Sampling Scale

High spatial heterogeneity of soil influences the diversity of microbes and their function. Spatial heterogeneity in soil microbial communities occurs at many scales, from soil particles (e.g., soil microaggregates) to the plant rhizosphere, to field plots, and to the ecosystem and global levels. In order to improve our knowledge of how microbial community structure influences ecosystem function, we must obtain more quantitative knowledge of the interaction between microbes, plant residues, and soil management at a variety of spatial scales.


We have come a long way in developing our understanding of microbial ecology, but have many milestones yet to meet. Molecular tools offer unparalleled opportunities to characterize microbes in culture and directly from field soils. These tools are allowing us to ask questions at much larger geographic scales than has been possible previously. We are now able to examine such issues as how micro-bial populations vary across soil types and climatic zones, in association with plant roots and between various plant species, and in response to soil management or soil pollution. We are now just seeing the tip of the iceberg in terms of soil microbial diversity with the use of molecular approaches. The amount of work that remains is daunting, yet exciting as so much remains to be discovered. Recent techniques developed for the study of microbial populations, such as T-RFLP and

DGGE, allow access to the very large proportion of organisms that are present in the soil and which remain unculturable under laboratory conditions. Other techniques, such as RFLP analysis of isotopically labeled, amplified nifH, amoA, nirS, and pmoA sequences or SIP will allow us to target, with high specificity, organisms or groups of organisms responsible for specific functions in soil, particularly those involved in key transformations in the C, N, and S nutrient cycles. These types of technical developments open new horizons of research and applications that will allow a far more complete and less biased view of microbial diversity and function in soils.


Amann, R. I., and Ludwig, W. (2000). Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology. FEMS Microbiol. Rev. 24, 555-565.

Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143-169.

Amann, R. I., Krumholz, L., and Stahl, D. A. (1990). Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172, 762-770.

Binnerup, S. J., Bloem, J., Hansen, B. M., Wolters, W., Veninga, M., and Hansen, M. (2001). Ribosomal RNA content in microcolony forming soil bacteria measured by quantitative 16S rRNA hybridization and image analysis. FEMS Microbiol. Ecol. 37, 231-237.

Bringhurst, R. M., Cardon, Z. G., and Gage, D. J. (2002). Galactosides in the rhizosphere: Utilization by Sinorhizobium meliloti and development of a biosensor. Proc. Natl. Acad. Sci. 98, 4540-4545.

Bruns, M. A., and Buckley, D. H. (2002). Isolation and purification of microbial community nucleic acids from environmental samples. In "Manual of Environmental Microbiology." 2nd ed. (C. J. Hurst, ed.). ASM Press, Washington, DC.

Campbell, N. A., and Reece, J. B. (2005). "Biology." 7th ed. Pearson, Benjamin-Cummings, San Francisco.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-895.

Clement, B. G., Kehl, L. E., DeBord, L., and Kitts, C. L. (1998). Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J. Microbiol. Methods 31, 135-142.

Cole, J. R., Chai, B., Farris, R. J., Wang, Q., Kulam, S. A., McGarrell, D. M., Garrity, G. M., and Tiedje, J. M. (2005). The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res. 33(database issue), D294-D296. doi: 10.1093/nar/gki038.

Denef, V. J., Park, J., Rodriques, J. L. M., Tsoi, T. V., Hashsham, S. A., and Tiedje, J. M. (2003). Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities. Environ. Microbiol. 5, 933-943.

Devare, M., Jones, C. M., and Thies, J. E. (2004). Effects of CRW transgenic corn and tefluthrin on the soil microbial community: biomass, activity, and diversity. J. Environ. Qual. 33, 837-843.

Dewit, M. Y. L., and Klatser, P. R. (1994). Mycobacterium leprae isolates from different sources have identical sequences of the spacer regions between the 16S and 23S ribosomal RNA genes. Microbiology 140, 1983-1987.

Dumont, M. G., and Murrell, J. C. (2005). Stable isotope probing—linking microbial identity to function. Nat. Rev. 3, 499-504.

