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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.

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.

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.

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to help safeguard such resources for increasing human populations on a global scale. Here, we find that Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, rice, barley, maize, and oat, providing protection against Fusarium head blight, stripe rust, and rice blast. Protection is also provided by disabled S. sclerotiorum strains harboring a hypovirulence virus. The disabled strain DT-8 promoted wheat yields by 4–18% in the field and consistently reduced Fusarium disease by 40–60% across multiple field trials. We term the host-dependent trophism of S. sclerotiorum, destructively pathogenic or mutualistically endophytic, as schizotrophism. As a biotroph, S. sclerotiorum modified the expression of wheat genes involved in disease resistance and photosynthesis and increased the level of IAA. Our study shows that a broad-spectrum pathogen of one group of plants may be employed as a biocontrol agent in a different group of plants where they can be utilized as beneficial microorganisms while avoiding the risk of in-field release of pathogens. Our study also raises provocative questions about the potential role of schizotrophic endophytes in natural ecosystems.

Sclerotial development is a vital stage in the life cycles of many fungal plant pathogens. In this study, the protein Ss-CAD, which contains three conserved domains of cinnamyl alcohol dehydrogenase (CAD), was found to be required for sclerotial development in Sclerotinia sclerotiorum. Ss-CAD was significantly upregulated during early stage of sclerotial development. In Ss-CAD-silenced strains, sclerotial development was abnormal. In these silenced strains, formation of sclerotia was delayed or sclerotia yield was reduced, whereas hyphal growth and virulence were normal. Nox1, Nox2, and NoxR, which encode reactive oxygen species (ROS)-generating NADPH oxidases, were downregulated in Ss-CAD-silenced strains. NoxR-silenced strains displayed similar defects during sclerotial formation as Ss-CAD-silenced strains. Treatment of Ss-CAD-silenced strains with exogenous oxidants or NADPH restored normal sclerotial development. Sclerogenesis in Ss-CAD-silenced strains could also be recovered through Nox1 overexpression. The results suggest that Ss-CAD is linked to the NADPH oxidase pathways to affect sclerotial development in S. sclerotiorum.

The symbiosis of endophytes and plants is universal in nature. However, how endophytes grow in plants is not entirely clear. Previously, we reported that a virus-infected fungal pathogen could grow in plants as an endophyte. In this study, we utilized Sclerotinia sclerotiorum strain DT-8, a virus-mediated endophyte, to investigate the mechanism of symbiosis with rapeseed by dual unique molecular identifier-RNA sequencing (dual-UMI RNA-seq). We found that the expressions of genes encoding S. sclerotiorum amylase/glucoamylase, glucose transporters, and rapeseed s ugars w ill e ventually be e xported t ransporter 11 (SWEET11) were upregulated. It suggested that strain DT-8 might utilize plant starch as a nutrient. The defense systems of rapeseed were also activated, such as production of reactive oxygen species, phenylpropanoids, and brassinin, to control the growth of strain DT-8, while strain DT-8 counteracted host suppression by producing effector-like proteins, detoxification enzymes, and antioxidant components. Moreover, rapeseed also upregulated pectate lyase and pectinesterase genes to facilitate the colonization by strain DT-8. Our findings provide novel insights into the interaction of virus-mediated endophytes and their hosts that warrant further study. IMPORTANCE Although endophytes are widespread in nature, the interactions between endophytes and their hosts are still not fully understood. Members of a unique class of endophytes, the virus-mediated endophytic fungi, are continuously being discovered and have received wide attention. In this study, we investigated the interaction between a mycovirus-mediated endophytic fungus and its host rapeseed by using dual-UMI RNA-seq. According to the dual-UMI RNA-seq results, an aerial view of symbiotic mechanism under balanced regulation was suggested. This research expands our understanding of the symbiotic mechanisms of virus-fungus-plant interactions and could establish a foundation for the further development of practical application with virus-mediated hypovirulent 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.

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum.

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum.

