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.

Transmission of Oyster Mushroom Spherical Virus to Progeny via Basidiospores and Horizontally to a New Host Pleurotus floridanus.

Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically through sporulation in natural settings. Oyster mushroom spherical virus (OMSV) is a mycovirus that infects Pleurotus ostreatus, with horizontal transmission via hyphal anastomosis. However, whether OMSV can be vertically transmitted is unclear. This study aimed to investigate the transmission characteristics of OMSV to progeny via basidiospores and horizontally to a new host. A total of 37 single-basidiospore offspring were obtained from OMSV-infected P. ostreatus and Pleurotus pulmonarius for Western blot detection of OMSV. The OMSV-carrying rate among monokaryotic isolates was 19% in P. ostreatus and 44% in P. pulmonarius. Then, OMSV-free and OMSV-infected monokaryotic isolates were selected for hybridization with harvested dikaryotic progeny strains. Western blot analyses of the offspring revealed that the OMSV transmission efficiency was 50% in P. ostreatus and 75% in P. pulmonarius, indicating vertical transmission via sexual basidiospores. Furthermore, we observed the horizontal transfer of OMSV from P. pulmonarius to Pleurotus floridanus. OMSV infection in P. floridanus resulted in significant inhibition of mycelial growth and yield loss. This study was novel in reporting the vertical transmission of OMSV through basidiospores, and its infection and pathogenicity in a new host P. floridanus.

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.

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 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.

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.

Plants interfere with non-self recognition of a phytopathogenic fungus via proline accumulation to facilitate mycovirus transmission.

Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission.

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

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

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.

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

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

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.

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.

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

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

Characterization of a novel gammapartitivirus infecting the phytopathogenic fungus Pyricularia oryzae.

In this study, we identified a novel double-strand RNA (dsRNA) mycovirus in Pyricularia oryzae, designated "Magnaporthe oryzae partitivirus 4" (MoPV4). The genome of MoPV4 consists of a dsRNA-1 segment encoding an RNA-dependent RNA polymerase (RdRP) and a dsRNA-2 segment encoding a capsid protein (CP). Phylogenetic analysis indicated that MoPV4 belongs to the genus Gammapartitivirus within family Partitiviridae. The particles of MoPV4 are isometric with a diameter of about 32.4 nm. Three-dimensional structure predictions indicated that the RdRP of MoPV4 forms a classical right-handed conformation, while the CP has a reclining-V shape.

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.

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.

Identification of a negative-strand RNA virus with natural plant and fungal hosts.

The presence of viruses that spread to both plant and fungal populations in nature has posed intriguingly scientific question. We found a negative-strand RNA virus related to members of the family Phenuiviridae, named Valsa mali negative-strand RNA virus 1 (VmNSRV1), which induced strong hypovirulence and was prevalent in a population of the phytopathogenic fungus of apple Valsa canker (Valsa mali) infecting apple orchards in the Shaanxi Province of China. Intriguingly, VmNSRV1 encodes a protein with a viral cell-to-cell movement function in plant tissue. Mechanical leaf inoculation showed that VmNSRV1 could systemically infect plants. Moreover, VmNSRV1 was detected in 24 out of 139 apple trees tested in orchards in Shaanxi Province. Fungal inoculation experiments showed that VmNSRV1 could be bidirectionally transmitted between apple plants and V. mali, and VmNSRV1 infection in plants reduced the development of fungal lesions on leaves. Additionally, the nucleocapsid protein encoded by VmNSRV1 is associated with and rearranged lipid droplets in both fungal and plant cells. VmNSRV1 represents a virus that has adapted and spread to both plant and fungal hosts and shuttles between these two organisms in nature (phyto-mycovirus) and is potential to be utilized for the biocontrol method against plant fungal diseases. This finding presents further insights into the virus evolution and adaptation encompassing both plant and fungal hosts.