Micetismos: Parte 2: Síndromes con sintomatología demorada y latencia muy larga / Mushroom poisonings. Part 2: Delayed-onset syndromes with very long latency time

En este trabajo se analizan los micetismos menos comunes, caracterizados por la aparición demorada de síntomas y por tiempos de latencia muy largos. Estas intoxicaciones son provocadas por especies de hongos ectomicorrícicos poco comunes. Se analizan: a) Toxíndrome nefrotóxico demorado o retrasado. Micetismo orellánico o por orellanina. b) Toxíndrome con rabdomiólisis. Micetismos por Tricholoma equestre y Tricholoma terreum. c) Toxíndrome encefalopático o neurotóxico retrasado. Micetismo por Pleurocybella porrigens. d) Toxíndrome cardiovascular. Micetismo por Trogia venenata. Se discuten el tiempo de incubación, las características sintomatológicas, el curso clínico, las toxinas responsables de cada micetismo y su mecanismo de acción, y el tratamiento que ha resultado efectivo para la recuperación de los pacientes.

Less common mycetisms, characterized by delayed-onset and very long latency times are analyzed. These intoxications are caused by rare ectomycorrhizal fungal species. The following syndromes have been taken into account: (a) Delayed nephrotoxic syndrome. Orellanic mycetism or mycetism due to orellanin. (b) Syndrome with rhabdomyolysis. Mycetisms by Tricholoma equestre, and Tricholoma terreum. (c) Delayed encephalopathic or neurotoxic syndrome. Mycetism by Pleurocybella porrigens. (d) Cardiovascular syndrome. Mycetism by Trogia venenata. The incubation time, symptom characteristics, clinical course, toxins responsible for each mycetism and their mechanism of action, and treatment that has shown to be effective for patient recovery are discussed.

Neste trabalho, analisam-se micetismos menos comuns, caracterizados pelo aparecimento retardado de sintomas e tempos de latência muito longos. Essas intoxicações são causadas por espécies raras de fungos ectomicorrízicos. São analisados os seguintes: (a) síndrome de toxicidade nefrotóxica retardada ou atrasada. Micetismo orelânico ou micetismo por orelanina. (b) Toxíndrome com rabdomiólise. Micetismo por Tricholoma equestre e Tricholoma terreum. (c) Toxicidade tardia encefalopática ou neurotóxica. Micetismo por Pleurocybella porrigens. (d) Síndrome cardiovascular. Miceticismo por Trogia venenata. O tempo de incubação, as características sintomatológicas, o curso clínico, as toxinas responsáveis por cada micetismo e seu mecanismo de ação e o tratamento efetivo para a recuperação dos pacientes são discutidos.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Spore Dispersion of Tricholoma matsutake at a Pinus densiflora Stand in Korea

The spore of Tricholoma matsutake is considered to be the starting point of the mushroom growth cycle, but the mechanism of mycelial development from the spore stage is not yet clarified. In this study, we tried to measure how far the spores of T. matsutake disperse from a fruiting body located at a Pinus densiflora stand in Korea. We established 16 slide glasses coated with glycerin near a fruiting body in four directions separated by four different distance intervals within a mushroom productive stand after removing all other fruiting bodies from three plots. The number of dispersed spores increased with time from the first day (475 spores/cm2) to the fourth day (836 spores/cm2) after the pileus opened. The number of spores dispersed downward was about 1.5 times greater than that dispersed toward the ridge. The number of dispersed spores decreased exponentially as the distance from each fruiting body increased. More than 95% of the spores dropped within a meter from the fruiting body, with 75% dropping within 0.5 m. Even so, the number of spores dispersed over 5 m from the fruiting body was more than 50 million when considering the total number of spores produced by a fruiting body is about 5 billion.

Ergosterol and Water Changes in Tricholoma matsutake Soil Colony during the Mushroom Fruiting Season

The purpose of this study is to understand spatio-temporal changes of active fungal biomass and water in Tricholoma matsutake soil colonies during the mushroom fruiting season. The active fungal biomass was estimated by analyzing ergosterol content at four different points within four replicated locations in a single circular T. matsutake colony at Ssanggok valley in the Sogri Mt. National Park in Korea during 2003 to 2005. The four points were the ahead of the colony, the front edge of the colony and 20 cm and 40 cm back from the front edge of the colony. Ergosterol content was 0.0 to 0.7 microg per gram dried soil at the ahead, 2.5 to 4.8 microg at the front edge, 0.5 to 1.8 microg at the 20 cm back and 0.3 to 0.8 microg at the 40 cm back. The ergosterol content was very high at the front edge where the T. matsutake hyphae were most active. However, ergosterol content did not significantly change during the fruiting season, September to October. Soil water contents were lower at the front edge and 20 cm back from the front edge of the colony than at the ahead and 40 cm back during the fruiting season. Soil water content ranged from 12 to 19% at the ahead, 10 to 11% at the edge, 9 to 11% at the 20 cm back and 11 to 15% at the 40 cm back. Our results suggest that the active front edge of the T. matsutake soil colony could be managed in terms of water relation and T. matsutake ectomycorrhizal root development.

Development of Specific Primer for Tricholoma matsutake

In this study, in an effort to develop a method for the molecular detection of Tricholoma matsutake in Korea from other closely related Tricholomataceae, a species-specific PCR primer pair, TmF and TmR, was designed using nuclear ribosomal intertranscribed spacer (ITS) sequences. The DTmF and DTmR sequences were 5'-CCTGACGCCAATCTTTTCA-3' and 5'-GGAGAGCAGACTTGTGAGCA-3', respectively. The PCR primers reliably amplified only the ITS sequences of T. matsutake, and not those of other species used in this study.

