Giardiasis is a parasitic intestinal infection of both animals and humans, caused by the enteric protozoan Giardia duodenalis (syn. G. lamblia and G. intestinalis). To date, symbiont RNA viruses of G. duodenalis: Giardia lamblia virus (GLV) and Giardia duodenalis RNA virus-2 (GdRV-2) have been reported. Here, we report the complete genome sequence of a novel virus identified in a human isolate of G. duodenalis using fragmented and primer-ligated double-stranded RNA sequencing. This virus, designated the Non-GLV Giardia duodenalis ISE strain (Noggi) virus, is genetically distinct from GLV and GdRV-2 and likely represents the third symbiont virus in Giardia parasites.

Abstract Recent extensive metatranscriptome mining vastly expanded the range of apparently covalently closed circular (ccc) RNA replicons. A notable family of such replicons is Obelisks, ~1 kilobase (kb) cccRNAs encoding a protein with a unique fold, Oblin-1, and detected in diverse metatranscriptomes. To identify potential cccRNAs in a sequence similarity–independent manner, we adopt the Fragmented and primer-Ligated DsRNA Sequencing (FLDS) method to selectively sequence double-stranded (ds) RNAs, replicative intermediates of RNA replicons. We focus on candidates with predicted extensive intramolecular base-pairing, a hallmark of viroid-like elements. Using FLDS, we explore metatranscriptomes from acidic hot springs in Japan and discover a distinct family of Obelisks apparently associated with thermoacidophilic bacteria (Hot spring Obelisks, HsObs). Despite lacking sequence similarity to known Oblins, HsObs share key features, including ~1 kb genome size, rod-like RNA secondary structure, and the predicted fold of the encoded protein, HsOblin. A comprehensive metatranscriptome search for Oblin-1 and HsOblin homologs expands Obelisk diversity about two-fold, revealing multiple subfamilies sharing the same core fold,. some of which are also predicted to encode additional small proteins with simple alpha-helical folds. These findings highlight Obelisks as widespread and overlooked components of microbial ecosystems, expanding understanding of viroid-like RNA replicon diversity and evolution.

Katsuobushi is a traditional processed seafood product used in Japanese-style cooking, and when it is produced through fermentation by fungi, it is called karebushi. The fungi involved in katsuobushi fermentation are collectively referred to as katsuobushi molds. We previously discovered seven novel viruses from katsuobushi molds and determined their genome sequences. However, our previous explorations used only nine fungal strains available from culture collections, leaving the diversity of viruses infecting fungi involved in katsuobushi fermentation unclear. Therefore, in this study, we aimed to isolate fungi from commercially available karebushi and clarify the prevalence of viruses in the isolates. Karebushi produced by three manufacturers was obtained, and 30 fungal strains (including Aspergillus spp.) were isolated from each. Double-stranded RNA (dsRNA) fractions were prepared from the mycelia of the isolated strains. Electrophoresis suggested that a relatively high proportion of the isolates harbored dsRNA elements consistent with RNA virus infection (30-70% per manufacturer; 59% overall). Furthermore, dsRNA sequencing identified four novel viruses in isolates of Aspergillus chevalieri and Aspergillus montevidensis: a beny-like virus, a gammapartitivirus, a narnavirus, and a victorivirus, in addition to two previously reported viruses. Notably, this represents the first report of a beny-like virus in Aspergillus spp. This study provides insights into the diversity of viruses infecting fungi involved in katsuobushi fermentation.

In the present study, a Pakistani isolate of a known chrysovirus was identified in Penicillium rubens strain S25 tentatively named as Penicillium rubens chrysovirus 1 (PrCV1). PrCV1 has five genomic segments, ranging from 0.5 to 3.7 kbp in length. Analysis of the RNA-dependent RNA polymerase (RdRP) amino acid sequence revealed a 99.28% identity with the genome of Penicillium chrysogenum chrysovirus, suggesting a close evolutionary relationship. Notably, a fifth segment, absent in P. chrysogenum, was identified and is described here for the first time in P. rubens. Sequence analysis showed that this fifth segment is 514 bp in length and has a 5'-terminus identical to the other four segments but it differs in the Box-1 region and lacks the characteristic CAA repeats.

