Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified under the newly formed Genomoviridae family. Here, we determine the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fruticola , two economically important members of family Sclerotiniaceae, and Fusarium oxysporum . SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea , and M. fruticola . SlaGemV-1 also replicates in Spodoptera frugiperda insect cells, but not in Caenorhabditis elegans nor plants. By expressing viral-encoded genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. Importance Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant pathogenic fungi and Fall armyworm (S. frugiperda , Sf9) insect cells, but not a model nematode, C. elegans , or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species under family Sclerotiniaceae, including Sclerotinia sclerotiorum , Botrytis cinerea , Monilinia fruticola . The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenome can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Deoxynivalenol (DON) toxin production during the infection of F. graminearum in small grain crops is one of the most harmful virulence factors associated with economic losses. Metatranscriptome sequencing and RT-qPCR traced back that the only mycovirus infecting an F. graminearum isolate, designated as Fg-4-2, was a novel strain of Fusarium graminearum virus 1 (FgV1), designated as FgV1-SD4. The isolate Fg-4-2 showed significantly reduced virulence against wheat compared to the virus-free culture, designated as isolate Fg-4-1, which was obtained by deep freezing and single conidial germination. Notably, no DON accumulation was detected in the harvested wheat seeds infected by Fg-4-2, whereas ~18 ppm DON was detected in seeds infected by Fg-4-1. Comparison of the genome sequence of FgV1-SD4 with other identified strains of FgV1, i.e., FgV1-DK21 and FgV1-ch, indicates mutations on ORF-2 and the 3′-UTR in the genome that might be associated with hypovirulence. This mycovirus strain alone and specific genetic components of FgV1-SD4 can be further optimized to be developed as a biocontrol agent to reduce Fusarium head blight and to lower the DON accumulation levels in small grain crops due to this fungal disease.