CN110904005B - Fomesafen degrading bacterium and application thereof - Google Patents
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Abstract
The invention relates to a sulfafurazone degrading bacterium which is classified and named as sinorhizobium (A)Sinorhizobiumsp.) W16 with the preservation number of CCTCC NO: m2019915. The degrading bacteria sieve is selected from soybean root nodules, can stably degrade fomesafen, can be applied to farmlands applying fomesafen for treatment and restoration, and is more beneficial to development and application compared with other soil bacteria in the prior art.
Description
Technical Field
The invention belongs to the field of environmental microorganisms, and particularly relates to fomesafen degrading bacteria and application thereof.
Background
Fomesafen (Fomesafen) is a diphenyl ether herbicide and is widely applied to the control of broadleaf weeds in fields of leguminous crops such as soybeans, peanuts and the like. The half-life period of fomesafen in soil is longer, and reaches 100-240 days. Since the 90 s of the 20 th century, the large-area use of fomesafen causes the problem of pesticide residue in farmlands for planting leguminous crops to become increasingly serious. The residue of large amounts of herbicide in the field can have a series of effects: firstly, the yield of leguminous crops and the standing seedlings of succeeding crops are influenced; ② resistant weeds are generated; thirdly, the ecological environment of the soil is destroyed and the soil function is influenced by influencing the activity of soil microorganisms and soil animals; and fourthly, the water enters the human body through drinking water or a soil-plant system through a food chain, and the health of the human body is harmed. Therefore, the remediation of the herbicide pollution of the farmland soil of the leguminous crops and the improvement of the crop yield become environmental problems to be solved urgently.
The existing restoration technology applied to restoration of herbicide-polluted soil mainly comprises physical restoration, chemical restoration, biological restoration and the like. The bioremediation method has a long remediation period for high-concentration organic contaminated soil, but is an optimal method for remedying the soil with low and medium pollution levels such as herbicide pollution of the farmland due to the advantages of economy, environmental protection, difficult damage to an ecological system and the like. The microorganism-plant combined remediation technology can overcome the defects that microorganism remediation is easily influenced by the environment and is difficult to colonize, the incomplete efficiency of a plant remediation and degradation enzyme system is low, and the like, and gradually becomes the trend of the development of the organic contaminated soil bioremediation technology. A plurality of researchers at home and abroad adopt a microorganism-plant combined remediation technology to restore the medium-low level organic polluted soil to obtain a good effect.
Researchers have separated fomesafen degrading bacteria in farmland and obtained the bacteria belonging to the group comprisingShigella、Pseudomonas、LysinibacillusFomesafen degrading bacteria belonging to the genus of isophthora. However, at present, the means of inoculating efficient degradation microorganisms are still mostly adopted for repairing fomesafen soil, and the process of repairing fomesafen polluted soil by utilizing microorganisms faces the problems that the survival and degradation activities of microorganisms are easily affected by the environment and the colonization is difficult. Therefore, the obtained fomesafen degrading rhizobia capable of symbiotic with the leguminous crops has important significance for alleviating phytotoxicity and increasing the yield of the leguminous crops in fields.
Disclosure of Invention
The invention aims to provide fomesafen degrading bacteria and application thereof aiming at the defects in the prior art.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect, the invention provides a sulfafurazone degrading bacterium which is classified and named as Sinorhizobium (A)Sinorhizobiumsp.) W16 belonging to gram-negative bacteria, deposited under the accession number: CCTCC NO: m2019915.
In a second aspect, the invention provides a method for culturing the fomesafen degrading bacteria, which comprises the steps of inoculating the fomesafen degrading bacteria to a culture medium, and culturing at 30 ℃.
Further, the culture medium is a yeast-mannitol (YMA) culture medium, and comprises the following components: 10.0 g/L mannitol, 0.4 g/L yeast powder, 0.5 g/L K 2 HPO 4 ，0.2 g/L MgSO 4 ·7H 2 O，0.1 g/L NaCl；pH 7.2。
In a third aspect, the invention provides an application of the fomesafen degrading bacterium in fomesafen degradation, which comprises the following steps: activating fomesafen degrading bacteria, preparing seed liquid, and inoculating the seed liquid into an inorganic salt culture medium containing fomesafen for degradation.
