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.

Full-length genome characterization of a novel mitovirus isolated from the root rot fungus Armillaria mellea.

Members of the genus Armillaria belong to the group of pathogenic and facultative saprotrophic fungi that are generally known as one of the causative agents of white root rot in infected plants including deciduous and evergreen trees and shrubs. Although several single-stranded RNA mycoviruses were previously described in different Armillaria species, there is no report on mitoviruses (one of the simplest RNA viruses of fungal hosts) known to infect Armillaria taxa. In this study, a new mitovirus denominated "Armillaria mellea mitovirus 1" (AmMV1) was identified in the sporophore samples of Armillaria mellea, commonly known as honey mushroom. AmMV1 has a genome length of 4440 nucleotides and a G + C content of 48%. It encompasses a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp). Comparison through BLASTp analysis revealed that the RdRp domain of AmMV1 shares a sequence identity ranging from 33.43% to 43.27% with RdRp domains of Duamitovirus genus members, having the highest similarity (43.27%) to Rhizoctonia solani mitovirus 94. According to phylogenetic analysis, AmMV1 is classified as a member of the genus Duamitovirus belonging to the Mitoviridae family. This marks the initial instance of a mitovirus identified in Armillaria spp..

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.

Identification and complete genome sequencing of a novel betapartitivirus naturally infecting the mycorrhizal desert truffle Terfezia claveryi.

Viruses that naturally infect fungal species and capable of establishing mycorrhizae are largely unknown. In this study, we identified and characterized a new partitivirus inhabiting the ascomycete, mycorrhizal desert truffle species Terfezia claveryi, and named it "Terfezia claveryi partitivirus 1" (TcPV1). The entire genome of TcPV1, sequenced by both high throughput sequencing of the total dsRNA extracts and by Sanger sequencing of the RLM-RACE PCR products comprised two dsRNA segments of 2404 bp and 2374 bp, respectively. Both dsRNA genome segments harbored a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. The BLASTp search of the RdRp and CP sequences revealed the highest sequence identities (41.92% and 24.13% identity, respectively) to those of Bipolaris maydis partitivirus 2 and Plasmopara viticola lesion associated partitivirus 5. Molecular phylogenetic analyses of the RdRp sequence showed that TcPV1 fall within a clade composed entirely of members of the genus Betapartitivirus, belonging to the family Partitiviridae. In light of this molecular evidence, TcPV1 is a new member of the genus Betapartitivirus. This is the first report of a new partitivirus hosted by the ascomycete, mycorrhizal fungus T. claveryi.

Molecular characterization of the complete genome of a novel partitivirus hosted by the saprobic mushroom Leucocybe candicans.

Virus communities of uncultivated fungi stay largely unknown. In the current study, we characterized a new partitivirus species detected in the basidiomycetous, saprobic mushroom Leucocybe candicans, named "Leucocybe candicans partitivirus 1" (LcPV1). The full-length genome of LcPV1, determined using deep sequencing and RLM-RACE approaches, consists of two dsRNA segments with each having the same size of 1984 bp. Both dsRNA genome segments comprise a single open reading frame (ORF), encoding an RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP show the highest identity (50.09% and 35.71% similarity, respectively) to those of partitiviruses reported from an oomycetous, plant pathogenic, stramenopile algae Plasmopara viticola and basidiomycetous, plant pathogenic fungus Ceratobasidium sp., respectively. Phylogenetic analyses performed based on the RdRp and CP sequences revealed that LcPV1 falls within a cluster that includes different alphapartitivirus species from the family Partitiviridae. In this study, we propose that LcPV1 is a new member of a species belonging to the genus Alphapartitivirus. To our knowledge, this is the first study reporting on a new fungal virus (mycovirus) identified in the basidiomycetous, saprobic mushroom Leucocybe candicans.

Molecular characterization of a new mitovirus hosted by the ectomycorrhizal fungus Albatrellopsis flettii.

