Phytopathogenic Fungus Sclerotinia Sclerotiorum Research Articles

Structural characterization of silver nanoparticles produced by biogenic synthesis using SAXS

Nanotechnology applied to the agricultural sector has highlighted in recent decades, making important contributions, including systems for pest control as biogenic nanoparticles. These nanoparticles are used to control phytopathogens, demonstrating the need to understand its composition, mechanisms of action and toxicity. Their capping of biomolecules, derived from the organism used in the synthesis, contributes to their stability and biological activity. Ag nanoparticles were produced by the fungus Trichoderma harzianum in aqueous solutions containing silver nitrate as a precursor for the silver nanoparticles. Some of the samples were exposed to the phytopathogenic fungus Sclerotinia sclerotiorum responsible for the white mold. After preparation, a fraction of the samples was submitted to physico-chemical processes to remove organic cap layer on nanoparticles surface formed during the preparation process. In this study we determined the effect of the phytopathogenic fungus and cap removal process in the average radius, radius dispersion, number density of the nanoparticles using small angle X-ray scattering (SAXS), where we considered their almost spherical shape in aqueous solution obtained by the biogenic route. The SAXS data analyses suggest that the presence of the pathogenic fungus results in a diminution of number and total volume of Ag NPs without significant effects on average radius and radius dispersion. Our results also indicate that the physic-chemical process to remove the organic cap surrounding the Ag NPs leads to a decrease in the fraction of the smaller nanoparticles.

Design and Synthesis of Novel Diphenyl Ether Carboxamide Derivatives To Control the Phytopathogenic Fungus Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum has led to serious losses in the yields of oilseed rape and other crops every year. Here, we designed and synthesized a series of carboxamide derivatives containing a diphenyl ether skeleton by adopting the scaffold splicing strategy. From the results of the mycelium growth inhibition experiment, inhibition rates of compounds 4j and 4i showed more than 80% to control S. sclerotiorum at a dose of 50 μg/mL, which is close to that of the positive control (flubeneteram, 95%). Then, the results of a structure-activity relationship study showed that the benzyl scaffold was very important for antifungal activity and that introducing a halogen atom on the benzyl ring would improve antifungal activity. Furthermore, the results of an in vitro activity test suggested that these novel compounds can inhibit the activity of succinate dehydrogenase (SDH), and the binding mode of 4j with SDH was basically similar to that of the flutolanil derivative. Morphological observation of mycelium revealed that compound 4j could cause a damage on the mycelial morphology and cell structure of S. sclerotiorum, resulting in inhibition of the growth of mycelia. Furthermore, in vivo antifungal activity assessment of 4j displayed a good control of S. sclerotiorum (>97%) with a result similar to that of the positive control at a concentration of 200 mg/L. Thus, the diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further development.

Characterization of a newly identified RNA segment derived from the genome of Sclerotinia sclerotiorum reovirus 1.

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.

Characterization of a novel botoulivirus isolated from the phytopathogenic fungus Sclerotinia sclerotiorum.

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.

Influence of the capping of biogenic silver nanoparticles on their toxicity and mechanism of action towards Sclerotinia sclerotiorum

BackgroundBiogenic nanoparticles possess a capping of biomolecules derived from the organism employed in the synthesis, which contributes to their stability and biological activity. These nanoparticles have been highlighted for the control of phytopathogens, so there is a need to understand their composition, mechanisms of action, and toxicity. This study aimed to investigate the importance of the capping and compare the effects of capped and uncapped biogenic silver nanoparticles synthesized using the filtrate of Trichoderma harzianum against the phytopathogenic fungus Sclerotinia sclerotiorum. Capping removal, investigation of the composition of the capping and physico-chemical characterization of the capped and uncapped nanoparticles were performed. The effects of the nanoparticles on S. sclerotiorum were evaluated in vitro. Cytotoxicity and genotoxicity of the nanoparticles on different cell lines and its effects on nontarget microorganisms were also investigated.ResultsThe capped and uncapped nanoparticles showed spherical morphology, with greater diameter of the uncapped ones. Functional groups of biomolecules, protein bands and the hydrolytic enzymes NAGase, β-1,3-glucanase, chitinase and acid protease from T. harzianum were detected in the capping. The capped nanoparticles showed great inhibitory potential against S. sclerotiorum, while the uncapped nanoparticles were ineffective. There was no difference in cytotoxicity comparing capped and uncapped nanoparticles, however higher genotoxicity of the uncapped nanoparticles was observed towards the cell lines. Regarding the effects on nontarget microorganisms, in the minimal inhibitory concentration assay only the capped nanoparticles inhibited microorganisms of agricultural importance, while in the molecular analysis of the soil microbiota there were major changes in the soils exposed to the uncapped nanoparticles.ConclusionsThe results suggest that the capping played an important role in controlling nanoparticle size and contributed to the biological activity of the nanoparticles against S. sclerotiorum. This study opens perspectives for investigations concerning the application of these nanoparticles for the control of phytopathogens.

