Deep sequencing analysis of the mycovirome in Botrytis cinerea

Deep sequencing on an Illumina HiScan SQ system was used to obtain a comprehensive characterization of the virome and identify novel viruses in Botrytis cinerea (Botryotinia fuckeliana), the fungal pathogen causing grey mould on several crops. Double stranded RNAs (dsRNAs) was 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 obtained from de novo assembly of the short reads were analysed by sequence similarity searching into nucleotide collection of viruses 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 the analysed pools of dsRNA samples. They included mycoviruses already identified in B. cinerea, such as Botrytis virus F, Botrytis cinerea mitovirus 1, Botryotinia fuckeliana partitivirus 1 and totivirus 1, together with viruses reported in other fungal species, such as Sclerotinia sclerotiorum (mitovirus 3, mitovirus 4, hypovirus 2, dsRNA mycovirus-L) and Fusarium graminearum (mycovirus 4), or associated to host plants, such as the grapevine-associated narnavirus 1, totivirus 1 and partitivirus 2. The remaining 14 sequences were novel viruses homologous to the coding sequences of partitiviruses, mitoviruses, hypoviruses, totiviruses, endornaviruses, umbraviruses or negative-stranded RNA viruses. 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.