Use of pseudomonas knackmussii WHP-AP2

Abstract

The present invention relates to a use of Pseudomonas knackmussii WHP-AP2. The Pseudomonas knackmussii WHP-AP2 has a deposit number of GDMCC NO: 64331. The Pseudomonas knackmussii WHP-AP2 of the present invention can quickly and efficiently degrade acephate at a degradation rate of 96% or more, can degrade acephate having a wide range of pH values, is resistant to high-concentration acephate, and has good application prospects in the control of environmental pollution in acephate soil, water sources, etc.

Description

Application of Pseudomonas kuribda WHP-AP2

[0001] This invention claims priority to Chinese Patent Application No. 202410780815X, filed on June 17, 2024, entitled "Application of Pseudomonas kuribsiella pneumoniae WHP-AP2", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of microbial technology and relates to Pseudomonas kuribica, specifically the application of Pseudomonas kuribica WHP-AP2. Background Technology

[0003] Acephate is a broad-spectrum organophosphorus insecticide with stomach poison and contact action, and it can kill eggs. It also has some fumigation effect and is a slow-acting insecticide. Its initial effect is slow, with significant effects after 2-3 days. Due to its low toxicity, it was once widely used as a substitute for the highly toxic organophosphorus pesticide methamidophos in crops such as vegetables, tea trees, tobacco, fruit trees, cotton, cabbage, wheat, rice, and rapeseed to control various chewing and piercing-sucking pests and mites. However, residue testing has revealed the presence of small amounts of methamidophos in its formulations. Furthermore, its poor stability means that both the formulation and degradation within crops can release methamidophos, leading to methamidophos residues in agricultural products and soil. Countries and regions such as the EU, New Zealand, and Brazil have already implemented bans or restrictions on its use. Acephate is hydrophilic and has weak soil adsorption at 25°C. Therefore, under normal environmental conditions, it can contaminate groundwater with its active ingredient, acephate, and may harm aquatic organisms. A study has shown that acephate is mobile in almost all soils. Compared to other organophosphorus pesticides, acephate is relatively stable in terms of toxicity and chemical properties, but high concentrations are harmful to humans. Acetaminophen inhibits the central nervous system by suppressing the activity of acetylcholinesterase in insects and humans, leading to muscle weakness and encephalopathy, ultimately resulting in death.

[0004] Studies have found that biodegradation is an effective way to remove pesticide residues. Compared with traditional physical and chemical methods, biodegradation has advantages such as high efficiency, low cost, and safety, and has received widespread attention in recent years. Acephate-degrading strains use acephate as their sole source of carbon, phosphorus, and energy, thus playing a role in protecting the ecological environment. Therefore, exploring the influencing factors of microbial degradation of acephate and identifying strains that can efficiently degrade acephate can provide a theoretical basis for targeted regulation of influencing factors in the microbial control of acephate pollution. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide the application of Pseudomonas kuribsiella pneumoniae WHP-AP2, which can rapidly and efficiently degrade acephate with a degradation rate of over 96%.

[0006] The technical solutions for achieving the above objectives include, for example, those mentioned above.

[0007] The first aspect of the present invention is the use of Pseudomonas knackmussii WHP-AP2 in the preparation of acephate degrading agents, wherein the preservation number of Pseudomonas knackmussii WHP-AP2 is GDMCC NO: 64331.

[0008] A second aspect of the present invention is to provide the application of Pseudomonas knackmussii WHP-AP2 in the degradation of acephate, wherein the preservation number of Pseudomonas knackmussii WHP-AP2 is GDMCC NO: 64331.

[0009] The third aspect of this invention is the application of Pseudomonas knackmussii WHP-AP2 in the remediation of acephate pollution in soil, wherein the preservation number of Pseudomonas knackmussii WHP-AP2 is GDMCC NO: 64331.

[0010] The fourth aspect of the present invention is the application of Pseudomonas knackmussii WHP-AP2 in the treatment of acetamiprid pollution in water, wherein the preservation number of Pseudomonas knackmussii WHP-AP2 is GDMCC NO: 64331.

[0011] A fifth aspect of the present invention is to provide a biological agent, the active ingredient of which includes Pseudomonas knackmussii WHP-AP2 as described above.

[0012] A sixth aspect of the present invention is to provide a method for the degradation of acephate, comprising the following steps: adding Pseudomonas knackmussii WHP-AP2 as described above to a sample containing acephate.

[0013] In some of these embodiments, the pH value of the *Pseudomonas knackmussii* WHP-AP2 strain that degrades acephate is 3–11.

[0014] In some of these embodiments, the pH value of the *Pseudomonas knackmussii* WHP-AP2 strain that degrades acephate is 5–9.