Morphological and protein alterations in Sclerotinia sclerotiorum (Lib.) de Bary after exposure to volatile organic compounds of Trichoderma spp.

Trichoderma fungi have an important potential for the control of plant diseases due to their various mechanisms of action, such as the production of volatile organic compounds (VOCs). The phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary causes a disease called white mold, sclerotinia rot or sclerotinia wilt, in several plant species. The objective of this work was to evaluate the mycelial morphology and protein profile of S. sclerotiorum after exposure to VOCs produced by six Trichoderma strains. The assays were performed in the laboratory using inverted plate tests with evaluation of mycelial growth inhibition, microscopic observation of hyphae and MALDI-TOF analysis of S. sclerotiorum protein profile. The best results were obtained from T. azevedoi CEN1241, with up to 35% inhibition of mycelial growth and the greatest inhibitory effect on the production of differential proteins present in S. sclerotiorum control treatment. All tested Trichoderma strains decreased the width of the pathogen’s hyphae. The diversity of Trichoderma species and VOCs produced by strains of this fungus may enable the development of strategies for the use of such compounds in plant disease control. Considering this biological activity, strain CEN1241 shows potential as a biocontrol agent of white mold.

The host generalist phytopathogenic fungus Sclerotinia sclerotiorum differentially expresses multiple metabolic enzymes on two different plant hosts

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen that infects upwards of 400 plant species, including several economically important crops. The molecular processes that underpin broad host range necrotrophy are not fully understood. This study used RNA sequencing to assess whether S. sclerotiorum genes are differentially expressed in response to infection of the two different host crops canola (Brassica napus) and lupin (Lupinus angustifolius). A total of 10,864 of the 11,130 genes in the S. sclerotiorum genome were expressed. Of these, 628 were upregulated in planta relative to in vitro on at least one host, suggesting involvement in the broader infection process. Among these genes were predicted carbohydrate-active enzymes (CAZYmes) and secondary metabolites. A considerably smaller group of 53 genes were differentially expressed between the two plant hosts. Of these host-specific genes, only six were either CAZymes, secondary metabolites or putative effectors. The remaining genes represented a diverse range of functional categories, including several associated with the metabolism and efflux of xenobiotic compounds, such as cytochrome P450s, metal-beta-lactamases, tannases and major facilitator superfamily transporters. These results suggest that S. sclerotiorum may regulate the expression of detoxification-related genes in response to phytotoxins produced by the different host species. To date, this is the first comparative whole transcriptome analysis of S. sclerotiorum during infection of different hosts.

Morphological and molecular aspects of sclerotial development in the phytopathogenic fungus Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary produces a resistance structure called sclerotium, which guarantees its survival in soil for long periods. Morphological and melanization aspects during sclerotial development were evaluated by microscopy and qRT-PCR techniques. S. sclerotiorum produces sclerotia with different phases of maturation and melanization during growth in PDA medium. Using scanning electron microscopy we observed that there are no structural differences in the three stages of formation of melanized and non-melanized sclerotium. Through histochemical analysis we observed that the melanized sclerotium accumulates more glycogen and produces less protein than non-melanized sclerotia. Melanin was most commonly found in the rind of melanized sclerotia, and the highest concentration of lipofucsins was found in non-melanized sclerotia. These molecules are products of the lipid peroxidation pathway and are associated with oxidative stress during differentiation in fungi. The expression of histidine kinase (shk) and adenylate cyclase (sac) genes in melanized and non-melanized sclerotiawere also evaluated. The higher gene expression of shk and lesser expression of sac in non-melanized sclerotiais an indication of the participation of cell signaling in the development of these structures. The higher expression of polyketide synthase (pks), tyrosinase (tyr) and laccase (lac) in non-melanized sclerotia suggested that S. sclerotiorum can use the DHN and L-dopa pathways to produce melanin. Expression studies of the enzymes chitin synthase and glucan synthase suggest that this process occurs along with the formation of melanin. This is interesting since polysaccharides, such as chitin and β-1,3-glucan, serve as a scaffold to which the melanin granules are cross-linked.