Edel-Hermann, W., Dreumont, C., Perez-Piqueres, A., and Steinberg, C. (2004). Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils. FEMS Microbiol. Ecol. 47, 397-404.

Ekins, R., and Chu, F. W. (1999). Microarrays: their origins and applications. Trends Biotechnol. 17, 217-218.

Errampalli, D., Leung, K., Cassidy, M. B., Kostrzynska, M., Blears, M., Lee, H., and Trevors, J. T. (1999). Applications of the green fluorescent protein as a molecular marker in environmental microorganisms. J. Microbiol. Methods 35, 187-199.

Espejo, R. T., and Romero, J. (1998). PAGE analysis of the heteroduplexes formed between PCR-amplified 16S rRNA genes: estimation of sequence similarity and rDNA complexity. Microbiology 144, 1611-1617.

Felske, A., Engelen, B., Nübel, U., and Backhaus, H. (1996). Direct ribosome isolation from soil to extract bacterial rRNA for community analysis. Appl. Environ. Microbiol. 62, 4162-4167.

Filion, M., St-Arnaud, M., and Jabaji-Hare, S. H. (2003). Direct quantification of fungal DNA from soil substrate using real-time PCR. J. Microbiol. Methods 53, 67-76.

Gans, J., Wolinsky, M., and Dunbar, J. (2005). Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309, 1387-1390.

Griffiths, R. I., Manefield, M., Ostle, N., McNamara, N., O'Donnell, A. G., Bailey, M. J., and Whiteley, A. S. (2004). 13CO2 pulse labelling of plants in tandem with stable isotope probing: methodological considerations for examining microbial function in the rhizosphere. J. Microbiol. Methods 58, 119-129.

Grüntzig, V., Stres, B., Ayala del Río, H. L., and Tiedje, J. M. (2002). Improved protocol for T-RFLP analysis by capillary electrophoresis. Center for Microbial Ecology, Michigan State Univ., East Lansing (

Guschin, D. Y., Mobarry, B. K., Proudnikow, D., Stahl, D. A., Rittmann, B. E., and Mirzabekov, A. D. (1997). Oligonucleotide microchips and genosensors for determinative and environmental studies in microbiology. Appl. Environ. Microbiol. 63, 2397-2402.

Henry, S., Baudoin, E., Lopez-Gutierrez, J. C., Martin-Laurent, F., Baumann, A., and Philippot, L. (2004). Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR. J. Microbiol. Methods 59, 327-335.

Hermansson, A., and Lindgren, P. E. 2001. Quantification of ammonia-oxidizing bacteria in arable soil by real-time PCR. Appl. Environ. Microbiol. 67, 972-976.

Heuer, H., Hartung, K., Wieland, G., Kramer, I., and Smalla, K. (1999). Polynucleotide probes that target a hypervariable region of 16S rRNA genes to identify bacterial isolates corresponding to bands of community fingerprints. Appl. Environ. Microbiol. 65, 1045-1049.

Horz, H. P., Yimga, M. T., and Liesack, W. (2001). Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and riboso-mal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling. Appl. Environ. Microbiol. 67, 4177-4185.

Jones, C. M., and Thies, J. E. (2006). Soil microbial community analysis using two-dimensional polyacrylamide gel electrophoresis of the bacterial ribosomal internal transcribed spacer region. J. Microbiol. Methods. In press.

Kolb, S., Knief, C., Stubner, S., and Conrad, R. (2003). Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays. Appl. Environ. Microbiol. 69, 2423-2429.

Kowalchuk, G.A., de Bruijn, F. J., Head, I. M., Akkermans, A. D. L., and van Elsas, J. D. (2003). "Molecular Microbial Ecology Manual" 2nd ed. Kluwer Academic Publishers, The Netherlands.

Landeweert, R., Veenman, C., Kuyper, T. W., Fritze, H., Wernars, K., and Smit, E. (2003). Quantification of ectomycorrhizal mycelium in soil by real-time PCR compared to conventional quantification techniques. FEMS Microbiol. Ecol. 45, 283-292.

