A novel betapartitivirus isolated from Cordyceps militaris, an edible-medicinal mushroom.

In this study, we identified a novel partitivirus, named "Cordyceps militaris partitivirus 1" (CmPV1), in Cordyceps militaris strain RCEF7506. The complete genome of CmPV1 comprises two segments, dsRNA1 and dsRNA2, each encoding a single protein. dsRNA1 (2,206 bp) encodes an RNA-dependent RNA polymerase (RdRp), and dsRNA2 (2,256 bp) encodes a coat protein (CP). Sequence analysis revealed that dsRNA1 has the highest similarity to that of Bipolaris maydis partitivirus 2 (BmPV2), whereas dsRNA2 shows the highest similarity to human blood-associated partitivirus (HuBPV). Phylogenetic analysis based on RdRp sequences suggests that CmPV1 is a new member of the genus Betapartitivirus of the family Partitiviridae. This is the first documentation of a betapartitivirus infecting the entomopathogenic fungus C. militaris.

Identification of a novel member of the genus Laulavirus (family Phenuiviridae) from the entomopathogenic ascomycete fungus Cordyceps javanica.

The virus family Phenuiviridae (order Hareavirales, comprising segmented negative-sense single stranded RNA viruses) has highly diverse members that are known to infect animals, plants, protozoans, and fungi. In this study, we identified a novel phenuivirus infecting a strain of the entomopathogenic fungus Cordyceps javanica isolated from a small brown plant hopper (Laodelphax striatellus), and this virus was tentatively named "Cordyceps javanica negative-strand RNA virus 1" (CjNRSV1). The CjNRSV1 genome consists of three negative-sense single stranded RNA segments (RNA1-3) with lengths of 7252, 2401, and 1117 nt, respectively. The 3'- and 5'-terminal regions of the RNA1, 2, and 3 segments have identical sequences, and the termini of the RNA segments are complementary to each other, reflecting a common characteristic of viruses in the order Hareavirales. RNA1 encodes a large protein (∼274 kDa) containing a conserved domain for the bunyavirus RNA-dependent RNA polymerase (RdRP) superfamily, with 57-80% identity to the RdRP encoded by phenuiviruses in the genus Laulavirus. RNA2 encodes a protein (∼79 kDa) showing sequence similarity (47-63% identity) to the movement protein (MP, a plant viral cell-to-cell movement protein)-like protein (MP-L) encoded by RNA2 of laulaviruses. RNA3 encodes a protein (∼28 kDa) with a conserved domain of the phenuivirid nucleocapsid protein superfamily. Phylogenetic analysis using the RdRPs of various phenuiviruses and other unclassified phenuiviruses showed CjNRSV1 to be grouped with established members of the genus Laulavirus. Our results suggest that CjNRSV1 is a novel fungus-infecting member of the genus Laulavirus in the family Phenuiviridae.

Complete genome sequence of a novel botourmiavirus infecting the fungus Phomopsis asparagi.

Here, we report a novel ourmia-like mycovirus, named "Phomopsis asparagi magoulivirus 1" (PaMV1), derived from the phytopathogenic fungus Phomopsis asparagi. The genome of PaMV1 consists of a positive-sense single-stranded RNA (+ ssRNA) that is 2,639 nucleotides in length, with a GC content of 57.13%. It contains a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) consisting of 686 amino acids with a molecular mass of 78.57 kDa. Phylogenetic analysis based on RdRp sequences revealed that PaMV1 grouped together with Diaporthe gulyae magoulivirus 1 (DgMV1) in a distinct clade. Sequence comparisons and phylogenetic analysis suggest that PaMV1 is a novel member of the genus Magoulivirus, family Botourmiaviridae.

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.

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.

Characterization of Two Novel Single-Stranded RNA Viruses from Agroathelia rolfsii, the Causal Agent of Peanut Stem Rot.

Peanut stem rot is a soil-borne disease caused by Agroathelia rolfsii. It occurs widely and seriously affects the peanut yield in most peanut-producing areas. The mycoviruses that induce the hypovirulence of some plant pathogenic fungi are potential resources for the biological control of fungal diseases in plants. Thus far, few mycoviruses have been found in A. rolfsii. In this study, two mitoviruses, namely, Agroathelia rolfsii mitovirus 1 (ArMV1) and Agroathelia rolfsii mitovirus 2 (ArMV2), were identified from the weakly virulent A. rolfsii strain GP3-1, and they were also found in other A. rolfsii isolates. High amounts of ArMV1 and ArMV2in the mycelium could reduce the virulence of A. rolfsii strains. This is the first report on the existence of mitoviruses in A. rolfsii. The results of this study may provide insights into the classification and evolution of mitoviruses in A. rolfsii and enable the exploration of the use of mycoviruses as biocontrol agents for the control of peanut stem rot.