A Novel RNA Virus Related to Sobemoviruses Confers Hypovirulence on the Phytopathogenic Fungus Sclerotinia sclerotiorum

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

Hormetic Effects of Mixtures of Dimethachlone and Prochloraz on Sclerotinia sclerotiorum.

Previous studies showed that dimethachlone has significant hormetic effects on phytopathogenic fungus Sclerotinia sclerotiorum. The present study investigated hormetic effects of mixtures of dimethachlone and prochloraz on mycelial growth and virulence of two dimethachlone-resistant isolates of S. sclerotiorum. The stimulatory dimethachlone dosage range was around 1 to 100 μg/ml in potato dextrose agar (PDA) medium for mycelial growth of the two isolates, and dimethachlone at 10 and 50 μg/ml had the maximum percent stimulations of 80.6 and 19.3% for isolates JMS14 and HLJ4, respectively. Prochloraz at 0.0003 and 0.002 μg/ml had the maximum percent stimulations of 9.3 and 11.1% for isolates JMS14 and HLJ4, respectively. However, dimethachlone and prochloraz mixed at their respective stimulatory concentrations had the maximum percent stimulations of 48.1 and 9.3% for isolates JMS14 and HLJ4, respectively. After the mycelia with increased and inhibited growth on fungicide-amended PDA were subcultured on PDA without fungicide, mycelial growth for the second generation increased compared with the nontreated control. After the mycelia grown on fungicide-amended PDA were inoculated on rapeseed leaves, the amplitude of virulence stimulation was much greater than that of mycelial growth on PDA amended with fungicide, and the inhibited mycelia also showed substantially increased virulence on leaves. The mixture of dimethachlone at 100 μg/ml and prochloraz at 0.03 μg/ml in PDA inhibited mycelial growth of isolate JMS14 by 59.4%; however, after the inhibited mycelia were inoculated on rapeseed leaves, virulence was stimulated by 69.0%. Spraying sublethal doses of dimethachlone and prochloraz on rapeseed leaves also exhibited significant stimulatory effects on virulence. For isolate JMS14, the stimulatory concentration ranges for dimethachlone and prochloraz were around 1 to 600 μg/ml and 0.0003 to 0.18 μg/ml, respectively. The fitted curve of virulence stimulation for the mixture of dimethachlone and prochloraz shifted to the left on the x axis, denoting dose-additive interactions between the two fungicides with regard to virulence stimulation. Spraying dimethachlone alone at 10 to 50 μg/ml had significant stimulations on virulence, whereas prochloraz alone at 10 to 50 μg/ml had significant inhibitory effects on virulence, and the mixture of dimethachlone and prochloraz at the concentration ratio of 1:1 had greater inhibitory effects than prochloraz alone, indicating dose-additive interactions for the inhibitory effects. Dimethachlone and prochloraz and their mixtures increased tolerance of mycelia to hydrogen peroxide. Dimethachlone significantly increased, whereas prochloraz reduced mycelial cell membrane permeability, and the mixture of the two fungicides had effect-additive interactions with respect to effects on cell membrane permeability. These studies will advance our understanding of hormesis of fungicide mixtures.

Endophytic Bacillus spp. from medicinal plants inhibit mycelial growth of Sclerotinia sclerotiorum and promote plant growth.

Plant growth-promoting bacteria that are also capable of suppressing plant pathogenic fungi play an important role in sustainable agriculture. There is a critical need for conducting research to discover, characterize and evaluate the efficacy of new strains of such bacteria in controlling highly aggressive plant pathogens. In this study, we isolated endophytic bacteria from medicinal plants of Bangladesh and evaluated their antagonistic capacity against an important phytopathogenic fungus Sclerotinia sclerotiorum. Growth-promoting effects of those isolates on cucumber and rice seedlings were also assessed. Among 16 morphologically distinct isolates, BDR-2, BRtL-2 and BCL-1 significantly inhibited the growth of S. sclerotiorum through induction of characteristic morphological alterations in hyphae and reduction of mycelial dry weight. When cucumber and rice seeds were treated with these endophytic bacteria, seven isolates (BCL-1, BDL-1, BRtL-2, BRtL-3, BDR-1, BDR-2 and BBoS-1) enhanced seed germination, seedling vigor, seedling growth and number of roots per plant at a varying level compared to untreated controls. All isolates produced high levels of indole-3-acetic acid (6 to 63 μg/mL) in vitro. Two most potential isolates, BDR-2 and BRtL-2, were identified as Bacillus amyloliquefaciens and B. subtilis, respectively, based on the 16S rRNA gene sequencing. These results suggest that endophytic Bacillus species from native medicinal plants have great potential for being used as natural plant growth promoter and biopesticides in sustainable crop production.

