Complex transmission of partiti-, ambi- and ourmiaviruses in the forest pathogen Heterobasidion parviporum.

Utilizing Heterobasidion partitivirus 13 strain an1 (HetPV13-an1) and 15 strain pa1 (HetPV15-pa1) in co-infection is considered a potential biocontrol approach against Heterobasidion root and butt rot. Both partitiviruses mediate debilitating effects in most Heterobasidion host isolates and are generally transmitted efficiently between host strains. In this investigation, we conducted transmission experiments in the laboratory (in vitro) using several H. parviporum isolates to test whether using dual partitivirus infections is a more efficient way of transmitting viruses to new hosts compared to using single partitivirus infections, and whether co-occurring single-stranded RNA (ssRNA) viruses are co-transmitted during the process. The results showed that H. parviporum donors carrying both partitiviruses, HetPV13-an1 and HetPV15-pa1, transmitted HetPV15-pa1 more efficiently to recipients than the same donors infected with only HetPV15-pa1. In contrast, the transmission of HetPV13-an1 did not differ significantly between donors infected with both or only one partitivirus. Altogether, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high on artificial media. Moreover, the transmission of the ssRNA viruses Heterobasidion ourmia-like virus 1(HetOlV1-pa7) and 4 (HetOlV4-an1) as well as Heterobasidion ambi-like virus 3 (HetAlV3-pa4) across different recipients were found to be variable. This study demonstrated for the first time the transmission of ambi- and ourmiaviruses between H. parviporum isolates in dual cultures and showed that H. parviporum mycelia can be cured of these ssRNA viruses using heat treatment.

Virus transmission frequencies in the pine root rot pathogen Heterobasidion annosum.

The combined use of Heterobasidion partitiviruses 13 and 15 (HetPV13-an1 and HetPV15-pa1) is considered a promising biocontrol approach against Heterobasidion root and butt rot. In a previous study, the transmission frequency of HetPV15-pa1 was found to be higher from a double partitivirus-infected donor than from a single partitivirus-infected donor. In this study, we included a wider array of recipient isolates to assess whether the phenomenon is widespread across different host strains and conducted transmission experiments on artificial media (in vitro) using a total of 45 different H. annosum donor-recipient pairs. In addition to investigating whether double partitivirus infection improves the transmission of HetPV13-an1 and HetPV15-pa1, we examined for the first time how efficiently co-infecting ssRNA viruses are concomitantly transmitted with the partitiviruses, and whether pre-existing ssRNA viruses in the recipients affect virus transmission. Generally, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high from both single partitivirus-infected and double partitivirus-infected donors to most of the H. annosum recipient strains, with few exceptions. However, in contrast to previous experiments, the transmission frequency was not higher from the double partitivirus-infected donors. Also, ourmiavirus was transmitted between H. annosum strains, but the presence of another ourmiavirus in the recipient might affect the efficacy.

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.

Transfection of entomopathogenic Metarhizium species with a mycovirus confers hypervirulence against two lepidopteran pests.

Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.

Molecular Characterization of the First Partitivirus from a Causal Agent of Salvia miltiorrhiza Dry Rot.

Root rot as a result of Salvia miltiorrhiza is a common root disease caused by Fusarium spp., which has become one of the main diseases affecting the production of S. miltiorrhiza. Currently, several hypovirulence-related mycoviruses have been identified in many phytopathogenic fungi, including Fusarium spp., which show potential as biological controls. In this study, we report a new mycovirus, Fusarium oxysporum partitivirus 1 (FoPV1), isolated from F. oxysporum strain FCR51, which is a causal agent of S. miltiorrhiza dry rot. The FoPV1 genome contains two double-stranded RNA segments (dsRNA1 and dsRNA2). The size of dsRNA1 is 1773 bp, and it encodes a putative RNA-dependent RNA polymerase (RdRp). The dsRNA2 is 1570 bp in length, encoding a putative capsid protein (CP). Multiple sequence alignments and phylogenetic analyses based on the amino acid sequences of the RdRp and the CP proteins indicated that FoPV1 appears to be a new member of the family Partitiviridae that is related to members of the genus Gammapartitivirus. Pathogenicity assay showed that FoPV1 confers hypervirulence to its host, F. oxysporum. This is the first report of a partitivirus infecting F. oxysporum and the first hypovirulence-related mycovirus from the causal agent of S. miltiorrhiza dry rot.

