New strain for degrading pesticide and application thereof

Abstract

The invention relates to a novel bacterial strain capable of degrading pesticides, in particular to a novel bacterial strain Acinetobacter oryzae F9-1, and a preparation method of the novel bacterial strain Acinetobacter oryzae F9-1. A new strain Acinetobacter oryzae F9-1 is obtained by separation, screening and identification from a soil sample in Hami region of Xinjiang, the strain is subjected to physiological and biochemical characteristic detection, the new strain F9-1 shows that the bacterial colony is small, the surface is smooth, the new strain F9-1 is white and semitransparent on an NB plate, the results of Gram staining, catalase and indole experiments and the like are positive, and the results of oxidase, V.P tests, urease and the like are negative. The strain F9-1 can hydrolyze hydrogen peroxide; l-alanine, lactic acid, L-aspartic acid, quinic acid and the like can be utilized, the strain is sensitive to 8% NaCl, lincomycin, nalidixic acid, chloramphenicol and the like, fluridone and pendimethalin can be degraded, and the strain has the advantages of being environmentally friendly, efficient, economical, sustainable and the like and is an effective means for solving the pesticide pollution problem.

Description

Technical Field

[0001] The invention relates to the technical field of microbial strains and their applications, and specifically to a new strain for degrading pesticides and its application technology. Background Art

[0002] Xinjiang is the main cotton producing area and advantage area in my country. Weeds in cotton fields are one of the main reasons affecting cotton production and output, and are an important factor restricting the sustainable development of cotton. Longcaojing, a fluridone suspension with an effective content of 42%, is a pyrrolidone soil-sealing herbicide widely used in Xinjiang cotton fields in recent years. It is usually used in combination with pendimethalin to prevent and control annual broad-leaved and grass weeds such as nightshade, quinoa and barnyard grass in cotton fields. The effective period is about 60 days, and it is safe for cotton germination and growth. The chemical name of fluridone is 1-methyl-3-phenyl-5-(3-trifluoromethylphenyl)-4(1H)-pyridone, which mainly acts on plant lycopene dehydrogenase, causing chlorophyll loss, inhibiting photosynthesis, and gradually bleaching and dying weeds, thereby playing a weed-killing role.

[0003] Since Xinjiang's cotton fields are planted with other crops in succession, the residual period of fluazifop-butyl in the soil is relatively long, and there is a certain potential risk of phytotoxicity to subsequent crops. Some research has been done at home and abroad on the safety of rotation crops after the use of fluazifop-butyl in cotton fields. Zhang Zhen et al. conducted a safety study on fluazifop-butyl on common local crops in Xinjiang and found that the safety of fluazifop-butyl on different crops was in the following order from high to low: seed squash = sweet potato > peanut > oil sunflower = corn = wheat > melon > melon > watermelon > tomato > pepper; Hill et al. conducted a safety study on fluazifop-butyl on common local subsequent crops of wheat, corn, soybean, rice, sorghum and sunflower in four irrigated cotton areas in Arkansas, USA in 2016, and the results showed that wheat was relatively sensitive; Guo Shijian et al. [5] Longcaojing treatment had no obvious phytotoxicity to seed zucchini, melon, cantaloupe, and sunflower, and had no significant impact on their yields; it had certain phytotoxicity symptoms to corn, wheat, and tomatoes. Elsie and Mohammed found that after applying flupyralidone in the soil, some of the drug residues produced would cause phytotoxicity to the corn seedlings planted subsequently, seriously affecting their growth.

[0004] According to statistics, Longcaojing has been used as a herbicide in more than 15 million mu of cotton fields in Xinjiang. The large-scale input of this pesticide may cause widespread pollution of the soil environment. Pesticides have the characteristics of bioaccumulation and biomagnification, which seriously threaten human health and agricultural and ecological security. Therefore, studying the pollution status of Longcaojing in the soil and its degradation behavior is of great significance to the sustainable development of Xinjiang agriculture. The methods of pesticide residue degradation mainly include physical degradation, chemical degradation and biological degradation. Among them, physical and chemical remediation technologies are not the best methods for repairing and controlling soil pesticide residue pollution due to their shortcomings such as the possibility of destroying the native soil ecosystem to a certain extent, large investment and secondary pollution. Bioremediation is considered to be a method of degrading toxic pollutants using active microbial cells or their enzymes. It has the advantages of being economical, efficient and green, and is increasingly used to degrade pesticide residues, gradually becoming a potential alternative to traditional technologies.

[0005] Domestic and foreign researchers have conducted extensive research on the degradation of pesticide residues by microorganisms. Studies have found that microorganisms capable of degrading pesticide residues are widely distributed in bacteria, including Pseudomonas ( Pseudonomonas ), Arthrobacter ( Arthrobacter ), Bacillus ( Bacillus) , Candida tropicalis ( Candida tropicalis ), Corynebacterium ( Corynebacterium ), Coleoptera Monospora ( Sphingonomas) , Burkholder ( Burkholderiales ), Rhodococcus ( Rhodococcus ruber) etc., and some fungi (Fungus Mortierella) also have the ability to degrade pesticide residues. Liang B et al.

