Novel pesticide-degrading bacterial strain and application thereof

Abstract

The application relates to a new pesticide-degrading strain, in particular to a strain New bacteria Acinetobacter oryzae F9-1 . A strain F9-1 is obtained by separation, screening and identification from a soil sample in Hami City of Xinjiang Uygur Autonomous Region New bacteria Acinetobacter oryzae F9- 1 The strain is subjected to physiological and biochemical characteristic detection, the strain F9-1 shows small colonies, smooth surface, white semi-transparency on NB plates, and the results of gram staining, contact enzyme and indole experiment are positive, and the results of oxidase, V.P test and urease are negative. The strain F9-1 can hydrolyze hydrogen peroxide, can utilize L-alanine, lactic acid, L-aspartic acid and quinine acid, is sensitive to 8% NaCl, lincomycin, naphthylidine ketone acid and chloromycetin, and can degrade fluazifop-butyl and pendimethalin, and has the advantages of environmental protection, high efficiency, economy and sustainability, and is an effective means for solving the problem of pesticide pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial strains and their applications, and in particular to a new pesticide-degrading strain and its application technical field. BACKGROUND

[0002] Xinjiang Uygur Autonomous Region is the main and dominant area of cotton production in China, and cotton field weeds are one of the main reasons affecting the yield of cotton production and an important factor restricting the sustainable development of cotton. Longcaojing, fluridone suspension agent with an effective content of 42%, is a pyrrolidone soil sealing herbicide widely used in cotton fields in Xinjiang Uygur Autonomous Region in recent years. It is usually used in combination with pendimethalin to prevent and control cotton field weeds such as Solanum nigrum, Chenopodium glaucum and Echinochloa crus-galli, annual broadleaf and gramineous weeds, and has a safe period of about 60 days, and is safe to cotton emergence and growth. The chemical name of fluridone is 1-methyl-3-phenyl-5-(3-trifluoromethylphenyl)-4(1H)-pyridinone, which mainly acts on plant lycopene dehydrogenase, causing chlorophyll loss, inhibiting photosynthesis, and gradually whitening and dying of weeds, thereby playing a herbicidal role.

[0003] Due to the continuous cropping of cotton fields in Xinjiang Uygur Autonomous Region and the planting of other different crops, fluridone has a long residual period in the soil, and there is a certain potential phytotoxicity risk to the subsequent rotation crops. Some research has been done on the safety of rotation crops after the use of fluridone in cotton fields at home and abroad. Zhang Zhen et al. conducted a safety study of fluridone on common crops in Xinjiang Uygur Autonomous Region, and found that the safety of fluridone to different crops from high to low was seed pumpkin = sweet potato > peanut > oil sunflower = corn = wheat > melon > melon > watermelon > tomato > pepper; Hill et al. 2016 studied the safety of fluridone to common subsequent crops such as wheat, corn, soybean, rice, sorghum and sunflower in four irrigated cotton areas in Arkansas, USA, and found that wheat was relatively sensitive; Guo Shijian et al. [5] Longcaojing treatment had no obvious phytotoxicity to seed pumpkin, melon, Hami melon and oil sunflower, and had no obvious effect on their yield; it had certain phytotoxicity symptoms to corn, wheat and tomato. Elsie and Mohammed found that after applying fluridone in the soil, part of the drug residues produced would cause phytotoxicity to the subsequent corn seedlings, seriously affecting the growth of corn seedlings.

[0004] According to statistics, Longcaojing, as a herbicide, has been used in more than 15 million mu of cotton fields in Xinjiang Uygur Autonomous Region. The large amount of pesticide input may cause widespread soil pollution. Pesticides have the characteristics of biological accumulation and biological amplification, which seriously threaten human health and agricultural and ecological safety. Therefore, it is of great significance to study the pollution status and degradation behavior of Longcaojing in soil for the sustainable development of agriculture in Xinjiang Uygur Autonomous Region. The main methods of pesticide residue degradation are physical degradation, chemical degradation and biological degradation. Among them, physical and chemical remediation technologies may destroy the original soil ecosystem to some extent, require large investment, cause secondary pollution and other shortcomings, and are not the optimal method for remediation of soil pesticide residue pollution. Biological remediation is considered a method of using active microbial cells or their enzymes to degrade toxic pollutants, and has the advantages of economy, efficiency and greenness, and is more and more used for degradation of pesticide residues, and gradually becomes a potential substitute for traditional technology.

[0005] Domestic and foreign researchers have carried out a lot of research work on microbial degradation of pesticide residues. Studies have found that microorganisms with pesticide residue degradation are widely distributed in bacteria, including Pseudomonas (Pseudomonas sp. Pseudonomonas ), Arthrobacter (Arthrobacter sp. Arthrobacter ), Bacillus (Bacillus sp. Bacillus , Candida tropicalis (Candida tropicalis Candida tropicalis ), Corynebacterium (Corynebacterium sp. Corynebacterium ), Monasporus (Monasporus sp. Sphingonomas , Burkholderia (Burkholderia sp. Burkholderiales ), Rhodococcus (Rhodococcus sp. Rhodococcus ruber , and some fungi (Fungus Mortierella) also have the ability to degrade pesticide residues. Liang B et al.