Clarireedia jacksonii is a phytopathogenic fungus responsible for dollar spot disease in turfgrass worldwide. In this study, we characterized the complete genome sequences of three novel mycoviruses isolated from C. jacksonii isolate MBCT-836 using next-generation sequencing and the fragmented and primer-ligated dsRNA sequencing (FLDS) method. Two of these viruses, designated Clarireedia jacksonii mitovirus 1 (CjMV1) and Clarireedia jacksonii mitovirus 2 (CjMV2), possess positive-sense single-stranded RNA genomes of 2,575 bp and 2,856 bp, respectively. Both viruses contain a single open reading frame that utilizes the mitochondrial genetic code and encodes an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis placed CjMV1 and CjMV2 within the genera Unuamitovirus and Duamitovirus, respectively, in the family Mitoviridae. The third virus, Clarireedia jacksonii negative-stranded RNA virus 1 (CjNSV1), features a bisegmented negative-sense RNA genome consisting of a large segment (7,961 nt) encoding an RdRp with a conserved Bunya_RdRp domain, and a small segment (1,444 nt) encoding a protein showing homology to bunyavirus nucleocapsid proteins. Phylogenetic analysis revealed that CjNSV1 clusters with members of the proposed family Sclerobunyaviridae within the order Bunyavirales. To our knowledge, this study provides the first report of complete genome sequences of mycoviruses infecting C. jacksonii, expanding our understanding of the mycovirosphere in economically significant turfgrass pathogens.

Fungi are frequently infected with viruses called mycoviruses. Some mycoviruses have shown potential as biocontrol agents because they can weaken the virulence of fungal plant pathogens. Intensive research has been conducted on the diversity of viruses infecting plant pathogens and the functions of such viruses. In contrast, the diversity of viruses infecting fungi that are not plant pathogens is less well understood. We previously identified a mycovirus of the family Partitiviridae, Aspergillus sulphureus partitivirus 1 (AsuPV1), from an Aspergillus sulphureus strain isolated from fermented dried bonito. In the present study, we conducted a detailed phylogenetic analysis of AsuPV1 and determined its molecular characteristics. The phylogenetic analysis revealed that AsuPV1 belongs to the proposed subgroup I within the genus Gammapartitivirus. Virus particles of AsuPV1 were isolated by density gradient centrifugation, and molecular analyses indicated that each of its three genome segments is encapsulated in capsids composed of the putative viral coat protein. AsuPV1 is the second virus with a tri-segmented genome to be found in a member of Gammapartitivirus subgroup I, after Penicillium stoloniferum virus F. A phenotypic comparison between the parental A. sulphureus strain and isogenic virus-free isolates indicated that AsuPV1 enhances conidium production in its host. This study provides insight into the diversity and molecular characteristics of partitiviruses with a tri-segmented genome.

Fungi produce a wide variety of compounds, especially those that exhibit biological activity. Such compounds may aid the survival of fungi in the environment or may contribute to host infection for pathogenic species. Regarding dermatophytes, which affect a large number of patients worldwide, studies on metabolites that exhibit biological activity are scarce. In this study, to gain insight into the interaction with skin microbiota at the site of infection, we searched for compounds that exhibit antibacterial activity among the metabolites of Trichophyton rubrum. We rediscovered viomellein, a red pigment, as a potent antibacterial compound and identified its biosynthetic gene (vio) cluster by RNA-sequencing and gene deletion analyses. Sequential reconstruction of the vio cluster genes in Aspergillus oryzae revealed the biosynthetic pathway for viomellein via nor-toralactone, semivioxanthin, and vioxanthin production. The vio gene cluster is widely conserved among dermatophytes and is also present in some Aspergillus and Penicillium species. Consistent with the results, viomellein and its structural analogs, xanthomegnin, and vioxanthin, were shown to be produced by most dermatophyte species. These results suggest that dermatophytes can produce diverse naphthopyranone compounds, some of which have strong growth inhibitory effects against bacteria. This study provides a previously unknown molecular entity for antibiotic production by dermatophytes and provides insight into the interaction between commensal bacteria and dermatophytes.IMPORTANCEDermatophytes are widespread human pathogens in the world, but the mechanisms of infection have been little studied. Although bacterial density at the site of infection is abundant, interaction between dermatophytes and the bacterial community has not yet been studied. Here, to understand the infection ecology of dermatophytes, we searched for antimicrobial substances that would be effective against the dermal bacterial community. We discovered viomellein, which exhibits strong antibacterial activity against gram-positive bacteria such as Staphylococcus aureus, and its biosynthetic genes are shared not only by dermatophytes but also by other fungi. Since many dermatophytes showed the ability to produce viomellein, it is likely that this is the initial infection strategy of dermatophytes, which has been a mystery for long.