Lane, D. J. (1991). 16S/23S rRNA sequencing. In "Nucleic Acid Techniques in Bacterial Systematics" (E. Stackebrandt and M. Goodfellow, eds.), pp. 115-175. Wiley, Chichester.

Lee, N., Nielsen, P. H., Andreasen, K. H., Juretschko, S., Nielsen, J. L., Schleifer, K.-H., and Wagner, M. (1999). Combination of fluorescent in situ hybridization and microautoradiography—a new tool for structure-function analyses in microbial ecology. Appl. Environ. Microbiol. 65, 1289-1297.

Li, Y., Dick, W. A., and Tuovinen, O. H. (2004). Fluorescence microscopy for visualization of soil microorganisms—a review. Biol. Fertil. Soils 39, 301-311.

Liu, W. T., Marsh, T. L., Cheng, H., and Forney, L. (1997). Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63, 4516-4522.

Liu, W.-T., and Stahl, D. A. (2002). Molecular approaches for the measurement of density, diversity and phylogeny. In "Manual of Environmental Microbiology." 2nd ed. (C. J. Hurst ed.). ASM Press, Washington, DC.

Loy, A., Horn, M., and Wagner, M. (2003). ProbeBase: an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res. 31, 514-516.

Madani, M., Subbotin, S. A., and Moens, M. (2005). Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using real-time PCR with SYBR green I dye. Mol. Cell. Probes 19, 81-86.

Manz, W., Amann, R., Ludwig, W., Wagner, M., Schleifer, K.H. (1992). Phylogenetic oligodeoxy-nucleotide probes for the major subclasses of proteobacteria - Problems and solutions. Syst. Appl. Microbiol. 15, 593-600.

McDonald, I. R., Radajewski, S., and Murrell, J. C. (2005). Stable isotope probing of nucleic acids in methanotrophs and methylotrophs: a review. Org. Geochem. 36, 779-787.

Moter, A., and Gobel, U. B. (2000). Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J. Microbiol. Methods 41, 85-112.

Mullis, K. B., and Faloona, F. A. (1987). Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol. 155, 335-350.

Muyzer, G., and Smalla, K. (1998). Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 73, 127-141.

Navarro, E., Simonet, P., Normand, P., and Bardin, R. (1992). Characterisation of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer. Arch. Microbiol. 157, 107-115.

Neufeld, J. D., Yu, Z., Lam, W., and Mohn, W. W. (2004). Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities. Environ. Microbiol. 6, 131-144.

Nicol, G. W., and Schleper, C. (2006). Ammonia-oxidising Chrenarchaeota: important players in the nitrogen cycle? Trends Microbiol. 14, 207-212.

Ouverney, C. C., and Fuhrman, J. A. (1999). Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65, 1746-1752.

Poly, F., Ranjard, L., Nazaret, S., Gourbiere, F., and Monrozier, L. J. (2001). Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties. Appl. Environ. Microbiol. 67, 2255-2262.

Ponsonnet, C., and Nesme, X. (1994). Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions. Arch. Microbiol. 161, 300-309.

Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992). Primary structure of the Aequorea victoria green fluorescent protein. Gene 111, 229-233.

Radajewski, S., Ineson, P., Parekh, N. R., and Murrell, J. C. (2000). Stable-isotope probing as a tool in microbial ecology. Nature 403, 646-649.

Radajewski, S., McDonald, I. R., and Murrell, J. C. (2003). Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms. Curr. Opin. Biotech. 14, 296-302.

Rangel-Castro, J. I., Killham, K., Ostle, N., Nicol, G. W., Anderson, I. C., Scrimgeour, C. M., Ineson, P., Meharg, A., and Prosser J. I. (2005). Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms. Environ. Microbiol. 7, 828-838.