[0015] The *Pseudomonas knackmussii* WHP-AP2 described in this invention has been deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), a depositary unit designated by the State Intellectual Property Office of China. The deposit date was January 29, 2024, and the accession number was GDMCC NO: 64331.

[0016] The present invention has the following beneficial effects:

[0017] This invention obtained a strain of *Pseudomonas knackmussii* WHP-AP2 through screening, which can rapidly and efficiently degrade acephate, achieving a degradation rate of over 96%. Furthermore, it has a wide pH range for acephate degradation, is tolerant to high concentrations of acephate, and can grow and survive in high-concentration acephate environments while still possessing the ability to degrade acephate. Therefore, it shows great promise for the remediation of acephate-contaminated soil and water. Attached Figure Description

[0018] Figure 1 is a transmission electron microscope image and a colony morphology diagram of Pseudomonas knackmussii WHP-AP2 of the present invention.

[0019] Figure 2 is a phylogenetic tree diagram constructed from the 16S rRNA sequence of Pseudomonas knackmussii WHP-AP2 of this invention.

[0020] Figure 3 shows the change in the degradation rate of acephate by Pseudomonas knackmussii WHP-AP2 at different pH values ​​over time.

[0021] Figure 4 shows the degradation of different concentrations of acephate by Pseudomonas knackmussii WHP-AP2 over time. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0023] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0025] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0026] The present invention will be further described in detail below with reference to specific embodiments.

[0027] Example 1: Isolation and screening of acephate-degrading bacteria WHP-AP2

[0028] Farmland soil samples were collected, and an environment rich in acephate pesticide was created by adding acephate to the soil samples for enrichment culture. Soil samples were added to liquid culture medium, and acephate was added to achieve an initial acephate concentration of 100 mg / L and a soil concentration of 100 g / L. The samples were incubated on a shaker for seven days, and then sequentially transferred to liquid culture media with higher acephate concentrations: 200 mg / L, 400 mg / L, and 800 mg / L. This was repeated for three generations. In the fourth generation, the enrichment culture was diluted and plated.

[0029] The enriched culture medium was diluted using a tenfold serial dilution method, resulting in a concentration of 10... -1 10-2 10 -3 10 -4 10 -5 0.1 ml of each diluted solution was evenly spread onto solid selective medium (15 g / L agar was added to the liquid medium). The inoculated plates were inverted and cultured in a 30°C constant temperature anaerobic incubator until new colonies grew. Single colonies were randomly selected for verification of acephate degradation function. The strain with better degradation efficiency was selected and purified in fresh liquid to obtain a pure strain named WHP-AP2. The bacterial solution was stored in 30% sterile glycerol at -80°C.

[0030] The liquid culture medium used above mainly includes: MgSO4·7H2O 0.2g / L, CaCl2·2H2O 0.01g / L, FeSO4·7H2O 0.001g / L, and 1ml / L of trace elements and vitamins.

[0031] Example 2: Strain Identification

[0032] (1) Morphological identification

[0033] The colony morphology of the WHP-AP2 strain screened in Example 1 was observed on solid agar plates (Figure 1). The WHP-AP2 strain grew on LB medium, and the colonies were round, light yellow, with neat edges, and about 1 mm in diameter. Microscopic morphological observation of the colonies was performed using an optical microscope (Figure 1). The results showed that the bacterial cells were short elliptical, about 1-2 μm long and about 0.4 μm wide.

[0034] (2) Molecular biological identification

[0035] DNA extraction from the strain: The bacterial strain preserved in Example 1 was inoculated onto liquid culture medium and cultured in a 30°C anaerobic incubator in the dark. After 24 hours of culture, the culture medium was removed, centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded to collect the precipitated bacterial cells. Total DNA of the isolated strain was extracted using the EZNATM Bacterial DNA Kit (Omega, USA), and the extracted DNA was stored at -20°C.

[0036] PCR amplification: The 16S rRNA gene of the bacteria was amplified using universal primers 27F (sequence 5′-AGAGTTTGATC MTGGCTCAG-3′, SEQ ID NO.1) and 1492R (sequence 5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO.2).

[0037] The amplification conditions were: 94℃ pre-denaturation for 4 minutes; 94℃ denaturation for 30 seconds; 55℃ annealing for 45 seconds; and 72℃ extension for 60 seconds, for a total of 30 cycles.

[0038] Gel electrophoresis: The PCR amplification products were detected by 1.0% agarose gel electrophoresis (1×TAE electrophoresis buffer, 150V / cm, approximately 30 min) and sent to BGI Genomics for sequencing. The sequence of its 16S rRNA gene is shown in SEQ ID NO.3 below:

[0039] The sequences were submitted to the EzBioCloud database (http: / / www.ezbiocloud.net / ) for homology comparison. Similarity information of the target strain was analyzed, and the taxonomic position of the strain was determined. Based on the alignment results, relevant sequences were selected, and a phylogenetic tree was constructed using the Mega 6.0NJ method. The constructed 16S rRNA phylogenetic tree is shown in Figure 2. The results show that the sequence has a similarity of over 99% to the 16S rDNA gene sequence of *Pseudomonas knackmussii* B13, thus confirming that strain WHP-AP2 belongs to the genus *Pseudomonas* (*Pseudomonas knackmussii*).