The Effects of Mycovirus BmPV36 on the Cell Structure and Transcription of Bipolaris maydis.

Bipolaris maydis partitivirus 36 (BmPV36) is a mycovirus that can significantly reduce the virulence of the host Bipolaris maydis, but its hypovirulence mechanism is not clear. To investigate the response of B. maydis to BmPV36, the effects of BmPV36 on host cell structure and gene expression were studied via transmission electron microscopy and transcriptome sequencing using BmPV36-carrying and virus-free mycelium on the second and fifth culture. The results of transmission electron microscopy showed that the cell wall microfibrils of B. maydis were shortened, the cell membrane was broken, and membrane-bound vesicles and vacuoles appeared in the cells after carrying BmPV36. Transcriptome sequencing results showed that after carrying BmPV36, B. maydis membrane-related genes were significantly up-regulated, but membrane transport-related genes were significantly down-regulated. Genes related to carbohydrate macromolecule polysaccharide metabolic and catabolic processes were significantly down-regulated, as were genes related to the synthesis of toxins and cell wall degrading enzymes. Therefore, we speculated that BmPV36 reduces the virulence of B. maydis by destroying the host's cell structure, inhibiting the synthesis of toxins and cell wall degrading enzymes, and reducing cell metabolism. Gaining insights into the hypovirulence mechanism of mycoviruses will provide environmentally friendly strategies for the control of fungal diseases.

A novel partitivirus conferring hypovirulence by affecting vesicle transport in the fungus Colletotrichum.

Colletotrichum spp. are economically important phytopathogenic fungi that cause anthracnose in a variety of plant species worldwide. Hypovirulence-associated mycoviruses provide new options for the biological control of plant fungal diseases. Here, we found a novel partitivirus from Colletotrichum alienum and named it Colletotrichum alienum partitivirus 1 (CaPV1). CaPV1 contained two dsRNA segments encoding an RNA-dependent RNA polymerase and a capsid protein and was classified under the genus Gammapartitivirus of the family Partitiviridae. CaPV1 significantly decreased host virulence, mycelial growth, appressorial development, and appressorium turgor but increased conidial production with abnormal morphology. In addition, CaPV1 could be successfully transfected into other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, and caused hypovirulence, indicating the broad application potential of this virus. CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum. Notably, some genes related to vesicle transport in the CaPV1-infected strain were downregulated, consistent with the impaired endocytosis pathway in this fungus. When the Rab gene CaRab7, which is associated with endocytosis in vesicle transport, was knocked out, the virulence of the mutants was reduced. Overall, our findings demonstrated that CaPV1 has the potential to control anthracnose caused by Colletotrichum, and the mechanism by which Colletotrichum induces hypovirulence is caused by affecting vesicle transport.IMPORTANCEColletotrichum is a kind of economically important phytopathogenic fungi that cause anthracnose disease in a variety of plant species worldwide. We found a novel mycovirus of the Gammapartitivirus genus and Partitiviridae family from the phytopathogenic fungus Colletotrichum alienum and named it CaPV1. This study revealed that CaPV1 infection significantly decreased host virulence and fitness by affecting mycelial growth, appressorial development, and appressorium turgor. In addition, CaPV1 could also infect other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, by viral particle transfection and resulting in hypovirulence of these Colletotrichum species. Transcriptomic analysis showed that CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum, especially the genes involved in vesicle transport. Moreover, endocytosis and gene knockout assays demonstrated that the mechanism underlying CaPV1-induced hypovirulence is, at least in part, caused by affecting the vesicle transport of the host fungus. This study provided insights into the mechanisms underlying the pathogenesis of Colletotrichum species and mycovirus-fungus interactions, linking the role of mycovirus and fungus vesicle transport systems in shaping fungal pathogenicity.

Galbut Virus Infection Minimally Influences Drosophila melanogaster Fitness Traits in a Strain and Sex-Dependent Manner.