[20] Isolated from enrichment cultures of soil samples Brevundimonas sp. After 5 days of cultivation in a flask, the strain degraded about 80% of 50 mg / L lactofenazole. The strain HME-24 isolated by Hu et al. had a degradation rate of 96.7% for 50 mg / L lactofenazole within 72 h. Saez et al.

[22] Studies have demonstrated the degradation effect of Streptomyces on lindane in soil.

[0006] At present, there are relatively few studies on the degradation of flupyralid residues. Yan Na studied the degradation behavior of flupyralid in soil and environment and found that the photolysis of flupyralid 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 will increase the number of microbial populations and make them more active, thereby accelerating the degradation rate of pesticides in soil. Wickham studied the degradation of flupyralid in water and sediments under different intensities of ultraviolet rays and temperatures. The results showed that the greater the intensity of ultraviolet rays, the faster the degradation of flupyralid in water, but the effect of ultraviolet exposure on the degradation of flupyralid in sediments was weak, and the degradation of flupyralid slowed down at low temperatures. Summary of the invention

[0007] The present invention aims to enrich the bacterial strain resources of pesticide-degrading microorganisms in my country. The present invention aims to screen efficient pesticide-degrading strains from cotton field soils that have been long-term applied with Longcaojing, and to study their degradation characteristics, in order to provide a theoretical basis for the treatment and restoration of pesticide-contaminated soils, and to provide a new strain for degrading pesticides, specifically a new strain Acinetobacter oryzae F9-1 The invention separates and screens new bacteria from soil samples in Xinjiang County. Acinetobacter oryzae F9-1 The strain was tested for physiological and biochemical characteristics. On the NB plate, the colonies were small, smooth, and white and translucent. The results of Gram staining, catalase, and indole tests were positive, while the results of oxidase, VP test, and urease were negative. The strain F9-1 ​​can hydrolyze hydrogen peroxide; it can utilize L-alanine, lactic acid, L-aspartic acid, quinic acid, etc. It is sensitive to 8% NaCl, lincomycin, nalidixic acid, chloramphenicol, etc., and can degrade fluazifop-butyl and pendimethalin. It 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 invention is implemented through the following technical solutions.

[0009] The present invention provides a new bacterium Acinetobacter oryzae F9-1, Acinetobacter oryzae The F9-1 ​​strain is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the strain collection number CGMCCNO.31742.

[0010] In some parts of this application, the "new bacteria" Acinetobacter oryzae F9-1" is referred to as " Acinetobacter oryzae F9-1" or "strain F9-1" The novel bacteria provided by the present invention Acinetobacter oryzae F9-1It was isolated and screened from soil samples in the new area. The 16SrDNA gene sequence phylogenetic analysis and morphological analysis showed that strain F9-1 ​​belongs to Acinetobacter milleri ( Acinetobacter oryzae.sp ), named Acinetobacter oryzae F9-1. The strain gene was sequenced and the obtained sequence was subjected to BLAST comparison analysis on the NCBI website. The 16S rDNA gene sequence of strain F9-1 ​​was Acinetobacter oryzae B23 (GU954428) has the highest homology, with a similarity of 98.27%. In the phylogenetic tree constructed with the 16Sr RNA gene sequence, the 16Sr RNA sequence of strain F9-1 ​​is similar to Acinetobacter oryzae The strains B23 (GU954428) and B23 (GU954428) are the closest relatives, but they are not on the same branch, indicating that the strain F9-1 ​​has a very high support rate as a new strain and has excellent stability in the evolutionary tree. The comprehensive determination of the similarity and homology of the strains confirms that the obtained Acinetobacter The strain number within the genus is F9-1, which is a typical new strain.

[0011] The present invention provides a new strain of bacteria Acinetobacter oryzae F9-1 was identified at the molecular level by the above-mentioned bacterial species system well known in the art, combined with morphological identification analysis, and the present invention relates to a new bacterium Acinetobacter oryzae F9-1 has a small colony, a smooth surface, and is white and translucent. The results of Gram staining, catalase, and indole tests are positive, and the results of oxidase, VP test, and urease are negative. Strain F9-1 ​​cannot hydrolyze starch, but can hydrolyze hydrogen peroxide; it can utilize L-alanine, lactic acid, L-aspartic acid, quinic acid, etc., but cannot utilize sucrose, D-fructose, gelatin, etc.; it is sensitive to 8% NaCl, lincomycin, nalidixic acid, chloramphenicol, etc., but insensitive to troleandomycin, tetrazolium violet, and minocycline. By comparing the strains, it is different from the neighboring strains. Acinetobacter oryzae There are clear differences in B23.