In the application of the invention, the inorganic salt culture medium comprises the following components: fomesafen, 0.9 g/L KH 2 PO 4 ，6.5 g/L NaHPO 4 ·12H 2 O，0.4 g/L (NH 4 ) 2 SO 4 ，0.2 g/L MgSO 4 ·7H 2 O, 1 mL of Ferrari trace element solution, and the pH value is 7.2; the Ferrari trace element solution is as follows: 500 mg/L EDTA.2Na, 5 mg/L MnSO 4 ·H 2 O，5 mg/L Na 2 MoO 4 ·2H 2 O，30 mg/L H 3 BO 4 ，5 mg/L CuSO 4 ·5H 2 O，10 mg/L ZnSO 4 ·7H 2 O，24 mg/L CoSO 4 ·7H 2 O，50 mg/L Ca(OH) 2 。
The content of fomesafen in the inorganic salt culture medium is 10-50 mg/L.
The preparation method of the seed liquid comprises the following steps: inoculating fomesafen degrading bacteria to YMA culture medium, culturing at 30 ℃ and 150 rpm until the last logarithmic growth stage of the strain, and obtaining seed liquid.
The inoculation amount of the seed liquid is 5%; the degradation conditions are as follows: 30 ℃ and pH 7.2.
The invention screens out a strain W16 of Sinorhizobium from soybean root nodule, can stably degrade fomesafen, can be applied to farmlands applying fomesafen for treatment and restoration, and is more beneficial to development and application compared with other soil bacteria in the prior art.
Drawings
FIG. 1 shows the colony morphology of Rhizobium degradans W16 according to the present invention.
FIG. 2 is a diagram showing the phylogenetic relationship of Rhizobium degradans W16 in the present invention.
FIG. 3 is a liquid chromatogram and a standard curve of fomesafen extracted from the degradation culture solution in the present invention.
FIG. 4 is a graph showing the change of the degradation rate of the strain W16 under different fomesafen concentrations.
The biological material of the present invention is classified and named as Sinorhizobium (A)Sinorhizobiumsp.) W16, which has been deposited in China Center for Type Culture Collection (CCTCC) at 11.11.2019 with the following deposition numbers: CCTCC NO: m2019915, address: wuhan in China.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
The invention provides a fomesafen degrading bacterium which is named as Sinorhizobium by classificationSinorhizobiumsp.) W16 strain with the following accession number: CCTCC NO: m2019915.
Note: the discovery of researchers in 2003SinorhizobiumAndEnsifethe similarity of the 16S rRNA gene sequences of different strains in the r genus ranges from 97.9% to 99.9%, the strains are recommended to be combined into one genus, and the international committee for bacteria classification adjudges in 2009EnsiferThe name of the legal genus is shown as,Sinorhizobiumfor synonyms, currently in the official journal of taxonomy- "InternWhen a new species belonging to the phylogenetic branch of the two genera is published in the scientific Journal of Systematic and evolution Microbiology "(IJSEM), it is necessary to useEnsiferAs the generic name. A review article published in 2016 by the institute of Rhizobium research, university of agriculture, China, and the institute of biological institute of agriculture, China, suggested that if isolated from root nodules, it can form nodules with legumes to fix nitrogen, and can also form nodules with legumes to fix nitrogenSinorhizobiumSome approaches in the background, in the study of the work related to nodulation nitrogen fixation, published articles in the journal of non-ijses SinorhizobiumSo that the reader can more easily understand the contents of the article without ambiguity. Therefore, the Rhizobium degradans in the present invention is considered to belong toSinorhizobiumBelongs to the field of medicine.
The strain belongs to gram-negative bacteria and is named as W16, and the morphological characteristics of the strain are as follows: the diameter of the colony is about 0.2-0.5 cm, the colony on the LB plate is white and round, the edge is neat, and the surface is wet and sticky. The optimal growth conditions of the strain are as follows: pH7.2, temperature 30 ℃.
