Snake River alfalfa virus, a persistent virus infecting alfalfa (Medicago sativa L.) in Washington State, USA.

Here we report an occurrence of Snake River alfalfa virus (SRAV) in Washington state, USA. SRAV was recently identified in alfalfa (Medicago sativa L.) plants and western flower thrips in south-central Idaho and proposed to be a first flavi-like virus identified in a plant host. We argue that the SRAV, based on its prevalence in alfalfa plants, readily detectable dsRNA, genome structure, presence in alfalfa seeds, and seed-mediated transmission is a persistent new virus distantly resembling members of the family Endornaviridae.

Discovery of Cucumis melo endornavirus by deep sequencing of human stool samples in Brazil.

The nearly complete genome sequences of two Cucumis melo endornavirus (CmEV) strains were obtained using deep sequencing while investigating fecal samples for the presence of gastroenteritis viruses. The Brazilian CmEV BRA/TO-23 (aa positions 116-5027) and BRA/TO-74 (aa positions 26-5057) strains were nearly identical to the reference CmEV CL-01 (USA) and SJ1 (South Korea) strains, showing 97% and 98% of nucleotide and amino acid identity, respectively. Endornaviruses are not known to be associated with human disease and their presence may simply reflect recent dietary consumption. Metagenomic analyses offered an opportunity to identify for the first time in Brazil a newly described endornavirus species.

Endornaviruses: persistent dsRNA viruses with symbiotic properties in diverse eukaryotes.

Endornaviruses are unique, persistent, double-stranded RNA (dsRNA) viruses with symbiotic properties that infect diverse eukaryotes, such as plants, fungi, and oomycetes. Endornaviruses contain a linear dsRNA genome of approximately 10 to 17 kbp in length and are classified in the family Endornaviridae, which consists of two genera, Alphaendornavirus and Betaendornavirus. The endornaviruses encode a single long open reading frame encoding approximately 3200 to 5800 amino acid residues of conserved viral RNA helicase and RNA-dependent RNA polymerase domains, and some endornaviruses contain a site-specific nick in the coding strand of their dsRNA genome. Acute plant viruses propagate rapidly and systemically, eventually killing the host plant, and are then transmitted horizontally. In contrast, plant endornaviruses have several common persistent (symbiotic) properties: a stable low copy number in the host plant, no obvious effect on the host plant, and efficient vertical transmission via gametes. Plant endornaviruses are likely maintained within host plants for hundreds of generations, so the host must stringently regulate their propagation. Whereas RNA silencing functions as a defense system against acute viruses in plants, it may be necessary for the persistent infection (symbiotic life cycle) of endornaviruses. This process includes the stringent regulation of low virus copy number (steady replication before every host cell division) and efficient vertical transmission of the virus to the next generation.

Four closely related endornaviruses each with a low incidence in the phytopathogenic fungi Exserohilum turcicum or Bipolaris maydis.

Endornaviruses are known to occur widely in plants, fungi, and oomycetes, but our understanding of their diversity and distribution is limited. In this study, we report the discovery of four endornaviruses tentatively named Setosphaeria turcica endornavirus 1 (StEV1), Setosphaeria turcica endornavirus 2 (StEV2), Bipolaris maydis endornavirus 1 (BmEV1), and Bipolaris maydis endornavirus 2 (BmEV2). StEV1 and StEV2 infect Exserohilum turcicum, while BmEV1 and BmEV2 infect Bipolaris maydis. The four viruses encode a polyprotein with less than 40 % amino acid sequence identity to other known endornaviruses, indicating that they are novel, previously undescribed endornaviruses. However, StEV1 and BmEV1 share a sequence identity of 78 % at the full-genome level and 87 % at the polyprotein level, suggesting that they may belong to the same species. Our study also found that each of the four endornaviruses has an incidence of approximately 3.5 % to 5.5 % in E. turcicum or B. maydis. Interestingly, BmEV1 and BmEV2 were found to be unable to transmit between hosts of different vegetative incompatibility groups, which may explain their low incidence.

