Rhizopogon roseolus (Corda) Th. M. Fr., known as “shouro” in Japanese, is an edible ectomycorrhizal mushroom. To successfully cultivate ectomycorrhizal mushrooms, inoculation and propagat on methods must be developed and isolates with superior traits must be selected. To advance successful cultivation, a cross-breeding system is desired to improve agronomic traits. The establishment of a cross-breeding system requires basic information on the life cycle of the mushroom. Pinus thunbergii seedlings were inoculated with several strains of R. roseolus using the Petri dish technique and their mycorrhizal abilities were assessed. Superior strains that rapidly colonized the roots of P. thunbergii and produced a large number of ectomycorrhizas were selected. An inoculation method using mycelial homogenates was found to be effective at producing ectomycorrhizas on seedlings. When the mycelial homogenate was inoculated on pine seedlings in a greenhouse, fruiting bodies were produced 3 － 4 months after the inoculation. Microscopic observation after HCl-Giemsa staining revealed distinct patterns of meiosis, post-meiotic mitosis, and nuclear migration. Electron microscopic studies showed that the spore-discharge mechanism was statismospory, and showed that the major ultrastructural change in basidiospore concerned in the spore wall. Variability in the rate of spore germination was observed between individual fruiting bodies. The peak germination rate was recorded for spores from fruiting bodies showing beige color. A higher spore germination rate was observed on a five-fold dilution of modified Melin-Norkrans medium. When the primary mycelial strain derived from the basdiospore was inoculated on pine seedlings, ectomycorrhizas were formed. A detection medium was developed for the rapid observation of clamp connection formation as an indicator of secondary mycelia. When tissue culture and basidiospore isolates were cultured on this detection medium, clamp connection formation was consistently observed in the tissue culture isolates, but never in the basidiospore isolates. The basic information regarding the life cycle of R. roseolus obtained from this study will contribute to the development of a cross-breeding system for ectomycorrhizal mushrooms.

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To develop artificial cultivation of Tricholoma matsutake, it is necessary to establish stable culture conditions under which mycelia can spread quickly. However, an advantageous solid-state culture method for this fungus has not yet been identified. We developed a solid-state culture medium using barley and vermiculite to obtain a large amount of T. matsutake mycelia in a short time, and we estimated the T. matsutake fungal biomass in this artificial medium. We determined optimal conditions for mycelial biomass quantification through measurement of n-acetylglucosamine (GlcNAc) concentration in the mycelia. For degrading dry T. matsutake mycelia, 1.0% Yatalase and 0.5% Cellulase “ONOZUKA” RS solution provided optimal degradation conditions, and 139 μg GlcNAc per 10 mg of dried mycelia was produced. Subsequently, T. matsutake Z-1, NBRC 30605, and strain No. 115 were tested and demonstrated good growth using medium with barley:vermiculite composition of 2:1 (w/w). After 35 days of cultivation, T. matsutake Z-1, NBRC 30605, and strain No. 115 produced 215.1, 254.0, and 266.7 mg biomass/flask, respectively. By both visual observation and measurement of GlcNAc content in colonized substrate block, a 2:1 barley:vermiculite composition was demonstrated to be the optimum medium for the culture of T. matsutake mycelia.

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Humidity control is considered to be very important in fruiting body formation during the cultivation of the mushroom Lentinula edodes. In this study, visualization of moisture transport after primordial formation was conducted using a fluorescent dye, Rhodamine B. Results showed that fluorescent dye sprayed on the mycelial block did not migrate into the fruiting body. On the other hand, fluorescent dye injected at the base of the fruiting body primordium was transported to the upper part of the stipe, while fluorescent dye dispersed on the primordium was delivered into the pileus of the fruiting body. These results suggested that a large amount of moisture may be taken into the pileus of the fruiting body from the air at the latter stage of fruiting body formation.

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