Leptosphaeria biglobosa is a phytopathogenic ascomycete of Brassica napus that causes phoma stem canker/blackleg. A new double-stranded RNA (dsRNA) mycovirus from this fungus has been fully characterized. The virus genome has five dsRNA segments, ranging in length from 1,180 bp to 2,402 bp. Each dsRNA has a single open reading frame flanked by 5′ and 3′ untranslated regions. The proteins encoded by dsRNAs 1 and 3, an RNA-dependent RNA polymerase (RdRP) and a methyltransferase, respectively, have significant similarity to those of Plasmopara viticola lesion associated polymycovirus 1. The proline-alanine-serine-rich protein encoded by dsRNA 5 is similar to that of Erysiphe necator associated polymycovirus 1. The proteins encoded by dsRNAs 2 and 4 have significant similarity to those of a mycovirus identified in Alternaria sp. FA0703. Phylogenetic analysis based on RdRP sequences showed that this virus clusters with members of the family Polymycoviridae. Based on these observations, this virus, which we have named "Leptosphaeria biglobosa polymycovirus 1", should be classified as a member of the family Polymycoviridae. This is the first report of a polymycovirus in L. biglobosa.

Extensive metatranscriptome mining has recently vastly expanded the range of covalently closed circular (ccc) RNA replicons. A notable group of such replicons are Obelisks, cccRNAs of about 1 kilobase (kb) encoding a protein with a unique fold, Oblin-1, and detected in a broad variety of metatranscriptomes, in particular, those from the human gastrointestinal tract. We adopted Fragmented and primer-Ligated DsRNA Sequencing (FLDS) method to selectively sequence double-stranded (ds) RNAs, the replicative intermediates of RNA replicons, and to identify cccRNAs among the resulting sequences. From these data, we selected cccRNAs with predicted extensive intramolecular base-pairing, a hallmark of viroid-like elements. ch We employed FLDS to explore metatranscriptomes from acidic hot springs in Japan and discovered a distinct family of Obelisks probably associated with thermoacidophilic bacteria (Hot spring Obelisks, HsObs). The proteins encoded by HsObs, HsOblins, show no significant sequence similarity to previously identified Oblin-1 proteins, but are predicted to adopt a closely similar structure. A comprehensive search of metagenomes for Oblin-1 and HsOblin homologs substantially expanded this family of Obelisk-encoded proteins revealing several distinct subfamilies that share the same core fold. A cccRNA encoding an HsOblin homolog was also detected in a Yellowstone hot spring metatranscriptome. Apart from Oblin-1, some subfamilies of Obelisks were predicted to encode additional small proteins with simple alpha-helical folds.

Unlike the well-known acute or chronic animal and plant RNA viruses, most fungal RNA viruses (RNA mycoviruses) have a persistent life cycle. They lack an extracellular infection route and coexist with their hosts for a lifetime. RNA mycoviruses affect various biological properties of host fungi and are widespread across all major fungal groups. The frequency of fungal isolates harboring RNA mycoviruses ranges from a few percent to several tens of percent, indicating their high persistence within ecosystems. However, the dynamics of RNA mycoviruses and the maintenance mechanisms within host populations remain largely unknown. Here, we developed an experimental laboratory model system to examine the dynamics of mycoviruses within clonal host populations and to clarify the mechanisms. In our experimental system, we used two mycoviruses infecting Aspergillus fumigatus. Using this system, we discovered that viral prevalence changed according to environmental conditions (temperature or fungicide exposure). The driving forces behind these prevalence changes were viral horizontal transmission between isogenic strains through hyphal fusion, viral loss, and viral impact on host growth. Our findings indicated that the dynamics of mycoviruses are similar to those of plasmids in bacterial populations. This analogy implied an uncovered role of mycoviruses as a switching factor for fungal adaptation to changing environments.IMPORTANCEWhile RNA viruses are generally known for their ability to infect cells from the extracellular environment, a substantial diversity of RNA viruses, particularly those that persistently infect fungi, lack such infectivity. The ecological success and evolutionary maintenance of these persistent RNA viruses remain poorly understood. In this study, we experimentally demonstrate how a non-infectious RNA virus is transmitted and stably maintained within specific fungal host lineages. The revealed mechanism resembles the inheritance strategies of plasmids, highlighting a fundamentally different viral lifestyle that does not rely on extracellular horizontal transmission. These findings advance our understanding of virus-host interactions beyond the classical infection model and shed light on the evolutionary flexibility of RNA viruses.