[20] isolated from the enrichment culture of soil samples Brevundimonas sp. After 5 days of culture in a flask, the strain degraded about 80% of 50 mg / L lactofen. Hu et al. isolated strain HME-24, which degraded 96.7% of 50 mg / L lactofen in 72 h. Saez et al.

[22] 's study proved the degradation effect of Streptomyces on lindane in soil.

[0006] At present, the research on the degradation of fluridone residue is relatively less, and Yan Na studies the degradation behavior of fluridone in soil and environment, finds that the photolysis of fluridone in different pH buffer solutions, different light sources, different water bodies and different initial concentrations is difficult to photolyze, high organic matter content and suitable moisture can increase the number of microbial population and make its activity stronger, thereby accelerating the soil degradation rate of the pesticide. Wickham studies the degradation of fluridone in water and sediments under different intensity of ultraviolet and temperature, and the results show that the greater the intensity of ultraviolet, the faster the degradation of fluridone in water, but the effect of ultraviolet exposure on the degradation of fluridone in sediments is weak, and the degradation of fluridone slows down at low temperature. SUMMARY

[0007] In order to enrich the strain resources of pesticide degradation microorganisms in China, the present application aims to screen high-efficiency pesticide-degrading strains from the soil of a cotton field where fluridone has been applied for a long time, and to study the degradation characteristics thereof, so as to provide a theoretical basis for the treatment and remediation of pesticide-contaminated soil and a new strain for degrading pesticides. Acinetobacter oryzae F9-1 and its application, the present application isolates and screens a new strain Acinetobacter oryzae F9-1 from soil samples, and detects the physiological and biochemical characteristics of the strain, which shows small colonies, smooth surface, white translucent, positive results of gram staining, contact enzyme and indole experiment, and negative results of oxidase, V.P test and urease. The strain F9-1 can hydrolyze hydrogen peroxide, utilize L-alanine, lactic acid, L-aspartic acid and quinine acid, is sensitive to 8% NaCl, lincomycin, naphthylidone acid and chloramphenicol, and can degrade fluridone and pendimethalin, and has the advantages of environmental protection, high efficiency, economy and sustainability, and is an effective means to solve the problem of pesticide pollution.

[0008] In order to achieve the above technical effects, the present application realizes the technical scheme as follows.

[0009] The present application provides a new strain Acinetobacter oryzae F9-1. Acinetobacter oryzae The strain F9-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the strain preservation number is CGMCC NO.31742.

[0010] In the present application, the new strain Acinetobacter oryzae F9-1 is referred to as Acinetobacter oryzae F9-1 or strain F9-1.

[0011] The present application provides a new strain Acinetobacter oryzae F9-1It was isolated and screened from soil samples in a new area. Phylogenetic and morphological analysis of the 16S rDNA gene sequence confirmed that strain F9-1 ​​belongs to Acinetobacter mibricinum (…). Acinetobacter oryzae.sp ), named Acinetobacter oryzae F9-1. Gene sequencing of this strain was performed, and the resulting sequences were compared using BLAST on the NCBI website. The 16S rDNA gene sequence of strain F9-1 ​​was consistent with... Acinetobacter oryzae B23 (GU954428) showed the highest homology, with a similarity of 96.67%. In the phylogenetic tree constructed using the 16S rRNA gene sequence, the 16S rRNA sequence of strain F9-1 ​​was most similar to... Acinetobacter oryzae The B23 (GU954428) strain is the most closely related, but not on the same branch, indicating that strain F9-1 ​​has extremely high support as a new species and exhibits excellent stability in the phylogenetic tree. Through a comprehensive assessment of species similarity and homology, the obtained strain was confirmed. Acinetobacter The strain numbered F9-1 ​​within the genus is a typical new strain.

[0012] The new strain provided by this invention Acinetobacter oryzae F9-1, having undergone molecular-level identification based on the aforementioned well-known and recognized strain systems in the art, combined with morphological identification analysis, relates to a novel microorganism. Acinetobacter oryzae F9-1 colonies are small, smooth, white, and translucent. Gram staining, catalase, and indole tests are positive, while oxidase, VP, and urease tests are negative. Strain F9-1 ​​cannot hydrolyze starch but can hydrolyze hydrogen peroxide; it can utilize L-alanine, lactic acid, L-aspartic acid, and quinic acid, but cannot utilize sucrose, D-fructose, or gelatin; it is sensitive to 8% NaCl, lincomycin, nalidixic acid, and chloramphenicol, but not to acetomycin, tetrazolium violet, and dimethylaminetetracycline. Through strain comparison, it is similar to neighboring strains... Acinetobacter oryzae B23 shows significant differences.

