淡水湖泊T4类浮游病毒遗传多样性研究
摘要
浮游病毒是淡水水体种类和数目最多的生物类群,论文通过三种方法对武汉淡水湖泊中浮游病毒的多样性进行了研究。
RAPD方法：通过不同湖泊的带型进行比较,能大致得到关于各水体病毒多样性的初步认识：对同一季节而言,随着营养水平的增加,种类和数量都呈现增加的趋势；通过对各湖泊冬夏两季抽动比较,发现每个湖泊则表现出不同的变化趋势。
DGGE方法：根据11个湖泊冬夏两季样品基于g23基因的T4类浮游病毒的研究,发现夏季水样较易得到高效率的PCR产物,且其电泳图谱上显示出的条带也更多。DGGE结果也表明,随着营养水平的增高,病毒种类也呈现增加的趋势：在夏季电泳图上,可以显示出明显的相同条带,以及特异性条带。
宏基因组法能得到关于各水体T4类浮游病毒的序列信息。从11个不同营养水平的湖泊的冬、夏两季样品中,共得到312条序列。将这些湖泊分为贫／中营养、轻度富营养、中度富营养以及重度富营养四组,组内发散度分析发现,营养水平的差异和季节变化都将对浮游病毒多样性产生影响；所有序列建立系统进化树,发现有7个特殊类群,即C1～C7,通过对这7个类群与部分其他无规律序列及海洋序列的进行系统进化分析,发现人为干扰也会对病毒群落结构产生影响。
Virioplankton is the most diverse and abundant living form in freshwaters, and in this study, we checked its diversity in freshwater lakes of Wuhan mainly with 3 different methods.
RAPD method:comparing profiles from different lakes, we know the general virioplankton diversity of different samples. To the same season, with the increase of nutrition, virioplankton seems to be more diverse and abundant. Each lake shows different trend from winter to summer.
DGGE method:we amplified g23 gene of 22 samples (11 lakes, in winter and summer) to identify T4 virioplankton diversity, and get the following results. More efficient PCR products can be easily got in summer samples, and the bands from electrophoresis image of summer samplesare much more than bands of winter samples. The same result with RAPD is that diversity of virioplankton is higher in eutrophic samples. Summer image showed significant universal and special bands.
Metagenome method:312 sequences were aquired from 11 different nutrient condition lakes in summer and winter. We divide all the lakes into 4 different goups according to their nutrient level. Average evolutionary divergence over sequence pairs within groups alalysis showde that nutrient level and seasonal change can do great to virioplankton community structure in lakes. Furthermore, the phylogenetic analysis of all the sequences revealed 7 special clusters, C1-C7, and proved that anthropology forcing could influence the genetic diversity of T4 phage in some freshwater lakes.
RAPD方法：通过不同湖泊的带型进行比较,能大致得到关于各水体病毒多样性的初步认识：对同一季节而言,随着营养水平的增加,种类和数量都呈现增加的趋势；通过对各湖泊冬夏两季抽动比较,发现每个湖泊则表现出不同的变化趋势。
DGGE方法：根据11个湖泊冬夏两季样品基于g23基因的T4类浮游病毒的研究,发现夏季水样较易得到高效率的PCR产物,且其电泳图谱上显示出的条带也更多。DGGE结果也表明,随着营养水平的增高,病毒种类也呈现增加的趋势：在夏季电泳图上,可以显示出明显的相同条带,以及特异性条带。
宏基因组法能得到关于各水体T4类浮游病毒的序列信息。从11个不同营养水平的湖泊的冬、夏两季样品中,共得到312条序列。将这些湖泊分为贫／中营养、轻度富营养、中度富营养以及重度富营养四组,组内发散度分析发现,营养水平的差异和季节变化都将对浮游病毒多样性产生影响；所有序列建立系统进化树,发现有7个特殊类群,即C1～C7,通过对这7个类群与部分其他无规律序列及海洋序列的进行系统进化分析,发现人为干扰也会对病毒群落结构产生影响。
Virioplankton is the most diverse and abundant living form in freshwaters, and in this study, we checked its diversity in freshwater lakes of Wuhan mainly with 3 different methods.
RAPD method:comparing profiles from different lakes, we know the general virioplankton diversity of different samples. To the same season, with the increase of nutrition, virioplankton seems to be more diverse and abundant. Each lake shows different trend from winter to summer.
DGGE method:we amplified g23 gene of 22 samples (11 lakes, in winter and summer) to identify T4 virioplankton diversity, and get the following results. More efficient PCR products can be easily got in summer samples, and the bands from electrophoresis image of summer samplesare much more than bands of winter samples. The same result with RAPD is that diversity of virioplankton is higher in eutrophic samples. Summer image showed significant universal and special bands.
Metagenome method:312 sequences were aquired from 11 different nutrient condition lakes in summer and winter. We divide all the lakes into 4 different goups according to their nutrient level. Average evolutionary divergence over sequence pairs within groups alalysis showde that nutrient level and seasonal change can do great to virioplankton community structure in lakes. Furthermore, the phylogenetic analysis of all the sequences revealed 7 special clusters, C1-C7, and proved that anthropology forcing could influence the genetic diversity of T4 phage in some freshwater lakes.