A Novel Strain of Fusarium oxysporum Alternavirus 1 Isolated from Fusarium oxysporum f. sp. melonis Strain T-BJ17 Confers Hypovirulence and Increases the Sensitivity of Its Host Fungus to Difenoconazole and Pydiflumetofen.

In the current study, a novel strain of Fusarium oxysporum alternavirus 1 (FoAV1) was identified from the Fusarium oxysporum f. sp. melonis (FOM) strain T-BJ17 and was designated as Fusarium oxysporum alternavirus 1-FOM (FoAV1-FOM). Its genome consists of four dsRNA segments of 3515 bp (dsRNA1), 2663 bp (dsRNA2), 2368 bp (dsRNA3), and 1776 bp (dsRNA4) in length. Open reading frame 1 (ORF1) in dsRNA1 was found to encode a putative RNA-dependent RNA polymerase (RdRp), whose amino acid sequence was 99.02% identical to that of its counterpart in FoAV1; while ORF2 in dsRNA2, ORF3 in dsRNA3, and ORF4 in dsRNA4 were all found to encode hypothetical proteins. Strain T-BJ17-VF, which was verified to FoAV1-FOM-free, was obtained using single-hyphal-tip culture combined with high-temperature treatment to eliminate FoAV1-FOM from strain T-BJ17. The colony growth rate, ability to produce spores, and virulence of strain T-BJ17 were significantly lower than those of T-BJ17-VF, while the dry weight of the mycelial biomass and the sensitivity to difenoconazole and pydiflumetofen of strain T-BJ17 were greater than those of T-BJ17-VF. FoAV1-FOM was capable of 100% vertical transmission via spores. To our knowledge, this is the first time that an alternavirus has infected FOM, and this is the first report of hypovirulence and increased sensitivity to difenoconazole and pydiflumetofen induced by FoAV1-FOM infection in FOM.

Complete Genome Analyses of a Novel Flexivirus with Unique Genome Organization and Three Endornaviruses Hosted by the Mycorrhizal Fungus Terfezia claveryi.

The extensive use of high-throughput sequencing (HTS) has significantly advanced and transformed our comprehension of virus diversity, especially in intricate settings like soil and biological specimens. In this study, we delved into mycovirus sequence surveys within mycorrhizal fungus species Terfezia claveryi, through employing HTS with total double-stranded RNA (dsRNA) extracts. Our findings revealed the presence of four distinct members from the Alsuviricetes class, one flexivirus designated as Terfezia claveryi flexivirus 1 (TcFV1) and three endornaviruses (TcEV1, TcEV2, and TcEV3) in two different T. claveryi isolates. TcFV1, a member of the order Tymovirales, exhibits a unique genome structure and sequence features. Through in-depth analyses, we found that it shares sequence similarities with other deltaflexiviruses and challenges existing Deltaflexiviridae classification. The discovery of TcFV1 adds to the genomic plasticity of mycoviruses within the Tymovirales order, shedding light on their evolutionary adaptations. Additionally, the three newly discovered endornaviruses (TcEV1, TcEV2, and TcEV3) in T. claveryi exhibited limited sequence similarities with other endornaviruses and distinctive features, including conserved domains like DEAD-like helicase, ATPases Associated with Diverse Cellular Activities (AAA ATPase), and RNA dependent RNA polymerase (RdRp), indicating their classification as members of new species within the Alphaendornavirus genus. In conclusion, this research emphasizes the importance of exploring viral diversity in uncultivated fungi, bridging knowledge gaps in mycovirus ecology. The discoveries of a novel flexivirus with unique genome organization and endornaviruses in T. claveryi broaden our comprehension of mycovirus diversity and evolution, highlighting the need for continued investigations into viral populations in wild fungi.

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia.

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

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.

Ambiviricota, a novel ribovirian phylum for viruses with viroid-like properties.

Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as 'ambiviruses,' were described. 'Ambiviruses' have single-stranded RNA genomes of about 4-5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known 'ambiviruses,' and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, 'ambivirus' genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations-features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as 'epsilonviruses,' 'zetaviruses,' and some 'obelisks'). As a first step toward the formal classification of 'ambiviruses,' the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences.

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.

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.

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Fusarium oxysporum f. sp. melonis strain T-SD3.

