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A new positive-sense single-stranded RNA (+ssRNA) mycovirus, Verticillium dahliae magoulivirus 1 (VdMoV1), was isolated from two strains (2-19 and XLZ70) of Verticillium dahliae. The complete genome of VdMoV1 is 2303 nucleotides (nt) in length and has a large open reading frame (nt positions from 61 to 1938) encoding an RNA-dependent RNA polymerase (RdRp). A multiple sequence alignment indicated that the central region of the RdRp encoded by VdMoV1 contains eight typical viral RdRp motifs. BLASTp analysis demonstrated that VdMoV1 has the highest sequence identity (86.88%) to Bremia lactucae associated ourmia-like virus 2 (BlaOLV2). Phylogenetic analysis revealed that VdMoV1 is a new member of the genus Magoulivirus. As far as we know, VdMoV1 is the first reported member of the family Botourmiaviridae infecting V. dahliae.

A significant number of mycoviruses have been identified that are related to plant viruses, but their evolutionary relationships are largely unexplored. A fusarivirus, Rhizoctonia solani fusarivirus 4 (RsFV4), was identified in phytopathogenic fungus Rhizoctonia solani (R. solani) strain XY74 co-infected by an alphaendornavirus. RsFV4 had a genome of 10,833 ​nt (excluding the poly-A tail), and consisted of four non-overlapping open reading frames (ORFs). ORF1 encodes an 825 aa protein containing a conserved helicase domain (Hel1). ORF3 encodes 1550 aa protein with two conserved domains, namely an RNA-dependent RNA polymerase (RdRp) and another helicase (Hel2). The ORF2 and ORF4 likely encode two hypothetical proteins (520 and 542 aa) with unknown functions. The phylogenetic analysis based on Hel2 and RdRp suggest that RsFV4 was positioned within the fusarivirus group, but formed an independent branch with three previously reported fusariviruses of R. solani. Notably, the Hel1 and its relatives were phylogenetically closer to helicases of potyviruses and hypoviruses than fusariviruses, suggesting fusarivirus Hel1 formed an evolutionary link between these three virus groups. This finding provides evidence of the occurrence of a horizontal gene transfer or recombination event between mycoviruses and plant viruses or between mycoviruses. Our findings are likely to enhance the understanding of virus evolution and diversity.

Rhizosphere microbiota is important for plant growth and health. Domestication is a process to select suitable plants to satisfy the needs of humans, which may have great impacts on the interaction between the host and its rhizosphere microbiota. Rapeseed (Brassica napus) is an important oilseed crop derived from the hybridization between Brassica rapa and Brassica oleracea ~7500 years ago. However, variations in rhizosphere microbiota along with rapeseed domestication remain poorly understood. Here, we characterized the composition and structure of the rhizosphere microbiota among diverse rapeseed accessions, including ten B. napus, two B. rapa, and three B. oleracea accessions through bacterial 16S rRNA gene sequencing. B. napus exhibited a higher Shannon index and different bacterial relative abundance compared with its wild relatives in rhizosphere microbiota. Moreover, artificial synthetic B. napus lines G3D001 and No.2127 showed significantly different rhizosphere microbiota diversity and composition from other B. napus accessions and their ancestors. The core rhizosphere microbiota of B. napus and its wild relatives was also described. FAPROTAX annotation predicted that the synthetic B. napus lines had more abundant pathways related to nitrogen metabolism, and the co-occurrence network results demonstrated that Rhodoplanes acted as hub nodes to promote nitrogen metabolism in the synthetic B. napus lines. This study provides new insights into the impacts of rapeseed domestication on the diversity and community structure of rhizosphere microbiota, which may highlight the contribution of rhizosphere microbiota to plant health.

Fungal segmented negative-sense RNA viruses (SNSRVs) have been frequently found. Only the large segment encoding RNA-dependent RNA polymerase (RdRp) has been reported in most fungal SNSRVs, except for a few fungal SNSRVs reported to encode nucleocapsids, nonstructural proteins, or movement proteins.

Sclerotinia sclerotiorum is an ascomycetous fungus and hosts various mycoviruses. In this study, a novel fungal alphaflexivirus with a special genomic structure, named Sclerotinia sclerotiorum alphaflexivirus 1 (SsAFV1), was cloned from a hypovirulent strain, AHS31. Strain AHS31 was also co-infected with two botourmiaviruses and two mitoviruses. The complete genome of SsAFV1 comprised 6939 bases with four open reading frames (ORFs), a conserved 5′-untranslated region (UTR), and a poly(A) tail in the 3′ terminal; the ORF1 and ORF3 encoded a replicase and a coat protein (CP), respectively, while the function of the proteins encoded by ORF2 and ORF4 was unknown. The virion of SsAFV1 was flexuous filamentous 480–510 nm in length and 9–10 nm in diameter. The results of the alignment and the phylogenetic analysis showed that SsAFV1 is related to allexivirus and botrexvirus, such as Garlic virus X of the genus Allexivirus and Botrytis virus X of the genus Botrevirus, both with 44% amino-acid (aa) identity of replicase. Thus, SsAFV1 is a novel virus and a new genus, Sclerotexvirus, is proposed to accommodate this novel alphaflexivirus.

Sclerotinia sclerotiorum reovirus 1 (SsReV1) was previously reported to infect hypovirulent strain SCH941 of the phytopathogenic fungus Sclerotinia sclerotiorum and to contain 11 double-stranded RNA (dsRNA) segments (S1-S11). Here, we report that SsReV1 is actually composed of 12 dsRNA segments instead of 11. The full-length nucleotide sequence of the twelfth segment (S12) was determined using a combination of RACE and high-throughput sequencing methods. S12 is 1217 nucleotides in length and has highly conserved terminal sequences that resemble those of the other 11 segments of SsReV1. S12 contains a single open reading frame encoding a protein (VP12) of 311 amino acids. Although regular BLAST analysis did not reveal any similarity of VP12 to known sequences, it was found to be homologous to the VP11 of Colorado tick fever virus of the genus Coltivirus when a hidden-Markov-model-based HHpred analysis was performed. A single-protoplast regeneration experiment suggested that S12 and S2 were maintained or lost in parallel. In summary, the SsReV1 genome consists of 12 dsRNA segments.

A novel double-stranded RNA virus was isolated and identified from Beauveria bassiana Vuillemin, derived from the muscardine cadaver of an Ostrinia furnacalis larva in China. The virus contains six dsRNAs, and each viral dsRNA contains only one open reading frame (ORF). As in other polymycoviruses, dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA3 encodes a methyltransferase (MTR), and dsRNA4 encodes a proline-alanine-serine-rich protein. A BLASTp search revealed that the viral RdRp domain showed 79.43%, 79.04%, and 59.05% sequence identity to Beauveria bassiana polymycovirus 2 and 3 (BbPmV-2, BbPmV-3) and Magnaporthe oryzae polymycovirus 1 (MoPmV-1), respectively. Phylogenetic analysis based on RdRp sequences showed that the phylogenetically closest relatives of this virus are BbPmV-2, BbPmV-3, and MoPmV-1. This virus, along with previously ill-defined polymycoviruses (BbPmV-2 and BbPmV-3), appears to belong to an as-yet-unestablished species. The findings further suggest that the virus is a new member of the genus Polymycovirus within the family Polymycoviridae, and we have named it "Beauveria bassiana polymycovirus 4" (BbPmV-4). However, the sixth dsRNA is a defective RNA with the same sequence as that of dsRNA4 except for a deletion of 312 bp from nt 185 to nt 496, but it still contains a complete ORF. To our knowledge, this is the first report of the existence of a defective RNA in a polymycovirus.

