| Fomesafen,a member in the category of diphenylethers,has been used to control the growth of broadleaf weeds in bean fields,particularly soybean and peanut fields.Because of its high herbicidal activity and low application rates,fomesafen has been used widely as a herbicide in soybean fields since its introduction to China.Due to its long-lived persistence,phytotoxicity and negative effects on crop rotation,fomesafen has been listed as a soil contaminant in recent years.This thesis reported the isolation and characterization of a bacterial strain named BY-1 that has the ability to use fomesafen as sole carbon source for growth.On the basis of the results of the phylogenetic analysis of 16Sr RNA,BY-1T showed high similarities to Pseudomonas luteola IAM 13000T(99.5%)and Pseudomonas duriflava HR2T(97.3%);whereas lower than 97.0%16S rRNA gene sequence similarity was observed with other Pseudomonas species.DNA-DNA hybridization indicated that strain BY-1 showed relatively low DNA-DNA relatedness to Pseudomonas luteola IAM 13000-T(29±3.1%)and Pseudomonas durifava HR2T(21±1.5%).BY-1 Cells are gram-staining-negative,aerobic,oxidase-negative,catalase-positive,non-spore-forming and flagellated.They are short rods,0.6-0.8μm in width and 2.8-3.2 μm in length.After 3 days of incubation on TSA,colonies grow to 1.0-2.0 mm in diameter,and are yellowish,opaque,convex,pigmentless,and circular with entire edges.C18:1(39.4%),C16:0(21.9%),and Summed features 3(C16:1ω6c and/or C161ω7c)(17.7%)are the predominant cellular fatty acids.The predominant ubiquinon is Q-9.The phospholipid profile is composed of diphosphatidylglycerol,phosphatidylglycerol and phosphatidylethanolamine.The genomic DNA G+C content is 55.3 mol%.Therefore,we indentify strain BY-1 is a novel species in Pseudomonas genus,and renamed it as Pseudomonas zeshuii sp..nov.BY-1T(= KACC 15471T=ACCC 05688T).BY-1 is able to to use fomesafen as sole carbon source.Up to 88.7%of 50 mg·L-1 fomesafen was degraded by this bacterium in MM medium within 3 days.The degradation efficeincy reduces as the concentration of fomesafen increases.The optimal temperature and pH of degradation are 30℃ and 7.0 respectively.The addition of 1 mmol·L-1Ca2+ and Fe3+ promote degradation,while the addition of Co2+、Mn2+ and Cu2+ inhibite the degradation.The crude enzyme extract from BY-1 bacterial cells showed that the degradative enzymes are cytoplasmic,constitutively expressed,and needs no induction.The best reaction system was as follows:incubation 50μL crude enzyme in 5 mL total volum in pH7.0 for 10 min at 30℃,the reaction duration was 60 min.During the degradation,five metabolites were detected and identified by LC-MS and MS/MS.Indicate the structure type and speculate the pathway.The primary degradation pathway of fomesafen might be the reduction of the nitro group to an amino group followed by the acetylation of the amino derivative,dechlorination and cleavage of the S-N bond.The final resultant is N-[4-(difluromethyl phenoxyl)-2-formylanino phenyl]acetamide.Vaccinate BY-1 to the soil polluted by 50 mg kg-1 fomesafen,the degradation efficiency was 73.4%in 30 days.The degradation is influenced by pH,temperature of the soil,and water content,degradation is optimal under pH7,and 30℃,with water content 20%-30%.Using fluorescence-labeled BY-1pTR,we investigated the remediation of fomesafen polluted soil by BY-1 and the factors affecting the degradation in the soil as well.The use of fomesafen greatly impacts the microbial diversity in the soil and the bioactivity of various enzymes.Through the analysis by DGGE,the addition of BY-1 recovered the altered microbial diversity by fomesafen to some extent and didn’t inhibit indigenous microorganisms,pot experiment indicate that partial remission the phytotoxicity of maize by fomesafen.The results of enzymological studies indicated that the fomesafen could inhibit the enzyme activities of dehydrogenase and saccharase intensively.There is a wave action of activation first and inhibition after for catalase,urease and polyphenol oxidase in soil.BY-1 could alleviates the inhibitory effect of fomesafen on various enzymes in the soil,and restored to near CK level. |
