Arthrobacter sp. And application thereof

Abstract

The invention discloses a strain of pseudoarthrobacter sp. F13, and the preservation number of the pseudoarthrobacter sp. F13 in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M 20241866; the strain F13 is applied to degradation of procymidone, the result shows that the pseudoarthrobacter F13 can efficiently degrade procymidone, germplasm resources and diversity of microbial degradation strains are enriched, and a new way is provided for treatment of pesticide residue pollution.

Description

Technical Field

[0001] The invention belongs to the field of environmental treatment and microbial technology, and specifically relates to a strain capable of efficiently degrading a procymidone fungicide and application thereof. Background Art

[0002] Dicarboximide fungicides such as procymidone have the advantages of high efficiency, low toxicity and broad-spectrum antibacterial, and have been widely used in agriculture, such as tomatoes, wheat and celery. However, long-term and large-dose application has caused its residues in the environment and food, and its impact on human health has attracted attention in recent years.

[0003] At present, most studies at home and abroad focus on residue detection in crops and soil environments, while there are few studies on the biodegradation of procymidone. Jin Huding and others isolated a strain that can promote the degradation of procymidone in a greenhouse where vegetables are grown and pesticides are sprayed. Methylobacterium sp. T32-1, and verified the degradation performance. It was found that after the strain was added with the aqueous solution of procymidone, the procymidone was gradually degraded, and the degradation rate reached 77.2% on the 14th day; Zhang et al. Providencia stuartii JD and Brevundimonas naejangsanensis The co-culture of two bacteria J3 significantly improved the biodegradation efficiency of fusaritolide in brown soil, but the mixed bacteria model has the potential risk of unstable effects.

[0004] It is known that Pseudoarthrobacter has a certain potential in the remediation of environmental pollution. Pseudoarthrobacter strains can degrade several phenolic compounds, including polycyclic aromatic hydrocarbons (PAHs), phenol, 2,4,6-trinitrotoluene (TNT), crude oil and various benzene compounds, phthalates (PAEs). At present, the application of Pseudoarthrobacter strains in the degradation of dicarboximide fungicides has not been reported. Summary of the invention

[0005] The present invention provides a pseudoarthrobacter ( Pseudarthrobacter sp.) F13, which was deposited in the China Center for Type Culture Collection on August 28, 2024, with the deposit number CCTCC NO: M 20241866, and the deposit address is Wuhan University, Wuhan, China.

[0006] Another object of the present invention is to treat the above-mentioned Pseudomonas aeruginosa ( Pseudarthrobacter sp.) F13 is used in the degradation of carbendazim.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions: 1. The soil collected from the vegetable greenhouse where pesticides are applied all year round was crushed and sieved. The sieved soil was added to the MSM liquid culture medium containing 50 mg / L procymidone. After constant temperature shaking culture at 37°C and 150 rpm for 8 days, it was allowed to stand. The supernatant culture solution was transferred to the newly sterilized MSM liquid culture medium containing 100 mg / L procymidone. The domestication and enrichment culture was continued at 37°C and 150 rpm. After enrichment and subculture for 4-8 times, the last enrichment culture solution was taken for gradient dilution and applied to the liquid culture medium containing 50 mg / L procymidone. The strain F13 was obtained by inoculating the obtained purified colonies into an MSM liquid medium containing 50 mg / L procymidone, culturing at 37°C until a single colony with regular colonies grew out, and then streaking the single colony on an MSM solid medium containing 50 mg / L procymidone to obtain a purified strain; the obtained purified colonies were inoculated into an MSM liquid medium containing 50 mg / L procymidone, and cultured at 37°C and 150 rpm for 6 days, and the culture fluid was collected. The degradation ability of the strain on procymidone was detected by high performance liquid chromatography (HPLC), and finally the strain F13 was obtained; 2. Identification of strain F13 (1) Morphological characteristics of strain F13: On LB solid medium, F13 is round, with a smooth, slightly moist surface, regular edges and a bulge in the center; Gram staining shows that it is a Gram-negative bacterium, and the cells are round and short rods under a microscope; (2) Molecular identification The genomic DNA of strain F13 was extracted by CATB method, and the extracted genome was used as a template for PCR amplification using 16S rDNA bacterial universal primers. After sequencing the PCR product, the sequencing results were compared with the sequences on NCBI. Combined with the morphological characteristics and molecular identification results, the strain was finally identified as a strain of the genus Pseudoarthrobacter ( Pseudarthrobacter sp.), named Pseudarthrobacter sp. F13; 3. Pseudomonas aeruginosa F13 was used to degrade procymidone. The experimental results showed that the degradation rate of procymidone by this strain reached 97.0%; the optimal degradation conditions of strain F13 were temperature 30℃ and pH 9.0.