Sclerotinia sclerotiorum ourmiavirus 17 (SsOV17) was isolated from the hypovirulent strain GF3 of Sclerotinia sclerotiorum. The genome of SsOV17 is 2,802 nt in length and contains a single long open reading frame (ORF) flanked by a short structured 5'-untranslated region (5'-UTR) (28 nt) and a long 3'-UTR (788 nt), respectively. The ORF encodes a protein with 663 amino acids and a predicted molecular mass of 75.0 kDa. A BLASTp search indicated that the protein encoded by SsOV17 is closely related to the putative RNA-dependent RNA polymerase (RdRp) of Sclerotinia sclerotiorum ourmiavirus 13 (71% identity). A multiple sequence alignment indicated that eight conserved amino acid motifs were present in the RdRp conserved region of SsOV17. Phylogenetic analysis demonstrated that SsOV17 clustered with members of the genus Botoulivirus.

Rhabdoviruses are ubiquitous and diverse viruses that propagate owing to bidirectional interactions with their vertebrate, arthropod, and plant hosts, and some of them could pose global health or agricultural threats. However, rhabdoviruses have rarely been reported in fungi. Here, two newly identified fungal rhabdoviruses, Rhizoctonia solani rhabdovirus 1 (RsRhV1) and RsRhV2, were discovered and molecularly characterized from the phytopathogenic fungus Rhizoctonia solani. The genomic organizations of RsRhV1 and RsRhV2 are 11,716 and 11,496 nucleotides (nt) in length, respectively, and consist of five open reading frames (ORFs) (ORFs I to V). ORF I, ORF IV, and ORF V encode the viral nucleocapsid (N), glycoprotein (G), and RNA polymerase (L), respectively. The putative protein encoded by ORF III has a lower level of identity with the matrix protein of rhabdoviruses. ORF II encodes a hypothetical protein with unknown function. Phylogenetic trees based on multiple alignments of N, L, and G proteins revealed that RsRhV1 and RsRhV2 are new members of the family Rhabdoviridae, but they form an independent evolutionary branch significantly distinct from other known nonfungal rhabdoviruses, suggesting that they represent a novel viral evolutionary lineage within Rhabdoviridae. Compared to strains lacking rhabdoviruses, strains harboring RsRhV2 and RsRhV1 showed hypervirulence, suggesting that RsRhV1 and RsRhV2 might be associated with the virulence of R. solani. Taken together, this study enriches our understanding of the diversity and host range of rhabdoviruses. IMPORTANCE Mycoviruses have been attracting an increasing amount of attention due to their impact on important medical, agricultural, and industrial fungi. Rhabdoviruses are prevalent across a wide spectrum of hosts, from plants to invertebrates and vertebrates. This study molecularly characterized two novel rhabdoviruses from four Rhizoctonia solani strains, based on their genomic structures, transcription strategy, phylogenetic relationships, and biological impact on their host. Our study makes a significant contribution to the literature because it not only enriches the mycovirus database but also expands the known host range of rhabdoviruses. It also offers insight into the evolutionary linkage between animal viruses and mycoviruses and the transmission of viruses from one host to another. Our study will also help expand the contemporary knowledge of the classification of rhabdoviruses, as well as providing a new model to study rhabdovirus-host interactions, which will benefit the agriculture and medical areas of human welfare.

RNA viruses usually have linear genomes and are encapsidated by their own capsids. Here, we newly identified four mycoviruses and two previously reported mycoviruses (a fungal reovirus and a botybirnavirus) in the hypovirulent strain SCH941 of Sclerotinia sclerotiorum. One of the newly discovered mycoviruses, Sclerotinia sclerotiorum yadokarivirus 1 (SsYkV1), with a nonsegmented positive-sense single-stranded RNA (+ssRNA) genome, was molecularly characterized. SsYkV1 is 5,256 nucleotides (nt) in length, excluding the poly(A) structure, and has a large open reading frame that putatively encodes a polyprotein with the RNA-dependent RNA polymerase (RdRp) domain and a 2A-like motif. SsYkV1 was phylogenetically positioned into the family Yadokariviridae and was most closely related to Rosellinia necatrix yadokarivirus 2 (RnYkV2), with 40.55% identity (78% coverage). Although SsYkV1 does not encode its own capsid protein, the RNA and RdRp of SsYkV1 are trans-encapsidated in virions of Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3), a bisegmented double-stranded RNA (dsRNA) mycovirus within the genus Botybirnavirus. In this way, SsYkV1 likely replicates inside the heterocapsid comprised of the SsBV3 capsid protein, like a dsRNA virus. SsYkV1 has a limited impact on the biological features of S. sclerotiorum. This study represents an example of a yadokarivirus trans-encapsidated by an unrelated dsRNA virus, which greatly deepens our knowledge and understanding of the unique life cycles of RNA viruses. IMPORTANCE RNA viruses typically encase their linear genomes in their own capsids. However, a capsidless +ssRNA virus (RnYkV1) highjacks the capsid of a nonsegmented dsRNA virus for the trans-encapsidation of its own RNA and RdRp. RnYkV1 belongs to the family Yadokariviridae, which already contains more than a dozen mycoviruses. However, it is unknown whether other yadokariviruses except RnYkV1 are also hosted by a heterocapsid, although dsRNA viruses with capsid proteins were detected in fungi harboring yadokarivirus. It is noteworthy that almost all presumed partner dsRNA viruses of yadokariviruses belong to the order Ghabrivirales (most probably a totivirus or toti-like virus). Here, we found a capsidless +ssRNA mycovirus, SsYkV1, from hypovirulent strain SCH941 of S. sclerotiorum, and the RNA and RdRp of this mycovirus are trans-encapsidated in virions of a bisegmented dsRNA virus within the free-floating genus Botybirnavirus. Our results greatly expand our knowledge of the unique life cycles of RNA viruses.