[0012] The above strains were identified, and the strains were identified at the molecular level and morphologically verified by the well-known strain system in the art. The strain number was F9-1. Acinetobacter oryzae A typical new species in the category Acinetobacter oryzae F9-1, the strain has been deposited in the Budapest Treaty International Microbiological Depository: China General Microbiological Culture Collection Center (CGMCC) before the application date. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Deposit Number: CGMCC NO.31742, Deposit Date: August 26, 2024.

[0013] The new bacteria Acinetobacter oryzaeThe gene sequence of F9-1 ​​is shown in SEQ ID NO:1.

[0014] The novel bacteria of the present invention Acinetobacter oryzae The isolation medium of F9-1 ​​is: liquid medium with 50-300 mg / L flupyridone MSM, and the medium is composed of 0.2 g MgSO4·7H2O; 0.1 g K2HPO4; (NH 4）2 SO4 0.1g; CaSO4 0.04g; FeSO4·7H2O 0.001g; deionized water 1L; pH 7.3.

[0015] The novel bacteria of the present invention Acinetobacter oryzae The purification medium of F9-1 ​​is: MSM solid plate with 50 mg / L pendimethalin as the sole carbon source, and the culture medium is MgSO4·7H2O 0.2 g; K2HPO4 0.1 g; (NH 4）2 SO4 0.1g; CaSO4 0.04g; FeSO4·7H2O 0.001g; deionized water 1L; pH 7.3.

[0016] At the same time, a new bacterium provided by the present invention Acinetobacter oryzae The preparation method of F9-1 ​​fermentation broth is prepared by the following steps: washing the activated new bacteria with sterile water Acinetobacter oryzae The spores on the F9-1 ​​plate were used to obtain a spore suspension, which was inoculated into a fermentation medium at a volume ratio of 5%, and fermented at 25°C and 150 rpm for 7 days to obtain a fermentation broth of strain F9-1.

[0017] Furthermore, the present invention provides a new bacterium Acinetobacter oryzae Application of F9-1 ​​in degradation of pesticides.

[0018] The new bacteria Acinetobacter oryzae Application of F9-1 ​​in the degradation of fluazifop-butyl.

[0019] The new bacteria Acinetobacter oryzae Application of F9-1 ​​in the degradation of pendimethalin.

[0020] By implementing the above specific technical solutions provided by the present invention and implementing the content of the present invention, the following beneficial effects can be obtained: (1) The present invention provides a new bacterium that has been scientifically identified and can degrade pesticides Acinetobacter oryzae F9-1 A new strain of bacteria was obtained by isolating, screening and identifying soil samples from Hami, Xinjiang. Acinetobacter oryzae F9-1The strain was tested for physiological and biochemical characteristics. The new strain F9-1 ​​showed small colonies, smooth surface, white and translucent on the NB plate. The results of Gram staining, catalase, indole test, etc. were positive, and the results of oxidase, VP test, urease, etc. were negative. The strain F9-1 ​​can hydrolyze hydrogen peroxide; it can use L-alanine, lactic acid, L-aspartic acid, quinic acid, etc., and is sensitive to 8% NaCl, lincomycin, nalidixic acid, chloramphenicol, etc., and can degrade flupyralidone and pendimethalin. It has the advantages of environmental protection, high efficiency, economy and sustainability, and is an effective means to solve the problem of pesticide pollution.

[0021] (2) By applying the new bacteria provided in this application Acinetobacter oryzae F9-1, under the optimal degradation conditions of pH 7, temperature 35°C, inoculation amount 3%, and initial concentration of fluazifop-butyl 100 mg / L, the OD600 of the strain reached 1.3 on day 0-3, and the strain grew rapidly, and 100 mg / L fluazifop-butyl could be degraded to 72.24 mg / L. On day 4-7, the strain grew slowly and the degradation rate was slow. On day 7, the residual amount of fluazifop-butyl was 36.29 mg / L, indicating that the new strain provided by the present application Acinetobacter oryzae F9-1 can effectively degrade fluazifop-butyl. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is the growth of the degrading strain on the pendimethalin plate.

[0023] Figure A shows the culture medium of the degradation strain; Figure B shows the growth of the degradation strain in different types of culture medium.

[0024] Figure 2 Shown is the liquid phase diagram of flupyralidone standard.

[0025] Figure 3 Shown is a standard curve for flupyralidone.

[0026] Figure 4 Shown are the UV-visible scanning spectrum and standard curve of pendimethalin; Figure A shows the UV-visible scanning spectrum of pendimethalin; Figure B shows the standard curve of pendimethalin.

[0027] Figure 5 New bacteria shown Acinetobacter oryzae F9-1 colony morphology and Gram staining.