Further, the strain is sent to Beijing Optimalaceae New Biotechnology Limited for sequencing, and the sequence of 16S rRNA of the strain is determined to be shown in SEQ ID NO: 1, the gene sequence is registered in a national center for biotechnology information (http:// www.ncbi.nlm.nih.gov) to carry out nucleotide sequence Blast comparison to obtain a plurality of nucleotide sequences with homology with the gene sequence of related strains, which shows that the 16S rRNA sequence of the strain W16 and the genus Jian (B) (I) areEnsifer) Rhizobium zhonghua (A)Sinorhizobium) The homology of the gene sequence is more than 99 percent, and the separated strain is identified as the sinorhizobium genus: (a)Sinorhizobium sp.）。
The bacterial preparation provided by the invention is a yeast-mannitol (YMA) culture medium of the strain W16.
The YMA medium comprises the following components: each liter contains 10.0 g mannitol, 0.4 g yeast powder, 0.5 g K 2 HPO 4 ，0.2 g MgSO 4 ·7H 2 O，0.1 g NaCl；pH 7.2。
The degradation performance of the strain W16 on fomesafen is illustrated below.
Examples the fomesafenThe inorganic salt medium with the only carbon source comprises the following components: 10 mg/20 mg/50 mg of fluorine-containing sulfadiazine ether and 0.9 g KH per liter 2 PO 4 ，6.5 g NaHPO 4 ·12H 2 O，0.4 g (NH 4 ) 2 SO 4 ，0.2 g MgSO 4 ·7H 2 O and 1 mL of Ferrari trace element solution, and the pH value is 7.2; the Ferrari trace element solution is as follows: each liter contains 500 mg EDTA-2 Na, 5 mg MnSO 4 ·H 2 O，5 mg Na 2 MoO 4 ·2H 2 O，30 mg H 3 BO 4 ，5 mg CuSO 4 ·5H 2 O，10 mg ZnSO 4 ·7H 2 O，24 mg CoSO 4 ·7H 2 O，50 mg Ca(OH) 2 。
The preparation method comprises the following steps:
dissolving fomesafen in methanol to prepare 1000 mg L -1 Taking a proper amount of the mother liquor in an aseptic operation table, adding the mother liquor into an aseptic triangular flask after methanol volatilizes, and adding the inorganic salt culture medium sterilized at high temperature and high pressure to ensure that the concentration of the fomesafen is 10 mg L -1 /20 mg L -1 /50 mg L -1 。
Inoculating Chinese rhizobium W16 in inorganic salt culture medium with only carbon source of fomesafen, culturing at 30 deg.C and 150 rpm for 7 d, strain W16 to 10 mg L -1 /20 mg L -1 /50 mg L -1 The degradation rates of fomesafen are respectively 52.9%, 43.6% and 35.4%, which shows that the rhizobium has stable degradation effect on fomesafen and has good application prospect.
Example 1
Isolation and screening of Rhizobium W16
The strain is separated from soybean nodules in a soybean farmland applied with fomesafen with twice field concentration, and the specific separation method is as follows:
(1) Surface disinfection of the nodule: collecting soybean roots (including root nodules), washing the surface of the plant with running water to remove dirt, and sucking water; shearing root nodule with sterile scissors, soaking in 75% ethanol for 30 s, washing with sterile water for 3 times, soaking in 0.3% sodium hypochlorite for 6 min, and washing with sterile water for 5 timesStep one, carrying out surface disinfection on the root nodules; crushing the root nodule with sterile tweezers, adding the root nodule content into 100 mL of fomesafen sole carbon source inorganic salt culture medium (10 mg L of fomesafen) -1 ）。
(2) Enrichment culture: and (3) carrying out shake culture on the culture solution for 5 d under the conditions of 30 ℃ and 150 rpm, inoculating 5 mL of enriched culture solution into a fresh 100 mL of fomesafen unique carbon source inorganic salt culture medium, continuing to carry out culture for 5 d, and repeating the transfer for 5 times. The initial concentration of fomesafen is 10 mg L -1 The concentration of fomesafen is increased by 10 mg L each time -1 The final concentration of fomesafen is 60 mg L -1 。
(3) Separation and purification: the sixth generation of the enriched culture solution is added with 10 -4 、10 -5 、10 -6 Diluting by multiple times, respectively taking 100 muL of diluent and coating the diluent on the surface of the product containing 20 mg L of diluent -1 And (3) carrying out inverted culture on a fomesafen inorganic salt culture medium plate at 30 ℃ for 3-4 days, picking out a single colony, and carrying out streaking purification for 3 times.
