玉米根际土壤中大环内酯类抗性基因的分布特征
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摘要
环境中的抗生素残留将胁迫农作物根际土壤微生物产生耐药性,使得这些农作物的根际土壤成为抗性基因转移的"热区"。采用陆生微宇宙系统,模拟玉米生长过程中大环内酯类抗生素进入植物根际土壤后相应抗性基因的分布情况。利用实时荧光定量PCR(qPCR)技术分析了63 d内玉米根际土壤中大环内酯类抗生素抗性基因(erms)的相对丰度(erms∶16S rDNA,即erms绝对浓度与16S rDNA绝对浓度的比值)。结果显示:土壤中不同抗性基因相对丰度存在较明显的差异,整体表现为ermF>ermX>ermB>ermC。7 d时,非根际(BK)土壤中ermB、ermC、ermF和ermX相对丰度分别为4.72×10~(-2)、1.98×10~(-3)、7.13×10~(-1)和1.75×10~(-2),而63 d时则分别为1.74×10~(-3)、3.24×10~(-4)、3.53×10~(-3)和2.28×10~(-3),基因相对丰度增幅分别为-96.3%、-83.6%、-99.5%和-87.0%;根际(RH)土壤中ermB、ermC、ermF和ermX相对丰度增幅分别为-88.3%、103.0%、-88.6%和71.5%,暗示玉米根际可能具有促进土壤中抗性基因ermC和ermX增殖或转移的作用。不同深度土层中erms相对丰度由大到小依次为0～0.2、>0.2～0.4和>0.4～0.6 m,表明erms在土壤中具有向下迁移的特性,且随着土壤深度的增加,相对丰度呈递减趋势;与未种植玉米(CK)土壤相比,种植玉米(MA)土壤中erms检出率和相对丰度较高,表明玉米的根际环境有利于不同类型erms的富集以及在剖面土壤中的纵向迁移。
Antibiotic residues in crop soils create selection pressure on the occurrence and dissemination of antibiotic resistance genes(ARGs), rendering rhizosphere soils "hot spots" for ARGs. In the present study, the soil microbiome was monitored for macrolide resistance genes(erms) arising from the rhizosphere in maize during different crop growth periods over a duration of 63 days using real-time polymerase chain reaction(PCR) technology. The relative abundance(erms∶16 S rDNA) of erms in the soil was in the order of ermF > ermX > ermB > ermC. The relative abundance of ermF, ermX, ermB, and ermC in bulk soil were 4.72 × 10~(-2), 1.98 × 10~(-3), 7.13 × 10~(-1), and 1.75×10~(-2), respectively, on D7, whereas, on D63, the relative abundance were 1.74 × 10~(-3), 3.24 × 10~(-4), 3.53 × 10~(-3) and 2.28 × 10~(-3), respectively. The corresponding gene proliferation rates in bulk soil were-96.3%,-83.6%,-99.5%, and-87.0%, respectively, compared to-88.3%, 103.0%,-88.6%, and 71.5%, respectively, in rhizosphere soil. These results suggest that the maize rhizosphere had a significant effect on the proliferation or transfer of ermC and ermX. The relative abundance of erms at different soil depths was in the order of 0-0.2, > 0.2-0.4, and > 0.4-0.6 m, indicating that the resistance genes had a downward migration tendency, with relative abundance of resistance genes decreasing with an increase in soil depth. The detection rate of resistance genes at corresponding depths of soil for the same sample was in the order of RH > CK, suggesting that the presence of the plant rhizosphere not only promotes an increase in resistance gene abundance in the soil but also promotes vertical migration in the soil profile.
Antibiotic residues in crop soils create selection pressure on the occurrence and dissemination of antibiotic resistance genes(ARGs), rendering rhizosphere soils "hot spots" for ARGs. In the present study, the soil microbiome was monitored for macrolide resistance genes(erms) arising from the rhizosphere in maize during different crop growth periods over a duration of 63 days using real-time polymerase chain reaction(PCR) technology. The relative abundance(erms∶16 S rDNA) of erms in the soil was in the order of ermF > ermX > ermB > ermC. The relative abundance of ermF, ermX, ermB, and ermC in bulk soil were 4.72 × 10~(-2), 1.98 × 10~(-3), 7.13 × 10~(-1), and 1.75×10~(-2), respectively, on D7, whereas, on D63, the relative abundance were 1.74 × 10~(-3), 3.24 × 10~(-4), 3.53 × 10~(-3) and 2.28 × 10~(-3), respectively. The corresponding gene proliferation rates in bulk soil were-96.3%,-83.6%,-99.5%, and-87.0%, respectively, compared to-88.3%, 103.0%,-88.6%, and 71.5%, respectively, in rhizosphere soil. These results suggest that the maize rhizosphere had a significant effect on the proliferation or transfer of ermC and ermX. The relative abundance of erms at different soil depths was in the order of 0-0.2, > 0.2-0.4, and > 0.4-0.6 m, indicating that the resistance genes had a downward migration tendency, with relative abundance of resistance genes decreasing with an increase in soil depth. The detection rate of resistance genes at corresponding depths of soil for the same sample was in the order of RH > CK, suggesting that the presence of the plant rhizosphere not only promotes an increase in resistance gene abundance in the soil but also promotes vertical migration in the soil profile.