A novel mitovirus was identified in Fusarium oxysporum f. sp. melonis strain T-SD3 and designated as "Fusarium oxysporum mitovirus 3" (FoMV3). The virus was isolated from diseased muskmelon plants with the typical symptom of fusarium wilt. The complete genome of FoMV3 is 2269 nt in length with a predicted AU content of 61.40% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a polypeptide of 679 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain with a molecular mass of 77.39 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5'-untranslated region (UTR) and 3'-UTR of FoMV3 were predicted to fold into stem-loop structures. BLASTp analysis revealed that the RdRp of FoMV3 shared the highest aa sequence identity (83.85%) with that of Fusarium asiaticum mitovirus 5 (FaMV5, a member of the family Mitoviridae) infecting F. asiaticum, the causal agent of wheat fusarium head blight. Phylogenetic analysis further suggested that FoMV3 is a new member of the genus Unuamitovirus within the family Mitoviridae. This is the first report of a new mitovirus associated with F. oxysporum f. sp. melonis.

Molecular characterization of a single-negative-stranded RNA virus from the rice blast fungus Magnaporthe oryzae isolate NJ39.

A novel negative-sense single-stranded RNA mycovirus, designated as "Magnaporthe oryzae mymonavirus 1" (MoMNV1), was identified in the rice blast fungus Magnaporthe oryzae isolate NJ39. MoMNV1 has a single genomic RNA segment consisting of 10,515 nucleotides, which contains six open reading frames. The largest open reading frame contains 5837 bases and encodes an RNA replicase. The six open reading frames have no overlap and are arranged linearly on the genome, but the spacing of the genes is small, with a maximum of 315 bases and a minimum of 80 bases. Genome comparison and phylogenetic analysis indicated that MoMNV1 is a new member of the genus Penicillimonavirus of the family Mymonaviridae.

Antarctica's hidden mycoviral treasures in fungi isolated from mosses: A first genomic approach.

This study investigates the presence of mycoviruses in Antarctic fungi and elucidates their evolutionary relationships. To achieve this, we aligned mycoviral gene sequences with genomes of previously sequenced Antarctic endophytic fungi, made available by our research group and accessible via Joint Genome Institute. Our findings reveal that the most prevalent genetic regions in all endophytic fungi are homologous to Partitiviruses, Baculoviridae, and Phycodnaviridae. These regions display evidence of positive selection pressure, suggesting genetic diversity and the accumulation of nonsynonymous mutations. This phenomenon implies a crucial role for these regions in the adaptation and survival of these fungi in the challenging Antarctic ecosystems. The presence of mycoviruses in Antarctic endophytic fungi may indicate shared survival strategies between the virus and its host, shedding light on their evolutionary dynamics. This study underscores the significance of exploring mycoviruses within endophytic fungi and their contributions to genetic diversity. Future research avenues could delve into the functional implications of these conserved mycoviral genetic regions in Antarctic endophytic fungi, providing a comprehensive understanding of this intriguing association and genomic retention of viral region in fungi.

Comparative Analysis of Viromes Identified in Multiple Macrofungi.

Macrofungi play important roles in the soil elemental cycle of terrestrial ecosystems. Fungal viruses are common in filamentous fungi, and some of them can affect the growth and development of hosts. However, the composition and evolution of macrofungal viruses are understudied. In this study, ninety strains of Trametes versicolor, Coprinellus micaceus, Amanita strobiliformis, and Trametes hirsuta were collected in China. Four mixed pools were generated by combining equal quantities of total RNA from each strain, according to the fungal species, and then subjected to RNA sequencing. The sequences were assembled, annotated, and then used for phylogenetic analysis. Twenty novel viruses or viral fragments were characterized from the four species of macrofungi. Based on the phylogenetic analysis, most of the viral contigs were classified into ten viral families or orders: Barnaviridae, Benyviridae, Botourmiaviridae, Deltaflexiviridae, Fusariviridae, Hypoviridae, Totiviridae, Mitoviridae, Mymonaviridae, and Bunyavirales. Of these, ambi-like viruses with circular genomes were widely distributed among the studied species. Furthermore, the number and overall abundance of viruses in these four species of macrofungi (Basidiomycota) were found to be much lower than those in broad-host phytopathogenic fungi (Ascomycota: Sclerotinia sclerotiorum, and Botrytis cinerea). By employing metatranscriptomic analysis in this study, for the first time, we demonstrated the presence of multiple mycoviruses in Amanita strobiliformis, Coprinellus micaceus, Trametes hirsute, and Trametes versicolor, significantly contributing to research on mycoviruses in macrofungi.

Fusarium sacchari hypovirus 1, a Member of Hypoviridae with Virulence Attenuation Capacity in Phytopathogenic Fusarium Species.

