Soybean root nodule endogenous fomesafen degrading bacterium and application thereof

Abstract

The invention relates to a soybean root nodule endogenous fomesafen degrading bacterium and application thereof. The classification name of the fomesafen degrading bacterium is Mycobacterium sp. W109, and the preservation number of the fomesafen degrading bacterium is CCTCC (China Center for Type Culture Collection) NO: M 20251729. According to the invention, a fomesafen degrading bacterium is separated from soybean root nodules, the bacterium can stably degrade fomesafen, and the variety of the fomesafen degrading bacterium is expanded.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology, specifically relating to a strain of endophytic flusulfanilamide-degrading bacteria in soybean root nodules and its application. Background Technology

[0002] Fomesafen is a diphenyl ether herbicide widely used in leguminous crop fields. It has a long residual period in the soil, with a half-life of 100-240 days. Long-term use has led to increasingly serious problems with herbicide residues in the soil, directly inhibiting the growth of current and subsequent leguminous crops, resulting in reduced yields, damaging the soil micro-ecosystem, and posing a risk to human health through the food chain.

[0003] Currently, microbial-phytoremediation technology is widely studied for the remediation of soils contaminated by herbicides due to its good environmental compatibility. This technology aims to integrate the degradation capabilities of microorganisms with the root system activity of plants to increase the colonization capacity of degrading bacteria in the environment and overcome the limitations of single remediation models. In recent years, researchers have screened microorganisms capable of degrading diphenyl ether herbicides using various methods, including bacteria, fungi, and actinomycetes. Research on bacteria has been the most extensive, while research on actinomycetes has been the least, and studies on actinomycetes mainly focus on the degradation of ethoxysulfuron. However, the degradation enzyme systems of individual microorganisms are limited, and there are differences between different diphenyl ether herbicides. Most microorganisms can only degrade a specific diphenyl ether herbicide. Therefore, developing different types of diphenyl ether herbicide-degrading microorganisms remains a current research direction and can also support the development of compound microbial agents. Summary of the Invention

[0004] The primary objective of this invention is to address the aforementioned deficiencies in the prior art by providing a strain of endophytic flusulfanilamide-degrading bacteria in soybean root nodules.

[0005] The first objective of this invention can be achieved through the following technical solutions: A strain of flusulfanilamide-degrading bacteria endophytic in soybean root nodules, characterized by its classification as Mycobacterium ( Mycobacterium sp.)W109, collection number CCTCC NO: M 20251729.

[0006] The present invention further provides a method for culturing the above-mentioned flusulfanilamide-degrading bacteria.

[0007] In some embodiments of the present invention, the flusulfanilamide-degrading bacteria are cultured in LB medium.

[0008] In some embodiments of the present invention, the flusulfanilamide-degrading bacteria are cultured under neutral pH conditions.

[0009] In some embodiments of the present invention, the flusulfanilamide-degrading bacteria are cultured at 25-35°C.

[0010] The present invention further provides the application of the above-mentioned flusulfanil degrading bacteria in flusulfanil degradation.

[0011] In some embodiments of the present invention, the seed liquid of the flumethrin-degrading bacteria is inoculated into a flumethrin-containing culture medium; or it is applied to a flumethrin-containing farmland.

[0012] In some embodiments of the present invention, the seed culture of the flusulfanil degrading bacteria is inoculated into an inorganic salt culture medium containing flusulfanil to carry out flusulfanil degradation.

[0013] In some embodiments of the present invention, the content of flusulfanilamide in the inorganic salt culture medium is 10-50 mg / L.

[0014] In some embodiments of the present invention, the flusulfanilamide-degrading bacteria are cultured in LB medium to the end of the logarithmic growth phase, and then washed with PBS to obtain seed culture.

[0015] In some embodiments of the present invention, the flusulfanil degrading bacteria degrade flusulfanil under neutral conditions at 25-35°C.

[0016] This invention isolates a flusulfanilamide-degrading bacterium from soybean root nodules. This bacterium is classified as Mycobacterium and is effective against 5 mg / L of flusulfanilamide. -1 / 50 mg L -1 The degradation rates of flusulfanil reached 76% and 62% respectively, demonstrating stable degradation of flusulfanil and expanding the range of flusulfanil-degrading bacteria. Attached Figure Description

[0017] Figure 1 This is a diagram showing the colony morphology and phylogenetic relationship of strain W109 in this invention.

[0018] Figure 2 This refers to the degradation rate of flusulfanilamide by strain W109 under pure culture conditions in this invention.

