New insights on the mycovirome of Botrytis cinerea by high-throughput sequencing

Mycoviruses infecting isolates of the grey mould fungus Botrytis cinerea (Botryotinia fuckeliana) have been reported by several authors, but just few of them have been fully characterized at the molecular level. A metagenomic approach using a high-throughput sequencing technology (Illumina) was applied to obtain a comprehensive characterization of the B. cinerea virome and identify novel viruses in the pathogen. Double stranded RNAs (dsRNAs) segments with sizes ranging from 0.6 to 5.0 kbp were extracted and purified from 12 pooled mycelia from a total of 60 isolates, collected from different host plants and geographical areas, and sequenced to generate about 64 million of high-quality short sequence single reads (50 bp). Contigs (0.4-14.0 kbp) obtained from de novo assembly of the short reads, were analysed by sequence similarity searching into nucleotide database of viruses at NCBI to identify putative mycoviral sequences. A great abundance and diversity of viruses infecting B. cinerea were found. At least 23 different viruses were detected and identified in one or more of the analysed pools of dsRNA samples. They included mycoviruses already identified in B. cinerea, such as Botrytis virus F, Botrytis cinerea mitovirus 1, mitovirus 2, mitovirus 3, mitovirus 4, Botryotinia fuckeliana partitivirus 1 and totivirus 1, together with viruses reported in other fungal species, such as Sclerotinia sclerotiorum (mitovirus 3, mitovirus 4, hypovirus 1, hypovirus 2, dsRNA mycovirus-L) and Fusarium graminearum (mycovirus 4), or associated to host plants, such as the grapevine-associated narnavirus 1 and totivirus 1. The remaining 8 sequences were novel viruses showing similarity (27-67%) to the coding sequences of mycoviruses from different viral families or still unclassified mycoviruses. Sequence analysis of the identified viruses evidenced a remarkable variability in viral genomic sequences. For selected viruses, the results were validated by PCR and Sanger sequencing. This study expands the current knowledge on mycoviruses in B. cinerea. The availability of complete sequences of the viruses infecting the fungus will serve as a basis for further investigations aimed at improving our understanding of mycoviruses properties and to explore their potential as biological control agents against grey mould.