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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 and Botrytis cinerea are typical necrotrophic pathogens that can attack more than 700 and 3000 plant species, respectively, and cause huge economic losses across numerous crops. In particular, the absence of resistant cultivars makes the stem rot because of S. sclerotiorum the major threat of rapeseed (Brassica napus) worldwide along with Botrytis. Previously, we identified an effector‐like protein (SsSSVP1) from S. sclerotiorum and a homologue of SsSSVP1 on B. cinerea genome and found that SsSSVP1 could interact with BnQCR8 of rapeseed, a subunit of the cytochrome b‐c1 complex. In this study, we found that BnQCR8 has eight homologous copies in rapeseed cultivar Westar and reduced the copy number of BnQCR8 using CRISPR/Cas9 to improve rapeseed resistance against S. sclerotiorum. Mutants with one or more copies of BnQCR8 edited showed strong resistance against S. sclerotiorum and B. cinerea. BnQCR8‐edited mutants did not show significant difference from Westar in terms of respiration and agronomic traits tested, including the plant shape, flowering time, silique size, seed number, thousand seed weight and seed oil content. These traits make it possible to use these mutants directly for commercial production. Our study highlights a common gene for breeding of rapeseed to unravel the key hindrance of rapeseed production caused by S. sclerotiorum and B. cinerea. In contrast to previously established methodologies, our findings provide a novel strategy to develop crops with high resistance against multiple pathogens by editing only a single gene that encodes the common target of pathogen effectors.

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.

Codon usage bias (CUB) could reflect co-evolutionary changes between viruses and hosts in contrast to plant and animal viruses, and the systematic analysis of codon usage among the mycoviruses that infect plant pathogenic fungi is limited. We performed an extensive analysis of codon usage patterns among 98 characterized RNA mycoviruses from eight phytopathogenic fungi. The GC and GC3s contents of mycoviruses have a wide variation from 29.35% to 64.62% and 24.32% to 97.13%, respectively. Mycoviral CUB is weak, and natural selection plays a major role in the formation of mycoviral codon usage pattern. In this study, we demonstrated that the codon usage of mycoviruses is similar to that of some host genes, especially those involved in RNA biosynthetic process and transcription, suggesting that CUB is a potential evolutionary mechanism that mycoviruses adapt to in their hosts.

Plasmodiophora brassicae is an important biotrophic eukaryotic plant pathogen and a member of the rhizarian protists. This biotrophic pathogen causes clubroot in cruciferous plants via novel intracellular mechanisms that are markedly different from those of other biotrophic organisms. To date, genomes from six single spore isolates of P. brassicae have been sequenced. An accurate description of the evolutionary status of this biotrophic protist, however, remains lacking. Here, we determined the draft genome of the P. brassicae ZJ-1 strain. A total of 10,951 protein-coding genes were identified from a 24.1 Mb genome sequence. We applied a comparative genomics approach to prove the Rhizaria supergroup is an independent branch in the eukaryotic evolutionary tree. We also found that the GPCR signaling pathway, the versatile signal transduction to multiple intracellular signaling cascades in response to extracellular signals in eukaryotes, is significantly enriched in P. brassicae-expanded and P. brassicae-specific gene sets. Additionally, treatment with a GPCR inhibitor relieved the symptoms of clubroot and significantly suppressed the development of plasmodia. Our findings suggest that GPCR signal transduction pathways play important roles in the growth, development, and pathogenicity of P. brassicae.

Background The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. Results Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. Conclusions Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

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.

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.

Botrytis cinerea, a ubiquitous necrotrophic plant-pathogenic fungus, is responsible for grey mold and rot disease in a very wide range of plant species. Subtilisin-like proteases (or subtilases) are a very diverse family of serine proteases present in many organisms and are reported to have a broad spectrum of biological functions. Here, we identified two genes encoding subtilisin-like proteases (Bcser1 and Bcser2) in the genome of B. cinerea, both of which contain an inhibitor I9 domain and a peptidase S8 domain. The expression levels of Bcser1 and Bcser2 increased during the sclerotial forming stage, as well as during a later stage of hyphal infection on Arabidopsis thaliana leaves, but the up-regulation of Bcser1 was significantly higher than that of Bcser2. Interestingly, deletion of Bcser1 had no effect on the fungal development or virulence of B. cinerea. However, deletion of Bcser2 or double deletion of Bcser1 and Bcser2 severely impaired the hyphal growth, sclerotial formation and conidiation of B. cinerea. We also found that ∆Bcser2 and ∆Bcser1/2 could not form complete infection cushions and then lost the ability to infect intact plant leaves of Arabidopsis and tomato but could infect wounded plant tissues. Taken together, our results indicate that the subtilisin-like protease Bcser2 is crucial for the sclerotial formation, conidiation, and virulence of B. cinerea.

