Pseudacidaminococcus sp. and application thereof

Abstract

This invention discloses a strain of *Arthrobacter* (…). Pseudarthrobacter The strain F13, with accession number CCTCC NO:M 20241866 at the China Center for Type Culture Collection, was used in this invention to degrade procymidone. The results showed that the *Pseudomonas* F13 strain of this invention can efficiently degrade procymidone. This invention enriches the germplasm resources and diversity of microbial degrading strains and provides a new approach for the control of pesticide residue pollution.

Description

Technical Field

[0001] This invention belongs to the fields of environmental treatment and microbial technology, specifically relating to a strain capable of efficiently degrading iprodione fungicide and its application. Background Technology

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

[0003] Currently, most domestic and international research focuses on the detection of residues in crops and soil environments, while research on the biodegradation of iprodione is relatively limited. Jin Huding et al. isolated a strain that promotes the degradation of iprodione in a greenhouse where vegetables were grown and pesticides were sprayed. Methylobacterium sp. T32-1, in verifying its degradation performance, showed that after the addition of iprodione aqueous solution, iprodione was gradually degraded by this strain, reaching a degradation rate of 77.2% by day 14; Zhang et al. studied... Providencia stuartii JD and Brevundimonas naejangsanensis The co-culture of two bacteria in J3 significantly improved the biodegradation efficiency of iprodione in brown soil, but the mixed-bacterial model has the potential risk of unstable effects.

[0004] The genus *Pseudomonas* is known to have potential in the remediation of environmental pollution. Strains of *Pseudomonas* can degrade several phenolic compounds, including polycyclic aromatic hydrocarbons (PAHs), phenol, 2,4,6-trinitrotoluene (TNT), crude oil, various benzene compounds, and phthalates (PAEs). Currently, there are no reports on the application of *Pseudomonas* strains in the degradation of diformylimide bactericides. Summary of the Invention

[0005] This invention provides a pseudoarthobacterium ( Pseudarthrobacter sp.) F13, which was deposited at the China Center for Type Culture Collection on August 28, 2024, with accession number CCTCC NO: M 20241866, deposited at Wuhan University, Wuhan, China.

[0006] Another object of the present invention is to use the above-mentioned pseudoarthobacterium ( Pseudarthrobacter sp.) F13 is used in the degradation of iprodione.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] 1. Soil collected from vegetable greenhouses where pesticides have been applied year-round was pulverized and sieved. The sieved soil was added to MSM liquid medium containing 50 mg / L iprodione and incubated at 37°C with shaking at 150 rpm for 8 days. After standing, the supernatant was transferred to freshly sterilized MSM liquid medium containing 100 mg / L iprodione and continued enrichment culture at 37°C with shaking at 150 rpm. After 4-8 subcultures, the last enriched culture was serially diluted and spread onto medium containing 50 mg / L iprodione. On L-propargyl MSM solid medium, the culture was carried out at 37℃ until regular single colonies grew. Single colonies were picked and streaked onto MSM solid medium containing 50 mg / L propargyl for purification to obtain the purified strain. Multiple purified colonies were inoculated into MSM liquid medium containing 50 mg / L propargyl and cultured at 37℃ and 150 rpm for 6 days. The culture medium was collected, and the degradation ability of the strain to propargyl was detected by high performance liquid chromatography (HPLC) to finally obtain strain F13.

[0009] 2. Identification of strain F13

[0010] (1) Morphological characteristics of strain F13: On LB solid medium, F13 is round, smooth and slightly moist, with regular edges and a bulging center; Gram staining shows that it is a Gram-negative bacterium, and the cells are short rods under a microscope;

[0011] (2) Molecular identification

[0012] Genomic DNA was extracted from strain F13 using the CATB method. Using the extracted genome as a template, PCR amplification was performed using universal 16S rDNA primers. After sequencing the PCR products, the sequencing results were compared with sequences on NCBI. Combined with morphological characteristics and molecular identification results, the strain was ultimately identified as a *Pseudomonas* genus strain. Pseudarthrobacter sp.), named Pseudarthrobacter sp. F13;

[0013] 3. When Pseudomonas F13 was applied to degrade iprodione, the experimental results showed that the degradation rate of iprodione by this strain reached 97.0%; the optimal degradation conditions for strain F13 were a temperature of 30℃ and a pH of 9.0.

