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Estimates of viral abundance in soils are strongly influenced by extraction and enumeration methods

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Abstract

Viruses are highly abundant in temperate soils, ranging from 107 to 109 g−1, and outnumbering soil bacteria from 5- to over 1,000-fold. In order to determine the potential impacts of viruses on soil microbial communities, it is important to establish reliable methods for comparing changes in viral abundances within and across soil samples. The goals of this study were to optimize extraction-enumeration methods to accurately determine viral abundances in a range of soil types, to evaluate the feasibility of simultaneously enumerating bacterial cells and virus particles using a single extraction procedure, and to assess the utility of flow cytometry (FCM) for enumerating virus particles in soil extracts. Comparisons of extraction approaches indicated that sonication or blender extraction of soils with potassium citrate buffer yielded the highest viral abundances for most soil types. Combined viral and bacterial extractions underestimate abundances compared to separately-optimized extractions for each. Flow cytometric counts were anywhere between 350- and 1,400-fold higher than epifluorescence microscopy (EFM)-based counts for the same soil. Trends in viral abundance across soil types were different from those via EFM, and different relationships between viral abundance and soil properties were observed depending on the enumeration method. Thus, FCM is not currently recommended for enumeration of viruses in soil extracts. Based on EFM results, soil moisture and organic matter content were the most important factors determining viral abundance in soils.

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Acknowledgments

We wish to thank Brent J. Callaway and Kimy A. Javier for assistance with field sampling and collecting environmental data, and Elizabeth L. Adams and Kirk J. Czymmek for expert advice on flow cytometry. Thanks also to Matt Saxton for reading the manuscript. This work was supported by a grant to KEW from the Jeffress Memorial Trust (J-988), a Cummings Memorial Summer Scholarship to KAC, and a core facility grant to RRH and KEW by the National Science Foundation EPSCoR (grant no. EPS-081425) from the National Center for Research Resources.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Biology, College of William & Mary, Williamsburg, VA, 23187, USA

    Kurt E. Williamson, Krysten A. Corzo, Jasmyn M. Buckingham & Charleton P. Thompson

  2. Department of Chemistry, College of William & Mary, Williamsburg, VA, 23187, USA

    Camelia L. Drissi

  3. Delaware Environmental Institute, University of Delaware, Newark, DE, 19716, USA

    Rebekah R. Helton

  4. Department of Biology, 3035 Integrated Science Center, College of William and Mary, Williamsburg, VA, 23185, USA

    Kurt E. Williamson

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  1. Kurt E. Williamson
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  2. Krysten A. Corzo
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Figure S1

Comparison of sample dilutions and heating treatment prior to flow cytometric analysis. Not all treatments were performed for all soils. Open bars, forest soil; stippled bars, wetland soil; hatched bars, clay soil; light grey bars, dune sand; dark grey bars, wildflower soil. Error bars indicate SD (n = 6) (JPEG 37 kb)

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Figure S2

Principal components analysis of variation in soil viral abundance based on EFM. VA, viral abundance; CEC, cation exchange capacity; %W, gravimetric water content; %OM, percent organic matter (JPEG 148 kb)

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Figure S3

Principal components analysis of variation in soil viral abundance based on FCM. VA, viral abundance; CEC, cation exchange capacity; %W, gravimetric water content; %OM, percent organic matter (JPEG 159 kb)

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Figure S4

Comparison of FCM and EFM for enumeration of phage T4. Error bars indicate range (n = 3) (JPEG 9 kb)

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Figure S5

Example epifluorescence micrographs. All extracts were filtered through 0.22 μm prior to staining with SYBR Gold. a, b Forest soil blender extraction with potassium citrate buffer; c, d, Forest soil blender extraction with sodium deoxycholate. Scale bars indicate 20 μm. Virus particles appear as small, bright, symmetrical points (indicated by arrows), whereas background debris is generally amorphous and variable in fluorescence intensity (JPEG 1362 kb)

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Figure S6

Typical flow cytograms of Forest soil, blender extraction with potassium citrate buffer; extracts were filtered through 0.22 μm prior to staining with SYBR Gold. a Soil extract without dilution; b, soil extract with 1:250 dilution; c, soil extract with heat treatment and 1:250 dilution (JPEG 44 kb)

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Williamson, K.E., Corzo, K.A., Drissi, C.L. et al. Estimates of viral abundance in soils are strongly influenced by extraction and enumeration methods. Biol Fertil Soils 49, 857–869 (2013). https://doi.org/10.1007/s00374-013-0780-z

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