Pseudomonas LH-2024W capable of efficiently degrading bromadiolone and application of pseudomonas LH-2024W

Abstract

The invention provides a pseudomonas LH-2024W capable of efficiently degrading bromadiolone and application of the pseudomonas LH-2024W, and belongs to the technical field of microorganisms and environmental remediation. The strain is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is March 4, 2025, and the preservation number is CCTCC M 2025375, the strain is named as Pseudomonas wadenensis, and the strain is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC M 2025375. The strain has higher and more stable degradation efficiency on rodenticide bromadiolone, the degradation efficiency is as high as 94.15%, and compared with other types of pseudomonas, the strain has obvious advantages. The invention determines that the LH-2024W has optimal degradation efficiency when the pH is 7, the temperature is 30 DEG C and the bacterium concentration is 1%. According to the method, LH-2024W is utilized to decompose bromadiolone, so that the method not only has the advantages of ecological environmental protection, high safety and the like, but also does not cause secondary pollution.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Pseudomonas LH-2024W capable of efficiently degrading bromadiolone and application thereof. Background Art

[0002] Rodents pose a threat to public health, important habitats, native plants and animals, crops, food and water supplies. Due to the serious rodent infestation, my country uses a large amount of the rodenticide bromadiolone. However, bromadiolone may cause social and environmental safety problems. While killing rats, the bromadiolone remaining in the environment may pose a potential health risk to some invertebrates, fish, wild birds and mammals, and even human health, which may even be life-threatening in severe cases. Bromadiolone usually has a strong physiological persistence in the liver of vertebrates, allowing rodents exposed to bromadiolone to continue to survive for several days and eat poisoned baits. Predatory animals eating poisoned rodents will cause secondary poisoning of predators, leading to the death of non-target species. It is reported that many non-target mammals and birds have been contaminated by bromadiolone, including direct ingestion of poisoned baits and indirect poisoning transmitted through the food chain. In addition, when the rodenticide bromadiolone is applied to eliminate or control rodents, it will enter the soil environment through its runoff and dissolution, causing pollution. Therefore, it is of great significance to study the degradation of bromadiolone in the environment.

[0003] Although the use of bromadiolone will pollute the soil environment, there is currently no special treatment method for the degradation of bromadiolone. The development of microbial agents from the natural soil environment to decompose bromadiolone not only has the advantages of ecological protection and high safety, but also will not cause secondary pollution. The development and application of this method will provide new ideas and ways to solve the problem of bromadiolone pollution.

[0004] Species of the genus Pseudomonas are present in almost all hydrocarbon-contaminated areas and play a special role in the biodegradation of these exogenous substances, as this genus has the potential to decompose various hydrocarbons and phenolic compounds, using them as the sole carbon source. Pseudomonas may play an important role in the degradation of bromadiolone and contribute to environmental protection. Summary of the invention

[0005] The purpose of the present invention is to provide a safe and environmentally friendly microbial agent for decomposing the soil environment contaminated by bromadiolone to reduce the potential risks to animal and human health.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Pseudomonas wadenswilerensis LH-2024W strain capable of efficiently degrading bromadiolone. The strain is deposited in China Center for Type Culture Collection, with a deposit address of Wuhan University, Wuhan, China. The deposit date is March 4, 2025, and the deposit number is CCTCC M 2025375.

[0008] The invention also provides a bromadiolone degradation agent with Pseudomonas wadenswilerensis LH-2024W as a main active ingredient.

[0009] The invention also provides the use of the Pseudomonas wadenswilerensis LH-2024W or the bromadiolone degradation agent in degrading bromadiolone.

[0010] Preferably, the use as claimed in claim 3 is characterized in that, when degrading bromadiolone, the live bacteria concentration of Pseudomonas wadenswilerensis LH-2024W is 0.5-2%, the pH is 6.8-7.2, and the degradation temperature is 28-30°C.

