A specimen was collected on roots of unknown trees beneath the log of Pinus densiflora, among the conifer forests of 30-year-old trees mixed with other deciduous trees such as Betula platyphylla, Cerastrus orbiculatus, Ligustrum tschonoskii, Rhododendron japonicum, Swida controvers. Although Vitis coignetiae is present in this forest, no individual was found nearby the collection site of the fungus, so it seems likely plausible that the host was not Vitis. The material was air-dried at 23°C for 24 hours. A small amount of spores were picked up by a fine needle and transferred to plates of malt extract agar (MEA; Nissui, Tokyo, Japan), cornmeal agar (CMA; Nissui), and half-strength malt extract-yeast extract agar (MEYE; malt extract 3 g; yeast extract 3 g, peptone 5 g, dextrose 10 g, agar 15 g, DW 1 L). Germination was observed under a light microscope to obtain a pure culture. The specimen and isolate were deposited in the mycological herbarium of the National Museum of Nature and Science (TNS) and the National Institute of Technology and Evaluation, Biological Resource Center (NBRC). The color names and codes in the description followed the Pantone color bridge (Pantone Inc., Carlstadt, NJ, USA), adopting the CYMK color system. Additional specimens collected in Japan were investigated in the TNS fungal herbarium.

The isolates were incubated in 2% malt extract broth for 2 weeks, and the mycelium was harvested. Approximately 50 mg of mycelium was mechanically lysed by a Qiagen Tissue Lyser Kit (Qiagen Inc., Mississauga, ON, Canada), using ceramic beads following the manufacturer’s instructions. The DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc.) following the manufacturer’s instructions. To amplify the internal transcribed spacer (ITS1 and ITS2) and 5.8S rDNA regions (ITS-5.8S), the primer pair ITS1F and ITS4 (White et al. 1990) was used. To amplify the D1-D2 region of 28S rDNA (nLSU rDNA; D1-D2), the primer pair NL1 and NL4 (O’Donnell 1993) was used. The DNA was amplified using 40-μl PCR reactions containing 0.2 μM of each primer, 1 unit of TaKaRa Ex Taq DNA polymerase (Takara Bio, Otsu, Japan), and a deoxynucleoside triphosphate (dNTP) mixture containing 2.5 mM of each dNTP and ExTaq buffer containing 2 mM MgCl₂. PCR was carried out using a Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). The DNA was denatured for 3 min at 95 °C, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 2 min, followed by final extension at 72 °C for 10 min. Residual primers, and unincorporated dNTPs were removed to prevent the inhibition of the following sequencing reaction using a ExoSAP-IT purification kit (USB Corp., Cleveland, OH, USA). Total DNA samples extracted for the present study were deposited in the Center for Molecular Biodiversity Research in National Museum of Nature and Science (ED16207). The sequencing reaction was carried out using a BigDye terminator cycle sequencing kit (Applied Biosystems) following the manufacturer’s instruction using the same primers as those used for DNA amplification. Sequencing was conducted using an ABI 3130x Genetic Analyzer (Applied Biosystems).

The available sequences for Roesleria subterranea were obtained from GenBank (Table 1). The obtained sequence data for the ITS-5.8S region were aligned by Clustal W (Thompson et al. 1994) and edited manually when necessary using BioEdit v.7.0.5.2 (Hall 1999). Phylogenetic analysis was conducted by a maximum parsimony (MP) method. An MP heuristic search was carried out using the PAUP* version 4b10 (Swofford 2002) with 1,000 replications, each with the option of random sequence additions. Branch swapping by tree bisection-reconnection (TBR) and MulTrees were in effect. Support for the individual nodes was tested with bootstrap analysis under the equally weighted parsimony criterion. The bootstrap analysis was based on 1000 bootstrap replicates using the heuristic search option (TBR and MulTrees options, on) of ten replicates with random addition sequences. Based on the previous analysis (Kirchmair et al. 2008), Hymenoscyphus epiphyllus (Pers.) Rehm ex Kauffman (AY348580), H. immutabilis (Fuckel) Dennis (AY348584), and H. scutula (Pers.) W. Phillips (AY789432) were used as outgroups. The trees were visualized using the Treeview program, version 1.6.6 (Page 1996). The alignments were deposited into TreeBASE as TB2:S11877.