[0013] The above-mentioned strain identification, including molecular-level identification and morphological verification using well-known and recognized strain systems in the field, confirmed that the obtained strain numbered F9-1 ​​is... Acinetobacter oryzae This belongs to a typical new strain within the scope. Acinetobacter oryzae F9-1, this strain was deposited prior to the application date at the Budapest Treaty International Collection Unit for Microorganisms: China General Microbiological Culture Collection Center (CGMCC). Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Accession number: CGMCC NO.31742. Deposit date: August 26, 2024.

[0014] The above-mentioned new bacteria Acinetobacter oryzaeThe gene sequence of F9-1 is shown as SEQ ID NO: 1.

[0015] The new bacteria Acinetobacter oryzae The separation culture medium of F9-1 is: 50-300 mg / L fluridone MSM liquid medium is added, and the culture medium uses MgSO4.7H2O 0.2 g; K2HPO4 0.1 g; (NH 4）2 SO4 0.1 g; CaSO4 0.04 g; FeSO4.7H2O 0.001 g; deionized water 1 L; pH 7.3.

[0016] The new bacteria Acinetobacter oryzae The purification culture medium of F9-1 is: 50 mg / L dimethenamid is used as the only carbon source on the MSM solid plate, and the culture medium uses MgSO4.7H2O 0.2 g; K2HPO4 0.1 g; (NH 4）2 SO4 0.1 g; CaSO4 0.04 g; FeSO4.7H2O 0.001 g; deionized water 1 L; pH 7.3.

[0017] Meanwhile, the new bacteria Acinetobacter oryzae The preparation method of the fermentation liquor of F9-1, the fermentation liquor is prepared through the following steps: rinsing the activated new bacteria Acinetobacter oryzae F9-1 plate spores with sterile water to obtain a spore suspension, inoculating the F9-1 spore suspension into a fermentation culture medium at a volume ratio of 5% inoculation amount, and fermenting and culturing at 25 DEG C and 150 rpm for 7 days to obtain the fermentation liquor of strain F9-1.

[0018] Further, the new bacteria Acinetobacter oryzae The application of F9-1 in degrading pesticides.

[0019] The new bacteria Acinetobacter oryzae The application of F9-1 in degrading fluridone.

[0020] The new bacteria Acinetobacter oryzae The application of F9-1 in degrading dimethenamid.

[0021] The following beneficial effects can be obtained by implementing the above specific technical solutions of the present application:

[0022] (1) The present application provides a new bacteria for degrading pesticides which is obtained through scientific identification. Acinetobacter oryzae F9-1 F9-1 The new bacteria is obtained through separation, screening and identification from soil samples. Acinetobacter oryzaeThe strain is detected in physiological and biochemical characteristics. The new strain F9-1 is small in colony, smooth in surface, white and semi-transparent on NB plate, positive in Gram staining, contact enzyme and indole experiment, and negative in oxidase, V.P test and urease. The strain F9-1 can hydrolyze hydrogen peroxide, utilize L-alanine, lactic acid, L-aspartic acid and quinine acid, and is sensitive to 8% NaCl, lincomycin, naphthyl ketone acid and chloramphenicol, and can degrade flufenacet and pendimethalin, has the advantages of environmental protection, high efficiency, economy and sustainability, and is an effective means to solve the problem of pesticide pollution.

[0023] (2) by applying the new strain F9-1 provided in the application Acinetobacter oryzae Under the optimal degradation conditions of pH 7, temperature 35 DEG C, inoculation amount 3%, and initial concentration of flufenacet 100 mg / L, the strain reaches OD600 of 1.3 in 0-3 days, grows rapidly, and degrades 100 mg / L flufenacet to 72.24 mg / L, the growth speed of the strain is slow in 4-7 days, the degradation rate is slow, and the residual amount of flufenacet is 36.29 mg / L in the 7th day, which shows that the new strain F9-1 provided in the application can effectively degrade flufenacet. Figure 1 BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 2 Fig. 1 shows the growth of the degradation strain on pendimethalin plate.

[0025] Fig. A shows the culture medium of the degradation strain, and Fig. B shows the growth of the degradation strain in different types of culture medium.

[0026] Figure 3 Fig. 3 shows the liquid chromatogram of flufenacet standard.

[0027] Figure 4 Fig. 4 shows the standard curve of flufenacet.

[0028] Figure 5 Fig. 5 shows the UV-visible light scanning spectrum and standard curve of pendimethalin;

[0029] Fig. A shows the UV-visible light scanning spectrum of pendimethalin, and Fig. B shows the standard curve of pendimethalin.

[0030] Acinetobacter oryzae Fig. 7 shows the colony morphology and Gram staining of the new strain F9-1. Acinetobacter oryzae Fig. A shows the colony morphology of the new strain F9-1, and Fig. B shows the Gram staining of the new strain F9-1.

[0031] Fig. A shows the colony morphology of the new strain F9-1, and Fig. B shows the Gram staining of the new strain F9-1. Acinetobacter oryzae Figure 6 Fig. A shows the colony morphology of the new strain F9-1, and Fig. B shows the Gram staining of the new strain F9-1.

