Takahiro Yagame

Ectomycorrhizal Inocybe species associate with the mycoheterotrophic orchid Epipogium aphyllum but not its asexual propagules.

BACKGROUND AND AIMS Epipogium aphyllum is a Eurasian achlorophyllous, mycoheterotrophic forest orchid. Due to its rarity, it is often protected, and its biology is poorly known. The identity and pattern of colonization of fungal associates providing carbon to this orchid have not been studied previously. METHODS Using samples from 34 individuals from 18 populations in Japan, Russia and France, the following were investigated: (a) colonization patterns of fungal associates of E. aphyllum by microscopy; (b) their identity by PCR amplification of nuclear ribosomal ITS carried out on rhizome fragments and hyphal pelotons. RESULTS AND CONCLUSIONS Microscopic investigations revealed that thick rhizomes were densely colonized by fungi bearing clamp-connections and dolipores, i.e. basidiomycetes. Molecular analysis identified Inocybe species as exclusive symbionts of 75 % of the plants investigated and, more rarely, other basidiomycetes (Hebeloma, Xerocomus, Lactarius, Thelephora species). Additionally, ascomycetes, probably endophytes or parasites, were sometimes present. Although E. aphyllum associates with diverse species from Inocybe subgenera Mallocybe and Inocybe sensu stricto, no evidence for cryptic speciation in E. aphyllum was found. Since basidiomycetes colonizing the orchid are ectomycorrhizal, surrounding trees are probably the ultimate carbon source. Accordingly, in one population, ectomycorrhizae sampled around an individual orchid revealed the same fungus on 11.2 % of tree roots investigated. Conversely, long, thin stolons bearing bulbils indicated active asexual multiplication, but these propagules were not colonized by fungi. These findings are discussed in the framework of ecology and evolution of mycoheterotrophy.

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza‐forming Ceratobasidiaceae fungi

• We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon. • We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a (13)C and (15)N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. • In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%. • This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.

Ceratobasidiaceae mycorrhizal fungi isolated from nonphotosynthetic orchid Chamaegastrodia sikokiana

Mycorrhizal fungi were isolated from the nonphotosynthetic orchid Chamaegastrodia sikokiana and identified as members of Ceratobasidiaceae by phylogenetic analysis of the internal transcribed spacer (ITS) region of ribosomal deoxyribonucleic acid. The ITS sequences were similar among geographically separated samples obtained from Mt. Kiyosumi in Chiba Prefecture and Mt. Yokokura in Kochi Prefecture. One of the isolated fungi, KI1-2, formed ectomycorrhiza on seedlings of Abies firma in pot culture, suggesting that tripartite symbiosis exists among C. sikokiana, mycorrhizal fungi, and A. firma in nature, and carbon compounds are supplied from A. firma to C. sikokiana through the hyphae of the mycorrhizal fungi. To our knowledge, this is the second study to suggest the involvement of Ceratobasidiaceae fungi in tripartite symbiosis with achlorophyllous orchids and photosynthetic host plants.

Specific arbuscular mycorrhizal fungi associated with non-photosynthetic Petrosavia sakuraii (Petrosaviaceae).

Mycorrhizal fungi in roots of the achlorophyllous Petrosavia sakuraii (Petrosaviaceae) were identified by molecular methods. Habitats examined were plantations of the Japanese cypress Chamaecyparis obtusa in Honshu, an evergreen broad-leaved forest in Amami Island in Japan and a mixed deciduous and evergreen forest in China. Aseptate hyphal coils were observed in root cortical cells of P. sakuraii, suggesting Paris-type arbuscular mycorrhiza (AM). Furthermore, hyphal coils that had degenerated to amorphous clumps were found in various layers of the root cortex. Despite extensive sampling of P. sakuraii from various sites in Japan and China, most of the obtained AM fungal sequences of the nuclear small subunit ribosomal RNA gene were nearly identical and phylogenetic analysis revealed that they formed a single clade in the Glomus group A lineage. This suggests that the symbiotic relationship is highly specific. AM fungi of P. sakuraii were phylogenetically different from those previously detected in the roots of some mycoheterotrophic plants. In a habitat in C. obtusa plantation, approximately half of the AM fungi detected in roots of C. obtusa surrounding P. sakuraii belonged to the same clade as that of P. sakuraii. This indicates that particular AM fungi are selected by P. sakuraii from diverse indigenous AM fungi. The same AM fungi can colonize both plant species, and photosynthates of C. obtusa may be supplied to P. sakuraii through a shared AM fungal mycelial network. Although C. obtusa plantations are widely distributed throughout Japan, P. petrosavia is a rare plant species, probably because of its high specificity towards particular AM fungi.

