Spore germination and ectomycorrhizae formation of Tricholoma matsutake on pine root systems with previously established ectomycorrhizae from a dikaryotic mycelial isolate of T. matsutake.

In vitro ectomycorrhizal synthesis of Tricholoma matsutake with host plants has been widely conducted to elucidate fungal symbiotic properties for future cultivation practices. Here, we report on the importance of basidiospore inocula for this fungus to provide ectomycorrhizal seedlings in vitro. Ectomycorrhizal pine seedlings synthesized in vitro with cultured mycelium of T. matsutake (isolate #45 or #84) in a 250-mL culture vessel (soil volume) were transplanted to a large 1-L culture vessel. Fresh basidiospores of this fungus were aseptically inoculated on the ectomycorrhizal root system. The ectomycorrhizal seedlings in the 1-L vessel were grown for 9 months, and some plants were further grown for 6 more months under non-aseptic conditions in 4.1-L jars. The ectomycorrhizal seedlings previously inoculated with isolate #84 in the 1-L vessel showed significant ectomycorrhizal biomass (mycorrhizal root length) after spore inoculation. The ectomycorrhizal seedlings in the 4.1-L vessel showed large shiro structures (> 10 cm in diameter). PCR amplification of intergenic spacer 1 of the rRNA gene and long terminal repeat retroelement of T. matsutake in ectomycorrhizal root tips in both the 1-L vessels and 4.1-L jars revealed the presence of amplicons of the previously inoculated culture isolate of T. matsutake and the new genet(s) that established via germination of the inoculated basidiospores. This is the first report that inoculated basidiospores of T. matsutake germinated and colonized the host root to generate ectomycorrhizae in vitro.

Repeated fruiting of Japanese golden chanterelle in pot culture with host seedlings.

Yellow chanterelles are among the most popular wild edible ectomycorrhizal mushrooms worldwide. The representative European golden chanterelle, Cantharellus cibarius, has only once been reported to fruit under greenhouse conditions, due to the difficulty of establishing pure culture. Recently, we developed a new technique for establishing a pure culture of a Japanese golden chanterelle (Cantharellus anzutake), and conducted in vitro ectomycorrhizal synthesis using established strains and Pinus densiflora. Acclimated pine mycorrhizal seedlings colonized with C. anzutake in a pot system under laboratory conditions produced small but distinct basidiomata with developed basidiospores. C. anzutake mycorrhizae were established on Quercus serrata seedlings by inoculation of mycorrhizal root tips of the fungus synthesized on P. densiflora. A scaled-up C. anzutake-host system in larger pots (4 L soil volume) exhibited repeated fruiting at 20-24 °C under continuous light illumination at 150 µmol m-2 s-1 during a 2-year incubation period. Therefore, a C. anzutake cultivation trial is practical under controlled environmental conditions.