[0032] Fig. A shows the colony morphology of the new strain F9-1, and Fig. B shows the Gram staining of the new strain F9-1.​Acinetobacter oryzae The new bacteria is shown Acinetobacter oryzae Figure 16S rDNA identification result of F9-1.

[0033] The new bacteria is shown Acinetobacter oryzae Figure 16S rDNA electrophoresis detection result of F9-1; the new bacteria is shown Figure 7 Figure phylogenetic tree of F9-1 based on 16S rDNA sequence.

[0034] Acinetobacter oryzae The new bacteria is shown Figure 8 Figure growth curve of F9-1.

[0035] Acinetobacter oryzae The new bacteria is shown Figure 9 Figure degradation characteristic of F9-1.

[0036] Figure A shows the effect of pH on the degradation of dimethenamid by strain F9-1; Figure B shows the effect of temperature on the degradation of dimethenamid by strain F9-1; Figure C shows the effect of inoculation amount on the degradation of dimethenamid by strain F9-1; and Figure D shows the effect of initial concentration on the degradation of dimethenamid by strain F9-1.

[0037] Acinetobacter oryzae The new bacteria is shown Figure 1 Figure degradation curve of F9-1. DETAILED DESCRIPTION

[0038] The following examples are provided to illustrate the present application, but the present application is not limited to the following examples. All raw and auxiliary materials used in the present application, and the culture method of the selected strain are well known in the art. The % used in the present application is mass percentage, unless otherwise specified.

[0039] In order to better explain the present application, the main content of the present application is further illustrated by the following specific examples, but the content of the present application is not limited to the following examples. If not specifically indicated, the technical means used in the examples is the conventional means well known to those skilled in the art. The fluridone standard (98.7%), dimethenamid technical, pendimethalin technical, acetonitrile, magnesium sulfate, ferrous sulfate, anhydrous calcium chloride, dipotassium hydrogen phosphate, sodium chloride, calcium sulfate, potassium dihydrogen phosphate, sodium hydroxide, phosphoric acid, anhydrous ethanol and related culture medium raw materials used in the present application are commercially available.

[0040] The NA culture medium used in the present application comprises: 10.0 g of proteose peptone; 3.0 g of beef extract; 5.0 g of sodium chloride; 15.0 g of agar; 1 L of deionized water; and pH 7.3.

[0041] The KB culture medium in the application adopts: hydrolyzed proteose peptone 20.0 g; dipotassium hydrogen phosphate 1.5 g; magnesium sulfate 1.5 g; agar 15.0 g; deionized water 1 L; pH 7.3.

[0042] The actinomyces culture medium in the application adopts: potassium nitrate 1.0 g; potassium dihydrogen phosphate 0.5 g; magnesium sulfate 0.1 g; ferrous sulfate 0.01 g; sodium chloride 0.5 g; soluble starch 20.0 g; agar 15.0 g; deionized water 1 L; pH 7.3.

[0043] The R2A culture medium in the application adopts: tryptone 0.25 g; acid hydrolyzed casein 0.5 g; yeast extract powder 0.5 g; soluble starch 0.5 g; dipotassium hydrogen phosphate 0.3 g; magnesium sulfate 0.1 g; sodium pyruvate 0.3 g; agar 12.0 g; proteose peptone 0.25 g; glucose 0.5 g; deionized water 1 L; pH 7.3.

[0044] The TSA culture medium in the application adopts: tryptone 15.0; soybean peptone 5.0; sodium chloride 30.0; agar 15.0; deionized water 1 L; pH 7.3.

[0045] The Bengal red culture medium in the application adopts: proteose peptone 5.0 g; glucose 10.0 g; potassium dihydrogen phosphate 1.0 g; magnesium sulfate 0.5 g; agar 20.0 g; Bengal red 0.033 g; chloramphenicol 0.1 g; deionized water 1 L; pH 7.3.

[0046] Example 1: Screening of flumioxazin-degrading strains

[0047] I. Materials and methods

[0048] 1. Test materials

[0049] (1) Test soil

[0050] The soil of a cotton field which has been applied with Longcaojing herbicide for many years was collected, 5-point sampling method was used, the soil sample was collected at a depth of 10-20 cm, dried, ground and passed through a 850 μm sieve, and stored at 4 ℃ for standby.

[0051] Table 1: Sample collection

[0052]

[0053] (2) Test agents

[0054] Flumioxazin standard (98.7%), Longcaojing technical material (42%), pendimethalin technical material, acetonitrile, magnesium sulfate, ferrous sulfate, anhydrous calcium chloride, dipotassium hydrogen phosphate, sodium chloride, calcium sulfate, potassium dihydrogen phosphate, sodium hydroxide, phosphoric acid, anhydrous ethanol.

