CN111088180B - Pyrazosulfuron-ethyl degradation strain BI-1 and application thereof - Google Patents
Pyrazosulfuron-ethyl degradation strain BI-1 and application thereof Download PDFInfo
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Abstract
The invention provides a pyrazosulfuron-ethyl degrading strain BI-1 and application thereof, and the preservation number of the strain is CCTCC M2019586. On the one hand, the high-efficiency degradation strain BI-1 obtained by separation has stable degradation property and strong environmental adaptability, has strong degradation capability in soil and water environments, and is suitable for popularization and use. On the other hand, the fermentation process for preparing the degradation microbial inoculum, and the settings of pH, ventilation, rotating speed and temperature ensure the activity of the strain and the number of viable bacteria after fermentation, and can be more effectively applied to the environmental pollution of the pyrazosulfuron-ethyl.
Description
Technical Field
The invention relates to a pyrazosulfuron-ethyl degradation strain BI-1 and application thereof.
Background
Sulfonylurea herbicides are mesityleneThe national dupont developed a high-efficiency, broad-spectrum herbicide in the 70 s of the 20 th century, currently the second largest class of herbicides, with global sales exceeding $ 23 billion, inferior to amino acid herbicides. The herbicide can inhibit synthesis of branched-chain amino acids (valine, leucine and isoleucine) by inhibiting activity of acetolactate synthase (ALS) in plants, thereby achieving weeding effect. Pyrazosulfuron-ethyl is a representative variety of sulfonylurea herbicides, and is widely used in China because of its high efficiency, low dosage per unit area, and good control effect on broadleaf weeds, sedge weeds and other malignant weeds in paddy fields. But the residual half-life of the pyrazosulfuron-ethyl is long, the pyrazosulfuron-ethyl is not easy to degrade in farmland soil, and the pyrazosulfuron-ethyl is not suitable for being used and is easy to cause phytotoxicity to various crops such as cucumber, corn, late rice and the like. 10% pyrazosulfuron-ethyl wettable powder is applied 4-5 d after direct seeding in paddy field, and the dosage exceeds 300g/hm 2 At the same time, the rice plants after 3 days can show phytotoxicity symptoms such as growth retardation, yellowing, few white roots and the like. Rape is sensitive to pyrazosulfuron, and the error Shi Bimi sulfuron in rape field can cause irrecoverable phytotoxicity, and other crops must be destroyed and cultivated for seed changing. The pyrazosulfuron ethyl residue also affects the community structure of soil microorganisms, and simultaneously reduces the oxidizing ability of the soil.
Pyrazosulfuron-ethyl is a representative species of sulfonylurea herbicides, and is widely used for chemical control of farmland weeds. However, along with the prolongation of the service life and the continuous increase of the application area, the residues in farmland soil are more and more serious, the damage to the soil ecological environment caused by the sensitive crop of the aftercrop is easily caused, and how to effectively remove the pyrazosulfuron-ethyl residues in the soil becomes a practical problem to be solved.
Microbial degradation is an environment-friendly, economical and practical repair means currently accepted. In recent years, a plurality of microorganisms capable of degrading pesticides are separated and screened by people, but the microbial degradation of the pyrazosulfuron is less in research report, and the strain degradation efficiency is lower, so that the further development and application of the microbial remediation technology of the pyrazosulfuron polluted environment are restricted.
Disclosure of Invention
The invention aims to provide a pyrazosulfuron-ethyl degradation strain BI-1 and application thereof, so that the pyrazosulfuron-ethyl degradation efficiency is safely and rapidly improved.
In particular, the invention provides a pyrazosulfuron-ethyl degrading strain BI-1, the preservation number of which is CCTCC M2019586.
The pyrazosulfuron-ethyl degrading strain BI-1 microbial inoculum is obtained by performing expansion culture on the pyrazosulfuron-ethyl degrading strain BI-1CCTCC M2019586.
