Transmission of Oyster Mushroom Spherical Virus to Progeny via Basidiospores and Horizontally to a New Host Pleurotus floridanus.

Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically through sporulation in natural settings. Oyster mushroom spherical virus (OMSV) is a mycovirus that infects Pleurotus ostreatus, with horizontal transmission via hyphal anastomosis. However, whether OMSV can be vertically transmitted is unclear. This study aimed to investigate the transmission characteristics of OMSV to progeny via basidiospores and horizontally to a new host. A total of 37 single-basidiospore offspring were obtained from OMSV-infected P. ostreatus and Pleurotus pulmonarius for Western blot detection of OMSV. The OMSV-carrying rate among monokaryotic isolates was 19% in P. ostreatus and 44% in P. pulmonarius. Then, OMSV-free and OMSV-infected monokaryotic isolates were selected for hybridization with harvested dikaryotic progeny strains. Western blot analyses of the offspring revealed that the OMSV transmission efficiency was 50% in P. ostreatus and 75% in P. pulmonarius, indicating vertical transmission via sexual basidiospores. Furthermore, we observed the horizontal transfer of OMSV from P. pulmonarius to Pleurotus floridanus. OMSV infection in P. floridanus resulted in significant inhibition of mycelial growth and yield loss. This study was novel in reporting the vertical transmission of OMSV through basidiospores, and its infection and pathogenicity in a new host P. floridanus.

The PoV mycovirus affects extracellular enzyme expression and fruiting body yield in the oyster mushroom, Pleurotus ostreatus.

Isogenic virus-cured and virus-infected fungal strains were previously obtained and compared to investigate mycoviral diseases and, specifically, the influence of viral infection on the vegetative growth of Pleurotus ostreatus. The present study demonstrated that infection with mycovirus PoV-ASI2792 (PoV) caused phenotypic and physiological changes in fungal cells and mycelia. The microscopically determined growth rate of the virus-infected strain was lower than that of the virus-cured strain, due to the conglomerate phenomenon during the mycelial growth process. An exploration of the viral effects of PoV on fruiting bodies yield showed significantly lower than that on virus-cured P. ostreatus. A colorimetric assay of polyphenol oxidase activity in the strains showed very weak activity in the virus-infected strain. To estimate the activity levels of enzymes related to the growth and fruiting body formation, the relative expression levels of genes encoding various extracellular enzymes such as Carbohydrate-Active Enzymes (CAZymes) were measured by quantitative RT-PCR. The expression levels of the assayed genes were significantly lower in virus-infected than in virus-cured P. ostreatus. Together, these results indicate that PoV infection affects the spawn growth and fruiting body formation of P. ostreatus via decreased expression and activity of some extracellular enzymes including lignocellulolytic enzymes.

[Morphology and structural peculiarities of Basidiomycetes pathogens].

The materials of studies of morphology and structural peculiarities of viruses, fungi and bacteria, which affect Basidiomycetes under biotechnology process and nature biocenosis conditions are given. The analysis of infection development in button mushroom (Agaricus bisporus) (J.Lge) Imbach and in oyster mushroom (Pleurotus ostreatus Kumm.), which served as model objects in the experiments of various levels of complexity has been carried out. Other kinds of edible and medicinal mushrooms, which were a source of biochemical fractions to form biologicals were investigated.

Curing viruses in Pleurotus ostreatus by growth on a limited nutrient medium containing cAMP and rifamycin.

Oyster mushroom spherical virus (OMSV) and oyster mushroom isometric virus (OMIV) are the causative agents of a fruiting body deformation disease in the edible mushroom Pleurotus ostreatus. The curing of these mycoviruses was facilitated by a serial transfer of infected mycelia onto a limited nutrient medium containing 1mM of cAMP and 75 µg/ml of rifamycin (cAMP-rifamycin plate). The mycelia were grown on cAMP-rifamycin plates for 5 successive passages. ELISA and RT-PCR showed that the amount of mycoviruses inside the mycelia decreased significantly with increasing numbers of passages. The mycelia became free of viruses after 5 successive passages. Cultivation of the virus-cured mycelia on a mushroom compost medium produced a normal harvest, whereas the spawn infected with viruses failed to produce any fruiting bodies.

Isolation of new Pseudomonas tolaasii bacteriophages and genomic investigation of the lytic phage BF7.

Sixteen lytic bacteriophages that infect Pseudomonas tolaasii LMG 2342(T) were isolated from smashed sporocarps of oyster mushroom (Pleurotus ostreatus) showing necrotic symptoms. On the basis of the host range investigation of the phages, they have wide infection abilities against the genus Pseudomonas, mainly in the case of phages Bf3, Bf7, Bf10, and Bf15. Molecular investigations have revealed that they all have dsDNA genomes about 40 kbp in size. Identical restriction patterns resulting from restriction enzyme analysis suggest that the isolates probably belong to the same phage species. However, there was a difference between these phage isolates in their infecting abilities. Phage isolate Bf7 was investigated and characterized more deeply. Morphological characterization of Bf7 by transmission electron microscopy (TEM) has shown that it has a short, noncontractile tail, an icosahedral phage head, and the size is about 60 nm in diameter, suggesting that it belongs to the Podoviridae family. Complete genome sequence analysis of the Bf7 phage isolate revealed a 40 058 bp genome, 58.4% G+C content, 46 open reading frames encoding different proteins showing homology to proteins of the bacteriophage Caulobacter crescentus φCd1 from the Podoviridae family. On the basis of these results and comparative genomic studies, we classified the Bf7 phage to the subfamily of Autographivirinae, φKMV-like phages.