Galbut virus (family Partitiviridae) infects Drosophila melanogaster and can be transmitted vertically from infected mothers or infected fathers with near perfect efficiency. This form of super-Mendelian inheritance should drive infection to 100% prevalence, and indeed, galbut virus is ubiquitous in wild D. melanogaster populations. However, on average, only about 60% of individual flies are infected. One possible explanation for this is that a subset of flies are resistant to infection. Although galbut virus-infected flies appear healthy, infection may be sufficiently costly to drive selection for resistant hosts, thereby decreasing overall prevalence. To test this hypothesis, we quantified a variety of fitness-related traits in galbut virus-infected flies from two lines from the Drosophila Genetic Reference Panel (DGRP). Galbut virus-infected flies had no difference in average lifespan and total offspring production compared to their uninfected counterparts. Galbut virus-infected DGRP-517 flies pupated and eclosed faster than their uninfected counterparts. Some galbut virus-infected flies exhibited altered sensitivity to viral, bacterial, and fungal pathogens. The microbiome composition of flies was not measurably perturbed by galbut virus infection. Differences in phenotype attributable to galbut virus infection varied as a function of fly sex and DGRP strain, and differences attributable to infection status were dwarfed by larger differences attributable to strain and sex. Thus, galbut virus infection does produce measurable phenotypic changes, with changes being minor, offsetting, and possibly net-negative.

A Satellite dsRNA Attenuates the Induction of Helper Virus-Mediated Symptoms in Aspergillus flavus.

Aspergillus flavus is an important fungal pathogen of animals and plants. Previously, we reported a novel partitivirus, Aspergillus flavus partitivirus 1 (AfPV1), infecting A. flavus. In this study, we obtained a small double-stranded (ds) RNA segment (734 bp), which is a satellite RNA of the helper virus, AfPV1. The presence of AfPV1 altered the colony morphology, decreased the number of conidiophores, created significantly larger vacuoles, and caused more sensitivity to osmotic, oxidative, and UV stresses in A. flavus, but the small RNA segment could attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus. Moreover, AfPV1 infection reduced the pathogenicity of A. flavus in corn (Zea mays), honeycomb moth (Galleria mellonella), mice (Mus musculus), and the adhesion of conidia to host epithelial cells, and increased conidial death by macrophages. However, the small RNA segment could also attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus, perhaps by reducing the genomic accumulation of the helper virus AfPV1 in A. flavus. We used this model to investigate transcriptional genes regulated by AfPV1 and the small RNA segment in A. flavus, and their role in generating different phenotypes. We found that the pathways of the genes regulated by AfPV1 in its host were similar to those of retroviral viruses. Therefore, some pathways may be of benefit to non-retroviral viral integration or endogenization into the genomes of its host. Moreover, some potential antiviral substances were also found in A. flavus using this system.

Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1-3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.

The Diversity of Viral Community in Invasive Fruit Flies (Bactrocera and Zeugodacus) Revealed by Meta-transcriptomics.

RNA viruses are extremely diverse and rapidly evolving in various organisms. Our knowledge on viral evolution with interacted hosts in the manner of ecology is still limited. In the agricultural ecosystem, invasive insect species are posing a great threat to sustainable crop production. Among them, fruit flies (Diptera: Tephritidae Bactrocera and Zeugodacus) are destructive to fruits and vegetables, which are also closely related and often share similar ecological niches. Thus, they are ideal models for investigating RNA virome dynamics in host species. Using meta-transcriptomics, we found 39 viral sequences in samples from 12 fly species. These viral species represented the diversity of the viromes including Dicistroviridae, negev-like virus clades, Thika virus clades, Solemoviridae, Narnaviridae, Nodaviridae, Iflaviridae, Orthomyxoviridae, Bunyavirales, Partitiviridae, and Reoviridae. In particular, dicistrovirus, negev-like virus, orthomyxovirus, and orbivirus were common in over four of the fly species, which suggests a positive interaction between fly viromes that exist under the same ecological conditions. For most of the viruses, the virus-derived small RNAs displayed significantly high peaks in 21 nt and were symmetrically distributed throughout the viral genome. These results suggest that infection by these viruses can activate the host's RNAi immunity. Our study provides RNA virome diversity and evidence on their infection activity in ecologically associated invasive fruit fly species, which could help our understanding of interactions between complex species and viruses.

Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1.

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

RdRp or RT, That is the Question.