The preparation method of the pyrazosulfuron-ethyl degradation strain BI-1 microbial inoculum comprises the following steps:
s1: activating the BI-1 strain on an R2A culture medium, and inoculating the strain on a test tube inclined plane for standby;
s2: selecting strain on the inclined plane, inoculating the strain into a shake flask containing an R2A culture medium, and shake culturing to a logarithmic phase;
s3: sterilizing at 115 ℃ under high pressure and moist heat after feeding, cooling to 30 ℃, inoculating the cultured shake flask strain into a fermentation tank according to 10% of inoculation amount, controlling the fermentation process according to a set fermentation environment, and ensuring that the number of thalli reaches more than 10 hundred million/mL after fermentation;
s4: and (5) discharging the culture solution from the tank after fermentation, and packaging the culture solution into liquid dosage forms by packaging bottles to obtain the pyrazosulfuron-ethyl degradation strain BI-1 microbial inoculum.
Preferably, according to the preparation method, the fermentation environment is: the temperature of the fermentation tank after inoculation is controlled at 30 ℃, and the ventilation rate of sterile air in the culture process is 1:0.6, the stirring speed is 180 rpm, the pH is 7.3, the feeding is carried out every 24 hours, and the culture time of the whole process flow is 96 hours.
Preferably, according to the preparation method, the formula of the R2A culture medium is as follows: yeast extract 0.5, peptone 0.5, casein 0.5, glucose 0.5, soluble starch 0.5, sodium pyruvate 0.3, K 2 HPO 4 0.3，MgSO 4 0.05, pH 7.2-7.5, in g/L.
The pyrazosulfuron degrading strain BI-1 or the pyrazosulfuron degrading strain BI-1 microbial inoculum is applied to preparation of pyrazosulfuron degrading agent.
Primer for amplifying the gene sequence of the pyrazosulfuron-ethyl degrading strain BI-116S rRNA, which is characterized in that the following primer is used for PCR amplification:
the upstream primer is 5'-AGAGTTTGATCCTGGCTCAG-3';
the downstream primer was 5'-TACCTTGTTACGACTT-3'.
Aiming at the practical problems and demands in production practice, the invention aims to separate and obtain a high-efficiency degradation strain of pyrazosulfuron, enrich germplasm resources, develop and develop a novel residual degradation microbial inoculum of pyrazosulfuron, reduce the residual quantity of pyrazosulfuron by more than 90 percent by using the microbial inoculum, and have lower production and use costs and wide application prospects.
The pyrazosulfuron-ethyl degrading bacteria disclosed by the invention are separated from farmland soil for long-term application of pyrazosulfuron-ethyl, and have good degradation effect on pyrazosulfuron-ethyl. The strain BI-1 is added to the culture medium for reaction for 3 days, 50mg/L of pyrazosulfuron ethyl can be completely degraded, and the strain is also a first strain degrading pyrazosulfuron ethyl in Chengangzhanghangella, so that ideal germplasm resources can be provided for repairing pyrazosulfuron ethyl polluted soil.
Compared with the traditional physicochemical repair, the microbial strain is used for repairing the pyrazosulfuron-ethyl polluted soil, the bacterial liquid extraction process is quicker, the field application becomes effective quickly, the secondary pollution to the environment can be avoided, and the method is more scientific and environment-friendly.
The preparation process disclosed by the invention has the advantages of simple flow, wide sources of raw materials, low separation and acquisition cost, good removal effect and easiness in large-scale popularization. The optimal growth and degradation temperature and pH of the strain BI-1 are respectively 30 ℃ and 7.0, the adaptability to the complex field restoration environment is strong, and the restoration effect is more remarkable. The strain BI-1 has a degradation effect on pyrazosulfuron-ethyl in soil and a recovery effect on corn growth, solves the problem of pyrazosulfuron-ethyl pollution in agricultural activities, and can produce green agricultural products without pesticide toxicity.
The above, as well as additional objectives, advantages, and features of the present invention will become apparent to those skilled in the art from the following detailed description of a specific embodiment of the present invention when read in conjunction with the accompanying drawings.
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Some specific embodiments of the invention will be described in detail hereinafter by way of example and not by way of limitation with reference to the accompanying drawings. The same reference numbers will be used throughout the drawings to refer to the same or like parts or portions. It will be appreciated by those skilled in the art that the drawings are not necessarily drawn to scale. In the accompanying drawings:
FIG. 1 is a graph showing colony morphology of BI-1 on R2A medium in the present invention;
FIG. 2 is a phylogenetic tree of the 16S rRNA genes of BI-1 according to the present invention;
FIG. 3 is a liquid chromatogram of the degradation effect of strain BI-1 on pyrazosulfuron, A: pyrazosulfuron-ethyl; b: pyrazosulfuron + BI-1;
FIG. 4 is the effect of temperature, pH and heavy metal ions on strain BI-1 degrading pyrazosulfuron.