The complete genome of a novel mycovirus, Albatrellopsis flettii mitovirus 1 (AfMV1), hosted by the basidiomycetous ectomycorrhizal fungus Albatrellopsis flettii (Morse ex Pouzar) Audet, was sequenced and analyzed. The full-length cDNA sequence, obtained from a dsRNA replication intermediate of the AfMV1 genome, is 3037 bp in length with a predicted G+C content of 40.66%. Sequence analysis revealed that a single large open reading frame (ORF) is present on the positive strand when the mold mitochondrial genetic code is applied. The single ORF encodes a putative RNA-dependent RNA polymerase of 859 amino acids with a predicted molecular weight of 97.05 kDa that shares the closest similarity with the corresponding protein of Entomophthora muscae mitovirus 7, with 43.38% sequence identity. Phylogenetic analysis showed that AfMV1 could be classified as a new member of the genus Mitovirus within the family Mitoviridae. This is the first report of the complete genome sequence of a new mitovirus, AfMV1, isolated from the basidiomycetous ectomycorrhizal fungus A. flettii.

Molecular characterization of a novel partitivirus hosted by the false morel mushroom Gyromitra esculenta.

Virus populations of uncultivated fungi remain scarcely studied. In the present study, we characterized a new partitivirus isolated from the false morel mushroom Gyromitra esculenta, named "Gyromitra esculenta partitivirus 1" (GePV1). The complete genome of GePV1, whose sequence was determined by combining high-throughput sequencing and RLM-RACE approaches, comprises two dsRNA segments of 1971 bp and 1799 bp, respectively. Each dsRNA genome segment contains a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. The sequences of the RdRp and CP exhibited the highest similarity (69.77% and 47.00% identity, respectively) to those of Rosellinia necatrix partitivirus 2 (RnPV2). Phylogenetic analysis based on the CP and RdRp sequences demonstrated that GePV1 clusters within a clade that includes members of the genus Alphapartitivirus, family Partitiviridae. We propose that GePV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified in the false morel mushroom Gyromitra esculenta.

Full-length genome characterization of a novel alphapartitivirus detected in the ectomycorrhizal fungus Hygrophorus penarioides.

Virus populations of ectomycorrhizal fungi remain poorly studied. In the present study, we characterized a new partitivirus isolated from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides, named "Hygrophorus penarioides partitivirus 1" (HpPV1). The whole genome of HpPV1, determined by merging deep sequencing and RLM-RACE approaches, comprised two dsRNA segments of 2053 bp and 2072 bp, respectively. Both dsRNA genome segments included a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP exhibits the highest similarity (67.49% and 75.61% identity, respectively) to those of partitiviruses identified from an ascomycetous ectomycorrhizal fungus Sarcosphaera coronaria. Phylogenetic analyses performed based on the CP and RdRp sequences demonstrated that HpPV1 clusters within a clade that includes members of the genus Alphapartitivirus, belonging to the family Partitiviridae. Here, we propose that HpPV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides.

Full-length genome characterization of a new fusagravirus hosted by the spring orange peel fungus Caloscypha fulgens.

Mycoviruses widely exist in diverse lineages of fungi, yet there are only a few studies on mycovirus infection in uncultivated fungi. We here report the presence of a dsRNA mycovirus in saprotrophic spring orange peel fungus Caloscypha fulgens. A novel dsRNA virus, named "Caloscypha fulgens fusagravirus 1" (CfFV1), was isolated from a single ascocarp of C. fulgens, and its molecular features were revealed. The full-length cDNA of CfFV1 comprises 9,548 nucleotides with a calculated GC content of 47.9% and two discontinuous open reading frames (ORF 1 and 2). A-1 ribosomal frameshift region with two distinctive elements, including a canonical slippery heptanucleotide (AAAAAAC) and a pseudoknot structure, predicted as a Recoding Stimulatory Element, was detected in the junction region of ORF1 and ORF2. The deduced amino acid sequence of ORF1 and ORF2 showed the highest similarity to the putative structural protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 4 (RnFGV4). Genome organization, sequence similarity, and phylogenetic analysis indicate that this virus belongs to a new member of the proposed family Fusagraviridae. This is the first report of the presence of a mycovirus in the spring orange peel fungus C. fulgens. Keywords: mycovirus; dsRNA; proposed Fusagraviridae; uncultivated fungi; Caloscypha fulgens.

Complete genome sequence of a novel mitovirus from the ectomycorrhizal fungus Geopora sumneriana.

