A novel alphapartitivirus from binucleate Rhizoctonia fumigata AG-Ba isolate C-314 Baishi.

The full-length nucleotide sequence and genome organization of a novel mycovirus designated as "Rhizoctonia fumigata partitivirus 1" (RfPV1) from Rhizoctonia fumigata AG-Ba strain C-314 Baishi was determined. The genome of RfPV1 consists of two double-stranded RNAs (dsRNAs): dsRNA1 (2003 bp) and dsRNA2 (1802 bp). Each of the two dsRNAs contains one open reading frame, coding for a putative RNA-dependent RNA polymerase and a coat protein, respectively. The 5' untranslated regions (UTRs) of both dsRNAs were conserved, and the 3'-UTRs of the two dsRNAs had interrupted poly(A) tails, similar to other partitiviruses. Phylogenetic analysis indicated that RfPV1 is a new species in the genus Alphapartitivirus, family Partitiviridae.

Genome Organizations and Functional Analyses of a Novel Gammapartitivirus from Rhizoctonia solani AG-1 IA Strain D122.

Here, we describe a novel double-stranded (ds) RNA mycovirus designated Rhizoctonia solani dsRNA virus 5 (RsRV5) from strain D122 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight. The RsRV5 genome consists of two segments of dsRNA (dsRNA-1, 1894 bp and dsRNA-2, 1755 bp), each possessing a single open reading frame (ORF). Sequence alignments and phylogenetic analyses showed that RsRV5 is a new member of the genus Gammapartitivirus in the family Partitiviridae. Transmission electron microscope (TEM) images revealed that RsRV5 has isometric viral particles with a diameter of approximately 20 nm. The mycovirus RsRV5 was successfully removed from strain D122 by using the protoplast regeneration technique, thus resulting in derivative isogenic RsRV5-cured strain D122-P being obtained. RsRV5-cured strain D122-P possessed the traits of accelerated mycelial growth rate, increased sclerotia production and enhanced pathogenicity to rice leaves compared with wild type RsRV5-infection strain D122. Transcriptome analysis showed that three genes were differentially expressed between two isogenic strains, D122 and D122-P. These findings provided new insights into the molecular mechanism of the interaction between RsRV5 and its host, D122 of R. solani AG-1 IA.

Molecular characterization of a novel mycovirus isolated from Rhizoctonia solani AG-1 IA strain 9-11.

The complete genome sequence of a double-stranded RNA (dsRNA) virus, Rhizoctonia solani dsRNA virus 11 (RsRV11), isolated from Rhizoctonia solani AG-1 IA strain 9-11 was determined. The RsRV11 genome is 9,555 bp in length and contains three conserved domains: structural maintenance of chromosomes (SMC) superfamily, phosphoribulokinase (PRK), and RNA-dependent RNA polymerase (RdRp). The RsRV11 genome has two non-overlapping open reading frames (ORFs). ORF1 is predicted to encode a 204.12-kDa protein that shares low but significant amino acid sequence similarity with a putative protein encoded by Rhizoctonia solani RNA virus HN008 (RsRV-HN008). ORF2 potentially encodes a 132.41-kDa protein that contains the conserved domain of the RdRp. Phylogenetic analysis indicated that RsRV11 clustered with RsRV-HN008 in a separate clade from other virus families. This implies that RsRV11 and RsRV-HN008 should be included in a new mycovirus taxon close to the family Megabirnaviridae and that RsRV11 is a new mycovirus.

A novel putative betapartitivirus isolated from the plant-pathogenic fungus Rhizoctonia solani.

In this study, we describe the genome sequence of a novel double-stranded RNA (dsRNA) mycovirus, designated as "Rhizoctonia solani partitivirus 15" (RsPV15), from the phytopathogenic fungus Rhizoctonia solani. RsPV15 consists of two genomic double-stranded RNA segments, dsRNA-1 and dsRNA-2, which are 2433 bp and 2350 bp long, respectively. Each of the dsRNA segments contains a single open reading frame, encoding the putative RNA-dependent RNA polymerase and coat protein, respectively. Homology searches and phylogenetic analysis suggested that RsPV15 is a new member of the genus Betapartitivirus within the family Partitiviridae.

Full genome sequence of a new mitovirus from the phytopathogenic fungus Rhizoctonia solani.

