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Phylogenetic placements and cultural characteristics of Tuber species isolated from ectomycorrhizas
2021 年 62 巻 2 号 p. 124-131
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2021 年 62 巻 2 号 p. 124-131
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.
Species of the genus Tuber (Pezizales, Ascomycota) are hypogeous fungi, chiefly common in the Northern Hemisphere, and form ectomycorrhizal (EcM) associations with a variety of woody plants, such as Pinaceae, Fagaceae, Myrtaceae, and Salicaceae(Bonito & Smith, 2016). The genus consists of greater than 180 species (Bonito, Gryganskyi, Trappe, & Vilgalys, 2010), and the number is still increasing because novel species are continually reported from regions where the diversity of the hypogeous genus has not been well explored (e.g., Guevara-Guerrero et al., 2018; Kinoshita et al., 2018a). All known Tuber species form tuberous fruiting bodies, referred as “true truffle” below ground, and discharge spores frequently with the help of mycophagous animals by attracting them with distinct aroma (Urban, 2016). So Tuber species have essential roles in the nutrient cycling as root-associated mutualists and in the food web as food resources in forest ecosystems. Furthermore, various Tuber species, including European white truffle T. magnatum as well as black truffle T. melanosporum, have substantial market value as delicacies because of their favorable aromas. With interest in ecology and systematics and demand for industrial use, Tuber species have been the subject of vast researches.
Pure cultured mycelia are essential resources not only for understanding basic features of the Tuber species including biological (Sbrana, Nuti, & Giovannetti, 2007), physiological (Saltarelli et al., 1998; Ceccaroli, Saltarelli, Cesari, Zambonelli, & Stocchi, 2001), and genetic properties (e.g., Martin et al., 2010) but also for their industrial applications including mushroom cultivation (Iotti, Piattoni, Leonardi, Hall, & Zambonelli, 2016; Leonardi, Murat, Puliga, Iotti, & Zambonelli, 2020). Generally, isolation and maintenance of pure cultures of Tuber are hard. However, various Tuber species including T. aestivum, T. borchii, T. brumale, T. himalayense, T. japonicum, T. longispinosum T. macrosporum, T. maculatum, T. melanosporum and T. rufum have been isolated from gleba of ascomata (Iotti, Amicucci, Stocchi, & Zambonelli, 2002; Le Roux et al., 2016; Nakano et al., 2020). Furthermore, Mischiati and Fontana (1993) described that T. magnatum could be isolated even from EcM root tips, which can be effective isolation sources to acquire pure mycelial cultures when no ascomata were acquired from field surveys. These results stipulate that pure cultures of Tuber species can be acquired from both ascomata and EcM roots, but the relevance of the isolation methods is not well documented and is largely unknown for a majority of Tuber species. In addition, we have quite limited information regarding the cultural characteristics of Tuber species because of the scarcity of the prior descriptional studies (Iotti et al., 2002).
Abies sachalinensis (Fr.Schmidt) Masters (Pinaceae) is a widely distributed tree species in Hokkaido, a northern island in Japan, and in the southern Kuril Islands and Sakhalin Island in Russia (Nakamura & Krestov, 2005). In Japan, several studies described that a variety of basidiomycetes and ascomycetes members, including Tuber species, are associated with A. sachalinensis (e.g., Matsuoka, Mori, Kawaguchi, Hobara, & Osono, 2016; Miyamoto, Narimatsu, & Nara, 2018; Matsuoka et al., 2020). Kinoshita et al. (2011) also documented that A. sachalinensis is likely among the EcM tree species that harbor several Japanese truffle species since their ascomata were occasionally sampled from forests including the fir trees. During our surveys of EcM fungal communities in A. sachalinensis plantations in Hokkaido, no ascomata of Tuber species have been found, but morphologically Tuber-like EcM roots were often observed in the roots of A. sachalinensis. In this study, we made an attempt to acquire pure cultures of Tuber from EcM roots of A. sachalinensis to understand flora of culturable Japanese truffle species and to check if EcM roots are appropriate resources for acquiring pure cultures of the taxa. The identities of pure cultures were validated using phylogeny based on the sequences of internal transcribed spacer (ITS) within ribosomal DNA (rDNA), and macroscopic and microscopic characteristics of the cultured mycelia were reported.
