Reexamining the Mycovirome of Botrytis spp.

Botrytis species cause gray mold disease in more than 200 crops worldwide. To control this disease, chemical fungicides are usually applied. However, more sustainable control alternatives should be explored, such as the use of hypovirulent mycovirus-infected fungal strains. To determine the mycovirome of two Botrytis species, B. cinerea and B. prunorum, we reanalyzed RNA-Seq and small RNA-Seq data using different assembly programs and an updated viral database, aiming to identify new mycoviruses that were previously not described in the same dataset. New mycoviruses were identified, including those previously reported to infect or be associated with B. cinerea and Plasmopara viticola, such as Botrytis cinerea alpha-like virus 1 and Plasmopara viticola lesion-associated ourmia-like virus 80. Additionally, two novel narnaviruses, not previously identified infecting Botrytis species, have been characterized, tentatively named Botrytis cinerea narnavirus 1 and Botrytis narnavirus 1. The analysis of small RNAs suggested that all identified mycoviruses were targeted by the antiviral fungal mechanism, regardless of the viral genome type. In conclusion, the enlarged list of newly found viruses and the application of different bioinformatics approaches have enabled the identification of novel mycoviruses not previously described in Botrytis species, expanding the already extensive list.

Mycologists and Virologists Align: Proposing Botrytis cinerea for Global Mycovirus Studies.

Mycoviruses are highly genetically diverse and can significantly change their fungal host's phenotype, yet they are generally under-described in genotypic and biological studies. We propose Botrytis cinerea as a model mycovirus system in which to develop a deeper understanding of mycovirus epidemiology including diversity, impact, and the associated cellular biology of the host and virus interaction. Over 100 mycoviruses have been described in this fungal host. B. cinerea is an ideal model fungus for mycovirology as it has highly tractable characteristics-it is easy to culture, has a worldwide distribution, infects a wide range of host plants, can be transformed and gene-edited, and has an existing depth of biological resources including annotated genomes, transcriptomes, and isolates with gene knockouts. Focusing on a model system for mycoviruses will enable the research community to address deep research questions that cannot be answered in a non-systematic manner. Since B. cinerea is a major plant pathogen, new insights may have immediate utility as well as creating new knowledge that complements and extends the knowledge of mycovirus interactions in other fungi, alone or with their respective plant hosts. In this review, we set out some of the critical steps required to develop B. cinerea as a model mycovirus system and how this may be used in the future.

ICTV Virus Taxonomy Profile: Fusariviridae 2024.

Fusariviridae is a family of mono-segmented, positive-sense RNA viruses with genome sizes of 5.9-10.7 kb. Most genomic RNAs are bicistronic, but exceptions have up to four predicted ORFs. In bicistronic genomes, the 5'-proximal ORF codes for a single protein with both RNA-directed RNA polymerase (RdRP) and RNA helicase (Hel) domains; little is known about the protein encoded by the second ORF. Fusarivirids do not appear to form virions. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Fusariviridae, which is available at ictv.global/report/fusariviridae.

Construction of a Mycoviral Infectious Clone for Reverse Genetics in Botrytis cinerea.

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.

Determination of the Mycovirome of a Necrotrophic Fungus.

Next-generation sequencing (NGS) of total RNA has allowed the detection of novel viruses infecting different hosts, such as fungi, increasing our knowledge on virus horizontal transfer events among different hosts, virus diversity, and virus evolution. Here, we describe the detailed protocols for the isolation of the plant pathogenic fungus Botrytis cinerea, from grapevine plants showing symptoms of the mold gray disease, the culture and maintenance of the isolated B. cinerea strains, the extraction of total RNA from B. cinerea strains for NGS, the bioinformatics pipeline designed and followed to detect mycoviruses in the sequenced samples, and the validation of the in silico detected mycoviruses by different approaches.

Molecular characterization of a tetra segmented ssDNA virus infecting Botrytis cinerea worldwide.

BACKGROUND: Family Genomoviridae was recently established, and only a few mycoviruses have been described and characterized, and almost all of them (Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, Fusarium graminearum gemyptripvirus 1 and Botrytis cinerea gemydayirivirus 1) induced hypovirulence in their host. Botrytis cinerea ssDNA virus 1 (BcssDV1), a tetrasegmented single-stranded DNA virus infecting the fungus Botrytis cinerea, has been molecularly characterized in this work. METHODS: BcssDV1 was detected in Spanish and Italian B. cinerea field isolates obtained from grapevine. BcssDV1 variants genomes were molecularly characterized via NGS and Sanger sequencing. Nucleotide and amino acid sequences were used for diversity and phylogenetic analysis. Prediction of protein tertiary structures and putative associated functions were performed by AlphaFold2 and DALI. RESULTS: BcssDV1 is a tetrasegmented single-stranded DNA virus. The mycovirus was composed by four genomic segments of approximately 1.7 Kb each, which are DNA-A, DNA-B, and DNA-C and DNA-D, that coded, respectively, for the rolling-circle replication initiation protein (Rep), capsid protein (CP) and two hypothetical proteins. BcssDV1 was present in several Italian and Spanish regions with high incidence and low variability among the different viral variants. DNA-A and DNA-D were found to be the more conserved genomic segments among variants, while DNA-B and DNA-C segments were shown to be the most variable ones. Tertiary structures of the proteins encoded by each segment suggested specific functions associated with each of them. CONCLUSIONS: This study presented the first complete sequencing and characterization of a tetrasegmented ssDNA mycovirus, its incidence in Spain and Italy, its presence in other countries and its high conservation among regions.