[0055] II. Test method

[0056] 1. Enrichment and domestication of flumioxazin-degrading strains

[0057] In 500 mL conical flasks, 200 mL of MSM liquid medium was added and wet heat sterilized. 50 mg / L of flumioxazin was added, and then 10 g of soil sample was added, and cultured at 30°C, 200 r / min on a shaking table for 7 days; 5 mL of the first culture solution was added to 100 mg / L flumioxazin MSM liquid medium, and placed in a 30°C, 200 r / min shaking table for 7 days; 5 mL of the second culture solution was added to 150 mg / L flumioxazin MSM liquid medium, and placed in a 30°C, 200 r / min shaking table for 7 days, and so on until the flumioxazin concentration was 300 mg / L. 1 mL of the degraded bacterial solution after transfer was taken in a 1.5 mL pipette and diluted to 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 Coated on NA, KB, TAS, R2A, modified Gao No. 1, and Bengal red medium, and inverted in a 30°C constant temperature incubator for 48 h. After the colonies grew, single colonies with consistent color and morphology were selected for purification culture on selective MSM medium.

[0058] 2. Re-screening of flumioxazin-degrading strains

[0059] The strains obtained by enrichment and domestication were inoculated on MSM solid plates with 50 mg / L pendimethalin as the sole carbon source, and the growth was observed after 30°C culture for 48 h. Strains with good growth were selected. The selected strains were inoculated in MSM liquid medium with 50 mg / L pendimethalin as the sole carbon source, and cultured at 30°C, 200 r / min for 5 days. The supernatant was taken to detect the residual pendimethalin content, and strains with the ability to degrade pendimethalin were selected.

[0060] The strains with the ability to degrade pendimethalin were placed in a medium with 100 mg / L flumioxazin as the sole carbon source, and cultured at 30°C, 200 r / min for 24 h. The supernatant was taken to detect the residual flumioxazin, and strains with good degradation effect were selected as the subsequent research objects.

[0061] III. Results and analysis

[0062] 1. Screening of flumioxazin-degrading strains

[0063] The bacterial liquid obtained by enrichment and domestication was diluted and coated on different screening plates, and single colonies with different morphology, size and color were selected on 200 mg / L flumioxazin MSM solid medium to obtain 163 degradation bacteria. The strains were from NA plate 66, TSA plate 21, R2A plate 16, KB plate 17, modified Gao No. 1 plate 41, and Bengal red plate 2.

[0064] Table 2: Results of enrichment and domestication of flumioxazin degrading strains

[0065]

[0066] 2. Re-screening of flumioxazin degrading strains

[0067] The 163 different degradation bacteria obtained by enrichment and domestication were placed in MSM solid medium containing 50 mg / L dimethenamid as substrate, and 53 strains with good growth were obtained. Among them, 20 strains were from NA plate, 2 strains from TAS, 1 strain from R2A, 11 strains from KB, 18 strains from modified Gao No. 1, and 2 strains from Bengal red. The growth of related strains on the plate and the growth of strains in different media are shown in FIGS. A and B. Figure 2

[0068] Example 2: Identification of degrading strains

[0069] I. Test method

[0070] 1. Identification of degrading strains

[0071] Morphological identification: inoculate the strain into solid plate, draw line at 37℃ for 24-48 h, pick out single colony, evenly smear on glass slide for Gram staining, and observe under microscope;

[0072] Physiology and biochemistry: refer to "Common Bacteria System Identification Manual" and "Berger System Bacteriology Manual" to preliminarily identify the isolated strains;

[0073] Molecular biology identification: use Shanghai Shengong Company kit to extract strain genomic DNA as template, and use 16S rDNA universal primer for PCR amplification reaction. The universal primer is: 27F (5'-AGAGTTT GATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACG ACTT-3'). 25 μL system: template 0.5 μL, primer 0.5 μL each, 2×Taq PCR Mix premix 12.5 μL, ddH2O 11 μL.

[0074] ​PCR reaction conditions: 95 °C pre-denaturation 3 min; 95 °C denaturation 30 s, 55 °C annealing 30 s, 72 °C extension 1 min, a total of 30 cycles; finally 72 °C extension 10 min. After the reaction, the PCR reaction product was detected by 1.2% agarose gel electrophoresis. After detecting the size of the amplified fragment by agarose gel electrophoresis, sequencing was completed by Shanghai Biotech Co., Ltd. The sequencing results were compared with the 16S rRNA sequence in NCBI using Blast software. The phylogenetic tree of the strain based on the 16S rRNA gene sequence was established by the neighbor-joining method in MEGA7.0.

[0075] 2. Detection of fluridone

[0076] Preparation of standard solution: accurately weigh 50.00±0.10 mg of fluridone standard into a 50 mL volumetric flask, dissolve with acetonitrile to obtain a 1000 μg / mL standard stock solution. Dilute the 1000 μg / mL standard solution to 40, 80, 120, 160, 200 μg / mL standard solutions. Determine the standard working solution by HPLC, and perform regression analysis on the fluridone chromatographic peak area of the standard working solution and its corresponding concentration to obtain a standard curve;

[0077] Detection of fluridone: take 5 mL of the degradation bacteria solution, centrifuge at 8000 rpm for 10 min, take the supernatant, add 5 mL of acetonitrile, vortex extract for 10 min; then add 2 g of sodium chloride, vortex vigorously for 1 min, then centrifuge at 4000 r / min for 5 min; take the supernatant obtained above through an organic filter membrane with a pore size of 0.22 μm to obtain a sample solution; determine the chromatographic peak area of fluridone in the sample solution by HPLC, and substitute it into the standard curve to calculate the concentration and content of fluridone in the sample solution.

