RESUMEN
BACKGROUND: In 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: The results suggest that LeV represents a novel family of mycoviruses, found thus far only among the basidiomycetes.
Asunto(s)
Genoma Viral , Virus ARN/genética , Virus ARN/aislamiento & purificación , ARN Viral/genética , Hongos Shiitake/virología , Biblioteca de Genes , Microscopía de Fuerza Atómica , Microscopía Electrónica , Datos de Secuencia Molecular , Filogenia , Virus ARN/ultraestructura , ARN Bicatenario/genética , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
BACKGROUND: A 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). RESULTS: Purified 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. CONCLUSIONS: Cap 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.
Asunto(s)
Flammulina/virología , Virus ARN/genética , Virus ARN/aislamiento & purificación , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Análisis por Conglomerados , Genoma Viral , Japón , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Filogenia , Virus ARN/clasificación , ARN Bicatenario/genética , ARN Bicatenario/aislamiento & purificación , ARN Viral/genética , ARN Viral/aislamiento & purificación , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Análisis de Secuencia de ADN , Homología de Secuencia , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
To elucidate the structure-activity relationships of saponin on fruiting body induction in Pleurotus ostreatus, monodesmosidic saponins comprising a triterpenoid and glucose residues were synthesized. Betulin, a triterpenoid present in Birch bark, was used as the aglycon. The fruiting body induction activity increased with the number of glucose residues, from one to four. The most active betulinic saponin was betulin-3-yl beta-D-(4-O-beta-D-maltotriosyl)-glucoside.
Asunto(s)
Cuerpos Fructíferos de los Hongos/efectos de los fármacos , Pleurotus/efectos de los fármacos , Saponinas/farmacología , Triterpenos/farmacología , Cuerpos Fructíferos de los Hongos/química , Conformación Molecular , Pleurotus/química , Saponinas/síntesis química , Saponinas/química , Estereoisomerismo , Relación Estructura-Actividad , Triterpenos/síntesis química , Triterpenos/químicaRESUMEN
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.
Asunto(s)
Agaricales/crecimiento & desarrollo , Azul de Bromotimol/metabolismo , Colorimetría/métodos , Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Microbiología Industrial/métodos , Agaricales/aislamiento & purificación , Medios de Cultivo , Japón , Lactosa/metabolismoRESUMEN
Amphipathic glucose derivatives 3-O-octyl- and 3-O-decyl-D-glucose were synthesized, and the ability to induce fruit bodies on Pleurotus ostreatus was examined. Surfactants such as CHAPS, CHAPSO, MEGA-8, MEGA-9, and MEGA-10 were also assayed. The result was that both glucose derivatives could stimulate fruiting on P. ostreatus, while none of the surfactants assayed in this study stimulated the fruiting of P. ostreatus. These results suggest that sugar moiety is necessary for amphipathic compounds to work as a fruiting signal for P. ostreatus.
Asunto(s)
Cuerpos Fructíferos de los Hongos/efectos de los fármacos , Glucosa/análogos & derivados , Pleurotus/efectos de los fármacos , Ácidos Cólicos/farmacología , Glucosa/química , Glucosa/farmacología , Pleurotus/crecimiento & desarrollo , Sorbitol/análogos & derivados , Sorbitol/farmacología , Relación Estructura-Actividad , Tensoactivos/farmacologíaRESUMEN
To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 alpha-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated.
Asunto(s)
Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Pleurotus/crecimiento & desarrollo , Técnica del ADN Polimorfo Amplificado Aleatorio , Cuerpos Fructíferos de los Hongos/genética , Cuerpos Fructíferos de los Hongos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Datos de Secuencia Molecular , Pleurotus/genética , Pleurotus/metabolismo , Análisis de Secuencia de ADNRESUMEN
Uracil auxotroph of Pleurotus ostreatus was transformed to prototrophy by means of particle bombardment. Five transformants were obtained under three conditions differing in the two parameters of target distance and helium pressure. The transformation frequency was one transformant per microg of DNA. In the transformants, plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of P. ostreatus by particle bombardment.