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.

Botrytis cinerea, a ubiquitous necrotrophic plant-pathogenic fungus, is responsible for grey mold and rot disease in a very wide range of plant species. Subtilisin-like proteases (or subtilases) are a very diverse family of serine proteases present in many organisms and are reported to have a broad spectrum of biological functions. Here, we identified two genes encoding subtilisin-like proteases (Bcser1 and Bcser2) in the genome of B. cinerea, both of which contain an inhibitor I9 domain and a peptidase S8 domain. The expression levels of Bcser1 and Bcser2 increased during the sclerotial forming stage, as well as during a later stage of hyphal infection on Arabidopsis thaliana leaves, but the up-regulation of Bcser1 was significantly higher than that of Bcser2. Interestingly, deletion of Bcser1 had no effect on the fungal development or virulence of B. cinerea. However, deletion of Bcser2 or double deletion of Bcser1 and Bcser2 severely impaired the hyphal growth, sclerotial formation and conidiation of B. cinerea. We also found that ∆Bcser2 and ∆Bcser1/2 could not form complete infection cushions and then lost the ability to infect intact plant leaves of Arabidopsis and tomato but could infect wounded plant tissues. Taken together, our results indicate that the subtilisin-like protease Bcser2 is crucial for the sclerotial formation, conidiation, and virulence of B. cinerea.

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.

Background The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. Results Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. Conclusions Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae. Electronic supplementary material The online version of this article (10.1186/s12864-018-5307-4) contains supplementary material, which is available to authorized users.

Chromatin remodeling is mediated in part by post-translational acetylation/deacetylation modifications of histones. Histone acetyltransferases (HATs), e.g., members of the GNAT/MYST superfamily, activate gene transcription via promotion of euchromatin formation. Here, we characterized a GNAT family HAT, Spt10 (BbSpt10), in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. Targeted gene knockout of BbSpt10 resulted in impaired asexual development and morphogenesis, reduced abilities to utilize various carbon/nitrogen sources, reduced tolerance to heat, fungicides, and DNA damage stress, and attenuated virulence. The ΔBbSpt10 mutant showed disrupted cell cycle development and abnormal hyphal septation patterns. Transcriptome analyses of wild type and ΔBbSpt10 cells revealed the differential expression of 373 genes, including 153 downregulated and 220 upregulated genes. Bioinformatic analyses revealed downregulated genes to be enriched in pathways involved in amino acid metabolism, cellular transportation, cell type differentiation, and virulence, while upregulated genes were enriched in carbon/nitrogen metabolism, lipid metabolism, DNA process, and cell rescue, defense, and virulence. Downregulated virulence genes included hydrophobins, cellular transporters (ABC and MFS multidrug transporters) and cytochrome P450 detoxification genes. These data indicated broad effects of BbSpt10 on fungal development, multi-stress response, and virulence.

Summary Cerato-platanin proteins (CPs), which are secreted by filamentous fungi, are phytotoxic to host plants, but their functions have not been well defined to date. Here we characterized a CP (SsCP1) from the necrotrophic phytopathogen Sclerotinia sclerotiorum. Sscp1 transcripts accumulated during plant infection, and deletion of Sscp1 significantly reduced virulence. SsCP1 could induce significant cell death when expressed in Nicotiana benthamiana. Using yeast two-hybrid, GST pull-down, co-immunoprecipitation and bimolecular florescence complementation, we found that SsCP1 interacts with PR1 in the apoplast to facilitate infection by S. sclerotiorum. Overexpressing PR1 enhanced resistance to the wild-type strain, but not to the Sscp1 knockout strain of S. sclerotiorum. Sscp1-expressing transgenic plants showed increased concentrations of salicylic acid (SA) and higher levels of resistance to several plant pathogens (namely Botrytis cinerea, Alternaria brassicicola and Golovinomyces orontii). Our results suggest that SsCP1 is important for virulence of S. sclerotiorum and that it can be recognized by plants to trigger plant defense responses. Our results also suggest that the SA signaling pathway is involved in CP-mediated plant defense .

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.