引文
[1]Wommack K E, Colwell R R. Virioplankton:Viruses in Aquatic Ecosystems [J]. Microbiology and Molecular Biology Reviews,2000,64(1):69-114.
[2]Bergh O, orsheim K Y. Bratbak B G, et al. High abundance of viruses found in aquatic environments [J]. Nature,1989,340:467-468.
[3]Wommack K E, Ravel J, Hill R T, et al. Hybridization analysis of Chesapeake Bay virioplankton [J]. Appl. Environ. Microbiol,1999,65:241-250.
[4]Wommack K E, Ravel J, Hill R T, et al. Population dynamics of Chesapeake Bay virioplankton:total-community analysis by pulse-field gel electrophoresis[J]. Appl. Environ. Microbiol,1999,65(1):231-240.
[5]Enumeration of virus particles by electron micrography. Sharp, G. D. Proc. Soc. Exp. Biol. Med.1949,70:54-59.
[6]Cochlan W P, Wikner J, Steward G F, et al. Spatial distribution of viruses, bacteria, and chlorophyll a in neritic, oceanic and estuarine environments [J]. Mar. Ecol. Prog. Ser.1993,92:77-87.
[7]Hara S, Terauchi K, Koike I, Abundance of viruses in marine waters:assessment by epifluorescence and transmission electron microscopy[J]. Appl. Environ. Microbiol,1991,57:2731-2734.
[8]Wommack K E, Hill R T, Kessel M, et al. Distribution of viruses in the Chesapeake Bay [J]. Appl. Environ. Microbiol,1992,58:2965-2970.
[9]Proctor L E.Advances in the study of marine viruses [J].Microsc. Res, Tech. 1997,37:136-161.
[10]Carlucci A F, Prameter D. An evaluation of factors affecting the survival of Escherichia coli n sea water [J]. Ⅳ. Bacteriophages. Appl. Microbiol. 1960,8:254-256.
[11]Weinbauer, M. G. Ecology of prokaryotic viruses. FEMS Microbiol. Rev.2004,28: 127-181.
[12]Demuth J, Neve H, Witzel K P, Direct electron microscopy study on the morphological diversity of bacteriophage populations in Lake Plu β see [J], Appl. Environ. Microbiol.1993,59:3378-3384.
[13]Weinbauer, M. G., D.Fuks, and P. Peuzzi. Distribution of viruses and dissolved DNA along a coastal trophic gradient in the northern Adriatic Sea [J]. Appl. Envrion. Microbiol.1993.59:4074-4082.
[14]Proctor, L, M., and Fuhrman, J. A. Viral Mortality of Marine Bacteria and Cyanobacteria [J]. Nature.1990,343:60-62.
[15]Cochran P K, Paul J H. Seasonal abundance of lysogenic bacteria in a subtropical estuary [J]. Appl. Environ. Microbiol.1998,64:2308.
[16]Suttle, C. A. Viruses in the sea. Nature[J].2005,437:356-361.
[17]Fuhrman, J. A. Marine viruses and their biogeochemical and ecological effects[J]. Nature.1999,399:541-548.
[18]Wilhelm, S. W.& Suttle, C. A. Viruses and nutrient cycles in the sea[J]. Bioscience. 1999,49:781-788.
[19]Suttle, C. A. Marine viruses-major players in the global ecosystem[J]. Nature RevMicrobiol.2007,5:801-812.
[20]Noble R. T., Fuhrman J. A. Virus decay and its causes in coastal waters[J]. Appl. Envrion. Microbiol.1997.63:77-83.
[21]Suttle, C. A. and Chen F.. Mechanisms and rates of deay of marine viruses in seawater[J]. Appl. Envrion. Microbiol.1992.58:3721-3729.
[22]Hedal M. and Bratbak G..Production and decay of viruses in aquatic environments[J]. Mar. Ecol. Prog. Ser.1991,72:205-212.
[23]Garza D. R. and Suttle C. A.. The effect of cyanophages on the mortality of Synechococcus spp. and selection for UV resistant viral communities[J]. Microb. Ecol.,1998,36:281
[24]Cottrell M.T., Suttle C.A.. Dynamics of a lytic virus infecting the photosynthetic marine picoflagellate. Micromonas pusilla[J].Limnol Oceanogr,1995,40:730-739.
[25]Milligan, K. L. D. and Cosper, E.M.Isolation of virus capable of lysing the Brown Tidemicroalga, Aureococcus anophagefferens[J]. Science,1994,266:805-807.
[26]Suttle, C. A. and Chan. A. M.Viruses infecting the marine Prymnesiophyte Chrysochromulina spp.:isolation, preliminary characterization and natural abundance[J].Mar. Ecol. Prog. Ser.1995,118:275-282.
[27]Malin G,Wilson W.H., Bratbak G, Liss P., Mann N.H., Elevated production dimethylsulfide of resulting from viral infection of cultures of Phaeocystis pouchetii[J]. Limnol Oceanography,1998,43:1389-1393.
[28]Fuhrman, J. Bacterioplankton roles in cycling of organic matter:the microbial food web,1992, p.361-383. In P. G Falkowski, and A. D. Woodhead (ed.), Primary productivity and biogeochemical cycles in the sea. Plenum Press, New York, N.Y.