Detailed Description
The invention provides a pyrazosulfuron-ethyl degrading strain BI-1, gram-negative bacteria and strict aerobic bacteria. The cells are in the shape of short rods, have no flagella, have no motility and do not produce spores. On R2A solid culture medium, after 4 days of culture at 30 ℃, the colony diameter is 1.0-2.0mm, white, round and smooth in edge. The growth of strain BI-1 occurs at 15-40℃with an optimum temperature of 30 ℃. The strain can be grown at pH6.0-8.0 (optimum pH 7.0) and 0-3% NaCl (optimum 2%). The 16S rDNA sequences of strain BI-1 were aligned in the EzBioCloud database (https:// www.ezbiocloud.net), and the results showed that the strain BI-1 was closest to the genus Chengangzhangella, with the highest similarity to the 16S rDNA of Chenggangzhangella methanolivorans reaching 99.79% and no more than 98% similarity to the 16S rDNA of other strains. The construction of the evolutionary tree by the Neighbor-Joining method revealed that strain BI-1 was located inside Chengangzhanghangella and was associated with Chenggangzhangellamethanolivorans CHL1 T Forming a sub-branch. The strain BI-1 was identified as a bacterium of the genus Chengangzhanghangella, which is the first strain in the genus to degrade pyrazosulfuron, in combination with the physiological and biochemical characteristics of BI-1. The Chinese typical culture collection is preserved in the year 7 and 26 of 2019, with the preservation number: cctccc M2019586.
Example 1
1. Obtain the pyrazosulfuron-ethyl degradation strain BI-1.
10g of farmland soil sample with pyrazosulfuron-ethyl applied for a long time is taken, added into 100mL of basic salt culture medium containing 50mg/L pyrazosulfuron-ethyl, placed in a shaking table and cultured for 7d at 30 ℃ under 160 rpm. After every 7d, 10mL of the culture broth was transferred to fresh medium. And (3) continuously transferring for 3 times, measuring degradation effect, absorbing 0.2mL of enrichment liquid with degradation capability, coating the enrichment liquid on a 50mg/L pyrazosulfuron-ethyl basic salt solid culture medium plate, culturing at 30 ℃, picking single bacterial colony when the single bacterial colony appears on the plate, transferring the single bacterial colony into a liquid culture medium test tube containing 50mg/L pyrazosulfuron-ethyl, culturing at 30 ℃ under 160rpm for 3d, and verifying degradation effect of single bacteria. The degradation effect of the strain on pyrazosulfuron-ethyl is determined by High Performance Liquid Chromatography (HPLC), namely 25% HCl is added to the reaction solution to adjust the pH to 2.0-3.0, equal volume of dichloromethane is added, vigorous vortex oscillation is carried out for 2min, the upper water phase is removed after standing and layering, excessive anhydrous sodium sulfate is added in the organic phase to remove excessive moisture, 1mL of the organic phase is taken to be placed in a 1.5mL centrifuge tube, the centrifuge tube is placed in a fume hood until the solvent is completely volatilized, then 0.2mL of methanol is used for concentration and resuspension, and after filtration by a 0.22 mu m nylon filter membrane, HPLC (Shimadzu RID-10A) is used for determination and analysis. High performance liquid chromatography conditions: chromatographic columns, C18 reverse phase columns (250 mm. Times.4.6 mm. Times.5 μm, agilent Technologies, palo Alto, calif., USA); mobile phase, acetonitrile: water: acetic acid (60:40:0.5); column temperature, 40 ℃; flow rate of 1.0 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Detection wavelength, 250nm; sample injection amount, 20. Mu.L.