Rondon, M. R., August, P. R., Betterman, A. D., Brady, S. F., Grossman, T. H., Liles, M. R., Loiacono, K. A., Lynch, B. A., MacNiel, I. A., Minor, C., Tiong, C. L., Gilman, M., Osburne, M. S., Clardy, J., Handelsman, J., and Goodman, R. M. (2000). Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66, 2541-2547.

Roslev, P., Iversen, N., and Henriksen, K. (1998). Direct fingerprinting of metabolically active bacteria in environmental samples by substrate specific radiolabelling and lipid analysis. J. Microbiol. Methods 31, 99-111.

Rotthauwe, J.-H., Witzel, K.-P., and Liesack, W. (1997). The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl. Environ. Microbiol. 63, 4704-4712. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA-polymerase. Science 239, 487-491. Sambrook, J., Fritsch, E. F., and Maniatis, T. A. (1989). "Molecular Cloning: A Laboratory Manual."

2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors.

Proc. Natl. Acad. Sci. USA 74, 5463-5467. Schleper, C., Jurgens, G., and Jonuscheit, M. (2005). Genomic studies of uncultivated archaea. Nat.

Rev. Microbiol. 3, 479-488. Stephen, J. R., Kowalchuk, G. A., Bruns, M. A. V., McCaig, A. E., Phillips, C.J., Embley, T.M., and Prosser, J. I. (1998). Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenic probing. Appl. Environ. Microbiol. 64, 2958-2965. Stuurman, N., Bras, C. P., Schlaman, H. R. M., Wijfjes, A. H. M., Bloemberg, G., and Spaink, H. P. (2000). Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants. Mol. Plant-Microbe Interact. 13, 1163-1169. Thies, J. E. (2006). Soil microbial community analysis using T-RFLP: Separating signals from the noise.

Soil Sci. Soc. Amer. J. In press. Torsvik, V., Daae, F.L., Sandaa, R.-A., 0vreas, L. (1998). Novel techniques for analysing microbial diversity in natural and perturbed environments. J. Bacteriol. 64, 53-62. Torsvik, V., Goksoyr, J., and Daae, F. L. (1990). High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782-787.

Treonis, A. M., Ostle, N. J., Stott, A. W., Primrose, R., Grayston, S. J., and Ineson, P. (2004). Identification of groups of metabolically-active rhizosphere microorganisms by stable isotope probing of PLFAs. Soil Biol. Biochem. 36, 533-537. Turpeinen, R., Kairesalo, T., and Haggblom, M. M. (2004). Microbial community structure and activity in arsenic-, chromium- and copper-contaminated soils. FEMS Microbiol. Ecol. 47, 39-50. Versalovic, J., Koeuth, T., and Lupski, J. R. (1991). Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acid. Res. 19, 6823-6831. Versalovic, J., Schneider, M., de Bruijn, F. J., and Lupski, J. R. (1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cell. Biol. 5, 25-40. Wellington, E. M. H., Berry, A., and Krsek, M. (2003). Resolving functional diversity in relation to microbial community structure in soil: exploiting genomics and stable isotope probing. Curr. Opin. Microbiol. 6, 295-301. Wintzingerode, F. V., Gobel, U. V., and Stackebrandt, E. (1997). Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21, 213-229. Woese, C. R., Kandler, O., and Wheelis, M. L. (1990). Towards a natural system of organisms: proposal for the domains Archaea, Bacteria and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576-4579. Wu, L. Y., Thompson, D. K., Li, G. S., Hurt, R. A., Tiedje, J. M., and Zhou, J. Z. (2001). Development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl. Environ. Microbiol. 67, 5780-5790. Zheng, D., Alm, E. W., Stahl, D. A. and Raskin, L. (1996). Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies. Appl. Environ. Microbiol. 62, 4504-4513. Zhou, J. Z. (2003). Microarrays for bacterial detection and microbial community analysis. Curr. Opin. Microbiol. 6, 288-294.

Zhou, J., and Thompson, D. K. (2002). Challenges in applying microarrays to environmental studies. Curr. Opin. Biotechnol. 13, 204-207.

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