Genetic diversity and phylogenetic characteristics of viruses in lily plants in Beijing.

Lily (Lilium) is an important bulbous perennial herb that is frequently infected by one or more viruses. To investigate the diversity of lily viruses, lilies with virus-like symptoms in Beijing were collected to perform small RNA deep sequencing. Then, the 12 complete and six nearly full-length viral genomes, including six known viruses and two novel viruses were determined. Based on sequence and phylogenetic analyses, two novel viruses were considered to be members of the genera Alphaendornavirus (Endornaviridae) and Polerovirus (Solemoviridae). These two novel viruses were provisionally named lily-associated alphaendornavirus 1 (LaEV-1) and lily-associated polerovirus 1 (LaPV-1). Based on sequence, phylogenetic and recombination analyses, strawberry latent ringspot virus (SLRSV) in the genus Stralarivirus (Secoviridae) was identified for the first time in China, and shown to exhibit the highest nucleotide (nt) diversity among the available full-length SLRSV genome sequences, with the highest identities of 79.5% for RNA1 and 80.9% for RNA2. Interestingly, the protease cofactor region in RNA1 was 752 aa in length, whereas those of the other 27 characterized isolates ranged from 700-719 aa in length. The genome sequences of lily virus A (Potyvirus), lily virus X (Potexvirus), and plantago asiatica mosaic virus (Potexvirus) exhibited varying degrees of sequence diversity at the nucleotide level compared with their corresponding characterized isolates. In addition, plantago asiatica mosaic virus (PlAMV) tended to cluster on a host species-basis. One identified lily mottle virus (Potyvirus) isolate was detected as a recombinant, and which clustered in a different group with four other isolates. Seven identified lily symptomless virus (Carlavirus) isolates, including one recombinant, were clustered into three clades. Our results revealed the genetic diversity of lily-infecting viruses, and sequence insertion, host species and recombination are factors that likely contribute to this diversity. Collectively, our results provide useful information regarding the control of viral disease in lily.

Grapevine Endophyte Endornavirus and Two New Endornaviruses Found Associated with Grapevines (Vitis vinifera L.) in Idaho, USA.

Five virus genomes, ranging between 12.0 and 12.3 kb in length and identified as endornaviruses, were discovered through a high-throughput sequencing (HTS) analysis of the total RNA samples extracted from two wine grape cultivars collected in the State of Idaho. One was found in a declining Chardonnay vine and was determined to be a local isolate of grapevine endophyte endornavirus (GEEV), and four others represented two novel endornaviruses named grapevine endornavirus 1 (GEV1) and grapevine endornavirus 2 (GEV2). All three virus genomes span a large, single open reading frame encoding polyproteins with easily identifiable helicase (HEL) and RNA-dependent RNA polymerase (RdRP) domains, while the GEV2 polyprotein also contains a glycosyltransferase domain. The GEV1 genome found in an asymptomatic Cabernet franc vine was related to, but distinct from, GEEV: the 5'-proximal, 4.7 kb segment of the GEV1 genome had a 72% identical nucleotide sequence to that of GEEV, while the rest of the genome displayed no significant similarity to the GEEV nucleotide sequence. Nevertheless, the amino acid sequence of the RdRP domain of GEV1 exhibited the closest affinity to the RdRP of GEEV. GEV2 was found in declining Chardonnay and asymptomatic Cabernet franc vines as three genetic variants exhibiting a 91.9-99.8% nucleotide sequence identity among each other; its RdRP had the closest affinity to the Shahe endorna-like virus 1 found in termites. In phylogenetic analyses, the RdRP and HEL domains of the GEV1 and GEV2 polyproteins were placed in two separate clades inside the large lineage of alphaendornaviruses, showing an affinity to GEEV and Phaseolus vulgaris endornavirus 1, respectively.

