Neonicotinoid pesticide-degrading bacterium b27 and application thereof

Abstract

The application discloses a neonicotinoid pesticide degrading bacterium B27 and application thereof, the neonicotinoid pesticide degrading bacterium B27 is identified as Flexivirga sp., has been preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:M 2022981.A new strain Flexivirga sp.B27 is obtained by screening and separating from soil where earthworms move, and can be used for degrading neonicotinoid pesticides imidacloprid, acetamiprid and nitenpyram; the degrading bacterium B27 can be used for degrading residual neonicotinoid pesticides in soil or water environment, solves the pollution and residual problems caused by neonicotinoid pesticides to soil or water environment, and enriches new species resources of Flexivirga genus neonicotinoid pesticide degrading bacteria.

Description

Technical Field

[0001] This invention relates to the microbial remediation of environmental pollution, specifically to a neonicotinoid insecticide degrading bacterium B27 and its applications. Background Technology

[0002] Neonicotinic insecticides are a class of compounds with pharmacodynamic groups such as nitromimine, nitrosoimine, and tricyanacetyl. They paralyze and kill insects by blocking the normal transmission of the central nervous system. Major types include imidacloprid, thiamethoxam, thiamethoxam, dinotefuran, and acetamiprid. They are widely used in the control of various agricultural pests, accounting for about one-third of the global insecticide market. When the probability of crop pests and diseases is high, neonicotinic insecticides are applied in larger quantities and more frequently. Repeated use of imidacloprid leads to accumulation and has a high leaching and runoff potential, accumulating in the soil and entering surface water and groundwater through surface runoff or infiltration. Residues of neonicotinic insecticides such as imidacloprid can be detected in most rivers worldwide. These residues not only harm humans through the food chain via agricultural products but also pollute groundwater through soil leaching, poisoning aquatic products. The residues also affect beneficial organisms and natural enemies of pests in the environment. Imidacloprid is a low-toxicity neonicotinoid insecticide, but it is highly toxic to bees. When beneficial organisms and natural enemies of pests are also eliminated, harmful organisms develop resistance to pesticides and become the dominant species.

[0003] Microorganisms are the primary factor in eliminating neonicotinoid pesticide residues in soil. In contaminated soil and water, microorganisms readily come into contact with pesticide residues, thus enabling them to degrade them. Soil bacteria, in particular, are characterized by rapid growth and a direct promoting effect on pesticide degradation. They can utilize their natural growth and reproduction to degrade residual neonicotinoid pesticides in soil or water, ultimately achieving soil remediation. Therefore, screening for neonicotinoid pesticide-degrading bacteria is of great significance. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a neonicotinoid insecticide degrading bacterium B27, which can efficiently degrade neonicotinoid insecticides.

[0005] Another objective of this invention is to provide a degrading agent produced by the degrading strain and its application. The degrading agent prepared by the strain of this invention can degrade neonicotinoid insecticides such as imidacloprid, acetamiprid, and nitenpyram. This agent can achieve a degradation rate of over 71% for imidacloprid, acetamiprid, and nitenpyram residues in soil or water environments within a short period of time. The degrading agent of this invention can be produced using general fermentation equipment in the fermentation industry, and has the advantages of low production cost, convenient use, and good removal effect.

[0006] Technical solution: In order to achieve the above objectives, the present invention provides a neonicotinoid insecticide degrading bacterium B27, which has been identified as a new species of *Flexivirga* sp. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022981 and deposit date of June 27, 2022.

[0007] The application of the degrading bacterium B27 described in this invention in the degradation of neonicotinoid insecticides.

[0008] The application of the degrading bacterium B27 in simultaneously degrading neonicotinoid insecticides in soil or water environments.

[0009] The neonicotinoid insecticide is one or more of imidacloprid, acetamiprid, and nitenpyram.

[0010] The degradation conditions are: natural pH and temperature of 28–37°C.

