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

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.