[0011] The present invention provides a strain of Pseudomonas wadenswilerensis LH-2024W, which has a high and relatively stable degradation efficiency for the rodenticide bromadiolone, and the degradation efficiency within 5 days is as high as 94.15%, which has obvious advantages over other types of Pseudomonas such as Pseudomonas vancouverensis and Pseudomonas mandelii. The present invention studies the degradation conditions of LH-2024W and determines that LH-2024W has the best degradation efficiency when the pH is 7, the temperature is 30°C, and the bacterial concentration is 1%. Developing microbial agents from natural soil environments to decompose bromadiolone not only has the advantages of ecological environmental protection and high safety, but also does not cause secondary pollution, and provides new ideas and approaches for solving the bromadiolone pollution problem. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The degradation comparison of LH-2024W and other strains in MSM medium;

[0013] Figure 2 This is the colony morphology of LH-2024W after being cultured on TSA plates containing bromadiolone for 24 hours;

[0014] Figure 3 This is the phylogenetic tree of LH-2024W;

[0015] Figure 4 The degradation of bromadiolone by LH-2024W at different bacterial concentrations;

[0016] Figure 5 The degradation of bromadiolone by LH-2024W at different pH values;

[0017] Figure 6 The degradation of bromadiolone by LH-2024W at different temperatures.

[0018] Collection Instructions

[0019] Pseudomonas wadenswilerensis LH-2024W, the strain is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is March 4, 2025, and the deposit number is CCTCC M2025375. DETAILED DESCRIPTION

[0020] In the present application, the formula of the tryptone soy agar (TSA) medium is (per liter): tryptone (15 g), soy peptone (5 g), sodium chloride (5 g) and agar powder (15 g);

[0021] The formula of LB solid medium is: tryptone (10 g), yeast powder (5 g), sodium chloride (10 g) and agar powder (15 g);

[0022] The formula of tryptic soy broth (TSB) is (per liter): tryptic soy broth (17 g), soybean papain hydrolysate (3 g), sodium chloride (5 g), potassium dihydrogen phosphate (2.5 g) and glucose (2.5 g).

[0023] Inorganic salt components in basal salt medium (MSM) (per liter): disodium hydrogen phosphate (2800 mg), potassium dihydrogen phosphate (1000 mg), ammonium sulfate (500 mg), EDTA disodium (0.5 mg), ferrous sulfate (heptahydrate) (0.2 mg), zinc sulfate heptahydrate (0.01 mg), manganese chloride tetrahydrate (0,003 mg), boric acid (0.03 mg), cobalt chloride hexahydrate (0.02 mg), cupric chloride dihydrate (0.001 mg), nickel chloride hexahydrate (0.002 mg), sodium molybdate dihydrate (0,003 mg), magnesium chloride hexahydrate (100 mg), calcium salt tetrahydrate (50 mg).

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1 Screening and identification of strains

[0026] (1) Soil was collected from Menyuan County, Qinghai Province, and placed in sterile ziplock bags and transported back to the laboratory. 10 g of soil sample was placed in 90 mL of sterile water, shaken for 10 min, and allowed to stand for 30 s. 100 μL of the sample was taken and placed in a 1.5 mL centrifuge tube containing 900 μL of sterile water to make 10 -1 The solution was mixed by pipetting, and then diluted in the same way to obtain 10 -2 and 10 -3 Solution;

[0027] (2) The obtained 10 -1 , 10 -2 and 10 -3 The solution was streaked and cultured on TSA (tryptone soy agar) and LB solid medium containing 0.005% bromadiolone to obtain single colonies, which were then sequenced with 16S rDNA. The 16S rDNA sequences were compared with BLAST, and a total of 17 strains were identified.

[0028] (3) The 17 isolated strains were activated and cultured in 100 mL TSB liquid medium (trypticase soy broth) containing bromadiolone (concentration 0.005%) at 30°C and 180 rpm for 24 h. 1 mL of the bacterial solution was taken and centrifuged at 8000 rpm for 1 min for a total of 3 times. The bacterial cells were then washed twice with physiological saline to obtain bacterial cells, which were diluted with physiological saline and measured using a UV-visible spectrophotometer at a wavelength of 600 nm (OD 600 ) was used to adjust the optical density of the bacteria to 0.6-1.0 to prepare the inoculum solution.

[0029] (4) The inoculated bacterial solution was inoculated into 200 mL of MSM medium (basal salt medium) containing 0.005% bromadiolone, and the mixture was shaken at 30°C and 180 rpm for 5 days. 10 mL of samples were taken at the beginning and the fifth day to measure the concentration of bromadiolone, and the degradation of bromadiolone by different strains was obtained. The results are as follows: Figure 1 The experimental group and the control group were repeated 3 times:

[0030] Experimental group: add inoculum solution to make the concentration of live bacteria in the culture medium 1%;

[0031] Control group: No bacteria were added, but 2 mL of normal saline was added.

