Flumetralin low-temperature degradation bacteria, bacterial agent and application

Abstract

A kind of fomesafen low-temperature degradation bacteria, bacterial agent and application, it relates to the field of microorganism, specifically to a kind of herbicide fomesafen low-temperature degradation bacteria, bacterial agent comprising the degradation bacteria, and the application of the degradation bacteria and its bacterial agent in degrading fomesafen.It solves the problem that there is lack of microorganism strain capable of efficiently degrading fomesafen under low temperature condition in prior art, which restricts the biological remediation of fomesafen residue in cold region farmland soil.The present application is a kind of herbicide fomesafen low-temperature degradation bacteria, and the degradation bacteria is Arthrobacter FH1.The strain can efficiently degrade fomesafen under 15 DEG C low-temperature condition, and has degradation capacity at multiple temperatures of 4 DEG C-35 DEG C.The present application also provides bacterial agent comprising the strain and its application in degrading fomesafen.The present application is suitable for cold region farmland soil remediation, fomesafen pollution treatment, low-temperature biological remediation and other fields.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a low-temperature degrading bacterium for the herbicide flusulfanil, an inoculum containing the degrading bacterium, and the application of the degrading bacterium and its inoculum in the degradation of flusulfanil. Background Technology

[0002] Flusulfanil is a diphenyl ether herbicide primarily used in soybean fields to control broadleaf weeds, and it also has a suppressive effect on grass weeds. Because this herbicide degrades slowly, with a half-life ranging from 45 days to one year, long-term use leads to its gradual accumulation in the soil, posing a potential threat to subsequent sensitive crops and human health.

[0003] Currently, there are reports on the microbial degradation of flusulfanilamide, and the degrading bacteria identified include Bacillus (…). Bacillus (sp.), etc. However, most existing degrading bacteria are room-temperature adapted, and their metabolic activity is significantly inhibited at low temperatures below 15°C, with a 7-day degradation rate generally below 20%, which is insufficient to meet the remediation needs of low-temperature regions. In addition, traditional screening methods for degrading bacteria usually use enrichment culture conditions of 25-30°C, which is not conducive to effectively enriching low-temperature adapted microorganisms, resulting in insufficient exploration of low-temperature degrading bacteria resources.

[0004] In summary, the core problem facing existing technologies is the lack of microbial strains capable of efficiently degrading flumethrin under low-temperature conditions, which restricts the bioremediation of flumethrin residues in farmland soils in cold regions. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Arthrobacter* capable of efficiently degrading flusulfanilamide under low-temperature conditions. Arthrobacter sp.) FH1, containing this Arthrobacterium (sp.) FH1, Arthrobacter The bacterial agent of sp.) FH1, and the Arthrobacter (sp.) Arthrobacter Application of sp.) FH1 and its inoculant in the degradation of flusulfanilamide.

[0006] A low-temperature degrading bacterium for flusulfanilamide, wherein the low-temperature degrading bacterium is Arthrobacter ( Arthrobacter sp.)FH1, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2026314, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on February 2, 2026.

[0007] The application of the flusulfanil-degrading bacteria in the degradation of flusulfanil.

[0008] In a further preferred embodiment, the application temperature is 4℃~35℃.

[0009] An inoculum containing the flusulfanilamide low-temperature degrading bacteria.

[0010] The application of the microbial agent in the degradation of flusulfanilamide.

[0011] In a further preferred embodiment, the application temperature is 4℃~35℃.

[0012] A cold-region soil remediation agent comprising the aforementioned flusulfanilamide low-temperature degrading bacteria.

[0013] The beneficial effects of this invention compared to the prior art are as follows: This invention obtains a strain of Arthrobacter (Bacillus) by isolating and screening from soybean farmland soil in cold regions. Arthrobacter FH1 sp. achieved efficient degradation of flusulfanilamide under low temperature conditions, solving the technical problem that the metabolic activity of existing room temperature degrading bacteria is significantly inhibited and the degradation rate is generally less than 20% at temperatures below 15°C.