Summary Sclerotinia sclerotiorum is a devastating necrotrophic fungal pathogen that infects over 400 species of plants worldwide. Reactive oxygen species (ROS) modulations are critical for the pathogenic development of S. sclerotiorum. The fungus applies enzymatic and non‐enzymatic antioxidants to cope with the oxidative stress during the infection processes. Survival factor 1 was identified and characterized to promote survival under conditions of oxidative stress in Saccharomyes cerevisiae. In this research, a gene named SsSvf1 was predicted to encode a survival factor 1 homologue in S. sclerotiorum. SsSvf1 transcripts showed high expression levels in hyphae under oxidative stress. Silencing of SsSvf1 resulted in increased sensitivity to oxidative stress in culture and increased levels of intracellular ROS. Transcripts of SsSvf1 showed a dramatic increase during the initial stage of infection and the gene‐silenced strains displayed reduced virulence on oilseed rape and Arabidopsis thaliana. Inhibition of plant ROS production partially restores virulence of SsSvf1 gene‐silenced strains. SsSvf1 gene‐silenced strains exhibited normal oxalate production, but were impaired in compound appressorium formation and cell wall integrity. The results suggest that SsSvf1 is involved in coping with ROS during fungal‐host interactions and plays a crucial role in the pathogenicity of S. sclerotiorum.

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.

Plant disease is one of the most important causes of crop losses worldwide. The effective control of plant disease is related to food security. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum leads to serious yield losses in rapeseed (Brassica napus) production. Hypovirulent strain DT-8 of S. sclerotiorum, infected with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), has the potential to control SSR. In this study, we found rapeseed bio-priming with strain DT-8 could significantly decrease the disease severity of SSR and increase yield in the field. After bio-priming, strain DT-8 could be detected on the aerial part of the rapeseed plant. By 16S rRNA gene and internal transcribed spacer (ITS) sequencing technique, the microbiome on different parts of the SSR lesion on bioprimed and non-bioprimed rapeseed stem was determined. The results indicated that SSR and bio-priming treatment could influence the structure and composition of fungal and bacterial communities. Bio-priming treatment could reduce the total abundance of possible plant pathogens and enhance the connectivity and robustness of the interaction network at the genus level. This might be one of the mechanisms that rapeseed bioprimed with strain DT-8 had excellent tolerance on SSR. It might be another possible mechanism of biocontrol and will provide a theoretical guide for agricultural practical production.

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.

Coniothyrium minitans is a mycoparasite of the notorious plant pathogen Sclerotinia sclerotiorum. To further understand the parasitism of C. minitans, we assembled and analysed its genome and performed transcriptome analyses. The genome of C. minitans strain ZS-1 was assembled into 350 scaffolds and had a size of 39.8 Mb. A total of 11 437 predicted genes and proteins were annotated, and 30.8 % of the blast hits matched proteins encoded by another member of the Pleosporales, Paraphaeosphaeria sporulosa, a worldwide soilborne fungus with biocontrol ability. The transcriptome of strain ZS-1 during the early interaction with S. sclerotiorum at 0, 4 and 12 h was analysed. The detected expressed genes were involved in responses to host defenses, including cell-wall-degrading enzymes, transporters, secretory proteins and secondary metabolite productions. Seventeen differentially expressed genes (DEGs) of fungal cell-wall-degrading enzymes (FCWDs) were up-regulated during parasitism, with only one down-regulated. Most of the monocarboxylate transporter genes of the major facilitator superfamily and all the detected ABC transporters, especially the heavy metal transporters, were significantly up-regulated. Approximately 8 % of the 11 437 proteins in C. minitans were predicted to be secretory proteins with catalytic activity. In the molecular function category, hydrolase activity, peptidase activity and serine hydrolase activity were enriched. Most genes involved in serine hydrolase activity were significantly up-regulated. This genomic analysis and genome-wide expression study demonstrates that the mycoparasitism process of C. minitans is complex and a broad range of proteins are deployed by C. minitans to successfully invade its host. Our study provides insights into the mechanisms of the mycoparasitism between C. minitans and S. sclerotiorum and identifies potential secondary metabolites from C. minitans for application as a biocontrol agent.

