BackgroundA mycovirus previously identified in brown discolored fruiting bodies of the cultivated mushroom Flammulina velutipes was characterized. We tentatively named the virus the F. velutipes browning virus (FvBV).ResultsPurified FvBV particles contained two dsRNA genomes (dsRNA1 and 2). The complete sequence of dsRNA1 was 1,915 bp long, containing a single open reading frame (ORF) that encoded 580 amino acids of a putative 66-kDa RNA-dependent RNA polymerase (RdRp). dsRNA2 was 1,730 bp long containing a single ORF encoding 541 amino acids of a putative 60-kDa coat protein (CP1). Phylogenetic analysis of the RdRp sequences revealed FvBV to be a Partitivirus, most closely related to Chondrostereum purpureum cryptic virus. An RT-PCR assay was developed for the amplification of a 495-bp cDNA fragment from dsRNA encoding the CP1. When wild F. velutipes isolated from various parts of Japan were examined by RT-PCR assay, three isolates from the central region of Japan contained FvBV. One wild strain infected with FvBV was isolated in Nagano prefecture, where brown discoloration of white cultivated strains has occurred. Fruiting bodies produced by virus-harboring and virus-free F. velutipes were compared.ConclusionsCap color of the fruiting bodies of F. velutipes that contained Partitivirus FvBV was darker than FvBV-free fruiting bodies. The use of RT-PCR enabled association of FvBV and dark brown color of the fruiting body produced by F. velutipes strains.

BackgroundIn the 1970s, mycoviruses were identified that infected the edible mushroom Lentinula edodes (shiitake), but they were not regarded as causal agents for mushroom diseases. None of their genes has been sequenced. In this study, the dsRNA genome of a mycovirus recently found in a shiitake commercial strain was sequenced and its molecular structure was characterized.MethodsA cDNA library was constructed from a dsRNA purified from the fruiting body of L. edodes. The virus was tentatively named L. edodes mycovirus HKB (LeV). Based on the deduced RNA-dependent RNA polymerase (RdRp) sequence, phylogenetic analysis of LeV was conducted. Because no virion particles associated with the dsRNA were observed by electron microscopic observation, atomic force microscopy (AFM) observation was chosen for achieving molecular imaging of the virus.ResultsThe 11,282-bp genome of LeV was obtained. The genome encoded two open reading frames (ORFs). ORF1 coded for a hypothetical protein and ORF2 for a putative RdRp, respectively. In addition, a region coding for a NUDIX domain was present in ORF1. There was a 62-bp intergenic region between ORF1 and RdRp. Similarity with coat protein of mycoviruses was not found within the whole sequence. Based on phylogenetic analysis of the putative RdRp sequence, LeV grouped into a clade with dsRNA found in the basidiomycetes Phlebiopsis gigantea and Helicobasidium mompa. The clade was placed apart from the Totiviridae and Chrysoviridae families. As suggested from the genome sequence, AFM revealed that the structure of LeV was linear unencapsidated dsRNA.ConclusionsThe results suggest that LeV represents a novel family of mycoviruses, found thus far only among the basidiomycetes.

Degeneration of cultivated strains of Flammulina velutipes is a serious problem. We developed a simple colorimetric method to detect degenerate strains by using a liquid medium supplemented with bromothymol blue and lactose. The ability of a strain to develop normal mushrooms could be determined by the color of the medium.The primary objective of this study was to develop a simple method for detecting degenerate Flammulina velutipes (Enokitake) cultures. Cultural degeneration of cultivated strains of Enokitake similar to the degeneration observed for Agaricus bisporus (1, 2) has become a serious problem in Japan. Previous efforts to evaluate the fruiting potential of Enokitake have been made using isozyme electrophoresis profiles, randomly amplified polymorphic DNA, and mitochondrial DNA restriction fragment length polymorphism, but there was no clear correlation between the results of these experiments and the degenerate symptoms. Normal and degenerate strains differ in the ability to decolorize synthetic dyes, such as bromothymol blue (BTB), which changes from blue-green to yellow. The underlying cause of degeneration is not known, so a simple bioassay that distinguishes degenerate and productive strains of Enokitake would be of use for both researchers and commercial spawn producers.Commercial cultivation of Enokitake began in Japan in the 1930s with sawdust and rice bran as the substrate. In 2003, the total yield of Enokitake was 110,185 tons, which is much greater than the 65,363 tons of Lentinula edodes (Shiitake) or the 5,210 tons of Pleurotus ostreatus produced in Japan (annual statistics for mushroom production in Japan from the Forestry Agency [http://www.rinya.maff.go.jp/puresu/h16-8gatu /0805tokusan.htm]). The degenerate symptoms of Enokitake reported in the 1980s were malformed fruiting bodies, reduced numbers of primordia, and in some cases complete loss of fruiting body development. However, degenerate mycelia are morphologically indistinguishable from normal mycelia, and the symptoms of degeneration are not apparent until the final stage of mushroom cultivation, which may result in major financial losses.Four cultivars (TK,YO, JB, and G5) developed at Nagano Vegetable and Ornamental Experiment Station were used in the present study. The first symptom of degeneration usually is the development of undifferentiated, callus-like tissue in normal fruiting bodies. TKd, YOd, and JBd were isolated from malformed portions of fruiting bodies. TKd mycelia can produce malformed abnormal fruiting bodies, but TKm and JBm are even more severely degenerate and can develop very few primordia. Field strains in our culture collection that produced normal fruiting bodies on potato dextrose agar (Nissui, Tokyo, Japan) (FV wild a, FV wild b, and FV wild c) or that formed no fruiting bodies (FV wild d and FV wild e) also were examined. None of the strains used in this study contained detectable double-stranded RNAs (3).Each strain was inoculated onto malt extract agar (10 g/liter malt extract, 18 g/liter agar) in 9-cm ...

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