[0019] The biological material described in this invention is classified and named Mycobacterium ( Mycobacterium sp.) W109 was deposited on July 30, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number: CCTCC NO: M20251729, address: Wuhan, China. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Example 1

[0021] Isolation and screening of W109 Strain was isolated from soybean nodule in soybean farmland which was applied with twice field concentration of fomesafen, and the specific isolation method was as follows: (1) Surface disinfection of nodule: soybean roots (including nodules) were collected, the plant surface dirt was washed with running water, and the water was absorbed; the nodule part was cut with sterile scissors, and the nodule was surface disinfected according to the steps of 75% ethanol soaking for 30 s - sterile water washing for 3 times - 0.3% sodium hypochlorite soaking for 6 min - sterile water washing for 5 times; the nodule content was crushed with sterile forceps and added into 100 mL fomesafen only carbon source inorganic salt medium (containing 10 mg L -1 ).

[0022] (2) Enrichment culture: the above culture solution was cultured at 30°C, 150 rpm for 5 d, and 5 mL of the enrichment culture was inoculated into fresh 100 mL inorganic salt medium with fomesafen as the only carbon source, and the culture was continued for 5 d, and the transfer was repeated 5 times. The initial concentration of fomesafen was 10 mg L -1 , the concentration of fomesafen was increased by 10 mg L -1 each time, and the final concentration of fomesafen was 60 mg L -1 .

[0023] (3) Isolation and purification: the sixth generation of enrichment culture was diluted by 10 -4 , 10 -5 , 10 -6 times, respectively, 100 μL of the diluted solution was spread on the inorganic salt medium plate containing 20 mg L -1 fomesafen, and cultured at 30°C for 3-4 days, and single colonies were picked and purified by streaking for 3 times. Example 2

[0024] Classification and identification of strain W109 The classification and identification of strain W109 was carried out by 16S rRNA gene sequence analysis method, and the specific steps were as follows: (1) Extraction of total bacterial DNA: the genomic DNA of strain W109 was extracted by using MiniBEST Bacterial Genomic DNA Extraction Kit of TaKaRa.

[0025] (2) The amplification of 16S rRNA gene sequence of strain W109: the universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') / 1492R (5'-CGGYTACCTTGTTACGACTT-3') were used, and 2xTaq Master Mix from Nanjing Novogene Bioinformatics Technology Co., Ltd. was used for the preparation of PCR premix, and the system was as follows: 2xTaq Master Mix 25 μL, 1 μL of each of F and R primers, 1 μL of template DNA, and dd H2O was added to 50 μL; the reaction cycle parameters were as follows: pre-denaturation at 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 35 cycles; total extension at 72°C for 10 min.

[0026] (3) Cloning and sequencing analysis of the PCR product: the strain was sent to General Biosystems (Anhui) Co., Ltd. for sequencing, and the 16S rRNA gene sequence of the strain was subjected to nucleotide sequence alignment on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov), and a number of nucleotide sequences with homology to the gene sequences of related strains were obtained, indicating that the 16S rRNA sequence of strain W109 had a homology of more than 99% with the gene sequence of Mycobacterium (Mycobacterium) Mycobacterium ), and the isolated strain was identified as Mycobacterium sp. Mycobacterium .

[0027] (4) The morphological characteristics of strain W109 were identified as follows: Sinorhizobium W109 belongs to actinomycetes, and the colony diameter is about 0.1-0.2 cm, the colony on the LB plate is white, round, with an intact edge, and the surface is moist. The optimal growth conditions of the strain are as follows: pH 7.0 and temperature 30°C. Example 3

[0028] Analysis of the degradation ability of strain W109 to different concentrations of mefenacet (1) Liquid degradation test: mefenacet was dissolved in methanol to prepare a mefenacet stock solution with a concentration of 1000 mg L -1 , and an appropriate amount of the stock solution was taken in a sterile flask, and after the methanol was volatilized, the above-mentioned inorganic salt medium after high-temperature high-pressure sterilization was added, so that the concentration of mefenacet was 5 mg L -1 / 50 mg L -1W109 bacteria liquid stored in glycerol tube was streaked on LB plate under sterile conditions and cultured at 30°C until single colony formation, and a single colony was inoculated in LB liquid medium (ingredients: 10.0 g / L peptone, 5.0 g / L yeast powder, 10.0 g / L NaCl, pH 7.0) and cultured at 30°C, 150 rpm until the end of the logarithmic growth of the strain, and then the seed liquid was obtained after washing with PBS, and was transferred into the above-mentioned inorganic salt medium with fomesafen as the sole carbon source at an inoculation amount of 5%, and was placed in a 30°C, 150 rpm shaking culture for 7 days.