A double-stranded RNA (dsRNA) segment was extracted from the ectomycorrhizal fungus Geopora sumneriana (Cooke) M. Torre, and its full-length cDNA sequence, comprising 3146 nucleotides, was determined. Sequence analysis revealed the presence of a large open reading frame (ORF) on the positive strand of this dsRNA segment when the mold mitochondrial genetic code was applied. The ORF encodes a putative RNA-dependent RNA polymerase (RdRp), which shares the highest degree of similarity with Tuber excavatum mitovirus, with 37.52% identity. This dsRNA segment represents the genome replication intermediate of a novel mitovirus that was tentatively designated as "Geopora sumneriana mitovirus 1" (GsMV1). Phylogenetic analysis further suggested that GsMV1 is a member of the family Narnaviridae. This is the first study reporting on a mitovirus genome sequence in the ectomycorrhizal fungus G. sumneriana.

Determination of anti-oxidative, anti-microbial activity and heavy metal contents of Leucoagaricus leucothites.

Mushrooms, a treasure of diverse bioactive scaffolds, have been widely admired due to their nutritional and medicinal significance all over the world. The current study intended to evaluate the therapeutic potentiality of an edible mushroom, Leucoagaricus leucothites (Vittad.) Wasser. Thus, anti-oxidant potential of L. leucothites was determined using DPPH assay and for the determination of anti-microbial potential agar dilution procedure was followed. TOS (total oxidant status), TAS (total anti-oxidant status), and OSI (oxidative stress index) values were evaluated utilizing Rel Assay Kits. For the assessment of heavy metal contents, wet decomposition approach with atomic absorption spectrophotometry was adopted. Screening of phytochemicals present in ethanolic extract of L. leucothites were determined by HPLC. TAS, TOS and OSI values were found to be 8.291mmol/L, 10.797µmol/L and 0.130 respectively. Our results declared that heavy metal contents are generally in the safe range. Phytochemical analysis of L. leucothites has affirmed the presence of important phenolics such as gallic acid, catechin, and hesperidin. Investigations on anti-oxidant and anti-microbial potential of L. leucothites has uncovered the fact that this naturally occurring, biologically active, and therapeutically effective mushroom specie has natural borne anti-oxidant and anti-microbial potential and it would be worthwhile to use it for nutritional as well as medicinal purpose.

A Case Study: What Doses of Amanita phalloides and Amatoxins Are Lethal to Humans?

There are few data estimating the human lethal dose of amatoxins or of the toxin level present in ingested raw poisonous mushrooms. Here, we present a patient who intentionally ingested several wild collected mushrooms to assess whether they were poisonous. Nearly 1 day after ingestion, during which the patient had nausea and vomiting, he presented at the emergency department. His transaminase levels started to increase starting from hour 48 and peaking at hour 72 (alanine aminotransferase 2496 IU/L; aspartate aminotransferase 1777 IU/L). A toxin analysis was carried out on the mushrooms that the patient said he had ingested. With reversed-phase high-performance liquid chromatography analysis, an uptake of approximately 21.3 mg amatoxin from nearly 50 g mushroom was calculated; it consisted of 11.9 mg alpha amanitin, 8.4 mg beta amanitin, and 1 mg gamma amanitin. In the urine sample taken on day 4, 2.7 ng/mL alpha amanitin and 1.25 ng/mL beta amanitin were found, and there was no gamma amanitin. Our findings suggest that the patient ingested approximately 0.32 mg/kg amatoxin, and fortunately recovered after serious hepatotoxicity developed.

A Case Study: Rare Lepiota brunneoincarnata Poisoning.

Amatoxin poisoning from the genus Lepiota may have a deadly outcome, although this is not seen as often as it is from the genus Amanita. In this report, we present a patient who was poisoned by a sublethal dose of Lepiota brunneoincarnata mushrooms. The patient was hospitalized 12 hours after eating the mushrooms. The patient's transaminase levels increased dramatically starting on day 4. Aspartate transaminase peaked at 78 hours. Starting at 1265 IU/L, alanine transaminase peaked at 90 hours at 5124 IU/L. The patient was discharged on day 8 to outpatient care, and his transaminase levels returned to normal ranges in the subsequent days. A toxin analysis was carried out on the mushrooms that the patient claimed to have eaten. Using reversed-phase high-performance liquid chromatography analysis, an uptake of approximately 19.9 mg of amatoxin from nearly 30 g of mushrooms was calculated. This consisted of 10.59 mg of α-amanitin, 9.18 mg of ß-amanitin, and 0.16 mg of γ-amanitin. In conclusion, we present a patient from Turkey who was poisoned by L. brunneoincarnata mushrooms.