[29]Bratbak, G., Heldal, M., Norland, S. et al. Viruses as partners in spring bloom microbial trophodynamics[J]. Appl. Environ. Microbiol.,1990,56:1400-1405.
[30]Fuhrman J.A.,Suttle C.A, Viruses in marine planktonic ystems[J].Oceanography. 1993,6:51-63.
[31]Paul, J. H., Rose, J. B., Jiang, S. C. et al. Distribution of viral abundance in the reef environment of Key Largo, Florida[J]. Appl. Environ. Microbiol.1993,59:718-724.
[32]Baas Becking, L. G. M. Geobiologie of Inleiding tot de Milieukunde. (Van Stockum & Zoon,1934).
[33]Pedros-Alio, C. Marine microbial diversity:can it be determined[J].Trends Microbiol.2006,14:257-263.
[34]Martiny, J. B. H. et al. Microbial biogeography:putting microorganisms on the map[J]. Nature Rev. Microbiol.2006,4:102-112.
[35]Foissner, W. Biogeography and dispersal of micro-organisms:a review emphasizing protists[J]. Acta Protozool.2006,45:111-136.
[36]Ramette, A.& Tiedje, J. M. Biogeography:an emerging cornerstone for understanding prokaryotic diversity, ecology, and evolution[J]. Microb. Ecol. 2007,53:197-207.
[37]Fierer, N.. From the Environment to Organisms and Genomes and Back (ed. Zengler, K.) 95-115 (ASM Press,2008).
[38]Jiang, S. C., and J. H. Paul. Seasonal and diel abundance of viruses and occurrence of lysogeny/bacteriocinogeny in the marine environment[J]. Mar. Ecol. Prog. Ser., 1994,104:163-172.
[39]Weinbauer, M. G., D. Fuks, S. Puskaric, et al. Diel, seasonal, and depth-related variability of viruses and dissolved DNA in the Northern Adriatic sea[J]. Microb. Ecol,1995,30:25-41.
[40]Boehme, J., M. E. Frischer, S. C. Jiang, et al. Viruses, bacterioplankton, and phytoplankton in the southeastern Gulf of Mexico:distribution and contribution to oceanic DNA pools[J]. Mar. Ecol. Prog. Ser.1993,97:1-10.
[41]Weinbauer, M. G., D. Fuks, P. Peuzzi.. Distribution of viruses and dissolved DNA along a coastal trophic gradient in the northern Adriatic Sea[J].Appl.Environ. Microbiol.1993.59:4074-4082.
[42]Maranger, R., and D. F. Bird. Viral abundance in aquatic systems:acomparison between marine and fresh waters[J]. Mar. Ecol. Prog. Ser,1995,121:1-3.
[43]D. Stopar1, A. erne1, et al. Viral Abundance and a High Proportion of Lysogens Suggest That Viruses Are Important Members of the Microbial Community in the Gulf of Trieste[J]. Microbial Ecology. Microb. Ecol,2003,47:249-256.
[44]Hewson,I., Fuhrman,J.A., Dennison,W.C. Virus-like particle distribution and abundance in sediments and overlying waters along eutrophication gradients in two subtropical estuaries[J].Limnol. Oceanogr.,2001,46:1734-1746.
[45]C. Corinaldesi, E. Crevatin, P. Del Negro, et al. Large-Scale Spatial Distribution of Virioplankton in the Adriatic Sea:Testing the Trophic State Control Hypothesis[J]. Appl. Envir. Microbiol.,2003,69(5):2664-2673.
[46]Suttle C. A., Cyanophages and their role in the ecology of cyanobacteria. In:Brian A, Malcolm P, eds. The ecology of cyanobacteria. Netherlands Dordrecht:Kluwer academic publishers,2000.563-589.
[47]Hara, S., I. Koike, K. Terauchi, et al. Abundance of viruses in deep oceanic waters[J]. Mar. Ecol. Prog. Ser.,1996,145:26-277.
[48]Hennes, K. P., M. Simon. Significance of bacteriophages for controlling bacterio-plankton growth in a mesotrophic lake[J].Appl. Envir. Microb.1995,61:333-340.
[49]Kepner, R. L., R. A. Wharton, and C. A. Suttle. Viruses in Antarctic lakes[J]. Limnol. Oceanogr.1998,43:1754.
[50]Tuomi, P., T. Torsvik, M. Heldal, et al. Bacterial population dynamics in a meromictic lake[J]. Appl. Environ. Microbiol,1997,63:2181-2188.
[51]Steward, G F., D. C. Smith, and F. Azam. Abundance and production of bacteria and viruses in the Bering and Chukchi seas[J]. Mar. Ecol. Prog. Ser.,1996,131:287-300.
[52]Weinbauer, M. G., D. Fuks, S. Puskaric, et al. Diel, seasonal, and depth-related variability of viruses and dissolved DNA in the Northern Adriatic sea[J]. Microb. Ecol,1995,30:25-41.
[53]Guixa-Boixereu, N., D. Vasque, J. P. Gasol, et al. Distribution of viruses and their potential effect on bacterioplankton in a oligotrophic marine system[J]. Aquat. Microb. Ecol.,1999,19:205-213.
