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The most important advances in our understanding of the viral life cycle, such as genome replication, packaging, transmission, and host interactions, have been made via the development of viral infectious full-length clones. Here, we describe the detailed protocols for the construction of an infectious clone derived from Botrytis virus F (BVF), a mycoflexivirus infecting the plant pathogenic fungus Botrytis cinerea, the determination of the complete sequence of the cloned mycovirus, the preparation of fungal protoplasts, and the transfection of protoplasts using transcripts derived from the BVF infectious clone.

Botrytis virus F (BVF) is a positive-sense, single-stranded RNA (+ssRNA) virus within the Gammaflexiviridae family of the plant-pathogenic fungus Botrytis cinerea. In this study, the complete sequence of a BVF strain isolated from B. cinerea collected from grapevine fields in Spain was analyzed. This virus, in this work BVF-V448, has a genome of 6827 nt in length, excluding the poly(A) tail, with two open reading frames encoding an RNA dependent RNA polymerase (RdRP) and a coat protein (CP). The 5′-and 3′-terminal regions of the genome were determined by rapid amplification of cDNA ends (RACE). Furthermore, a yet undetected subgenomic RNA species in BVF-V448 was identified, indicating that the CP is expressed via 3′ coterminal subgenomic RNAs (sgRNAs). We also report the successful construction of the first BVF full-length cDNA clone and synthesized in vitro RNA transcripts using the T7 polymerase, which could efficiently transfect two different strains of B. cinerea, B05.10 and Pi258.9. The levels of growth in culture and virulence on plants of BVF-V448 transfected strains were comparable to BVF-free strains. The infectious clones generated in this work provide a useful tool for the future development of an efficient BVF foreign gene expression vector and a virus-induced gene silencing (VIGS) vector as a biological agent for the control of B. cinerea.

Botrytis cinerea is one of the most important plant-pathogenic fungus. Products based on microorganisms can be used in biocontrol strategies alternative to chemical control, and mycoviruses have been explored as putative biological agents in such approaches. Here, we have explored the mycovirome of B. cinerea isolates from grapevine of Italy and Spain to increase the knowledge about mycoviral diversity and evolution, and to search for new widely distributed mycoviruses that could be active ingredients in biological products to control this hazardous fungus. A total of 248 B. cinerea field isolates were used for our metatranscriptomic study. Ninety-two mycoviruses were identified: 62 new mycoviral species constituting putative novel viral genera and families. Of these mycoviruses, 57 had a positive-sense single-stranded RNA (ssRNA) genome, 19 contained a double-stranded RNA (dsRNA) genome, 15 had a negative-sense ssRNA genome, and 1 contained a single-stranded DNA (ssDNA) genome. In general, ssRNA mycoviruses were widely distributed in all sampled regions, the ssDNA mycovirus was more frequently found in Spain, and dsRNA mycoviruses were scattered in some pools of both countries. Some of the identified mycoviruses belong to clades that have never been found associated with Botrytis species: Botrytis-infecting narnaviruses; alpha-like, umbra-like, and tymo-like ssRNA+ mycoviruses; trisegmented ssRNA- mycovirus; bisegmented and tetrasegmented dsRNA mycoviruses; and finally, an ssDNA mycovirus. Among the results obtained in this massive mycovirus screening, the discovery of novel bisegmented viruses, phylogenetically related to narnaviruses, is remarkable.IMPORTANCE The results obtained here have expanded our knowledge of mycoviral diversity, horizontal transfers, and putative cross-kingdom events. To date, this study presents the most extensive and wide diversity collection of mycoviruses infecting the necrotrophic fungus B. cinerea The collection included all types of

The obligatory biotrophic oomycetes Plasmopara viticola is the causal agent of downy mildew, a destructive disease of grapevine worldwide. So far, chemical fungicides are widely employed to limit this pathogen, but their adverse effects are stimulating the quest for environmentally friendly alternative approaches. Here, we report on the search for mycoviruses that might be later developed as biocontrol agents for this pathogen. Symptomatic leaves were collected from various regions in Spain and Italy and mycelia associated to leaf lesions was harvested. Total RNA extractions were depleted of rRNA and metatranscriptomes were generated using a high-throughput sequencing approach. The virome associated to leaf lesions was then characterized through a bioinformatic pipeline relying on blast searches against current viral databases. Here, we present an inventory of 283 new RNA viruses: 222 positive strand RNA viruses, 29 negative strand RNA viruses, 27 double-stranded RNA viruses and 5 ORFan virus RdRP segments, which could not be reliably assigned to any existing group in the Riboviria. In addition to ORFan viruses, we found other surprising new evolutionary trajectories in this wide inventory of viruses. The most represented viruses in our collection are those in phylum Lenarviricota, and, among them, a group of mycovirus segments distantly related to narnaviruses, but characterized by a polymerase palm domain lacking subdomain C, with the putative GDD catalytic triad. We also provided evidence of a strict association between two RNA segments that form a new mycovirus clade of positive strand RNA in the phylum Kitrinoviricota, order Martellivirales. In the phylum Negarnaviricota, we report for the first time in the order Mononegavirales a clade of viruses that is ambisense, a feature that so far was present only in the order Bunyavirales. Furthermore, in the same phylum we detected the widespread occurrence and abundant accumulation in our libraries of a distinct mycov

In this study, we analysed a total of 16 libraries from over 150 grapevine leaf and grape samples infected with Plasmopara viticola (downy mildew of grapevine) to characterise the virome associated to this oomycete. Samples were collected in five distinct regions in Italy and in four different regions in Spain, representative of different pedoclimatic conditions and different grapevine cultivars during 2018 growing season. Due to the metagenomics nature of the samples (containing at least both downy mildew hyphae and spores, and grapevine cells residues), we were able to assemble several plant viruses and a few possible novel plant virus genomes with our in silico analysis. We detected several plant virus variants already reported in grapevine, and a putative new ilarvirus previously unreported in grapevine. Furthermore, we characterised three new phenui-like viruses (in the order Bunyavirales), one of which shares some commonalities with plant coguviruses. Finally, we report a new strict association of three viral segments (one flavi-like and two virga-like) that we propose to be a new virus taxon named jivivirus.