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ABSTRACT: In order to estimate how diverse the mating types in Pleurotus eryngii from different regions are, pairings between monokaryons derived from inter- and intragroups were done. Sixteen and 15 alleles were identified at loci A and B from the 12 strains. In the P. eryngii KNR2312, widely used for commercial production, four mating loci, A3, A4, B3, and B4, were determined. Those loci, except A3, were found in 4 strains out of 12 strains. To improve breeding efficiency, especially in mating type determination, RAPD and BSA were performed to screen for a mating type specific marker. The SCAR marker 13- 2(2100) was developed based on the RAPD-derived sequence typing B3 locus. The sequence analysis of 13-2(2100) revealed that it contained a conserved domain, the STE3 superfamily, and consensus sequences like the TATA box and GC box. It seems likely that the SCAR marker region is a part of the pheromone receptor gene.
Journal of Microbiology and Biotechnology 09/2012; 22(9):1177-84. · 1.38 Impact Factor
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ABSTRACT: The retrotransposon marY1 is a gypsy family retroelement, which is detected ubiquitously within the fungal taxonomic groups in which mushrooms are included. To utilize marY1 as a molecular marker for the DNA fingerprinting of mushrooms, oligonucleotides marY1-LTR-L and marY1-LTR-R were designed on the basis of highly conserved regions from the multiple sequence alignment of 30 marY1 sequences retrieved from a nucleotide sequence database. In accordance with Retrotransposon Microsatellite Amplified Polymorphism (REMAP) fingerprinting methodology, the two oligonucleotides were utilized together with the short sequence repeat primers UBC807 and UBC818 for polymerase chain reaction using templates from different mushroom genomic DNAs. Among the tested oligonucleotides, the marY1-LTR-L and UBC807 primer set yielded the greatest amount of abundance and variation in terms of DNA band numbers and patterns. This method was successfully applied to 10 mushroom species, and the primer set successfully discriminate
Mycobiology. 09/2008; 36(3):161-166.
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ABSTRACT: 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.
Analytical Biochemistry 04/2008; 374(2):313-7. · 3.00 Impact Factor
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ABSTRACT: 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.
Journal of Virological Methods 04/2008; 148(1-2):120-4. · 2.01 Impact Factor
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ABSTRACT: A database application, namely MushBase, has been built based on Microsoft Access in order to store and manage different kinds of data about mushroom biological information of species, strains and their physiological characteristics such as geometries and growth condition(s). In addition, it is also designed to store another group of information that is experimental data about mushroom classification by Random Amplification of Polymorphic DNA (RAPD). These two groups of information are stored and managed in the way so that it is convenient to retrieve each group of data and to cross-refer between them as well.
Mycobiology. 09/2007; 35(3):154-156.
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ABSTRACT: Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99% sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200-4000bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.
Mycological Research 07/2007; 111(Pt 6):710-5. · 2.81 Impact Factor
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ABSTRACT: 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.
Biotechnology Letters 02/2007; 29(1):129-35. · 1.68 Impact Factor
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ABSTRACT: 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.
Virology 10/2003; 314(1):9-15. · 3.35 Impact Factor