[54]Bettarel,Y, Sime-Ngando,T., et al. Virioplankton and microbial communities in aquatic systems:a seasonal study in two lakes of differing trophy[J]. Freshwater Biology,2003,48:810-822.
[55]Mathias, C. B., A. K. T. Kirschner, and B. Velimirov. Seasonal variations of virus abundance and viral control of the bacterial production in a backwater system of the Danube river[J].Appl. Environ. Microbiol,1995,61:3734-3740.
[56]Farnell-Jackson E.A. and Ward A.K. Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland[J]. Freshwater Biology,2003,48: 841-851.
[57]赵淑敏,田勇琴.三种浓缩方法在水体病毒分离中的比较研究[J].环境与健康杂志,1996,13(3)：120-122。
[58]尤风兴,刘秉辉,张玲妹等.水标本中脊瘤灰质炎病毒3种浓缩方法的比较及改进方法的建立[J].病毒学杂志,1991,6(2)：125-128.
[59]Suttle C. A. Enumeration and isolation of viruses[A]. Aquatic microbial ecology[C]. Boca Raton:Lewis Publishers,1993,121-134.
[60]Suttle C. A. Community structure:viruses[A]. Manual of environmental micro-biology[C]. Washington DC:ASM Press,1997,272-277.
[61]Marie D, Brussard CP, Thyrhaug R, et al. Enumeration of marine viruses in culture and natural samples by flow cytometry[J]. Appl. Environ.Microbiol,1999,65:45-52.
[62]Noble R T, Fuhrman J A. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria[J]. Aquat. Microb. Ecol,1998,14:113-118.
[63]Steward GF, Wikner J, Cochlan WP, et al. Estimation of virus production in the sea[J]. Method development. Mar. Microb. Food Webs,1992,6:57-78.
[64]Chen, F., Lu, J. R., Binder, B. J. et al. Application of digital image analysis and flow cytomehy to enumerate marine viruses stained with SYBR Gold[J]. Applied and Environmenal Microbiology,2001,67:539-545.
[65]Chen F, Suttle C A, Short S M. Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes[J]. Appl. Environ. Microbiol,1996,62(8):2869-2874.
[66]Chen F, Suttle CA. Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes[J]. Virology,1996,219(1):170-178.
[67]Hambly E, Tetart F, Desplats C, et al. A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2[J]. Proc. Natl. Acad. Sci,2001,98(20):11411-11416.
[68]Chen F, Suttle C A. Amplification of DNA polymerase gene fragments from viruses infecting microalgae[J].Appl. Environ. Microbiol,1995,61(4):1274-1278
[69]Short S M, Suttle C A.. Use of the polymerase chain reaction and denaturing gradient gel electrophoresis to study diversity in natural virus communities [J]. Hydrobiology,1999,401:19-32.
[70]罗文清,鞠川,程凯等.一种浓缩噬藻体的反冲超滤技术[J].中国病毒学,2003,18(4)：397-400.
[71]Filee, J., Tetart, F., Suttle, C. A., et al. Marine T4-typebacteriophages, a ubiquitous component of the dark matter of the biosphere[J]. Proc. Natl. Acad. Sci. USA,2005, 102:12471-12476.
[72]Desplats C, H. M. Krisch. The diversity and evolution of the T4-type bacteriophages [J].Research in Microbiology.2003,154:259-267.
[73]Tetart.F, C. D, M. K, C. M, H. A, et al. Phylogeny of the Major Head and Tail Genes of the Wide-Ranging T4-Type Bacteriophages[J]. Journal of Bacteriology,2001, 358-366.
[74]Breitbart. M, J. H. Miyake, Forest R. Global distribution of nearly identical phage-encoded DNA sequences. FEMS Microbiology Letters,2004,236:249-256.
[75]Fischer SG, Lerman LS. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis [J]. Cell,1979,16 (1):191-200.
[76]Lerman LS, Fischer SG, Hurley I, et al. Annu Rev Biophys Bioeng,1984,13:399 399-23.
[77]Ursula Dorigo, Stephan Jacquet. Cyanophage Diversity, Inferred from g20 Gene Analyses, in the Largest Natural Lake in France, Lake Bourget[J]. Appl. Environ. Microbiol,2004,70(2):1017-1022.
[78]Zhong, Y., F. Chen, S. W. Wilhelm, L. Poorvin, et al. Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20[J]. Appl. Environ. Microbiol.,2002,68: 1576-1584.
[79]Sullivan, M. B., J. B. Waterbury, and S. W. Chisholm. Cyanophages infecting the
oceanic cyanobacterium Prochlorococcus[J]. Nature,2003,424:1047-1051.
[80]Becker, S., M. Fahrbach, et al. Quantitative tracing, by Taq nuclease assays, of a Synechococcus ecotype in a highly diversified natural population[J]. Appl. Environ. Microbiol.,2002,68:4486-4494.
[81]Urbach, E., D. J. Scanlan, D. L. Distel, et al. Rapid diversification of marine picoplankton with dissimilar light harvesting structures inferred from sequences of Prochlorococcus and Synechococcus(cyanobacteria) [J]. J. Mol. Evol.,1998, 46:188-201.