Proper protein secretion is critical for fungal development and pathogenesis. However, the potential roles of proteins involved in the early secretory pathway are largely undescribed in filamentous fungi. p24 proteins are cargo receptors that cycle between the endoplasmic reticulum (ER) and Golgi apparatus in the early secretory pathway and recruit cargo proteins to nascent vesicles. This study characterized the function of two p24 family proteins (SsEmp24 and SsErv25) in a phytopathogenic fungus, Sclerotinia sclerotiorum. Both SsEmp24 and SsErv25 were upregulated during the early stages of S. sclerotiorum infection. ΔSsEmp24 mutant and ΔSsErv25 mutant displayed abnormal vegetative growth and sclerotium formation, were defective in infection cushion formation, and showed lower virulence on host plants. ΔSsEmp24 mutant had a more severe abnormal phenotype than ΔSsErv25 mutant, implying that SsEmp24 could play a central role in the early secretory pathway. Similar to their Saccharomyces cerevisiae counterparts, SsEmp24 interacted with SsErv25 and predominantly colocalized in the ER or nuclear envelope. The absence of SsEmp24 or SsErv25 led to defective in protein secretion in S. sclerotiorum, including the pathogenicity-related extracellular hydrolytic enzymes and effectors. It is proposed that SsEmp24 and SsErv25, components in the early secretory pathway, are involved in modulating morphogenesis and pathogenicity in S. sclerotiorum by mediating protein secretion. IMPORTANCE Understanding the reproduction and pathogenesis mechanism of phytopathogens could provide new opinions to effectively control fungal diseases. Although it has been known that effectors and extracellular hydrolytic enzymes secreted by phytopathogenic fungi play important roles in fungus-host interactions, the secretion system for the delivery of virulence factors to the host is still largely undescribed. Although the role of the early secretory pathway-associated p24 proteins in S. cerevisiae has been well characterized, the function of these proteins in filamentous fungi was scarcely known prior to this study. The present research provides evidence that p24 proteins participate in the reproduction and pathogenesis of phytopathogenic fungi through the mediation of protein secretion. This research advances our understanding of p24 proteins in filamentous phytopathogenic fungi. In addition, the candidate cargos of the two p24 proteins, SsEmp24 and SsErv25, were screened out by comparative proteomics, which could aid the identification of novel development and virulence-associated factors in phytopathogenic fungi.

A novel positive single-stranded RNA virus, Sclerotinia sclerotiorum hypovirus 9 (SsHV9), was identified in the plant-pathogenic Sclerotinia sclerotiorum strain GB375, which was associated with a garden bean plant in the United States. The complete genome of SsHV9 is 14,067 nucleotides in length, excluding the poly(A) tail. It has a single large open reading frame encoding a putative polyprotein (4,196 amino acids), which is predicted to contain a papain-like protease, a protein of unknown function, an RNA-dependent RNA polymerase, and an RNA helicase. Phylogenetic analysis based on a multiple alignment of amino acid sequences of polyproteins that suggested SsHV9 belongs to the proposed genus "Alphahypovirus" in the family Hypoviridae.

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation., Author summary Identification of mycoviruses in phytopathogenic fungi is necessary for understanding the origin of viruses and developing virocontrol strategies to protect plants. Nine mycoviruses with RNA genomes were identified in a hypovirulent strain of Sclerotinia sclerotiorum and were classified into eight potential viral families, suggesting that the composition of mycoviral communities was complex in this single fungal strain. They included four previously characterized mycoviruses and three distant relatives of known mycoviruses, as well as the first reports of a deltaflexivirus with a tripartite genome, and a fungal rhabdovirus. In addition, we found an endornavirus associated with hypovirulence in a phytopathogenic fungus. Our study makes a significant contribution because it not only expands the diversity-related knowledge of mycoviruses and potential virocontrol agents, but also provides new insights into mycovirus evolution.

Mycovirus infection is a universal phenomenon in the major fungus groups. So far, however, mycoviruses have not been described in the phytopathogenic fungus Leptosphaeria biglobosa, the causal agent of phoma stem canker (blackleg). Here, we report the complete genome sequence of a novel mitovirus, Leptosphaeria biglobosa mitovirus 1 (LbMV1), isolated from the strain H3-38 of L. biglobosa. The LbMV1 genome comprises 2568 nucleotides with a low G+C content of 30%. Using the mitochondrial genetic code, the genome of LbMV1 was found to contain a single large open reading frame that encodes a predicted protein of 756 amino acids. Multiple alignment of the predicted protein sequence showed the highest similarity (51% identity) to the putative RNA-dependent RNA polymerase of Ophiostoma mitovirus 4. In addition, phylogenetic analysis grouped LbMV1 within the genus Mitovirus of the family Narnaviridae. Our results suggest that the double-stranded RNA found in strain H3-38 is the replicating genome of the mitovirus LbMV1, its deletion represents the first evidence of a mycovirus infecting L. biglobosa.

Mycovirus diversity is generally analyzed from isolates of fungal culture isolates at a single point in time as a snapshot. The stability of mycovirus composition within the same geographical location over time remains unclear. Not knowing how the population fluctuates in the field can be a source of unpredictability in the successful application of virocontrol. To better understand the changes over time, we monitored the interannual dynamics and abundance of mycoviruses infecting Sclerotinia sclerotiorum at a rapeseed-growing field for three years. We found that the virome in S. sclerotiorum harbors unique mycovirus compositions each year. In total, sixty-eight mycoviruses were identified, among which twenty-four were detected in all three successive years. These twenty-four mycoviruses can be classified as the members of the core virome in this S. sclerotiorum population, which show persistence and relatively high transmissibility under field conditions. Nearly two-thirds of the mycoviruses have positive-sense, single-stranded RNA genomes and were found consistently across all three years. Moreover, twenty-eight mycoviruses are newly described, including four novel, multi-segmented narnaviruses, and four unique bunyaviruses. Overall, the newly discovered mycoviruses in this study belong to as many as twenty families, into which eight were first identified in S. sclerotiorum, demonstrating evolutionarily diverse viromes. Our findings not only shed light on the annual variation of mycovirus diversity but also provide important virus evolutionary clues.

Nidovirales, which accommodates viruses with the largest RNA genomes, includes the notorious coronaviruses; however, the evolutionary route for nidoviruses is not well understood. We have characterized a positive-sense (+) single-stranded (ss) RNA mycovirus, Rhizoctonia solani hypovirus 2 (RsHV2), from the phytopathogenic fungus Rhizoctonia solani. RsHV2 has the largest RNA genome size of 22,219 nucleotides, excluding the poly(A) tail, in all known mycoviruses, and contains two open reading frames (ORF1 and ORF2). ORF1 encodes a protein of 2,009 amino acid (aa) that includes a conserved helicase domain belonging to helicase superfamily I (SFI). In contrast, ORF2 encodes a polyprotein of 4459 aa containing the hallmark genes of hypoviruses. The latter includes a helicase belonging to SFII. Following phylogenetic analysis, the ORF1-encoded helicase (Hel1) unexpectedly clustered in an independent evolutionary branch together with nidovirus helicases, including coronaviruses, and bacteria helicases. Thus, Hel1 presence indicates the occurrence of horizontal gene transfer between viruses and bacteria. These findings also suggest that RsHV2 is most likely a recombinant arising between hypoviruses and nidoviruses.

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.

