2022 Volume 63 Issue 1 Pages 39-44
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.
Species of the genus Tuber (Pezizales, Ascomycota) are ectomycorrhizal (EcM) fungi that form symbiotic associations with the roots of various tree species including Pinaceae, Betulaceae, Fagaceae, Myrtaceae, and Salicaceae (Bonito & Smith, 2016). In Japan, 20 species of Tuber have been recorded to date (Kinoshita, Sasaki, & Nara, 2011). Of these, intensive research has been undertaken to understand the biological characteristics of the three species T. japonicum, T. himalayense, and T. longispinosum (Kinoshita, Obase, & Yamanaka, 2018; Shimokawa et al., 2019; Furusawa et al., 2020; Nakamura et al., 2020; Nakano et al., 2020). However, other species of Tuber are less studied, except for descriptions of their ascomata and phylogenetic position (Kinoshita et al., 2011; Kinoshita, Sasaki, & Nara, 2016; Kinoshita, Sasaki, Orihara, Nakajima, & Nara, 2021). Currently, little is known about their biological characteristics, such as the morphological and anatomical characteristics of their ectomycorrhizas.
Puberulum is the most species-diverse clade in the genus Tuber (Bonito et al., 2013; Bonito & Smith, 2016) and is widely distributed across Europe, Asia, North America, South America, and North Africa (Lancellotti, Iotti, Zambonelli, & Franceschini, 2016). In Japan, 11 species (i.e., 55% of Tuber species in Japan) belong to this clade. Most of these species show low similarity (<95%) of their nuclear ribosomal RNA gene (nrDNA) internal transcribed spacer (ITS) sequence with Tuber species found in other countries (Kinoshita et al., 2011), suggesting that most species in this fungal group are endemic to Japan. The odor characteristics of these truffles have not been reported, and it is unknown whether they are edible. The morphology of ectomycorrhizas synthesized using pure culture mycelia or spore inoculum or collected in the field has been described for several species of the Puberulum clade, namely the T. anniae species complex with Pinus sylvestris (Wang et al., 2013), T. borchii with several conifer and broad-leafed seedlings (e.g., Giomaro et al., 2000; Lancellotti, Iotti, Zambonelli, & Franceschini, 2014), T. liyuanum with Castanea mollissima and Pinus armandii (Wan et al., 2016), T. oligospermum with Quercus robur (Boutahir, Iotti, & Piattoni, 2013), T. puberulum with Picea abies (Blaschke, 1988), T. cf. separans with Fagus grandifolia var. mexicana (Ramos, Garay-Serrano, César, Montoya, & Bandala, 2018), and undescribed Tuber species with Alnus acuminate (Ramos et al., 2018). However, no studies have examined the ectomycorrhizas formed by Tuber species of the Puberulum clade in Japan.
In a previous study, pure culture strains of undescribed Tuber species of the Puberulum clade were obtained from EcM root tips in Abies sachalinensis plantations in Hokkaido, Japan (Obase, Yamanaka, Kinoshita, Tamai, & Yamanaka, 2021). In this study, conifer seedlings were inoculated with Tuber mycelial strains in vitro, and the morphological and anatomical characteristics of their ectomycorrhizas were described.
One to three strains were selected from five undescribed species of the Puberulum clade [denoted as TuSp16 strains OBASE00033, OBASE00043, and OBASE00077; TuSp17 strain OBASE00018; TuSp18 strains OBASE00012, OBASE00029, and OBASE00133; TuSpKOA strain OBASE00117; and TuSpKOB strain OBASE00087 in Obase et al. (2021)] (Table 1). All of these strains were isolated from EcM root tips of Abies sachalinensis in Hokkaido, Japan, and their phylogenetic positions were confirmed by ITS barcoding in a previous study (Obase et al., 2021). The pairwise distances of the ITS sequences between strains were found to be 0.3%-1.2% in TuSp16 and TuSp18. The fungal cultures were maintained on yeast-glucose (YG) agar plates (Tanaka & Nara, 2009) stored at 23- 25 °C in the dark.
