Mitoviruses are small vertically transmitted RNA viruses found in fungi, plants and animals. Taxonomically, a total of 105 species and 4 genera have been formally recognized by ICTV, and recently, 18 new putative species have been included in a new proposed genus. Transcriptomic and metatranscriptomic studies are a major source of countless new virus-like sequences that are continually being added to open databases and these may be good sources for identifying new putative mitoviruses. The search for mitovirus-like sequences in the NCBI databases resulted in the discovery of more than one hundred new putative mitoviruses, with important implications for taxonomy and also for the evolutionary scenario. Here, we propose the inclusion of four new putative members to the genus Kvaramitovirus, and the existence of a new large basally divergent lineage composed of 144 members that lack internal UGA codons (subfamily “Arkeomitovirinae”), a feature not shared by the vast majority of mitoviruses. Finally, a taxonomic categorization proposal and a detailed description of the evolutionary history of mitoviruses were carried out. This in silico study supports the hypothesis of the existence of a basally divergent lineage that could have had an impact on the early evolutionary history of mitoviruses.

The evolutionary history of crop pathogens is shaped by a complex interaction of natural and anthropogenic factors. The fungus Colletotrichum graminicola causes maize anthracnose which results in significant yield losses worldwide. We conducted a comprehensive investigation into the evolutionary genomics of C. graminicola using a collection of 212 isolates from 17 countries across five continents. Genomic analyses supported the existence of three geographically isolated genetic lineages, with a significant pattern of isolation by distance. We identified two distinct gene flow patterns, driven by short- and long-distance dispersal, likely resulting from the natural spread of the pathogen and the exchange of contaminated seeds. We present evidence of genetic introgression between lineages, suggesting a long history of recombination. We identified significant recombination events coalescing at distinct points in time, with the North American lineage displaying evidence of the most ancient recombination. Demographic modelling has indicated that North America is an intermediate between Brazil, Europe and an ancestral, unsampled source population, which is hypothesised to be Mesoamerican. Our analyses revealed that the global genomic structure of C. graminicola is shaped by geographic differentiation driven by long-distance migration and a long history of recombination and introgression. We show historical relationships amongst these lineages, identifying a potential route for fungal spread, with the North American population emerging ancestrally, followed sequentially by the Brazilian and European populations. Our research indicates that the European lineage is more virulent, which has implications for the potential emergence of new outbreaks of maize anthracnose in Europe.

Fusarium verticillioides is a globally prevalent phytopathogenic fungus responsible for multiple diseases in maize and a major producer of the mycotoxin fumonisin B1 (FB1), a highly toxic fungal secondary metabolite (FSM). The histone code, which includes reversible modifications such as acetylation and methylation, plays a critical role in regulating chromatin structure and gene expression. In fungi, di- and tri-methylation of histone H3 at lysine 9 (H3K9me2/3) serves as a key epigenetic mark associated with heterochromatin formation and transcriptional repression. In this study, we identified and characterized a putative heterochromatin protein 1 (HP1) family member in F. verticillioides, designated FvHP1, based on conserved domain architecture and phylogenetic analyses. FvHP1 retains essential residues required for H3K9me2/3 recognition, supporting its functional conservation within the HP1 protein family. Phenotypic analysis of the ΔFvHP1 mutant revealed impaired vegetative growth, reduced conidiation and virulence, and altered FB1 mycotoxin production. Additionally, the accumulation of red pigment in the mutant was linked to the deregulation of secondary metabolism, specifically the overproduction of fusarubin-type naphthoquinones, such as 8-O-methylnectriafurone. These results support the role of FvHP1 in facultative heterochromatin-mediated repression of sub-telomeric biosynthetic gene clusters, including the pigment-associated PGL1 cluster. Our findings provide new insights into the epigenetic regulation of fungal pathogenicity and metabolite production, as well as the first evidence of a functional HP1 homolog in F. verticillioides.