In a survey of mycoviruses in Fusarium species that cause sugarcane Pokkah boeng disease, twelve Fusarium strains from three Fusarium species (F. sacchari, F. andiyazi, and F. solani) were found to contain Fusarium sacchari hypovirus 1 (FsHV1), which we reported previously. The genomes of these variants range from 13,966 to 13,983 nucleotides, with 98.6% to 99.9% nucleotide sequence identity and 98.70% to 99.9% protein sequence similarity. Phylogenetic analysis placed these FsHV1 variants within the Alphahypovirus cluster of Hypoviridae. Intriguingly, no clear correlation was found between the geographic origin and host specificity of these viral variants. Additionally, six out of the twelve variants displayed segmental deletions of 1.5 to 1.8 kilobases, suggesting the existence of defective viral dsRNA. The presence of defective viral dsRNA led to a two-thirds reduction in the dsRNA of the wild-type viral genome, yet a tenfold increase in the total viral dsRNA content. To standardize virulence across natural strains, all FsHV1 strains were transferred into a single, virus-free Fusarium recipient strain, FZ06-VF, via mycelial fusion. Strains of Fusarium carrying FsHV1 exhibited suppressed pigment synthesis, diminished microspore production, and a marked decrease in virulence. Inoculation tests revealed varying capacities among different FsHV1 variants to modulate fungal virulence, with the strain harboring the FsHV1-FSA1 showing the lowest virulence, with a disease severity index (DSI) of 3.33, and the FsHV1-FS1 the highest (DSI = 17.66). The identification of highly virulent FsHV1 variants holds promise for the development of biocontrol agents for Pokkah boeng management.

Characterization of Mycoviruses in Armillaria ostoyae and A. cepistipes in the Czech Republic.

Members of the genus Armillaria are widespread forest pathogens against which effective protection has not yet been developed. Due to their longevity and the creation of large-scale cloning of Armillaria individuals, the use of mycoviruses as biocontrol agents (BCAs) against these pathogens could be an effective alternative. This work describes the detection and characterization of viruses in Armillaria spp. collected in the Czech Republic through the application of stranded total RNA sequencing. A total of five single-stranded RNA viruses were detected in Armillaria ostoyae and A. cepistipes, including viruses of the family Tymoviridae and four viruses belonging to the recently described "ambivirus" group with a circular ambisense genome arrangement. Both hammerhead (HHRz) and hairpin (HpRz) ribozymes were detected in all the ambiviricot sequences. Armillaria viruses were compared through phylogenetic analysis and confirmed their specific host by direct RT-PCR. One virus appears to infect both Armillaria species, suggesting the occurrence of interspecies transmission in nature.

Molecular characterization of the complete genome of a novel ormycovirus infecting the ectomycorrhizal fungus Hortiboletus rubellus.

Advancements in high-throughput sequencing and the development of new bioinformatics tools for large-scale data analysis play a crucial role in uncovering virus diversity and enhancing our understanding of virus evolution. The discovery of the ormycovirus clades, a group of RNA viruses that are phylogenetically distinct from all known Riboviria members and are found in fungi, highlights the value of these tools for the discovery of novel viruses. The aim of this study was to examine viral populations in fungal hosts to gain insights into the diversity, evolution, and classification of these viruses. Here, we report the molecular characterization of a newly discovered ormycovirus, which we have named "Hortiboletus rubellus ormycovirus 1" (HrOMV1), that was found in the ectomycorrhizal fungus Hortiboletus rubellus. The bipartite genome of HrOMV1, whose nucleotide sequence was determined by HTS and RLM-RACE, consists of two RNA segments (RNA1 and RNA2) that exhibit similarity to those of previously studied ormycoviruses in their organization and the proteins they encode. The presence of upstream, in-frame AUG triplets in the 5' termini of both RNA segments suggests that HrOMV1, like certain other ormycoviruses, employs a non-canonical translation initiation strategy. Phylogenetic analysis showed that HrOMV1 is positioned within the gammaormycovirus clade. Its putative RNA-dependent RNA polymerase (RdRp) exhibits sequence similarity to those of other gammaormycovirus members, the most similarity to that of Termitomyces ormycovirus 1, with 33.05% sequence identity. This protein was found to contain conserved motifs that are crucial for RNA replication, including the distinctive GDQ catalytic triad observed in gammaormycovirus RdRps. The results of this study underscore the significance of investigating the ecological role of mycoviruses in mycorrhizal fungi. This is the first report of an ormycovirus infecting a member of the ectomycorrhizal genus Hortiboletus.