Role of filamentous fungi in migration of radioactive cesium in the Fukushima forest soil environment.

The fate of radioactive Cs deposited after the Fukushima nuclear power plant accident and its associated radiological impacts are largely dependent on its mobility from surface soils to forest ecosystems. We measured the accumulation of radioactive Cs in the fruit bodies of wild fungi in a forest at Iitate, Fukushima, Japan. The transfer factors (TFs) of radioactive Cs from soil to the fruit bodies of wild fungi were between 10-2 and 102, a range similar to that reported for the fruit bodies collected in Europe after the Chernobyl accident and in parts of Japan contaminated by the nuclear bomb test fallout. Comparison of the TFs of wild mushroom and those of fungal hyphae of 704 stock strains grown on agar medium containing nutrients and 137Cs showed that the TFs of wild mushroom were lower. The TF was less than 0.1 after the addition of the minerals zeolite, vermiculite, phlogopite, smectite, or illite of 1.0% weight to the agar medium. These results indicate that the presence of minerals decreases Cs uptake by fungi grown on the agar medium, and filamentous fungi still accumulate radioactive Cs even when minerals are present in the medium.

A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, "matsutake" mushrooms along densely developed mycelia, called "shiro," in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.

Ectomycorrhizas in vitro between Tricholoma matsutake, a basidiomycete that associates with Pinaceae, and Betula platyphylla var. japonica, an early-successional birch species, in cool-temperate forests.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates with Pinaceae in the Northern Hemisphere and produces prized "matsutake" mushrooms. We questioned whether the symbiont could associate with a birch that is an early-successional species in boreal, cool-temperate, or subalpine forests. In the present study, we demonstrated that T. matsutake can form typical ectomycorrhizas with Betula platyphylla var. japonica; the associations included a Hartig net and a thin but distinct fungal sheath, as well as the rhizospheric mycelial aggregate "shiro" that is required for fruiting in nature. The in vitro shiro also emitted a characteristic aroma. This is the first report of an ectomycorrhizal formation between T. matsutake and a deciduous broad-leaved tree in the boreal or cool-temperate zones that T. matsutake naturally inhabits.

The host ranges of conifer-associated Tricholoma matsutake, Fagaceae-associated T. bakamatsutake and T. fulvocastaneum are wider in vitro than in nature.

Tricholoma matsutake is the most commercially important edible mushroom in pine forests in Japan. Tricholoma bakamatsutake and T. fulvocastaneum, species closely related to T. matsutake, occur in Fagaceae forests. We examined ectomycorrhizal (EM) formation by these Tricholoma species by in vitro synthesis among seven strains (two of T. matsutake, four of T. bakamatsutake, one of T. fulvocastaneum) and axenic plants of pine (Pinus densiflora) and oak (Quercus serrata, Q. phillyraeoides). All strains, except for one of T. matsutake, formed EM associations with both pine and oak. Plant growth and mycelial development were differently affected by EM formation depending on the plant-fungus combination.

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa.

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or "shiro". We questioned whether this phenomenon could have occurred because of plant-microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant-fungal symbiosis.

Mobile DNA distributions refine the phylogeny of "matsutake" mushrooms, Tricholoma sect. Caligata.

"Matsutake" mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.

Phylogenetic relationship and species delimitation of matsutake and allied species based on multilocus phylogeny and haplotype analyses.

Tricholoma matsutake (S. Ito & S. Imai) Singer and its allied species are referred to as matsutake worldwide and are the most economically important edible mushrooms in Japan. They are widely distributed in the northern hemisphere and established an ectomycorrhizal relationship with conifer and broadleaf trees. To clarify relationships among T. matsutake and its allies, and to delimit phylogenetic species, we analyzed multilocus datasets (ITS, megB1, tef, gpd) with samples that were correctly identified based on morphological characteristics. Phylogenetic analyses clearly identified four major groups: matsutake, T. bakamatsutake, T. fulvocastaneum and T. caligatum; the latter three species were outside the matsutake group. The haplotype analyses and median-joining haplotype network analyses showed that the matsutake group included four closely related but clearly distinct taxa (T. matsutake, T. anatolicum, Tricholoma sp. from Mexico and T. magnivelare) from different geographical regions; these were considered to be distinct phylogenetic species.

megB1, a novel macroevolutionary genomic marker of the fungal phylum Basidiomycota.

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.