The RNA-dependent RNA polymerase (RdRp) of all known double-stranded RNA viruses is located within the viral particle and is responsible for the transcription and replication of the viral genome. Through an RT-PCR assay, we determined that purified virions, in vitro translated RdRp proteins, and purified recombinant RdRp proteins of partitiviruses also have reverse transcriptase (RT) function. We show that partitivirus RdRps 1) synthesized DNA from homologous and heterologous dsRNA templates; 2) are active using both ssRNA and dsRNA templates; and 3) are active at lower temperatures compared to an optimal reaction temperature of commercial RT enzymes. This finding poses an intriguing question: why do partitiviruses, with dsRNA genomes, have a polymerase with RT functions? In comparison, 3Dpol, the RdRp of poliovirus, did not show any RT activity. Our findings lead us to propose a new evolutionary model for RNA viruses where the RdRp of dsRNA viruses could be the ancestor of RdRps.

A Novel Partitivirus That Confer Hypovirulence to the Plant Pathogenic Fungus Colletotrichum liriopes.

Here, we report a novel double-stranded RNA virus designated Colletotrichum liriopes partitivirus 1 (ClPV1) from the plant pathogenic fungus C. liriopes. ClPV1 genome has two double stranded RNAs (dsRNAs), named as dsRNA 1 and dsRNA 2, which in the lengths of 1,807 and 1,706 bp, respectively. The dsRNA 1 and dsRNA 2 encoded proteins showing significant amino acid (aa) sequence identity to the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) of partitiviruses, respectively. Phylogenetic analysis using the aa sequences of RdRp and CP indicated that ClPV1 was grouped to members of the putative Epsilonpartitivirus genus in the Partitiviridae family. Spherical viral particles in approximately 35 nm in diameter and packaging the ClPV1 genome were isolated. Virus elimination and virus transfection with purified viral particles, and biological comparison revealed that ClPV1 could reduce the virulence and conidia production of C. liriopes. To the best of our knowledge, this is the first report of mycovirus in C. liriopes fungus.

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest.

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which-Partitiviridae and Mitoviridae-were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin-two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

Co-infection of a novel fusagravirus and a partitivirus in a Korean isolate of Rosellinia necatrix KACC40168.

We report here the presence of dsRNA mycoviruses in a Korean isolate of Rosellinia necatrix. A multiple band pattern of double-stranded RNA (dsRNA) from R. necatrix suggested mixed mycovirus infection. Next-generation sequencing analysis of purified dsRNAs indicated the presence of two dsRNA mycoviruses related to the members of families "Fusagraviridae" (proposed) and Partitiviridae. The first dsRNA virus revealed that the complete genome sequence was 8868 bp in size and contained two large open reading frames (ORFs 1 and 2), overlapped by 22 bp containing a canonical (- 1) slippery heptanucelotide sequence of UUUAAAC. The deduced amino acid sequence of ORF1 and ORF2 showed highest similarity to the hypothetical protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 3 (RnFGV3). Phylogenetic analysis showed that this dsRNA virus clustered with RnFGV3 and other fusagraviruses. Gene organization, sequence similarity, and phylogenetic analysis indicate that this virus seems to belong to a novel species of "Fusagraviridae", which we have named Rosellinia necatrix fusagravirus 4. The second virus has two dsRNA segments with sizes of 1907 bp and 1918 bp, each of which encoded a single ORF showing highest similarity to the RdRp and capsid protein of known members of Partitiviridae. Evaluation of genome structure, sequence similarity, and phylogeny indicate this to be a new member of the genus Alphapartitivirus in the family Partitiviridae, hereafter designated as Rosellinia necatrix partitivirus 26. This is the first report of the presence of a fusagravirus in an Asian R. necatrix isolate and of its mixed infection with a partitivirus.

Diverged and Active Partitiviruses in Lichen.

The lichen is a microbial consortium that mainly consists of fungi and either algae (Viridiplantae) or cyanobacteria. This structure also contains other bacteria, fungi, and viruses. However, RNA virus diversity associated with lichens is still unknown. Here, we analyzed RNA virus diversity in a lichen dominated by fungi and algae using dsRNA-seq technology and revealed that partitiviruses were dominant and active in the microbial consortium. The Partitiviridae sequences found in this study were classified into two genera, which have both plant- and fungi-infecting partitiviruses. This observation suggests that the lichen provides an opportunity for horizontal transfer of these partitiviruses among microbes that form the lichen consortium.

Population Structure of Double-Stranded RNA Mycoviruses That Infect the Rice Blast Fungus Magnaporthe oryzae in Japan.