And (3) taking single bacterial colonies of the degradation strain, streaking the bacterial colonies onto an R2A solid culture medium, culturing the bacterial colonies for 5d at 30 ℃, and observing the characteristics of the bacterial colonies, such as size, color, edge, protrusion, transparency and the like. Fresh cultures cultured for 5d on the R2A solid culture medium are selected and inoculated into the R2A liquid culture medium, and shake culture is carried out for 48h at the temperature of 30 ℃ to obtain seed liquid. The cultured degrading bacteria seed liquid is transferred into a fresh R2A liquid culture medium according to the inoculum size of 2% (v/v) and evenly mixed. Respectively culturing in water bath at 4deg.C, 10deg.C, 15deg.C, 20deg.C, 25deg.C, 30deg.C, 35deg.C, 45deg.C, 50deg.C and 60deg.C, and measuring growth at 24 hr and 48 hr. Sodium chloride concentrations of 0.0%, 2.0%, 3.5%, 5.0%, 7.0%, 9.0%, 10.0%, 11.0% and 12.0% of R2A medium were transferred at an inoculum size of 2% (v/v), and were recorded at 30℃for growth conditions of 24 hours and 48 hours, respectively. Sterile R2A medium was adjusted to pH 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 8.5, 9.0, 10.0, 11.0, 12.0, 13.0 with sterile 1mol/L HCl and 1mol/L NaOH, seed solutions were inoculated at 2% inoculum size, incubated at 30℃and recorded at growth status of 24h and 48h, respectively. The strain is identified by conventional physiological and biochemical assay methods. The experimental steps are described in the specification.
A strain of degrading single bacteria is obtained through verification and is named as BI-1. Strain BI-1 is a gram-negative bacterium, strictly aerobic. The cells are in the shape of short rods, have no flagella, have no motility and do not produce spores. As shown in FIG. 1, the colony diameter was 1.0-2.0mm, white, round, and smooth at the edge after 4 days of incubation at 30℃on R2A solid medium. The growth of strain BI-1 occurs at 15-40℃with an optimum temperature of 30 ℃. The strain can be grown at pH6.0-8.0 (optimum pH 7.0) and 0-3% NaCl (optimum 2%). Oxidase, catalase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, acid phosphatase, urease reaction positive, naphthol-AS-BI-phosphohydrolase, alpha-chymotrypsin, alpha-galactosidase, beta-glucuronidase, alpha-glucosidase, beta-glucosidase, N-acetyl-beta-aminoglucosidase, alpha-mannosidase and beta-fucosidase reaction negative. H 2 S is generated, the hydrolysis reaction of tween 20 and tween 60 is negative, and the hydrolysis of tween 40 is positive. The strain can grow by using D-gluconic acid, glucuronic acid, D-gluconic acid and D-malic acid.
Example 2
2. Extracting genome DNA of strain BI-1, performing PCR amplification and determining genus species
The invention adopts a high-salt method to extract the genome DNA of the strain BI-1: culturing activated strain of strain BI-1 on R2A solid plate by streaking, culturing at 30deg.C for 5d, selecting single colony into R2A liquid culture medium, and culturing at 30deg.C and 160rpm to logarithmic phase; centrifuging at 12000rpm for 5min, collecting thallus into a 2mL sterile centrifuge tube, adding 1.0mL TE buffer to wash BI-1 thallus, and adding 1.0mL TE buffer to re-suspend the thallus after the thallus is washed until no culture medium residue exists; lysozyme was added to a final concentration of 20mg/mL, and after 2h incubation at 37℃8. Mu.L proteinase K (20 mg/mL) and 50. Mu.L 10% SDS were added, and clarified (or overnight to clarify at 37 ℃) in a water bath of 65℃for 2 h; adding one third volume of saturated NaCl solution, fully shaking and uniformly mixing for 15s, carrying out water bath at 65 ℃ for 10min, centrifuging at 12000rpm for 10min, carefully transferring the supernatant into a clean sterile centrifuge tube, extracting with equal volume of phenol, chloroform and isoamyl alcohol (25:24:1) until the interface is free of protein, and collecting the supernatant; transferring the supernatant to another clean sterile centrifuge tube, adding 3/5 times of isopropanol, and mixing upside down to precipitate; the precipitated DNA is picked out by a clean capillary tube, then washed by 70 percent ethanol, and placed at an ultra-clean typhoon mouth to volatilize the ethanol completely; finally, 50. Mu.L of sterile water was used to dissolve the DNA and stored in a-20℃refrigerator.