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota).

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

ICTV Virus Taxonomy Profile: Hypoviridae 2023.

Hypoviridae is a family of capsidless viruses with positive-sense RNA genomes of 7.3-18.3 kb that possess either a single large open reading frame (ORF) or two ORFs. The ORFs appear to be translated from genomic RNA by non-canonical mechanisms, i.e. internal ribosome entry site- and stop/restart translation. This family includes the genera Alphahypovirus, Betahypovirus, Gammahypovirus, Deltahypovirus, Epsilonhypovirus, Zetahypovirus, Thetahypovirus and Etahypovirus. Hypovirids have been detected in ascomycetous and basidiomycetous filamentous fungi and are considered to replicate in host, Golgi apparatus-derived, lipid vesicles that contain virus dsRNA as the replicative form. Some hypovirids induce hypovirulence to host fungi, while others do not. This is a summary of the ICTV report on the family Hypoviridae, which is available at www.ictv.global/report/hypoviridae.

Mycoviruses as a part of the global virome: Diversity, evolutionary links and lifestyle.

Knowledge of mycovirus diversity, evolution, horizontal gene transfer and shared ancestry with viruses infecting distantly related hosts, such as plants and arthropods, has increased vastly during the last few years due to advances in the high throughput sequencing methodologies. This also has enabled the discovery of novel mycoviruses with previously unknown genome types, mainly new positive and negative single-stranded RNA mycoviruses ((+) ssRNA and (-) ssRNA) and single-stranded DNA mycoviruses (ssDNA), and has increased our knowledge of double-stranded RNA mycoviruses (dsRNA), which in the past were thought to be the most common viruses infecting fungi. Fungi and oomycetes (Stramenopila) share similar lifestyles and also have similar viromes. Hypothesis about the origin and cross-kingdom transmission events of viruses have been raised and are supported by phylogenetic analysis and by the discovery of natural exchange of viruses between different hosts during virus-fungus coinfection in planta. In this review we make a compilation of the current information on the genome organization, diversity and taxonomy of mycoviruses, discussing their possible origins. Our focus is in recent findings suggesting the expansion of the host range of many viral taxa previously considered to be exclusively fungal, but we also address factors affecting virus transmissibility and coexistence in single fungal or oomycete isolates, as well as the development of synthetic mycoviruses and their use in investigating mycovirus replication cycles and pathogenicity.

2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

ICTV Virus Taxonomy Profile: Mymonaviridae 2022.

Typical members of the family Mymonaviridae produce filamentous, enveloped virions containing a single molecule of linear, negative-sense RNA of about about 10 kb, but some may not produce any virions. The family includes several genera, some with multiple species. Mymonavirids usually infect filamentous fungi, but a few have been identified associated with insects, oomycetes or plants. At least one virus, Sclerotinia sclerotiorum negative-stranded RNA virus 1, induces hypovirulence in its fungal host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Mymonaviridae, which is available at ictv.global/report/mymonaviridae.

Construction and Characterization of a Botrytis Virus F 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.

Going Viral: Virus-Based Biological Control Agents for Plant Protection.

The most economically important biotic stresses in crop production are caused by fungi, oomycetes, insects, viruses, and bacteria. Often chemical control is still the most commonly used method to manage them. However, the development of resistance in the different pathogens/pests, the putative damage on the natural ecosystem, the toxic residues in the field, and, thus, the contamination of the environment have stimulated the search for saferalternatives such as the use of biological control agents (BCAs). Among BCAs, viruses, a major driver for controlling host populations and evolution, are somewhat underused, mostly because of regulatory hurdles that make the cost of registration of such host-specific BCAs not affordable in comparison with the limited potential market. Here, we provide a comprehensive overview of the state of the art of virus-based BCAs against fungi, bacteria, viruses, and insects, with a specific focus on new approaches that rely on not only the direct biocidal virus component but also the complex ecological interactions between viruses and their hosts that do not necessarily result in direct damage to the host.

2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Novel Mycoviruses Discovered in the Mycovirome of a Necrotrophic Fungus.

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 mycoviruses, with dsRNA, ssRNA+, ssRNA-, and ssDNA genomes, most of which were discovered here, and some of which were previously reported as infecting B. cinerea or other plant-pathogenic fungi. Some of these mycoviruses are reported for the first time here associated with B. cinerea, as a trisegmented ssRNA- mycovirus and as an ssDNA mycovirus, but even more remarkablly, we also describe here four novel bisegmented viruses (binarnaviruses) not previously described in nature. The present findings significantly contribute to general knowledge in virology and more particularly in the field of mycovirology.

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

ICTV Virus Taxonomy Profile: Botourmiaviridae.

The family Botourmiaviridae includes viruses infecting plants and filamentous fungi containing a positive-sense, ssRNA genome that can be mono- or multi-segmented. Genera in the family include: Ourmiavirus (plant viruses), and Botoulivirus, Magoulivirus and Scleroulivirus (fungal viruses). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Botourmiaviridae, which is available at ictv.global/report/botourmiaviridae.

ICTV Virus Taxonomy Profile: Mymonaviridae.

Members of the family Mymonaviridae produce filamentous, enveloped virions containing a single molecule of linear, negative-sense RNA of ≈10 kb. The family currently includes a single genus, Sclerotimonavirus. Mymonaviruses usually infect filamentous fungi, and one virus, Sclerotinia sclerotiorum negative-stranded RNA virus 1, induces hypovirulence in the fungal host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Mymonaviridae, which is available at ictv.global/report/mymonaviridae.