[0028] Figure A shows a new bacterium Acinetobacter oryzae F9-1 Colony morphology; Figure B shows a new bacterium Acinetobacter oryzae F9-1 Gram staining image.

[0029] Figure 6New bacteria shown Acinetobacter oryzae Figure 16S rDNA identification results of F9-1.

[0030] Figure A shows a new bacterium Acinetobacter oryzae The results of 16S rDNA electrophoresis of F9-1; Figure B shows the new strain Acinetobacter oryzae Phylogenetic tree of F9-1 ​​constructed based on 16S rDNA sequence.

[0031] Figure 7 New bacteria shown Acinetobacter oryzae F9-1 growth curve chart.

[0032] Figure 8 New bacteria shown Acinetobacter oryzae F9-1 degradation characteristics diagram.

[0033] Figure A shows the effect of pH on the degradation of Longcaojing by strain F9-1; Figure B shows the effect of temperature on the degradation of Longcaojing by strain F9-1; Figure C shows the effect of inoculation size on the degradation of Longcaojing by strain F9-1; Figure D shows the effect of initial concentration on the degradation of Longcaojing by strain F9-1.

[0034] Figure 9 New bacteria shown Acinetobacter oryzae F9-1 degradation curve. DETAILED DESCRIPTION

[0035] The present invention is described below with reference to the following examples, but the present invention is not limited to the following examples. All raw and auxiliary materials used in the present invention, as well as the selected bacterial strain culture methods are well known in the art, and the % involved in the present invention are all mass volume percentages, unless otherwise specified.

[0036] In order to better explain the present invention, the main content of the present invention is further explained in conjunction with specific examples below, but the content of the present invention is not limited to the following examples. If not otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the fluazifop standard (98.7%), chloranil technical (42%), 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 this application are all commercially available products.

[0037] The NA culture medium of the present invention comprises: 10.0 g of 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.

[0038] The KB culture medium of the present invention comprises: 20.0 g of hydrolyzed peptone; 1.5 g of dipotassium hydrogen phosphate; 1.5 g of magnesium sulfate; 15.0 g of agar; 1 L of deionized water; and pH 7.3.

[0039] The actinomycete culture medium of the present invention adopts: 1.0g potassium nitrate; 0.5g potassium dihydrogen phosphate; 0.1g magnesium sulfate; 0.01g ferrous sulfate; 0.5g sodium chloride; 20.0g soluble starch; 15.0g agar; 1L deionized water; pH 7.3.

[0040] The R2A culture medium of the present invention adopts: 0.25g of tryptone; 0.5g of acid hydrolyzed casein; 0.5g of yeast extract powder; 0.5g of soluble starch; 0.3g of dipotassium hydrogen phosphate; 0.1g of magnesium sulfate; 0.3g of sodium pyruvate; 12.0g of agar; 0.25g of peptone; 0.5g of glucose; 1L of deionized water; and pH 7.3.

[0041] The TSA culture medium of the present invention adopts: 15.0% tryptone, 5.0% soytone, 30.0% sodium chloride, 15.0% agar, 1L deionized water, and pH 7.3.

[0042] The rose Bengal culture medium of the present invention adopts: 5.0g of peptone, 10.0g of glucose, 1.0g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 20.0g of agar, 0.033g of rose Bengal, 0.1g of chloramphenicol, 1L of deionized water, and pH 7.3.

[0043] Example 1: Screening of flupyralidone-degrading strains 1. Materials and Methods 1. Test materials (1) Test soil Soil was collected from cotton fields where the herbicide Longcaojing was applied all year round. The five-point sampling method was used. The soil samples were collected at a depth of 10-20 cm, air-dried, ground and sieved through an 850 μm sieve, and stored at 4 ℃ for later use.

[0044] Table 1: Sample collection

[0045] (2) Experimental drugs Flupyralid standard (98.7%), chlorpyrifos technical (42%), 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.

[0046] 2. Test methods 1. Enrichment and domestication of flupyralidone-degrading strains Add 200 mL of MSM liquid culture medium to a 500 mL conical flask and sterilize it with wet heat. Add 50 mg / L flupyridone and 10 g of soil sample, and culture in a shaker at 30 ℃ and 200 r / min for 7 days; take 5 mL of the first culture solution and add it to the MSM liquid culture medium with 100 mg / L flupyridone, and culture it in a shaker at 30 ℃ and 200 r / min for 7 days; take 5 mL of the second culture solution and add it to the MSM liquid culture medium with 150 mg / L flupyridone, and culture it in a shaker at 30 ℃ and 200 r / min for 7 days, and so on until the flupyridone concentration reaches 300 mg / L. Take 1 mL of the degraded bacterial solution after transfer and dilute it to 10 in a 1.5 mL pipette. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 Spread on NA, KB, TAS, R2A, improved Gao's No. 1, and Bengal red culture media, inverted in a 30℃ constant temperature incubator and culture for 48 hours. After the colonies grow, pick single colonies with consistent color and morphology for purification and culture on selective MSM culture medium.