Majority of the EcM root samples were incidentally sampled during the research on the effects of retention forestry on EcM fungal diversity in artificial forests of A. sachalinensis, where large-scale manipulative experiments known as “the Retention Experiment for plantation Forestry in Sorachi, Hokkaido (REFRESH)” are undertaken in the Sorachi region, Hokkaido, Japan (Yamaura et al., 2018). The research site (43°34ʼ51"–36ʼ29"N, 142°07ʼ10"–59"E) was located on the foot of Mt. Irumukeppu (864 m above sea level), where most plantations of A. sachalinensis were established in the years 1961–1980. A soil core (about 7 cm cube) beneath a seedling (5–30 cm high) of A. sachalinensis was taken to collect the root systems of the seedling and/or adjacent mature trees of A. sachalinensis in various experimental plots where mature trees of A. sachalinensis or broad-leaved trees were retained in various manners (Yamaura et al., 2018). In this study, EcM root tips that remained following the processing of root samples for the survey were subjected to fungal isolation. Additional EcM root samples were collected in an artificial forest of A. sachalinensis in Tomakomai (42°41'2.00"N, 141°35'55.24"E) and Hiyama regions (41°46'2.05"N, 140°8'50.31"E). One or three 30 × 10 cm soil blocks were taken in Hiyama and Tomakomai, respectively. All soil samples were maintained in a refrigerator at 4 °C for 1 mo at longest until further analysis.
2.2. Isolation from Tuber-like EcM root tipsEcM root tips were morphotyped using a dissecting microscope (SSZ-B, Kyowa Optical Co., Ltd., Japan), and then those that have classic morphological features of Tuber (specifically pale-yellow to brown, smooth, often with densely or sparsely emanating needle-shaped cystidia on surface, e.g., Kinoshita, Obase, & Yamanaka, 2018b) were sampled for fungal isolation. Various Tuber-like EcM root tips (1–3 mm long each) were placed into a 2.0 mL microtube with about 1.0 mL of tap water and then vortexed with maximum speed (Vortex-Genie 2, Scientific Industries, NY, USA) for 60 s to remove adhering soils. Then, tap water with contaminants (i.e., soil particles) was rigorously removed with the use of the plastic dropper. The remaining EcM root tips were washed with 1.0 mL tap water in the same manner with four or five repeats. EcM root tips were then individually surface-sterilized by soaking into 20 mL of 30% H2O2 for 1–5 s and then placed into 20 mL of sterilized deionized water for a few minutes. Nine to 12 EcM root tips were placed, at equal intervals, on each plastic dish (9 cm diam), which were filled with 15 mL of modified Melin-Norkrans (MMN) agar medium (Marx, 1969; pH was not adjusted and it was around 6.5) supplemented with streptomycin (150 mg/L) and chloramphenicol (150 mg/L) which were sterilized with filters (Minisart NML, 16534K, Sartorius, Göttingen, Germany), and then incubated in dark at 25 °C.
EcM root tips, from which hyaline or a bit light brownish, smooth, relatively thick, septate and simply branched hyphae emanated with low density 1–10 d following incubation, were transferred from MMN agar plate with the mycelia on fresh MMN agar plates individually and incubated in dark at 25 °C. We further subcultured those isolated mycelia without EcM root tips as soon as possible since root endophytes occasionally inhabited inside the EcM roots and they begin to grow on MMN agar following mycelial growth of Tuber, causing contamination. Pure culture strains were maintained on MMN agar plates in the dark at 25 °C in Microbial Ecology Laboratory, Department of Mushroom Science and Forest Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Japan.
