Molecular characterization of a new endornavirus inhabiting the ectomycorrhizal fungus Hygrophorus penarioides.

Viruses hosted by uncultivated fungi have been poorly studied. We carried out studies to characterize a large dsRNA segment (~20 kbp) detected in the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides. The dsRNA was gel-purified and its randomly amplified cDNA fragments were used for high throughput sequencing (HTS). Reads were de novo assembled and BLASTx analysis revealed sequence similarity to viruses of the family Endornaviridae. The 5' and 3' terminal sequences of the dsRNA segment were determined by performing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The full-length cDNA sequence of the putative endornavirus comprises 16,785 nt and contains a single, long open reading frame which encodes for a polyprotein of 5522 aa with conserved domains for cysteine-rich region, helicase, glycosyltransferase, and RNA-dependent RNA polymerase. The virus was named Hygrophorus penarioides endornavirus 1 (HpEnV1). A BLASTp search performed using the polyprotein sequence revealed that the most closely related, fully sequenced endornavirus to HpEnV1 is Ceratobasidium endornavirus B.

Full-length genome characterization of a novel alphapartitivirus detected in the ectomycorrhizal fungus Hygrophorus penarioides.

Virus populations of ectomycorrhizal fungi remain poorly studied. In the present study, we characterized a new partitivirus isolated from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides, named "Hygrophorus penarioides partitivirus 1" (HpPV1). The whole genome of HpPV1, determined by merging deep sequencing and RLM-RACE approaches, comprised two dsRNA segments of 2053 bp and 2072 bp, respectively. Both dsRNA genome segments included a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP exhibits the highest similarity (67.49% and 75.61% identity, respectively) to those of partitiviruses identified from an ascomycetous ectomycorrhizal fungus Sarcosphaera coronaria. Phylogenetic analyses performed based on the CP and RdRp sequences demonstrated that HpPV1 clusters within a clade that includes members of the genus Alphapartitivirus, belonging to the family Partitiviridae. Here, we propose that HpPV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides.

Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages.

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonastolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonasagarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.

Spread and morphological-structural properties of plant rhabdoviruses and similar pathogens in Basidiomycetes.

Long-term studies of spread of rhabdoviruses which indicated their harmfulness in different plant species under conditions of environmental factors were first discussed. Their harmfulness to different plant species under environmental conditions was shown. A comparative description of rhabdoviruses with similar pathogens of the mushrooms is carried out. Thus the main focus was on the morphology and structure of the pathogens. These data are extremely important in the study of distribution of the rhabdovirus on crops in different regions.

A novel mycovirus from Clitocybe odora.

The Ninth Report of the International Committee on Taxonomy of Viruses (ICTV) reports only a few species whose members replicate in fungi. Most of these mycoviruses are described to replicate in phytopathogenic and commercially cultivated fungi. A few reports describe virus-like symptoms and virus-like particles in non-cultivated basidiocarps such as Boletus edulis, Laccaria spp. and Cantharellus spp. However, viral sequences from non-cultivated Agaricomycotina are not available yet. In this report, I present a partial sequence of a virus found in Clitocybe odora (Bull.:Fr.) P. Kumm var. odora coding for a putative RNA-dependent RNA polymerase (RdRp) and a small 20-kDa ORF that may encode a coat protein (CP). The sequence of the putative RdRp (ORF 1) of C. odora clusters with those of the Tanathephorus cucumeris virus RdRp and the Tuber aestivum mitovirus RdRp. In addition to sequence homology, Tanathephorus cucumeris virus shows a similar codon usage and TA content in the 5'- and 3' non-translated regions, but it does not encode a putative CP. A viral DNA form proposed for Tanathephorus cucumeris virus was not found in Clitocybe odora. This viral sequence does not fit into any of the existing virus taxa.

The enigma of double-stranded RNA (dsRNA) associated with mushroom virus X (MVX).

New variants of pathogenic fungal viruses are emerging and they are enigmatic in revealing their molecular identity and of their origin. Double-stranded RNAs, some in non-encapsidated forms are increasingly becoming causal agents for sporadic diseases and are consistently associated with a complex profile of dsRNAs, presumably of (multiple) viral origin present in the host while the same are conspicuously absent in healthy (looking) counterparts. The emergence of an unusual Agaricus bisporus mushroom 'patch disease' first reported in 1996, later termed as 'mushroom virus X' (MVX), exhibited a wide range of symptoms (e.g. barren patches beside healthy looking mushrooms, arrested pins, premature opening, brown, off-colour and distortions in shape). A variable compendium of novel 26 (dsRNA) elements, ranging in sizes between 20.2kb to 0.64kb, several of them (-17/26) in non-encapsidated form have been shown to occur in the diseased mushroom fruiting bodies and are thought to comprise multiple viruses. Ten years on, this devastating disease is now more widespread and prevalent in a number of European countries (e.g. The Netherlands, Ireland) ranging from occasional to severe outbreaks leading to crop losses. Impressive data has been accumulated on the symptoms, but the potential aetiological sources, biochemical and molecular characterizations corresponding to the symptoms vis-a-vis MVX linked dsRNAs still remain either elusive or unclear. We have overviewed mainly the molecular findings of research groups working on MVX in these countries together with our own work on MVX in Northern Ireland. To date, the results reviewed suggest that with the exception of 4 low molecular weight dsRNA bands (sizes 2.0. 1.8, 0.8 and 0.6 kb) which consistently were found synchronous to mushroom off-colour/browning symptoms in the UK and The Netherlands, other individual MVX dsRNAs or their banding patterns clearly lack credible relationship with other symptoms of the MVX disease complex. The issues in the molecular characterisation of the MVX dsRNAs include the disparate results on the molecular sequences obtained for some of these by the different research groups, the varying molecular methods or approaches adopted by them for deciphering the nucleotide sequences of the novel dsRNAs that are different from previously encountered mushroom viruses. The future outlook and general consensus among mushroom researchers worldwide is for an urgent need to recruit international taskforce and re-focus on clarifying the symptom vis-a-vis dsRNAs in the enigmatic MVX disease complex. As crossing the cellular membrane is a key step to infection process, we have also attempted to draw parallels with other viruses in terms of the potential cell entry mechanisms for MVX dsRNAs. In the light of MVX disease and A. bisporus being a commercial crop worldwide in agri-food markets, and taking cue from its nearest Basidiomycete model mushroom, Coprinus cinereus whose genome mapping is completed, we also propose that it may be timely for the international research groups to renew efforts to prop up a network for sequencing the host A. bisporus mushroom genome (-38 MB) for a better understanding of host-pathogen relationships.

Double-stranded RNA transmission through basidiospores of Heterobasidion annosum.

A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14% of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10-84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P < 0.05); germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant; germination was 3 and 10% for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.

Double-stranded RNA and virus-like particles in the edible basidiomycete Flammulina velutipes (Enokitake).

One of the commercial strains of Flammulina velutipes was analyzed for the presence of double-stranded RNA (dsRNA) elements to examine the underlying mechanism of strain degeneration. As a result, two dsRNA elements sized 1.9 and 1.8 kb were detected in mycelium derived from spontaneously brown-colored fruit body. They were not detected in the normal strains or in fruiting-impaired degenerative isolates. The dsRNAs were not in the nuclear or mitochondrial fractions, but were located in the cytoplasmic fraction. The presence of virus-like particles of ca. 50 nm diameter associated with the dsRNAs was confirmed by electron microscopic observation.