[0047] 2. Rescreening of flupyralidone-degrading strains The enriched and domesticated strains were inoculated on MSM solid plates with 50 mg / L pendimethalin as the sole carbon source, cultured at 30°C for 48 hours, and the growth was observed to select strains with good growth. The selected strains were inoculated in MSM liquid medium with 50 mg / L pendimethalin as the sole carbon source, cultured at 30°C, 200 r / min for 5 days, and the supernatant was taken to detect the residual pendimethalin content, and strains with the ability to degrade pendimethalin were screened.

[0048] The bacterial strains capable of degrading pendimethalin were placed in a culture medium with 100 mg / L fluazifop as the sole carbon source at 30°C and 200 r / min. After culturing for 24 hours, the supernatant was taken to detect the residual amount of fluazifop, and the strains with good degradation effect were selected as the subsequent research objects.

[0049] 3. Results and Analysis 1. Screening of flupyralidone-degrading strains The bacterial solution obtained after enrichment and domestication was diluted and coated on different screening plates, and single colonies with different colony morphology, size, color, etc. were selected on the MSM solid medium containing 200 mg / L flupyridone, and 163 degrading bacteria were obtained. The strains were 66 strains on NA plates, 21 strains on TSA plates, 16 strains on R2A plates, 17 strains on KB plates, 41 strains on improved Gao's No. 1 plates, and 2 strains on Bengal red plates.

[0050] Table 2: Results of enrichment and acclimatization of flupyralidone-degrading strains

[0051] 2. Rescreening of flupyralidone-degrading strains The 163 strains of different degradation bacteria obtained by enrichment and domestication were placed in MSM solid containing 50 mg / L pendimethalin as substrate, and 53 strains with good growth conditions were obtained. Among them, there were 20 strains from NA plates, 2 strains from TAS, 1 strain from R2A, 11 strains from KB, 18 strains from improved Gao's No. 1, and 2 strains from Bengal Red. The growth of related strains on plates and the growth of strains in different culture media are shown in the attached Figure 1 As shown in Figures A and B.

[0052] Example 2: Identification of degradation strains 1. Test methods 1. Identification of degradation strains Morphological identification: Inoculate the strain into a solid plate, draw a line, and culture at 37°C for 24-48 hours. Pick out a single colony, spread it evenly on a slide, perform Gram staining, and observe under a microscope. Physiology and biochemistry: refer to the Manual of Identification of Common Bacterial Systems and Bergey's Manual of Systematic Bacteriology to conduct preliminary identification of isolated strains; Molecular biological identification: The genomic DNA of the strain was extracted using the kit of Shanghai Bioengineering Company as a template, and the universal primers of 16SrDNA were used for PCR amplification reaction. The universal primers are: 27F (5′-AGAGTTT GATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACG ACTT-3′). The 25μL system is: 0.5μL template, 0.5μL primers each, 12.5μL 2×Taq PCR Mix premix, 11μL ddH2O.

[0053] PCR reaction conditions: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, for a total of 30 cycles; and finally 72℃ extension for 10min. After the reaction, 1.2% agarose gel electrophoresis was used to detect the PCR reaction products. After the size of the amplified fragment was detected by agarose gel electrophoresis, the sequencing work was completed by Shanghai Bioengineering Company. 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 using the neighbor-joining method in MEGA7.0.

[0054] 2. Detection of Flupyralidone Preparation of standard solution: Accurately weigh 50.00±0.10mg of flupyralidone standard in a 50mL volumetric flask and dissolve it in acetonitrile to obtain a 1000μg / mL standard stock solution. Dilute the 1000μg / mL standard solution to prepare 40, 80, 120, 160, and 200μg / mL standard solutions. Perform HPLC determination on the standard working solution, and perform regression analysis on the corresponding concentration of the flupyralidone chromatographic peak area of ​​the standard working solution to obtain a standard curve; Detection of fluazifop-butyl: take 5 mL of degradation bacterial solution, centrifuge at 8000 rpm for 10 min, take the supernatant, add 5 mL of acetonitrile, and vortex extract for 10 min; then add 2 g of sodium chloride, vortex vigorously for 1 min, and centrifuge at 4000 r / min for 5 min; take the supernatant obtained above and pass it through an organic filter membrane with a pore size of 0.22 μm to obtain a sample solution; the obtained sample solution is subjected to HPLC determination, the chromatographic peak area of ​​fluazifop-butyl in the sample solution is measured, and the concentration and content of fluazifop-butyl in the sample solution are calculated by substituting it into the standard curve of the step. HPLC conditions: Chromatographic column: 250 mm × 4.6 mm (id) stainless steel column, filled with Lichrospher C18, 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; 3. Detection of Pendimethalin Preparation of standard curve: Take pendimethalin standard and dissolve it in dichloromethane to prepare a solution with a mass concentration of 50 mg / L, and perform full wavelength scanning analysis to determine the maximum absorption peak. Set 9 mass concentration gradients, namely 10, 20, 30, 40, and 50 mg / L, and use ultraviolet spectrophotometry to measure the absorbance of solutions with different mass concentrations. Draw a standard curve with pendimethalin mass concentration as the horizontal axis and absorbance as the vertical axis, and find the equation of the standard curve.