TaqMan Multiplex Real-Time qPCR assays for the detection and quantification of Barley yellow dwarf virus, Wheat dwarf virus and Wheat streak mosaic virus and the study of their interactions

The phytopathogenic fungus Sclerotinia sclerotiorum forms dormant structures (termed sclerotia) that germinate myceliogenically under certain environmental conditions. During myceliogenic germination, sclerotia produce hyphae, which can infect leaves or stems of host plants directly from the ground; this is termed basal infection. This study determined which abiotic conditions were most important for promoting myceliogenic germination of sclerotia in vitro. A high sclerotium hydration level and low incubation temperature (15°C) improved mycelial growth in the presence of a nutrient source. Sclerotia incubated without a nutrient source on moist sand, vigorously myceliogenically germinated most frequently (63%) when they had been previously imbibed and then conditioned at −20°C. By far the most consistent amount of vigorous myceliogenic germination (>75%) was produced when sclerotia were heat-dried before being submerged in water. The hyphae of these sclerotia were shown to infect and proliferate on leaves of intact Brassica napus plants. This research provides a better understanding of the abiotic conditions that are likely to increase the risk of basal infection by S. sclerotiorum.

Plant extracellular vesicles are incorporated by a fungal pathogen and inhibit its growth.

Extracellular vesicles (EV) are membrane particles released by cells into their environment and are considered to be key players in intercellular communication. EV are produced by all domains of life but limited knowledge about EV in plants is available, although their implication in plant defense has been suggested. We have characterized sunflower EV and tested whether they could interact with fungal cells. EV were isolated from extracellular fluids of seedlings and characterized by transmission electron microscopy and proteomic analysis. These nanovesicles appeared to be enriched in cell wall remodeling enzymes and defense proteins. Membrane-labeled EV were prepared and their uptake by the phytopathogenic fungus Sclerotinia sclerotiorum was verified. Functional tests further evaluated the ability of EV to affect fungal growth. Spores treated with plant EV showed growth inhibition, morphological changes, and cell death. Conclusive evidence on the existence of plant EV is presented and we demonstrate their ability to interact with and kill fungal cells. Our results introduce the concept of cell-to-cell communication through EV in plants.

New insights into reovirus evolution: implications from a newly characterized mycoreovirus.

We performed molecular cloning and complete genome sequencing of a novel mycoreovirus, Sclerotinia sclerotiorumreovirus 1 (SsReV1), isolated from an isolate of the phytopathogenic fungus Sclerotinia sclerotiorum. SsReV1 has a genome of 28 055 bp and is composed of 11 double-stranded RNA segments. With a combination of unique molecular features, virion shape and composition, and phylogenetic analysis, SsReV1 is significantly distinct from all known reoviruses and defines a novel genus in the family Reoviridae. Interestingly, two conserved domains, double-stranded RNA binding motif (dsRBM, Pfam 00 035) and reovirus sigma C capsid protein (Reo_σC, pfam04 582), were identified in the genome of SsReV1, which are widespread in diverse virus lineages. Sequence comparison and phylogenetic analysis revealed that multiple cross-family horizontal gene transfer (HGT) events could occur between reoviruses and double-stranded DNA viruses, single-stranded RNA viruses and even cellular organisms. Interestingly, the dsRBM of SsReV1 was phylogenetically related to dsRNA-binding proteins of some insects, but not reoviruses. These results indicated that SsReV1 is a new taxonomic representative in Reoviridae, which provides new insights into the diversity and global ecology of reoviruses and other segmented double-stranded RNA viruses. More importantly, the present results provided evidence indicating that reoviruses indeed have HGT events with other virus lineages on a large scale and that HGT may serve as an important driving factor that plays a key role in the evolution of reoviruses.