Various viruses infect Magnaporthe oryzae (syn. Pyricularia oryzae), which is a well-studied fungus that causes rice blast disease. Most research has focused on the discovery of new viruses and the hypovirulence-associated traits conferred by them. Therefore, the diversity and prevalence of viruses in wild fungal populations have not been explored. We conducted a comprehensive screening of M. oryzae mycoviruses from various regions in Japan using double-stranded RNA (dsRNA) electrophoresis and RT-PCR assays. We detected three mycoviruses, Magnaporthe oryzae virus 2 (MoV2), Magnaporthe oryzae chrysovirus 1 (MoCV1), and Magnaporthe oryzae partitivirus 1 (MoPV1), among 127 of the 194 M. oryzae strains screened. The most prevalent virus was MoPV1 (58.8%), which often co-infected in a single fungal strain together with MoV2 or MoCV1. MoV2 and MoCV1 were found in 22.7 and 10.8% of strains, respectively, and they were usually distributed in different regions so that mixed-infection with these two mycoviruses was extremely rare. The predominance of MoPV1 in M. oryzae is supported by significant negative values from neutrality tests, which indicate that the population size of MoPV1 tends to increase. Population genetic analyses revealed high nucleotide diversity and the presence of phylogenetically diverse subpopulations among the MoV2 isolates. This was not the case for MoPV1. Furthermore, studies of a virus-cured M. oryzae strain revealed that MoV2 does not cause any abnormalities or symptoms in its host. However, a leaf sheath inoculation assay showed that its presence slightly increased the speed of mycelial growth, compared with virus-free mycelia. These results demonstrate that M. oryzae in Japan harbors diverse dsRNA mycovirus communities with wide variations in their population structures among different viruses.

Partitiviruses Infecting Drosophila melanogaster and Aedes aegypti Exhibit Efficient Biparental Vertical Transmission.

Partitiviruses are segmented, multipartite double-stranded RNA (dsRNA) viruses that until recently were only known to infect fungi, plants, and protozoans. Metagenomic surveys have revealed that partitivirus-like sequences are also commonly associated with arthropods. One arthropod-associated partitivirus, galbut virus, is common in wild populations of Drosophila melanogaster To begin to understand the processes that underlie this virus's high global prevalence, we established colonies of wild-caught infected flies. Infection remained at stably high levels over 3 years, with between 63 and 100% of individual flies infected. Galbut virus infects fly cells and replicates in tissues throughout infected adults, including reproductive tissues and the gut epithelium. We detected no evidence of horizontal transmission via ingestion, but vertical transmission from either infected females or infected males was ∼100% efficient. Vertical transmission of a related partitivirus, verdadero virus, that we discovered in a laboratory colony of Aedes aegypti mosquitoes was similarly efficient. This suggests that efficient biparental vertical transmission may be a feature of at least a subset of insect-infecting partitiviruses. To study the impact of galbut virus infection free from the confounding effect of other viruses, we generated an inbred line of flies with galbut virus as the only detectable virus infection. We were able to transmit infection experimentally via microinjection of homogenate from these galbut-only flies. This sets the stage for experiments to understand the biological impact and possible utility of partitiviruses infecting model organisms and disease vectors.IMPORTANCE Galbut virus is a recently discovered partitivirus that is extraordinarily common in wild populations of the model organism Drosophila melanogaster Like for most viruses discovered through metagenomics, most of the basic biological questions about this virus remain unanswered. We found that galbut virus, along with a closely related partitivirus found in Aedes aegypti mosquitoes, is transmitted from infected females or males to offspring with ∼100% efficiency and can be maintained in laboratory colonies over years. This efficient transmission mechanism likely underlies the successful spread of these viruses through insect populations. We created Drosophila lines that contained galbut virus as the only virus infection and showed that these flies can be used as a source for experimental infections. This provides insight into how arthropod-infecting partitiviruses may be maintained in nature and sets the stage for exploration of their biology and potential utility.

Diverse Partitiviruses From the Phytopathogenic Fungus, Rosellinia necatrix.

Partitiviruses (dsRNA viruses, family Partitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, Rosellinia necatrix, only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 R. necatrix strains belonging to 15 new species, for which "Rosellinia necatrix partitivirus 11-Rosellinia necatrix partitivirus 25" were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, Alphapartitivirus, and Betapartitivirus. Some partitiviruses were transfected into reference strains of the natural host, R. necatrix, and an experimental host, Cryphonectria parasitica, using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of R. necatrix and C. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed.