Neonicotinoid insecticide degrading bacterium B27 and application thereof

Abstract

The invention discloses a neonicotinoid insecticide degrading strain B27 and application thereof, the neonicotinoid insecticide degrading strain B27 is identified as Flexivirga sp. And is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 2022981. According to the invention, a new strain Flexivirga sp.B27 is separated and screened from soil in which earthworms move, and can be used for degrading neonicotinoid insecticides such as imidacloprid, acetamiprid and nitenpyram; the degrading bacterium B27 disclosed by the invention can be used for degrading residual neonicotinoid insecticides in soil or a water body environment, so that the problem of pollution residues of the neonicotinoid insecticides to the soil or the water body environment is solved, and new resources of the degrading bacterium of the Flexivirga neonicotinoid insecticides are enriched.

Description

TECHNICAL FIELD

[0001] The present application relates to environmental pollution microbial remediation, and in particular to a new neonicotinoid insecticide degrading bacteria B27 and its application. BACKGROUND

[0002] Neonicotinoid insecticides are a class of compounds with nitro imine, nitroso imine, tricyanoacetyl and other pharmacodynamic groups. It can block the normal conduction of the central nervous system of insects, leading to paralysis and death of pests. The main types include imidacloprid, thiamethoxam, clothianidin, dinotefuran and nitenpyram, etc. They are widely used in various types of farmland pest control, accounting for about one third of the global insecticide market. When the probability of crop disease and pest occurrence is high, the amount of neonicotinoid insecticide used is large, and the frequency of use is high. The repeated use of imidacloprid leads to accumulation, and it has a high potential for leaching and runoff. It can accumulate in the soil and enter surface water and groundwater through surface runoff or infiltration. Neonicotinoid insecticides such as imidacloprid can be detected in water bodies of major rivers in the world. The residues not only cause harm to the human body through the food chain in agricultural products, but also cause pollution to groundwater through soil leaching, and harm to aquatic products. The residues also affect beneficial organisms, pest natural enemies, etc. in the environment. Imidacloprid is a low-toxicity neonicotinoid insecticide, but it is highly toxic to bees. When beneficial organisms and pest natural enemies are also eliminated, harmful organisms become dominant species.

[0003] Microorganisms are the main factors for the elimination of neonicotinoid insecticide residues in soil. For contaminated soil and water, microorganisms can easily contact with the residues of pesticides, thereby exerting their degradation function. Among them, bacteria in the soil have the characteristics of fast growth and direct promotion of pesticide degradation, and can utilize the natural growth and reproduction of bacteria to degrade the residues of neonicotinoid insecticides in the soil or water environment, and ultimately achieve the purpose of soil remediation. Therefore, it is of great significance to screen and obtain degrading bacteria that can degrade neonicotinoid insecticides. SUMMARY

[0004] The present application provides a neonicotinoid insecticide degrading bacteria B27, which can efficiently degrade neonicotinoid insecticides.

[0005] Another object of the present application is to provide a degrading bacterial agent produced by the degrading strain and its application. The degrading bacterial agent prepared by the strain of the present application can degrade neonicotinoid insecticides such as imidacloprid, acetamiprid and nitenpyram. The bacterial agent can degrade more than 71% of the residues of imidacloprid, acetamiprid and nitenpyram in the soil or water environment in a short time. The degrading bacterial agent of the present application can be produced by general fermentation equipment in the fermentation industry, and has the advantages of low production cost, easy use and good removal effect.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the neonicotinoid pesticide degrading bacteria B27 of the application is identified as Flexivirga sp., and is a new species, the strain has been preserved in China typical culture preservation center, the preservation number is CCTCC NO: M 2022981, and the preservation date is: June 27, 2022.

[0007] The application of the degrading bacteria B27 in degrading neonicotinoid pesticides.

[0008] Among them, the application of the degrading bacteria B27 in simultaneously degrading neonicotinoid pesticides in soil or water environment.

[0009] Among them, the neonicotinoid pesticide is one or more of imidacloprid, acetamiprid and nitenpyram.

[0010] Among them, the degradation condition is that the pH is natural and the temperature is 28-37 DEG C.

[0011] The application of the degrading bacteria B27 produced by the degrading bacteria B27 in degrading neonicotinoid pesticides.

[0012] Among them, the fermentation of the bacterial agent comprises the following steps:

[0013] 1) the strain B27 is activated;

[0014] 2) the activated bacterial liquid is inoculated into the culture medium of the seed tank for culture, and the seed liquid is prepared in the logarithmic growth phase;

[0015] 3) the seed liquid is inoculated into the culture medium of the production tank for culture; after fermentation, the culture liquid is discharged from the tank and is divided into liquid agents.

[0016] The application of the degrading bacteria B27 produced by the degrading bacteria B27 in degrading neonicotinoid pesticides.

[0017] Among them, the application of the degrading bacteria B27 in simultaneously degrading neonicotinoid pesticides in soil or water environment.

[0018] Further, the neonicotinoid pesticide degraded by the degrading bacteria is one or more of imidacloprid, acetamiprid and nitenpyram.

[0019] The application provides a high-efficiency neonicotinoid insecticide degrading strain B27 which is screened and separated from earthworm active soil through domestication and treatment of earthworms, and provides resource guarantee and scientific basis for relieving neonicotinoid insecticide poisoning in soil and water environment by using the degrading bacteria.

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

[0021] The application provides a new species strain Flexivirga sp. B27 which is screened and separated from earthworm active soil, and can be used for degrading neonicotinoid insecticides, especially degrading residual neonicotinoid insecticides imidacloprid, acetamiprid and nitenpyram in soil or water environment. The strain B27 degrading agent can be used for degrading residual neonicotinoid insecticides imidacloprid, acetamiprid and nitenpyram in soil or water environment, and can degrade more than 71% of residual neonicotinoid insecticides imidacloprid, acetamiprid and nitenpyram in soil or water environment in a short time. The application can effectively utilize biological means to solve the pollution and residual problems of neonicotinoid insecticides to soil or water environment.

[0022] The new species strain B27 provided by the application is Flexivirga sp., and the degrading agent thereof can be produced by using general fermentation equipment in the fermentation industry, has low production cost, is convenient to use, has high activity, and can efficiently degrade neonicotinoid insecticides imidacloprid, acetamiprid and nitenpyram. The application conforms to the green and safe environmental protection concept, and realizes the maximization of resource utilization of soil microorganisms. The application has important significance for reducing pesticide phytotoxicity, improving the added value of agricultural products, and producing pollution-free vegetables and green food. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a colony photograph of the strain B27;

[0024] Figure 2 It is an evolutionary tree diagram of the strain B27;

[0025] Figure 3 It is an HPLC analysis spectrum of the strain B27 degrading imidacloprid (initial imidacloprid concentration is 50 mg / L. A: CK; B: treatment). DETAILED DESCRIPTION

[0026] The application is further described below in combination with the drawings and examples.

[0027] The raw materials and reagents in the present application are commercially available unless otherwise specified.

[0028] Example 1

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

[0030] The degradation strain for experimental enrichment screening was derived from the soil where earthworms were active. The earthworms were from the tropical ecological agricultural base of the Chinese Academy of Tropical Agricultural Sciences, and the soil used for domesticating earthworms was collected from Haikou City. The earthworms that were adapted to the experimental soil and had clean intestines were placed in soil containing imidacloprid at a concentration of 2.0 mg·L -1 . After 28 days, 5 g of the soil where the earthworms were active was weighed and added to 100 mL of a basic salt medium containing imidacloprid at a concentration of 80 mg·L -1 as the sole carbon source, and the mixture was cultured at 30°C and 150 rpm for 7 days. The culture solution was transferred to the same medium at a volume ratio of 5%, and the transfer was repeated three times in succession. Finally, the enrichment liquid was gradiently diluted, and 0.02 mL of the enrichment liquid at a dilution of 10 -3 to 10 -6 was spread on a basic salt solid plate containing imidacloprid at a concentration of 80 mg·L -1 as the sole carbon source. After being cultured at 30°C for 72 h, single colonies that grew on the plate were picked and purified, and then the purified colonies were inoculated in a basic salt medium containing imidacloprid, acetamiprid and nitenpyram at a concentration of 80 mg·L -1 as the sole carbon source, respectively. After being cultured at 30°C and 150 rpm for 72 h, the degradation effects of the strains on imidacloprid, acetamiprid and nitenpyram were verified by high-performance liquid chromatography. The formula of the basic salt medium (1 L) was as follows: 1.0 g of NH4NO3, 1.0 g of NaCl, 1.5 g of K2HPO4, 0.5 g of KH2PO4, and 0.2 g of MgSO4·7H2O; the pH was 7.0; and 18.0 g of agar was added to the solid medium.