Here, we describe a novel mycovirus, Auricularia heimuer fusarivirus 1 (AhFV1), isolated from the edible fungus Auricularia heimuer strain CCMJ1296. The virus has a single-stranded positive-sense [+ssRNA] genome of 7,127 nucleotides containing two overlapping open reading frames (ORFs) and a poly(A) tail. The large ORF1 encodes a polyprotein of 1,637 amino acids (aa) with conserved RNA-dependent RNA polymerase (RdRp) and DEAD-like helicase superfamily (DEXDc) domains. ORF2 encodes a putative 633-aa protein with unknown function. A BLAST search showed that the nucleotide sequence of the AhFV1 genome is 41.28% identical to that of Sclerotium rolfsii fusarivirus 2 and 40.49% identical to that of Sclerotium rolfsii fusarivirus 1. Phylogenetic analysis based on RdRp and helicase (Hel) sequences indicated that AhFV1 is related to unclassified mycoviruses and other fusariviruses. Our data suggest that AhFV1 should be classified as a member of the newly proposed family "Fusariviridae". This is the second virus and the first full genome sequence of a fusarivirus from A. heimuer.

We previously identified Sclerotinia sclerotiorum negative-stranded virus 1 (SsNSRV-1), the first (&minus, ) ssRNA mycovirus, associated with hypovirulence of its fungal host Sclerotinia sclerotiorum. In this study, functional analysis of Open Reading Frame &Iota, (ORF &Iota, ) of SsNSRV-1 was performed. The integration and expression of ORF &Iota, led to defects in hyphal tips, vegetative growth, and virulence of the mutant strains of S. sclerotiorum. Further, differentially expressed genes (DEGs) responding to the expression of ORF &Iota, were identified by transcriptome analysis. In all, 686 DEGs consisted of 267 up-regulated genes and 419 down-regulated genes. DEGs reprogramed by ORF &Iota, were relevant to secretory proteins, pathogenicity, transcription, transmembrane transport, protein biosynthesis, modification, and metabolism. Alternative splicing was also detected in all mutant strains, but not in hypovirulent strain AH98, which was co-infected by SsNSRV-1 and Sclerotinia sclerotiorum hypovirus 1 (SsHV-1). Thus, the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host. Taken together, the results suggested that protein ORF &Iota, could regulate the transcription, translation, and modification of host genes in order to facilitate viral proliferation and reduce the virulence of the host. Therefore, ORF &Iota, may be a potential gene used for the prevention of S. sclerotiorum.

The family Botourmiaviridae includes viruses infecting plants and filamentous fungi containing a positive-sense, ssRNA genome that can be mono-or multi-segmented. Genera in the family include: Ourmiavirus (plant viruses), and Botoulivirus, Magoulivirus and Scleroulivirus (fungal viruses). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Botourmiaviridae, which is available at ictv. global/ report/ botourmiaviridae.

Recently, an increasing number of ourmia-like viruses have been found in fungi; however, the features of these viruses remain unknown. Here, we report a novel ourmia-like virus isolated from Sclerotinia sclerotiorum. This virus, named S. sclerotiorum ourmia-like virus 4 (SsOLV4), has a genome 2,982 nt in length with a G-pentamer (GGGGG) at the 5'-terminus and a C-pentamer (CCCCC) at the 3'-terminus. The SsOLV4 genome has only one large putative open reading frame (ORF) predicted with both standard codes and mitochondrial codes and encodes an RNA-dependent RNA polymerase (RdRp). SsOLV4 is closely phylogenetically related to Pyricularia oryzae ourmia-like virus 1, with 42% identity between the RdRp amino acid sequences. We constructed full-length cDNA of SsOLV4 and synthesized RNA in vitro using the T7 RNA polymerase. The synthesized RNA could transfect S. sclerotiorum protoplasts efficiently. We further found that viral RNA could infect mycelia when mixed with PEG buffer. Our study suggests that a novel genus in family Botourmiaviridae should be established for SsOLV4 and other related viruses and demonstrates that one single-stranded RNA segment encoding RdRp is sufficient for ourmia-like viruses in fungi.

Hypoviruses are positive-sense single-stranded RNA mycovirus that infect filamentous fungi. However, hypoviruses have not been reported in Bipolaris oryzae, an important phytopathogenic fungus in water bamboo and rice. Here, we report the characterization of a novel hypovirus, tentatively named "Bipolaris oryzae hypovirus 1" (BoHV1), isolated from strain ES35 of B. oryzae infecting water bamboo. The complete genome of BoHV1 consists of 13,596 nucleotides and a poly(A) tail at the 3' end. BoHV1 has single open reading frame (ORF) and encodes a putative polyprotein (4,218 amino acids) containing four potential conserved domains for a papain-like protease, a protein of unknown function (DUF3525), RNA-dependent RNA polymerase (RdRp), and helicase. Phylogenetic analysis of the polyprotein, RdRp, and helicase domains suggested that BoHV1 belongs to the genus Hypovirus within the family Hypoviridae. This is the first report of the presence of a hypovirus in the phytopathogenic fungus B. oryzae.

A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family “Alternaviridae”. Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family “Alternaviridae” members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1—and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family “Alternaviridae”. Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family “Alternaviridae”, and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.

Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically via sexual/asexual spores. Previously, we reported that a gemycircularvirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), could infect its fungal host extracellularly. Here, we discovered that SsHADV-1 could infect a mycophagous insect, Lycoriella ingenua, and use it as a transmission vector. Virus acquired by larvae feeding on colonies of a virus-infected strain of S. sclerotiorum was replicated and retained in larvae, pupae, adults, and eggs. Virus could be transmitted to insect offspring when larvae were injected with virus particles and allowed to feed on a nonhost fungus. Virus replication in insect cells was further confirmed by inoculating Spodoptera frugiperda cells with virus particles and analyzing with RT-PCR, Northern blot, immunofluorescence, and flow cytometry assays. Larvae could transmit virus once they acquired virus by feeding on virus-infected fungal colony. Offspring larvae hatched from viruliferous eggs were virus carriers and could also successfully transmit virus. Virus transmission between insect and fungus also occurred on rapeseed plants. Virus-infected isolates produced less repellent volatile substances to attract adults of L. ingenua. Furthermore, L. ingenua was easily observed on Sclerotinia lesions in rapeseed fields, and viruliferous adults were captured from fields either sprayed with a virus-infected fungal strain or nonsprayed. Our findings may facilitate the exploration of mycoviruses for control of fungal diseases and enhance our understanding of the ecology of SsHADV-1 and other newly emerging SsHADV-1–like viruses, which were recently found to be widespread in various niches including human HIV-infected blood, human and animal feces, insects, plants, and even sewage.

Via virome sequencing, six viruses were detected from Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae, one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae. Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3&prime, untranslated region (3&prime, UTR) and 5&prime, UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae. Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 (MOBV5, MOBV6, and MOBV7), respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae, and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae.

