Greetings from virologists to mycologists: A review outlining viruses that live in fungi.

Viruses are genetic elements that parasitize self-replicating cells. Therefore, organisms parasitized by viruses are not limited to animals and plants but also include microorganisms. Among these, viruses that parasitize fungi are known as mycoviruses. Mycoviruses with an RNA genome persistently replicate inside fungal cells and coevolve with their host cells, similar to a cellular organelle. Within host cells, mycoviruses can modulate various fungal characteristics and activities, including pathogenicity and the production of enzymes and secondary metabolites. In this review, we provide an overview of the mycovirus research field as introduction to fungal researchers. Recognition of all genetic elements in fungi aids towards better understanding and control of fungi, and makes fungi a significant model system for studying microorganisms containing multiple genetic elements.

Diversity and impact of single-stranded RNA viruses in Czech Heterobasidion populations.

Heterobasidion annosum sensu lato comprises some of the most devastating pathogens of conifers. Exploring virocontrol as a potential strategy to mitigate economic losses caused by these fungi holds promise for the future. In this study, we conducted a comprehensive screening for viruses in 98 H. annosum s.l. specimens from different regions of Czechia aiming to identify viruses inducing hypovirulence. Initial examination for dsRNA presence was followed by RNA-seq analyses using pooled RNA libraries constructed from H. annosum and Heterobasidion parviporum, with diverse bioinformatic pipelines employed for virus discovery. Our study uncovered 25 distinct ssRNA viruses, including two ourmia-like viruses, one mitovirus, one fusarivirus, one tobamo-like virus, one cogu-like virus, one bisegmented narna-like virus and one segment of another narna-like virus, and 17 ambi-like viruses, for which hairpin and hammerhead ribozymes were detected. Coinfections of up to 10 viruses were observed in six Heterobasidion isolates, whereas another six harbored a single virus. Seventy-three percent of the isolates analyzed by RNA-seq were virus-free. These findings show that the virome of Heterobasidion populations in Czechia is highly diverse and differs from that in the boreal region. We further investigated the host effects of certain identified viruses through comparisons of the mycelial growth rate and proteomic analyses and found that certain tested viruses caused growth reductions of up to 22% and significant alterations in the host proteome profile. Their intraspecific transmission rates ranged from 0% to 33%. Further studies are needed to fully understand the biocontrol potential of these viruses in planta.IMPORTANCEHeterobasidion annosum sensu lato is a major pathogen causing significant damage to conifer forests, resulting in substantial economic losses. This study is significant as it explores the potential of using viruses (virocontrol) to combat these fungal pathogens. By identifying and characterizing a diverse array of viruses in H. annosum populations from Czechia, the research opens new avenues for biocontrol strategies. The discovery of 25 distinct ssRNA viruses, some of which reduce fungal growth and alter proteome profiles, suggests that these viruses could be harnessed to mitigate the impact of Heterobasidion. Understanding the interactions between these viruses and their fungal hosts is crucial for developing effective, environmentally friendly methods to protect conifer forests and maintain ecosystem health. This study lays the groundwork for future research on the application of mycoviruses in forest disease management.

A pooled mycoviral resource in a strain of Rhizoctonia solani are regulators of fungal virulence.

Rhizoctonia solani is a widespread and devastating soil-borne plant fungal pathogen that causes diseases, including rice sheath blight, which are difficult to control. Some mycoviruses are potential biocontrol agents for the control of fungal diseases. In order to investigate the factors that influence the virulence of R. solani and search for mycoviruses with the potential for biocontrol of R. solani, a rice-infecting R. solani strain, ZJXD1-1, was isolated and confirmed to contain eight mycoviruses via dsRNA extraction and high-throughput sequencing. The identified mycoviruses belong to families of Endornaviridae (RsEV11 and RsEV12) and Mitoviridae (RsMV125 to RsMV129), and an unclassified Toti-like clade (RsTLV1). The C39 domain in RsEV12, which shares a close evolutionary relationship with bacteria, is observed for the first time in a mycovirus. Strains with different virus combinations were obtained through viral horizontal transfer, and pathogenicity test deduced that the Endornaviruses RsEV11 and RsEV12, and Mitovirus RsMV129 might potentially enhance the pathogenicity of R. solani, while RsMV125 might reduce the virulence or interfere with the function of other Mitoviruses. Furthermore, virus curing via protoplast regeneration and viral horizontal transfer demonstrated that RsMV129 is the causal agent of R. solani hypervirulence. Overall, our study provided the resource pool of viruses that may contribute to the discovery of new biocontrol agents against R. solani and enhance our understanding of the pathogenesis of R. solani regulated by mycoviruses.