[0014] This invention provides a solution containing Arthrobacter ( Arthrobacter The FH1 microbial agent (sp.) enables convenient application and stable preservation of this low-temperature degrading bacterium, providing a directly applicable product form for the bioremediation of flumethrin residues in farmland soils in cold regions.

[0015] This invention utilizes Arthrobacter ( Arthrobacter When sp.) FH1 or its inoculum is applied to degrade flusulfanilamide, it can achieve efficient removal of flusulfanilamide after 7 days of cultivation at 15℃ and 180 rpm, providing an effective bioremediation method for the treatment of herbicide pollution in cold agricultural areas of northern China.

[0016] This invention verifies Arthrobacter ( Arthrobacter The ability of sp.)FH1 to degrade flusulfanil in a temperature range of 4℃ to 35℃ demonstrates the applicability of this strain over a wide temperature range, making it suitable not only for low-temperature environments but also for degradation under normal temperature conditions.

[0017] The flusulfanil low-temperature degrading bacteria and their inoculants described in this invention are applicable to fields such as farmland soil remediation in cold regions, flusulfanil pollution control, and low-temperature bioremediation. Attached Figure Description

[0018] Figure 1 It is the Arthrobacterium described in Embodiment 1 ( Arthrobacter Colony morphology of sp. FH1 on inorganic salt medium; Figure 2 It is the Arthrobacterium described in Embodiment 2 ( Arthrobacter Developmental tree of sp.)FH1; Figure 3 It is the Arthrobacterium described in Embodiment 3 (Arthrobacter OD600 values ​​of sp.) FH1 with flusulfanil as the sole carbon source at low temperature; Figure 4 It is the Arthrobacterium described in Embodiment 3 ( Arthrobacter Degradation efficiency of FH1 for flusulfanilamide under low temperature conditions (sp.) Figure 5 It is the Arthrobacterium described in Embodiment 4 ( Arthrobacter OD600 values ​​of sp.) FH1 with flusulfanil as the sole carbon source under different temperature conditions; Figure 6 It is the Arthrobacterium described in Embodiment 4 ( Arthrobacter Degradation efficiency of FH1 at different temperatures for flusulfanilamide (sp.) Detailed Implementation

[0019] Various embodiments of the present invention will now be clearly and completely described with reference to the accompanying drawings. The embodiments described with reference to the drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0020] The low-temperature degrading bacteria for flusulfanilamide described in this invention are Arthrobacter ( Arthrobacter sp.)FH1, deposited at the China Center for Type Culture Collection (Wuhan University), with accession number CCTCC NO: M 2026314, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on February 2, 2026.

[0021] Implementation Method 1: Arthrobacter ( Arthrobacter Acquisition of sp.)FH1.

[0022] The arthrobacterium ( Arthrobacter FH1 (sp.) was isolated and screened from soybean farmland soil in Jiamusi City, Heilongjiang Province in May 2025.

[0023] Arthrobacter ( Arthrobacter Separation method of sp.) FH1: Accurately weigh 5 g of soil and place it in a 150 mL Erlenmeyer flask containing 45 mL of sterile physiological saline. Then, place the flask in a shaker and shake at 15°C and 180 rpm for 1 hour, followed by standing for 1 hour. Next, take 5 mL of the supernatant and add it to 50 mL of sterile inorganic salt medium, and incubate with shaking at 15°C and 180 rpm. After seven days of incubation, take 10 mL of the bacterial suspension and place it in a sterile centrifuge tube, centrifuging at 4°C and 5000 rpm for 10 min. Then, resuspend the bacterial suspension with 2 mL of sterile physiological saline and centrifuge for another 2 min, repeating this process three times. Finally, take 1 mL of the bacterial suspension and add it to 50 mL of sterile inorganic salt medium for subculturing and enrichment, continuing the subculturing process for 10 generations.