Various mycoviruses have been isolated from Sclerotinia sclerotiorum. Here, we identified a viral RNA sequence contig, representing a novel virus, Sclerotinia sclerotiorum deltaflexivirus 2 (SsDFV2), from an RNA_Seq database. We found that SsDFV2 was harbored in the hypovirulent strain, 228, which grew slowly on potato dextrose agar, produced a few sclerotia, and could not induce typical lesions on detached rapeseed (Brassica napus) leaves. Strain 228 was also infected by Botrytis porri RNA Virus 1 (BpRV1), a virus originally isolated from Botrytis porri. The genome of SsDFV2 comprised 6711 nucleotides, excluding the poly (A) tail, and contained a single large predicted open reading frame encoding a putative viral RNA replicase. Phylogenetic analysis demonstrated that SsDFV2 is closely related to viruses in the family Deltaflexiviridae, however, it also differs significantly from members of this family, suggesting that it may represent a new species. Further we determined that SsDFV2 could be efficiently transmitted to host vegetative incompatible individuals by dual culture. To our best knowledge, this is the first report that a (+) ssRNA mycovirus can overcome the transmission limitations of the vegetative incompatibility system, a phenomenon that may facilitate the potential use of mycoviruses for the control of crop fungal diseases.

Megabirnaviridae is a family of non-enveloped spherical viruses with dsRNA genomes of two linear segments, each of 7.2-8.9 kbp, comprising 16.1 kbp in total. The genus Megabirnavirus includes the species Rosellinia necatrix megabirnavirus 1, the exemplar isolate of which infects the white root rot fungus (Rosellinia necatrix) to which it confers hypovirulence. Megabirnaviruses are characterized by their bisegmented genome with large 5'-untranslated regions (1.6 kb) upstream of both 5'-proximal coding strand ORFs, and large protrusions on the particle surface. This is a summary of the ICTV Report on the family Megabirnaviridae, which is available at ictv.global/report/megabirnaviridae.

The Partitiviridae is a family of small, isometric, non-enveloped viruses with bisegmented double-stranded (ds) RNA genomes of 3–4.8 kbp. The two genome segments are individually encapsidated. The family has five genera, with characteristic hosts for members of each genus: either plants or fungi for genera Alphapartitivirus and Betapartitivirus, fungi for genus Gammapartitivirus, plants for genus Deltapartitivirus and protozoa for genus Cryspovirus. Partitiviruses are transmitted intracellularly via seeds (plants), oocysts (protozoa) or hyphal anastomosis, cell division and sporogenesis (fungi); there are no known natural vectors. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Partitiviridae, which is available at www.ictv.global/report/partitiviridae.

Two double-stranded RNA (dsRNA) mycoviruses were found in isolate QSP5 of the rice blast fungus Magnaporthe oryzae. Sequence analysis of the two dsRNA mycoviruses revealed that one is closely related to Magnaporthe oryzae virus 2 (MoV2), and the other one is related to Magnaporthe oryzae chrysovirus 1-A (MoCV1-A). Therefore, they were named Magnaporthe oryzae virus 3 (MoV3) and Magnaporthe oryzae chrysovirus 1-C (MoCV1-C), respectively. In this paper, the molecular and structural characteristics of MoV3 were analyzed in detail. The full genome sequence (5181 bp) of MoV3 was obtained by cDNA cloning. Sequence analysis indicated that MoV3 has two overlapping open reading frames (ORF1 and ORF2). The 5'-proximal ORF1 encodes a putative coat protein (CP) with a molecular weight of 80,939 Da; the 3'-proximal ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp) with a molecular weight of 90,506 Da. The stop codon of ORF1 overlaps the start codon of ORF2, with the tetranucleotide sequence AUGA, which is characteristic of members of the genus Victorivirus of the family Totiviridae. Phylogenetic analysis of RdRp and CP further supported the view that MoV3, a novel mycovirus, belongs to the genus Victorivirus of the family Totiviridae.

Recent studies have demonstrated that a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum. Here, we report the molecular characterization of a newly identified mycovirus, Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), which was isolated from a sclerotia-defective strain JMTJ14 of S. sclerotiorum. Excluding a poly (A) tail, the genome of SsFV1 comprises 7754 nucleotides (nts) in length with 83 and 418 nts for 5'- and 3'-untranslated regions, respectively. SsFV1 has four non-overlapping open reading frames (ORFs): ORF1 encodes a 191 kDa polyprotein (1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase (RdRp) and helicase domains, the other three ORFs encode three putative hypothetical proteins of unknown function. Phylogenetic analysis, based on RdRp and Helicase domains, indicated that SsFV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, SsFV1 is markedly different from FgV1 and RnFV1 in genome organization and nucleotide sequence. SsFV1 was transmitted successfully to two vegetatively incompatible virus-free strains. SsFV1 is not responsible for the abnormal phenotype of strain JMTJ14.

A double-stranded RNA (dsRNA) segment was isolated from a hypovirulent strain, HC025, of Sclerotinia sclerotiorum. The complete nucleotide sequence of the dsRNA was determined to be 2530 bp in length. Using the fungal mitochondrial genetic code, the positive strand of the dsRNA was found to contain a single large open reading frame (ORF) with the characteristic conserved motifs of the RNA-dependent RNA polymerase (RdRp). BLAST analysis revealed that RdRp shares 74% sequence identity with Sclerotinia sclerotiorum mitovirus 1 (SsMV1/KL-1). The positive strand of the dsRNA could be folded into potentially stable stem-loop structures at both the 5' and 3' terminal sequences. Moreover, the 5' and 3' terminal sequences were inverted complementary sequences and formed a panhandle structure. These results reveal that this dsRNA segment represents the replicative form of a mitovirus that is a strain of SsMV1 from the genus Mitovirus in the family Narnaviridae and was tentatively designated as Sclerotinia sclerotiorum mitovirus 1 (SsMV1/HC025). Sequence comparison and phylogenetic analysis suggest that mitovirus RdRp gene was evolutionarily related to plant mitochondrial genome. Our results demonstrate that SsMV1/HC025 infection exerted obvious effects on host biological properties. Hypovirulence feature and SsMV1/HC025 could be co-transmitted from hypovirulent strains to other virulent strains via hyphal contact. Thus, SsMV1/HC025 related to plant mitochondrial gene confers hypovirulence on S. sclerotiorum.

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.

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases., Author summary Self/non-self recognition, as the most fundamental immune system, is an essential part in response to nonself host defense in fungi, plants and animals against pathogens or molecular parasites. Vegetative incompatibility as a conspecific nonself system in filamentous fungi results in PCD when cells of incompatible strains contact each other, which also restricts the spread of molecular parasites. The hypovirulence-associated Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), was found to function as a potent inhibitor of G protein signaling pathway, ROS production and vegetative incompatibility-mediated PCD. This SsMYRV4-mediated unique feature enables horizontal transmission of heterologous mycoviruses among vegetatively incompatible S. sclerotiorum individuals in an interaction system (SsMYRV4/S. sclerotiorum) to create a bridge donor strain for mycovirus spread under natural conditions. Furthermore, this virus-mediated suppression of host non-self recognition provides a potential strategy for virus spread that can enhance our understanding of virus ecology.