| Taxaa | Hostb | Strain IDs (Accession numbers) | Mycorrhizal systems | Mantle | Cystidia | ||||||
| Length ×width in max. | Color | Surface | Outer layerc,d | Inner layerd | Septa (near base) | Diameter at base, length | |||||
| TuSp16 | P.d. | OBASE00033 (LC556127) OBASE00043 (LC556128) OBASE00077 (LC556129) |
4.8 × 6.4 mm | Pale yellow (PE) to brown (B) | Sparsely (S) to densely (D) short spiny | Type D/E; 3-8 µm diam Type M/Q; 4-9.5 × 4.5-13.5 µm |
Pseudoparenchymatous (Pp); 4.5-14 × 4.5-17 µm | Mono- to tri-septate | 2.5-5 µm, 55-108 µm |
||
| A.s. | 6.9 × 9.7 mm | PE to B | S | Type D/E; 2.5-9 µm diam Type M/Q; 6-15 × 8-21.5 µm |
Pp; 6-21.5 × 6.5-24.5 µm | Mono- or di-septate | 2.5-4 µm, 60-118 µm |
||||
| TuSp17 | P.d. | OBASE00018 (LC556131) | 2.7 × 2.9 mm | PE to B | S | Type D/E; 3-8 µm diam Type M/Q; 7.5-12 × 10-15.5 µm |
Pp; 6-13.5 × 8.5-15 µm | Mono- to tri-septate | 2.5-4 µm, 75-115 µm |
||
| A.s. | 3.9 × 2.6 mm | PE to light brown (LB) | S | Type D/E; 2.5-10 µm diam Type M/Q; 6-12 × 9-18 µm |
Pp; 6-13.5 × 10.5-20.5 µm | Mono- to tri-septate | 3-5.5 µm, 78-122 µm |
||||
| TuSp18 | P.d. | OBASE00012 (= LC556132e) OBASE00029 (LC556133) OBASE00133 (LC556134) |
6.9 × 6.8 mm | PE to B | S to D | Type D/E; 3-8.5 µm diam Type M/Q; 5-11.5 × 6.5-17.5 µm |
Pp; 4-14.5 × 5-22 µm | Mono- or di-septate | 2.5-6.5 µm 58-130 µm |
||
| A.s. | 13.2 × 4.2 mm | PE to B | D | Type D/E; 3.5-8 µm diam Type M/Q; 7-11.5 × 7-16.5 µm |
Pp; 5-17 × 7-21 µm | Mono- or di-septate | 2.5-6.5 µm, 70-103 µm |
||||
| TuSpKOA | P.d. | OBASE00117 (LC556137) | 5.0 × 4.0 mm | PE to LB | S to D | Type D/E; 3-6 µm diam | Pp; 4-9 × 5-15.5 µm | Mono- or di-septate | 2.5-5 µm, 55-85 µm |
||
| A.s. | 7.6 × 4.2 mm | PE to B | S to D | Type D/E; 2-6 µm diam Type M/Q; 5-9 × 6-14 µm |
Pp; 8-13 × 9-23 µm | Mono-septate | 3-5 µm, 58-83 µm |
||||
| TuSpKOB | P.d. | OBASE00087 (= LC556138e) | 5.6 × 2.8 mm | PE to LB | S to D | Type D/E; 2.5-6 µm diam Type M/Q; 5-10 × 5.5-13 µm |
Pp; 6-11.5 × 6.5-15.5 µm | Mono- to tri-septate | 3-5 µm, 65-113 µm |
||
| A.s. | n.a. | PE to B | S | Type D/E; 3-5.5 µm diam | Pp; 5.5-11.5 × 8.5-21 µm | Mono- or di-septate | 4-5 µm, 65-103 µm |
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n.a.; not analyzed
a Names of taxa were refered from Obase et al. (2021)
b P.d.; P. densiflora, A.s.; A. sachalinensis
c Mantle type is refered from Agerer and Rambold (2004-2021). Mantles which have transitional features of two types (e.g., type A and B) were described as “type A/B”.
d The short side × long side are shown.
e Sequences of the strains were not deposited in the GenBank database, but identical with those of the accession numbers.