A double-stranded RNA (dsRNA) segment was identified in Rhizoctonia solani anastomosis group (AG)-2-2IIIB, the primary causal agent of Rhizoctonia crown and root rot of sugar beet. The dsRNA segment represented the genome replication intermediate of a new mitovirus that was tentatively designated as "Rhizoctonia solani mitovirus 39" (RsMV-39). The complete sequence of the dsRNA was 2805 bp in length with 61.9% A+U content. Using either the fungal mitochondrial or universal genetic code, a protein of 840 amino acids containing an RNA-dependent RNA polymerase (RdRp) domain was predicted with a molecular mass of 94.46 kDa. BLASTp analysis revealed that the RdRp domain of RsMV-39 had 43.55% to 72.96% sequence identity to viruses in the genus Mitovirus, and was the most similar (72.96% identical) to that of Ceratobasidium mitovirus A (CbMV-A). Phylogenetic analysis based on RdRp domains clearly showed that RsMV-39 is a member of a distinct species in the genus Mitovirus of the family Mitoviridae. This is the first full genome sequence of a mycovirus associated with R. solani AG-2-2IIIB.

Viruses Infecting the Plant Pathogenic Fungus Rhizoctonia solani.

The cosmopolitan fungus Rhizoctonia solani has a wide host range and is the causal agent of numerous crop diseases, leading to significant economic losses. To date, no cultivars showing complete resistance to R. solani have been identified and it is imperative to develop a strategy to control the spread of the disease. Fungal viruses, or mycoviruses, are widespread in all major groups of fungi and next-generation sequencing (NGS) is currently the most efficient approach for their identification. An increasing number of novel mycoviruses are being reported, including double-stranded (ds) RNA, circular single-stranded (ss) DNA, negative sense (-)ssRNA, and positive sense (+)ssRNA viruses. The majority of mycovirus infections are cryptic with no obvious symptoms on the hosts; however, some mycoviruses may alter fungal host pathogenicity resulting in hypervirulence or hypovirulence and are therefore potential biological control agents that could be used to combat fungal diseases. R. solani harbors a range of dsRNA and ssRNA viruses, either belonging to established families, such as Endornaviridae, Tymoviridae, Partitiviridae, and Narnaviridae, or unclassified, and some of them have been associated with hypervirulence or hypovirulence. Here we discuss in depth the molecular features of known viruses infecting R. solani and their potential as biological control agents.

Characterization of three novel betapartitiviruses co-infecting the phytopathogenic fungus Rhizoctonia solani.

In this study, we isolated and characterized seven dsRNA elements, designed as dsRNA-1 to 7, from a Rhizoctonia solani strain. Sequence analysis indicated that there were at least three novel mycoviruses co-infected in this fungal strain, termed Rhizoctonia solani partitivirus 6 (RsPV6), Rhizoctonia solani partitivirus 7 (RsPV7), and Rhizoctonia solani partitivirus 8 (RsPV8), respectively. RsPV6 contained three dsRNA segments, dsRNA-1, 6 and 7. DsRNA-1 encoded a RNA-dependent RNA polymerase (RdRp), whereas the proteins encoded by dsRNA-6 and 7 showed no detectable sequence similarity with any known viral proteins in the database. RsPV7 had the genome segments of dsRNA-2 and 5, encoding proteins of RdRp and capsid protein, respectively. RsPV8 containing the genomes of dsRNA-3 and 4 also encoded a RdRp and a protein with unknown function. RdRp-based phylogenetic analysis revealed that all the three viruses were phylogenetically related to members of the genus Betapartitivirus in the family Partitiviridae. In addition, the three viruses could be horizontal co-transmitted via hyphal contact between R. solani strains and cause no apparent phenotypic alteration to their fungal host. These findings provided new insights into the virus taxonomy of the family Partitiviridae and expanded our understanding of viral diversity in R. solani fungus.

Extreme Diversity of Mycoviruses Present in Isolates of Rhizoctonia solani AG2-2 LP From Zoysia japonica From Brazil.