Successful full-length genomic cloning and characterization of site-specific nick structures of Phytophthora endornaviruses 2 and 3 in yeast, Saccharomyces cerevisiae.

Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5' and 3' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3. We have successfully constructed full-length cDNA clones of the entire RNA genomes of PEV2 and PEV3 using a homologous recombination system in the yeast, Saccharomyces cerevisiae. These full-length cDNA sequences were ligated downstream of a constitutive expression promoter (TDH3) or a galactose-inducing promoter (GAL1) in the shuttle vector to enable the production of the full-length RNA transcripts of PEV2 and PEV3 in yeast cells. Interestingly, a 1.0 kb RNA fragment from the PEV3 positive-strand transcript was also detected with a 5'-region RNA probe, indicating that site-specific cleavage also occurred in yeast cells. Further, when PEV2 or PEV3 mRNA was overexpressed under the GAL1 promoter, yeast cell growth was suppressed. A fusion protein combining EGFP to the N-terminus of the full-length PEV2 ORF or C-terminus of the full-length PEV3 ORF was expressed, and allowed PEV2 and PEV3 ORFs to be successfully visualized in yeast cells. Expression of the fusion protein also revealed presence of heterogeneous bodies in the cells.

Genome Characterization and Phylogenetic Analysis of a Novel Endornavirus That Infects Fungal Pathogen Sclerotinia sclerotiorum.

Endornaviruses are capsidless linear (+) ssRNA viruses in the family Endornaviridae. In this study, Scelrotinia sclerotiorum endornavirus 11 (SsEV11), a novel endornavirus infecting hypovirulent Sclerotinia sclerotiorum strain XY79, was identified and cloned using virome sequencing analysis and rapid amplification of cDNA ends (RACE) techniques. The full-length genome of SsEV11 is 11906 nt in length with a large ORF, which encodes a large polyprotein of 3928 amino acid residues, containing a viral methyltransferase domain, a cysteine-rich region, a putative DEADc, a viral helicase domain, and an RNA-dependent RNA polymerase (RdRp) 2 domain. The 5' and 3' untranslated regions (UTR) are 31 nt and 90 nt, respectively. According to the BLAST result of the nucleotide sequence, SsEV11 shows the highest identity (45%) with Sclerotinia minor endornavirus 1 (SmEV1). Phylogenetic analysis based on amino acid sequence of RdRp demonstrated that SsEV11 clusters to endornavirus and has a close relationship with Betaendornavirus. Phylogenetic analysis based on the sequence of endornaviral RdRp domain indicated that there were three large clusters in the phylogenetic tree. Combining the results of alignment analysis, Cluster I at least has five subclusters including typical members of Alphaendornavirus and many unclassified endornaviruses that isolated from fungi, oomycetes, algae, and insects; Cluster II also has five subclusters including typical members of Betaendornavirus, SsEV11, and other unclassified viruses that infected fungi; Cluster III includes many endorna-like viruses that infect nematodes, mites, and insects. Viruses in Cluster I and Cluster II are close to each other and relatively distant to those in Cluster III. Our study characterized a novel betaendornavirus, SsEV11, infected fungal pathogen S. sclerotiorum, and suggested that notable phylogenetic diverse exists in endornaviruses. In addition, at least, one novel genus, Gammaendornavirus, should be established to accommodate those endorna-like viruses in Cluster III.

Small RNA Analyses of a Ceratobasidium Isolate Infected with Three Endornaviruses.

