Akira Ohta

Production of fruit-bodies of a mycorrhizal fungus, Lyophyllum shimeji, in pure culture

Cultivation of mycorrhizal fungus,Lyophyllum shimeji, was examined using selected strains capable of forming primordia in pure culture. Mycelia grew fastest on barley grains containing synthetic liquid medium. The primordia readily formed in test-tubes after lowering the incubation temperature from 23°C to 15°C. The co-existence of pine seedlings had no promotive effect on primordium formation. Fruit-bodies formed on a medium consisting of barley, beech sawdust, and liquid synthetic nutrients in 500-ml glass bottles. Mature fruit-bodies produced basidiospores. The spores thus produced could germinate on an agar medium and formed mycelial colonies. Thereby, the life cycle inL. shimeji was accomplished in pure culture without using the host plant.

Genetic mosaics in the massive persisting rhizosphere colony "shiro" of the ectomycorrhizal basidiomycete Tricholoma matsutake.

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.

Ability of ectomycorrhizal fungi to utilize starch and related substrates

Basidiomycetous fungi of 55 strains of 33 species in 15 genera which are thought to be ectomycorrhizal were grown on starch and related substrates as a sole carbon source, and their ability to utilize these substrates was determined. Mycellial weights of the fungi grown on agar media containing starch and amylose varied between the strains from 1.1 to 94.9 mg/flask and from 0.4 to 93.3mg/flask, respectively. Mycelial growth rates ranged from 0 to 1.17 mm/d on barley grain medium and from 0 to 2.03 mm/d on rice grain medium; the highest rate corresponded to about half of the average of reference wood-rotting fungi. Most of the mycorrhizal fungi that grew well on amylose gave higher growth rates on barley. Several strains inLyophyllum, Hebeloma, Sarcodon, andTricholoma grew well on both glucose and starch media.

Traceability of Asian Matsutake, Specialty Mushrooms Produced by the Ectomycorrhizal Basidiomycete Tricholoma matsutake, on the Basis of Retroelement-Based DNA Markers

ABSTRACT The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies, matsutake, in forests. Here we report a PCR system targeting retroelement integration sites to differentiate among individual Asian isolates of T. matsutake based on their geographical origins, such as Japan, the area of South Korea through North Korea, the northeastern provinces of China, and the area of the southwestern provinces of China through Bhutan. The overall misjudgment rate of the analytical system was approximately 5% based on 95 samples of T. matsutake examined including those from cultures and from commodities. We also provide evidence that T. matsutake isolates grown throughout the Far East, including the northeastern provinces of China, are closely related to each other while distinct from those in the area of the southwestern provinces of China through Bhutan. The method allows us to trace back geographical origins of Asian matsutake, thus contributing to food safety, appropriate tariffs, and proper price setting.

Fruit-body production of two ectomycorrhizal fungi in the genus Hebeloma in pure culture

Fruit-body production of two ectomycorrhizal fungi,Hebeloma radicosum andhebeloma sp. (nagaenosugitakedamashi in Japanese), in pure culture was examined. First, nutrients that promote mycelial growth of the fungi when added to the basal medium consisting of barley grains and sawdust were determined. Then the fungi were cultivated to produce fruit-bodies in larger-scale media containing additional nutrients selected for each fungus. Mature fruit-bodies bearing basidiospores were formed after incubation at 22°C for 35–42 d, followed by incubation at 17°C for 21–32 d.

Detection of Tricholoma matsutake by specific ITS primers

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA of ectomycorrhizal fungi were analysed and a specific PCR primer pair for Tricholoma matsutake was designed. Using the primer pair, part of the ITS region of T. matsutake (400 bp) was amplified, but no amplified fragment was detected for other ectomycorrhizal fungi. PCR was performed on DNA extracted from mycorrhizas of T. matsutake and Shiro soil (extramatrical mycelium of T. matsutake and soil complexes) and the 400 bp fragments were also specifically amplified. This indicates that presence of T. matsutake at any of its life stages can be easily confirmed by PCR typing.

Some cultural characteristics of mycelia of a mycorrhizal fungus, Lyophyllum shimeji

Cultural characteristics of 45 strains ofLyophyllum shimeji and 10 strains of three related species were determined. The average optimum temperature for mycelial growth ofL. shimeji on a medium consisting of rye grains was 24.9°C, slightly higher than those forL. fumosum andL. decastes. The average mycelial growth rate of theL. shimeji strains, each at its optimum temperature, was 2.0 mm/day, almost the same as that ofL. decastes and 2 times greater than those ofL. semitale andL. fumosum. All strains ofL. shimeji could grow on beech and pine sawdust, but none could significantly decompose beech wood blocks. Of the 45 strains ofL. shimeji, 3 strains had ability to form primordia on the rye grain medium without the host plant.

Bipolar incompatibility system of an ectomycorrhizal basidiomycete, Rhizopogon rubescens

The mating systems of most ectomycorrhizal fungi have not been elucidated because of two reasons. One is the difficulty of obtaining homokaryotic isolates for mating tests caused by the low germination rate of basidiospores, and another is the difficulty of checking dikaryotization caused by the absence or inconsistent production of clamp connections on heterokaryotic mycelia under laboratory conditions. Basidiospore germination of a few ectomycorrhizal fungi has been induced by living roots of their host plants. Based on this information, we examined methods to obtain homokaryotic isolates of Rhizopogon rubescens using its host plant, Pinus thunbergii. The basidiospores of R. rubescens appeared to germinate well on an agar plate, on which axenic pine seedlings were grown in advance to induce germination, even when the seedlings were removed from the plate at the time of spore inoculation. To enhance the production rate of clamp connections on the heterokaryotic mycelia of R. rubescens, the culture medium composition was modified. The pH of the medium was critical for the production of clamp connections, and the optimal pH was higher for the production of clamp connections than for mycelial growth. These findings made it possible to conduct mating tests, and we found that the mating system of R. rubescens is bipolar with a multiallelic mating type factor.

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.

Fruit-body production of an ectomycorrhizal fungus in genus Boletus in pure culture

Mycelial growth and fruit-body production of an ectomycorrhizal Boletus sp. were examined in pure culture. Mycelia of the strain Bo1 grew well on a medium consisting of sawdust and barley grains. Mature fruit bodies bearing basidiospores were produced after incubation at 22°C for 90 days in the dark, followed by incubation at 26°C for 30–46 days under conditions of high humidity and illumination. The addition of porous stone as a casing on the medium increased fruit-body yield. Deposited spores germinated well on an agar medium and formed mycelial colonies, thus completing the life cycle of Bo1 without a host plant and under axenic conditions. The ability of Bo1 to form ectomycorrhizas was confirmed by axenic resynthesis of mycorrhizas on Quercus serrata. Cultured fruit bodies of Bo1 resembled Gyroporus castaneus and Boletus subcinnamomeus, but its taxonomic position was not elucidated at the species level.