[0011] The present invention relates to a degradation agent produced using a new species of degradation bacteria, B27.

[0012] The fermentation of the microbial agent includes the following steps:

[0013] 1) Activate strain B27;

[0014] 2) Inoculate the activated bacterial solution into the culture medium of the seed tank and culture until the logarithmic growth phase to obtain the seed solution;

[0015] 3) Inoculate the seed culture into the culture medium in the production tank for cultivation; after fermentation, the culture medium is discharged from the tank and packaged into liquid form.

[0016] The application of the degrading microbial agent described in this invention in the degradation of neonicotinoid insecticides.

[0017] The application of the degrading microbial agent in simultaneously degrading neonicotinoid insecticides in soil or water environments.

[0018] Furthermore, the neonicotinoid insecticides degraded by the degrading microbial agent are one or more of imidacloprid, acetamiprid, and nitenpyram.

[0019] This invention, through the domestication and treatment of earthworms, screened and isolated a highly efficient neonicotinoid insecticide-degrading strain B27 from the soil in which earthworms live. This provides resource security and scientific basis for using degrading bacteria to alleviate the toxicity of neonicotinoid insecticides in soil and water environments. The strain B27 isolated and screened by this invention can not only effectively degrade neonicotinoid insecticides such as imidacloprid, acetamiprid, and acetamiprid, but more importantly, comparison revealed that this strain is a novel species within the genus *Flexivir*, possessing significant value in the fields of novel species and neonicotinoid insecticide degradation.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0021] This invention isolates and screens a new strain of *Flexivirgasp.* B27 from soil inhabited by earthworms. This strain can be used to degrade neonicotinoid insecticides, especially imidacloprid, acetamiprid, and nitenpyram residual neonicotinoid insecticides in soil or water environments. The strain B27 of this invention can degrade residual neonicotinoid insecticides such as imidacloprid, acetamiprid, and nitenpyram in soil or water environments, achieving a degradation rate of over 71% in a short time. This invention effectively utilizes biological methods to solve the pollution and residue problems caused by neonicotinoid insecticides in soil or water environments.

[0022] The novel strain B27 provided by this invention is *Flexivirga* sp., whose degrading agent can be produced using general fermentation equipment in the fermentation industry. It features low production cost, ease of use, high activity, and efficient degradation of neonicotinoid insecticides such as imidacloprid, acetamiprid, and acetamiprid. Furthermore, this invention aligns with the principles of green and safe environmental protection, maximizing the utilization of soil microbial resources. It is of great significance for reducing pesticide damage, increasing the added value of agricultural products, and producing pollution-free vegetables and green food. Attached Figure Description

[0023] Figure 1 Photograph of bacterial colonies of strain B27;

[0024] Figure 2 This is a phylogenetic tree diagram of strain B27;

[0025] Figure 3 HPLC chromatogram of imidacloprid degradation by strain B27 (initial imidacloprid concentration 50 mg / L. A: CK; B: treatment). Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0028] Example 1

[0029] Screening, isolation and identification of the new species B27:

[0030] The degrading strains selected for enrichment and screening in the experiment were derived from the soil in which earthworms lived. The earthworms were sourced from the Tropical Ecological Agriculture Base of the Chinese Academy of Tropical Agricultural Sciences, and the soil used for acclimatizing the earthworms was collected from Haikou City. Earthworms that had adapted to the experimental soil and undergone intestinal cleansing were placed in an environment containing 2.0 mg / L of imidacloprid. -1 The earthworms were grown in the soil, and after 28 days, 5g of the soil containing the earthworms was weighed and added to a solution containing 80mg·L⁻¹. -1 In 100 mL of basal salt medium with imidacloprid as the sole carbon source, the culture was incubated at 30 °C and 150 rpm for 7 days. The culture was then transferred to the same medium at a 5% (v / v) inoculum, and this process was repeated three times. Finally, the enrichment solution was serially diluted, and 10 μL of each solution was collected. -3 ~10 -6 0.02 mL of the enrichment solution at dilution was spread onto a substrate containing 80 mg·L⁻¹ -1 On a basic salt solid plate with imidacloprid as the sole carbon source, after 72 hours of incubation at 30°C, single colonies grown on the plate were picked, purified, and inoculated onto plates containing 80 mg·L⁻¹ of imidacloprid. -1 The degradation effects of imidacloprid, acetamiprid, and nitenpyram on these three substances were verified by high-performance liquid chromatography (HPLC) after incubation at 30°C and 150 rpm for 72 h in a basal salt medium with imidacloprid, acetamiprid, and nitenpyram as the sole carbon source. The basal salt medium formula (1 L) was: 1.0 g NH4NO3, 1.0 g NaCl, 1.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4·7H2O; pH 7.0; and 18.0 g agar was added to the solid medium.