引文
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[13] 程燕,周军英,续卫利,等.克百威、乐果在陆生微宇宙土芯中的降解和淋溶研究[J].农药科学与管理,2012,33(8):29-34.[CHENG Yan,ZHOU Jun-ying,XU Wei-li,et al.Degradation and Leaching of Carbofuran and Dimethoate in Terrestrial Soil-Core Microcosm[J].Pesticide Science and Administration,2012,33(8):29-34.]
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[16] POPOWSKA M,RZECZYCKA M,MIERNIK A,et al.Influence of Soil Use on Prevalence of Tetracycline,Streptomycin,and Erythromycin Resistance and Associated Resistance Genes[J].Antimicrobial Agents and Chemotherapy,2012,56(3):1434-1443.
[17] HEUER H,FOCKS A,LAMSH?FT M,et al.Fate of Sulfadiazine Administered to Pigs and Its Quantitative Effect on the Dynamics of Bacterial Resistance Genes in Manure and Manured Soil[J].Soil Biology and Biochemistry,2008,40(7):1892-1900.
[18] LI X Z,RUI J P,XIONG J B,et al.Functional Potential of Soil Microbial Communities in the Maize Rhizosphere[J].PLoS One,2014,9(11):e112609.DOI:10.1371/journal.pone.0112609.
[19] DOORNBOS R F,VAN LOON L C,BAKKER P A H M.Impact of Root Exudates and Plant Defense Signaling on Bacterial Communities in the Rhizosphere:A Review[J].Agronomy for Sustainable Development,2012,32(1):227-243.
[20] BRANDT K K,SJ?HOLM O R,KROGH K A,et al.Increased Pollution-Induced Bacterial Community Tolerance to Sulfadiazine in Soil Hotspots Amended With Artificial Root Exudates[J].Environmental Science & Technology,2009,43(8):2963-2968.
[21] JECHALKE S,HEUER H,SIEMENS J,et al.Fate and Effects of Veterinary Antibiotics in Soil[J].Trends in Microbiology,2014,22(9):536-545.
[22] CHEN C,YANG K,YU C N,et al.Influence of Redox Conditions on the Microbial Degradation of Polychlorinated Biphenyls in Different Niches of Rice Paddy Fields[J].Soil Biology and Biochemistry,2014,78:307-315.
[23] RYSZ M,MANSFIELD W R,FORTNER J D,et al.Tetracycline Resistance Gene Maintenance Under Varying Bacterial Growth Rate,Substrate and Oxygen Availability,and Tetracycline Concentration[J].Environmental Science & Technology,2013,47(13):6995-7001.
[2] MARON D,SMITH Y S,NACHMAN K E.Restrictions on Antimicrobial Use in Food Animal Production:An International Regulatory and Economic Survey[J].Globalization and Health,2013,9(1):48.DOI:10.1186/1744-8603-9-48.
[3] JECHALKE S,FOCKS A,ROSENDAHL I,et al.Structural and Functional Response of the Soil Bacterial Community to Application of Manure From Difloxacin-Treated Pigs[J].FEMS Microbiology Ecology,2014,87(1):78-88.
[4] HEUER H,SCHMITT H,SMALLA K.Antibiotic Resistance Gene Spread Due to Manure Application on Agricultural Fields[J].Current Opinion in Microbiology,2011,14(3):236-243.
[5] FAHRENFELD N,KNOWLTON K,KROMETIS L A,et al.Effect of Manure Application on Abundance of Antibiotic Resistance Genes and Their Attenuation Rates in Soil:Field-Scale Mass Balance Approach[J].Environmental Science & Technology,2014,48(5):2643-2650.
[6] REICHEL R,ROSENDAHL I,PEETERS E T H M,et al.Effects of Slurry From Sulfadiazine-(SDZ) and Difloxacin-(DIF) Medicated Pigs on the Structural Diversity of Microorganisms in Bulk and Rhizosphere Soil[J].Soil Biology and Biochemistry,2013,62:82-91.