Lepiota castanea mushroom growing in Turkiye does not contain phallotoxins and amatoxins.

The number of poisoning cases caused by the Lepiota genus is globally increasing. This genus has more poisonous species than the Amanita genus, and many Lepiota species can cause severe toxicity and death if ingested. As recognized in the literature, L. castanea is a toxic species containing amatoxin. Although crude analytical methods have shown that L. castanea contains amatoxins, more recent and sensitive analyses suggest otherwise. Toxin concentrations can vary even among the same fungal species due to geographical and climatic differences. Therefore, this confusion can be resolved by analyzing L. castanea toxins from different geographical regions. This study aimed to demonstrate the toxin levels of L. castanea collected from forests in different regions of Turkiye (Istanbul and Kocaeli) using sensitive methods. The collected mushrooms were analyzed for alpha amanitin, beta amanitin, gamma amanitin, amanin, phallacidin, and phalloidin levels using RP-HPLC-UV and LC-ESI-MS/MS methods. L. castanea mushroom was found to be free of amatoxin and phallotoxin. Our study revealed for the first time that L. castanea mushrooms from different geographical regions of Turkiye do not contain amatoxin and phallotoxin. Supporting these findings with new studies from different parts of the world would be appropriate.

Fatty acid compositions of six wild edible mushroom species.

The fatty acids of six wild edible mushroom species (Boletus reticulatus, Flammulina velutipes var. velutipes, Lactarius salmonicolor, Pleurotus ostreatus, Polyporus squamosus, and Russula anthracina) collected from different regions from Anatolia were determined. The fatty acids were identified and quantified by gas chromatography and studied using fruit bodies. Fatty acid composition varied among species. The dominant fatty acid in fruit bodies of all mushrooms was cis-linoleic acid (18 : 2). Percentage of cis-linoleic acid in species varied from 22.39% to 65.29%. The other major fatty acids were, respectively, cis-oleic, palmitic, and stearic acids. Fatty acids analysis of the mushrooms showed that the unsaturated fatty acids were at higher concentrations than saturated fatty acids.

Diverse partitiviruses hosted by the ectomycorrhizal agaric Hebeloma mesophaeum and the natural transmission of a partitivirus between phylogenetically distant, sympatric fungi.

Mycorrhizal fungi host diverse mycoviruses that contribute to our understanding of their diversity and evolution. Here we report on the identification and complete genome characterization of three novel partitiviruses naturally infecting the ectomycorrhizal fungus Hebeloma mesophaeum. During NGS derived viral sequence analyses, we identified a partitivirus that is conspecific with the previously reported partitivirus (LcPV1) described from a saprotrophic fungus Leucocybe candicans. The two distinct fungal specimens inhabited the same vicinity of a campus garden. RdRp sequences encoded by the LcPV1 isolates from both host fungi was found to be identical. Bio-tracking studies revealed that viral loads of LcPV1 drop significantly in L. candicans but not in H. mesophaeum within four years period. The physical proximity of the mycelial networks of both fungal specimens implied the occurrence of a virus transmission event with unknown mechanism. Nature of this virus transmission was discussed in relation to transient interspecific mycelial contact hypothesis.

Anatoluin A and B isolated from medicinal Tricholoma anatolicum are new cytotoxic ergostanoids against the most common cancers.

Tricholoma anatolicum is an edible mushroom from the matsutake group growing under Cedar trees. Bioactivity-guided fractionation of Tricholoma anatolicum afforded two new (1 and 2), three known ergosterols (3-6), and four known (6-9) compounds. Structures were identified as anatoluin A (1), anatoluin B (2), 5α,6α-epoxy-ergosta-7,22-dien,3ß-ol (3), ergosterol-endoperoxide (4), ergosterol,3ß-ol (5), 3,5-dihydroxyfuran-2(5H)-one (6), mannitol (7), turanose (8), fumaric acid (9) using spectroscopic techniques. The cytotoxic activity of extract and isolated compounds was performed using MTT assay against MCF7, HT29, H1299, and HeLa cancerous cell lines while toxicity against PDF and L929 fibroblast healthy cell lines. The lipid peroxidation inhibitory and ABTS•+ scavenging activities were used to determine antioxidant activity. The polar extracts exhibited significant cytotoxic activity. The more perfect is that the extracts and isolated compounds (1-5) were inactive against PDF and L929 healthy cell lines. Compounds 1-3 and 4 exhibited noticeable cytotoxic activity, while 1-5 moderately inhibited lipid peroxidation.

