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.

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.

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.

Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain.

Liberibacter is a bacterial group causing different diseases and disorders in plants. Among liberibacters, Candidatus Liberibacter solanaceraum (CLso) produces disorders in several species mainly within Apiaceae and Solanaceae families. CLso isolates are usually grouped in defined haplotypes according to single nucleotide polymorphisms in genes associated with ribosomal elements. In order to characterize more precisely isolates of CLso identified in potato in Spain, a Multilocus Sequence Analysis (MLSA) was applied. This methodology was validated by a complete analysis of ten housekeeping genes that showed an absence of positive selection and a nearly neutral mechanism for their evolution. Most of the analysis performed with single housekeeping genes, as well as MLSA, grouped together isolates of CLso detected in potato crops in Spain within the haplotype E, undistinguishable from those infecting carrots, parsnips or celery. Moreover, the information from these housekeeping genes was used to estimate the evolutionary divergence among the different CLso by using the concatenated sequences of the genes assayed. Data obtained on the divergence among CLso haplotypes support the hypothesis of evolutionary events connected with different hosts, in different geographic areas, and possibly associated with different vectors. Our results demonstrate the absence in Spain of CLso isolates molecularly classified as haplotypes A and B, traditionally considered causal agents of zebra chip in potato, as well as the uncertain possibility of the present haplotype to produce major disease outbreaks in potato that may depend on many factors that should be further evaluated in future works.