The Hypoviridae, comprising one genus, Hypovirus, is a family of capsidless viruses with positive-sense, ssRNA genomes of 9.1-12.7 kb that possess either a single large ORF or two ORFs. The ORFs appear to be translated from genomic RNA by non-canonical mechanisms, i.e. internal ribosome entry site-mediated and stop/restart translation. Hypoviruses have been detected in ascomycetous or basidiomycetous filamentous fungi, and are considered to be replicated in host Golgi-derived, lipid vesicles that contain their dsRNA as a replicative form. Some hypoviruses induce hypovirulence to host fungi, while others do not. This is a summary of the current ICTV report on the taxonomy of the Hypoviridae, which is available at www.ictv.global/report/hypoviridae.

Infection by diverse mycoviruses is a common phenomenon in Sclerotinia sclerotiorum. In this study, the full genome of a single-stranded RNA mycovirus, tentatively named Hubei sclerotinia RNA virus 1 (HuSRV1), was determined in the hypovirulent strain 277 of S. sclerotiorum. The HuSRV1 genome is 4492 nucleotides (nt) long and lacks a poly (A) tail at the 3ˊ- terminus. Sequence analyses showed that the HuSRV1 genome contains four putative open reading frames (ORFs). ORF1a was presumed to encode a protein with a conserved protease domain and a transmembrane domain. This protein is 27% identical to the P2a protein encoded by the subterranean clover mottle virus. ORF1b encodes a protein containing a conserved RNA-dependent RNA polymerase (RdRp) domain, which may be translated into a fusion protein by a -1 ribosome frameshift. This protein is 45.9% identical to P2b encoded by the sowbane mosaic virus. ORF2 was found to encode a putative coat protein, which shares 23% identical to the coat protein encoded by the olive mild mosaic virus. ORF3 was presumed to encode a putative protein with an unknown function. Evolutionary relation analyses indicated that HuSRV1 is related to members within Sobemovirus, but forms a unique phylogenetic branch, suggesting that HuSRV1 represents a new member within Solemoviridae. HuSRV1 virions, approximately 30 nm in diameter, were purified from strain 277. The purified virions were successfully introduced into virulent strain Ep-1PNA367, resulting in a new hypovirulent strain, which confirmed that HuSRV1 confers hypovirulence on S. sclerotiorum.

We previously determined that virions of Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 (SsHADV-1) could directly infect hyphae of Sclerotinia sclerotiorum, resulting in hypovirulence of the fungal host. However, the molecular mechanisms of SsHADV-1 virions disruption of the fungal cell wall barrier and entrance into the host cell are still unclear. To investigate the early response of S. sclerotiorum to SsHADV-1 infection, S. sclerotiorum hyphae were inoculated with purified SsHADV-1 virions. The pre- and post-infection hyphae were collected at one&ndash, three hours post-inoculation for transcriptome analysis. Further, bioinformatic analysis showed that differentially expressed genes (DEGs) regulated by SsHADV-1 infection were identified in S. sclerotiorum. In total, 187 genes were differentially expressed, consisting of more up-regulated (114) than down-regulated (73) genes. The identified DEGs were involved in several important pathways. Metabolic processes, biosynthesis of antibiotics, and secondary metabolites were the most affected categories in S. sclerotiorum upon SsHADV-1 infection. Cell structure analysis suggested that 26% of the total DEGs were related to membrane tissues. Furthermore, 10 and 27 DEGs were predicted to be located in the cell membrane and mitochondria, respectively. Gene ontology enrichment analyses of the DEGs were performed, followed by functional annotation of the genes. Interestingly, one third of the annotated functional DEGs could be involved in the Ras-small G protein signal transduction pathway. These results revealed that SsHADV-1 virions may be able to bind host membrane proteins and influence signal transduction through Ras-small G protein-coupled receptors during early infection, providing new insight towards the molecular mechanisms of virions infection in S. sclerotiorum.

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.

Background Sclerotinia sclerotiorum, a notorious plant fungal pathogen, causes yield loss of many crops and vegetables, and is a natural host of a diverse viruses with positive-sense RNA (+ssRNA), negative-sense RNA (−ssRNA), double-stranded RNA (dsRNA), or DNA genomes. Mixed-infection with multiple related or unrelated mycoviruses is a common phenomenon in S. sclerotiorum. However, a single strain co-infected with dsRNA and + ssRNA viruses has not been reported in S. sclerotiorum. Results We report two unrelated viruses, Sclerotinia sclerotiorum botybirnavirus 2 (SsBRV2) with a bipartite dsRNA genome and Sclerotinia sclerotiorum mitovirus 4 (SsMV4/AH16) with a + ssRNA genome, which were originally detected in a single hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 has a typical genome of mitovirus and is a strain of mitovirus SsMV4. The genome of SsBRV2 consists of two separated dsRNA segments. The large dsRNA segment is 6159 bp in length and only has a single open reading frame (ORF) encoding a putative 1868-aa polyprotein with a conserved RNA dependent RNA polymerase (RdRp) domain. The small dsRNA segment is 5872 bp in length and encodes a putative 1778-aa protein. Phylogenetic analysis using RdRp conserved domain sequences revealed that SsBRV2 is phylogenetically related to the previously reported three bipartite viruses SsBRV1, Botrytis porri RNA virus 1 (BpRV1), and soybean leaf-associated botybirnavirus 1 (SlaBRV1). Electron microscopy demonstrated that SsBRV2 forms rigid spherical virions with a diameter of approximately 40 nm in infected mycelia. The virion of SsBRV2 was successfully introduced into a virus-free strain, which provides conclusive evidence that SsBRV2 confers hypovirulence on phytopathogenic fungus S. sclerotiorum. Conclusions A bisegmented dsRNA virus (SsBRV2/AH16) and a nonsegmented + ssRNA virus (SsMV4/AH16) were characterized in a hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 is a strain of a reported mitovirus, whereas SsBRV2 is a new botybirnavirus. SsBRV2 is the causal agent of hypovirulence on S. sclerotiorum. Our findings supplied a first evidence that a single S. sclerotiorum strain is co-infected by dsRNA and + ssRNA mycoviruses. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0550-2) contains supplementary material, which is available to authorized users.

Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen Sclerotinia sclerotiorum was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1∆SP could induce significant plant cell death and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1∆SP interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C38 and C44) in SsSSVP1∆SP had significant effects on its homo-dimer formation, SsSSVP1∆SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1∆SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1∆SP might be caused by the SsSSVP1∆SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of S. sclerotiorum. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction., Author Summary To resist biotrophic and hemibiotrophic phytopathogens, plants utilize an innate immune system, mediated through nucleotide binding (NB)-leucine rich repeat (LRR) proteins, to respond to effectors, most of which are small secreted proteins. Hypersensitive responses (HRs) resulting from this type of interaction can effectively restrain the expansion of biotrophic or hemibiotrophic phytopathogens in plant tissues. However, it is not effective against typical necrotrophs with remarkably broad host range, such as S. sclerotiorum, because these necrotrophs have long been thought to just simply kill hosts and complete their life cycles using nutrients derived mostly from dead plant tissues. This type of phytopathogen-plant interaction obviously does not comply with the gene-for-gene or inversed gene-for-gene relationship. The results in present study show that SsSSVP1 of S. sclerotiorum functions as an effector in pathogen-plant interactions. SsSSVP1 is dramatically induced during infection, and required for the full virulence of S. sclerotiorum. SsSSVP1 can be internalized by plant cells after being secreted from fungal cells in the absence of a pathogen during infection. Furthermore, SsSSVP1∆SP interacts with QCR8, a subunit of cytochrome b-c1 complex, and disturbs the localization of QCR8 in mitochondria, which may disable its biological function. The nonfunctionalization of QCR8 caused significant plant cell death. Hence, SsSSVP1 acts as an effector to manipulate the host cell physiology to facilitate the colonization of S. sclerotiorum. Obviously, this is a completely different interaction model from the gene-for-gene or inversed gene-for-gene paradigm. These findings suggest that the pathogenesis of S. sclerotiorum is more subtle and complex than previously appreciated and highlight the significance to investigate the interaction models between the host-non-specific necrotrophs and their hosts.