[0040] Example 3: Study on the degradation of acephate by strain WHP-AP2

[0041] The preparation of liquid culture medium and the preparation of bacterial suspension are the same as in Example 1;

[0042] WHP-AP2 cultured in aerobic LB medium to the logarithmic growth phase, then inoculated at a 1% (v / v) inoculation rate into fresh MSM medium containing 100 mg / L acephate at different pH values ​​(4, 5, 6, 7, 8, 9, 10). The media were incubated in the dark at 30°C on a shaker at 180 rpm. Acetaminophen content was measured every 12 hours. 3 ml of the filtrate was filtered through a 0.22 μm filter, and the acephate content was determined using a high-performance liquid chromatograph (Agilent). The acephate degradation rate curve over time was recorded and plotted. The acephate degradation rate was calculated using the following formula: (acephate content at detection point - initial acephate content) / initial acephate content * 100%.

[0043] As shown in Figure 3, WHP-AP2 can degrade acephate under pH conditions ranging from 4 to 10, with the optimal pH range for acephate degradation being 5 to 9, and the degradation rate of acephate reaching over 96%.

[0044] Example 4: Study on the tolerance concentration of acephate to strain WHP-AP2

[0045] The preparation of liquid culture medium and the preparation of bacterial suspension are the same as in Example 1;

[0046] WHP-AP2 was cultured in aerobic LB medium to the logarithmic growth phase. Then, at a 1% (volume percentage) inoculum, it was inoculated into MSM medium at pH 7 containing 100 mg / L, 200 mg / L, 400 mg / L, 800 mg / L, and 1600 mg / L acephate, respectively. The medium was incubated in the dark at 30°C on a shaker at 180 rpm. Samples were taken every 12 hours, and 3 ml of the sample was filtered through a 0.22 μm filter. The acephate content of the filtrate was determined using high-performance liquid chromatography (HPLC). The degradation rate of acephate was calculated using the same formula as described in Example 3.

[0047] Figure 4 shows the degradation effect of WHP-AP2 on acephate at different concentrations. Calculations showed that the degradation rate of acephate by this bacterium exceeded 93% under concentrations ranging from 100 mg / L to 800 mg / L. Furthermore, even with a concentration of 1600 mg / L acephate, WHP-AP2 could still grow and survive, and still possessed a certain degradation ability for acephate (degradation rate close to 20%), indicating that this bacterium has tolerance to high concentrations of acephate.

[0048] In summary, this invention provides a strain of Pseudomonas knackmussii WHP-AP2 that can rapidly and efficiently degrade acephate with a degradation rate of over 96%, and is tolerant to high concentrations of acephate. It has great application potential in the treatment of acephate pollution in the environment.

[0049] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of Pseudomonas knackmussii WHP-AP2 for the preparation of a degrader of acephate, characterized in that, The Pseudomonas knackmussii WHP-AP2 has a preservation number of GDMCC NO: 64331.

2. Use of Pseudomonas knackmussii WHP-AP2 for the degradation of acephate, characterized in that, The Pseudomonas knackmussii WHP-AP2 has a preservation number of GDMCC NO: 64331.

3. Use of Pseudomonas knackmussii WHP-AP2 for remediating soil contaminated with acephate, characterized in that, The Pseudomonas knackmussii WHP-AP2 has a preservation number of GDMCC NO: 64331.

4. Use of Pseudomonas knackmussii WHP-AP2 for the remediation of water contaminated with acephate, characterized in that, The Pseudomonas knackmussii WHP-AP2 has a preservation number of GDMCC NO: 64331.

5. A biological agent, characterized in that, The active ingredient of the biological agent includes Pseudomonas knackmussii WHP-AP2, and the Pseudomonas knackmussii WHP-AP2 has a preservation number of GDMCC NO: 64331.

6. A method of degrading acephate, characterized by, The method comprises the following steps: Pseudomonas knackmussii WHP-AP2 is added into a sample containing acephate, and the Pseudomonas knackmussii WHP-AP2 has a preservation number of GDMCC NO: 64331.

7. The method of degradation of acephate according to claim 6, wherein, The Pseudomonas knackmussii WHP-AP2 degrades acephate at a pH value of 3-11.

8. The method of degradation of acephate according to claim 7, wherein, The Pseudomonas knackmussii WHP-AP2 degrades acephate at a pH value of 5-9.

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