Coniothyrium minitans is a mycoparasite of the notorious plant pathogen Sclerotinia sclerotiorum. To further understand the parasitism of C. minitans, we assembled and analysed its genome and performed transcriptome analyses. The genome of C. minitans strain ZS-1 was assembled into 350 scaffolds and had a size of 39.8 Mb. A total of 11 437 predicted genes and proteins were annotated, and 30.8 % of the blast hits matched proteins encoded by another member of the Pleosporales, Paraphaeosphaeria sporulosa, a worldwide soilborne fungus with biocontrol ability. The transcriptome of strain ZS-1 during the early interaction with S. sclerotiorum at 0, 4 and 12 h was analysed. The detected expressed genes were involved in responses to host defenses, including cell-wall-degrading enzymes, transporters, secretory proteins and secondary metabolite productions. Seventeen differentially expressed genes (DEGs) of fungal cell-wall-degrading enzymes (FCWDs) were up-regulated during parasitism, with only one down-regulated. Most of the monocarboxylate transporter genes of the major facilitator superfamily and all the detected ABC transporters, especially the heavy metal transporters, were significantly up-regulated. Approximately 8 % of the 11 437 proteins in C. minitans were predicted to be secretory proteins with catalytic activity. In the molecular function category, hydrolase activity, peptidase activity and serine hydrolase activity were enriched. Most genes involved in serine hydrolase activity were significantly up-regulated. This genomic analysis and genome-wide expression study demonstrates that the mycoparasitism process of C. minitans is complex and a broad range of proteins are deployed by C. minitans to successfully invade its host. Our study provides insights into the mechanisms of the mycoparasitism between C. minitans and S. sclerotiorum and identifies potential secondary metabolites from C. minitans for application as a biocontrol agent.

SsITL, a secretory protein of the necrotrophic phytopathogen Sclerotinia sclerotiorum, was previously reported to suppress host immunity at the early stages of infection. However, the molecular mechanism that SsITL uses to inhibit plant defence against S. sclerotiorum has not yet been elucidated. Here, we report that SsITL interacted with a chloroplast‐localized calcium‐sensing receptor, CAS, in chloroplasts. We found that CAS is a positive regulator of the salicylic acid signalling pathway in plant immunity to S. sclerotiorum and CAS‐mediated resistance against S. sclerotiorum depends on Ca2+ signalling. Furthermore, we showed that SsITL could interfere with the plant salicylic acid (SA) signalling pathway and SsITL‐expressing transgenic plants were more susceptible to S. sclerotiorum. However, truncated SsITLs (SsITL‐NT1 or SsITL‐CT1) that lost the ability to interact with CAS do not affect plant resistance to S. sclerotiorum. Taken together, our findings reveal that SsITL inhibits SA accumulation during the early stage of infection by interacting with CAS and then facilitating the infection by S. sclerotiorum., The secretory protein SsITL inhibits salicylic acid accumulation during the early stage of infection by interacting with CAS (calcium‐sensing receptor) and then facilitating S. sclerotiorum infection.

Coniothyrium minitans is an important mycoparasite of the notorious phytopathogenic fungus Sclerotinia sclerotiorum. The mycoparasitism system of C. minitans-S. sclerotiorum is unique and important in probing fungi and fungal interactions. Here, we report a conidiation-deficient mutant, ZS-1TN1961, which was screened from a transfer DNA (T-DNA) insertional library of C. minitans. A single-copy gene, encoding a protein with high sequence similarity to Aim24 (altered inheritance of mitochondria protein 24) in Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Gene replacement and complementation experiments confirmed that mutants lacking CmAim24 exhibited significantly reduced conidial production and germination as well as reduced sclerotial mycoparasitic ability. Furthermore, cellular localization assays showed that CmAim24 localized to mitochondria, and abnormal mitochondria were observed in the ΔCmAim24 mutant. The ΔCmAim24 mutant exhibited significant accumulation of reactive oxygen species (ROS) and a reduced ATP content in mycelia. In summary, our results suggest that CmAim24 plays a key role in mitochondrial architecture and function, conidiogenesis, and mycoparasitism in C. minitans. IMPORTANCE Aim24 proteins are involved in mitochondrial biogenesis and accumulate between the two membranes of a mitochondrion. Their function in prokaryotes and filamentous fungi is as yet unknown. In the present study, we characterized an Aim24 protein, CmAim24, in the mycoparasite Coniothyrium minitans and proved its critical role in mitochondrial morphology and function, conidiogenesis, conidial germination, and mycoparasitism to S. sclerotiorum.

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.