A transmissible disease of the plant pathogenic fungus Helminthosporium victoriae, the causal agent of Victoria blight of oats, was reported more than 50 years ago. Diseased, but not normal, isolates, of H. victoriae contain two distinct viruses designated according to their sedimentation values as victorivirus Helminthosporium victoriae virus 190S (HvV190S) and chrysovirus Helminthosporium victoriae 145S (HvV145S). Although a viral etiology of the disease was previously proposed, conclusive evidence was lacking. Here we present unequivocal evidence based on transfecting virus-free H. victoriae protoplasts with purified virus particles showing that HvV190S is essential for disease development. Furthermore, we show an expansion of the host range of HvV190S to include Cryphonectria parasitica and we also show similarity in a subset of phenotypic traits between HvV190S-infected RNA silencing deficient mutant (Δdcl-2) of C. parasitica and a strain of H. victoriae. In virulence assays on detached American chestnut branches and Red Delicious apple fruits, HvV190S-infected C. parasitica strain Δdcl-2 was markedly less virulent than wild type and virus-free Δdcl-2 C. parasitica strains. Furthermore, the hypovirulent HvV190S-infected C. parasitica Δdcl-2 strain exhibited strong antifungal activity in dual culture with the plant pathogenic fungus Sclerotinia sclerotiorum. No such inhibitory activity was observed in comparable dual cultures with wild type and virus-free Δdcl-2 C. parasitica strains. The discovery that infection with HvV190S induced a hypovirulent phenotype in a heterologous plant pathogenic host is very significant since it might be possible to convert other economically important plant pathogenic fungi to hypovirulence using HvV190S.

A transmissible disease of the plant pathogenic fungus Helminthosporium victoriae, the causal agent of Victoria blight of oats, was reported more than 50 years ago. Diseased, but not normal, isolates, of H. victoriae contain two distinct viruses designated according to their sedimentation values as victorivirus Helminthosporium victoriae virus 190S (HvV190S) and chrysovirus Helminthosporium victoriae 145S (HvV145S). Although a viral etiology of the disease was previously proposed, conclusive evidence was lacking. Here we present unequivocal evidence based on transfecting virus-free H. victoriae protoplasts with purified virus particles showing that HvV190S is essential for disease development. Furthermore, we show an expansion of the host range of HvV190S to include Cryphonectria parasitica and we also show similarity in a subset of phenotypic traits between HvV190S-infected RNA silencing deficient mutant (Δdcl-2) of C. parasitica and a strain of H. victoriae. In virulence assays on detached American chestnut branches and Red Delicious apple fruits, HvV190S-infected C. parasitica strain Δdcl-2 was markedly less virulent than wild type and virus-free Δdcl-2 C. parasitica strains. Furthermore, the hypovirulent HvV190S-infected C. parasitica Δdcl-2 strain exhibited strong antifungal activity in dual culture with the plant pathogenic fungus Sclerotinia sclerotiorum. No such inhibitory activity was observed in comparable dual cultures with wild type and virus-free Δdcl-2 C. parasitica strains. The discovery that infection with HvV190S induced a hypovirulent phenotype in a heterologous plant pathogenic host is very significant since it might be possible to convert other economically important plant pathogenic fungi to hypovirulence using HvV190S.

Three dsRNA segments, two similarly sized at 9.5kbp and a third one of approximately 3.6kbp, were extracted from a hypovirulent strain SZ-150 of Sclerotinia sclerotiorum. The complete cDNA sequence of one of the two large dsRNA segment (10398bp, excluding the poly (A) tail) reveals a single ORF that encodes a polyprotein with conserved domains of putative papain-like protease, UDP glucose/sterol glycosyltransferase, RNA-dependent RNA polymerase and viral RNA Helicase. This virus is closely related to Cryphonectria hypovirus (CHV) 3/GH2 and CHV4/SR2 in the family Hypoviridae and designated as Sclerotinia sclerotiorum hypovirus 1 (SsHV1/SZ-150). The satellite-like 3.6kbp dsRNA segment (S-dsRNA) shares high sequence identity with the 5′-UTR of SsHV1/SZ-150. SsHV1/SZ-150 alone is not the primary causal agent for hypovirulence of strain SZ-150 since strains without the S-dsRNA show normal phenotype. This is the first report of a naturally occurring hypovirus that infects a fungus other than Cryphonectria parasitica.

Mycoviruses are viruses that infect fungi and have the potential to control fungal diseases of crops when associated with hypovirulence. Typically, mycoviruses have double-stranded (ds) or single-stranded (ss) RNA genomes. No mycoviruses with DNA genomes have previously been reported. Here, we describe a hypovirulence-associated circular ssDNA mycovirus from the plant pathogenic fungus Sclerotinia sclerotiorum . The genome of this ssDNA virus, named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), is 2166 nt, coding for a replication initiation protein (Rep) and a coat protein (CP). Although phylogenetic analysis of Rep showed that SsHADV-1 is related to geminiviruses, it is notably distinct from geminiviruses both in genome organization and particle morphology. Polyethylene glycol-mediated transfection of fungal protoplasts was successful with either purified SsHADV-1 particles or viral DNA isolated directly from infected mycelium. The discovery of an ssDNA mycovirus enhances the potential of exploring fungal viruses as valuable tools for molecular manipulation of fungi and for plant disease control and expands our knowledge of global virus ecology and evolution.