[0031] Verification method for the degradation effect of neonicotinoid insecticides: Dichloromethane was added to the culture medium at a 1:1 volume ratio, and after vigorous shaking and standing, the upper aqueous phase was removed. Anhydrous Na2SO4 was added to remove a small amount of water. 1 mL of the organic phase was dried and dissolved in an equal volume of methanol. After filtration through 0.22 μm, the solution was analyzed by high-performance liquid chromatography (HPLC). HPLC conditions: mobile phase: water:methanol (60:40, volume ratio); C18-WP column (5 μm × 4.6 mm × 250 mm); column temperature: room temperature; detection wavelength: 270 nm; injection volume: 10 μL; flow rate: 1.0 mL·min. -1 External standard method: quantification based on peak area.

[0032] In this embodiment, an aerobic degrading bacterium capable of degrading neonicotinoid insecticides was isolated from the enrichment solution and named B27. This bacterium showed significant degradation effects on certain concentrations of imidacloprid, acetamiprid, and acetamiprid within a certain time period. The bacterial colonies were milky white and moist. Figure 1 Gram-positive. Under a transmission microscope, the bacterial cells are spherical, without flagella, and do not form spores. Catalase test positive.

[0033] Using the genomic DNA of strain B27 as a template, PCR amplification was performed using universal primers for bacterial 16S rRNA gene sequencing, yielding a 16S rDNA gene sequence of approximately 1482 bp, as shown in SEQ ID No: 1. Comparison with the EzBioCloud database (www.EzBioCloud.net) showed that strain B27 was most homologous to *Flexivirga* sp. strains and most homologous to *Flexivirga alba* ST13. T (AB539735) is most closely related, with 96.89% 16S rDNA gene similarity. A phylogenetic tree can be constructed. Figure 2 Whole-genome sequencing was performed on the strain, which showed high homology with Flexivirgaalba ST13. T Genome alignment revealed that the highest values ​​for average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) were 83.0%, 83.2%, and 25.2%, respectively, all below the species identification thresholds of 95%, 95%, and 70%. Based on morphological and physiological-biochemical characteristics, strain B27 was preliminarily identified as a new species of *Flexivirga sp.*, named *Flexivirga sp.* B27. This strain was deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2022981, on June 27, 2022.

[0034] Example 2

[0035] The degradation effect of strain B27 on imidacloprid in basal salt medium:

[0036] Add 20 and 50 mg·L to the basal salt medium (same as in Example 1), respectively. -1Imidacloprid was inoculated into bacterial culture of strain B27 in the logarithmic growth phase at a 1% (v / v) inoculum. Simultaneously, 20 and 50 mg·L⁻¹ mg·L⁻¹ were added to the basal salt medium, respectively. -1 Imidacloprid was inoculated at a 1% (v / v) inactivated strain B27 as a control. The mixture was cultured at 30°C with shaking at 150 rpm. Samples were taken periodically, and the degradation of imidacloprid by the strain was detected by high-performance liquid chromatography (HPLC) using the method described in Example 1. Strain B27 showed degradation of 20 mg·L⁻¹ imidacloprid within 48 hours. -1 Imidacloprid degradation rate can reach over 71%, at 50 mg·L⁻¹. -1 The degradation rate of imidacloprid can reach over 25%. The strain showed a resistance rate of 50 mg·L⁻¹. -1 The HPLC chromatograms of the degradation results are as follows: Figure 3 As shown in Table 1, the degradation rate is as follows.