[0024] Arthrobacter ( Arthrobacter Purification method of sp.) FH1: After passage, the microorganisms were collected by centrifugation and resuspended in physiological saline. The bacterial culture was then diluted to 10⁻⁶. -6 The culture dish was then spread onto a solid inorganic salt medium supplemented with agar using a sterile spreader. The dish was inverted and placed in an incubator at 15°C for static incubation. Once single colonies had grown, [further details needed]. Figure 1 As shown, the strain was purified twice on LB solid medium using the streak plate method to obtain a single strain. A portion of the single colonies were used for subsequent experiments, while the other portion was mixed with glycerol and stored at -80°C for long-term preservation.

[0025] Implementation Method 2: Arthrobacter ( Arthrobacter Identification of sp.) FH1.

[0026] DNA was extracted from the strain isolated in Example 1 using an alkaline lysis method. 16S rDNA amplification was performed using primers 27F / 1492R and P1 / P6, followed by sequencing. Sequence alignment was performed using the GenBank database. The results showed that this strain was related to… Arthrobacter sp. CMU6 (DQ985470) belongs to the same branch, with a similarity of over 99%, and is named Arthrobacter (…). Arthrobacter sp.)FH1.

[0027] The arthrobacterium ( Arthrobacter The 16S rDNA gene sequence of *Sp.* FH1 is shown in SEQ ID NO: 1, and is 1363 bp in length. Nucleotide BLAST was used to compare its 16S rDNA sequence with other available sequences in the GenBank database. BLAST analysis results showed that this strain is similar to *Arthrobacter* (…). ArthrobacterThis strain is highly related to *Arthrobacter* sp. Based on 16S rDNA sequences from the GenBank database, a phylogenetic tree was constructed using the neighbor-joining method. The results showed that this strain is located in the same branch as related strains of the genus *Arthrobacter*, and it was named *Arthrobacter* sp. Arthrobacter sp.) FH1. Among them, Arthrobacter ( Arthrobacter The developmental tree of sp.)FH1 is as follows Figure 2 As shown.

[0028] Implementation Method 3: Arthrobacter ( Arthrobacter Culture of sp.) FH1.

[0029] The Arthrobacterium ( Arthrobacter sp.) FH1 was inoculated into LB liquid medium and cultured at 15°C for 3 days. Then, the cells were obtained by centrifugation at 12000 rpm for 10 min, and washed and resuspended using sterile physiological saline. The bacterial concentration was diluted to 10-1. 7 CFU·mL -1 The bacterial suspension was inoculated at a ratio of 1:100 (V / V) onto sterile substrate containing flusulfanilamide (10 mg·mL⁻¹). -1 The bacteria were cultured in an inorganic salt medium at 15°C and 180 rpm for 7 days. The OD600 value and the concentration of flusulfanilamide were then measured.

[0030] Figure 3 Arthrobacter ( Arthrobacter The OD600 value of *Sp.* FH1 strain with flufenoxuron as the sole carbon source under low temperature conditions is shown in the figure. After culturing at 15°C for 7 days, the OD600 value of the blank control group (CK) was 0.0370. Inoculation with *Arthrobacter* (sp.) Arthrobacter The OD600 value of the experimental group of *Arthrobacter sp.* FH1 was 0.0547. The increase in OD600 value indicates that *Arthrobacter sp.* (sp.) Arthrobacter The FH1 strain was able to grow and metabolize using flusulfanil as the sole carbon source under low-temperature conditions, further confirming the degradation effect of flusulfanil by this strain.

