Search

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or "shiro," which produces the prized "matsutake" mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140 days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or "false matsutake" naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host-plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.

Molecular studies on the evolution and systematics of fungi have been established primarily based on the neutral theory by analyzing neutral mutations in some defined segments of housekeeping genes as genetic markers. Such an approach is, however, hardly applicable for analyzing ancient evolutionary radiations. In the present study, we looked for DNA sequences characterizing higher taxa, and discovered a unique macroevolutionary genomic marker, megB1, that specifies the phylum Basidiomycota. megB1 is an approximately 500-bp DNA element, which is defined by terminal sequences and five internal segments conserved throughout the phylum. megB1 resides on the rDNA intergenic spacer 1 (IGS1) from 27 species of 10 Basidiomycota genera examined. While megB1 was not found in IGS1 from the other 92 species of the 27 Basidiomycota genera, several genera representing them carry megB1 in some other genomic regions. No known taxonomic criteria fit into the classification on the basis of whether megB1 resides on rDNA. Neighbor-joining analysis of the megB1 sequence, however, properly assigned species to their respective genera. Thus far, megB1 has not been found in any genomic or genetic databases currently available for other phyla. These results suggest that megB1 may have emerged upon the occurrence of Basidiomycota, and that this phylum evolved thereafter leaving this element conserved throughout their further differentiation. megB1 may be a novel genomic marker useful in the analysis of ancient through the latest evolutionary radiation in Basidiomycota.

Mycorrhizal basidiomycetes, Suillus grevillei and S. bovinus, were transformed by particle bombardment. We isolated eight and four transformants from S. grevillei and S. bovinus respectively under three conditions differing in terms of target distance and helium pressure. The transformation frequencies were 1.2 and 0.6 transformants/microg DNA. Plasmid DNAs were integrated into the genomic DNA of the transformants and stably maintained. This is the first description of the transformation of S. grevillei and S. bovinus by particle bombardment.

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5' gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies "matsutake" on a massive persisting rhizosphere aggregate of mycelia and mycorrhizas called "shiro." Using inter-retrotransposon amplified polymorphism analysis, we attempted to explore the potential diversity within the population of T. matsutake isolated from small Pinus densiflora woodlands located in various parts of Japan. In general, random phylogenetic relationship was noted among T. matsutake tested. The population from each limited sampling area was highly heterogeneous. Even some isolates from fruit bodies produced in the same shiro and those from spores in the same fruit bodies were found to be genetically diverse, indicating the occurrence of genetic mosaics in shiro. In a mosaic shiro, heterologous genets produced their fruit bodies concurrently. Data suggested that the dispersal of spores through sexual reproduction may have been more prevalent than generally accepted in T. matsutake to bring mosaicism and coordination of heterologous genets within the shiro. Implementation of management taking such diversity into consideration is urgently needed for the restoration of devastated matsutake fields in Japan. Exploration of individual clones in mosaic fungal resources that promote colonization and fruit body production is necessary for it.

The spawn of cultivated mushrooms are generally produced, propagated, and distributed to growers as a mycelial culture without genetic purification, in which phenotypic variants frequently occur. We investigated how heterologous mycelia present in a spawn influence fruit body production in the cultivated basidiomycete Pholiota nameko. The 'di-mon' dual cultivation of protoplast clones produced mosaic fruit bodies, which could result from the 'di-mon' mating. In the 'di-di' dual cultivation of heterologous strains with different fruiting times, authentic fruit bodies of each dikaryon and chimera showing a feature combining characteristics of the two dikaryons emerged simultaneously. Mycelia isolated from the chimera produced all three types of fruit bodies, indicating unlikeliness of the occurrence of anastomosis. These results suggest that mycelia colonized in the substrate interact with each other and coordinately promote fruit body production in P. nameko. This phenomenon masks a clonal variability that may be surfaced through multiplication and distribution of the spawn, occasionally bringing about abnormal fruiting.

We designed a polymerase chain reaction that allows us to clone two types of putative reverse transcriptase genes from 11 species of ectomycorrhizal basidiomycetes that belong to the genus Tricholoma. One corresponds to the putative gene of marY1, a long terminal repeat (LTR) retroelement from Tricholoma matsutake, and the other, marY2N, a LINE-like non-LTR (L1-like) retroelement from this fungus. Putative protein products predicted from nucleotide sequencing of cloned fragments were phylogenetically analyzed. marY1-like elements had a parallel phylogenetic relationship with no apparent correlation to a current taxonomic profile, while marY2N-like elements showed a vertical one in relation to host-plant species. Data suggest that marY1-like elements and marY2N-like elements have evolved independently, and the evolution of marY1-like elements could have occurred later than the evolution of marY2N-like elements in the species of Tricholoma.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom 'matsutake' in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia. [ABSTRACT FROM AUTHOR]