Novel and Diverse Mycoviruses in Hypovirulent Botrytis cinerea Isolates Infecting Raspberries and Strawberries

Botrytis cinerea causes significant economic losses in many crops, including vegetables, fruits, and ornamental plants, and its management is becoming increasingly difficult due to increasing fungicide resistance. Harnessing mycoviruses that reduce virulence in B. cinerea is a promising alternative. This research aims to characterize mycovirus diversity using high-throughput sequencing and identify potential hypovirulence-inducing mycoviruses. B. cinerea isolates were collected from raspberries, strawberries, and grapevine in the province of Quebec, Canada. Indicators of fungal fitness and pathogenicity criteria, including colony morphotype and lesion size, were evaluated. A double-stranded RNA (dsRNA) extraction protocol optimized for the detection of mycoviruses was used to sequence dsRNA from 45 isolates, most of which had reduced fitness and pathogenicity. Mycoviruses were identified in 98% of the selected isolates. A total of 94% of mycoviruses had positive-sense single-stranded RNA or dsRNA genomes, and 6% had negative-sense single-stranded RNA, single-stranded DNA, or reverse-transcribing single-stranded RNA genomes. A co-occurrence analysis revealed that several mycoviruses were significantly more frequent in B. cinerea isolates from either strawberry or raspberry and with specific colony morphotypes. Potential hypovirulence-inducing mycoviruses, including Botrytis cinerea mitovirus 1, Botrytis cinerea hypovirus 1, and Botrytis porri botybirnavirus 1 were identified. We also identified 62 unique novel genomic species representing new variants of known mycovirus species. Four putative novel mycovirus species were identified belonging to the families Endornaviridae, Botybirnaviridae, Peribunyaviridae, and the order Elliovirales. Two of these novel mycoviruses belonged to taxa known to produce viral particles, which is a promising feature for their use as biocontrol agents.