Insect hypovirulence-associated mycovirus confers entomopathogenic fungi with enhanced resistance against phytopathogens.

Mycoviruses can alter the biological characteristics of host fungi, including change virulence or pathogenicity of phytopathogens and entomopathogenic fungi (EPF). However, most studies on the mycoviruses found in EPF have focused on the effects of the viruses on the virulence of host fungi towards insect pests, with relatively few reports on the effects to the host fungi with regard to plant disease resistance in hosts. The present study investigated the effects of the mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) virus infection on host biological characteristics, evaluated antagonistic activity of BbCV2 against two phytopathogenic fungi (Sclerotinia sclerotiorum and Botrytis cinerea), and transcriptome analysis was used to reveal the interactions between viruses and hosts. Our results showed that BbCV2 virus infection increased B. bassiana's growth rate, spore production, and biomass, it also enhanced the capacity of host fungi and their metabolic products to inhibit phytopathogenic fungi. BbCV2 virus infection reduced the contents of the two pathogens in tomato plants significantly, and transcriptome analysis revealed that the genes related to competition for ecological niches and nutrition, mycoparasitism and secondary metabolites in B. bassiana were significantly up-regulated after viral infection. These findings indicated that the mycovirus infection is an important factor to enhance the ability of B. bassiana against plant disease after endophytic colonization. We suggest that mycovirus infection causes a positive effect on B. bassiana against phytopathogens, which should be considered as a potential strategy to promote the plant disease resistance of EPF.

Coat Proteins of the Novel Victoriviruses FaVV1 and FaVV2 Suppress Sexual Reproduction and Virulence in the Pathogen of Fusarium Head Blight.

Fusarium head blight (FHB), a disease inflicted by Fusarium graminearum and F. asiaticum, poses a growing threat to wheat in China, particularly in the face of climate change and evolving agricultural practices. This study unveiled the discovery of the victorivirus FgVV2 from the F. asiaticum strain F16176 and comprehensively characterized the function of the two victoriviruses FaVV1 and FaVV2 in virulence. Through comparative analysis with a virus-free strain, we established that these mycoviruses markedly repress the sexual reproduction and pathogenicity of their fungal hosts. Furthermore, we synthesized the coat protein (CP) genes CP1 from FaVV1 and CP2 from FaVV2, which were fused with the green fluorescent protein (GFP) gene and successfully expressed in Fusarium strains in wild-type isolates of F. asiaticum and F. graminearum. Similar to virus-infected strains, the transformed strains expressing CPs showed a significant decrease in perithecia formation and pathogenicity. Notably, CP2 exhibited a stronger inhibitory effect than CP1, yet the suppression of sexual reproduction in F. graminearum was less pronounced than that in F. asiaticum. Additionally, the pathogenicity of the F. asiaticum and F. graminearum strains expressing CP1 or CP2 was substantially diminished against wheat heads. The GFP-tagged CP1 and CP2 revealed distinct cellular localization patterns, suggesting various mechanisms of interaction with the host. The findings of this study provide a significant research foundation for the study of the interaction mechanisms between FaVV1 and FaVV2 with their hosts, as well as for the exploration and utilization of fungal viral resources.

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.

Mycoviruses from Aspergillus fungi involved in fermentation of dried bonito.

