Exploring the mycovirome: novel and diverse mycoviruses in Botrytis cinerea

Botrytis cinerea is a necrotrophic fungal pathogen that causes significant economic losses to many crops, including vegetables, fruits, and ornamental plants. The management of B. cinerea is difficult due to a rise in fungicide resistance. Harnessing mycoviruses that cause reduced virulence (hypovirulence) in B. cinerea is a promising alternative. Over 100 mycoviruses have been identified in Botrytis spp. to date, including several hypovirulence-inducing mycoviruses. This research aimed to further explore, for the first time in Canada, the mycovirome of B. cinerea and identify potential hypovirulence-inducing mycoviruses. Isolates of B. cinerea were collected from fruits and vegetables in the province of Quebec. Fitness and pathogenicity criteria, including sclerotia production, colony morphotype, and lesion size were evaluated. A double-stranded RNA (dsRNA) extraction protocol tailored to the detection of mycoviruses was used to sequence dsRNA from 45 isolates with low fitness/pathogenicity, and an in-house bioinformatics workflow was used to profile the mycovirome. Mycoviruses were identified in 44/45 isolates. Most of these had positive single-stranded RNA or dsRNA genomes, and a small number had negative single-stranded RNA, single-stranded DNA, or reverse transcriptase RNA genomes. Following deep analysis of RNA-dependent RNA polymerase and replication initiation proteins, a total of 62 unique contigs were identified belonging to new strains of mycovirus species. Furthermore, four putative novel mycovirus species belonging to Endornaviridae, Botybirnaviridae, Peribunyaviridae, and Bunyavirales taxa were identified. Several mycovirus species positively and/or negatively co-occurred with B. cinerea isolates collected from strawberry or raspberry. This study revealed a high degree of diversity in the mycovirome of B. cinerea. Species accumulation curve analysis indicated that, with the number of isolates characterized, we were unable to capture the full extent of expected diversity. Nevertheless, we identified potential hypovirulence-inducing mycoviruses, including Botrytis cinerea mitovirus 1, Botrytis cinerea hypovirus 1, and Botrytis porri botybirnavirus 1. Some of these novel mycoviruses belonged to taxa known to produce viral particles, which can be an interesting feature for their use as biocontrol agents (BCA). ImportanceThis study provides the first comprehensive profiling of mycoviruses infecting Botrytis cinerea in Canada, a significant step in understanding how these viruses can naturally limit crop disease. Due to growing resistance against conventional fungicides, new biological methods to control B. cinerea are crucial. By profiling mycoviruses in fungal samples collected in Quebec, we identified several novel viruses that appear to reduce the pathogenicity of B. cinerea. These viruses, known as hypovirulence-inducing mycoviruses, could be used to develop biocontrol agents (BCA), offering a more sustainable disease management alternative. Notably, we found virus families with extracellular potential, which may enable easier application as BCAs in agriculture. This research not only broadens the understanding of fungal virology but also holds promise for innovative, eco-friendly approaches to managing Botrytis cinerea in Canada.