[82]Crosbie, N. D., M. Pockl, and T. Weisse. Dispersal and phylogenetic diversity of nonmarine picocyanobacteria, inferred from 16S rRNA gene and cpcBA-intergenic spacer sequences analyses[J]. Appl. Environ. Microbiol.,2003,69:5716-5721.
[83]Wommack K E, Hill R T, Kessel M, et al. Distribution of viruses in the ChesapeakeBay[J].Appl. Environ. Microbiol.,1992,58:2965-2970.
[84]Steven W. Wilhelm, Matthew J. Carberry,Marine and Freshwater Cyanophages in a Laurentian Great Lake:Evidence from Infectivity Assays and Molecular Analyses of g20 Genes[J]. Appl. Environ. Microbiol.,2006,72(7),4957-4963.
[85]Takeshi Fujiia, Natsuko Nakayama. Novel capsid genes (g23) of T4-type bacteriophages in a Japanese paddy field[J]. Soil Biology & Biochemistry,2008, 40:1049-1058.
[86]Guanghua Wang, Motoki Hayashi. Survey of major capsid genes (g23) of T4-type bacteriophages in Japanese paddy field soils[J]. Soil Biology & Biochemistry,2009, 41:13-20.
[87]Chuan, Yap P, Fan, Yuan Y, Lua, Linda. Quantitative analysis of virus-like particle size and distribution by field-flow fractionation[J]. Biotechnol Bioeng,2008,99(6): 1425-1433.
[88]Winget, D. M.,Worrimack, K. E. Randomly amplified polymorphic DNA PCR as a tool for assessment of marine viral richness[J]. Appl. Environ. Microbiol.,2008,74(9): 2612-2618.
[89]Elizabeth A. Dinsdale, Robert A. Edwards,etc. Functional metagenomic profiling of nine biomes[J]. Nature,2008,452(3):629-633.
[90]Daniel H. Huson, Alexander F. Auch, etc. MEGAN analysis of metagenomic data[J]. Genome Res.2007,17:377-386.
[91]侯昆,程凯,许敏,赵以军.武汉东湖PP类噬藻体的遗传多样性研究[J].华中师范大学学报(自然科学版),2008,42(1)：93-96.
[92]叶新,程凯,许敏,等.PP类噬藻体的生态分布及系统进化研究[J].华中师范大学学报(自然科学版),2010,44(1)：109-114.
[93]Cochran P K, Paul J H, Seasonal abundance of lysogenic bacteria in a subtropical estuary [J].Appl. Environ. Microbiol.1998,64:2308.
[94]Levin B R, LipsitchM, Bonhoeffer S,1999, Population biology, evolution and infectious disease:convergence and synthesis [J].Science,283:806-809.
[2]Bergh O, orsheim K Y. Bratbak B G, et al. High abundance of viruses found in aquatic environments [J]. Nature,1989,340:467-468.
[3]Wommack K E, Ravel J, Hill R T, et al. Hybridization analysis of Chesapeake Bay virioplankton [J]. Appl. Environ. Microbiol,1999,65:241-250.
[4]Wommack K E, Ravel J, Hill R T, et al. Population dynamics of Chesapeake Bay virioplankton:total-community analysis by pulse-field gel electrophoresis[J]. Appl. Environ. Microbiol,1999,65(1):231-240.
[5]Enumeration of virus particles by electron micrography. Sharp, G. D. Proc. Soc. Exp. Biol. Med.1949,70:54-59.
[6]Cochlan W P, Wikner J, Steward G F, et al. Spatial distribution of viruses, bacteria, and chlorophyll a in neritic, oceanic and estuarine environments [J]. Mar. Ecol. Prog. Ser.1993,92:77-87.
[7]Hara S, Terauchi K, Koike I, Abundance of viruses in marine waters:assessment by epifluorescence and transmission electron microscopy[J]. Appl. Environ. Microbiol,1991,57:2731-2734.
[8]Wommack K E, Hill R T, Kessel M, et al. Distribution of viruses in the Chesapeake Bay [J]. Appl. Environ. Microbiol,1992,58:2965-2970.
[9]Proctor L E.Advances in the study of marine viruses [J].Microsc. Res, Tech. 1997,37:136-161.
[10]Carlucci A F, Prameter D. An evaluation of factors affecting the survival of Escherichia coli n sea water [J]. Ⅳ. Bacteriophages. Appl. Microbiol. 1960,8:254-256.
[11]Weinbauer, M. G. Ecology of prokaryotic viruses. FEMS Microbiol. Rev.2004,28: 127-181.
[12]Demuth J, Neve H, Witzel K P, Direct electron microscopy study on the morphological diversity of bacteriophage populations in Lake Plu β see [J], Appl. Environ. Microbiol.1993,59:3378-3384.
[13]Weinbauer, M. G., D.Fuks, and P. Peuzzi. Distribution of viruses and dissolved DNA along a coastal trophic gradient in the northern Adriatic Sea [J]. Appl. Envrion. Microbiol.1993.59:4074-4082.
[14]Proctor, L, M., and Fuhrman, J. A. Viral Mortality of Marine Bacteria and Cyanobacteria [J]. Nature.1990,343:60-62.