[0078] HPLC conditions: column: 250 mm x 4.6 mm (id) stainless steel column, packed with Lichrospher C18 filler, particle size 5 μm. Column temperature: 25 °C; detection wavelength: 310 nm; injection volume: 20 μL; mobile phase: acetonitrile-water (0.1% formic acid) (V:V=65:35); flow rate: 1.0 mL / min;

[0079] 3. Detection of pendimethalin

[0080] Preparation of the standard curve: Dimethoate standard was dissolved in dichloromethane to prepare solutions with a mass concentration of 50 mg / L, and full-wavelength scan analysis was performed to determine the maximum absorption peak. Nine mass concentration gradients were set up: 10, 20, 30, 40, and 50 mg / L. The absorbance of solutions with different mass concentrations was measured using ultraviolet spectrophotometry. A standard curve was plotted with dimethoate concentration on the x-axis and absorbance on the y-axis, and the equation of the standard curve was determined.

[0081] Dimethyl pendimethalin assay: Add an equal volume of dichloromethane to the dimethyl pendimethalin degradation culture medium, shake vigorously for 5 minutes to mix thoroughly, let stand until the aqueous phase and organic phase are completely separated, remove the upper aqueous phase, aspirate the lower organic phase, add excess anhydrous sodium sulfate to remove residual water, and use a UV-Vis spectrophotometer to scan the wavelength range of 200-600 nm to determine the wavelength absorption curve of dimethyl pendimethalin. The residual amount of dimethyl pendimethalin and the degradation status of dimethyl pendimethalin in the culture medium are determined based on the changes in the characteristic absorption peaks of ultraviolet and visible light of dimethyl pendimethalin.

[0082] II. Experimental Results

[0083] (1) Detection of flupyradifurone standard

[0084] Under the chromatographic conditions, a typical high-performance liquid chromatogram of flupyradifurone standard is shown in the appendix. Figure 3 As shown, the retention time was 2.843 min, slightly different from the 6.17 min retention time in the literature. The standard curves for flupyradifurone, plotted after determining the peak height at 310 nm for different concentrations of standards, are shown in the appendix. Figure 4 As shown, the standard curve exhibits good linear correlation with a correlation coefficient of 0.998, and can be used to detect the residual flupyradifurone content in the degrading bacterial solution.

[0085] (2) Testing of pendimethalin standard

[0086] Ultraviolet-visible scanning analysis of pendimethalin standards revealed a characteristic absorption peak at 430 nm within the 200-600 nm wavelength range. The absorbance of different concentrations of the standard was measured at 430 nm to obtain the pendimethalin standard curve (see Appendix). Figure 5 As shown, the linear correlation coefficient is 0.999, indicating a good correlation, which can be used to detect the residual pendimethalin content in the degrading bacterial solution.

[0087] After testing the degradation capacity of pendimethalin and flupyradifurone, eight strains that were tolerant and effective at degradation were finally obtained, as shown in Table 3. Among them, F9-1 ​​could degrade 82.43% of 50 mg / L pendimethalin within 7 days and 15.64% of 100 mg / L flupyradifurone within 24 hours. This strain was selected for subsequent experiments.

[0088] Table 3: Flupyradifurone screening strains

[0089]

[0090] (3) Morphological characteristics and physiological and biochemical characteristics of strain F9-1

[0091] The degradation strain F9-1 showed small colonies, smooth surface, white and translucent on NB plates, and the results of Gram staining, contact enzyme, and indole experiment were positive, and the results of oxidase, V.P experiment, and urease were negative. Strain F9-1 could not hydrolyze starch, but could hydrolyze hydrogen peroxide; could utilize L-alanine, lactic acid, L-aspartic acid, quinine acid, etc., but could not utilize sucrose, D-fructose, gelatin, etc.; was sensitive to 8% NaCl, lincomycin, naflicin, chloramphenicol, etc., and was not sensitive to acetylphthalide, tetrazolium purple, minocycline, etc. The determination results are shown in Table 5. Figure 6

[0092] Table 5: Physiological and biochemical characteristics of strain F9-1

[0093]

[0094] (4) 16S rDNA sequence analysis of strain F9-1

[0095] The genomic DNA of strain F9-1 was extracted by using a Shanghai Generay reagent kit, and the target DNA fragment of bacteria was amplified by using a bacterial 16S rDNA universal primer. The agarose gel electrophoresis diagram of the PCR product is shown in FIG. A of the accompanying drawings, and the size of the target DNA fragment is about 1500 bp. After the 16S rDNA gene fragment of strain F9-1 was sequenced, the sequence was recorded in the NCBI database for comparison, and the sequences with high similarity were selected for phylogenetic tree construction. The 16S rRNA gene sequence of strain F9-1 had the highest homology with B23 (GU954428), and the similarity was 96.67%, as shown in FIG. B of the accompanying drawings. Acinetobacter oryzae Figure 6 B23 (GU954428), and the similarity was 96.67%, as shown in FIG. B of the accompanying drawings. Combined with the morphological characteristics, physiological and biochemical properties of the isolated strain F9-1, and the 16S rDNA sequence comparison analysis, the isolated strain F9-1 was identified as Acinetobacter mitchellii (sp), and was named F9-1. Acinetobacter oryzae Acinetobacter oryzae Figure 6 F9-1.