Recent studies have shown that Sclerotinia sclerotiorum, an important plant pathogen fungus, harbors diverse mycoviruses. A new mycovirus, tentatively named as Sclerotinia sclerotiorum deltaflexivirus 1 (SsDFV1), was isolated from a S. sclerotiorum strain AX19 containing multiple dsRNA elements. The complete genome of SsDFV1 was shown to be 8178 nucleotides long excluding the poly (A) tail. SsDFV1 has a large putative open reading frame (ORF1) and three smaller ORFs (2-4). ORF1 encodes a putative methyltransferase-helicase-RdRp polyprotein of 2075 amino acids. ORFs (2-4) encode three putative small hypothetical proteins (

A variety of mycoviruses have been found in Sclerotinia sclerotiorum. In this study, we report a novel mycovirus Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1) that was originally isolated from the hypovirulent strain SCH941 of S. sclerotiorum. SsBRV1 has rigid spherical virions that are ~38 nm in diameter, and three dsRNA segments (dsRNA1, 2 and 3 with lengths of 6.4, 6.0 and 1.7 kbp, respectively) were packaged in the virions. dsRNA1 encodes a cap-pol fusion protein, and dsRNA2 encodes a polyprotein with unknown functions but contributes to the formation of virus particles. The dsRNA3 is dispensable and may be a satellite-like RNA (SatlRNA) of SsBRV1. Although phylogenetic analysis of the RdRp domain demonstrated that SsBRV1 is related to Botrytis porri RNA virus 1 (BpRV1) and Ustilago maydis dsRNA virus-H1 (UmV-H1), the structure proteins of SsBRV1 do not have any significant sequence similarities with other known viral proteins with the exception of those of BpRV1. SsBRV1 carrying dsRNA3 seems to have no obvious effects on the colony morphology, but can significantly reduce the growth rate and virulence of S. sclerotiorum. Notably, a growth hormone receptor binding domain (GHBP, Pfam12772) is detected in ORF2-encoded protein of SsBRV1, which have not been reported in any other viruses. These findings provide new insights into the virus taxonomy, virus evolution and the interactions between SsBRV1 and the fungal hosts.

Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, S clerotinia s clerotiorum m ega b irna v irus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum . Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5′-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1. IMPORTANCE Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus ( Sclerotinia sclerotiorum megabirnavirus 1 [SsMBV1]) that was isolated from an apparently hypovirulent strain, SX466, of Sclerotinia sclerotiorum . Although SsMBV1 is phylogenetically related to RnMBV1, SsMBV1 is markedly distinct from other reported megabirnaviruses with two features of VLPs and conserved domains. Our results convincingly showed that SsMBV1 is viable in the absence of L2-dsRNA/SsMBV1 (a potential large satellite-like RNA or genuine genomic virus component). More interestingly, we detected a conserved papain-like protease domain that commonly exists in ssRNA viruses, including members of the families Potyviridae and Hypoviridae . Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer might have occurred from an ssRNA virus to a dsRNA virus, which may provide new insights into the evolutionary history of dsRNA and ssRNA viruses.

Members of the family Partitiviridae have bisegmented double-stranded RNA (dsRNA) genomes and are not generally known to cause obvious symptoms in their natural hosts. An unusual partitivirus, Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), conferred hypovirulence on its natural plant-pathogenic fungal host, Sclerotinia sclerotiorum strain WF-1. Cellular organelles, including mitochondria, were severely damaged. Hypovirulence and associated traits of strain WF-1 and SsPV1/WF-1 were readily cotransmitted horizontally via hyphal contact to different vegetative compatibility groups of S. sclerotiorum and interspecifically to Sclerotinia nivalis and Sclerotinia minor . S. sclerotiorum strain 1980 transfected with purified SsPV1/WF-1 virions also exhibited hypovirulence and associated traits similar to those of strain WF-1. Moreover, introduction of purified SsPV1/WF-1 virions into strain KY-1 of Botrytis cinerea also resulted in reductions in virulence and mycelial growth and, unexpectedly, enhanced conidial production. However, virus infection suppressed hyphal growth of most germinating conidia of B. cinerea and was eventually lethal to infected hyphae, since very few new colonies could develop following germ tube formation. Taken together, our results support the conclusion that SsPV1/WF-1 causes hypovirulence in Sclerotinia spp. and B. cinerea . Cryo-EM (cryo-electron microscopy) reconstruction of the SsPV1 particle shows that it has a distinct structure with similarity to the closely related partitiviruses Fusarium poae virus 1 and Penicillium stoloniferum virus F. These findings provide new insights into partitivirus biological activities and clues about molecular interactions between partitiviruses and their hosts. IMPORTANCE Members of the Partitiviridae are believed to occur commonly in their phytopathogenic fungal and plant hosts. However, most partitiviruses examined so far appear to be associated with latent infections. Here we report a partitivirus, SsPV1/WF-1, that was isolated from a hypovirulent strain of Sclerotinia sclerotiorum and describe its biological and molecular features. We have demonstrated that SsPV1 confers hypovirulence. Furthermore, SsPV1 can infect and cause hypovirulence in Botrytis cinerea . Our study also suggests that SsPV1 has a vigorous ability to proliferate and spread via hyphal contact. SsPV1 can overcome vegetative incompatibility barriers and can be transmitted horizontally among different vegetative compatibility groups of S. sclerotiorum , even interspecifically. Cryo-EM reconstruction of SsPV1 shows that it has a distinct structure with similarity to closely related partitiviruses. Our studies exploit a novel system, SsPV1 and its hosts, which can provide the means to explore the mechanisms by which partitiviruses interact with their hosts.

Mycoviruses are widespread in nature and often occur with dsRNA and positive-stranded RNA genomes. Recently, strong evidence from RNA sequencing analysis suggested that negative-stranded (−)ssRNA viruses could infect fungi. Here we describe a (−)ssRNA virus, Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV-1), isolated from a hypovirulent strain of Sclerotinia sclerotiorum. The complete genome of SsNSRV-1 is 10,002 nt with six ORFs that are nonoverlapping and linearly arranged. Conserved gene-junction sequences that occur widely in mononegaviruses, (A/U)(U/A/C)UAUU(U/A)AA(U/G)AAAACUUAGG(A/U)(G/U), were identified between these ORFs. The analyses 5′ and 3′ rapid amplification of cDNA ends showed that all genes can be transcribed independently. ORF V encodes the largest protein that contains a conserved mononegaviral RNA-dependent RNA polymerase (RdRp) domain. Putative enveloped virion-like structures with filamentous morphology similar to members of Filoviridae were observed both in virion preparation samples and in ultrathin hyphal sections. The nucleocapsids are long, flexible, and helical; and are 22 nm in diameter and 200–2,000 nm in length. SDS/PAGE showed that the nucleocapsid possibly contains two nucleoproteins with different molecular masses, ∼43 kDa (p43) and ∼41 kDa (p41), and both are translated from ORF II. Purified SsNSRV-1 virions successfully transfected a virus-free strain of S. sclerotiorum and conferred hypovirulence. Phylogenetic analysis based on RdRp showed that SsNSRV-1 is clustered with viruses of Nyamiviridae and Bornaviridae. Moreover, SsNSRV-1 is widely distributed, as it has been detected in different regions of China. Our findings demonstrate that a (−)ssRNA virus can occur naturally in fungi and enhance our understanding of the ecology and evolution of (−)ssRNA viruses.

Partitiviruses constitute one of the nine currently recognized families of viruses with encapsidated, double-stranded (ds)RNA genomes. The partitivirus genome is bisegmented, and each genome segment is packaged inside a separate viral capsid. Different partitiviruses infect plants, fungi, or protozoa. Recent studies have at last shed light on the three-dimensional (3-D) structures of the virions of three, representative fungal partitiviruses. These structures include a number of distinctive features, allowing informative comparisons with the structures of dsRNA viruses from other families. The results and comparisons suggest several new conclusions about the functions, assembly, and evolution of these viruses.