Sclerotinia sclerotiorum, a notorious soil-borne pathogen of various important crops, produces numerous sclerotia to oversummer in the soil. Considering that sclerotia may also be attacked by other microbes in the soil, we hypothesized that sclerotia in soil may affect the community of soil microbes directly and/or indirectly. In this study, we inoculated sclerotia of S. sclerotiorum in soil collected from the field to observe changes in microbial diversity over three months using 16S rRNA and ITS2 sequencing techniques. Alpha diversity indices exhibited a decline in the diversity of microbial communities, while permanova results confirmed a significant difference in the microbial communities of sclerotia-amended and non-amended soil samples. In sclerotia-amended soil, fungal diversity showed enrichment of antagonists such as Clonostachys, Trichoderma, and Talaromyces and a drastic reduction in the plant pathogenic microbes compared to the non-amended soil. Sclerotia not only activated the antagonists but also enhanced the abundance of plant growth-promoting bacteria, such as Chitinophaga, Burkholderia, and Dyella. Moreover, the presence of sclerotia curtailed the growth of several notorious plant pathogenic fungi belonging to various genera such as Fusarium, Colletotrichum, Cladosporium, Athelia, Alternaria, and Macrophomina. Thus, we conclude that S. sclerotiorum when dormant in soil can reduce the diversity of soil microbes, including suppressing plant pathogens and enriching beneficial microbes. To the best of our knowledge, this is the first time a plant pathogen has been found in soil that can significantly suppress other pathogens. Our findings may provide novel cues to understand the ecology of crop pathogens in soil and maintaining soil conditions that could be beneficial for constructing a healthy soil microorganism community required for mitigating soil-borne diseases.

Mitogen-activated protein kinase (MAPK) cascades play a central role in cellular growth, proliferation, and survival. MAPK cascade genes have been extensively investigated in model plants, mammals, yeast, and fungi but are not characterized in Plasmodiophora brassicae, which causes clubroot disease in cruciferous plants. Here, we identified 7 PbMAPK, 3 PbMAPKK, and 9 PbMAPKKK genes in the P. brassicae genome. Transcriptional profiling analysis demonstrated that several MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) genes were preferentially expressed in three different zoosporic stages. Based on yeast two-hybrid assays, PbMAKKK7 interacted with PbMAKK3 and PbMAKK3 interacted with PbMAK1/PbMAK3. The PbMAKKK7-PbMAKK3-PbMAK1/PbMAK3 cascade may be present in P. brassicae. U0126, a potent and specific inhibitor of MAPKK, could inhibit the germination of P. brassicae resting spores. U0126 was used to treat the resting spores of P. brassicae and coinoculate rapeseed, and was proven to significantly relieve the severity of clubroot symptoms in the host plant and delay the life cycle of P. brassicae. These results suggest that MAPK signaling pathways may play important roles in P. brassicae growth, development, and pathogenicity.

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.

Clubroot caused by Plasmodiophora brassicae, is a severe disease of cruciferous crops that causes large hypertrophic galls in the roots. The plant microbiome is important for growth promotion and disease suppression. In this study, using 16S rRNA and internal transcribed spacer (ITS) sequencing techniques, we compared the endosphere microbiome of symptomatic and asymptomatic B. napus roots infected with P. brassicae collected from the same natural clubroot field. The results showed that the microbial population and its relative abundance in the asymptomatic roots was far higher than that in the symptomatic roots, and that many microorganisms in asymptomatic roots have biological control and plant growth promotion functions that may be related to clubroot symptoms. These results suggest the importance of the endosphere microbiome in clubroot disease and provide potential bio-control resources for its prevention.

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.

Clubroot disease is a serious threat to cruciferous plants worldwide, especially to oilseed rape. However, knowledge on pathogenic molecular mechanisms and host interaction is limited. We presume that the recognition between Arabidopsis thaliana and Plasmodiophora brassicae occurs at the early stage of infection and within a relatively short period. In this study, we demonstrated changes on gene expression and pathways in A. thaliana during early infection with P. brassicae using transcriptome analysis. We identified 1903 and 1359 DEGs at 24 and 48 hours post inoculation (hpi), respectively. Flavonoids and the lignin synthesis pathways were enhanced, glucosinolates, terpenoids and proanthocyanidins accumulated and many hormonal- and receptor-kinase related genes were expressed, caused by P. brassicae infection during its early phase. Therefore, the early interaction between A. thaliana and P. brassicae plays an important role in the entire infection process. The results provide a new contribution to a better understanding of the interaction between host plants and P. brassicae, as well as the development of future measures for the prevention of clubroot.

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.

