Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi.

The number of reported mycoviruses is increasing exponentially due to the current ability to detect mycoviruses using next-generation sequencing (NGS) approaches, with a large number of viral genomes built in-silico using data from fungal transcriptome projects. We decided to screen a collection of fungi originating from a specific marine environment (associated with the seagrass Posidonia oceanica) for the presence of mycoviruses: our findings reveal a wealth of diversity among these symbionts and this complexity will require further studies to address their specific role in this ecological niche. In specific, we identified twelve new virus species belonging to nine distinct lineages: they are members of megabirnavirus, totivirus, chrysovirus, partitivirus and five still undefined clades. We showed evidence of an endogenized virus ORF, and evidence of accumulation of dsRNA from metaviridae retroviral elements. We applied different techniques for detecting the presence of mycoviruses including (i) dsRNA extraction and cDNA cloning, (ii) small and total RNA sequencing through NGS techniques, (iii) rolling circle amplification (RCA) and total DNA extraction analyses, (iv) virus purifications and electron microscopy. We tried also to critically evaluate the intrinsic value and limitations of each of these techniques. Based on the samples we could compare directly, RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses. To our knowledge this is the first report on the virome of fungi isolated from marine environment. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: KT601099-KT601110; KT601114-KT601120; KT592305; KT950836-KT950841.