Wild mushrooms from Ilgaz Mountain National Park (Western Black Sea, Turkey): element concentrations and their health risk assessment.

The purpose of this study was to determine Fe, Cd, Cr, Se, P, Cu, Mn, Zn, Al, Ca, Mg, and K contents of some edible (Chlorophyllum rhacodes, Clavariadelphus truncatus, Clitocybe nebularis, Hydnum repandum, Hygrophorus pudorinus, Infundibulicybe gibba, Lactarius deliciosus, L. piperatus, L. salmonicolor, Macrolepiota mastoidea, Russula grata, Suillus granulatus, and Tricholoma imbricatum), inedible (Amanita pantherina, Geastrum triplex, Gloeophyllum sepiarium, Hypholoma fasciculare, Phellinus vorax, Pholiota limonella, Russula anthracina, and Tapinella atrotomentosa), and poisonous mushroom species (Amanita pantherina and Hypholoma fasciculare) collected from Ilgaz Mountain National Park (Western Black Sea, Turkey). The element contents of the mushrooms were determined to be 18.0-1239.1, 0.2-4.6, 0.1-3.4, 0.2-3.2, 1.0-8.9, 3.3-59.9, 3.7-220.4, 21.3-154.1, 6.4-754.3, 15.8-17,473.0, 413.0-5943.0, and 2803.0-24,490.0 mg·kg-1, respectively. In addition to metal contents, the daily intakes of metal (DIM) and Health Risk Index (HRI) values of edible mushrooms were also calculated. Both DIM and HRI values of mushroom species except L. salmanicolor, M. mastoidea, and R. grata were within the legal limits. However, it was determined that the Fe content of L. salmanicolor and M. mastoidea and Cd content of R. grata were above the legal limits.

Total Antioxidant, Antimicrobial, Antiproliferative Potentials and Element Contents of Wild Mushroom Candolleomyces candolleanus (Agaricomycetes) from Turkey.

Mushrooms are among the natural products that are widely used in the treatment of diseases. In our study, the biological activities of Candolleomyces candolleanus (Fr.) D. Wacht. & A. Melzer were determined. Within the scope of the study, ethanol and methanol extracts of the basidiomata of the mushroom were obtained. Total antioxidant, total oxidant, and oxidative stress values of the mushroom were determined using Rel Assay kits. Its antimicrobial activity was tested against standard bacterial and fungal strains by agar dilution method. Antiproliferative activity was measured against A549 lung cancer cell line by MTT assay. Element contents were determined using atomic absorption spectrometry. According to the findings, the TAS value of the mushroom was determined as 5.547 ± 0.173, the TOS value as 8.572 ± 0.217 and the OSI value as 0.155 ± 0.001. It was determined that the mushroom extract was effective at 25-400 µg/mL concentrations. Element contents were determined to be at normal levels. The antiproliferative activity of the extracts showed strong effects depending on the concentration increase. As a result, it was determined that the biological activities of the extracts of C. candolleanus were high.

Novel and diverse mycoviruses co-inhabiting the hypogeous ectomycorrhizal fungus Picoa juniperi.

Viruses hosted by ectomycorrhizal fungi remain poorly studied. In this study, we detected eight new fungal viruses co-infecting a single isolate of the hypogeous ectomycorrhizal fungus Picoa juniperi using high-throughput sequencing. Phylogenetic analysis of one identified virus abbreviated as PjMTV1 revealed its closest relatives as members of the newly proposed family "Megatotiviridae". Phylogenetic analyses of two identified viruses abbreviated as PjV1 and PjV2 showed that these viruses are associated with members of the proposed family "Fusagraviridae". Phylogenetic analysis of the identified one another virus abbreviated as PjYV1 demonstrated that this virus is related to the members of the proposed family Yadokariviridae. The remaining four identified virus-like contigs were determined as segments of the bipartite dsRNA mycoviruses from the family Partitiviridae. The mycoviruses reported in this study are the first viruses described in Picoa juniperi, and PjMTV1 characterized herein is the secondly reported member of the newly proposed family "Megatotiviridae".