Particle and naked RNA mycoviruses in industrially cultivated mushroom Pleurotus ostreatus in China.

Many cultivated mushroom strains, such as Pleurotus ostreatus TD300, displayed symptoms of degeneration. A spherical virus POSV and four dsRNA segments were extracted from mycelium of P. ostreatus TD300. POSV had a diameter of 23 nm and encapsidated a 2.5kb dsRNA segment with coat proteins whose molecular weights were 39 kDa and 30 kDa. Four dsRNA segments were 8.2 kb, 2.5 kb, 2.0 kb, and 1.1 kb in size, respectively. The 1.1 kb dsRNA segment often escaped detection. The cDNA and the amino acid sequences of the 8.2 kb dsRNA were homologous to those of RNA-dependent RNA polymerases (RDRP) of ssRNA oyster mushroom spherical virus (OMSV), and contained conserved motifs A to D which were almost identical to those in RDRP of OMSV. The cDNA and amino acid sequences of the 2.5 kb and 2.0 kb dsRNA segments were homologous to that of RDRP and capsid protein of dsRNA virus P. ostreatus virus 1 (PoV1), respectively. In particular, the amino acid sequence of 2.5 kb dsRNA segment had high identity with the conserved motifs A to C in RDRP of PoV1, a Partiviridae virus. After eliminating the viruses in P. ostreatus TD300, the symptoms of degeneration completely disappeared. The results reveal that P. ostreatus TD300 was at least infected by a particle virus POSV, and two naked viruses, one was a dsRNA virus with a 2.0 kb dsRNA segment, the other was an ssRNA virus whose replicating form of genome was an 8.2 kb dsRNA segment. Mycoviruses infection is a causative agent of mushroom strain degeneration.

[Comparisons of different methods for virus-elimination of edible fungi].

Four dsRNA bands were extracted from Pleurotus ostreatus TD300 by the dsRNA isolation technique with sizes of 8.2 kb, 2.5 kb, 2.1 kb, and 1.1 kb, respectively. Four virus-eliminated methods, i. e. hyphal tips cut (HTC), protoplast regeneration (PR), single spore hybridization (SSH), and frozen and lyophilized (FL), were applied to prepare virus-eliminated strains, and one virus-eliminated strain was selected for each virus-elimination method. The virus-eliminated strains were named as HTC8, PR15, FL01, and SSH11, respectively. There were low concentration of 8.2 kb dsRNA remained in HTC8, as well as low concentration of 8.2 kb and 2.5 kb dsRNA remained in FL01. However, no dsRNA remained in PR15 and SSH11. The hyphal growth rate and laccase activity of the virus-eliminated strains increased, especially HTC8 and PR15, whose hyphal growth rate was higher by 22.73% and 18.18%, and laccase activities higher by 145.83% and 134.38% than that of the original strain, respectively. The conclusion is that hyphal tips cut and protoplast regeneration are suitable to prepare virus-eliminated strains of edible fungi.

The identification of a novel Pleurotus ostreatus dsRNA virus and determination of the distribution of viruses in mushroom spores.

Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.

An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus.

The oyster mushroom spherical virus (OMSV) is a causative agent of dieback disease in the oyster mushroom, Pleurotus ostreatus. Outbreaks of this virus occasionally result in serious disease that is associated with hefty economic losses. Thus, the detection and removal of OMSV-infected spawn is considered to be a crucial step for the stable production of P. ostreatus. For the detection of OMSV, we attempted to generate monoclonal antibodies (mAbs) against an RNA polymerase domain (RPD) of an OMSV protein. In an effort to simplify the laborious multistep mAb screening process, we developed a protein microarray on a slide glass that is chemically modified with the RPD protein. The culture supernatants of 87 hybridoma cells, which were prepared from the fusion of RPD-immunized mouse spleen cells with myeloma cells, were spotted onto the RPD-coated microarray. The binding of mAb to RPD was detected via Alexa 488 dye-labeled anti-mouse immunoglobulin G (IgG) as a secondary antibody. Of 87 samples, 13 evidenced a significant level of fluorescence signal intensity. Subsequent immunoblot analysis revealed that the specificity of each mAb against RPD coincided with the corresponding fluorescence signal intensity, thereby indicating the effectiveness of the protein microarray in mAb screening.

Detection of the mycovirus OMSV in the edible mushroom, Pleurotus ostreatus, using an SPR biosensor chip.