[0096] (5) Sequence determination

[0097] ​​​​The PCR amplification product was sequenced after electrophoresis detection and purification, and the sequence length was 1392 bp. The sequencing results are shown in SEQ ID No: 1. BLAST homologous sequence retrieval was performed on NCBI. The Neighbor-Joining method was used to establish a phylogenetic tree (1000 times of repeated sampling) by using the commonly used MEGA 7.0 software in the art, and the results are shown in Figure 2. ​ The obtained sequence was analyzed by comparison on the NCBI website. It was found that the 16S rRNA gene sequence of the strain F9-1 had the highest homology with that of B23 (GU954428), and the similarity was 96.67%. In the phylogenetic tree constructed by the 16S rRNA gene sequence, the 16S rRNA sequence of the strain F9-1 was closest to that of the B23 (GU954428) strain, but not in the same branch, indicating that the support rate of the strain F9-1 as a new species was very high, and it had excellent stability in the phylogenetic tree. Through the comprehensive determination of the similarity and homology of the strain, it was confirmed that the obtained strain F9-1 belonged to a typical new species in the genus category. Acinetobacter oryzae Acinetobacter oryzae Acinetobacter

[0098] (6) Growth characteristic identification

[0099] The growth of the strain F9-1 at different time points is shown in Figure 1. Figure 7 As can be seen from the figure, the strain F9-1 grew slowly at 0-8 h, grew rapidly at 8-18 h, and grew slowly at 18-48 h. The growth amount reached the maximum at 20 h of culture. The growth of the strain F9-1 was in the form of "S". The inflection point of the "S" type appeared at 12 h, indicating that the growth rate of the strain was the largest at this time.

[0100] Example Three: Study on bacterial degradation characteristics

[0101] I. Test method

[0102] (1) Effect of pH on degradation of the strain F9-1 to monolinuron: 0.3 mL of bacterial suspension was added to 20 mL of MSM liquid medium containing monolinuron at a concentration of 100 mg / L at different pH (5.0, 6.0, 7.0, 8.0, 9.0), and the mixture was cultured in a 30℃, 150 rpm shaking incubator. After 7 days, the residual concentration of monolinuron was determined, and the degradation rate was calculated. At the same time, a control without adding bacteria was set, and the treatment and control were set in triplicate.

[0103] ​​​(2) Effect of temperature on the degradation of lansoprazole by strain F9-1: 0.3 mL of bacterial suspension of the degrading bacteria was added to 20 mL MSM liquid medium with a lansoprazole concentration of 100 mg / L. The medium was cultured at different temperatures of 25℃, 30℃, 35℃, 40℃ and 45℃ with shaking at 150 rpm. After 7 days, samples were taken to determine the residual concentration of lansoprazole and calculate its degradation rate. A control without bacteria was also set up. Both the treatment and the control were repeated 3 times.

[0104] (3) Effect of initial inoculum amount on the degradation of lansoprazole by strain F9-1: 0.2 mL, 0.6 mL, 1.0 mL, 1.4 mL, and 1.8 mL of the prepared bacterial suspension (OD600=1.0) were added to MSM liquid medium with a lansoprazole concentration of 100 mg / L, respectively, to make each culture system 20 mL. The culture was carried out at 37℃ and 150 rpm. After 7 days, the residual concentration of lansoprazole was measured and its degradation rate was calculated. A control without inoculum was also set up. Both the treatment and the control were repeated three times.

[0105] (4) Effect of initial concentration on the degradation of cyclophosphamide by strain F9-1: 0.3 mL of bacterial suspension of the degrading strain was added to 20 mL of MSM containing cyclophosphamide at initial concentrations of 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, and 400 mg / L. The mixture was incubated at 37℃ and 150 rpm. After 7 days, samples were taken to determine the residual concentration of cyclophosphamide, calculate the degradation rate, and count the number of viable bacteria to observe the strain's tolerance to cyclophosphamide. A control without bacterial suspension was set up, and both treatments and controls were replicated three times.

[0106] (5) Degradation curve determination of strain F9-1: 1.4 mL of the prepared bacterial suspension was added to MSM liquid medium with a concentration of 100 mg / L of Longcaojing and cultured at 35℃, pH=7.0 and 150 rpm. Samples were taken after 1, 2, 3, 4, 5, 6 and 7 days to determine the residual concentration of Longcaojing and calculate its degradation rate. At the same time, the OD600 value of the bacterial suspension was recorded daily.