[0037] Table 1. Degradation effect of strain B27 on imidacloprid in culture medium.

[0038]

[0039] Example 3

[0040] The degradation effect of strain B27 on acetamiprid and nitenpyram in basal salt medium:

[0041] Add 20 mg·L⁻¹ to the basal salt medium (same as in Example 1) respectively. -1 Acetamiprid and acetamiprid were inoculated into bacterial culture of strain B27 in the logarithmic growth phase at a 1% (v / v) inoculation rate. Simultaneously, 20 mg / L of each insecticide was added to the basal salt medium. -1 Acetamiprid and acetamiprid were inoculated at a 1% (v / v) inactivated strain B27 as Control 1 and Control 2. The cultures were incubated at 30°C with shaking at 150 rpm. Samples were taken periodically, and the degradation of acetamiprid and acetamiprid by the strain was detected by high-performance liquid chromatography using the method described in Example 1. Strain B27 showed degradation of acetamiprid and acetamiprid by 20 mg·L⁻¹ within 48 h. -1 Acetamiprid can be degraded at a rate of over 78% at 20 mg / L. -1 The degradation rate of acetamiprid can reach over 73%. The degradation rate of the strains is shown in Table 2.

[0042] Table 2. Degradation effect of strain B27 on acetamiprid and nitenpyram in culture medium.

[0043]

[0044] Example 4

[0045] Preparation method of strain B27 degrading agent

[0046] 1) Inoculate the B27 colonies on the plate into test tubes and culture for 18 hours. Inoculate the cultured test tube liquid into fermentation medium at a volume ratio of 1% and culture with shaking until the logarithmic phase to obtain the fermentation strain.

[0047] 2) The above-cultured fermentation strains were inoculated into a 5-liter seed tank with a liquid volume of 65% (based on the fermenter volume, the same below) at an inoculation rate of 5% (v / v, based on the volume of the culture medium). The sterile air ventilation rate was 1:0.8vvm, 200 rpm, and the culture temperature was 28℃. The culture was carried out until the logarithmic growth phase to obtain the seed liquid.

[0048] 3) Inoculate the seed culture at a rate of 5% (v / v, based on the volume of the culture medium, the same below) into the culture medium of a production tank with a volume of 65% and culture it. The stirring speed is 200 rpm, the culture temperature is 28℃, and the fermentation time is 36 h. After fermentation, the culture medium is discharged from the tank and directly packaged into liquid form in packaging bottles.

[0049] The fermentation medium, the seed tank medium, and the production tank medium all have the same formula: 0.1 wt% glucose, 1.0 wt% NaCl, 0.5 wt% peptone, 0.25 wt% yeast extract, and pH 7.0.

[0050] Example 5

[0051] Fermentation of degrading microbial agents:

[0052] 1) Inoculate the B27 colonies on the plate into test tubes and culture for 18 hours. Inoculate the cultured test tube solution into fermentation medium at a volume ratio of 1.5% and culture with shaking until the logarithmic phase to obtain the fermentation strain.

[0053] 2) The above-cultured fermentation strains were inoculated into a 5-liter seed tank with a liquid volume of 65% (based on the fermenter volume, the same below) at an inoculation rate of 8% (v / v, based on the volume of the culture medium). The sterile air ventilation rate was 1:0.8vvm, the stirring speed was 220 rpm, the culture temperature was 30℃, and the culture was carried out until the logarithmic growth phase to obtain the seed liquid.