A surface plasmon resonance (SPR) biosensor chip was developed for the rapid detection of the oyster mushroom spherical virus (OMSV), which causes a mushroom die-back disease, the symptoms of which include malformed fruiting bodies and retarded mycelial growth in the cultivated edible mushroom, Pleurotus ostreatus. An anti-OMSV monoclonal antibody (mAb) was generated initially using purified OMSV viral particles. For the fabrication of the biosensor chip, the anti-OMSV mAb was layered onto an activated carboxymethyl-dextran (CM-Dex) gold thin film. Analysis on the SPR angle shift showed that the bound mAb was 6.7 ng/mm2 of the chip surface. Subsequently, the biosensor chip was applied to the detection of OMSV in the mushroom mycelial extract. It detected specifically OMSV in the extract in a concentration-dependent manner. Finally, the biosensor chip was employed for the detection of OMSV in the mushroom fruiting bodies collected from 10 commercial farms. Among the tested samples, OMSV was found to infect fruiting bodies from a farmland, and this was confirmed further via immunoblot analysis and a TAS-ELISA assay. In conclusion, the SPR biosensor chip combined with an anti-OMSV mAb evidenced superior performance, particularly with regard to the prompt detection of OMSV infection.

Isolation and characterization of a novel mycovirus, PeSV, in Pleurotus eryngii and the development of a diagnostic system for it.

A novel mycovirus was isolated from a cultivated edible mushroom, Pleurotus eryngii, with severe epidemic symptoms. Purification of the virus was carried out by a sequential procedure of polyethylene glycol precipitation, differential centrifugation, and equilibrium centrifugation in a CsCl gradient. Nuclease digestion assay and protein analysis revealed that the virus consisted of a single-stranded RNA (ssRNA) genome of 7.8 kbp which was encapsulated by a coat protein of 22 kDa. Transmission electron microscope showed that it was spherical with a diameter of 31 nm. Since there was neither a previous report on discovery of a virus in P. eryngii, nor known mushroom viruses with similar characteristics, we concluded that this is a novel virus and thus have named it as P. e ryngii Spherical Virus (PeSV). Because of a diagnostic test would be helpful in preventing the PeSV-related disease outbreaks, we developed a triple antibody sandwich-ELISA (TAS-ELISA) system using anti-PeSV mouse monoclonal and anti-PeSV rabbit polyclonal antibodies. The TAS-ELISA system successfully detected less than 0.5 microg of the virus particles in 1 g diseased mushroom tissue collected from various commercial farms.

Isolation of a novel mycovirus OMIV in Pleurotus ostreatus and its detection using a triple antibody sandwich-ELISA.

A novel mycovirus was isolated from a diseased mushroom, Pleurotus ostreatus, using a purification procedure involving polyethylene glycol (PEG)-NaCl precipitation, differential centrifugation, and equilibrium centrifugation in a CsCl gradient. The virion was a 43 nm isometric virus encapsulating double-stranded RNA (dsRNA) genome of 2.1, 2.0, 1.9, and 1.7 kbp with a coat protein (CP) of 58 kDa. The new mycovirus was named Oyster Mushroom Isometric Virus (OMIV). A triple antibody sandwich-ELISA (TAS-ELISA) system was constructed to detect OMIV in the mushroom using an anti-OMIV mouse monoclonal antibody and an anti-OMIV rabbit polyclonal serum. The TAS-ELISA system was sensitive enough to allow detection of OMIV in the mushroom with the naked eye. It detected successfully virus particles from 0.6 mg of diseased tissue as well as 0.4 microg/ml purified virus preparation.

Complete nucleotide sequence and genome organization of a dsRNA partitivirus infecting Pleurotus ostreatus.

The nucleotide sequences of the genomic dsRNA mycovirus infecting Pleurotus ostreatus (P. ostreatus virus 1; PoV1) were determined and compared to the sequences of the other mycoviruses belonging to partitiviruses and totivirues. PoV1 dsRNA-1 and dsRNA-2 had genomes of 2296 and 2223 nucleotides, respectively. The purified virus preparations contained isometric particles of 28-30 nm in diameter, and also the same two dsRNAs were isolated from purified virus preparations. The sequences of PoV1 dsRNA-1 and dsRNA-2 had GC contents of 48.4 and 51.5%, respectively. dsRNA-1 had 78 and 97 nucleotides of 5'- and 3'-untranslated region (UTR) while dsRNA-2 had 114 and 198 nucleotides of 5'- and 3'-UTR, respectively. Computer analysis of putative open reading frame (ORF) shows that dsRNA-1 and dsRNA-2 contain a single ORF encoding proteins of 82.2 and 71.1 kDa that show high sequence identity with RNA-dependent RNA polymerase and capsid protein of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis they were found to form a distinct virus clade with partitiviruses, and were more distantly related to totiviruses.

Characterization of a novel single-stranded RNA mycovirus in Pleurotus ostreatus.

A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group.