Current Microbiology

, Volume 67, Issue 4, pp 423–430 | Cite as

Investigating the Diversity of Pseudomonas spp. in Soil Using Culture Dependent and Independent Techniques

  • Lili Li
  • Waleed Abu Al-Soud
  • Lasse Bergmark
  • Leise Riber
  • Lars H. Hansen
  • Jakob Magid
  • Søren J. Sørensen
Article

Abstract

Less than 1 % of bacterial populations present in environmental samples are culturable, meaning that cultivation will lead to an underestimation of total cell counts and total diversity. However, it is less clear whether this is also true for specific well-defined groups of bacteria for which selective culture media is available. In this study, we use culture dependent and independent techniques to describe whether isolation of Pseudomonas spp. on selective nutrient-poor NAA 1:100 agar-medium can reflect the full diversity, found by pyrosequencing, of the total soil Pseudomonas community in an urban waste field trial experiment. Approximately 3,600 bacterial colonies were isolated using nutrient-poor NAA 1:100 medium from soils treated with different fertilizers; (i) high N-level sewage sludge (SA), (ii) high N-level cattle manure (CMA), and (iii) unfertilized control soil (U). Based on Pseudomonas specific quantitative-PCR and Pseudomonas CFU counts, less than 4 % of Pseudomonas spp. were culturable using NAA 1:100 medium. The Pseudomonas selectivity and specificity of the culture medium were evaluated by 454 pyrosequencing of 16S rRNA gene amplicons generated using Bacteria- and Pseudomonas-specific primers. Pyrosequencing results showed that most isolates were Pseudomonas and that the culturable fraction of Pseudomonas spp. reflects most clusters of the total Pseudomonas diversity in soil. This indicates that NAA 1:100 medium is highly selective for Pseudomonas species, and reveals the ability of NAA 1:100 medium to culture mostly the dominant Pseudomonas species in soil.

Keywords

Pseudomonas Bacterial Primer Culturable Fraction Lauroyl Sarcosine Sodium Lauroyl Sarcosine 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We thank Annelise Kjøller for proofreading of the manuscript and technician Karin Vestberg for excellent help with laboratory work. The authors thank the Danish Research Centre for Organic Food and Farming (DARCOF), for supporting the project CRUCIAL (Closing the Rural Urban Nutrient Cycle), and the Center for Environmental and Agricultural Microbiology (CREAM), funded by The Villum Foundation.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Lili Li
    • 1
  • Waleed Abu Al-Soud
    • 1
  • Lasse Bergmark
    • 1
  • Leise Riber
    • 1
  • Lars H. Hansen
    • 1
  • Jakob Magid
    • 2
  • Søren J. Sørensen
    • 1
  1. 1.Section for Microbiology, Department of BiologyUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of Agriculture and Ecology, Faculty of Life SciencesUniversity of CopenhagenFrederiksberg CDenmark

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