Limited effect of short- to mid-term storage conditions on an Australian farmland soil RNA virome

Abstract

Soils represent one of the largest and most diverse reservoirs of microbial life on Earth, yet their associated RNA viruses remain underexplored compared to animal and aquatic systems. Viral discovery in soils has been further limited by technical hurdles, particularly obtaining sufficient yields of high-quality RNA for sequencing. To address this, we evaluated a range of storage and preservation strategies, including the use of commercial preservative solutions and ultra-cold snap-freezing, followed by standardised RNA extraction, sequencing, and virus discovery pipelines. This work aimed to establish minimum sample storage requirements that maintain RNA integrity, generate sufficient RNA sequencing data, and subsequently enable reliable soil virome characterisation. While no preservative solution proved effective, "neat" soil samples were stable at 2-8{degrees}C and -30{degrees}C for at least two weeks, and at -80{degrees}C for at least three months, with no measurable reduction in RNA quality, sequencing data, or viral abundance and diversity. From 32 resulting sequencing libraries, we identified 1,475 putative novel RNA viruses, with the majority belonging to the microbe-associated phylum Lenarviricota. Several novel viruses formed divergent clusters with other environmentally derived sequences distantly related to traditionally animal-associated families such as the Astroviridae and Picornaviridae. Furthermore, unique clusters within the Picobirnaviridae, Alsuvurucetes, Ghabrivirales, and Amabiliviricetes comprised exclusively Australian viruses, suggesting instances of region-specific evolution. Together, these findings highlight soils as rich reservoirs of RNA viral diversity and provide practical minimum standards for storage, expanding opportunities to investigate the ecological and evolutionary roles of RNA viruses in terrestrial systems. ImportanceRNA viruses are the most abundant and diverse biological entities on Earth and are likely present in all other organisms and ecosystems, including soil-dwelling invertebrates, microbes, and plants. Despite this, their diversity and role in soil systems remains largely unknown. Methodological challenges in preserving and extracting sufficient quantities of RNA from soils have hindered the study of these communities. Here, we identified 1,475 previously undescribed RNA viruses in Australian soils while systematically testing different preservation strategies. The significance of our research lies in the demonstration that snap-freezing soil is a viable and robust storage strategy for at least three months, while also highlighting the extraordinary scale of viral diversity present in terrestrial environments. This work establishes a foundation for reliable exploration of terrestrial RNA viruses, improving the accessibility of more remote environmental viromes and enabling future efforts to integrate them into broader models of microbial ecology and ecosystem function.