Identification and symbiotic ability of Psathyrellaceae fungi isolated from a photosynthetic orchid, Cremastra appendiculata (Orchidaceae)

UNLABELLED PREMISE OF THE STUDY Photosynthetic orchids found in highly shaded forests are often mixotrophic, receiving part of their carbon energy via ectomycorrhizal fungi that had originally received carbohydrate from trees. A photosynthetic orchid, Cremastra appendiculata, is also found under highly shaded forest, but our preliminary data suggested that its associated fungi were not ectomycorrhizal. We tested whether their relation is an unusual example of a mixotrophic orchid associating with saprotrophic fungi by direct detection of fungal DNAs in conjunction with isolation of the fungus in pure culture and experimental inoculation of orchid seeds with the fungus. • METHODS For isolated mycobionts of C. appendiculata plants, two regions of nuclear ribosomal DNA, the internal transcribed spacer (ITS) and the large subunit (LSU), were sequenced, and fruiting bodies of the one isolate, SI1-1 were induced. In addition, two fungal isolates, SI1-1 and KI1-1, were grown in symbiotic cultures with C. appendiculata to verify their status as mycobionts. • KEY RESULTS In phylogenetic analyses, all isolates clustered with fungi belonging to Coprinellus in Psathyrellaceae of Agaricales. Phylogenetic analyses of these DNA sequences showed that five fungal isolates from C. appendiculata, including SI1-1 and two mycobionts isolated from the mycoheterotrophic orchid Epipogium roseum, have very similar ITS sequences. Isolate SI1-1 was identified as Coprinellus domesticus based on the morphological characteristics of the fruiting body. Isolates SI1-1 and KI1-1 induced seed germination of C. appendiculata as mycobionts. • CONCLUSIONS This report is the first of a mycorrhizal symbiosis between a fungus in Psathyrellaceae and a photosynthetic orchid, revealing a new pathway to full mycoheterotrophy and contributing to our understanding of the evolution of mycoheterotrophy.

Effect of environmental gradient in coastal vegetation on communities of arbuscular mycorrhizal fungi associated with Ixeris repens (Asteraceae)

The community structure of arbuscular mycorrhizal (AM) fungi associated with Ixeris repens was studied in coastal vegetation near the Tottori sand dunes in Japan. I. repens produces roots from a subterranean stem growing near the soil surface which provides an opportunity to examine the effects of an environmental gradient related to distance from the sea on AM fungal communities at a regular soil depth. Based on partial sequences of the nuclear large subunit ribosomal RNA gene, AM fungi in root samples were divided into 17 phylotypes. Among these, five AM fungal phylotypes in Glomus and Diversispora were dominant near the seaward forefront of the vegetation. Redundancy analysis of the AM fungal community showed significant relationships between the distribution of phylotypes and environmental variables such as distance from the sea, water-soluble sodium in soil, and some coexisting plant species. These results suggest that environmental gradients in the coastal vegetation can be determinants of the AM fungal community.

Mycorrhizal diversity and specificity in Lecanorchis (Orchidaceae)

Lecanorchis is a nonphotosynthetic plant genus in Vanilloideae, Orchidaceae. Because of the distribution of many Lecanorchis taxa in various climate conditions, we hypothesized that mycorrhizal diversity and specificity are different among the different taxa of Lecanorchis. In the present study, identities of mycorrhizal fungi were examined for 90 individuals of 10 Lecanorchis taxa at 26 sites from Niigata to Okinawa Prefectures in Japan. Phylogenetic analyses of Lecanorchis taxa based on the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene (rDNA) divided the examined Lecanorchis taxa into three groups, groups A, B, and C. ITS rDNA sequences suggested that fungi associating with Lecanorchis were ectomycorrhiza-forming fungi in Lactarius, Russula, Atheliaceae, and Sebacina, with Lactarius and Russula dominant. Our results suggested some degree of mycorrhizal specialization among Lecanorchis taxa. Interestingly, the Lecanorchis group C had some specific relationships with Lactarius, whereas less specificity was found in the relationships with Russula. However, observed specificity results may be biased by geographic opportunity, and we suggest further research to assess whether Lecanorchis species are limited to the associations we observed.