[0008] Advantages and technical effects of the present invention: The present invention adopts field soil from a vegetable-growing area where pesticides have been applied for a long time and the efficacy of pesticides has been reduced, and obtains a microbial flora with the ability to degrade procymidone through continuous liquid propagation and enrichment and domestication. The cultivable microorganisms in the enriched flora are separated and purified, and the degradation ability is determined to obtain a strain F13 with the ability to degrade procymidone. The strain has great application value in the bioremediation of water and soil contaminated by procymidone, and provides a new approach to the control of pesticide residue pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1Schematic diagram of the colony morphology (left) and Gram staining (right) results of strain F13; Figure 2 is the phylogenetic tree of strain F13; Figure 3 is the growth curve of strain F13; Figure 4 The effect of culture temperature on the growth of strain F13; Figure 5 To investigate the effect of culture pH on the growth of strain F13; Figure 6 is the effect of the initial concentration of procymidone on the growth of strain F13; Figure 7 The degradation results of procymidone by strain F13 under different nitrogen source conditions; Figure 8 The degradation results of procymidone by strain F13 at different procymidone concentrations; Fig. 9 The degradation results of procymidone by strain F13 under different temperature conditions; Fig.10 The degradation results of carbendazim by strain F13 under different pH conditions. DETAILED DESCRIPTION

[0010] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified; In the following example, the MSM liquid culture medium formula is K2HPO4 0.2g, KH2PO4 0.8g, MgSO4⋅7H2O 0.2g, FeSO4⋅7H2O 0.005g, Na2MoO4⋅2H2O 0.003g, CaCl2 0.1g, (NH4)2SO4 0.5g, distilled water 1L, pH 7.0, sterilized at 121°C for 30 minutes; solid culture medium plus 2% agar powder; The separation medium was prepared by adding 50 mg / L procymidone to MSM medium and sterilizing at 115°C for 20 minutes.

[0011] Example 1: Isolation and purification of strain F13 1. Collect the soil from a vegetable greenhouse in a farmland in Yuxi County, Yunnan Province where the pesticide procymidone is used all year round, crush it through an 80-mesh sieve, take 5g of the sieved soil, add it to a 250mL conical flask containing 50mL MSM liquid culture medium containing 50mg / L procymidone, and culture it at 37℃ and 150rpm for 8 days. Let the conical flask stand, take the supernatant culture solution, and transfer it to the newly sterilized MSM liquid culture medium containing 50mg / L procymidone at a 10% inoculum amount. Continue to acclimate and enrich and culture at 37℃ and 150rpm for 8 days. After repeating the enrichment and subculture for 6 times, take the last enrichment culture solution for 10-fold gradient dilution and apply it on MSM solid medium containing 50mg / L procymidone. On the culture medium, the culture was carried out at a constant temperature of 37°C until a single colony with regular colonies grew. Then a single colony was picked and streaked on a solid culture medium containing 50 mg / L procymidone for purification, and 9 purified strains were obtained. The purified strains were inoculated into a liquid culture medium containing 50 mg / L procymidone in MSM, and cultured at 37°C and 150rpm for 6 days. The degradation effect of each strain on procymidone was determined. The results showed that the degradation rate of procymidone by strain F13 was the highest among these purified strains, at 40.06%.

[0012] 2. Identification of strain F13 (1) Morphological identification The strain F13 was inoculated on a solid LB plate and inverted for 3 days at 37°C. The colony morphology was observed. The colony of F13 was round, with a smooth, slightly moist surface, regular edges and a bulge in the center ( Figure 1 Left), Gram staining shows that it is a Gram-negative bacterium, and under the microscope, individual cells are short round rods ( Figure 1 right).

[0013] (2) The activated F13 strain was inoculated into 50 mL LB liquid medium and cultured at 37°C and 150 rpm for 2 days. The culture was centrifuged at 12,000 rpm for 5 minutes, and the bacteria were collected. The whole genomic DNA of the bacteria was extracted using the CATB method. The extracted genome was used as a template and 16S rDNA bacterial universal primers (27F 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R 5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification. The PCR amplification system was 2×Taq Plus Master Mix 12.5 μL, total DNA 10 ng, upstream and downstream primers 1 μL each (10 μmol / L), and ddH2O was added to 25 μL. The PCR amplification program was 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, and finally 72°C extension for 10 min, for a total of 35 cycles. The PCR amplification product was sequenced by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA sequence was compared with the NCBI database by BLAST online. Pseudarthrobacter The highest similarity, phylogenetic tree see Figure 2 , combined with morphological characteristics and molecular identification results, strain F13 was finally identified as Pseudomonas aeruginosa ( Pseudarthrobacter sp.).

[0014] Example 2: Physiological and biochemical identification of strain F13 The physiological and biochemical identification methods of strain F13 refer to the conventional methods in the Manual of Identification of Common Bacterial Systems, and the results are shown in Table 1; Table 1 Physiological and biochemical characteristics of strain F13

[0015] Note: “+” means positive, “-” means negative.