Isolates of three endornavirus species were identified co-infecting an unidentified species of Ceratobasidium, itself identified as a symbiont from within the roots of a wild plant of the terrestrial orchid Pterostylis vittata in Western Australia. Isogenic lines of the fungal isolate lacking all three mycoviruses were derived from the virus-infected isolate. To observe how presence of endornaviruses influenced gene expression in the fungal host, we sequenced fungus-derived small RNA species from the virus-infected and virus-free isogenic lines and compared them. The presence of mycoviruses influenced expression of small RNAs. Of the 3272 fungus-derived small RNA species identified, the expression of 9.1% (300 of 3272) of them were up-regulated, and 0.6% (18 of 3272) were down-regulated in the presence of the viruses. Fourteen novel micro-RNA-like RNAs (Cer-milRNAs) were predicted. Gene target prediction of the differentially expressed Cer-milRNAs was quite ambiguous; however, fungal genes involved in transcriptional regulation, catalysis, molecular binding, and metabolic activities such as gene expression, DNA metabolic processes and regulation activities were differentially expressed in the presence of the mycoviruses.

Persistent, and Asymptomatic Viral Infections and Whitefly-Transmitted Viruses Impacting Cantaloupe and Watermelon in Georgia, USA.

Cucurbits in Southeastern USA have experienced a drastic decline in production over the years due to the effect of economically important viruses, mainly those transmitted by the sweet potato whitefly (Bemisia tabaci Gennadius). In cucurbits, these viruses can be found as a single or mixed infection, thereby causing significant yield loss. During the spring of 2021, surveys were conducted to evaluate the incidence and distribution of viruses infecting cantaloupe (n = 80) and watermelon (n = 245) in Georgia. Symptomatic foliar tissues were collected from six counties and sRNA libraries were constructed from seven symptomatic samples. High throughput sequencing (HTS) analysis revealed the presence of three different new RNA viruses in Georgia: cucumis melo endornavirus (CmEV), cucumis melo amalgavirus (CmAV1), and cucumis melo cryptic virus (CmCV). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of CmEV and CmAV1 in 25% and 43% of the total samples tested, respectively. CmCV was not detected using RT-PCR. Watermelon crinkle leaf-associated virus 1 (WCLaV-1), recently reported in GA, was detected in 28% of the samples tested. Furthermore, RT-PCR and PCR analysis of 43 symptomatic leaf tissues collected from the fall-grown watermelon in 2019 revealed the presence of cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and cucurbit leaf crumple virus (CuLCrV) at 73%, 2%, and 81%, respectively. This finding broadens our knowledge of the prevalence of viruses in melons in the fall and spring, as well as the geographical expansion of the WCLaV-1 in GA, USA.

Molecular characterization of a new endornavirus inhabiting the ectomycorrhizal fungus Hygrophorus penarioides.

Viruses hosted by uncultivated fungi have been poorly studied. We carried out studies to characterize a large dsRNA segment (~20 kbp) detected in the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides. The dsRNA was gel-purified and its randomly amplified cDNA fragments were used for high throughput sequencing (HTS). Reads were de novo assembled and BLASTx analysis revealed sequence similarity to viruses of the family Endornaviridae. The 5' and 3' terminal sequences of the dsRNA segment were determined by performing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The full-length cDNA sequence of the putative endornavirus comprises 16,785 nt and contains a single, long open reading frame which encodes for a polyprotein of 5522 aa with conserved domains for cysteine-rich region, helicase, glycosyltransferase, and RNA-dependent RNA polymerase. The virus was named Hygrophorus penarioides endornavirus 1 (HpEnV1). A BLASTp search performed using the polyprotein sequence revealed that the most closely related, fully sequenced endornavirus to HpEnV1 is Ceratobasidium endornavirus B.

Genome structure and diversity of novel endornaviruses from wheat sharp eyespot pathogen Rhizoctonia cerealis.

