Phylogenetic placements and cultural characteristics of Tuber species isolated from ectomycorrhizas

Abstract

Pure cultures of Tuber were isolated from ectomycorrhizal root tips in Abies sachalinensis plantations in Hokkaido, Japan. Their phylogenetic relationships as well as vegetative hyphal characteristics on culture media were reported. Phylogenetic analysis based on the internal transcribed spacer within ribosomal DNA settled well-supported eight lineages within Puberulum, Latisporum, and Maculatum clades in Tuber. Three and one lineages were grouped with undescribed species of Puberulum clade in Japan and that of the Latisporum group in China, respectively. Two lineages were closely associated to but distinct from an undescribed species of Puberulum clade in Japan. One lineage did not group with any sequences in the International Nucleotide Sequence Database (INSD), proposing a new taxon in the Latisporum group. One lineage was grouped with T. foetidum in Maculatum clade. All strains in each lineage displayed yellowish white, thin, filamentous colonies on Melin-Norkrans agar medium. Various differences in morphological characteristics of hyphae on pure cultures of various strains were noted, but they were frequently uncommon among strains of the same taxa. Isolation from ectomycorrhizal root tips can be among the effective ways to acquire pure cultures of Tuber strains.

Keywords: Latisporum; Puberulum; ectomycorrhizal root; pure culture; truffles.

Fig. 1. – Phylogenetic diversity of pure cultured strains of Tuber acquired in this study and their allied species. Phylogenetic relationships were inferred with the use of the maximum likelihood method based on ITS sequences. Bootstrap values greater than 80% are designated at the nodes (1,000 replications). One phylogenetic tree was split into two parts and appointed each as (A) and (B).

Fig. 2. – Colonies of cultured Tuber spp. on MMN agar medium. A: TuSp16 (OBASE00113). B: TuSp17 (OBASE00016). C: TuSp18 (OBASE00012). D: TuSp2016A (OBASE00093). E: TuSpKOA (OBASE00117). F: TuSpKOC (OBASE00040). G, H: T. foetidum (OBASE00103) with (G) and without bacteria (H). Bars: 1 cm.

Fig. 3. – Morphological characteristics and branching pattern of the hyphae of cultured strains of Tuber spp. A, B: Hyphal aggregates in TuSp2016A strain OBASE00093 (A) and TuSpKOC strain OBASE00040 (B). C: Hyphal cells harbor vacuoles in TuSp18 strain OBASE00134. D: Swollen and vesicle-like hyphae in TuSp17 strain OBASE00016. E: Hyphal anastomosis in TuSp2016A strain OBASE00093 (arrows). F, G: Swollen hyphae at the hyphal anastomosis in TuSp17 strain OBASE00016. H: Normally septated and irregularly branched hyphae in TuSp18 strain OBASE00134. I: Normally septated and irregularly branched hyphae with swollen cells in T. foetidum strain OBASE00103 which is contaminated by bacteria. Bars: A, F 500 µm; B 100 µm; C–E, G–I 50 µm.