Fungi are exploited for fermentation of foods such as cheese, Japanese sake, and soy sauce. However, the diversity of viruses that infect fungi involved in food fermentation is poorly understood. Fermented dried bonito ("katsuobushi") is one of the most important processed marine products in Japan. Fungi involved in katsuobushi fermentation are called katsuobushi molds, and Aspergillus spp. have been reported to be dominant on the surface of katsuobushi during fermentation. Because various mycoviruses have been found in members of the genus Aspergillus, we hypothesized that katsuobushi molds are also infected with mycoviruses. Here, we describe seven novel mycoviruses belonging to six families (Chrysoviridae, Fusariviridae, Mitoviridae, Partitiviridae, Polymycoviridae, and Pseudototiviridae) from isolated katsuobushi molds (Aspergillus chevalieri and A. sulphureus) detected by fragmented and primer-ligated double-stranded RNA sequencing. Aspergillus chevalieri fusarivirus 1 has a unique bi-segmented genome, whereas other known fusariviruses have a single genomic segment. Phenotypic comparison between the parental A. chevalieri strain infected with Aspergillus chevalieri polymycovirus 1 (AchPmV1) and isogenic AchPmV1-free isolates indicated that AchPmV1 inhibits the early growth of the host. This study reveals the diversity of mycoviruses that infect katsuobushi molds, and provides insight into the effect of mycoviruses on fungi involved in fermentation.

The Expanding Mycovirome of Aspergilli.

Mycoviruses are viruses that infect fungi and are widespread across all major fungal taxa, exhibiting great biological diversity. Since their discovery in the 1960s, researchers have observed a myriad of fungal phenotypes altered due to mycoviral infection. In this review, we examine the nuanced world of mycoviruses in the context of the medically and agriculturally important fungal genus, Aspergillus. The advent of RNA sequencing has revealed a previous underestimate of viral prevalence in fungi, in particular linear single-stranded RNA viruses, and here we outline the diverse viral families known to date that contain mycoviruses infecting Aspergillus. Furthermore, we describe these novel mycoviruses, highlighting those with peculiar genome structures, such as a split RNA dependent RNA polymerase gene. Next, we delineate notable mycovirus-mediated phenotypes in Aspergillus, in particular reporting on observations of mycoviruses that affect their fungal host's virulence and explore how this may relate to virus-mediated decreased stress tolerance. Furthermore, mycovirus effects on microbial competition and antifungal resistance are discussed. The factors that influence the manifestation of these phenotypes, such as temperature, fungal life stage, and infection with multiple viruses, among others, are also evaluated. In addition, we attempt to elucidate the molecular mechanisms that underpin these phenotypes, examining how mycoviruses can be targets, triggers, and even suppressors of RNA silencing and how this can affect fungal gene expression and phenotypes. Finally, we highlight the potential therapeutic applications of mycoviruses and how, in an approach analogous to bacteriophage therapy, their ability to produce hypovirulence in Aspergillus might be used to attenuate invasive aspergillosis infections in humans.

Corrigendum: Characterization of a novel mitovirus infecting Melanconiella theae isolated from tea plants.

[This corrects the article DOI: 10.3389/fmicb.2021.757556.].

Metatranscriptomic Sequencing of Sheath Blight-Associated Isolates of Rhizoctonia solani Revealed Multi-Infection by Diverse Groups of RNA Viruses.

Rice sheath blight, caused by the soil-borne fungus Rhizoctonia solani (teleomorph: Thanatephorus cucumeris, Basidiomycota), is one of the most devastating phytopathogenic fungal diseases and causes yield loss. Here, we report on a very high prevalence (100%) of potential virus-associated double-stranded RNA (dsRNA) elements for a collection of 39 fungal strains of R. solani from the rice sheath blight samples from at least four major rice-growing areas in the Philippines and a reference isolate from the International Rice Research Institute, showing different colony phenotypes. Their dsRNA profiles suggested the presence of multiple viral infections among these Philippine R. solani populations. Using next-generation sequencing, the viral sequences of the three representative R. solani strains (Ilo-Rs-6, Tar-Rs-3, and Tar-Rs-5) from different rice-growing areas revealed the presence of at least 36 viruses or virus-like agents, with the Tar-Rs-3 strain harboring the largest number of viruses (at least 20 in total). These mycoviruses or their candidates are believed to have single-stranded RNA or dsRNA genomes and they belong to or are associated with the orders Martellivirales, Hepelivirales, Durnavirales, Cryppavirales, Ourlivirales, and Ghabrivirales based on their coding-complete RNA-dependent RNA polymerase sequences. The complete genome sequences of two novel RNA viruses belonging to the proposed family Phlegiviridae and family Mitoviridae were determined.