Rhizoctonia cerealis (teleomorph Ceratobasidium cereale) is a soil-borne plant pathogenic fungus that can cause sharp eyespot in wheat or yellow patch in grasses. In this study, 21 new endornavirus genomes were obtained from five R. cerealis strains through the high-throughput sequencing of viral double-stranded RNA. Eighteen viruses were identified as Alphaendornavirus, and three viruses were identified as new species of Betaendornavirus on the basis of the phylogenetic analysis of the deduced amino acid sequences of RNA-dependent RNA polymerase. Notably, 12 of the new alphaendornaviruses could encode two open reading frames (ORFs), which were a rare feature of Endornaviridae. The amino acid sequences encoded by ORF2 from different endornaviruses had very low identity, and their functions and evolution origins remained unclear. Different endornavirus species with remarkably different genome structures could be found in the same R. cerealis strain. This study indicated that endornaviruses are common in R. cerealis and display wide diversity. Betaendornaviruses were found in R. cerealis, and a new species was proposed. This study is the first to report that the endornaviruses from R. cerealis can encode two ORFs and enhances our understanding of the viruses in the Endornaviridae family.

Two Novel Endornaviruses Co-infecting a Phytophthora Pathogen of Asparagus officinalis Modulate the Developmental Stages and Fungicide Sensitivities of the Host Oomycete.

Two novel endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3) were found in isolates of a Phytophthora pathogen of asparagus collected in Japan. A molecular phylogenetic analysis indicated that PEV2 and PEV3 belong to the genus Alphaendornavirus. The PEV2 and PEV3 genomes consist of 14,345 and 13,810 bp, and they contain single open reading frames of 4,640 and 4,603 codons, respectively. Their polyproteins contain the conserved domains of an RNA helicase, a UDP-glycosyltransferase, and an RNA-dependent RNA polymerase, which are conserved in other alphaendornaviruses. PEV2 is closely related to Brown algae endornavirus 2, whereas PEV3 is closely related to Phytophthora endornavirus 1 (PEV1), which infects a Phytophthora sp. specific to Douglas fir. PEV2 and PEV3 were detected at high titers in two original Phytophthora sp. isolates, and we found a sub-isolate with low titers of the viruses during subculture. We used the high- and low-titer isolates to evaluate the effects of the viruses on the growth, development, and fungicide sensitivities of the Phytophthora sp. host. The high-titer isolates produced smaller mycelial colonies and much higher numbers of zoosporangia than the low-titer isolate. These results suggest that PEV2 and PEV3 inhibited hyphal growth and stimulated zoosporangium formation. The high-titer isolates were more sensitive than the low-titer isolate to the fungicides benthiavalicarb-isopropyl, famoxadone, and chlorothalonil. In contrast, the high-titer isolates displayed lower sensitivity to the fungicide metalaxyl (an inhibitor of RNA polymerase I) when compared with the low-titer isolate. These results indicate that persistent infection with PEV2 and PEV3 may potentially affect the fungicide sensitivities of the host oomycete.

Identification of a novel endornavirus in Geranium carolinianum and occurrence within three agroecosystems.

A putative endornavirus was detected in Carolina geranium (Geranium carolinianum) in Louisiana, USA. The virus was provisionally named Geranium carolinianum endornavirus 1 (GcEV1). The viral RNA was sequenced, and it consisted of 14,625 nt containing a single ORF coding a putative polyprotein of 4815 aa with conserved domains for a helicase 1, peptidase C97, glycosyl transferase GTB-type, and RNA-dependent RNA polymerase 2. The 5'end consisted of 130 nt while the 3'end consisted of 54 nt ending in nine cytosine residues. The closest relative to GcEV1 was Phaseolus vulgaris endornavirus 3. In phylogenetic analyses, GcEV1 clustered with members of the genus Alphaendornavirus. GcEV1 was detected in 57 of 60 G. carolinianum plants collected from three distinct agroecosystems. The virus was not detected in eight other species of the genus Geranium. There was no association of a particular phenotypic trait of the host with the presence or absence of the virus. GcEV1 was transmitted at a rate of 100% in seeds of a self-pollinated G. carolinianum plant.

Challenges to elucidating how endornaviruses influence fungal hosts: Creating mycovirus-free isogenic fungal lines and testing them.