BI-116S rRNA gene sequence (1451 bp)
GAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGCATCCTTCGGGGTGAGCGGCAGACGGGTGAGTAACGCGTGGGGATGTGCCTGGTGGTACGGAACAACTCATGGAAACGTGAGCTAATACCGTATAAGCCCTTTTGGGGAAAGATTTATCGCCACCAGATCAACCCGCGTTGGATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACTGGGGAAGATAATGACGGTACCCAGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACTAAGGGGGCTAGCGTTGTTCGGAATCACTGGGCGTAAAGCGCACGTAGGCGGAGTCTTAAGTCAGAGGTGAAATCCCAAGGCTCAACCTTGGAACTGCCTTTGATACTGGGTGTCTTGAGGTCGAGAGAGGTGAGTGGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACCAGTGGCGAAGGCGGCTCACTGGCTCGATTCTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTACCTGATACCCTGGTAGTCCACGCCGTAAACGATGGAAGCTAGCCGTTGGTCAGCATGCTGATCAGTGGCGCAGCTAACGCTTTAAGCTTCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCTTTGACATCCTGTGCCACTCAGAGAGATTTGAGGTTCCCTTCGGGGACGCAGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCTAGTTGCCAGCATTCAGTTGGGCACTCTAGGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGCAGCGAAGGGGTGACCCGGAGCTAATCTCCAGAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGTTGCGCTAACCCGCAAGGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAACCGTA
The primers used for the 16S rRNA gene sequence amplification reaction are a pair of universal primers. The upstream primer was 5'-AGAGTTTGATCCTGGCTCAG-3' and the downstream primer was 5'-TACCTTGTTACGACTT-3'. The PCR reaction system was 50. Mu.L, which included 10 XBuffer 5. Mu.L, dNTP (20 mmol/. Mu.L) 4. Mu.L, primers (25 pmol/. Mu.L) 1. Mu.L each, mg 2+ (25 mmol/L) 4. Mu.L, 1. Mu.L of cell DNA (about 50 ng/. Mu.L), 0.3. Mu.L of Taq DNA polymerase (5U/. Mu.L), and ultrapure water was added to 50. Mu.L. Reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s; annealing at 54 ℃ for 30s; extending at 72deg.C for 1min;30 cycles, extension at 72℃for 10min. The size of the amplified product (about 1.5 kb) was detected by agarose gel electrophoresis, and the 16S rRNA gene fragment was recovered by using a PCR recovery kit, and after T/A cloning, sequencing was performed. The sequencing results were compared with the 16S rRNA gene sequence in GenBank by on-line analysis (www.ezbiocloud.net) to construct a phylogenetic tree.
PCR amplification is carried out by taking total DNA of the strain BI-1 as a template and a general primer of the 16S rRNA gene, so as to obtain a BI-116S rRNA gene sequence with the total length of 1451 bp. The sequence is compared in EzBioCloud database (https:// www.ezbiocloud.net), and the result shows that the genetic relationship between the strain BI-1 and Chengangzhanghangella is nearest, wherein the sequence similarity with the 16S rRNA gene of Chenggangzhangella methanolivorans is highest and reaches 99.79%, and the sequence similarity with the 16S rRNA gene of the strain of other genera is not more than 98%. As shown in FIG. 2, construction of a phylogenetic tree of the strain BI-1 by the Neighbor-Joining method revealed that the strain BI-1 was located inside the Chengangzhanghangella phylogenetic tree and was identical to Chenggangzhangella methanolivorans CHL1 T Constitutes a sub-branch, and the strain BI-1 is identified as a member of the genus Chengangzhanghangella in combination with the physiological and biochemical characteristics of BI-1Bacteria are strain.
The strain BI-1 disclosed by the invention has been preserved in China Center for Type Culture Collection (CCTCC) at 7.26.2019, and the preservation number is as follows: cctccc M2019586. The strain is classified under the name Chengangzhanglangella sp.BI-1.
Example 3
3. Determining the influence of temperature, pH and metal ions on strain BI-1 degraded pyrazosulfuron
1. Effect of temperature on strain BI-1 degradation of pyrazosulfuron:
inoculating seed solution of strain BI-1 into 50mg/L of base salt liquid culture medium of pyrazosulfuron-ethyl at an inoculum size of 2% by volume, culturing at 20deg.C, 25deg.C, 30deg.C, 40deg.C, 45deg.C and 160rpm for 3d, detecting pyrazosulfuron-ethyl concentration, and calculating degradation rate.
2. Effect of pH on strain BI-1 degrading pyrazosulfuron:
the pH of the basal salt liquid medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, respectively, and 50mg/L pyrazosulfuron-ethyl was additionally added. Seed solution of strain BI-1 was added in an inoculum size of 2% by volume, cultured at 30℃and 160rpm for 3 days, and then the pyrazosulfuron-ethyl concentration was measured to calculate the degradation rate.