Plasmodiophora brassicae causes clubroot disease in cruciferous. In this report, lipid droplets were observed in the resting spores of P. brassicae. 295 lipid droplet-associated proteins were identified and categorized into nine groups. Transcriptome analysis of these proteins during three different zoosporic stages revealed differences in gene expression pattern. GO enrichment analysis revealed that these proteins associated with lipid droplets were mainly linked to biosynthesis and metabolism. GC-MS analysis revealed that lipid droplets contain seven types of free fatty acids: saturated fatty acids C16:0 and C18:0, and unsaturated fatty acids C18:1Δ9, C18:1Δ11, C18:2, C20:4 and C20:5. P. brassicae accumulated a large amount of triacylglycerols (TAGs). We systematically analyzed the putative proteins involved in TAG biosynthesis and its metabolic pathway. KEGG pathway analysis defined 3390 genes, including 167 genes involved in lipid metabolism. Transcriptome analysis revealed that 162 candidate enzymes involved in lipid metabolism were differential expressed. Our omics studies are the first to investigate the lipid droplet organelles in P. brassicae, providing a reference resource to study protist lipid droplets.

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.

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.

Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1 and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2 and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 (non-expresser of PR genes that regulate systemic acquired resistance (SAR)) mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms enhance the resistance to clubroot disease.

Histone methylation is widely present in animals, plants and fungi, and the methylation modification of histone H3 has important biological functions. Methylation of Lys9 of histone H3 (H3K9) has been proven to regulate chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. In this work, we investigated the functions of a H3K9 methyltransferase gene BcDIM5 in Botrytis cinerea, which contains a PreSET domain, a SET domain and a PostSET domain. Characterization of BcDIM5 knockout transformants showed that the hyphal growth rate and production of conidiophores and sclerotia were significantly reduced, while complementary transformation of BcDIM5 could restore the phenotypes to the levels of wild type. Pathogenicity assays revealed that BcDIM5 was essential for full virulence of B. cinerea. BcDIM5 knockout transformants exhibited decreased virulence, down-regulated expression of some pathogenic genes and drastically decreased H3K9 trimethylation level. However, knockout transformants of other two genes heterochromatin protein 1 (HP1) BcHP1 and DNA methyltransferase (DIM2) BcDIM2 did not exhibit significant change in the growth phenotype and virulence compared with the wild type. Our results indicate that H3K9 methyltransferase BcDIM5 is required for H3K9 trimethylation to regulate the development and virulence of B. cinerea.

Clubroot disease is a serious threat to cruciferous plants worldwide, especially to oilseed rape. However, knowledge on pathogenic molecular mechanisms and host interaction is limited. We presume that the recognition between Arabidopsis thaliana and Plasmodiophora brassicae occurs at the early stage of infection and within a relatively short period. In this study, we demonstrated changes on gene expression and pathways in A. thaliana during early infection with P. brassicae using transcriptome analysis. We identified 1,903 and 1,359 DEGs at 24 and 48 h post-inoculation (hpi), respectively. Flavonoids and the lignin synthesis pathways were enhanced, glucosinolates, terpenoids, and proanthocyanidins accumulated and many hormonal- and receptor-kinase related genes were expressed, caused by P. brassicae infection during its early phase. Therefore, the early interaction between A. thaliana and P. brassicae plays an important role in the entire infection process. The results provide a new contribution to a better understanding of the interaction between host plants and P. brassicae, as well as the development of future measures for the prevention of clubroot.

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.

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.

This paper reports the isolation from soil of Penicillium strain PY-1 with strong antagonistic activity against plant pathogenic fungi. On the basis of its morphological characteristics and the sequence of the ITS region, strain PY-1 was identified as P. oxalicum. Strain PY-1 produces antifungal substances that suppress the mycelial growth of Sclerotinia sclerotiorum and many other plant pathogenic fungi tested; the highest antagonistic activity was detected at 72 h when cultured in a 250-ml flask containing 80 ml potato dextrose broth. Compared with carbendazim, the relative activity of the antifungal substances produced by strain PY-1 was approximately 4 μg active ingredient (a.i.) per milliliter. The antifungal substances were extracted with ethyl acetate and further separated by high-performance liquid chromatography (HPLC); at least two active components were discovered. The ability to control plant disease with strain PY-1 was confirmed with S. sclerotiorum, a widespread pathogenic fungus that attacks rapeseed (Brassica napus) and other plants. Spores (106 or 107 ml−1) and filtrate (tenfold diluted or undiluted) of strain PY-1 could significantly suppress infection and/or the extent of infection by S. sclerotiorum of plants at seven-true-leaves stage. The potential of strain PY-1 for identifying new antibiotics to control fungal disease and for biological control of plant disease, for example oilseed rape stem rot, is discussed.