Fungal partner shifts during the evolution of mycoheterotrophy in Neottia

PREMISE OF THE STUDY Few previous studies have examined how mycobionts change during the evolution from autotrophy to mycoheterotrophy based on phylogenetic hypotheses. Neottia (Orchidaceae) comprises leafy species that are autotrophic and related leafless mycoheterotrophic species, and the phylogenetic relationships among them have been clarified. Accordingly, Neottia is a suitable taxon for investigating the question above. Here we clarified the diversity of mycobionts in Neottia plants and elucidated changes in the character of symbiotic associations during the evolution of mycoheterotrophy. METHODS We sequenced the internal transcribed spacer (ITS) regions of nuclear ribosomal (nr) DNA for mycobionts of Neottia plants. Furthermore, we selected one representative DNA sample from each fungal operational taxonomic unit (OTU) and used it to amplify the large subunit (LSU) nrDNA sequences. Phylogenetic analyses of Sebacinales (basidiomycetes), the dominant mycobiont of Neottia, were conducted and sample-based rarefaction curves generated for the observed mycobiont richness on each OTU. KEY RESULTS Leafy and leafless species in Neottia were associated with Sebacinales Group B and Sebacinales Group A, respectively. The composition and specificity level of fungal partners varied among Neottia species. CONCLUSIONS Fungal partner composition and specificity level changed with speciation in both leafy and leafless Neottia species. In particular, mycorrhizal associations likely shifted from Sebacinales Group B to Group A during the evolution from autotrophy to mycoheterotrophy. Partner shifts to Sebacinales Group A have also been reported in the evolution of mycoheterotrophy of other plant groups, suggesting that convergence to this fungal group occurs in association with the evolution of mycoheterotrophy.

Quantitative evaluation of protocorm growth and fungal colonization in Bletilla striata (Orchidaceae) reveals less-productive symbiosis with a non-native symbiotic fungus

BackgroundIn nature, orchid plants depend completely on symbiotic fungi for their nutrition at the germination and the subsequent seedling (protocorm) stages. However, only limited quantitative methods for evaluating the orchid–fungus interactions at the protocorm stage are currently available, which greatly constrains our understanding of the symbiosis. Here, we aimed to improve and integrate quantitative evaluations of the growth and fungal colonization in the protocorms of a terrestrial orchid, Blettila striata, growing on a plate medium.ResultsWe achieved both symbiotic and asymbiotic germinations for the terrestrial orchid B. striata. The protocorms produced by the two germination methods grew almost synchronously for the first three weeks. At week four, however, the length was significantly lower in the symbiotic protocorms. Interestingly, the dry weight of symbiotic protocorms did not significantly change during the growth period, which implies that there was only limited transfer of carbon compounds from the fungus to the protocorms in this relationship. Next, to evaluate the orchid–fungus interactions, we developed an ink-staining method to observe the hyphal coils in protocorms without preparing thin sections. Crushing the protocorm under the coverglass enables us to observe all hyphal coils in the protocorms with high resolution. For this observation, we established a criterion to categorize the stages of hyphal coils, depending on development and degradation. By counting the symbiotic cells within each stage, it was possible to quantitatively evaluate the orchid-fungus symbiosis.ConclusionsWe describe a method for quantitative evaluation of orchid-fungus symbiosis by integrating the measurements of plant growth and fungal colonization. The current study revealed that although fungal colonization was observed in the symbiotic protocorms, the weight of the protocorm did not significantly increase, which is probably due to the incompatibility of the fungus in this symbiosis. These results suggest that fungal colonization and nutrition transfer can be differentially regulated in the symbiosis. The evaluation methods developed in this study can be used to study various quantitative aspects of the orchid-fungus symbiosis.