Searching for mycoviruses in a collection of fungal endophytes isolated from the seagrass Posidonia oceanica

The Mycotheca Universitatis Taurinensis (MUT) preserves a wide collection of fungi (more than 5300 isolates belonging to about 1300 species). A subset of endophytic fungal species present in this collection were isolated from the seagrass Posidonia oceanica and previously characterized (Panno et al. 2013). The fungal isolates come from a P. oceanica meadow localized in Punta Manara close to Riva Trigoso Bay (Liguria, Italy). We have selected 80 isolates representative of some of the fungal taxa present in P. oceanica as endophytes and initially assessed their halophylic properties growing them on solid media in presence or absence of marine salts (3%). Growth data was recorded and used to select the most appropriate media for liquid culture. Each culture was harvested approximately at stationary phase growth, and lyophilized. About 200 mg of lyophilized mycelia samples were used for total nucleic acids and dsRNA purification. Total nucleic acid was digested with RNAse and used as template in a Rolling Cycle Amplification (RCA) protocol in order to determine the possible presence of circular ssDNA viruses as previously described (Haible et al. 2006). Samples positive for dsRNA presence were subjected to DNAse and prepared for sequencing using a NGS platform as previously described (Roossinck et al. 2010). At the moment we found 14 samples positive for dsRNA presence. So far we have confirmed the presence of mycovirus sequences in 7 isolates, and each contained virus sequences sufficiently distant from those deposited in the databases to warrant a "new species" status: all the viruses so far characterized molecularly belong to known taxonomic groups. Two more isolates were shown to contain abundant accumulation of dsRNA with homology to known fungal transposon/retro-transposon sequences. Five dsRNAs are still under characterization. So far only one sample was positive in RCA analysis (out of 43 tested) and we are currently characterizing the RCA product. All dsRNA positive samples were also subjected to a partial virus purification protocol and electron microscopic observation. Penicillium aurantiogriseum var. viridicatum MUT 4330 contained isodiametric particles of at least two different diameters. We screened colonies from single conidia progeny for presence/absence of each of the dsRNA bands present in the original 4330 isolate: so far we have derived at least two different dsRNA patterns that are isogenic for nuclear background and show different phenotype on Czapek Yeast Extract Agar (CYA). Future work will try to complete the molecular characterization of the viruses, and to assess their possible biological and ecological significance in the interaction with the fungal host, and the seagrass that hosts the fungi. We are also evaluating the possibility of using deep sequencing of siRNA fungal libraries to detect further viruses that might have escaped our search and to assemble virus genomes (Kreuze et al. 2009).