Co-infection of Four Novel Mycoviruses from Three Lineages Confers Hypovirulence on Phytopathogenic Fungus Ustilaginoidea virens.

Rice false smut caused by Ustilaginoidea virens has become one of the most important diseases of rice. Mycoviruses are viruses that can infect fungi with the potential to control fungal diseases. However, little is known about the biocontrol role of hypoviruses in U. virens. In this study, we revealed that the hypovirulence-associated U. virens strain Uv325 was co-infected by four novel mycoviruses from three lineages, designated Ustilaginoidea virens RNA virus 16 (UvRV16), Ustilaginoidea virens botourmiavirus virus 8 (UvBV8), Ustilaginoidea virens botourmiavirus virus 9 (UvBV9), and Ustilaginoidea virens narnavirus virus 13 (UvNV13), respectively. The U. virens strain co-infected by four mycoviruses showed slower growth rates, reduced conidial yield, and attenuated pigmentation. We demonstrated that UvRV16 was not only the major factor responsible for the hypovirulent phenotype in U. vriens, but also able to prevent U. virens to accumulate more mycotoxin, thereby weakening the inhibitory effects on rice seed germination and seedling growth. Additionally, we indicated that UvRV16 can disrupt the antiviral response of U. virens by suppressing the transcriptional expression of multiple genes involved in autophagy and RNA silencing. In conclusion, our study provided new insights into the biological control of rice false smut.

A novel hypovirulence-associated Hadaka virus 1 (HadV1-LA6) in Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum f. sp. cubense (Foc) poses a significant threat to banana crops as a lethal fungal pathogen. The global spread of Foc underscores the formidable challenges associated with traditional management methods in combating this pathogen. This study delves into the hypovirulence-associated mycovirus in Foc. From Foc strain LA6, we isolated and characterized a novel member of the Hadakaviridae family, named Hadaka virus 1 strain LA6 (HadV1-LA6). HadV1-LA6 comprises 10 genomic RNA segments, with RNA1 to RNA7 sharing 80.9%-95.0% amino acid sequence identity with known HadV1-7n, while RNA8 to RNA10 display significantly lower identity. HadV1-LA6 demonstrates horizontal transmission capabilities in an all-or-none fashion between different Foc strains via coculturing. Phenotypic comparisons highlight that HadV1-LA6 significantly reduces the growth rates of its host fungus under cell wall stress and oxidative stress conditions. Importantly, HadV1-LA6 attenuates Foc's virulence in detached leaves and banana plants. This study represents the first introduction of a novel hypovirulence-associated Hadaka virus 1 in Foc.IMPORTANCEFusarium wilt of banana (FWB) is a severe fungal disease caused by soil-borne Fusarium oxysporum f. sp. cubense (Foc). Among various strategies, biocontrol emerges as a safe, ecologically friendly, and cost-effective approach to managing FWB. In this study, we focus on exploring the potential of a novel hypovirulent member of hadakavirid, HadV1-LA6. Previous reports suggest that HadV1 shows no apparent effect on the host. However, through phenotypic assessments, we demonstrate that HadV1-LA6 significantly impedes the growth rates of its host fungus under stress conditions. More importantly, HadV1-LA6 exhibits a remarkable capacity to attenuate Foc's virulence in detached leaves and banana plants. Furthermore, HadV1-LA6 could be horizontally transmitted between different Foc strains, presenting a promising resource for revealing the molecular mechanism of the interaction between Hadaka virus 1 and its host.

Complete genome analysis of a novel hypovirus in the phytopathogenic fungus Monilinia fructicola.

Monilinia fructicola is one of the most devastating fungal diseases of rosaceous fruit crops, both in the field and postharvest, causing significant yield losses. Here, we report the discovery of a novel positive single-stranded RNA virus, Monilinia fructicola hypovirus 3 (MfHV3), in a strain (hf-1) of the phytopathogenic fungus Monilinia fructicola. The complete genome of MfHV3 is 9259 nucleotides (nt) in length and contains a single large open reading frame (ORF) from nt position 462 to 8411. This ORF encodes a polyprotein with three conserved domains, namely UDP-glycosyltransferase, RNA-dependent RNA polymerase (RdRp), and DEAD-like helicase. The MfHV3 polyprotein shares the highest similarity with Colletotrichum camelliae hypovirus 1. Phylogenetic analysis indicated that MfHV3 clustered with members of the genus Betahypovirus within the family Hypoviridae. Taken together, the results of genomic organization comparisons, amino acid sequence alignments, and phylogenetic analysis convincingly show that MfHV3 is a new member of the genus Betahypovirus, family Hypoviridae.