[0014] Advantages and technical effects of the present invention:

[0015] This invention utilizes field soil from vegetable growing areas where pesticides have been applied for a long time and whose efficacy has been reduced. Through continuous liquid subculturing and enrichment, a microbial community with iprodione degradation capabilities was obtained. The culturable microorganisms in the enriched community were isolated, purified, and their degradation capabilities were determined, resulting in strain F13 with iprodione degradation capabilities. This strain has significant application value in the bioremediation of iprodione-contaminated water and soil, providing a new approach to the treatment of pesticide residue pollution. Attached Figure Description

[0016] Figure 1 This is a schematic diagram showing the colony morphology (left) and Gram staining results (right) of strain F13;

[0017] Figure 2 Phylogenetic tree of strain F13;

[0018] Figure 3 The growth curve of strain F13;

[0019] Figure 4 The effect of culture temperature on the growth of strain F13;

[0020] Figure 5 To investigate the effect of pH on the growth of strain F13;

[0021] Figure 6 The effect of initial concentration of iprodione on the growth of strain F13;

[0022] Figure 7 The degradation results of procymidone by strain F13 under different nitrogen source conditions;

[0023] Figure 8 The degradation results of procymidone by strain F13 at different concentrations of procymidone;

[0024] Figure 9 The degradation results of procymidone by strain F13 under different temperature conditions;

[0025] Figure 10 The degradation results of procymidone by strain F13 under different pH conditions. Detailed Implementation

[0026] The following examples further illustrate the content of the present invention, but these examples do not limit the scope of protection of the present invention. Unless otherwise specified, the methods in the examples are conventional methods, and unless otherwise specified, the reagents used are conventional commercial reagents or reagents prepared according to conventional methods.

[0027] In the examples below, the MSM liquid culture medium formulation was as follows: 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, 1L distilled water, pH 7.0, sterilized at 121℃ for 30 minutes; the solid culture medium was supplemented with 2% agar powder.

[0028] The isolation medium was prepared by adding 50 mg / L iprodione to MSM medium and sterilizing at 115°C for 20 minutes.

[0029] Example 1: Isolation and purification of strain F13

[0030] 1. Soil from a vegetable greenhouse in Yuxi County, Yunnan Province, where the pesticide iprodione has been used year-round, was collected. The soil was pulverized and passed through an 80-mesh sieve. 5g of the sieved soil was added to a 250mL Erlenmeyer flask containing 50mL of MSM liquid medium (50mg / L iprodione). The flask was incubated at 37℃ and 150rpm with constant temperature shaking for 8 days. After allowing the flask to stand, the supernatant was collected and transferred at a 10% inoculum to freshly sterilized MSM liquid medium (50mg / L iprodione). The medium was further enriched and cultured at 37℃ and 150rpm for 8 days. This enrichment and subculturing process was repeated 6 times. The final enriched medium was then serially diluted 10-fold and spread onto MSM solid medium (50mg / L iprodione). On the culture medium, the cells were incubated at a constant temperature of 37℃ until regular single colonies grew. Single colonies were picked and streaked onto MSM solid medium containing 50 mg / L iprodione for purification, resulting in 9 purified strains. The purified strains were then inoculated into MSM liquid medium containing 50 mg / L iprodione and cultured at 37℃ and 150 rpm for 6 days. The degradation effect of each strain on iprodione was measured. The results showed that strain F13 had the highest degradation rate of iprodione among these purified strains, at 40.06%.

[0031] 2. Identification of strain F13

[0032] (1) Morphological identification

[0033] Strain strain F13 was inoculated onto LB agar plates and incubated upside down at 37°C for 3 days. Colony morphology was observed; F13 colonies were round, smooth, slightly moist, with regular edges and a bulging center. Figure 1 (Left), Gram staining shows it is a Gram-negative bacterium; under a microscope, individual cells appear as short, round rods. Figure 1 right).

[0034] (2) The activated F13 strain was inoculated into 50 mL of LB liquid medium and cultured at 37 °C and 150 rpm for 2 days with continuous shaking. After centrifugation at 12000 rpm for 5 minutes, the bacterial cells were collected and the whole genome DNA of the bacteria was extracted by CATB method. Using the extracted genome as a template, PCR amplification was performed using 16S rDNA bacterial universal primers (27F 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R 5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification system was 12.5 μL of 2×Taq Plus Master Mix, 10 ng of total DNA, 1 μL of each of the forward and reverse primers (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.

[0035] The PCR amplification products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The obtained 16S rDNA sequences were compared online using BLAST in the NCBI database. The F13 strain was compared with *Pseudomonas aeruginosa*. Pseudarthrobacter Highest similarity, see phylogenetic tree Figure 2 Based on morphological characteristics and molecular identification results, strain F13 was ultimately identified as *Pseudorobacter*. Pseudarthrobacter sp.).

[0036] Example 2: Physiological and biochemical identification of strain F13

[0037] The physiological and biochemical identification methods for strain F13 were based on the routine methods in the "Handbook of Systematic Identification of Common Bacteria", and the results are shown in Table 1.

[0038] Table 1. Physiological and biochemical characteristics of strain F13

[0039]

[0040] Note: "+" indicates positive, "-" indicates negative.

