Morphological characteristics of ectomycorrhizas formed by in vitro synthesis between conifer seedlings and Tuber mycelial strains of the Puberulum clade isolated in Japan

Seedlings of Pinus densiflora and Abies sachalinensis were inoculated with Tuber mycelial strains of the Puberulum clade in vitro to examine the morphological characteristics of their ectomycorrhizas. Axenically germinated seedlings were inoculated with the mycelia of five taxa from the Puberulum clade and grown in glass jars for 4 mo in an illuminated incubator. The seedlings were successfully colonized by the inoculated Tuber strains, as confirmed by the nuclear ribosomal internal transcribed spacer barcoding of the synthesized ectomycorrhizas. The ectomycorrhizas were characterized by a pale yellow to brown color, short needle-shaped cystidia, and net-like hyphal arrangement, and epidermoid cells on the mantle surface; notably, these features are similar to the ectomycorrhizas of various Puberulum clade members. As the ectomycorrhizas of different Tuber species are indistinguishable by morphological characters, molecular techniques are necessary to identify ectomycorrhizas formed by Tuber species within the Puberulum clade.