Zoysia japonica, in Brazil, is commonly infected by Rhizoctonia solani (R. solani) in humid and cool weather conditions. Eight isolates of R. solani, previously identified as belonging to the AG2-2 LP anastomosis group, isolated from samples from large path symptoms, were collected from three counties in São Paulo state (Brazil) and investigated for the presence of mycoviruses. After detection of double-strand RNA (dsRNA) in all samples, RNA_Seq analysis of ribosomal RNA-depleted total RNA from in vitro cultivated mycelia was performed. Forty-seven partial or complete viral unique RNA dependent-RNA polymerase (RdRp) sequences were obtained with a high prevalence of positive sense ssRNA viruses. Sequences were sufficiently different from the first match in BLAST searches suggesting that they all qualify as possible new viral species, except for one sequence showing an almost complete match with Rhizoctonia solani dsRNA virus 2, an alphapartitivirus. Surprisingly four large contigs of putative viral RNA could not be assigned to any existing clade of viruses present in the databases, but no DNA was detected corresponding to these fragments confirming their viral replicative nature. This is the first report on the occurrence of mycoviruses in R. solani AG2-2 LP in South America.

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.

First report of a novel alphapartitivirus in the basidiomycete Rhizoctonia oryzae-sativae.

Rhizoctonia oryzae-sativae is a soil-borne basidiomycete fungus that causes aggregate sheath spot disease on rice worldwide. Here, we report the complete genome sequence of a partitivirus designated as Rhizoctonia oryzae-sativae partitivirus 1 (RosPV1) infecting this fungus. The genome of RosPV1 consists of two double-stranded RNA (dsRNA) segments. The larger segment, designated as dsRNA-1 (1,961 bp), contains a single open reading frame (ORF) that encodes a putative polypeptide with a conserved RNA-dependent RNA polymerase (RdRp) domain. The smaller segment, dsRNA-2 (1,819 bp), also has a single ORF, which is predicted to encode the capsid protein (CP). BLAST searches and phylogenetic analyses suggested that RosPV1 is a representative member of a new species within the genus Alphapartitivirus. This is the first report of an alphapartitivirus infecting the fungus R. oryzae-sativae.

Complete Nucleotide Sequence of a Partitivirus from Rhizoctonia solani AG-1 IA Strain C24.

The complete genome of a novel double-stranded (ds) RNA mycovirus, named as Rhizoctonia solani partitivirus 5 (RsPV5), isolated from rice sheath blight fungus R. solani AG-1 IA strain C24, was sequenced and analysed. RsPV5 consists of two segments, dsRNA-1 (1899 nucleotides) and dsRNA-2 (1787 nucleotides). DsRNA-1 has an open reading frame (ORF) 1 that potentially codes for a protein of 584 amino acid (aa) containing the conserved motifs of a RNA-dependent RNA polymerase (RdRp), and dsRNA-2 also contains a ORF 2, encoding a putative capsid protein (CP) of 513 aa. Phylogenetic analysis revealed that RsPV5 clustered together with six other viruses in an independent clade of the genus Alphapartitivirus, indicating that RsPV5 was a new member of the genus Alphapartitivirus, within the family Partitiviridae.

Characterization of a novel dsRNA mycovirus isolated from strain A105 of Rhizoctonia solani AG-1 IA.

Rhizoctonia solani dsRNA virus 3 (RsRV3), a novel mycovirus, was isolated from the rice sheath blight pathogen Rhizoctonia solani AG-1 IA strain A105. The RsRV3 genome consists of two segments of dsRNA (dsRNA1, 1,890 bp and dsRNA2, 1,811 bp). DsRNA1 has a single open reading frame (ORF) with a putative conserved RNA-dependent RNA polymerase (RdRp) domain, and dsRNA2 comprises a single ORF, predicted to encode a coat protein. Purified viral particles of RsRV3 were isometric and measured approximately 20 nm in diameter by negative-stain transmission electron microscope (TEM). Phylogenetic analyses indicated that RsRV3 is highly similar to viruses taxonomically classified in the genus Alphapartitivirus, family Partitiviridae. Taken together, the integrative analyses of viral genomic organization, amino acid sequence alignments and phylogenetic analyses clearly demonstrate that the RsRV3 virus isolated from R. solani AG-1 IA strain A105 is classifiable as a new member of the genus Alphapartitivirus, family Partitiviridae.

Two alphapartitiviruses co-infecting a single isolate of the plant pathogenic fungus Rhizoctonia solani.

Seven dsRNA segments were detected from a single Rhizoctonia solani strain HG81. From the full-length cDNA sequences of four smaller dsRNA segments, the genomes of two related partitiviruses, designated as Rhizoctonia solani partitivirus 3 (RsPV3) and RsPV4, were determined. The genomes of RsPV3 and RsPV4 are both composed of two separate dsRNA segments, with each segment possessing a single open reading frame (ORF). ORF1 from RsPV3 and RsPV4 encodes a putative RNA-dependent RNA polymerase, while ORF2 of RsPV3 and RsPV4 encodes a putative capsid protein. RsPV3 and RsPV4 share high sequence identity with viruses classified within the genus Alphapartitivirus, family Partitiviridae.