[0107] II. Test Results

[0108] (1) The effect of pH on the degradation of flupyradifurone by strain F9-1 ​​is shown in Appendix. Figure 8 As shown in Figure A, strain F9-1 ​​effectively degraded flupyradifurone within a pH range of 5-9. After 7 days of cultivation, the degradation rates of flupyradifurone in treatments with pH values ​​of 5, 6, 7, 8, and 9 were 73.02%, 74.96%, 75.26%, 73.43%, and 72.28%, respectively. This indicates that the optimal pH range for flupyradifurone degradation by strain F9-1 ​​is 7-8. These results suggest that neutral or slightly alkaline conditions are favorable for the degradation of flupyradifurone by strain F9-1.

[0109] (2) The effect of temperature on the degradation of flupyradifurone by strain F9-1 ​​is shown in Appendix. Figure 8 As shown in Figure B, strain F9-1 ​​effectively degraded flupyradifurone at cultivation temperatures ranging from 25 to 45℃. After 7 days of cultivation, the degradation rates of flupyradifurone at cultivation temperatures of 25℃, 30℃, 35℃, 40℃, and 45℃ were 72.81%, 74.66%, 75.42%, 75.17%, and 72.40%, respectively. Therefore, 35-40℃ is the optimal temperature range for strain F9-1 ​​to degrade flupyradifurone.

[0110] (3) The effect of different initial inoculum amounts on the degradation of flupyradifurone by strain F9-1 ​​is shown in Appendix. Figure 8 As shown in Figure C, the inoculum amounts were 0.2 mL, 0.6 mL, 1.0 mL, 1.4 mL, and 1.8 mL (OD600=1.0), respectively. After 7 days of cultivation, the degradation rates of flupyradifurone were 68.41%, 69.46%, 71.63%, 69.15%, and 68.40%, respectively. This indicates that as the inoculum amount increased (0.1-1.0 mL), the degradation rate of flupyradifurone by strain F9-1 ​​gradually increased. When the inoculum amount exceeded 1.0 mL, the inoculum amount reached saturation for 100 mg / L flupyradifurone, and further increases in inoculum amount did not significantly change the degradation rate of flupyradifurone by strain F9-1.

[0111] (4) The degradation effect of strain F9-1 ​​on flupyradifurone at different initial concentrations is shown in Appendix. Figure 8 As shown in Figure D, after 7 days of cultivation, the degradation rates of flupyradifurone by strain F9-1 ​​were 57.98%, 74.66%, 70.21%, 64.92%, and 50.15% when the initial concentration of flupyradifurone was 50, 100, 200, 300, and 400 mg / L, respectively. This indicates that the degradation rate of flupyradifurone decreases with increasing initial concentration, under the same inoculum quantity. However, the viable cell count gradually decreased with increasing initial flupyradifurone concentration, reaching a maximum of 2.78 × 10⁻⁶ when the flupyradifurone concentration reached 100 mg / L. 7 When the concentration of flupyradifurone was 400 mg / L, the viable bacterial count was 1.93 × 10⁻⁶. 6 This indicates that under flupyridine-induced conditions, high concentrations of flupyridine have a certain inhibitory stress effect on the growth of the strain.

[0112] (5) Under the optimal degradation conditions of pH 7, temperature 35℃, inoculum size 3%, and initial flupyradifurone concentration 100 mg / L, the degradation curves of strain F9-1 ​​from day 1 to day 7 were plotted. See Appendix. Figure 9As shown, the strain reached 1.3 at OD600 in 0-3 days, the strain grew rapidly, and 100 mg / L fluridone was degraded to 72.24 mg / L, in 4-7 days, the strain grew slowly, the degradation rate was slow, and the fluridone residual amount was 36.29 mg / L on the 7th day.

[0113] The above-described embodiments are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only to represent selected embodiments of the application. All other embodiments obtained by relevant deduction and replacement made by those skilled in the art under the condition of the concept of the present application, without making creative labor, belong to the scope of protection of the present application.

Claims

1. A strain of Acinetobacter oryzae F9-1 that degrades pesticides, characterized in that, The Acinetobacter oryzae F9-1 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the strain preservation number is CGMCC NO. 31742. The 16S rRNA gene sequence of the Acinetobacter oryzae F9-1 strain is shown in SEQ ID NO:

1.

2. The Acinetobacter oryzae F9-1 for degrading pesticides according to claim 1 is applied in degrading pesticides.

3. The Acinetobacter oryzae F9-1 for degrading pesticides according to claim 1 is applied in degrading fluridone.

4. The Acinetobacter oryzae F9-1 for degrading pesticides according to claim 1 is applied in degrading pendimethalin.

5. A method of degrading fluridone, characterized by, The Acinetobacter oryzae F9-1 according to claim 1 is used for fermentation and degradation.

6. A method of degrading pendimethalin, characterized by, The Acinetobacter oryzae F9-1 according to claim 1 is used for fermentation and degradation.

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