[0032] like Figure 1, a total of 6 strains were able to degrade bromadiolone, among which Pseudomonas wadenswilerensis LH-2024W had the highest degradation efficiency, with the highest degradation rate within five days reaching 94.15%, followed by Pseudomonas jejuni with 94.14%, and the others did not reach 90%. The colony morphology of Pseudomonas wadenswilerensis LH-2024W on tryptic soy casein broth (TSB) was white, raised, opaque and smooth ( Figure 2 ). At the same time, 16S rDNA sequencing was performed on it, as shown in SEQ ID NO.1. The measured 16S rDNA sequence was compared by BLAST. The comparison results showed that the nucleotide sequence of 16S rDNA of strain Pseudomonas wadenswilerensis had greater than 99% homology with the nucleotide sequence of Pseudomonas. The phylogenetic tree is shown in Figure 3 shown.

[0033] 16S rDNA sequence of Pseudomonas wadenswilerensis LH-2024W (SEQ ID NO.1):

[0034]

[0035] Example 2 Study on degradation characteristics

[0036] Among the six strains, LH-2024W with the best degradation ability was selected for research on its degradation characteristics.

[0037] The strain LH-2024W was activated in TSB liquid medium and washed with physiological saline to obtain inoculum (the specific preparation method is the same as step (3) of Example 1). The bacterial solution OD 600 The value was adjusted to 0.6-1.0. Then different temperatures, pH values ​​and bacterial concentrations were set, and three parallel tests were set up. The cells were acclimated in MSM medium containing 0.005% bromadiolone for 5 days, and the initial and final concentrations of bromadiolone were measured to explore the degradation of bromadiolone by LH-2024W at different temperatures, pH values ​​and bacterial concentrations.

[0038] Experimental groups:

[0039] 1. Adjust the initial bacterial concentration in the culture medium to 0.5%, 1%, 2% and 3% respectively, and start culturing at a temperature of 30°C, a pH of 7 and a rotation speed of 180 rpm. The results are as follows: Figure 4 As shown in the figure, the degradation efficiency of bromadiolone showed a trend of first increasing and then decreasing with the increase of bacterial concentration in the culture medium. When the initial bacterial concentration in the culture medium was 1%, the degradation efficiency of bromadiolone reached the highest.

[0040] 2. Set different pH values: 5, 6, 7, 8, 9, and start culturing at a culture medium bacterial concentration of 1%, a temperature of 30°C, and a rotation speed of 180 rpm. Figure 5 As shown in the results, the degradation efficiency of bromadiolone reached the highest level at pH 7, while there was no significant degradation ability under other pH conditions.

[0041] 3. Set different temperature gradients: 20, 25, 30, 35 and 40°C, and start culturing at a medium bacterial concentration of 1%, pH 7, and a rotation speed of 180 rpm. Figure 6 As shown in the figure, the degradation efficiency of bromadiolone first increases and then decreases with the increase of culture temperature. When the culture temperature is 30℃, the degradation efficiency of bromadiolone reaches the highest, which is 94.15%.

[0042] In summary, it can be determined that the Pseudomonas wadenswilerensis LH-2024W provided by the present invention has the best degradation efficiency when the pH is 7, the temperature is 30°C, and the bacterial concentration is 1%, and has great application potential in the treatment of bromadiolone pollution and environmental restoration.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Pseudomonas wadenswilerensis LH-2024W strain that can efficiently degrade bromadiolone. The strain is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is March 4, 2025, and the deposit number is CCTCC M 2025375.

2. A bromadiolone degradation agent with Pseudomonas wadenswilerensis LH-2024W as claimed in claim 1 as the main active ingredient.

3. Use of the Pseudomonas wadenswilerensis LH-2024W described in claim 1 or the bromadiolone degrading agent described in claim 2 in degrading bromadiolone.

4. The use according to claim 3, characterized in that: When degrading bromadiolone, the concentration of live bacteria of Pseudomonas wadenswilerensis LH-2024W was 0.5-2%; pH 6.8-7.2; The degradation temperature is 28-30℃.

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