Proliferation of Tricholoma matsutake Mycelial Mats in Pine Forest Using Mass Liquid Inoculum

Two isolates of Tricholoma matsutake T-008 and T-034, preserved in Entomopathogenic Fungal Culture Collection (EFCC) of Korea, were used in the present study. The isolates had 100% Bootstrap homology with Tricholoma matsutake U62964 and T. matsutake AB188557 and AF309538 preserved in Gene Bank of NCBI. Mycelial growth of T. matsutake was highest in TMM and MYA at 25degrees C. The highest dry wt. of mycelium was obtained after 65 days of culture, when 6 mycelial discs were inoculated in 100 ml of broth in 250 ml shaking flask. Mycelial mats were observed in clumped condition at the inoculation sites of pine forest after two weeks of inoculation. After 5 months of inoculation, mycelia mats were observed growing inside soil and walls of a few inoculation sites, while mycelial mats growth up to 5~8 cm were observed in the roots of pine tree after 6 months. The survival rate of the inoculum was about 40% of the total inoculation sites. The survival rate was found below 20% when the mycelium was inoculated in the summer. The reasons for low survival rates of the mycelium were mainly due to dry season and the soil-borne small animals such as earthworm and mole. After one year of inoculation, no external difference was observed between the artificially inoculated mycelia and the naturally existing mycelia of T. matsutake. The present study showed that fruiting bodies of T. matsutake could be produced by artificial inoculation under the appropriate environmental conditions.

Mycorrhizal Formations and Seedling Growth of Pinus desiflora by in vitro Synthesis with the Inoculation of Ectomycorrhizal Fungi

The mycelia were directly isolated from eight species of fungal basidiocarps, confirmed to the ectomycorrhiza in the roots from the fields (forestry); Suillus bovinus, Paxillus involutus, Lactarius hysginus, Russula fragilis, Lepista nuda, Lyophyllum shimeji, Tricholoma matsutake, and Russula integra. The mycelia were pure-cultured with several transferring in various agars, and inoculated to the roots of pine (Pinus densiflora) seedling by in vitro method. After ten months growth under artificially aseptic conditions, all pine seedlings inoculated were stimulated at the growth-height, whereas those not inoculated were nearly dead. Also, the ramifications of ectomycorrhizal pine roots formed in the synthetic in vitro systems and were various according to the different mycelia. Synthesis of ectomycorrhiza were clearly confirmed in ten months growth, but not distinguished at this moment. It was clearly proved that the mycelia isolated caused the ectomycorrhizae in the roots of pine seedlings.

Characteristics of the Amylase and its Related Enzymes Produced by Ectomycorrhizal Fungus Tricholoma matsutake

Extracellular amylase properties were examined with the mycelium of Tricholoma matsutake isolated from ectomycorrhizal roots of Pinus densiflora. The molecular weights of alpha-amylase and glucoamylase were estimated as 34.2 kD and 11.5 kD, respectively, after eluted through Superdex 75 column. The optimum pH of the purified enzyme was found in a range of pH 5.0~6.0, with a peak at pH 5.0. The activities of these enzymes were stable from 4degrees C to 30degrees C. The alpha-amylase of T. matsutake readily hydrolyzed soluble starch and amylose-B, while it weakly hydrolyzed glycogen, dextrin, amylose and amylose-A. The main products of hydrolysis were confirmed to be glucose, maltose and maltotriose on the basis of the similarities in the thin layer chromatographic mobility.

Cloning and Phylogenetic Analysis of Chitin Synthase Genes from Tricholoma matsutake

Chitin synthases(UDP-N-acetyl-D-glucosamine: chitin 4-beta-N-acetyl-D-glucosaminyl transferase, EC 2.4.1.16) catalyze the synthesis of chitin from UDP-N-acetyl-D-glucosamine. Two zymogenic type of chitin synthase gene(TmCHS1 and TmCHS2) were amplified and its nucleotide sequences were determined. By the amino acid comparison and UPGMA tree grouping, TmChs1 and TmChs2 were classified as class II and class IV chitin synthases respectively. The class II type TmChs1 was grouped with others of Agaricales ectomycorrhizal mushroom. Additionally the phylogenetic tree was well adapted to Hymenomycete previously classified by morphological and physiological characteristics.

Ectomycorrhizal Roots Collected from the Bases of the four Edible Basidiocarps Around Mt. Wol Ak

The ectomycorrhizal roots were collected from the soils around the bases of basidiocarps of the four edible mushrooms in the stands of Pinus densiflora or Querus acutissima communities (Mt. Wol-Ak in Eastern Chung-Puk): The basidiocarps of Tricholoma matsutake (TM), Sarcodon asparatus (SA), S. imbricatum (SI), and Polyozellus multiplex (PM) are usually collected. The ectomycorrhizal roots of TM, PM and SI were related to the roots of P. densiflora, but the other to the roots of Q. acutissima in Korea. Particularly, the basidiocarps of PM were collected in the mixed stand of both P. densiflora and Q. acutissima. The morphologies of the ectomycorrhizal roots were observed to be the yellowish brown coral (dichromatous) or pyramid types in the roots of the pine, but dark brown un-branched sticks (roots) in the ends of ectomycorrhizal roots of Querus plants. The un-branched roots were covered with the dark mycelia (rhizomorph) around them. Therefore, the ectomyorrhizal roots of PM were observed to have two kinds of types; The single blackish un-branched roots were observed to be attached to the yellowish coral type roots. The bundles of TM mycelia were filled with cortical cells (in the roots of P. densiflora), but the mycelia of the other fungi (Aphylloporales) were massed between the cortical cells of P. densiflora or Q. acutissima. Their anatomical and gross features were considered to be simailar but very important in the ectomycorrhizal roots for these edible mushrooms.