2.3. ITS barcoding and phylogenetic analysisA mycelial fragment (about 2–3 mm square) was obtained from each pure culture with the use of a scalpel and placed individually in a 0.2 mL microtube (1-1599-03, AS ONE, Japan). Various cultures did not grow well enough to extract DNA and thus were removed from further analysis. DNA was extracted with the use of Extract-N-Amp™ Plant PCR Kit (XNAP2-1KT, Sigma-Aldrich, MO, USA) following a manufacturer instruction with some alteration. Mycelia were briefly disrupted in 30 µL of extraction solution with sterilized woody toothpick and then incubated for 10 min at 95°C with a thermal cycler (2720, Applied Biosystems, Tokyo, Japan). Following the cooling down to room temperature, 30 µL of dilution solution was added and vortexed to mix. The solution was once frozen at −20 °C to solidify agar, thawed, and briefly centrifuged with the use of a microcentrifuge (Multi Spin, Tomy, Japan). The suspension was utilized as a DNA template for polymerase chain reaction (PCR). When DNA extraction failed with the earlier methods, we utilized DNeasy Plant mini Kit (QIAGEN, Germany) following a manufacturer instruction. ITS region (ITS1–5.8S–ITS2) of rDNA was amplified by PCR with the use of TaKaRa Ex Taq (Takara Bio Inc., Japan) and a primer pair of ITS1F (Gardes & Bruns, 1993) and ITS4 (White, Bruns, Lee, & Taylor, 1990) following a manufacturer instruction. The PCR conditions were as follows: 30 cycles of 98 °C for 10 s, 54 °C for 30 s, and 72 °C for 1 min. The PCR products were purified with the use of Exonuclease I and Antarctic Phosphatase (New England Biolabs, Inc.) (Glenn & Schable, 2005). Sequencing reactions with the use of the primer ITS1F were entrusted to Eurofin Genomics (Tokyo, Japan). The DNA sequence data were then submitted to the DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/index.html) under the accession numbers LC556122–LC556140.
ITS sequences were also submitted to BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to deduce taxonomic positions of pure cultured mycelia. Sequences of Tuber species that belonged to the Puberulum clade, Latisporum group, and Maculatum clade (e.g., Bonito et al., 2013) were downloaded from the International Nucleotide Sequence Database (INSD) at the website of The National Center for Biotechnology Information (NCBI, http://blast.ncbi.nlm.nih.gov). Sequences of four Tuber species that belonged to Gibbosum clade were also downloaded as an outgroup. All sequences were aligned using MAFFT v.7 (Katoh & Standley, 2013) with default options, and vaguely aligned regions were removed using GBLOCKS 0.91b (Castresana, 2000; http://molevol.cmima.csic.es/castresana/Gblocks_server.html) with the least stringent setting. Maximum likelihood (ML) analysis was conducted with the use of MEGA v.7.0.20 (Kumar, Stecher, & Tamura, 2016). The general time reversible model incorporating invariant sites and a gamma distribution (GTR+I+G) was chosen as the suitable model based on the results of the corrected Akaike information criterion value in the MEGA and therefore applied to the ML analysis. Branch supports were analyzed with 1,000 bootstrap replications. Sequence alignment data and the relevant tree file are deposited at TreeBase (https://www.treebase.org/treebase-web/home.html) under the study ID S26681.
2.4. Morphological observation on pure culture strainsOne to four strains in each phylogenetic lineage were subjected to the macro- and microscopic observation on culture. Five mycelial disks (1 cm diam) were taken from inner edge of a 45–50-d-old culture on 20 mL MMN agar plate, and then they were inoculated individually on 20 mL MMN agar plates (9 cm diam) and incubated in the dark at 25 °C for 2 mo. Colony diameters were measured following 1 mo of inoculation, and hyphal growth rate (mm/mo) was also calculated. Mycelial colonies following 1 and 2 mo of inoculation were examined under a microscope (BX60, OLYMPUS, Japan), and images were captured using a digital camera (D5300, NIKON, Japan). Measurements were done with the use of PHOTORULER v.1.1.3.0 (http://www.inocybe.info/_userdata/ruler/PhotoRuler.html).
Around 500 EcM root tips from 31 soil samples were subjected to fungal isolation. At the first subculture process (i.e., transfer of EcM roots with extending mycelia onto another MMN plates), we obtained 192 cultures in total, but about one-third (64 cultures) failed in further hyphal growth on the subcultured agar plates (Supplementary Table S1). Sixteen cultures did not belong to Tuber and were removed from further analysis; five were likely Helvella species (90.0% similarity with Helvella macropus), three were closely related to Hydnotrya bailii (98.0% similarity), and the remaining eight were another taxon of Hydnotrya (92.8% similarity with Hydnotrya nigricans). Clear sequences could not be obtained from eight cultures possibly because of contamination or too small amount of fungal materials accessible for DNA extractions. Clear ITS sequences of Tuber were obtained from 104 cultures.
