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

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°C and −30°C for at least two weeks, and at −80°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.