[0055] Determination of pendimethalin: Add an equal volume of dichloromethane to the pendimethalin degradation culture medium to be tested, shake vigorously for 5 minutes to mix thoroughly, let stand until the aqueous phase and the organic phase are completely separated, remove the upper aqueous phase, absorb the lower organic phase, add excess anhydrous sodium sulfate to remove residual water, use a UV-visible spectrophotometer to scan the wavelength range of 200-600 nm to determine the wavelength absorption curve of pendimethalin, and determine the residual amount of pendimethalin and the degradation of pendimethalin in the culture medium based on the changes in the ultraviolet and visible light characteristic absorption peaks of pendimethalin.

[0056] 2. Experimental Results (1) Flupyralidone standard test Under the chromatographic conditions, the typical HPLC chromatogram of flupyralidone standard is shown in the attached Figure 2 As shown in the figure, the retention time is 2.843min, which is slightly different from the retention time of 6.17min in the literature. After measuring the peak height of the standard sample with different concentrations at 310nm, the standard curve of flupyralidone is drawn. See the attached Figure 3 As shown, the standard curve has good linear correlation, with a correlation coefficient of 0.998, and can be used to detect the residual content of fluazifop-butyl in the degradation bacterial solution.

[0057] (2) Pendimethalin standard test UV-visible light scanning was used to detect the standard product of pendimethalin. It was found that within the wavelength range of 200-600nm, there was a characteristic absorption peak of pendimethalin at a wavelength of 430nm. The absorbance of the standard product of different concentrations was measured at 430nm to obtain the standard curve of pendimethalin. See the attached Figure 4 As shown, the linear correlation coefficient is 0.999, which has a good correlation and can be used to detect the residual pendimethalin content in the degradation bacterial solution.

[0058] After the degradation ability test of pendimethalin and flupyralidone, 8 strains with tolerance and effective degradation were finally obtained, as shown in Table 3. Among them, F9-1 ​​could degrade 50 mg / L pendimethalin by 82.43% within 7 days and 100 mg / L flupyralidone by 15.64% within 24 hours, and this strain was selected for the next experiment.

[0059] Table 3: Flupyralidone screening strains

[0060] (3) Morphological and physiological and biochemical characteristics of strain F9-1 The degradation strain F9-1 ​​showed small colonies, smooth surface, white and translucent on the NB plate. The results of Gram staining, catalase, indole test, etc. were positive, and the results of oxidase, VP test, urease, etc. were negative. The strain F9-1 ​​cannot hydrolyze starch, but can hydrolyze hydrogen peroxide; it can utilize L-alanine, lactic acid, L-aspartic acid, quinic acid, etc., but cannot utilize sucrose, D-fructose, gelatin, etc.; it is sensitive to 8% NaCl, lincomycin, nalidixic acid, chloramphenicol, etc., but insensitive to troleandomycin, tetrazolium violet, minocycline, etc. The test results are shown in the attached Figure 5 As shown and recorded in Table 5.

[0061] Table 5: Physiological and biochemical characteristics of strain F9-1

[0062] (4) 16S rDNA sequence analysis of strain F9-1 The genomic DNA of strain F9-1 ​​was extracted using the Shanghai Biotech kit, and the bacterial 16S rDNA universal primers were used to amplify the bacterial target DNA fragment. The agarose gel electrophoresis of the PCR product is shown in the attached Figure 6 As shown in Figure A, the target DNA fragment is about 1500 bp in size. After sequencing the 16S rDNA gene fragment of strain F9-1, the sequence was entered into the NCBI database for comparison, and the sequences with high similarity were selected for phylogenetic tree construction. Acinetobacter oryzae B23 (GU954428) has the highest homology, with a similarity of 96.67%, see Appendix Figure 6 As shown in Figure B. Based on the morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence comparison analysis of the isolated strain F9-1, the isolated strain F9-1 ​​was identified as Acinetobacter milleri ( Acinetobacter oryzae .sp), named Acinetobacter oryzae F9-1. (5) Sequence determination The PCR amplification product was sequenced after electrophoresis detection and purification. The sequence length was 1392 bp. The sequencing result is shown in SEQ ID No: 1. BLAST homologous sequence search was performed on NCBI. The phylogenetic tree was established by the Neighbor-Joining method using MEGA 7.0 software commonly used in this field (repeated sampling 1000 times). The results are attached. Figure 6 As shown in Figure B, the obtained sequence was compared and analyzed on the NCBI website and it was found that the 16S rRNA gene sequence of strain F9-1 ​​was Acinetobacter oryzae B23 (GU954428) had the highest homology, with a similarity of 96.67%. In the phylogenetic tree constructed with 16Sr RNA gene sequences, the 16Sr RNA sequence of strain F9-1 ​​was similar to Acinetobacter oryzae The strains B23 (GU954428) and B23 (GU954428) are the closest relatives, but they are not on the same branch, indicating that the strain F9-1 ​​has a very high support rate as a new strain and has excellent stability in the evolutionary tree. The comprehensive determination of the similarity and homology of the strains confirms that the obtained Acinetobacter The strain number within the genus is F9-1, which is a typical new strain.