3.2. ITS phylogenyPhylogenetic analysis based on rDNA ITS region showed well-supported eight lineages within Puberulum, Latisporum, and Maculatum clades (Fig. 1). Thirty, five, and 40 cultures were grouped with each different undescribed species of Puberulum clade recorded in Japan (Tuber spp. 16, 17, and 18; Kinoshita et al., 2011), respectively; here each lineage was appointed as TuSp16, TuSp17, and TuSp18, respectively. Four and nine cultures were closely related to an undescribed species of Puberulum clade in Japan (Tuber sp. 15; Kinoshita et al., 2011), but ITS similarity was relatively lower among each other (around 96.7%) and therefore discriminated as distinct lineages and appointed as TuSpKOA and TuSpKOB, respectively. Four cultures were grouped with an undescribed species of the Latisporum group recorded in China (Tuber sp. 2016A, voucher HKAS95777) with high ITS similarity (98.2%) and thus designated as TuSp2016A. Eleven cultures were not grouped to any sequences derived from specimens in the INSD with high-sequence similarity and therefore designated as TuSpKOC. One culture was grouped with T. foetidum with high ITS similarity (98.0%) and therefore designated as T. foetidum.
All strains of Tuber displayed yellowish white, thin, flat, filamentous colonies on MMN agar medium plates but demonstrated remarkable differences in mycelial density and hyphal growth among lineages (Fig. 2, Table 1). All strains of TuSp18 and TuSp2016A showed a high rate of hyphal growth and spread all over the agar plates within 1 mo of incubation (> 37.5 mm/mo), displaying cobweb-like colonies with relatively high mycelial density. TuSpKOA and TuSp17 manifested intermediate (12.8–19.3 mm/mo) and low rate (7.0–7.6 mm/mo) of hyphal growth with low mycelial density. TuSp16 and TuSpKOC demonstrated a variable rate of hyphal growth among strains (8.9–24.4 mm/mo and 0.7–32.8 mm/mo, respectively) and even among replicates within a strain, e.g., 16.0–37.5 mm/mo and 9.8–25.5 mm/mo for strains OBASE00113 in TuSp16 and OBASE00099 in TuSpKOC. Three out of five mycelial disks from a T. foetidum strain OBASE00103 were contaminated by bacteria, but they yield a remarkably higher rate of hyphal growth (11.0–17.3 mm/mo) than mycelial disks without the contamination (2.0–2.5 mm/mo). A strain of TuSpKOB (OBASE00095) did not extend hyphae during the incubation test.
Lineage |
Cultured strain |
Hyphal growth (mm/mo) 1 |
Width of hyphae (µm) |
Hyphalaggregates |
Hyphae with oil droplets |
Swollen hyphae |
Irregulary branched hyphae |
TuSp16 |
OBASE00113 |
24.4±9.4 |
3.3–6.0 |
+ |
−/+ |
− |
− |
TuSp16 |
OBASE00047 |
16.4±2.4 |
3.1–5.9 |
− |
− |
− |
− |
TuSp16 |
OBASE00078 |
8.9±1.4 |
3.7–6.8 |
−/+ |
− |
− |
− |
TuSp17 |
OBASE00015 |
7.0±3.5 |
2.6–4.2 |
+ |
− |
− |
− |
TuSp17 |
OBASE00016 |
7.6±2.4 |
2.9–4.5 |
+ |
− |
−/+ |
−/+ |
TuSp18 |
OBASE00134 |
> 37.5 |
4.3–7.2 |
−/+ |
+ |
−/+ |
+ |
TuSp18 |
OBASE00029 |
> 37.5 |
4.5–7.3 |
+ |
−/+ |
−/+ |
+ |
TuSp18 |
OBASE00012 |
> 37.5 |
4.1–8.3 |
+ |
+ |
+ |
+ |
TuSp2016A |
OBASE00093 |
34.2±4.6 |
4.1–6.8 |
+ |
+ |
+ |
+ |
TuSpKOA |
OBASE00117 |
19.3±0.9 |
2.3–5.9 |
+ |
− |
+ |
−/+ |
TuSpKOA |
OBASE00056 |
12.8±2.1 |
3.6–6.8 |
−/+ |
− |
− |
− |
TuSpKOB 2 |
OBASE00095 |
0 |
(3.2–5.5) |
(+) |
(−) |
(−) |
(−) |
TuSpKOC |
OBASE00040 |
32.8±4.4 |
3.2–7.8 |
+ |
+ |
− |
+ |
TuSpKOC |
OBASE00026 |
0.7±1.5 |
3.3–4.3 |
− |
− |
− |
− |
TuSpKOC |
OBASE00001 |
5.6±4.1 |
2.6–7.2 |
+ |
− |
− |
+ |
TuSpKOC |
OBASE00099 |
20.9±6.5 |
3.2–6.5 |
+ |
− |
−/+ |
−/+ |
T. foetidum 3 |
OBASE00103 |
9.0±6.6 |
3.5–8.3 |
−[+] |
−[+] |
−[+] |
−[+] |
+, present; −, absent; −/+, absent at 1 mo of incubation but present following 2 mo of incubation. |