[15]Cochran P K, Paul J H. Seasonal abundance of lysogenic bacteria in a subtropical estuary [J]. Appl. Environ. Microbiol.1998,64:2308.
[16]Suttle, C. A. Viruses in the sea. Nature[J].2005,437:356-361.
[17]Fuhrman, J. A. Marine viruses and their biogeochemical and ecological effects[J]. Nature.1999,399:541-548.
[18]Wilhelm, S. W.& Suttle, C. A. Viruses and nutrient cycles in the sea[J]. Bioscience. 1999,49:781-788.
[19]Suttle, C. A. Marine viruses-major players in the global ecosystem[J]. Nature RevMicrobiol.2007,5:801-812.
[20]Noble R. T., Fuhrman J. A. Virus decay and its causes in coastal waters[J]. Appl. Envrion. Microbiol.1997.63:77-83.
[21]Suttle, C. A. and Chen F.. Mechanisms and rates of deay of marine viruses in seawater[J]. Appl. Envrion. Microbiol.1992.58:3721-3729.
[22]Hedal M. and Bratbak G..Production and decay of viruses in aquatic environments[J]. Mar. Ecol. Prog. Ser.1991,72:205-212.
[23]Garza D. R. and Suttle C. A.. The effect of cyanophages on the mortality of Synechococcus spp. and selection for UV resistant viral communities[J]. Microb. Ecol.,1998,36:281
[24]Cottrell M.T., Suttle C.A.. Dynamics of a lytic virus infecting the photosynthetic marine picoflagellate. Micromonas pusilla[J].Limnol Oceanogr,1995,40:730-739.
[25]Milligan, K. L. D. and Cosper, E.M.Isolation of virus capable of lysing the Brown Tidemicroalga, Aureococcus anophagefferens[J]. Science,1994,266:805-807.
[26]Suttle, C. A. and Chan. A. M.Viruses infecting the marine Prymnesiophyte Chrysochromulina spp.:isolation, preliminary characterization and natural abundance[J].Mar. Ecol. Prog. Ser.1995,118:275-282.
[27]Malin G,Wilson W.H., Bratbak G, Liss P., Mann N.H., Elevated production dimethylsulfide of resulting from viral infection of cultures of Phaeocystis pouchetii[J]. Limnol Oceanography,1998,43:1389-1393.
[28]Fuhrman, J. Bacterioplankton roles in cycling of organic matter:the microbial food web,1992, p.361-383. In P. G Falkowski, and A. D. Woodhead (ed.), Primary productivity and biogeochemical cycles in the sea. Plenum Press, New York, N.Y.
[29]Bratbak, G., Heldal, M., Norland, S. et al. Viruses as partners in spring bloom microbial trophodynamics[J]. Appl. Environ. Microbiol.,1990,56:1400-1405.
[30]Fuhrman J.A.,Suttle C.A, Viruses in marine planktonic ystems[J].Oceanography. 1993,6:51-63.
[31]Paul, J. H., Rose, J. B., Jiang, S. C. et al. Distribution of viral abundance in the reef environment of Key Largo, Florida[J]. Appl. Environ. Microbiol.1993,59:718-724.
[32]Baas Becking, L. G. M. Geobiologie of Inleiding tot de Milieukunde. (Van Stockum & Zoon,1934).
[33]Pedros-Alio, C. Marine microbial diversity:can it be determined[J].Trends Microbiol.2006,14:257-263.
[34]Martiny, J. B. H. et al. Microbial biogeography:putting microorganisms on the map[J]. Nature Rev. Microbiol.2006,4:102-112.
[35]Foissner, W. Biogeography and dispersal of micro-organisms:a review emphasizing protists[J]. Acta Protozool.2006,45:111-136.
[36]Ramette, A.& Tiedje, J. M. Biogeography:an emerging cornerstone for understanding prokaryotic diversity, ecology, and evolution[J]. Microb. Ecol. 2007,53:197-207.
[37]Fierer, N.. From the Environment to Organisms and Genomes and Back (ed. Zengler, K.) 95-115 (ASM Press,2008).
[38]Jiang, S. C., and J. H. Paul. Seasonal and diel abundance of viruses and occurrence of lysogeny/bacteriocinogeny in the marine environment[J]. Mar. Ecol. Prog. Ser., 1994,104:163-172.
[39]Weinbauer, M. G., D. Fuks, S. Puskaric, et al. Diel, seasonal, and depth-related variability of viruses and dissolved DNA in the Northern Adriatic sea[J]. Microb. Ecol,1995,30:25-41.
[40]Boehme, J., M. E. Frischer, S. C. Jiang, et al. Viruses, bacterioplankton, and phytoplankton in the southeastern Gulf of Mexico:distribution and contribution to oceanic DNA pools[J]. Mar. Ecol. Prog. Ser.1993,97:1-10.
[41]Weinbauer, M. G., D. Fuks, P. Peuzzi.. Distribution of viruses and dissolved DNA along a coastal trophic gradient in the northern Adriatic Sea[J].Appl.Environ. Microbiol.1993.59:4074-4082.
[42]Maranger, R., and D. F. Bird. Viral abundance in aquatic systems:acomparison between marine and fresh waters[J]. Mar. Ecol. Prog. Ser,1995,121:1-3.