[7] JECHALKE S,KOPMANN C,ROSENDAHL I,et al.Increased Abundance and Transferability of Resistance Genes After Field Application of Manure From Sulfadiazine-Treated Pigs[J].Applied and Environmental Microbiology,2013,79(5):1704-1711.
[8] KOPMANN C,JECHALKE S,ROSENDAHL I,et al.Abundance and Transferability of Antibiotic Resistance as Related to the Fate of Sulfadiazine in Maize Rhizosphere and Bulk Soil[J].FEMS Microbiology Ecology,2013,83(1):125-134.
[9] SHANKAR P R.Antimicrobial Resistance:Global Report on Surveillance[J].Australasian Medical Journal,2014,7(4):237.
[10] JOY S R,LI X,SNOW D D,et al.Fate of Antimicrobials and Antimicrobial Resistance Genes in Simulated Swine Manure Storage[J].Science of the Total Environment,2014,481:69-74.
[11] KNAPP C W,DOLFING J,EHLERT P A I,et al.Evidence of Increasing Antibiotic Resistance Gene Abundances in Archived Soils Since 1940[J].Environmental Science & Technology,2010,44(2):580-587.
[12] SU H C,PAN C G,YING G G,et al.Contamination Profiles of Antibiotic Resistance Genes in the Sediments at a Catchment Scale[J].Science of the Total Environment,2014,490:708-714.
[13] 程燕,周军英,续卫利,等.克百威、乐果在陆生微宇宙土芯中的降解和淋溶研究[J].农药科学与管理,2012,33(8):29-34.[CHENG Yan,ZHOU Jun-ying,XU Wei-li,et al.Degradation and Leaching of Carbofuran and Dimethoate in Terrestrial Soil-Core Microcosm[J].Pesticide Science and Administration,2012,33(8):29-34.]
[14] CHEN B,HAO L J,GUO X Y,et al.Prevalence of Antibiotic Resistance Genes of Wastewater and Surface Water in Livestock Farms of Jiangsu Province,China[J].Environmental Science and Pollution Research,2015,22(18):13950-13959.
[15] LANG K S,ANDERSON J M,SCHWARZ S,et al.Novel Florfenicol and Chloramphenicol Resistance Gene Discovered in Alaskan Soil by Using Functional Metagenomics[J].Applied and Environmental Microbiology,2010,76(15):5321-5326.
[16] POPOWSKA M,RZECZYCKA M,MIERNIK A,et al.Influence of Soil Use on Prevalence of Tetracycline,Streptomycin,and Erythromycin Resistance and Associated Resistance Genes[J].Antimicrobial Agents and Chemotherapy,2012,56(3):1434-1443.
[17] HEUER H,FOCKS A,LAMSH?FT M,et al.Fate of Sulfadiazine Administered to Pigs and Its Quantitative Effect on the Dynamics of Bacterial Resistance Genes in Manure and Manured Soil[J].Soil Biology and Biochemistry,2008,40(7):1892-1900.
[18] LI X Z,RUI J P,XIONG J B,et al.Functional Potential of Soil Microbial Communities in the Maize Rhizosphere[J].PLoS One,2014,9(11):e112609.DOI:10.1371/journal.pone.0112609.
[19] DOORNBOS R F,VAN LOON L C,BAKKER P A H M.Impact of Root Exudates and Plant Defense Signaling on Bacterial Communities in the Rhizosphere:A Review[J].Agronomy for Sustainable Development,2012,32(1):227-243.
[20] BRANDT K K,SJ?HOLM O R,KROGH K A,et al.Increased Pollution-Induced Bacterial Community Tolerance to Sulfadiazine in Soil Hotspots Amended With Artificial Root Exudates[J].Environmental Science & Technology,2009,43(8):2963-2968.
[21] JECHALKE S,HEUER H,SIEMENS J,et al.Fate and Effects of Veterinary Antibiotics in Soil[J].Trends in Microbiology,2014,22(9):536-545.
[22] CHEN C,YANG K,YU C N,et al.Influence of Redox Conditions on the Microbial Degradation of Polychlorinated Biphenyls in Different Niches of Rice Paddy Fields[J].Soil Biology and Biochemistry,2014,78:307-315.
[23] RYSZ M,MANSFIELD W R,FORTNER J D,et al.Tetracycline Resistance Gene Maintenance Under Varying Bacterial Growth Rate,Substrate and Oxygen Availability,and Tetracycline Concentration[J].Environmental Science & Technology,2013,47(13):6995-7001.
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