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1 Average ± standard deviation is designated. “>37.5” means that hyphae spread all over the culture plate within 1 mo of incubation. |
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2 Hyphae did not extend from mycelial disks, so hyphal characteristics were assessed with the use of a subculture of the strain and designated in parentheses. |
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3Hyphal characteristics found in cultures contaminated by bacteria are designated in brackets. |
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All strains of Tuber basically comprised hyaline to light grayish, filamentous with uniform width, smooth, septated, and simply branched hyphae and extended them both on the surface and into the agar medium plates. Nonetheless, some differences were observed in the morphological characteristics and branching pattern of the hyphae among strains (Table 1). Looped and aggregated hyphal structures, which was referred as “hyphal aggregates” in Iotti et al. (2002), were observed on the agar surface or on the aerial hyphae in all taxa of Tuber species, but they were not formed in some strains (Fig. 3A, B). Hyphal cells that harbor several or dozens of oily droplets were sometimes found in the several strains of TuSp16, TuSp18, TuSp2016A, and TuSpKOC (Fig. 3C). Hyphae that are septated in a short distance and swollen at the middle parts were found in several strains of TuSp17, TuSp18, TuSp2016A, TuSpKOA, and TuSpKOC (Fig. 3D). Hyphal anastomoses were frequently found in all strains (Fig. 3E). In the strain OBASE00016 for TuSp17, anastomosed parts in hyphae frequently accompanied swollen cells (Fig. 3F, G). Frequently septated and irregularly branched hyphae were found in the various strains of TuSp17, TuSp18, TuSp2016A, TuSpKOA, and TuSpKOC (Fig. 3H).
Morphological characteristics and the branching pattern of the hyphae were mainly different between contaminated and non-contaminated mycelial disks by bacteria in a strain of T. foetidum OBASE00103. Hyphae from non-contaminated mycelial disks did not show any specific features; nonetheless, those from contaminated disks showed frequently septated and irregularly shaped, occasionally swollen hyphae on the agar medium plates (Fig. 3I). Conidiogenesis was never observed in any strains.
This study recorded that EcM roots can be efficacious isolation sources for various Tuber species. Unexpectedly, fresh EcM root tips of Tuber were often observed and sometimes abundantly acquired from A. sachalinensis plantations. As a shortcoming for isolation sources, EcM roots are commonly rather hard to be manipulated since they are tiny and fragile. Nonetheless, it likely depends on the kinds of tree species; EcM roots of A. sachalinensis were large in size, about twice thicker than those of broad-leaved trees including Quercus and Betula, and tough (Kinoshita, Satomura, Hashimoto, & Horikoshi, 2007), therefore easy to be handled. Furthermore, Tuber EcM roots have smooth surface (e.g., Kinoshita et al., 2018b), and adhering soils on the surface can be removed easily by vortexing or pipetting with tap water; therefore brief surface sterilization is enough to remove contaminants. As another disadvantage, we can fail to deduce the fungal identities correlating the EcM roots from macro-morphological characteristics of the EcM roots; indeed, 16 strains acquired from Tuber-like EcM roots were identified to be other ascomycete taxa. Nonetheless, even just thinking about it, isolation using EcM roots can be among the effective ways to acquire pure cultures of Tuber.