[0063] (6) Identification of growth characteristics The growth of strain F9-1 ​​at different time points is shown in the attached Figure 7As shown in the figure, strain F9-1 ​​grew slowly from 0 to 8 h, grew rapidly from 8 to 18 h, and tended to grow slowly from 18 to 48 h. The growth reached its maximum at 20 h of cultivation. The growth of strain F9-1 ​​was "S" shaped, and the inflection point of the "S" shape appeared at 12 h, indicating that the strain had the highest growth rate at this time.

[0064] Example 3: Study on bacterial degradation characteristics 1. Test methods (1) Effect of pH on the degradation of chloranil by strain F9-1: 0.3 mL of bacterial suspension of the degrading bacteria was added to 20 mL of MSM liquid culture medium with different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0) containing 100 mg / L chloranil, and cultured in a shaker at 30°C and 150 rpm. After 7 days, samples were taken to determine the residual concentration of chloranil and calculate its degradation rate. A control without bacteria was set up at the same time, and the treatment and control were repeated 3 times.

[0065] (2) Effect of temperature on the degradation of chloranil by strain F9-1: 0.3 mL of bacterial suspension of the degrading bacteria was added to 20 mL of MSM liquid culture medium with a chloranil concentration of 100 mg / L. The culture was shaken at 150 rpm at different temperatures of 25°C, 30°C, 35°C, 40°C, and 45°C. After 7 days, samples were taken to determine the residual concentration of chloranil and calculate its degradation rate. A control without bacteria was set up at the same time. Both the treatment and the control were repeated 3 times.

[0066] (3) Effect of initial inoculum amount on the degradation of chloranil 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 the MSM liquid culture medium with a chloranil concentration of 100 mg / L, respectively, so that the culture system was 20 mL. The culture was carried out at 37 ° C and 150 rpm. After 7 days, samples were taken to determine the residual concentration of chloranil and calculate its degradation rate. At the same time, a control without bacteria was set up, and the treatment and control were repeated 3 times.

[0067] (4) Effect of initial concentration on the degradation of chloranil by strain F9-1: 0.3 mL of bacterial suspension of the degrading bacteria was cultured at 37°C and 150 rpm in 20 mL MSM with initial chloranil concentrations of 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, and 400 mg / L-1, and the residual concentration of chloranil was measured after 7 days, and its degradation rate was calculated. At the same time, the number of live bacteria was counted to observe the strain's tolerance to chloranil. A control without bacteria was set up, and the treatment and control were repeated 3 times.

[0068] (5) Degradation curve of strain F9-1: Add 1.4 mL of the prepared bacterial suspension to the MSM liquid culture medium with a concentration of 100 mg / L of chloranthate, and culture at 35°C, pH = 7.0, and 150 rpm. Take samples after 1, 2, 3, 4, 5, 6, and 7 days to determine the residual concentration of chloranthate, calculate its degradation rate, and record the OD600 value of the bacterial liquid every day.

[0069] 2. Test results (1) Effect of pH on the degradation of flupyralid by strain F9-1 Figure 8 As shown in Figure A. In the pH range of 5-9, strain F9-1 ​​can effectively degrade flupyralidone. After 7 days of cultivation, the degradation rates of flupyralidone in the treatments of pH 5, 6, 7, 8, and 9 were 73.02%, 74.96%, 75.26%, 73.43%, and 72.28%, respectively. It can be seen that the suitable pH value for strain F9-1 ​​to degrade flupyralidone is 7-8. These results show that neutral or weak alkalinity is conducive to the degradation of flupyralidone by strain F9-1. (2) The effect of temperature on the degradation of flupyralid by strain F9-1 ​​is shown in the attached Figure 8 As shown in Figure B. The strain F9-1 ​​can effectively degrade flupyralid at a culture temperature of 25-45°C. After 7 days of culture, the degradation rates of flupyralid were 72.81%, 74.66%, 75.42%, 75.17% and 72.40% at culture temperatures of 25°C, 30°C, 35°C, 40°C and 45°C, respectively. It can be seen that 35-40°C is the optimal temperature for strain F9-1 ​​to degrade flupyralid.

