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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.

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We acknowledge Masamichi Ichikawa for the supply of fresh Tricholoma matsutake samples in the spore inoculation experiment, and Hitoshi Murata in Forestry and Forest Products Research Institute for the technical advice in DNA analyses. We thank the members of Applied Mycology Laboratory, Shinshu University for their support of this study.


This study was supported in part by KAKENHI Grant Number 15H01751 from the Japan Society for the Promotion of Science (JSPS), and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan, “Technology development for the optimal use of forest resources.”

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Correspondence to Akiyoshi Yamada.

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Horimai, Y., Misawa, H., Suzuki, K. et al. Spore germination and ectomycorrhizae formation of Tricholoma matsutake on pine root systems with previously established ectomycorrhizae from a dikaryotic mycelial isolate of T. matsutake. Mycorrhiza 31, 335–347 (2021).

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  • Edible mycorrhizal mushrooms
  • Fungus–plant interaction
  • Genetic diversity
  • Pinus densiflora
  • Seedling growth