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.

Mycoviruses are viruses that infect fungi. A growing number of novel mycoviruses have expanded our knowledge of virology, particularly in taxonomy, ecology, and evolution. Recent progress in the study of mycoviruses has comprehensively improved our understanding of the properties of mycoviruses and has strengthened our confidence to explore hypovirulence-associated mycoviruses that control crop diseases. In this review, the advantages of using hypovirulence-associated mycoviruses to control crop diseases are discussed, and, as an example, the potential for Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) to control the stem rot of rapeseed (Brassica napus) is also introduced. Fungal vegetative incompatibility is likely to be the key factor that limits the wide utilization of mycoviruses to control crop diseases; however, there are suggested strategies for resolving this problem.

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.

Mycoviruses are thought not to be infectious as free particles and to lack an extracellular phase in their life cycles, limiting the broad use of hypovirulence-associated mycoviruses in controlling fungal disease. Here, we demonstrate that purified particles of a DNA mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), are infectious when applied extracellularly to its host Sclerotinia sclerotiorum . Virus particles isolated from an infected host can infect the hyphae of virus-free S. sclerotiorum directly when applied to hyphae grown on potato dextrose agar or sprayed on leaves of Arabidopsis thaliana and Brassica napus , regardless of vegetative compatibility affiliation. When applied to leaves, the virus can suppress the development of lesions. SsHADV-1 can also reduce disease severity and enhance rapeseed yield significantly under field conditions. SsHADV-1 has a narrow host range; it can infect Sclerotinia minor and Sclerotinia nivalis , sister species of S. sclerotiorum , and cause debilitation of these two fungi, but cannot infect or transfect other tested fungi, such as Botrytis cinerea , which shares the same family with S. sclerotiorum . Virus particles are likely to be very stable on the leaves of A. thaliana plants because viral DNA could be detected at 15 d postinoculation on unwounded leaves and at 10 d postinoculation on wounded leaves, respectively; however, this virus could not infect and move in plant cells. Our findings may prompt a reconsideration of the generalization that mycoviruses lack an extracellular phase in their life cycles and stimulate the search for other DNA mycoviruses with potential use as natural fungicides.

Background Sclerotinia sclerotiorum is a notorious plant fungal pathogen which spreads across the world. Hypovirulence is a phenomenon where the virulence of fungal pathogens is decreased, even lost, due to mycovirus infection. The potential of hypoviruses for biological control of the chestnut blight fungus (Cryphonectria parasitica) has attracted much interest, and has led to discovery of new hypovirulent strains in other fungi. Results A hypovirulent strain, strain XG36-1, was isolated from a typical lesion on the stem of rapeseed (Brassica napus) caused by Sclerotinia sclerotiorum. Strain XG36-1 grew on PDA very slowly (average 2.5 ± 0.1 mm/d) with sectoring, and developed abnormal colony morphology with few sclerotia. Unlike health strains (such as wildtype strain XG-13), it was unable to induce lesions on detached leaves of rapeseed. Sclerotia of strain XG36-1 produced apothecia rarely. A sexual progeny test showed that the phenotypes of all 104 sexual progeny were not different from wildtype strain XG-13 which shows normal phenotype of S. sclerotiorum, and protoplast regeneration tests showed that 25.5% of the regenerants of strain XG36-1 were recovered fully. Furthermore, the hypovirulence and its associated traits could be transmitted to XG36-1A34 R , a hygromycin-resistance gene labelled sexual progeny of strain XG36-1, by hyphal anastomosis. Transmission electron microscope (TEM) observation showed that the cytoplasm of strain XG36-1 was destroyed and granulated; the membranes of nuclei and mitochondria were disintegrated; and mitochondrial cristae were cavitated. Viral particles (about 40 nm) in hyphae of strain XG36-1, but not in its sexual progeny and wildtype strain XG-13, could be observed with TEM, and several virus-like particles were uniquely enveloped by single layer membrane in the cells of strain XG36-1. Furthermore, the viral particles could be co-transmitted with the hypovirulence traits through hyphal anastomosis. Conclusion Hypovirulence and its associated traits of strain XG36-1 could be mediated by a fungal virus. Currently, we could not know the characteristic of this virus, but it likely represent a new type of mycovirus in S. sclerotiorum, and possibly in fungi.