[0031] Verification method for the degradation effect of neonicotinoid insecticides: The culture solution was added with dichloromethane at a volume ratio of 1:1, and after being vigorously shaken and allowed to stand, the upper aqueous phase was removed, anhydrous Na2SO4 was added to remove a small amount of water, 1 mL of the organic phase was drawn and dried, an equal volume of methanol was added to dissolve the organic phase, the solution was filtered through a 0.22-μm filter, and then the filtered solution was detected by high-performance liquid chromatography (HPLC). The liquid chromatography conditions were as follows: the mobile phase was water:methanol (60:40, by volume), the C18-WP chromatographic column (5 μm×4.6 mm×250 mm) was used at room temperature, the detection wavelength was 270 nm, the injection amount was 10 μL, and the flow rate was 1.0 mL·min -1 . The peak area was quantified by the external standard method.

[0032] An aerobic degrading bacterium capable of degrading neonicotinoid insecticides was isolated from the enrichment liquid, named B27. The bacterium has obvious degrading effect on imidacloprid, acetamiprid and nitenpyramine within a certain time and at a certain concentration. The colony of the bacterium is milky white, wet Figure 1 Gram staining positive. Under transmission electron microscope, the bacterial cells are spherical, non-flagellated and do not form spores. Peroxidase test is positive.

[0033] The genomic DNA of the strain B27 was used as a template for PCR amplification with bacterial 16S rRNA gene sequence universal primers, and a 16S rDNA gene sequence with a length of about 1482 bp was obtained, as shown in SEQ ID No: 1. Alignment in EzBioCloud database (www.EzBioCloud.net) showed that the strain B27 had the closest homology with Flexivirga sp. strain, the closest phylogenetic relationship with Flexivirga alba ST13 T (AB539735), and the 16S rDNA gene similarity was 96.89%, and a phylogenetic tree was established Figure 2 The strain was subjected to whole genome sequencing, and the highest values of average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) were obtained by comparing the genome with the genomes of Flexivirga alba ST13 T , etc., which were 83.0%, 83.2% and 25.2%, respectively, lower than the identification thresholds of 95%, 95% and 70% for species. Combined with morphological and physiological and biochemical characteristics, the strain B27 was preliminarily identified as a new species of Flexivirga sp., named Flexivirga sp. B27. The strain was sent to China Center for Type Culture Collection (CCTCC) located in Wuhan, Wuhan University, and the preservation number was CCTCC NO: M 2022981, and the preservation date was 2022.6.27.

[0034] Example 2

[0035] Effect of strain B27 on imidacloprid degradation in basic salt medium:

[0036] The final concentration of imidacloprid added in the basic salt medium (same as in Example 1) was 20, 50 mg·L -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 bacterial degrading agent of strain B27

[0046] 1) The strain B27 colony on the plate is inoculated into a test tube for 18h culture, and the cultured test tube liquid is inoculated into the fermentation medium at a volume ratio of 1%, and is cultured in a logarithmic phase to prepare a fermentation seed;

[0047] 2) The above cultured fermentation seed is inoculated into a 5L seed tank with a medium volume of 65% (based on the volume of the fermentation tank, the same below) at a volume ratio of 5% (v / v, based on the volume of the medium) for culture, the sterile air flow is 1:0.8 vvm, the stirring speed is 200 rpm, the culture temperature is 28°C, and the culture is carried out to the logarithmic growth phase to prepare a seed liquid;

[0048] 3) The seed liquid is inoculated into a production tank with a medium volume of 65% at a volume ratio of 5% (v / v, based on the volume of the medium, the same below) for culture; the stirring speed is 200 rpm, the culture temperature is 28°C, and the fermentation time is 36h. After the fermentation is completed, the culture liquid is directly packed into a liquid agent by a packaging bottle.