As described for European truffle species by prior studies (Iotti et al., 2002; Le Roux et al., 2016), it was hard to maintain pure cultures of several Tuber species since they showed poor growth on MMN medium (e.g., TuSp17), and several cultures ceased to grow or largely decreased their hyphal growth with each subculture step. Hyphal growth rate on MMN was different among strains within a species (TuSp16 and TuSpKOC) and even among mycelial disks subcultured simultaneously from an agar medium plate (e.g., TuSp16 strain OBASE00113). It can be partly resolved by altering culture medium for subculturing as proposed in the earlier study (Le Roux et al., 2016). During subculturing, not all but several strains (e.g., TuSp16 strain OBASE00038), which did not grow well on MMN medium, demonstrated a remarkably better growth on either yeast-glucose medium (Tanaka & Nara, 2009, pH was not adjusted) and Nutrient Agar medium (Difco) (K. Obase, unpublished data). Further studies are required to validate which nutrient media are appropriate for maintaining each Tuber strain.
Bacterial contamination occasionally happens during subculturing of Tuber strains, affecting the survivorship of the strains positively or negatively (Sbrana et al., 2002). Tuber foetidum strain OBASE00103, which did not grow on MMN media with no bacterial contamination, demonstrated a moderate rate of hyphal growth (11.0–17.3 mm/mo) when contaminated by bacteria. The functions of bacteria on the Tuber strains are yet to be investigated; nevertheless, it likely ranges from symbiotic to antagonistic in accordance with the environmental conditions. Indeed, the bacteria on the strain OBASE00103 are advantageous on the hyphal growth of the strain on agar media, but the bacteria totally repress mycelial growth of the strain when they were co-incubated in MMN liquid media (K. Obase, unpublished data). Even on the agar media, bacterial cells occasionally grew along with hyphae and totally cover the Tuber mycelia; in this case, subsequent transfer of the mycelium to another fresh agar plate would often fail because of severe contamination by the bacteria (K. Obase, unpublished data). The facts deduce the difficulty in the use of advantageous bacteria on subculturing of Tuber strains, but the biological interaction between Tuber mycelia and the bacteria should give hints for the enhancement of mycelial growth (e.g., Obase, 2019) and improvement of success rate of subcultuing of Tuber on nutrient media in the future.
There were several differences in morphological characteristics of hyphae among pure cultures of various strains of Tuber species grown on MMN agar medium plates, and such difference was even present among strains of the same species. Therefore, these hyphal characteristics are unlikely available for delimiting of different species of Tuber, and molecular techniques including ITS phylogeny is inevitable to distinguish Tuber species, as suggested earlier by Iotti et al. (2002).
Strains of Puberulum clade, Latisporum group, and Maculatum clade were acquired from EcM roots in these study sites. It is unlikely that these lineages could be selectively isolated, since our ongoing studies revealed that members of the other lineages did not inhabit EcM roots in the A. sachalinensis forests (Obase et al. unpublished data). So, it is yet to be investigated if the isolation method with the use of EcM roots is appropriate to other lineages in Tuber. Also, it remains to be tested if the isolation method is also applicable to the EcM roots of other tree species such as Fagaceae and Betulaceae, which have thinner roots rather than A. sachalinensis and likely more sensitive to the treatments of surface-sterilization.
In Japan, 20 species of Tuber are noted, but nearly 40 species are estimated to be potentially present (Kinoshita et al., 2011). And most of those taxa display low similarities of ITS sequences with European and North American species, suggesting distinct flora of Tuber in each geographic region at continental scale. Establishment of pure cultures of Tuber species by isolation with the use of EcM roots should largely contribute to unearthing diversity, ecology, and biology of truffle, especially for not well explored Asian species in the future.
This study was partially supported by grant in aid for young scientists and encouraged research from Nippon Life Insurance Foundation to K. Obase. The authors deeply appreciate the members of the laboratories of Microbial Ecology and Forest Pathology in Forestry and Forest Products Research Institute for their support during the DNA analysis, and the editor in charge and two anonymous reviewers for valuable comments.
Disclosure
The authors declares no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.