(4) Rhizobium screening: the formula of the YMA Congo red solid medium is as follows: each liter contains 10.0 g mannitol, 0.4 g yeast powder, 0.5 g K 2 HPO 4 ，0.2 g MgSO 4 ·7H 2 O, 0.1 g of NaCl and 5 mL of 1% Congo red solution; the pH was 7.2. And (3) scribing the single colony obtained by purification on a YMA Congo red plate, wherein the colony grown on the Congo red culture medium is white due to the fact that symbiotic rhizobia generates a large amount of extracellular polysaccharide, and the colonies of other bacteria are red, and selecting the white single colony on the Congo red plate to obtain the target strain.
Example 2
Classification and identification of Rhizobium W16
The 16S rRNA gene sequence analysis method is adopted to classify and identify the strain W16, and the specific steps are as follows:
(1) extraction of total DNA of bacteria: the genomic DNA of the strain W16 was extracted using the bacterial genomic DNA extraction kit of TaKaRa MiniBEST.
(2) Amplification of rRNA Gene sequence of Strain W1616S: the universal primer 27F (5' -AGAGAGTTT) is adoptedGATCMTGGCTCAG-3 ')/1492R (5 ' -CGGYTACCTTGTTACGACTT-3 '), PCR premix was prepared using 2 XTaq Master Mix from Nanjing Novozam Biotechnology Ltd as follows: 2 XTaq Master Mix 25 muL, F, R primers are 1 muL each, template DNA is 1 muL, dd H 2 Supplementing and leveling to 50 mu L; reaction cycle parameters: pre-denaturation at 95 ℃ for 5 min; at 95 ℃ for 30 s, at 55 ℃ for 30 s, at 72 ℃ for 1 min, and circulating for 35 min; total extension 72 ℃ for 10 min.
(3) Clonal sequencing analysis of PCR products: the strain is sent to Beijing Optimalaceae New industry biotechnology limited company for sequencing, the 16S rRNA gene sequence of the strain is compared with the nucleotide sequence of the national center of biotechnology information (http:// www.ncbi.nlm.nih.gov) to obtain a plurality of nucleotide sequences with homology with the gene sequence of the related strain, which shows that the 16S rRNA sequence of the strain W16 and the Jian 'er (Jian' er) ((R) ())Ensifer) Rhizobium zhonghua (A)Sinorhizobium) The homology of the gene sequence is more than 99 percent, and the separated strain is identified as the sinorhizobium genus: (a)Sinorhizobium sp.）。
(4) The morphological characteristics of strain W16 were identified as follows:
the rhizobium meliloti W16 belongs to gram-negative bacteria, the diameter of a bacterial colony is about 0.2-0.5 cm, the bacterial colony on an LB plate is white and round, the edge is neat, and the surface is wet and viscous. The optimal growth conditions of the strain are as follows: pH7.2, temperature 30 ℃.