Mitoviruses, which are considered evolutionary relics of extinct alpha-proteobacteria RNA phages, represent one of the simplest self-replicating biological systems. This study aims to quantitatively describe genomes and identify potential genomic signatures that support the protein phylogenetic-based classification criterion. Genomic variables, such as mononucleotide and dinucleotide composition, codon usage bias, and minimal free energy derived from optimized predicted RNA secondary structure, were analyzed. From the values obtained, the main evolutionary pressures were discussed, indicating that natural selection plays a significant role in shaping mitovirus genomes. However, neutral evolution also makes a significant contribution. This study reveals a significant discovery of structural divergence in Kvaramitovirus. The energy minimization approach employed to study 2D folding in this study reveals a distinct spatial organization of their genomes, providing evidence for the hypothesis of a single evolutionary event of circularization in the most recent common ancestor of the lineage. This hypothesis was discussed in light of recent discoveries by other researchers that partially support the existence of mitoviruses with circular genomes. Finally, this study represents a significant advancement in the understanding of mitoviruses, as it quantitatively describes the nucleotide sequence at the family and genus taxonomic levels. Additionally, we provide hypotheses that can be experimentally validated to inspire new research and address the gaps in knowledge of this fascinating, basally divergent RNA virus lineage.

Mycoviruses represent promising tools for the biological control of phytopathogenic fungi. However, their application is often constrained by host specificity and low natural prevalence in certain pathogens. Fusarium verticillioides, a major maize pathogen and fumonisin B1 (FB1) producer, harbors very few known mycoviruses, a circumstance that limits the development of virus-based control strategies. In this study, we explored a virocontrol approach for F. verticillioides based on the use of a heterologous mycovirus, Cryphonectria hypovirus 1 (CHV1). For the artificial transfection of F. verticillioides with CHV1, we used an infectious cDNA clone of the virus and protoplast-mediated transformation. Through RT-PCR and double-stranded RNA (dsRNA) purification by chromatography on cellulose, it was confirmed that the CHV1 infectious cDNA clone is stably integrated into the fungal genome, giving rise to autonomous virus replication in the cytoplasm of F. verticillioides cells. Characterization of four independent transformants harboring CHV1 showed a significant reduction in FB1 production compared to the parental uninfected strain and only moderate alterations in vegetative growth. The CHV1-associated reduction in FB1 was confirmed in maize grown under both greenhouse and field conditions. Our findings provide evidence that heterologous mycoviruses can modulate mycotoxin production in F. verticillioides, thus expanding the pool of viral species that can be explored as potential biocontrol agents in this pathogen. Further work will be required to elucidate the underlying mechanisms of CHV1-associated downregulation of FB1 production in F. verticillioides and assess its potential application in virocontrol strategies.

The evolutionary history of crop pathogens is shaped by a complex interaction of natural and anthropogenic factors. The ascomycete fungus Colletotrichum graminicola causes maize anthracnose. The disease can result in significant yield losses and is also an important model for genetic studies. We conducted a comprehensive investigation into the evolutionary genomics of C. graminicola using a collection of 212 isolates from 17 countries. Genomic analyses supported the existence of three geographically isolated genetic lineages, with a significant pattern of isolation by distance. We identified two distinct gene flow patterns, driven by short and long-distance dispersion, likely resulting from the natural spread of the pathogen and the exchange of contaminated seeds. We present evidence of genetic introgression between lineages, suggesting a long history of recombination. We identified significant recombination events coalescing at distinct points in time, with the North American lineage displaying evidence of most ancient recombination. Demographic modeling indicated that North America is an intermediate between Brazil, Europe and an ancestral, unsampled source population, hypothesized to be Mesoamerican. Our analyses revealed that the global genomic structure of C. graminicola is shaped by geographic differentiation driven by long-distance migration and a long history of recombination and introgression. We show historical relationships among these lineages, identifying a potential route for fungal spread, with the North American population emerging ancestrally, followed sequentially by the Brazilian and European populations. Our research indicates that the European lineage is more virulent, which has implications for the potential emergence of new outbreaks of maize anthracnose in Europe.