Determining roles of mycoviruses in fungal biology is complicated, especially when fungi are co-infected with multiple viruses. Genetically identical (isogenic) fungal lines that are infected by and not infected by viruses must be created and compared. Here, we study an isolate of Ceratobasidium sp., a fungus isolated from pelotons in roots of a wild terrestrial orchid. The fungal isolate was co-infected with three distinct endornaviruses, isolates of Ceratobasidium endonarvirus B (CbEVB), Ceratobasidium endonarvirus C (CbEVC) and Ceratobasidium endonarvirus D (CbEVD). An experiment to reveal natural distribution of the three mycoviruses within a fungal colony revealed no sectoring; they were all evenly distributed throughout the colony. Hyphal tipping and treatments with one of five antibiotics (kanamycin, streptomycin, cycloheximide, rifampicin and ampicillin) were applied in attempts to 'cure' fungal lines of one, two or three of the viruses present. Surprisingly, the three mycoviruses responded differentially to each curing approach. The isolate of CbEVC was eliminated upon treatment with cycloheximide, but not with kanamycin or streptomycin, whereas the isolate of CbEVD did not respond to cycloheximide. The isolate of CbEVB was eliminated upon all treatments. In some cases, a virus was undetectable by species-specific RT-PCR assay after treatment, but when the fungus was cultured for a period on non-selective medium, the virus was detected again. Effects of mycoviruses on growth characteristics of isogenic fungal lines on two nutrient media were studied. Co-infection by the three viruses reduced mycelial growth rate on both media. In contrast, some fungal lines infected with one or two mycoviruses grew more rapidly than virus-free lines.

High-Throughput Sequencing Reveals Bell Pepper Endornavirus Infection in Pepper (Capsicum annum) in Slovakia and Enables Its Further Molecular Characterization.

Ribosomal RNA-depleted total RNAs from a sweet pepper plant (Capsicum annuum, labelled as N65) grown in western Slovakia and showing severe virus-like symptoms (chlorosis, mottling and deformation of leaf lamina) were subjected to high-throughput sequencing (HTS) on an Illumina MiSeq platform. The de novo assembly of ca. 5.5 million reads, followed by mapping to the reference sequences, revealed the coinfection of pepper by several viruses; i.e., cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), pepper cryptic virus 2 (PCV2) and bell pepper endornavirus (BPEV). A complete polyprotein-coding genomic sequence (14.6 kb) of BPEV isolate N65 was determined. A comparison of BPEV-N65 sequences with BPEV genomes available in GenBank showed 86.1% to 98.6% identity at the nucleotide level. The close phylogenetic relationship with isolates from India and China resulted in their distinct grouping compared to the other BPEV isolates. Further analysis has revealed the presence of BPEV in sweet or chili peppers obtained from various sources and locations in Slovakia (plants grown in gardens, greenhouse or retail shop). Additionally, the partial sequencing of two genomic portions from 15 BPEV isolates revealed that the Slovak isolates segregated into two molecular clusters, indicating a genetically distinct population (mean inter-group nucleotide divergence reaching 12.7% and 14.5%, respectively, based on the genomic region targeted). Due to the mix infections of BPEV-positive peppers by potato virus Y (PVY) and/or CMV, the potential role of individual viruses in the observed symptomatology could not be determined. This is the first evidence and characterization of BPEV from the central European region.

Molecular Characterization of a Novel Endornavirus Conferring Hypovirulence in Rice Sheath Blight Fungus Rhizoctonia solani AG-1 IA Strain GD-2.