Example 3
Analysis of degradation capability of strain W16 on fomesafen with different concentrations
(1) Liquid degradation test: dissolving fomesafen in methanol to prepare 1000 mg L -1 Taking a proper amount of the mother liquor in an aseptic operation table, adding the mother liquor into an aseptic triangular flask after methanol volatilizes, and adding the inorganic salt culture medium sterilized at high temperature and high pressure to ensure that the concentration of the fomesafen is 10 mg L -1 /20 mg L -1 /50 mg L -1 (ii) a Streaking the W16 bacterial liquid stored in glycerol tube on YMA plate under aseptic condition, culturing at 30 deg.C until single colony forms, selecting single colony, inoculating in YMA liquid culture medium, culturing at 30 deg.C and 150 rpm until the strain number is logarithmic to growthAnd in the long-term stage, obtaining seed liquid, inoculating the seed liquid into the inorganic salt culture medium which takes fomesafen as the only carbon source in an inoculation amount of 5 percent, and performing shake culture for 7 days at the temperature of 30 ℃ and the speed of 150 rpm.
(2) Extraction of fomesafen: mixing 3 mL of culture solution with equal amount of dichloromethane, vortexing and shaking for 1 min, and drying the organic phase with Na 2 SO 4 Removing water, adding 1 mL of organic phase solution into a glass test tube, blowing nitrogen until the solution is dry, adding 1 mL of acetonitrile for dissolution, filtering the solution through a 0.22 mu m organic filter membrane, and placing the solution in a refrigerator at 4 ℃ for detection.
(3) Determination of fomesafen: accurately weighing 0.0103 g (accurate to 0.0001 g) of fomesafen (99.7%) standard substance with electronic balance, dissolving with chromatographic pure acetonitrile, and diluting to 100 mL to obtain 100 mg L -1 Accurately transferring appropriate amount of stock solution, and diluting with acetonitrile to 1 mg L -1 ，5 mg L -1 ，10 mg L -1 ，20 mg L -1 ，50 mg L -1 ，100 mg L -1 ，200 mg L -1 The series of standard solutions of (1). Measuring fomesafen concentration by high performance liquid chromatography (ZORBAX SB-C) 18 The (4.6X 150 mm) reverse phase chromatographic column is a separation column, the column temperature is 30 ℃, the mobile phase is acetonitrile, water (0.5 percent phosphoric acid) =65:35 (v: v), the flow rate is 1.0 mL min -1 And detecting the wavelength of 290 nm, and the sample size of 20 muL. The retention time of fomesafen is 3.468 min.
As can be seen from FIG. 4, after 7 days of culture, strain W16 was added to 10 mg L -1 、20 mg L -1 、50 mg L -1 The degradation rates of fomesafen are respectively 52.9%, 43.6% and 35.4%, which shows that the rhizobia has more stable degradation effect on fomesafen.
Sequence listing
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tgggtgtcta gagtatggaa gaggtgagtg gaattccgag tgtagaggtg aaattcgtag 600
atattcggag gaacaccagt ggcgaaggcg gctcactggt ccattactga cgctgaggtg 660
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tgttagccgt cgggcagttt actgttcggt ggcgcagcta acgcattaaa cattccgcct 780
ggggagtacg gtcgcaagat taaaactcaa aggaattgac gggggcccgc acaagcggtg 840
gagcatgtgg tttaattcga agcaacgcgc agaaccttac cagcccttga catcccgatc 900
gcggattacg gagacgtttt ccttcagttc ggctggatcg gagacaggtg ctgcatggct 960
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Claims (2)
1. A sulfafurazone ether degrading bacterium is characterized in that the bacterium is classified and named as sinorhizobiumSinorhizobiumsp, W16 with a preservation number of CCTCC NO: m2019915.
2. The method for culturing fomesafen degrading bacteria according to claim 1, wherein the fomesafen degrading bacteria are inoculated to a culture medium and cultured at 30 ℃; the culture medium is a yeast-mannitol culture medium and comprises the following components: 10.0 g/L mannitol, 0.4 g/L yeast powder, 0.5 g/L K 2 HPO 4 ，0.2 g/L MgSO 4 ·7H 2 O，0.1 g/L NaCl；pH 7.2。
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| CN105861358A (en) * | 2016-04-07 | 2016-08-17 | 浙江大学 | Fomesafen degrading bacterium and application thereof |
| CN108841748A (en) * | 2018-06-28 | 2018-11-20 | 新疆农业大学 | Sinorhizobium nitrogen-fixing bacteria strain H6 and its application |
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