Phytopathogenic fungus hosts a plant virus: A naturally occurring cross-kingdom viral infection.

The transmission of viral infections between plant and fungal hosts has been suspected to occur, based on phylogenetic and other findings, but has not been directly observed in nature. Here, we report the discovery of a natural infection of the phytopathogenic fungus Rhizoctonia solani by a plant virus, cucumber mosaic virus (CMV). The CMV-infected R. solani strain was obtained from a potato plant growing in Inner Mongolia Province of China, and CMV infection was stable when this fungal strain was cultured in the laboratory. CMV was horizontally transmitted through hyphal anastomosis but not vertically through basidiospores. By inoculation via protoplast transfection with virions, a reference isolate of CMV replicated in R. solani and another phytopathogenic fungus, suggesting that some fungi can serve as alternative hosts to CMV. Importantly, in fungal inoculation experiments under laboratory conditions, R. solani could acquire CMV from an infected plant, as well as transmit the virus to an uninfected plant. This study presents evidence of the transfer of a virus between plant and fungus, and it further expands our understanding of plant-fungus interactions and the spread of plant viruses.

Complete genome sequence of a novel mitovirus from the phytopathogenic fungus Rhizoctonia oryzae-sativae.

A double-stranded RNA (dsRNA) segment was isolated from the filamentous phytopathogenic fungus Rhizoctonia oryzae-sativae and its full-length cDNA sequence (3038 nucleotides) was determined. Sequence analysis revealed that a large open reading frame (ORF) is present on the positive strand of this dsRNA segment when the mitochondrial genetic code was applied. The ORF encodes a putative RNA-dependent RNA polymerase, which shares the closest similarity with Rhizoctonia mitovirus 1 and Rhizophagus sp. RF1 mitovirus, with 43% and 29% identity, respectively. This dsRNA segment represents the replication form of a novel mitovirus that was temporarily designated Rhizoctonia oryzae-sativae mitovirus 1 (RoMV1). Phylogenetic analysis further suggested that RoMV1 belongs to the family Narnaviridae. This is the first study to report a mitovirus genome sequence in the phytopathogenic fungus R. oryzae-sativae.

Identification of a novel mycovirus isolated from Rhizoctonia solani (AG 2-2 IV) provides further information about genome plasticity within the order Tymovirales.

The complete genome of a novel mycovirus, named Rhizoctonia solani flexivirus 1 (RsFV-1), which infects an avirulent strain of Rhizoctonia solani AG 2-2 IV, was sequenced and analyzed. Its RNA genome consists of 10,621 nucleotides, excluding the poly-A tail, and encodes a single protein of 3477 amino acids. The identification of conserved motifs of methyltransferase, helicase and RNA-dependent RNA polymerase revealed its relatedness to members of the alphavirus-like superfamily of positive-strand RNA viruses. Phylogenetic analysis of these fused domains suggested that this virus should be assigned to the order Tymovirales. The recently described Fusarium graminearum deltaflexivirus 1 was found to be its closest relative. However, the whole genome, as well as the encoded protein of RsFV-1, is larger than that of other known members of the order Tymovirales, and unlike all other viruses belonging to this order, its methyltransferase domain is not located at the N-terminus of the replicase. Although genome diversity, as a result of recombination and gene loss, is a well-documented trait in members of the order Tymovirales, no related virus with a comparable genome alteration has been reported before. For these reasons, RsFV-1 broadens our perception about genome plasticity and diversity within the order Tymovirales.

Deep Sequencing Analysis Reveals the Mycoviral Diversity of the Virome of an Avirulent Isolate of Rhizoctonia solani AG-2-2 IV.