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

[0050] Example 5

[0051] Fermentation of the degrading agent:

[0052] 1) The strain B27 colony on the plate is inoculated into a test tube for 18h culture, and the cultured test tube liquid is inoculated into the fermentation medium at a volume ratio of 1.5%, and is cultured in a logarithmic phase to prepare a fermentation seed;

[0053] 2) The above cultured fermentation seed is inoculated into a 5L seed tank with a medium volume of 65% (based on the volume of the fermentation tank, the same below) at a volume ratio of 8% (v / v, based on the volume of the medium) for culture, the sterile air flow is 1:0.8 vvm, the stirring speed is 220 rpm, the culture temperature is 30°C, and the culture is carried out to the logarithmic growth phase to prepare a seed liquid;

[0054] 3) The seed liquid is inoculated into a production tank with a medium volume of 65% at a volume ratio of 8% (v / v, based on the volume of the medium, the same below) for culture; the stirring speed is 220 rpm, the culture temperature is 30°C, and the fermentation time is 24h. After the fermentation is completed, the culture liquid is directly packed into a liquid agent by a plastic packaging barrel.

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

[0056] Example 6

[0057] Preparation method of the degradation bacteria agent

[0058] The preparation method of the degradation bacteria agent in Example 6 is basically the same as that in Example 5, except that the aeration amount of sterile air in the culture process of the seed tank and the production tank in steps 2) and 3) 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 the strain B27 degradation bacteria agent on neonicotinoid insecticides in soil

[0061] The soil sample without neonicotinoid insecticides in the vegetable field is used as the test soil sample. The soil sample is passed through a 2mm sieve, and a certain amount of imidacloprid, acetamiprid and nitenpyram is evenly mixed into 500g of soil, so that the final concentration of imidacloprid, acetamiprid and nitenpyram in the soil is 10mg·kg -1 The fresh logarithmic phase bacterial liquid of the strain B27 is collected, washed with sterile deionized water for 3 times, and then suspended with sterile water. The concentration of the bacterial body is adjusted to about 1.0×10 9 cfu·mL -1 The inoculation amount is 5mL / 100g, which is inoculated into the above soil, and cultured in a constant temperature dark incubator at 30℃. The soil without inoculation is used as a control. The water holding capacity of the soil is maintained at 60% during the culture. After 10 days of culture, the residual amount is determined by high performance liquid chromatography. The results show that the degradation rates of the strain B27 on imidacloprid, acetamiprid and nitenpyram in the soil within 10 days are 71.04%, 73.51% and 71.27% respectively.

Claims

1. A strain of degrading bacteria B27, characterized in that, The B27 is identified as Flexivirga sp., which has been preserved in China Center for Type Culture Collection, with a preservation date of June 27, 2022, and a preservation number of CCTCC NO: M2022981.

2. Application of the degrading bacteria B27 in degrading neonicotinoid insecticides according to claim 1.

3. Use according to claim 2, characterized in that, Application of the degrading bacteria B27 in simultaneously degrading neonicotinoid insecticides in soil or water environment.

4. Use according to claim 2 or 3, characterized in that, The neonicotinoid insecticide is preferably one or more of imidacloprid, acetamiprid and nitenpyram.

5. Use according to claim 2 or 3, characterized in that, The degradation condition is natural pH and temperature of 28-37℃.

6. Application of the new species B27 in producing a neonicotinoid insecticide degrading bacteria agent according to claim 1.

7. The degrading microbial agent of claim 6, wherein, The fermentation of the bacteria agent includes the following steps: 1) inoculating the strain B27 into a culture medium for activation treatment; 2) inoculating the activated bacteria liquid into a culture medium in a seed tank for culture until the logarithmic growth phase to obtain a seed liquid; 3) inoculating the seed liquid into a culture medium in a production tank for culture; and after fermentation, the culture liquid is discharged from the tank and packaged into a liquid agent.

8. Application of the degrading bacteria agent in degrading neonicotinoid insecticides according to claim 6.

9. Use according to claim 8, characterized in that, Application of the degrading bacteria agent in degrading neonicotinoid insecticides in soil or water environment.

10. Use according to claim 8 or 9, characterized in that, The neonicotinoid insecticide is one or more of imidacloprid, acetamiprid and nitenpyram.

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