The order Martellivirales in the Riboviria realm includes seven established families. The viruses in this order have a single-stranded positive-sense RNA genome and infect animals, plants, or fungi. In this study, we characterized Aspergillus flavus vivivirus 1 (AfViV1), an RNA virus infecting Aspergillus flavus that presumably belongs to the proposed "Viviviridae" family in the Martellivirales order. In previous reports, multiple RNA-dependent RNA polymerase (RdRP) sequences related to "Viviviridae" were mainly identified from metatranscriptome data. However, their virological characteristics were not disclosed. Our analysis showed that the AfViV1 virion exhibited a rod-shaped structure with varying lengths and identified the coat protein (CP) encoded by RNA12 of AfViV1. Using the AfViV1-CP sequence, we detected several potential CP sequences from viruses in the suggested "Viviviridae" family based on sequence read archive (SRA) data. These data suggest that viruses in this family have similar rod-shaped structures. Interestingly, the AfViV1-CP amino acid sequence was not significantly similar to the known viral CP. However, the predicted structure was similar to rod-shaped viruses in the Potyviridae (Patatavirales) and Closteroviridae (Martellivirales) families (and orders). Our analysis describes the first multi-segmented fungal ssRNA virus with rod-shaped particles and expands the morphological diversity of fungal RNA viruses. Additionally, this study highlights the similarities between fungal and plant viruses, suggesting their deep relationships concerning host range, host adaptation, and more.

Metatranscriptome sequencing dramatically expanded the known diversity of the global RNA virome and, in particular, suggested several new candidate phyla in riboviruses. Using a double-stranded RNA (dsRNA) sequencing, here, we report five complete, bisegmented RNA genomes of a putative phylum group, paraxenoviruses, identified from marine environments. Phylogenetic analysis of the RNA-directed RNA polymerases of paraxenoviruses demonstrated their affinity with the ribovirus order Durnavirales within the class Duplopiviricetes of the phylum Pisuviricota. The order Durnavirales includes families Cystoviridae that consists of well-characterized dsRNA bacteriophages and less thoroughly studied Picobirnaviridae that are also suspected to infect bacteria. Consistently, modeling and analysis of the structure of the predicted capsid protein (CP) of several paraxenoviruses revealed similarity to picobirnavirus CP although the paraxenovirus CP is much larger and contains unique structural elaborations. Taken together, these affinities suggest that paraxenoviruses represent a distinct family within Durnavirales, which we provisionally name "Paraxenoviridae". Both genomic segments in Picobirnaviridae and "Paraxenoviridae" encompass multiple open reading frames, each preceded by a typical bacterial ribosome-binding site, strongly suggesting that these families consist of bacterial viruses. Search for homologs of paraxenovirus genes shows widespread distribution of this virus group in the global ocean, suggesting an important contribution to marine microbial ecosystems. Our findings further expand the diversity and ecological role of the bacterial RNA virome, reveal extensive structural variability of RNA viral CPs, and demonstrate the common ancestry of several distinct families of bacterial viruses with dsRNA genomes.

Fragmented and primer Ligated DsRNA Sequencing (FLDS) was used to reconstruct five complete, bisegmented RNA genomes of paraxenoviruses, a group of viruses that was previously identified in the ocean and that based on the analysis of partial genomes was proposed to represent a putative new phylum within the kingdom Orthornavirae of the realm Riboviria. Phylogenetic analysis of the RNA-directed RNA polymerases of paraxenoviruses demonstrated their affinity with the ribovirus order Durnavirales within the class Duplopiviricetes of the phylum Pisuviricota. The order Durnavirales includes families Cystoviridae that consists of well-characterized dsRNA bacteriophages and less thoroughly studied Picobirnaviridae that are also suspected to infect bacteria. Consistently, modeling and analysis of the structure of the predicted capsid protein (CP) of several paraxenoviruses revealed similarity to picobirnavirus CP although the paraxenovirus CP is much larger and contains unique structural elaborations. Taken together, these affinities suggest that paraxenoviruses represent a distinct family within Durnavirales, which we provisionally name "Paraxenoviridae". Both genomic segments in Picobirnaviridae and "Paraxenoviridae" encompass multiple open reading frames, each preceded by a typical bacterial ribosomebinding site, strongly suggesting that these families consist of bacterial viruses. Search for homologs of paraxenovirus genes shows widespread distribution of this virus group in the global ocean, suggesting a potential important contribution to marine microbial ecosystems. Our findings further expand the diversity and ecological role of the bacterial RNA virome, reveal extensive structural variability of RNA viral capsid proteins, and demonstrate the common ancestry of several distinct families of bacterial viruses with dsRNA genomes.

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

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.