The complete sequence and genome organization of a novel Endornavirus from the hypovirulent strain GD-2 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight, were identified using a deep sequencing approach and it was tentatively named as Rhizoctonia solani endornavirus 1 (RsEV1). It was composed of only one segment that was 19,936 bp in length and was found to be the longest endornavirus genome that has been reported so far. The RsEV1 genome contained two open reading frames (ORFs): ORF1 and ORF2. ORF1 contained a glycosyltransferase 1 domain and a conserved RNA-dependent RNA polymerase domain, whereas ORF2 encoded a conserved hypothetical protein. Phylogenetic analysis revealed that RsEV1 was phylogenetically a new endogenous RNA virus. A horizontal transmission experiment indicated that RsEV1 could be transmitted from the host fungal strain GD-2 to a virulent strain GD-118P and resulted in hypovirulence in the derivative isogenic strain GD-118P-V1. Metabolomic analysis showed that 32 metabolites were differentially expressed between GD-118P and its isogenic hypovirulent strain GD-118P-V1. The differential metabolites were mainly classified as organic acids, amino acids, carbohydrates, and the intermediate products of energy metabolism. Pathway annotation revealed that these 32 metabolites were mainly involved in pentose and glucuronate interconversions and glyoxylate, dicarboxylate, starch, and sucrose metabolism, and so on. Taken together, our results showed that RsEV1 is a novel Endornavirus, and the infection of virulent strain GD-118P by RsEV1 caused metabolic disorders and resulted in hypovirulence. The results of this study lay a foundation for the biocontrol of rice sheath blight caused by R. solani AG1-IA.

Viromes in Xylariaceae fungi infecting avocado in Spain.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

Genomic sequence of a novel endornavirus from Phaseolus vulgaris and occurrence in mixed infections with two other endornaviruses.

The Endornaviridae family includes viruses with ssRNA genome that infect plants, fungi, and oomycetes. Plant endornaviruses do not cause visible symptoms and are transmitted only vertically. Many common bean (Phaseolus vulgaris) genotypes have been reported to be infected by Phaseolus vulgaris endornavirus 1 and Phaseolus vulgaris endornavirus 2. Using next-generation sequencing, we obtained the RNA sequence of a third common bean endornavirus, which we named Phaseolus vulgaris endornavirus 3 (PvEV3). The complete sequence consisted of 15,205nt in length with a single open reading frame (ORF) coding for a polyprotein of 4932 aa, and untranslated regions of 344önt and 62önt at the 5' and 3' ends respectively. The polyprotein contained conserved protein domains including viral helicase 1, peptidase C97, glycosyltransferases of the GTB-type, and RdRp 2. The polyprotein shared 31% amino acid identity with the counterpart encoded by Hordeum vulgare endornavirus. A phylogenetic tree constructed with the RdRp sequences of PvEV3 and other endornaviruses placed PvEV3 in a clade with members of the genus Alphaendornavirus. PvEV3 was detected in cultivated and wild P. vulgaris genotypes as single and mixed infections with two other common bean endornaviruses. The natural occurrence of three distinct endornaviruses in a single plant species is unique and has not been reported in other plant-endornavirus systems.

Sclerotinia minor Endornavirus 1, a Novel Pathogenicity Debilitation-Associated Mycovirus with a Wide Spectrum of Horizontal Transmissibility.

Sclerotinia minor is a phytopathogenic fungus causing sclerotinia blight on many economically important crops. Here, we have characterized the biological and molecular properties of a novel endornavirus, Sclerotinia minor endornavirus 1 (SmEV1), isolated from the hypovirulent strain LC22 of S. minor. The genome of SmEV1 is 12,626 bp long with a single, large open reading frame (ORF), coding for a putative protein of 4020 amino acids. The putative protein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. The putative protein and the conserved domains are phylogenetically related to endornaviruses. SmEV1 does not contain a site-specific nick characteristic of most previously described endornaviruses. Hypovirulence and associated traits of strain LC22 and SmEV1 were readily cotransmitted horizontally via hyphal contact to isolates of different vegetative compatibility groups of S. minor. Additionally, SmEV1 in strain LC22 was found capable of being transmitted vertically through sclerotia. Furthermore, mycelium fragments of hypovirulent strain LC22 have a protective activity against attack by S. minor. Taken together, we concluded that SmEV1 is a novel hypovirulence-associated mycovirus with a wide spectrum of transmissibility, and has potential for biological control (virocontrol) of diseases caused by S. minor.