6 days, its germination rate, shoot length and root length and other indicators are observed and recorded.

[0124] 11. Enrichment, domestication, culture, isolation, and purification of highly efficient degrading bacteria

[0125] Enrichment culture was carried out on inorganic salt media with added diflubenzuron, and strains with transparent zones in the inorganic salt-added herbicide medium were screened. Further purification was carried out on LB solid medium, and 45 highly efficient degrading strains were isolated from the tested soil samples, as shown in Table 4. Among them, 12 strains were able to degrade diflubenzuron.

[0126] 12. Determination of growth curves of highly efficient degrading bacterial strains

[0127] The growth curve of the highly efficient bacterium strain of diflubenzuron after 72 hours is shown below. Figure 2 As shown, strains T2P21-1 and T13P21-1 showed a growth trend at an initial concentration of 50 mg / L of diflubenzuron, while strain T5P21-1 showed a growth trend at an initial concentration of 75 mg / L of diflubenzuron.

[0128] Table 4. Number of Highly Degrading Bacterial Strains

[0129]

[0130] 13. Determination of the recovery rate of highly efficient degrading bacterial strains

[0131] 13.1 Construction of the standard curve for highly efficient degrading bacterial strains

[0132] The standard curve of the diflubenzuron herbicide standard was plotted as follows: Figure 3 As shown. The linear regression equation for diflubenzuron is: y = 27969x + 20327, and its correlation coefficient is: R0 2 =0.9989.

[0133] 13.2 Determination of the recovery rate of highly efficient degrading bacterial strains

[0134] The recovery rates of diflubenzuron are shown in Table 5, ranging from 90.47% to 109.78%, 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.

[0135] Table 5. Recovery Rate of Highly Degradative Bacterial Strains Added

[0136]

[0137] 13.3 Determination of Degradation Rate of Highly Efficient Degrading Bacterial Strains

[0138] By assessing the degradation ability of degrading bacteria on diflubenzuron in inorganic salt culture media, strains with degradation effects were screened, and the results are as follows: Figure 4 As shown, strains T2P21-1 and T13P21-1 showed better degradation effects on diflubenzuron at an initial concentration of 50 mg / L within 5 days, with degradation rates of 96.87% and 96.58%, respectively; strain T5P21-1 showed a degradation rate of 68.3% on diflubenzuron at an initial concentration of 75 mg / L within 5 days.

[0139] 14. Identification of Highly Efficient Degrading Bacterial Strains

[0140] 14.1 Morphological identification of highly efficient degrading bacterial strains

[0141] Observe the colony morphology of strain T2P21-1 on LB medium, such as... Figure 5 As shown, the colonies are white, round, with smooth edges, a raised center, and are opaque. They stain red with Gram stain and are short, thick rod-shaped bacteria, indicating that the strain is a Gram-negative bacterium.

[0142] 14.2 Biochemical identification of highly efficient degrading bacterial strains

[0143] 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.

[0144] Table 6 Biochemical characteristics of highly efficient degrading bacterial strains

[0145]

[0146]

[0147] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0148] 14.3 Identification of 16S rDNA sequences of highly efficient degrading bacterial strains

[0149] 14.3.1 Agarose gel electrophoresis detection of 16S rDNA from highly efficient degrading strains

[0150] 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.

[0151] 14.3.216S rDNA nucleotide sequence determination results

[0152] Comparison results of strain T2P21-1 on NCBI Blast Figure 7 As shown, the results indicate that the 16S rDNA sequence of strain T2P21-1 has high homology with Klebsiella. Based on the morphological and biochemical identification results, the strain was identified as Klebsiella aerogenes and submitted to GenBank for accession number MW350091.

[0153] 15. Determination of growth characteristics of highly efficient degrading bacterial strains

[0154] 15.1 Growth determination of bacterium strains that are highly efficient at degrading diflubenzuron at different pH values ​​and inoculum sizes

[0155] The growth of diflubenzuron-efficient degrading bacteria strains under different pH values ​​and inoculum sizes was determined 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 T2P21-1 was 8. 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 T2P21-1 was 10%.

[0156] 15.2 Growth determination of bacterial strains that are highly efficient at degrading diflubenzuron using different carbon and nitrogen sources

[0157] The growth assay results of bacterial strains that are highly efficient at degrading diflubenzuron using 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 T2P21-1 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 T2P21-1 was ammonium sulfate.

[0158] 15.3 Determination of the effect of incubation time on the growth and degradation ability of diflubenzuron-efficient bacterial strains

[0159] The effects of incubation time on the growth and degradation ability of diflubenzuron-efficient bacterial strains are as follows: Figure 10 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 and degradation ability of diflubenzuron. The strain T2P21-1 was in the logarithmic growth phase after 12-36h of culture on a medium containing inorganic salts and diflubenzuron, with an absorbance of 0.36. After 72h, the concentration of diflubenzuron in strain T2P21-1 decreased from 50mg / L to 2.72mg / L, and the degradation rate reached 94.57%.