A single mycelial disk (1 cm2, 4-5 mm thick) of each Tuber strain was cut from a 1-2-mo-old subculture plate and then incubated in a 50 mL conical tube (352098, Falcon, Mexico) containing 20 mL of YG liquid medium for 30-45 d at 23 °C in the dark.
Pinus densiflora and Abies sachalinensis were chosen as the host tree species to be tested in the inoculation experiments because they are native to Japan, potential hosts for Japanese Tuber species (Kinoshita et al., 2011; Obase et al., 2021), and can be easily manipulated in in vitro experiments. Seeds of the two pine species were surface-sterilized with 30% hydrogen peroxide for 30 min, rinsed twice with sterilized distilled water, and then placed on water agar plates (1% agar in distilled water) in an illuminated incubator (FLI-2000; EYELA, Tokyo, Japan) programmed to provide 16 h of daylight (4,000 lux) at 23 °C and 8 h of darkness at 20 °C until germination.
A total of 250 mL of joint soil of turf (red ball earth; akadamatsuchi in Japanese; Joy Garden, Kanuma, Japan) and 100 mL of YG liquid medium were loaded into a 450 mL glass mayonnaise bottle with a TPX (polymethylpentene) cap (Sansyo Co., Ltd., Tokyo, Japan) with a hole measuring 1 cm diam at the center and sealed using a membrane seal (MilliSeal, FWMS01800, Merck Millipore). The glass bottles were autoclaved once at 121 °C for 30 min.
Subsequently, a single germinating conifer seedling (7-10-d-old after germination) was sown in the glass bottle. At the time of sowing, a mycelial colony of the Tuber strain incubated in the conical tubes was inoculated onto the root of the germinating seedling. The lower half of each bottle was covered with an aluminum foil to prevent the soil from being exposed to light. All seedlings were grown in an illuminated incubator programmed to provide 16 h of daylight (4,000 lux) at 23 °C and 8 h of darkness at 20 °C for 4 mo. All combinations of tree species and Tuber strains were tested for ectomycorrhizal synthesis with two replicates established per treatment.
Lateral roots were collected from seedlings and separated from the adhering soil by soaking and carefully washing them in tap water. EcM root tips were examined using a dissecting microscope (SZH-ILLB; OLYMPUS, Tokyo, Japan) and a differential interference contrast microscope (BX53; OLYMPUS) to characterize their macro-morphological characteristics (color, mycorrhizal system, surface texture of EcM roots, etc.) and anatomical characteristics (mantle type, shape of cystidia, etc.). A hand section of each EcM root tip was mounted with distilled water on a glass slide, and microscopic images were captured using a digital camera (D5300; Nikon, Tokyo, Japan). Descriptive terminology was based on Agerer and Rambold (2004-2021).
One EcM root tip (1-2 mm long) from each seedling was subjected to ITS barcoding. Each EcM root tip was placed in a 0.2 mL microtube (1-1599-03; AS ONE, Osaka, Japan), and DNA was extracted using an Extract-N-AmpTM Plant PCR Kit (XNAP2-1KT; Sigma-Aldrich, MO, USA) following the manufacturer's instructions with modification of the volume of extraction and dilution solutions (40 µL/sample). The ITS region (ITS1-5.8S-ITS2) of nrDNA was amplified by polymerase chain reaction (PCR) using an Extract-N-AmpTM Plant PCR Kit and the primer pair ITS1F (Gardes & Bruns, 1993) and ITS4 (White, Bruns, Lee, & Taylor, 1990) using a thermal cycler (2720; Applied Biosystems, Waltham, USA) following the manufacturer's instructions. The PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min and final extension at 72 °C for 10 min. The methods of purification and Sanger sequencing were the same as those described by Obase et al. (2021). The obtained ITS sequences were submitted to the DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/index.html) under the accession numbers LC653507-LC653523 and compared with those of inoculated mycelial strains to confirm their identity.
This study is the first to report the in vitro ectomycorrhization of five undescribed Japanese Tuber species in the Puberulum clade for two native conifer trees, namely P. densiflora and A. sachalinensis. All of the combinations of inoculated Tuber strains and conifer seedlings formed ectomycorrhizas, with the exception of the TuSp18 strain OBASE00012 on A. sachalinensis. The ITS sequences obtained from the representative EcM root tips were identical to those of the inoculated Tuber strains (Supplementary Table S1). It is unclear why ectomycorrhization failed in the above treatment; inoculation tests under different environmental conditions such as nutritional conditions in the soil substrate with more replicates would clarify the compatibility between Tuber strains and host tree species.