[0054] 3) Inoculate the seed culture at an inoculation rate of 8% (v / v, based on the volume of the culture medium, the same below) into the culture medium of a production tank with a liquid volume of 65% and culture it; the stirring speed is 220 rpm, the culture temperature is 30℃, and the fermentation time is 24 h. After fermentation, the culture medium is discharged from the tank and directly packaged into liquid form in plastic packaging buckets.

[0055] The fermentation medium, the seed tank medium, and the production tank medium have the same formula: 0.1 wt% glucose, 1.0 wt% NaCl, 0.5 wt% peptone, 0.25 wt% yeast extract, and pH 7.2.

[0056] Example 6

[0057] Preparation method of degrading bacterial agent

[0058] The preparation method of the degradation agent in Example 6 is basically the same as that in Example 5, except that in steps 2) and 3), the ventilation rate of sterile air in the seed tank and production tank is 1:0.9vvm, the stirring speed is 200rpm, the culture temperature is 32℃, and the fermentation time is 30h.

[0059] Example 7

[0060] Degradation test of neonicotinoid insecticides by strain B27 in soil

[0061] Soil samples from vegetable plots free of neonicotinoid insecticides were used as test samples. The soil samples were sieved through a 2mm sieve. Certain amounts of imidacloprid, acetamiprid, and nitenpyram were taken and evenly mixed into 500g of soil, respectively, to ensure a final concentration of 10 mg / kg for each of the three pesticides. -1 Fresh bacterial culture of strain B27 in the logarithmic phase was collected, and the cells were washed three times with sterile deionized water and then resuspended in sterile water to adjust the cell concentration to approximately 1.0 × 10⁻⁶. 9 cfu·mL -1 The inoculum was 5 mL / 100 g, and the mixture was incubated in a dark incubator at 30°C. Uninoculated soil served as a control. Soil moisture content was maintained at 60% during the incubation period. After 10 days, the residual amounts were determined using high-performance liquid chromatography (HPLC). The results showed that strain B27 achieved degradation rates of 71.04%, 73.51%, and 71.27% for imidacloprid, acetamiprid, and dinotefuran in the soil within 10 days.

Claims

1. A strain of degrading bacteria B27, characterized in that, B27 was identified as belonging to the genus Curvularia ( ). Flexivirga (sp.), deposited at the China Center for Type Culture Collection, dated June 27, 2022, accession number CCTCC NO: M2022981.

2. The application of the degrading bacterium B27 according to claim 1 in the degradation of neonicotinoid insecticides, wherein the neonicotinoid insecticide is one or more of imidacloprid, acetamiprid, and nitenpyram.

3. The application according to claim 2, characterized in that, Application of the degrading bacterium B27 in the simultaneous degradation of neonicotinoid insecticides in soil or water environments.

4. The application according to claim 2 or 3, characterized in that, The degradation conditions are: natural pH and temperature 28~37℃.

5. The application of the degrading bacterium B27 according to claim 1 in the preparation of a neonicotinoid insecticide degrading agent, wherein the neonicotinoid insecticide is one or more of imidacloprid, acetamiprid, and acetamiprid.

6. A biodegrading agent, characterized in that, The bacterial agent includes the degrading bacteria B27 according to claim 1, and the bacterial agent includes the following steps: 1) Inoculate strain B27 into the culture medium for activation treatment; 2) Inoculate the activated bacterial solution into the culture medium of the seed tank and culture until the logarithmic growth phase to obtain the seed solution; 3) Inoculate the seed culture into the culture medium in the production tank for cultivation; after fermentation, the culture medium is discharged from the tank and packaged into liquid form.

7. The application of the degrading microbial agent according to claim 6 in the degradation of neonicotinoid insecticides, wherein the neonicotinoid insecticide is one or more of imidacloprid, acetamiprid, and acetamiprid.

8. The application according to claim 7, characterized in that, The application of the degrading microbial agent in degrading neonicotinoid insecticides in soil or water environments.

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