[0041] Example 3: Detection of growth characteristics of strain F13

[0042] (1) Determination of growth curve of strain F13

[0043] Strain strain F13 was inoculated into 5 mL of LB liquid medium and cultured at 37 °C and 150 rpm for 12 h using a shaker. This inoculum was then added to 5 mL of LB liquid at a 1% (v / v) inoculation rate and cultured at 37 °C and 150 rpm. A blank control was included. The absorbance of the bacterial culture at 600 nm was measured every two hours using a UV spectrophotometer. The values ​​are plotted as follows: x-axis: time t, y-axis: OD. 600 Plot growth curves and observe the growth cycle of the strain. Results are shown below. Figure 3 As can be seen from the figure, the strain grows slowly in the first 2 hours of culture, which is the lag phase. Between 2 and 12 hours, the OD value of strain F13 changes drastically, entering the logarithmic growth phase. Between 24 and 72 hours, the growth slows down, and the strain is in the stationary phase.

[0044] (2) Effect of temperature on the growth of strain F13

[0045] Strain strain F13 was cultured in LB liquid medium to the logarithmic growth phase. A 1% inoculum was then added to test tubes containing 5 mL of LB medium and incubated at 20℃, 25℃, 30℃, 37℃, and 42℃ for 12 h at 150 rpm. The OD of the culture medium was then measured. 600 The value was used to determine the effect of culture temperature on the growth of the strain. Results are shown below. Figure 4 The optimal growth temperature for strain F13 is 30℃, and its growth is significantly inhibited when the temperature is above 37℃.

[0046] (3) Effect of pH on the growth of degrading bacteria F13

[0047] Seed culture of strain F13 in LB liquid medium was inoculated at a rate of 1% into 5 mL of LB liquid medium at pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. The cultures were incubated at 30°C and 150 rpm for 12 h, and the OD values ​​of the cultures were 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 conditions with a pH of 6.0-9.0, with the optimal growth pH being 7.0. When the pH is less than 4.0, the strain hardly grows.

[0048] (4) Effect of initial concentration of iprodione on the growth of strain F13

[0049] Strain strain F13 was inoculated into MSM liquid medium containing 25, 50, 75, and 100 mg / L iprodione at pH 7.0 and cultured at 30°C and 150 rpm for 7 days. The results are shown below. Figure 6Strain F13 grew well in medium containing 25-50 mg / L iprodione. When the iprodione concentration was as high as 75 mg / L, no obvious cell decay was observed during the entire degradation process, which indicates that iprodione has low cytotoxicity to the strain even at high concentrations.

[0050] Example 4: Degradation of iprodione by strain F13 and optimization of conditions

[0051] Add 50 mg / L iprodione and 10% (v / v) 10× concentrated cell suspension to 2 mL of MSM liquid medium, mix well, and treat the samples on a shaker at 30℃ and 150 rpm for 8 days. Add an equal volume of DMSO to the reaction mixture to stop the degradation reaction, and extract on a shaker (25℃, 220 rpm) for 30 min. Filter the extract through a 0.22 μm filter to obtain the culture filtrate, and analyze the metabolites by HPLC. A control group without cell suspension but with an equal amount of iprodione was set up, and a blank control group with an equal amount of sterile water was set up. All experiments were repeated three times.

[0052] 1. Nitrogen source

[0053] Yeast extract, threonine, L-phenylalanine, tyrosine, and tryptone were added to the above-mentioned culture media, respectively, with a nitrogen source concentration of 0.5%. The effect of nitrogen source on the degradation of iprodione was evaluated, and the results are shown in [Figure number missing]. Figure 7 In the culture medium supplemented with threonine and yeast extract, the degradation rate of iprodione was the highest, at 65.5% and 79.7%, respectively.

[0054] 2. Initial concentration of iprodione

[0055] The effects of initial concentrations of iprodione (25, 50, 75, and 100 mg / L) on iprodione degradation were evaluated in 2 mL of MSM liquid medium. Results are shown in [Figure number missing]. Figure 8 As the initial concentration of iprodione 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 ).

[0056] 3. Temperature

[0057] The degradation of iprodione was evaluated by treating the samples at 25, 30, or 37°C on a shaker at 150 rpm for 8 days. The results are shown in [Table missing]. Figure 9 At 25, 30, and 37°C, the hydrolysis rates of iprodione were 77.4%, 80.1%, and 56.5%, respectively. Figure 9 );

[0058] 4. pH value

[0059] The pH values ​​of MSM liquid culture medium were set to 5, 6, 7, 8, and 9 to evaluate the effect of pH on the degradation of iprodione. The results are shown in [Figure number missing]. Figure 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, with the highest degradation rate observed at pH 9.

Claims

1. A type of pseudoarthobacterium ( Pseudarthrobacter sp.)F13, whose accession number at the China Center for Type Culture Collection is CCTCC NO: M 20241866.

2. The application of the *Pseudomonas* F13 as described in claim 1 in the degradation of iprodione.

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