To contribute to the mitigation of global anthropogenic crises, engineering education needs to provide holistic views of these crises through collaborating with experts in various fields. This study designed three educational board games representing common structures among global anthropogenic issues and investigated their effects on the lessons learned by university students. Experts in Energy System Engineering, Virology, Philosophy of Education, Design Science, Agricultural Economics, and Law participated in this study. The 2nd, 3rd, and 4th authors of this study designed games named ‘Parasite Wars’, ‘The Dialogue with Future’, and ‘The House”. The concept of games, such as themes, roles and purposes of players, and lessons expected to be imparted to participants, largely differed among the games; each designer represented the same issues from very different perspectives. The results of test sessions with 60 students suggest that participants learned lessons relevant to the messages that the designers conveyed. Further, discussions and reviews for designing board games are useful to understand the perspectives and approaches of experts in other fields and reflect on the roles of their own disciplines in terms of sustainability education. Such interdisciplinary collaboration enables the design of educational content that complements diverse perspectives on global anthropogenic issues and has the potential to help promote a holistic view of such complex issues.

Mycoviruses have been described in all major fungal taxonomic groups. There has been much focus on commercially cultivated basidiomycetous macrofungi, while attention to viruses from ascomycetous macrofungi is lacking. Therefore, in this study, we conducted viral screening against fungal mycelia that were regenerated from ascomycetous macrofungi using agarose gel electrophoresis (AGE) and fragmented and primer-ligated dsRNA sequencing (FLDS). Among the 57 isolates, four isolates were detected with virus-like bands through screening with AGE, and subsequent FLDS analyses determined the viral sequences. Other isolates without virus-like bands in AGE were pooled to check for viral sequences. Using FLDS analysis, a total of seven new mycoviruses were identified, including two double-stranded RNA (dsRNA) viruses belonging to Quadriviridae and Partitiviridae, five positive-sense single-stranded RNA (ssRNA) viruses (three belonging to Mitoviridae, one belonging to Endornaviridae and one belonging to Virgaviridae). All viruses characterized in this study are novel species, and all the hosts are firstly reported to be infected by mycoviruses. These findings expand our knowledge of the diversity of mycoviruses from macrofungi in natural environments.

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.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus cultured. However, only two strains of HcRNAV have been registered with complete genome sequences (strains 34 and 109 for UA and CY types, respectively). To extend the genomic information of HcRNAV, we performed full-genome sequencing of an unsequenced strain of HcRNAV (strain A8) using the fragmented and primer-ligated double-stranded RNA (dsRNA) sequencing (FLDS) method. The complete genome of HcRNAV A8 with 4457 nucleotides (nt) was successfully determined, and sequence alignment of the major capsid protein gene suggested that A8 was a UA-type strain, consistent with its intraspecific host specificity. The complete sequence was found to be 80 nt longer at the 5′ terminus than the registered sequences of HcRNAV strains (34 and 109), suggesting that FLDS is more reliable for determining the terminal sequence than conventional methods (5’ Rapid Amplification of cDNA End). Our study contributes to a better understanding of dinoflagellate-infecting viruses with limited sequence data.

Rhizopus microsporus is a species in the order Mucorales that is known to cause mucormycosis, but it is poorly understood as a host of viruses. Here, we examined 25 clinical strains of R. microsporus for viral infection with a conventional double-stranded RNA (dsRNA) assay using agarose gel electrophoresis (AGE) and the recently established fragmented and primer-ligated dsRNA sequencing (FLDS) protocol. By AGE, five virus-infected strains were detected. Then, full-length genomic sequences of 12 novel RNA viruses were revealed by FLDS, which were related to the families Mitoviridae, Narnaviridae, and Endornaviridae, ill-defined groups of single-stranded RNA (ssRNA) viruses with similarity to the established families Virgaviridae and Phasmaviridae, and the proposed family "Ambiguiviridae." All the characterized viruses, except a potential phasmavirid with a negative-sense RNA genome, had positive-sense RNA genomes. One virus belonged to a previously established species within the family Mitoviridae, whereas the other 11 viruses represented new species or even new genera. These results show that the fungal pathogen R. microsporus harbors diverse RNA viruses and extend our understanding of the diversity of RNA viruses in the fungal order Mucorales, division Mucoromycota. Identifying RNA viruses from clinical isolates of R. microsporus may expand the repertoire of natural therapeutic agents for mucormycosis in the future.IMPORTANCEThe diversity of mycoviruses in fungal hosts in the division Mucoromycota has been underestimated, mainly within the species Rhizopus microsporus. Only five positive-sense RNA genomes had previously been discovered in this species. Because current sequencing methods poorly complete the termini of genomes, we used fragmented and primer-ligated double-stranded RNA sequencing to acquire the full-length genomes. Eleven novel mycoviruses were detected in this study, including the first negative-sense RNA genome reported in R. microsporus. Our findings extend the understanding of the viral diversity in clinical strains of Mucoromycota, may provide insights into the pathogenesis and ecology of this fungus, and may offer therapeutic options.