[0160] Validation of the degradation ability of highly efficient degrading bacterial strains using 16 bioassay methods

[0161] 16.1 Determination of Sensitive Concentrations for the Effects of Herbicides on Seed Germination of Subsequent Sensitive Crops

[0162] The results of the sensitive concentration test of the effect of diflubenzuron (P21) on maize seed germination are shown in Table 7. Compared with the group with 0 added herbicide, the sensitive concentration at which diflubenzuron had a significant difference in the effect of diflubenzuron on the germination rate, stem length and root length of maize seeds at the 5% level was 100 mg / L. Therefore, a concentration of 100 mg / L of diflubenzuron was selected for subsequent tests.

[0163] 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 diflubenzuron on maize seed germination rate, stem length, and root length are: y1 = -0.1987x + 93.305, and the correlation coefficient R0 is 1. 2 =0.9921; y2 = -0.0062x + 2.2393, correlation coefficient R 2 =0.9298; y3 = -0.0051x + 1.0804, correlation coefficient R 2 =0.9819, where y1, y2, y3, and x represent the germination rate of soybean seeds, stem length, root length, and concentration of diflubenzuron, respectively.

[0164] 16.2 Validation of the degradation ability of highly efficient degrading bacterial strains using bioassay methods

[0165] The changes in germination rate, stem length, and shoot length of seeds treated with diflubenzuron (P21) by its degrading strain T2P21-1 are shown in Table 8. Among them, the stem length of maize seeds in the initial diflubenzuron culture solution with an initial concentration of 100 mg / L was significantly lower than that of the control group. After treatment with its degrading strain T2P21-1, the germination rate of maize seeds was significantly higher than that of the control group. Compared with the control group, the stem length and root length showed an increasing trend, increasing by 15.65% and 14.95%, respectively.

[0166] Table 7. Results of bioassay on the sensitivity of subsequent crop seeds to herbicide concentrations.

[0167] 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.

[0168] Table 8 Seed germination test results after herbicide treatment with bacterial strains.

[0169]

[0170]

[0171] 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.

[0172] In summary, sulfonylurea herbicides are a new type of herbicide with good efficacy and high selectivity, and have been widely used for weed control in crops. However, due to the increasing application rate year by year, trace amounts of sulfonylurea herbicide residues in the soil can cause phytotoxicity problems in sensitive subsequent crops. Microbial degradation of herbicides is a good measure for remediating contaminated soil. This invention screened Klebsiella aerogenes strains from soil long-term contaminated with diflubenzuron to identify strains capable of degrading it. Strain T2P21-1 achieved a degradation rate of 96.87% for diflubenzuron at an initial concentration of 50 mg / L within 5 days. Although Zhang et al. found that Klebsiella jilinsis 2N3 can degrade more than 90% of chlorimuron-methyl, and Lin Yang et al. found that Klebsiella 2N3 can degrade thifensulfuron-methyl at 96.03%, and other studies have found that Klebsiella can degrade quinclorac acid, atrazine, phenanthrene, bifenthrin, polyacrylamide, and 2,2,6,6-tetramethylpiperidone (TMPD), this invention is the first report on the degradation of diflubenzuron.

[0173] This invention isolated strain T2P21-1, which uses diflubenzuron as the sole carbon and nitrogen source, from soil samples tested in Heilongjiang and Shandong provinces. Based on morphological observation, physiological and biochemical identification, and 16S rDNA sequence isotype comparison, the strain was identified as *Klebsiella aerogenes*, with the GenBank accession number MW350091. Strain T2P21-1 achieved a 96.87% degradation rate of diflubenzuron at an initial concentration of 50 mg / L within 5 days. The optimal growth conditions for strain T2P21-1 were: pH 8, inoculum size of 10%, and optimal external carbon and nitrogen sources of lactose and ammonium sulfate, respectively. After 72 hours, the diflubenzuron concentration decreased from 50 mg / L to 2.72 mg / L, achieving a degradation rate of 94.57%. The addition of degrading strains has a repairing effect on seed germination. In the initial concentration of 100 mg / L diflubenzuron culture medium, the stem length of maize seeds was significantly lower than that of the control group. After treatment with its degrading strain T2P21-1, the germination rate of maize seeds was significantly higher than that of the control group. Compared with the control group, the stem length and root length showed an increasing trend, increasing by 15.65% and 14.95%, respectively.

Claims

1. A strain of bacteria capable of remediating soil contaminated with diflubenzuron, characterized in that... It is Klebsiella pneumoniae ( Klebsiella aerogenes T2P21-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 21, 2021, and the accession number is CGMCC No. 24156.

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

3. The application of the strain with the function of remediating soil contaminated with diflubenzuron as described in claim 1, characterized in that... It is used as a remediation agent to promote seed germination in soil contaminated with diflubenzuron.

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