The ectomycorrhizas of the five undescribed Tuber species tested in the present study share the following morphological characteristics: pale yellow- to brown-colored, dichotomous for P. densiflora or irregularly pinnate, dichotomous-like EcM root systems for A. sachalinensis; straight to bent, cylindrical unramified ends; short, sparsely to densely distributed spines on the surface; a lack of or few emanating hyphae; and the absence of rhizomorphs (Table 1; Figs. 1, 2, 3; Supplementary Table S2). These macro-morphological characteristics are similar to those of other Tuber species of the Puberulum clade (Lancellotti et al., 2016), and no marked differences among the ectomycorrhizas of the different species in this clade were observed.
The anatomy of the outer mantle layer included the following features, which are also present in other species of Puberulum clade: hyphae arranged in a net-like manner with prominent cystidia [type D in Agerer and Rambold (2004-2021)], hyphae arranged with a repeated and squarrosely branched net-like pattern (type E), and epidermoid cells (type M) or epidermoid cells bearing a hyphal net (type Q). Notably, the mantles showed transitional features between the two types, i.e., transitional features between types D and E, referred to as type D/E, or included apparently different types in EcM root systems within a seedling, i.e., having both type D/E and type M/Q. For example, ectomycorrhizas of all Tuber species were repeatedly and squarrosely branched, frequently septate, and they occasionally had inflated hyphal junctions and showed a net-like hyphal arrangement with or without prominent cystidia on the outer mantle layer (type D/E); however, those inoculated with certain strains (e.g., TuSp16 strain OBASE00077 on P. densiflora) harbored mantle types with epidermoid cells with or without a hyphal net (type M/Q). Consistent with previous reports (Giomaro et al., 2000; Kovács & Jakucs, 2006; Boutahir et al., 2013), the mantle type varied even within the same species, and the size range of hyphal cells on the outer and inner mantle layers overlapped among different species. Thus, the anatomical characteristics of the mantle are unlikely to be useful for distinguishing Tuber species within the Puberulum clade under in vitro synthetic conditions.
According to previous studies, several characteristics of cystidia, such as length and cell wall thickness (Kovács & Jakucs, 2006); and presence or absence of ramification (Boutahir et al., 2013), septa (Kinoshita et al., 2019), and basal inflation (Boutahir et al., 2013), are useful to discriminate ectomycorrhizas of different Tuber species that produce whitish truffles, including species of the Puberulum, Maculatum, Rufum, and Japonicum clades. However, the cystidia of the five undescribed Tuber species tested in the present study harbored several common features: bristle-like, short, smooth, straight to bent, thin-walled, mono- or multi- (up to tri-) septate and hyaline, with no distinct differences among the species. Therefore, cystidia traits are not effective for separating Tuber species within the Puberulum clade, although they may occasionally be useful for discriminating ectomycorrhizas between Tuber species of other clades (e.g., Kinoshita et al., 2019).
The results of the present study indicate that it is difficult to discriminate these five Tuber species using the morphological and anatomical characteristics of the ectomycorrhizas under in vitro experimental condition. Further studies are needed to confirm if it is also applicable to ectomycorrhizas formed in fields because mantle structures and organizations of ectomycorrhizas can vary depending on the environmental conditions. On the other hands, these species can be clearly distinguished by their sequence homology, i.e., more than 3% dissimilarity in the ITS sequence between species (Obase et al., 2021). Thus, molecular techniques such as ITS barcoding are useful to accurately identify ectomycorrhizas formed by Tuber species within the Puberulum clade without concerning about developmental conditions of the ectomycorrhizas.
The author declares no conflicts of interest. All of the experiments undertaken in this study comply with the current Japanese laws.
This work was supported by JSPS KAKENHI Grant Number 21K05676 in Japan. The author thanks the members of the Laboratories of Microbial Ecology and Forest Pathology at the Forestry and Forest Products Research Institute for their support during the DNA analysis.