[0031] Figure 4 Arthrobacter ( Arthrobacter The degradation efficiency of flusulfanil by sp.) FH1 under low temperature conditions is shown in the figure. After culturing at 15℃ for 7 days, the degradation efficiency of flusulfanil in the blank control group (CK) was only 0.470%, indicating that the non-biodegradation of flusulfanil was negligible under the experimental conditions; inoculation with Arthrobacter ( Arthrobacter The degradation efficiency of the FH1 strain reached 28.496%, indicating that the strain has the ability to degrade flusulfanilamide under low temperature conditions.

[0032] Implementation Method 4: Arthrobacter ( ArthrobacterApplication of sp.)FH1.

[0033] The present invention uses Arthrobacterium ( Arthrobacter sp.) FH1 was inoculated into LB liquid medium and cultured at 15°C for 3 days. Then, the cells were obtained by centrifugation at 12000 rpm for 10 min, and washed and resuspended using sterile physiological saline. The bacterial concentration was diluted to 10-1. 7 CFU·mL -1 The bacterial suspension was inoculated at a ratio of 1:100 (V / V) onto sterile substrate containing flusulfanilamide (10 mg·mL⁻¹). -1 The bacteria were cultured in inorganic salt medium at temperatures of 4℃, 15℃, 25℃, and 35℃ at 180 rpm. Samples were taken after 7 days to determine the OD600 value and the concentration of flusulfanilamide.

[0034] Figure 5 Arthrobacter ( Arthrobacter OD600 values ​​of *Arthrobacter sp.* FH1 under different temperature conditions with flusulfanil as the sole carbon source. After culturing for 7 days at 4℃, 15℃, 25℃, and 35℃, the OD600 value of the blank control group (CK) was 0.0370, while the OD600 values ​​of the experimental groups were 0.0387, 0.0547, 0.0637, and 0.0587, respectively. The results indicate that *Arthrobacter sp.* FH1... Arthrobacter The FH1 sp. can grow and metabolize using flusulfanilamide as the sole carbon source at temperatures of 4℃, 15℃, 25℃ and 35℃, and its growth is more active at temperatures of 15℃, 25℃ and 35℃.

[0035] Figure 6 Arthrobacter ( Arthrobacter The degradation efficiency of *Arthrobacter sp.* FH1 against flusulfanilamide at different temperatures is shown in the figure. After culturing for 7 days at 4℃, 15℃, 25℃, and 35℃, the degradation efficiency of the blank control group (CK) was 0.470%, while the degradation efficiencies of the experimental groups were 10.352%, 28.496%, 32.320%, and 20.659%, respectively. The results indicate that *Arthrobacter sp.* FH1... Arthrobacter FH1 sp. can effectively degrade flusulfanilamide in a temperature range of 4℃ to 35℃, with the highest degradation efficiency at 15℃ to 25℃. Notably, it maintains a high degradation rate of 28.5% even at a low temperature of 15℃, which is significantly better than the existing room-temperature degrading bacteria, whose degradation rate is generally below 20% at temperatures below 15℃.

[0036] The arthrobacterium ( Arthrobacter The strain FH1 can efficiently degrade flumethrin under 15℃ cultivation conditions. The strain described in this invention has important technical value for solving the herbicide pollution problem in cold agricultural areas of northern China and ensuring the sustainable development of agriculture.

Claims

1. A low-temperature degrading bacterium for flusulfanilamide, characterized in that, The low-temperature degrading bacteria is Arthrobacter ( ) Arthrobacter sp.)FH1, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2026314, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on February 2, 2026.

2. The application of the flusulfanilamide low-temperature degrading bacteria according to claim 1 in the degradation of flusulfanilamide.

3. The application according to claim 2, characterized in that, The application temperature is 4℃~35℃.

4. A microbial agent comprising the flusulfanilamide low-temperature degrading bacteria as described in claim 1.

5. The application of the microbial agent according to claim 4 in the degradation of flusulfanilamide.

6. The application according to claim 5, characterized in that, The application temperature is 4℃~35℃.

7. A cold-region soil remediation agent comprising the flusulfanilamide low-temperature degrading bacteria as described in claim 1.

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