Cross-species transmission of a novel bisegmented orfanplasmovirus in the phytopathogenic fungus Exserohilum rostratum.

Mycoviruses have been found in various fungal species across different taxonomic groups, while no viruses have been reported yet in the fungus Exserohilum rostratum. In this study, a novel orfanplasmovirus, namely Exserohilum rostratum orfanplasmovirus 1 (ErOrfV1), was identified in the Exserohilum rostratum strain JZ1 from maize leaf. The complete genome of ErOrfV1 consists of two positive single-stranded RNA segments, encoding an RNA-dependent RNA polymerase and a hypothetical protein with unknown function, respectively. Phylogenetic analysis revealed that ErOrfV1 clusters with other orfanplasmoviruses, forming a distinct phyletic clade. A new family, Orfanplasmoviridae, is proposed to encompass this newly discovered ErOrfV1 and its associated orfanplasmoviruses. ErOrfV1 exhibits effective vertical transmission through conidia, as evidenced by its 100% presence in over 200 single conidium isolates. Moreover, it can be horizontally transmitted to Exserohilum turcicum. Additionally, the infection of ErOrfV1 is cryptic in E. turcicum because there were no significant differences in mycelial growth rate and colony morphology between ErOrfV1-infected and ErOrfV1-free strains. This study represents the inaugural report of a mycovirus in E. rostratum, as well as the first documentation of the biological and transmission characteristics of orfanplasmovirus. These discoveries significantly contribute to our understanding of orfanplasmovirus.

Molecular characterization of a new botybirnavirus that infects Alternaria sp. from tobacco.

The genus Alternaria comprises many important fungal pathogens that infect a wide variety of organisms. In this report, we present the discovery of a new double-stranded RNA (dsRNA) mycovirus called Alternaria botybirnavirus 2 (ABRV2) from a phytopathogenic strain, XC21-21C, of Alternaria sp. isolated from diseased tobacco leaves in China. The ABRV2 genome consists of two dsRNA components, namely dsRNA1 and dsRNA2, with lengths of 6,162 and 5,865 base pairs (bp), respectively. Each of these genomic dsRNAs is monocistronic, encoding hypothetical proteins of 201.6 kDa (P1) and 2193.3 kDa (P2). ABRV2 P1 and P2 share 50.54% and 63.13% amino acid sequence identity with the corresponding proteins encoded by dsRNA1 of Alternaria botybirnavirus 1 (ABRV1). Analysis of its genome organization and phylogenetic analysis revealed that ABRV2 is a new member of the genus Botybirnavirus.

The CfKOB1 gene related to cell apoptosis is required for pathogenicity and involved in mycovirus-induced hypovirulence in Colletotrichum fructicola.

Colletotrichum fructicola is a globally significant phytopathogenic fungus. Mycovirus-induced hypovirulence has great potential for biological control and study of fungal pathogenic mechanisms. We previously reported that the mycovirus Colletotrichum alienum partitivirus 1 (CaPV1) is associated with the hypovirulence of C. fructicola, and the present study aimed to further investigate a host factor and its roles in mycovirus-induced hypovirulence. A gene named CfKOB1, which encodes putative protein homologous to the ß-subunit of voltage-gated potassium channels and aldo-keto reductase, is downregulated upon CaPV1 infection and significantly upregulated during the early infection phase of Nicotiana benthamiana by C. fructicola. Deleting the CfKOB1 gene resulted in diminished vegetative growth, decreased production of asexual spores, hindered appressorium formation, reduced virulence, and altered tolerance to abiotic stresses. Transcriptome analysis revealed that CfKOB1 regulates many metabolic pathways as well as the cell cycle and apoptosis. Furthermore, enhanced apoptosis was observed in the ΔCfKOB1 mutants. Viral RNA accumulation was significantly increased in the CfKOB1 deletion mutant. Additionally, our findings demonstrated that CaPV1 infection in the WT strain also induced cell apoptosis. Collectively, these results highlight the diverse biological roles of the CfKOB1 gene in the fungus C. fructicola, while it also participates in mycovirus-induced hypovirulence by regulating apoptosis.