[0016] Example 3: Growth characteristic detection of strain F13 (1) Growth curve determination of strain F13 Inoculate strain F13 in 5 mL of LB liquid medium, shake and culture at 37°C, 150 rpm for 12 h, and use the inoculum as the inoculum. Inoculate the inoculum into 5 mL of LB liquid at a 1% (v / v) inoculation volume, and culture at 37°C, 150 r / min. Set up a blank control, and use a UV spectrophotometer to measure the absorbance of the bacterial solution at a wavelength of 600 nm every two hours. The horizontal axis is time t, and the vertical axis is OD 600 Draw a growth curve and observe the growth cycle of the strain. Figure 3 From the figure, we can see that the strain grows slowly in the first 2 hours of culture, which is the lag period. The OD value of strain F13 changes sharply from 2 to 12 hours, entering the logarithmic growth phase. It grows slowly from 24 to 72 hours and is in the stable period.

[0017] (2) Effect of temperature on the growth of strain F13 Strain F13 was cultured in LB liquid medium to the logarithmic growth phase, and inoculated into a test tube containing 5 mL LB medium at a 1% inoculum. The culture was cultured at 20°C, 25°C, 30°C, 37°C, and 42°C at 150 rpm for 12 h, and the OD of the culture solution was measured. 600 The results are shown in Figure 4 The optimum growth temperature of strain F13 is 30℃. When the temperature is higher than 37℃, the growth is obviously inhibited.

[0018] (3) Effect of pH on the growth of degradation bacteria F13 The seed solution of strain F13 cultured in LB liquid medium was inoculated into 5 mL of LB liquid medium with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 at a 1% inoculum, and cultured at 30°C and 150 rpm for 12 h. The OD of the culture solution was measured. 600 The value was used to determine the effect of culture pH on the growth of the strain; the results are shown in Figure 5 The strain grows well under the condition of pH 6.0-9.0, the optimal growth pH is 7.0, and when the pH is less than 4.0, the strain hardly grows.

[0019] (4) Effect of initial concentration of procymidone on the growth of strain F13 The strain F13 was inoculated into MSM liquid medium containing 25, 50, 75, and 100 mg / L procymidone at pH 7.0 and cultured in a shaking incubator at 30°C and 150 rpm for 7 days. Figure 6 The strain F13 grew better in the culture medium containing 25-50 mg / L of procymidone. When the procymidone concentration was as high as 75 mg / L, no obvious bacterial decay was observed during the entire degradation process, which indicated that procymidone had less cytotoxicity to the strain even at high concentrations.

[0020] Example 4: Degradation of Procymidone by Strain F13 and Optimization of Conditions 50 mg / L procymidone and 10% (v / v) 10× concentrated cell suspension were added to 2 mL of MSM liquid culture medium, and the samples were placed on a shaker at 30°C and 150 rpm for 8 days after mixing. An equal volume of DMSO was added to the reaction mixture to stop the degradation reaction. After extraction on a shaker (25°C, 220 rpm) for 30 minutes, the extract solution was filtered through a 0.22 μm filter to obtain the culture filtrate, and metabolite analysis was performed using HPLC. At the same time, a non-degradation control group was set up in which no cell suspension was added but an equal amount of procymidone was added, and a blank control group was set up in which an equal amount of sterilized water was added. All experiments were repeated three times.

[0021] 1. Nitrogen source Yeast extract, threonine, L-phenylalanine, tyrosine, and tryptone were added to the above culture medium, and the concentration of nitrogen source in the culture medium was 0.5%. The effect of nitrogen source on the degradation of procymidone was evaluated. The results are shown in Figure 7 , in the culture solution with threonine and yeast extract added, the degradation rate of procymidone was the highest, which was 65.5% and 79.7%, respectively; 2. Initial concentration of procymidone 25, 50, 75, and 100 mg / L of procymidone were added to 2 mL of MSM liquid culture medium to evaluate the effect of its initial concentration on the degradation of procymidone; the results are shown in Figure 8When the initial concentration of procymidone increased, the degradation rate decreased significantly. At 25, 50, 75 and 100 mg / L, the degradation rates were 100%, 78.3%, 40.3% and 32.5%, respectively ( Figure 8 ).

[0022] 3. Temperature The treatment was carried out at 25, 30 or 37℃ for 8 days on a shaker at 150rpm to evaluate the effect of temperature on the degradation of procymidone. The results are shown in Fig. 9 At 25, 30 and 37°C, the dissolution rates of procymidone were 77.4%, 80.1% and 56.5%, respectively ( Fig. 9 ); 4. pH value The pH of the MSM liquid culture medium was set to 5, 6, 7, 8, and 9, respectively, to evaluate the effect of pH on the degradation of procymidone. The results are shown in Fig.10 At pH values ​​of 5, 6, 7, 8 and 9, the degradation rates were 52.6%, 77.7%, 88.6%, 85.2% and 97.0%, respectively. The degradation of carbendazim was higher at pH value of 9.

Claims

1. A pseudoarthrobacter Pseudarthrobacter sp.) F13, and its deposit number in China Center for Type Culture Collection is CCTCC NO: M 20241866.

2. Use of the Pseudomonas aeruginosa F13 described in claim 1 in the degradation of procymidone.

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