[43]D. Stopar1, A. erne1, et al. Viral Abundance and a High Proportion of Lysogens Suggest That Viruses Are Important Members of the Microbial Community in the Gulf of Trieste[J]. Microbial Ecology. Microb. Ecol,2003,47:249-256.
[44]Hewson,I., Fuhrman,J.A., Dennison,W.C. Virus-like particle distribution and abundance in sediments and overlying waters along eutrophication gradients in two subtropical estuaries[J].Limnol. Oceanogr.,2001,46:1734-1746.
[45]C. Corinaldesi, E. Crevatin, P. Del Negro, et al. Large-Scale Spatial Distribution of Virioplankton in the Adriatic Sea:Testing the Trophic State Control Hypothesis[J]. Appl. Envir. Microbiol.,2003,69(5):2664-2673.
[46]Suttle C. A., Cyanophages and their role in the ecology of cyanobacteria. In:Brian A, Malcolm P, eds. The ecology of cyanobacteria. Netherlands Dordrecht:Kluwer academic publishers,2000.563-589.
[47]Hara, S., I. Koike, K. Terauchi, et al. Abundance of viruses in deep oceanic waters[J]. Mar. Ecol. Prog. Ser.,1996,145:26-277.
[48]Hennes, K. P., M. Simon. Significance of bacteriophages for controlling bacterio-plankton growth in a mesotrophic lake[J].Appl. Envir. Microb.1995,61:333-340.
[49]Kepner, R. L., R. A. Wharton, and C. A. Suttle. Viruses in Antarctic lakes[J]. Limnol. Oceanogr.1998,43:1754.
[50]Tuomi, P., T. Torsvik, M. Heldal, et al. Bacterial population dynamics in a meromictic lake[J]. Appl. Environ. Microbiol,1997,63:2181-2188.
[51]Steward, G F., D. C. Smith, and F. Azam. Abundance and production of bacteria and viruses in the Bering and Chukchi seas[J]. Mar. Ecol. Prog. Ser.,1996,131:287-300.
[52]Weinbauer, M. G., D. Fuks, S. Puskaric, et al. Diel, seasonal, and depth-related variability of viruses and dissolved DNA in the Northern Adriatic sea[J]. Microb. Ecol,1995,30:25-41.
[53]Guixa-Boixereu, N., D. Vasque, J. P. Gasol, et al. Distribution of viruses and their potential effect on bacterioplankton in a oligotrophic marine system[J]. Aquat. Microb. Ecol.,1999,19:205-213.
[54]Bettarel,Y, Sime-Ngando,T., et al. Virioplankton and microbial communities in aquatic systems:a seasonal study in two lakes of differing trophy[J]. Freshwater Biology,2003,48:810-822.
[55]Mathias, C. B., A. K. T. Kirschner, and B. Velimirov. Seasonal variations of virus abundance and viral control of the bacterial production in a backwater system of the Danube river[J].Appl. Environ. Microbiol,1995,61:3734-3740.
[56]Farnell-Jackson E.A. and Ward A.K. Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland[J]. Freshwater Biology,2003,48: 841-851.
[57]赵淑敏,田勇琴.三种浓缩方法在水体病毒分离中的比较研究[J].环境与健康杂志,1996,13(3)：120-122。
[58]尤风兴,刘秉辉,张玲妹等.水标本中脊瘤灰质炎病毒3种浓缩方法的比较及改进方法的建立[J].病毒学杂志,1991,6(2)：125-128.
[59]Suttle C. A. Enumeration and isolation of viruses[A]. Aquatic microbial ecology[C]. Boca Raton:Lewis Publishers,1993,121-134.
[60]Suttle C. A. Community structure:viruses[A]. Manual of environmental micro-biology[C]. Washington DC:ASM Press,1997,272-277.
[61]Marie D, Brussard CP, Thyrhaug R, et al. Enumeration of marine viruses in culture and natural samples by flow cytometry[J]. Appl. Environ.Microbiol,1999,65:45-52.
[62]Noble R T, Fuhrman J A. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria[J]. Aquat. Microb. Ecol,1998,14:113-118.
[63]Steward GF, Wikner J, Cochlan WP, et al. Estimation of virus production in the sea[J]. Method development. Mar. Microb. Food Webs,1992,6:57-78.
[64]Chen, F., Lu, J. R., Binder, B. J. et al. Application of digital image analysis and flow cytomehy to enumerate marine viruses stained with SYBR Gold[J]. Applied and Environmenal Microbiology,2001,67:539-545.
[65]Chen F, Suttle C A, Short S M. Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes[J]. Appl. Environ. Microbiol,1996,62(8):2869-2874.
[66]Chen F, Suttle CA. Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes[J]. Virology,1996,219(1):170-178.
[67]Hambly E, Tetart F, Desplats C, et al. A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2[J]. Proc. Natl. Acad. Sci,2001,98(20):11411-11416.
[68]Chen F, Suttle C A. Amplification of DNA polymerase gene fragments from viruses infecting microalgae[J].Appl. Environ. Microbiol,1995,61(4):1274-1278
[69]Short S M, Suttle C A.. Use of the polymerase chain reaction and denaturing gradient gel electrophoresis to study diversity in natural virus communities [J]. Hydrobiology,1999,401:19-32.