[0029] The inorganic salt medium with fomesafen as the sole carbon source described in the present embodiment has the following ingredients: 5 mg / 50 mg fomesafen, 0.9 g KH2PO4, 6.5 g NaHPO4·12H2O, 0.4 g (NH4)2SO4, 0.2 g MgSO4·7H2O and 1 mL Ferrari trace element solution per liter, pH 7.2; The Ferrari trace element solution contains 500 mg EDTA·2Na, 5 mg MnSO4·H2O, 5 g Na2MoO4·2H2O, 30 mg H3BO4, 5 mg CuSO4·5H2O, 10 mg ZnSO4·7H2O, 24 mg CoSO4·7H2O and 50 mg Ca(OH)2 per liter.

[0030] The preparation method is as follows: Dissolve fomesafen in methanol to prepare a fomesafen stock solution with a concentration of 1000 mg / L. -1 In a sterile operation table, take an appropriate amount of stock solution in a sterile flask, and after the methanol evaporates, add the above-mentioned inorganic salt medium after high-temperature high-pressure sterilization, so that the concentration of fomesafen is 5 mg / L -1 / 50 mg / L -1 .

[0031] (2) Extraction of fomesafen: mix 3 mL of culture solution with an equal amount of dichloromethane and vortex for 1 min, dry the organic phase over Na2SO4 to remove water, take 1 mL of organic phase solution, add 1 mL of acetonitrile, filter through a 0.22 µm organic filter membrane, and store in a 4°C refrigerator for testing.

[0032] (3) Determination of fomesafen: accurately weigh 0.0103 g (accurate to 0.0001 g) of fomesafen (99.7%) standard substance with an electronic balance, dissolve and dilute to 10 mL with chromatographically pure acetonitrile, and prepare a solution with a concentration of 1000 mg / L.-1 Accurately transfer appropriate amounts of the stock solution and dilute them with acetonitrile to prepare 1 mg / L solutions. -1 5 mg L -1 10 mg L -1 20 mg L -1 50mg L -1 100 mg L -1 200 mg L -1 A series of standard solutions were prepared. The concentration of flufenoxuron was determined by high-performance liquid chromatography (HPLC) using a ZORBAX SB-C... 18 The reverse-phase chromatographic column (4.6 × 150 mm) was used for separation, with a column temperature of 30℃ and a mobile phase of acetonitrile:water (0.5% phosphoric acid) = 65:35 (v:v), at a flow rate of 1.0 mL / min. -1 The detection wavelength was 290 nm, and the injection volume was 20 µL. The retention time of flusulfanilamide was 3.468 min.

[0033] Depend on Figure 2 It is evident that after 7 days of culture, strain W109 showed resistance to 5 mg L... -1 / 50 mg L -1 The degradation rates of flusulfanil and sulfanil reached 76% and 62%, respectively, indicating that strain W109 has a relatively stable degradation effect on flusulfanil.

Claims

1. A soybean endophytic strain of fomesafen-degrading bacteria, characterized in that, The classification name is Mycobacterium sp. Mycobacterium W109, with the preservation number of CCTCC NO: M 20251729.

2. The culture method of the bacteria degrading fomesafen according to claim 1, characterized by, The fluorochloridone-degrading bacteria are cultured in LB medium.

3. The culture method of the bacteria degrading fomesafen according to claim 1, characterized by, The fluorochloridone-degrading bacteria are cultured under neutral pH condition.

4. The culture method of the bacteria degrading fomesafen according to claim 1, characterized by, The fluorochloridone-degrading bacteria are cultured under 25-35℃ condition.

5. The fluorochloridone-degrading bacteria of claim 1 are used in fluorochloridone degradation.

6. Use according to claim 5, characterized in that, The seed liquid of the fluorochloridone-degrading bacteria is inoculated into the medium containing fluorochloridone; or is sprayed onto the farmland containing fluorochloridone.

7. Use according to claim 5, characterized in that, The seed liquid of the fluorochloridone-degrading bacteria is inoculated into the inorganic salt medium containing fluorochloridone, and fluorochloridone degradation is carried out.

8. Use according to claim 5, characterized in that, The fluorochloridone content in the inorganic salt medium is 10-50 mg / L.

9. Use according to claim 6 or 7, characterized in that, The fluorochloridone-degrading bacteria are cultured in LB medium to the end of logarithmic growth, and the seed liquid is obtained after washing with PBS.

10. Use according to claim 5, characterized in that, The fluorochloridone-degrading bacteria degrade fluorochloridone under 25-35℃ and neutral condition.

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