3. Influence of metal ions on bacterial strain BI-1 to degrade pyrazosulfuron-ethyl:
the strain BI-1 seed solution is inoculated into a liquid culture medium filled with 100mL of basic salt according to the inoculation amount of 2% volume ratio, and the concentration of the pyrazosulfuron-ethyl is 50mg/L. 1mmol/L heavy metal ion Ag is added into the culture medium respectively + 、Ni 2+ 、Cd 2+ 、Zn 2+ After culturing at 30℃and 160rpm for 3 days, the pyrazosulfuron-ethyl concentration was measured, and the degradation rate was calculated.
As shown in FIG. 3, under the laboratory condition, the strain BI-1 is added to the culture medium for reaction for 3 days, so that 50mg/L pyrazosulfuron ethyl can be completely degraded, and the strain which is the first strain in Chengganzhanghangella and degrades pyrazosulfuron ethyl can provide ideal germplasm resources for repairing pyrazosulfuron ethyl polluted soil. As shown in FIG. 4, the degradation rate of pyrazosulfuron-ethyl was highest in the medium having a temperature of 30℃and a pH of 7.0 and no addition of heavy metal ions. After Ag is added + 、Cd 2+ 、Zn 2+ 、Ni 2+ In the culture medium of (2), the degradation rates of the pyrazosulfuron-ethyl are 9.8%, 7.8%, 9.6% and 53.4%, respectively, which indicate heavy metal ion Ag + 、Cd 2+ 、Zn 2+ 、Ni 2 + Has different degrees of inhibition on the degradation of the strain.
Example 4
4. Obtain pyrazosulfuron-ethyl degradation strain BI-1 microbial inoculum
The strain is activated on an R2A culture medium and inoculated on a test tube inclined plane for standby. The strain on the inclined plane is selected and inoculated into a 1000mL shaking flask containing 200mL of R2A culture medium, and the strain is subjected to shaking culture until the strain reaches the logarithmic phase, so as to prepare an inoculation fermentation tank. 500 liters of fermentation tank, 400 liters of feed amount and the formula of the culture medium is (g/L): yeast extract 0.5, peptone 0.5, casein 0.5, glucose 0.5, soluble starch 0.5, sodium pyruvate 0.3, K 2 HPO 4 0.3，MgSO 4 0.05, pH 7.2-7.5. After the material is fed, sterilizing at 115 ℃ under high pressure and moist heat, cooling to 30 ℃, inoculating the cultured shake flask strain into a fermentation tank according to 10% of inoculation amount, controlling the temperature of the fermentation tank after inoculation at 30 ℃, and controlling the ventilation amount of sterile air in the culture process to be 1:0.6, the stirring speed is 180 rpm, the pH value is 7.3, the material is fed once every 24 hours, the culture time of the whole process flow is 96 hours, and the number of thalli is ensured to be more than 10 hundred million/mL after the fermentation is finished. And (5) discharging the culture solution from the tank after fermentation, and packaging the culture solution into liquid dosage forms by packaging bottles.
Example 5
5. Pyrosulfuron degrading strain BI-1 and application of microbial inoculum thereof in soil
Taking 450g of natural air-dried vegetable soil (pyrazosulfuron ethyl is not detected) sieved by a 20-mesh sieve into a pot, adding pyrazosulfuron ethyl to 3mg/kg, uniformly stirring, spraying water to enable the water content to reach 60% of the saturated water content, inoculating the prepared microbial inoculum with the inoculation amount of 0.01mL/g, uniformly stirring, additionally setting a treatment of only adding microbial inoculum and only adding pyrazosulfuron ethyl, taking untreated samples as blank control, and transplanting corn seedlings into pots with different treatments, wherein each treatment is set to be 4 times in parallel. Placing in artificial climate incubator (GXZ type intelligent illumination incubator, ningbo Jiangnan Instrument factory, GXZ-500D) for culturing at 30deg.C for 12 hr and 20deg.C for 20 days in dark period. And (5) measuring the concentration of pyrazosulfuron-ethyl in the soil, and the length of the stems and leaves, the length of the roots, the fresh weight of the stems and leaves and the fresh weight of the root system of the corn.