[70]罗文清,鞠川,程凯等.一种浓缩噬藻体的反冲超滤技术[J].中国病毒学,2003,18(4)：397-400.
[71]Filee, J., Tetart, F., Suttle, C. A., et al. Marine T4-typebacteriophages, a ubiquitous component of the dark matter of the biosphere[J]. Proc. Natl. Acad. Sci. USA,2005, 102:12471-12476.
[72]Desplats C, H. M. Krisch. The diversity and evolution of the T4-type bacteriophages [J].Research in Microbiology.2003,154:259-267.
[73]Tetart.F, C. D, M. K, C. M, H. A, et al. Phylogeny of the Major Head and Tail Genes of the Wide-Ranging T4-Type Bacteriophages[J]. Journal of Bacteriology,2001, 358-366.
[74]Breitbart. M, J. H. Miyake, Forest R. Global distribution of nearly identical phage-encoded DNA sequences. FEMS Microbiology Letters,2004,236:249-256.
[75]Fischer SG, Lerman LS. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis [J]. Cell,1979,16 (1):191-200.
[76]Lerman LS, Fischer SG, Hurley I, et al. Annu Rev Biophys Bioeng,1984,13:399 399-23.
[77]Ursula Dorigo, Stephan Jacquet. Cyanophage Diversity, Inferred from g20 Gene Analyses, in the Largest Natural Lake in France, Lake Bourget[J]. Appl. Environ. Microbiol,2004,70(2):1017-1022.
[78]Zhong, Y., F. Chen, S. W. Wilhelm, L. Poorvin, et al. Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20[J]. Appl. Environ. Microbiol.,2002,68: 1576-1584.
[79]Sullivan, M. B., J. B. Waterbury, and S. W. Chisholm. Cyanophages infecting the
oceanic cyanobacterium Prochlorococcus[J]. Nature,2003,424:1047-1051.
[80]Becker, S., M. Fahrbach, et al. Quantitative tracing, by Taq nuclease assays, of a Synechococcus ecotype in a highly diversified natural population[J]. Appl. Environ. Microbiol.,2002,68:4486-4494.
[81]Urbach, E., D. J. Scanlan, D. L. Distel, et al. Rapid diversification of marine picoplankton with dissimilar light harvesting structures inferred from sequences of Prochlorococcus and Synechococcus(cyanobacteria) [J]. J. Mol. Evol.,1998, 46:188-201.
[82]Crosbie, N. D., M. Pockl, and T. Weisse. Dispersal and phylogenetic diversity of nonmarine picocyanobacteria, inferred from 16S rRNA gene and cpcBA-intergenic spacer sequences analyses[J]. Appl. Environ. Microbiol.,2003,69:5716-5721.
[83]Wommack K E, Hill R T, Kessel M, et al. Distribution of viruses in the ChesapeakeBay[J].Appl. Environ. Microbiol.,1992,58:2965-2970.
[84]Steven W. Wilhelm, Matthew J. Carberry,Marine and Freshwater Cyanophages in a Laurentian Great Lake:Evidence from Infectivity Assays and Molecular Analyses of g20 Genes[J]. Appl. Environ. Microbiol.,2006,72(7),4957-4963.
[85]Takeshi Fujiia, Natsuko Nakayama. Novel capsid genes (g23) of T4-type bacteriophages in a Japanese paddy field[J]. Soil Biology & Biochemistry,2008, 40:1049-1058.
[86]Guanghua Wang, Motoki Hayashi. Survey of major capsid genes (g23) of T4-type bacteriophages in Japanese paddy field soils[J]. Soil Biology & Biochemistry,2009, 41:13-20.
[87]Chuan, Yap P, Fan, Yuan Y, Lua, Linda. Quantitative analysis of virus-like particle size and distribution by field-flow fractionation[J]. Biotechnol Bioeng,2008,99(6): 1425-1433.
[88]Winget, D. M.,Worrimack, K. E. Randomly amplified polymorphic DNA PCR as a tool for assessment of marine viral richness[J]. Appl. Environ. Microbiol.,2008,74(9): 2612-2618.
[89]Elizabeth A. Dinsdale, Robert A. Edwards,etc. Functional metagenomic profiling of nine biomes[J]. Nature,2008,452(3):629-633.
[90]Daniel H. Huson, Alexander F. Auch, etc. MEGAN analysis of metagenomic data[J]. Genome Res.2007,17:377-386.
[91]侯昆,程凯,许敏,赵以军.武汉东湖PP类噬藻体的遗传多样性研究[J].华中师范大学学报(自然科学版),2008,42(1)：93-96.
[92]叶新,程凯,许敏,等.PP类噬藻体的生态分布及系统进化研究[J].华中师范大学学报(自然科学版),2010,44(1)：109-114.
[93]Cochran P K, Paul J H, Seasonal abundance of lysogenic bacteria in a subtropical estuary [J].Appl. Environ. Microbiol.1998,64:2308.
[94]Levin B R, LipsitchM, Bonhoeffer S,1999, Population biology, evolution and infectious disease:convergence and synthesis [J].Science,283:806-809.