Tricholoma matsutake forms ectomycorrhizas with Pinus densiflora under field conditions. The present study aimed to test the ability of T. matsutake isolates to form mycorrhizas with aseptic seedlings of P. densiflora in vitro. Pine seeds were germinated aseptically on a nutrient agar medium, and pairs of 1-wk-old seedlings were transplanted into polymethylpentene bottles containing autoclaved sphagnum moss/vermiculite substrate. The substrate was saturated with nutrient medium containing glucose. At the same time, the bottles were inoculated with a T. matsutake isolate. Three mo after inoculation, the fungus formed a sheath and Hartig net on the pine lateral roots. Ectomycorrhizas were also confirmed on 4-6-mo-old seedlings which showed the same or slightly better growth than the control plants. These results indicate that cultured T. matsutake mycelium can form true ectomycorrhizas with P. densiflora seedlings in vitro.

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Mycorrhizal association of Tricholoma matsutake with Pinus densiflora was studied. A naturally established P. densiflora stand (age: ca. 45 yr) where occurrences of T. matsutake sporocarps had been confirmed was studied in Ibaraki Prefecture, Japan. Pine root systems connected with T. rnatsutake sporocarps via the fungal white mycelia were sampled in October 1997. The sampled pine roots were covered overall with white mycelia. Under a dissecting microscope, the mycelia were confirmed to form fungal sheaths on the lateral roots. Under a light microscope, transverse and longitudinal sections of these roots showed the presence of both fungal sheaths and Hartig nets, which are typical of ectomycorrhizas. The fungal sheath was ca. 1.5-20/~m in thickness, and felt prosenchymatous in texture. Hartig nets developed continuously at the cortex and extended to the boundary between cortical cells and endodermal cells. The same ectomycorrhizal morphotype on the pine was also recovered from inside the same mycelial colony (i.e., "shiro") of T. matsutake from winter to summer. These results suggest that T. rnatsutake has a perennial ectomycorrhizal association with P. densiflora.

Tricholoma matsutake was isolated into pure cultures from field samples of ectomycorrhizas on Pinus densiflora. The mycorrhizal tips were collected at different times of the year from a colony of T. matsutake in a P. densiflora stand, The mycorrhizal tips were continuously washed with sterilized distilled water and diluted Tween 80 solution, surface-sterilized with calcium hypochlorite solution, and inoculated on several kinds of nutrient agar media, Most of the mycorrhizal tips collected in winter and spring produced colonies that were morphologically similar to cultures of T. matsutake isolated from basidiocarps, The identity of isolates obtained from mycorrhizas was further confirmed to be T. matsutake based on fungal morphology and RFLP patterns of PCR amplified rDNA, The feasibility of T. bakamatsutake isolation into pure culture from ectomycorrhizas on Quercus serrata was also confirmed. These results indicated that mycelium of matsutake mushrooms can be isolated into pure culture from ectomycorrhizas at different times of the year, Mycorrhizas of both T. matsutake and T. bakamatsutake were not observed to have any specific association with soil fungi such as Mortierella spp,

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