Determination of pyrazosulfuron-ethyl concentration in soil: collecting soil sample, weighing 5g, placing into 50mL centrifuge tube, adding 10mL of extract (PBS: acetonitrile=8:2), centrifuging at 150rpm for 1h,4000g for 15min, collecting supernatant, re-suspending precipitate with 10mL of extract, repeating the above operation for 3 times, mixing the supernatants, adjusting pH to 2.5 with HCl, and purifying extract with Cleanert HXN solid phase extraction column. N for eluent 2 Blow dried, resuspended in 1mL of methanol, and filtered through a 0.22 μm nylon membrane and detected in the liquid phase. Liquid chromatography conditions: the model of the liquid chromatograph is Shimadzu RID-10A; the liquid chromatographic column is a C18 reversed phase column with the specification of 250mm multiplied by 4.6mm multiplied by 5 mu m; the mobile phase is acetonitrile, water and acetic acid (60:40:0.5); column temperature is 40 ℃; the flow rate is 1.0 mL-min -1 The method comprises the steps of carrying out a first treatment on the surface of the The detection wavelength was 250nm. And (3) comparing the peak area of the target peak of the sample with that of a standard product by using an external standard method to quantitatively detect the concentration of the pyrazosulfuron.
TABLE 1 repair effect of strain BI-1 on degradation and phytotoxicity of pyrazosulfuron in soil (20 days)
Note that: control: no strain BI-1 and pyrazosulfuron were added; BI-1: only strain BI-1 was added; pyrazosulfuron-ethyl: only pyrazosulfuron-ethyl is added; pyrazosulfuron + BI-1: pyrazosulfuron and strain BI-1 were added. The different letters represent a significant difference at the P <0.05 level (duncan test)
Pyrazosulfuron-ethyl is a high-efficiency herbicide, and a small amount of residues can cause serious phytotoxicity to sensitive crops. In the experiment, sensitive crop corn is used as an indicator crop, and the degradation effect of strain BI-1 on pyrazosulfuron in soil and the recovery effect of strain BI-1 on corn growth are measured. The results are shown in table 1, and the stem and leaf length, root length, stem and leaf fresh weight and root fresh weight of the corn can be obviously reduced due to the addition of the pyrazosulfuron ethyl, and the inhibition rates are 75.97%, 80.35%, 82.88% and 91.13%, respectively, which indicate that the pyrazosulfuron ethyl has obvious influence on the growth of the corn. When only strain BI-1 was inoculated in the soil, various indexes of corn growth were not significantly different from the control, indicating that strain BI-1 had no effect on corn growth. When the strain BI-1 is inoculated into the pyrazosulfuron-ethyl contaminated soil, the growth of the corn is obviously recovered. And meanwhile, the concentration of the pyrazosulfuron in the soil under different treatments is measured, and the result shows that the concentration of the pyrazosulfuron in the soil can still reach 2.18mg/kg after 20 days of the soil treated by only adding 3mg/kg of pyrazosulfuron, and the existence of the pyrazosulfuron in the soil can not be detected after 20 days of the soil treated by adding pyrazosulfuron and BI-1 bacterial suspension.
Meanwhile, the degradation effect of the strain BI-1 degrading bacterial agent on pyrazosulfuron in the water body is measured: taking 500mL of pyrazosulfuron-ethyl production factory wastewater, preliminarily filtering, adding pyrazosulfuron-ethyl Long Zhizhong with the concentration of 3mg/L, and adding the prepared BI-1 degrading microbial inoculum to the final concentration of 10 8 And (3) taking the water body added with the sterile water with the same volume as a control, placing the water body in a 30 ℃ incubator for constant temperature culture under the dark condition, sampling after 3 days to measure the residual quantity of pyrazosulfuron-ethyl in the water body, carrying out parallel experiments for 3 times on each group, measuring the residual quantity by utilizing high performance liquid chromatography, and calculating the average degradation rate. The results show that the pyrazosulfuron-ethyl can not be detected in the samples treated by adding the BI-1 microbial inoculum, and the pyrazosulfuron-ethyl is not degraded after the treatment, so that the BI-1 degradation microbial inoculum can effectively degrade the residual pyrazosulfuron-ethyl in the water body.