Mycoparasite Coniothyrium minitans parasitizes specifically the mycelia or sclerotia of Sclerotinia sclerotiorum, a worldwidely spread plant fungal pathogen causing serious diseases on crops. The interaction of C. minitans with S. sclerotiorum remains reciprocal and complex and little is known, especially on the side of the host (S. sclerotiorum). In this study, the early transcriptional response of S. sclerotiorum to the mycoparasitism by C. minitans was explored and the differentially expressed genes (DEGs) were analyzed. Based on GO ontology, KEGG pathway and fungal categories database, 887 up-regulated DEGs were enriched in the growth related function (i.e., rRNA processing, ribosome biogenesis, binding and transport), while the 546 down-regulated DEGs were enriched in the stress-related functions (i.e., oxidoreductase, response to stress and heat and the chorismate biosynthetic process). The expression of shikimate pathway and the biosynthesis of phenylalanine involving genes was significantly suppressed. Furthermore, 581 unenriched DEGs were explored in the parasitizing process and were mapped on the Pfam domains of redox enzymes, Alpha/Beta hydrolase, haloacid dehalogenase, and other universal conserved domain containing proteins. Thirty-two DEGs encoding candidate effectors, with 16 up-regulated and 16 down-regulated, were observed with diverse function. SS1G_11912 (encoding SsNEP2) was significantly up-regulated and may function in the parasitism. The involving of the shikimate pathway of phenylalanine biosynthesis and effector candidates were discussed. The results provide a basal understand on the interaction of S. sclerotiorum and C. minitans.

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 .

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.

BackgroundConiothyrium minitans is a mycoparasite of the notorious plant pathogen Sclerotinia sclerotiorum. To further understand the parasitism of C. minitans, here, we assembled and analyzed its genome by combining transcriptome data.ResultsThe genome of C. minitans strain ZS-1 was 39.77 Mb in 350 scaffolds. A total of 11437 predicted genes and proteins were annotated, and 30.8% of blast hits matched proteins encoded by Paraphaeosphaeria sporulosa, a worldwide soil fungus. The transcriptome of strain ZS-1 during the early interaction at 0 h, 4 h and 12 h with its host was analyzed. The detected expressed genes were involved in response to host defenses, including cell wall-degrading enzymes, transporters, secretory proteins and secondary metabolites. The fungal cell wall-degrading enzymes belonged to the GH16, GH18, and GH72 classes in CAZymes, and some were significantly up-regulated during mycoparasitism. Most of the monocarboxylate transporter genes of the major facilitator superfamily and all the detected ABC transporters, especially the heavy metal transporters, were significantly up-regulated. Approximately 8% of the 11437 proteins in C. minitans were predicted to be secretory proteins, with catalytic activity, hydrolase activity, peptidase activity and serine hydrolase activity enriched in molecular function. Most genes involved in serine hydrolase activity were significantly up-regulated during mycoparasitism.ConclusionThis assemble genome and genome-wide expression study demonstrate that the mycoparasitism process of C. minitans is complex and a series of genes or proteins would be deployed by C. minitans to invade successfully the host. Our study provides insights into the mechanisms of the mycoparasitism between C. minitans and S. sclerotiorum and clues to excavate active secondary metabolites from C. minitans.

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.

Coniothyrium minitans is an important sclerotial and hyphal parasite of the plant pathogen Sclerotinia sclerotiorum. Previously, a conidiation-deficient mutant, ZS-1N22225, was screened from a T-DNA insertional library of C. minitans. CmVps39, a homologue of Vam6p/Vps39p that plays a critical role in vacuolar morphogenesis in yeast, was disrupted by a T-DNA insertion in this mutant. CmVps39 is composed of 1071 amino acids with an amino-terminal citron homology domain and a central clathrin homology domain, as observed for other Vam6p/Vps39p family proteins. Abnormal fragmented vacuoles were observed in ΔCmVps39 under light microscopy and transmission electron microscopy, and ΔCmVps39 showed impairment in autophagy. ΔCmVps39 also exhibited significantly reduced hyphal development, poor conidiation and decreased sclerotial mycoparasitism. In addition, deletion of CmVps39 affected osmotic adaptation, pH homeostasis and cell wall integrity. Taken together, our results suggest that CmVps39 has an essential function in vacuolar morphology, autophagy, fungal development and mycoparasitism in C. minitans.

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.

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.