Characterization of two novel fusariviruses co-infecting a single isolate of phytopathogenic fungus Botrytis cinerea.

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate. The identified viruses were tentatively named Botrytis cinerea fusarivirus 9 (BcFV9) and Botrytis cinerea fusarivirus 3a (BcFV3a). Both viruses had a single-segmented (ssRNA) genome having a size of 6424 and 8370 nucleotides encoding two discontinuous open reading frames (ORFs). ORF-1 of both mycoviruses encodes for a polyprotein having a conserved domain of RNA-dependent RNA polymerase (RdRP) and a helicase domain (Hel) which function in RNA replication, while ORF2 encodes a hypothetical protein with an unknown function, respectively. Phylogenetic analysis indicated that BcFV9 made a clade with the genus Alphafusarivirus and BcFV3a fall in the genus Betafusarivirus in the family Fusariviridae. To our knowledge, this is the first report of two fusariviruses identified in isolates of B. cinerea from Pakistan. Both mycoviruses successfully transfected to a compatible strain of B. cinerea (Mst11). A comparison of virus-free (VF) and virus-infected (VI) isogenic lines showed the presence of these viruses was causing hypovirulence in infected strains. Virus-infected strains also had a small lesion size while testing the pathogenicity via apple assay.

Discovery of novel RNA viruses through analysis of fungi-associated next-generation sequencing data.

BACKGROUND: Like all other species, fungi are susceptible to infection by viruses. The diversity of fungal viruses has been rapidly expanding in recent years due to the availability of advanced sequencing technologies. However, compared to other virome studies, the research on fungi-associated viruses remains limited. RESULTS: In this study, we downloaded and analyzed over 200 public datasets from approximately 40 different Bioprojects to explore potential fungal-associated viral dark matter. A total of 12 novel viral sequences were identified, all of which are RNA viruses, with lengths ranging from 1,769 to 9,516 nucleotides. The amino acid sequence identity of all these viruses with any known virus is below 70%. Through phylogenetic analysis, these RNA viruses were classified into different orders or families, such as Mitoviridae, Benyviridae, Botourmiaviridae, Deltaflexiviridae, Mymonaviridae, Bunyavirales, and Partitiviridae. It is possible that these sequences represent new taxa at the level of family, genus, or species. Furthermore, a co-evolution analysis indicated that the evolutionary history of these viruses within their groups is largely driven by cross-species transmission events. CONCLUSIONS: These findings are of significant importance for understanding the diversity, evolution, and relationships between genome structure and function of fungal viruses. However, further investigation is needed to study their interactions.

Sensitivity of Globisporangium ultimum to the fungicide metalaxyl is enhanced by the infection with a toti-like mycovirus.

In recent years, numerous oomycete mycoviruses have been discovered; however, very few studies have focused on their effects on the host oomycete phenotype. In this study, we investigated the impact of toti-like Pythium ultimum RNA virus 2 (PuRV2) infection on the phytopathogenic soil-borne oomycete Globisporangium ultimum, which serves as a model species for Globisporangium and Pythium, specifically the UOP226 isolate in Japan. We generated a PuRV2-free isogenic line through hyphal tip isolation using high-temperature culture and subsequently compared the phenotypic characteristics and gene expression profiles of UOP226 and the PuRV2-free isogenic line. Our findings revealed that the metalaxyl sensitivity of UOP226 was greater than that of the PuRV2-free isogenic line, whereas the mycelial growth rate and colony morphology remained unchanged in the absence of the fungicide. Furthermore, transcriptome analyses using RNA-seq revealed significant downregulation of ABC-type transporter genes, which are involved in fungicide sensitivity, in UOP226. Our results suggest that PuRV2 infection influences the ecology of G. ultimum in agricultural ecosystems where metalaxyl is applied.

A circular single-stranded DNA mycovirus infects plants and confers broad-spectrum fungal resistance.

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21-26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1-P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.