The bacterial strain for efficiently degrading the pyrazosulfuron is obtained by separation and screening, the germplasm resources of microorganisms are enriched, the degradation efficiency is high, the properties are stable, the preparation method of the degradation microbial agent is provided, the residual pyrazosulfuron in soil and water can be effectively degraded, the method has the advantages of low production cost, convenience in use and good removal effect, is suitable for repairing the pyrazosulfuron polluted environment, and has important significance for protecting the ecological environment and protecting the physical health of human bodies.
By now it should be appreciated by those skilled in the art that while a number of exemplary embodiments of the invention have been shown and described herein in detail, many other variations or modifications of the invention consistent with the principles of the invention may be directly ascertained or inferred from the present disclosure without departing from the spirit and scope of the invention. Accordingly, the scope of the present invention should be understood and deemed to cover all such other variations or modifications.
Sequence listing
<110> Nanyang teaching and learning school
<120> a pyrazosulfuron-ethyl degrading strain BI-1 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> rRNA
<213> Pyrimsulfuron degrading Strain BI-116S rRNA Gene sequence ()
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Claims (6)
1. A pyrazosulfuron ethyl degradation strain Chengangzhanglangella sp.BI-1 has a preservation number of CCTCC M2019586.
2. The pyrazosulfuron-ethyl degrading strain BI-1 microbial inoculum is characterized in that the pyrazosulfuron-ethyl degrading strain BI-1CCTCC M2019586 is obtained after the amplification culture.
3. The method for preparing the pyrazosulfuron-ethyl degradation strain BI-1 microbial inoculum as claimed in claim 2, which is characterized by comprising the following steps:
s1: activating the BI-1 strain on an R2A culture medium, and inoculating the strain on a test tube inclined plane for standby;
s2: selecting strain on the inclined plane, inoculating the strain into a shake flask containing an R2A culture medium, and shake culturing to a logarithmic phase;
s3: sterilizing at 115 ℃ under high pressure and moist heat after feeding, cooling to 30 ℃, inoculating the cultured shake flask strain into a fermentation tank according to 10% of inoculation amount, controlling the fermentation process according to a set fermentation environment, and ensuring that the number of thalli reaches more than 10 hundred million/mL after fermentation;
s4: and (5) discharging the culture solution from the tank after fermentation, and packaging the culture solution into liquid dosage forms by packaging bottles to obtain the pyrazosulfuron-ethyl degradation strain BI-1 microbial inoculum.
4. A method of preparation according to claim 3, wherein the fermentation environment is: the temperature of the fermentation tank after inoculation is controlled at 30 ℃, and the ventilation rate of sterile air in the culture process is 1:0.6, the stirring speed is 180 rpm, the pH is 7.3, the feeding is carried out every 24 hours, and the culture time of the whole process flow is 96 hours.
5. The method of claim 3, wherein the R2A medium is formulated as follows: yeast extract 0.5, peptone 0.5, casein 0.5, glucose 0.5, soluble starch 0.5, sodium pyruvate 0.3, K 2 HPO 4 0.3，MgSO 4 0.05, pH 7.2-7.5, in g/L.
6. Use of a pyrazosulfuron ethyl degrading strain BI-1 as defined in claim 1 or a pyrazosulfuron ethyl degrading strain BI-1 microbial inoculum as defined in claim 2 for preparing a pyrazosulfuron ethyl degrading agent.
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| CN102321539A (en) * | 2011-08-26 | 2012-01-18 | 河北农业大学 | Fermentation and preparation of composite microbial agent for degrading sulfonylurea herbicide with high efficiency |
| CN106834149A (en) * | 2015-12-03 | 2017-06-13 | 中国科学院沈阳应用生态研究所 | Into the thermophilic methyl brevibacterium of firm Pseudomonas and its application in degraded sulfonylurea herbicide |
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| CN101948782A (en) * | 2010-09-01 | 2011-01-19 | 湖南省植物保护研究所 | Rhodopseudomonas spheroides strain and liquid inoculant thereof as well as preparation method and application thereof |
| CN102321539A (en) * | 2011-08-26 | 2012-01-18 | 河北农业大学 | Fermentation and preparation of composite microbial agent for degrading sulfonylurea herbicide with high efficiency |
| CN106834149A (en) * | 2015-12-03 | 2017-06-13 | 中国科学院沈阳应用生态研究所 | Into the thermophilic methyl brevibacterium of firm Pseudomonas and its application in degraded sulfonylurea herbicide |
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