The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion

Mitogen-activated protein kinases (MAPKs) play a central role in transferring signals and regulating gene expression in response to extracellular stimuli. An ortholog of the Saccharomyces cerevisiae cell wall integrity MAPK was identified in the phytopathogenic fungus Sclerotinia sclerotiorum. Disruption of the S. sclerotiorum Smk3 gene severely reduced virulence on intact host plant leaves but not on leaves stripped of cuticle wax. This was attributed to alterations in hyphal apical dominance leading to the inability to aggregate and form infection cushions. The mutation also caused loss of the ability to produce sclerotia, increased aerial hyphae formation, and altered hyphal hydrophobicity and cell wall integrity. Mutants had slower radial expansion rates on solid media but more tolerance to elevated temperatures. Loss of the SMK3 cell wall integrity MAPK appears to have impaired the ability of S. sclerotiorum to sense its surrounding environment, leading to misregulation of a variety of functions. Many of the phenotypes were similar to those observed in S. sclerotiorum adenylate cyclase and SMK1 MAPK mutants, suggesting that these signaling pathways co-regulate aspects of fungal growth, physiology, and pathogenicity.

The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum is a major disease of oilseed rape (Brassica napus). During infection, large, white/grey lesions form on the stems of the host plant, perturbing seed development and decreasing yield. Due to its ability to produce long‐term storage structures called sclerotia, S. sclerotiorum inoculum can persist for long periods in the soil. Current SSR control relies heavily on cultural practices and fungicide treatments. Cultural control practices aim to reduce the number of sclerotia in the soil or create conditions that are unfavourable for disease development. These methods of control are under increased pressure in some regions, as rotations tighten and inoculum levels increase. Despite their ability to efficiently kill S. sclerotiorum, preventative fungicides remain an expensive gamble for SSR control, as their effectiveness is highly dependent on the ability to predict the establishment of microscopic infections in the crop. Failure to correctly time fungicide applications can result in a substantial cost to the grower. This review describes the scientific literature pertaining to current SSR control practices. Furthermore, it details recent advances in alternative SSR control methods including the generation of resistant varieties through genetic modification and traditional breeding, and biocontrol. The review concludes with a future directive for SSR control on oilseed rape.

The Microbial Opsin Homolog Sop1 is involved in Sclerotinia sclerotiorum Development and Environmental Stress Response.

Microbial opsins play a crucial role in responses to various environmental signals. Here, we report that the microbial opsin homolog gene sop1 from the necrotrophic phytopathogenic fungus Sclerotinia sclerotiorum was dramatically up-regulated during infection and sclerotial development compared with the vegetative growth stage. Further, study showed that sop1 was essential for growth, sclerotial development and full virulence of S. sclerotiorum. Sop1-silenced transformants were more sensitive to high salt stress, fungicides and high osmotic stress. However, they were more tolerant to oxidative stress compared with the wild-type strain, suggesting that sop1 is involved in different stress responses and fungicide resistance, which plays a role in the environmental adaptability of S. sclerotiorum. Furthermore, a Delta blast search showed that microbial opsins are absent from the genomes of animals and most higher plants, indicating that sop1 is a potential drug target for disease control of S. sclerotiorum.

Molecular characterization of a bipartite double-stranded RNA virus and its satellite-like RNA co-infecting the phytopathogenic fungus Sclerotinia sclerotiorum.

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

A mitovirus related to plant mitochondrial gene confers hypovirulence on the phytopathogenic fungus Sclerotinia sclerotiorum

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

Characterisation of a novel hypovirus from Sclerotinia sclerotiorum potentially representing a new genus within the Hypoviridae

A novel mycovirus tentatively assigned the name Sclerotinia sclerotiorum hypovirus 2 (SsHV2/5472) was detected in the phytopathogenic fungus Sclerotinia sclerotiorum. The genome is 14581 nucleotides (nts) long, excluding the poly (A) tail. A papain-like cysteine protease (Pro), an RNA-dependent RNA polymerase (RdRp) and a helicase (Hel) domain were detected in the polyprotein. Phylogenetic analysis based on multiple alignments of the aa sequence of the polyprotein placed it in a distinct clade from Alphahypovirus and Betahypovirus. The distinct aa sequence plus the fact that SsHV2/5472 possesses the longest reported genome for a hypovirus, suggests that SsHV2/5472 may represent a new genus in the family Hypoviridae. Eliminating SsHV2/5472 from S. sclerotiorum significantly increased the virulence of the protoplast virus-free derivative 5472-P5, although SsHV/5472-containing isolates showed significant variation in their virulence. In addition, membrane-bound vesicles (25–50nm) were observed in ultrathin mycelial sections of SsHV2/5472 containing isolates but not in SsHV2/5472-free isolate.