Rhizoctonia solani represents an important plant pathogenic Basidiomycota species complex and the host of many different mycoviruses, as indicated by frequent detection of dsRNA elements in natural populations of the fungus. To date, eight different mycoviruses have been characterized in Rhizoctonia and some of them have been reported to modulate its virulence. DsRNA extracts of the avirulent R. solani isolate DC17 (AG2-2-IV) displayed a diverse pattern, indicating multiple infections with mycoviruses. Deep sequencing analysis of the dsRNA extract, converted to cDNA, revealed that this isolate harbors at least 17 different mycovirus species. Based on the alignment of the conserved RNA-dependent RNA-polymerase (RdRp) domain, this viral community included putative members of the families Narnaviridae, Endornaviridae, Partitiviridae and Megabirnaviridae as well as of the order Tymovirales. Furthermore, viruses, which could not be assigned to any existing family or order, but showed similarities to so far unassigned species like Sclerotinia sclerotiorum RNA virus L, Rhizoctonia solani dsRNA virus 1, Aspergillus foetidus slow virus 2 or Rhizoctonia fumigata virus 1, were identified. This is the first report of a fungal isolate infected by 17 different viral species and a valuable study case to explore the diversity of mycoviruses infecting R. solani.

Novel mitoviruses in Rhizoctonia solani AG-3PT infecting potato.

Double-stranded RNA (dsRNA) elements are ubiquitous in Rhizoctonia solani. Total dsRNA was randomly amplified from a R. solani isolate (RS002) belonging to anastomosis group-3PT (AG-3PT), associated with black scurf in potato. Assembly of resulting cDNA sequences identified a nearly complete genome of a novel virus related to the genus Mitovirus (family Narnaviridae), herein named Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002). The 2797 nucleotide partial genome of RMV-1-RS002 is A-U rich (59.06 %), and can be folded into stable stem-loop structures at 5' and 3' ends. Universal and mold mitochondrial codon usages revealed a large open reading frame in the genome, putatively encoding an 826 amino acid polypeptide, which has conserved motifs for mitoviral RNA-dependent RNA polymerase. The full length putative polypeptide shared 25.6 % sequence identity with the corresponding region of Tuber excavatum mitovirus (TeMV). The partial genome of a second mitovirus (proposed name Rhizoctonia mitovirus 2 RS002 (RMV-2-RS002)) was also amplified from RS002. A nearly identical copy of RMV-1-RS002 was detected in two additional AG-3PT isolates. These data indicate that multiple mitoviruses can exist in a single isolate of R. solani AG-3PT, and that mitoviruses such as RMV-1-RS002 are probably widespread in this pathogen. The roles of mitoviruses in the biology of R. solani AG-3PT remain unknown.

Molecular characterization of a novel mycovirus from Rhizoctonia fumigata AG-Ba isolate C-314 Baishi.

The complete genome sequence of a novel dsRNA virus isolated from Rhizoctonia fumigata AG-Ba isolate C-314 Baishi (designated as Rhizoctonia fumigata virus 1, RfV1) was determined. The RfV1 genome was 9,907 bp in length and contained two open reading frames (ORFs). ORF1 potentially coded for a 198.10-kDa protein (P1). P1 shared low but significant amino acid sequence similarity to the putative protein encoded by Lentinula edodes mycovirus (LeV) ORF1. P1 contained a NUDIX domain, which was also present in the putative proteins encoded by the ORF1s of LeV and Phlebiopsis gigantea large virus 1 (PgLV-1). ORF2 potentially coded for a 146.72-kDa protein (P2) that contained the conserved motifs of the RNA-dependent RNA polymerase (RdRp). ORF1 and ORF2 were overlapping, and it was predicted that ORF2 could be translated as a fusion with ORF1 via a ribosomal -1 frameshifting mechanism. Phylogenetic analysis indicated that RfV1 clustered with PgLV-1, LeV and Rosellinia necatrix megabirnavirus 1 (RnMBV1) in a separate clade independent of other virus genera. We propose that RfV1, along with PgLV-1 and LeV, should be grouped into a new viral genus related to the family Megabirnaviridae. This is the first report of the full-length genome sequence of a novel mycovirus isolated from R. fumigata.

Genome sequence of a novel mycovirus of Rhizoctonia solani, a plant pathogenic fungus.

Here we present the genome sequence of a novel dsRNA virus we designed as Rhizoctonia solani RNA virus HN008 (RsRV-HN008) from a filamentous fungus R. solani. Its genome (7596 nucleotides) contains two non-overlapping open reading frames (ORF1 and ORF2). ORF1 encoded a 128 kDa protein that showed no significant identity to any other virus sequence in the NCBI database. ORF2 encoded a protein with a molecular weight of 140 kDa and shared a low percentage of sequence identity to the RdRps of unclassified dsRNA viruses. Sequence analysis revealed that RsRV-HN008 may be a member of a novel unclassified family of mycoviruses.