[0070] (3) The effect of different initial inoculum doses on the degradation of flupyralid by strain F9-1 ​​is shown in the attached 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 culture, the degradation rates of flupyralidone were 68.41%, 69.46%, 71.63%, 69.15%, and 68.40%, respectively; this indicated that with the increase of the inoculum amount (0.1-1.0 mL), the degradation rate of flupyralidone by strain F9-1 ​​gradually increased; when the inoculum amount exceeded 1.0 mL, the bacterial amount had reached saturation for 100 mg / L flupyralidone, and the degradation rate of flupyralidone by strain F9-1 ​​did not change significantly with further increase of the inoculum amount.

[0071] (4) The degradation effect of strain F9-1 ​​on different initial concentrations of fluazifop-butyl is shown in the attached Figure 8As shown in Figure D. When the initial concentrations of fluazifop-butyl were 50, 100, 200, 300 and 400 mg / L, the degradation rates of fluazifop-butyl by strain F9-1 ​​were 57.98%, 74.66%, 70.21%, 64.92% and 50.15% respectively after 7 days of cultivation. This indicates that with the increase of initial concentration, the degradation rate of fluazifop-butyl decreased under the same inoculum amount. However, with the increase of initial concentration of fluazifop-butyl, the number of viable bacteria gradually decreased, and the maximum number of viable bacteria was 2.78×10- 7 , while when the concentration of flupyralidone was 400 mg / L, the number of viable bacteria was 1.93×10 6 This indicates that under the induction conditions of fluazifop-butyl, high concentration conditions have a certain inhibitory stress effect on the growth of the strain.

[0072] (5) Under the optimal degradation conditions of pH 7, temperature 35°C, inoculation size 3%, and initial concentration of flupyralidone 100 mg / L, the degradation curve of strain F9-1 ​​from 1 to 7 days was drawn. See Appendix. Fig. 9 As shown, the OD600 of the strain reached 1.3 on days 0-3, and the strain grew rapidly, degrading 100 mg / L fluazifop to 72.24 mg / L. On days 4-7, the strain grew slowly and the degradation rate was slow, with the residual amount of fluazifop being 36.29 mg / L on the 7th day.

[0073] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A new strain of bacteria that degrades pesticides Acinetobacter oryzae F9-1, characterized in that, Acinetobacter oryzae F9-1 is deposited in China General Microbiological Culture Collection Center (CGMCC) with the culture collection number of CGMCCNO.31742. The gene sequence of the Acinetobacter oryzae F9-1 ​​strain is shown in SEQ ID NO:

1.

2. Used to isolate a new bacterium that degrades pesticides as claimed in claim 1 Acinetobacter oryzae The culture medium of F9-1 ​​is characterized in that The separation medium is: a liquid medium with 50-300 mg / L flupyridone MSM added, and the medium uses 0.2 g MgSO4·7H2O; 0.1 g K2HPO4; (NH 4）2 SO4 0.1g; CaSO4 0.04g; FeSO4·7H2O0.001g; deionized water 1L; pH 7.

3.

3. Used for purifying the novel bacteria for degrading pesticides as claimed in claim 1 Acinetobacter oryzae The culture medium of F9-1 ​​is characterized in that The purification medium is: 50 mg / L pendimethalin as the sole carbon source on an MSM solid plate, the culture medium uses MgSO4·7H2O 0.2 g; K2HPO4 0.1 g; (NH 4）2 SO4 0.1g; CaSO4 0.04g; FeSO4·7H2O 0.001g; deionized water 1L; pH 7.

3.

4. The novel bacteria for degrading pesticides as claimed in claim 1 Acinetobacter oryzae F9-Application in degradation of pesticides.

5. The novel bacteria for degrading pesticides as claimed in claim 1 Acinetobacter oryzae F9-Application in the degradation of fluazifop-butyl.

6. The novel bacteria for degrading pesticides as claimed in claim 1 Acinetobacter oryzae F9-Application in the degradation of pendimethalin.

7. Or the method for isolating the new bacterium for degrading pesticides as claimed in claim 1 according to claim 2 Acinetobacter oryzae Application of F9-1 ​​culture medium in degradation of pesticides.

8. The novel bacterium for purifying the pesticide degradation bacterium according to claim 1 as claimed in claim 3 Acinetobacter oryzae Application of F9-1 ​​culture medium in degradation of pesticides.

9. A method for degrading flupyridamole, characterized in that: Using a new bacterium as claimed in claim 1 Acinetobacter oryzae F9-1 was subjected to fermentation degradation.

10. A method for degrading pendimethalin, characterized in that: Using a new bacterium as claimed in claim 1 Acinetobacter oryzae F9-1 was subjected to fermentation degradation.

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