The sequence of the D1-D2 region of 28S rDNA was almost identical to the available sequences from European specimens. The present sequence differed from EF608073, EF608074, and EF608075 at three, one, and four sites, respectively, among the 554 sites that were compared. The ITS-5.8S sequence was almost identical to the available sequences, and the identification based on morphology was confirmed.

In the MP analysis, we incorporated the available ITS-5.8S sequences of R. subterranea, using the three species of Hymenoscyphus used in Kirchmair et al. (2008) as outgroups. The aligned dataset included 466 characters, of which 28 characters were excluded from the analyses due to ambiguous alignment. The remaining 438 characters were used for the analysis, including a total of 23 parsimony-informative characters.

A single most parsimonious tree was obtained (Fig. 2). Within the tree, a monophyly of isolates from Vitis spp. from Germany, Italy and USA was strongly supported. The remaining group of this clade were found to be paraphyletic and composed of the isolates from various deciduous trees. The sequence obtained from the Japanese isolate (NBRC 108276) was included in this group (Fig. 2). Although the sequence of the latter group seemed to be identical in Fig. 2, the original sequence obtained from the Japanese isolate (AB628057) was not identical with several nucleotide differences to the other three. However, these differences were present only in the ambiguously aligned region, which was excluded from the analysis.

Although the tree topology depends on the choice of outgroup (data not shown), the monophyly of the species as a whole and the monophyly of the clade of isolates from Vitis (“Vitis-clade”) were highly stable (BS values were 100 in the present data and also in Kirchmair et al. (2008)). However, a group of isolates from other hosts besides Vitis (“non-Vitis group”) was paraphyletic. This result suggests that the host specificity was more important than the geographical distance as a factor for infraspecific genetic diversification.

All of the known isolates of the “Vitis clade” were obtained from cultivated grape samples. On the other hand, isolates of “non-Vitis grade” including our isolate were from the roots of Malus sylvesris, Populus sp. and other unknown wild deciduous trees. It is therefore hypothesized that isolates highly specific to Vitis are more widely distributed in the cultivated grape fields than is currently recognized. To clarify whether such a host specificity is present or not, inoculation experiments are indispensable. In addition, further isolates from various hosts and localities should be collected and examined to know the infraspecific relationships of the species.

Figure 1. 

Roesleria subterranea (TNS-F-38701). 1 Growing habits of two mature mazaedia on the root tip of an unidentified tree 2 Hymenium of mazaedium, showing protruding paraphyses 3 Enlarged hymenium showing developmental stages of asci and paraphyses 4 Mature ascus containing eight ascospores 5 Paraphysis 6 Discharged matured ascospores; on the right, a transversely one-septate spore is shown in side view 7 Germination of ascospores on MEYE, showing that germination tubes from one of the two-celled spores 2–6 Mounted in Meltzer’s solution 7 Mounted in lacto-aniline blue. Bars: 1 = 2 mm; 2 = 50 μm; 3–7 = 10 μm.

Figure 2. 

A single most parsimonious tree of Roesleria subterranea (ITS-5.8S sequences). Tree length = 31, CI = 0.8065, HI = 0.1935, RI = 0.8723, RC = 0.7035. The tree was constructed from a dataset of all available ITS-5.8S sequences of R. subterranea from various hosts and localities. The numbers on the branches indicate the bootstrap values (BP) of 1000 replications in maximum parsimony analysis when the BP exceeds 50%. The scale bar indicates the number of substitutions. Abbreviations indicate the localities of the isolates: AU, Austria; GE, German; IT, Italy; NE, Netherlands; US, USA; JP, Japan.