Soil viruses are expected to be pivotal members of soil ecosystems, and recent advances in viral size fraction metagenomic (viromic) approaches have substantially improved our ability to interrogate soil viral ecology. However, the first step of viromics relies on extraction buffers to effectively remove viral particles from the soil matrix for downstream analysis, and viral extraction efficiency at this stage could be affected by the interplay between viral particles, soils, and extraction buffer chemistry. Here, we investigated whether extraction buffer chemistry affected extractable viral community composition measured by viromics from different soil types, for both biological (samples collected 1 meter apart) and technical (subsamples from the same soil homogenate) replicates. We first investigated protein-supplemented phosphate-buffered saline pH (PPBS, pHs 4.5, 5.5, 6.5, and 7.5) on a forest, grassland and wetland soil that exhibited different soil edaphic properties, and then we tested different buffer chemistries (PPBS, Carbonated Buffer, Glycine, and Saline Magnesium) on just the wetland soil. Spatial distance, or where the sample was taken in the field (i.e., biological replicate), was the primary driver of extractable viral community composition across all buffers and soils tested. Differences in viral community composition according to extraction buffer properties were only observed in the grassland technical replicates at PPBS buffer pH 4.5, as well as in both the wetland technical and biological replicates treated with different buffer chemistries, but the effects of buffer chemistry were secondary to spatial distance in all cases where spatial distance was a factor. The lack of buffering capacity in the grassland soil technical replicates likely increased sorption of some viral particles at pH 4.5, but neither protein composition nor isoelectric point (both calculated bioinformatically) explained this phenomenon. Given that most soil viral ecological studies to date include sample collection schemes over distances much farther apart than the 1-meter distances considered here, results suggest that extraction buffer chemistry is likely of much lower importance than ecological considerations, such as spatial distance, in the design of future soil viral ecological investigations.

Exploring viral particle, soil, and extraction buffer physicochemical characteristics and their impacts on extractable viral communities