Autoactivation of mycorrhizal symbiosis signaling through gibberellin deactivation in orchid seed germination.

Orchids parasitically depend on external nutrients from mycorrhizal fungi for seed germination. Previous findings suggest that orchids utilize a genetic system of mutualistic arbuscular mycorrhizal (AM) symbiosis, in which the plant hormone gibberellin (GA) negatively affects fungal colonization and development, to establish parasitic symbiosis. Although GA generally promotes seed germination in photosynthetic plants, previous studies have reported low sensitivity of GA in seed germination of mycoheterotrophic orchids where mycorrhizal symbiosis occurs concurrently. To elucidate the connecting mechanisms of orchid seed germination and mycorrhizal symbiosis at the molecular level, we investigated the effect of GA on a hyacinth orchid (Bletilla striata) seed germination and mycorrhizal symbiosis using asymbiotic and symbiotic germination methods. Additionally, we compared the transcriptome profiles between asymbiotically and symbiotically germinated seeds. Exogenous GA negatively affected seed germination and fungal colonization, and endogenous bioactive GA was actively converted to the inactive form during seed germination. Transcriptome analysis showed that B. striata shared many of the induced genes between asymbiotically and symbiotically germinated seeds, including GA metabolism- and signaling-related genes and AM-specific marker homologs. Our study suggests that orchids have evolved in a manner that they do not use bioactive GA as a positive regulator of seed germination and instead autoactivate the mycorrhizal symbiosis pathway through GA inactivation to accept the fungal partner immediately during seed germination.

Ubiquitous arbuscular mycorrhizal fungi in the roots of herbaceous understory plants with hyphal degeneration in Colchicaceae and Gentianaceae.

Due to the loss of photosynthetic ability during evolution, some plant species rely on mycorrhizal fungi for their carbon source, and this nutritional strategy is known as mycoheterotrophy. Mycoheterotrophic plants forming Paris-type arbuscular mycorrhizas (AM) exhibit two distinctive mycorrhizal features: degeneration of fungal materials and specialization towards particular fungal lineages. To explore the possibility that some understory AM plants show partial mycoheterotrophy, i.e., both photosynthetic and mycoheterotrophic nutritional strategies, we investigated 13 green herbaceous plant species collected from five Japanese temperate forests. Following microscopic observation, degenerated hyphal coils were observed in four species: two Colchicaceae species, Disporum sessile and Disporum smilacinum, and two Gentianaceae species, Gentiana scabra and Swertia japonica. Through amplicon sequencing, however, we found that all examined plant species exhibited no specificity toward AM fungi. Several AM fungi were consistently found across most sites and all plant species studied. Because previous studies reported the detection of these AM fungi from various tree species in Japanese temperate forests, our findings suggest the presence of ubiquitous AM fungi in forest ecosystems. If the understory plants showing fungal degeneration exhibit partial mycoheterotrophy, they may obtain carbon compounds indirectly from a wide range of surrounding plants utilizing such ubiquitous AM fungi.

Impact of mycoheterotrophy on the growth of Gentiana zollingeri (Gentianaceae), as suggested by size variation, morphology, and 13C abundance of flowering shoots.

Gentiana zollingeri is an annual photosynthetic plant that employs a mycoheterotrophic growth strategy during its underground seedling stage (initial mycoheterotrophy). Notably, the morphological characteristics of its flowering shoots, such as shoot size, leaf size, and leaf color, are highly variable, and it was hypothesized that these variations may be linked to nutritional mode. The morphological characteristics of G. zollingeri individuals were thus investigated alongside environmental factors, 13C abundance, and diversity of colonizing arbuscular mycorrhizal (AM) fungi. The majority of G. zollingeri flowering individuals were found to exhibit a high affinity for the specific AM fungi that exclusively colonize roots of the mycoheterotrophic seedlings, while other phylogenetically diverse AM fungi could also be detected. The leaves to shoot dry weight ratio (leaf ratio) was negatively correlated with the canopy openness in the habitat, suggesting that leaf development is impeded in sunny conditions. Furthermore, the shoot weight of G. zollingeri was positively correlated with leaf 13C abundance. Given that 13C enrichment can provide indirect evidence of mycoheterotrophy in AM plants, the results suggest that the utilization of carbon obtained through mycoheterotrophy, at least during the underground seedling stage, is crucial for G. zollingeri.

Isolation and identification of an arbuscular mycorrhizal fungus specifically associated with mycoheterotrophic seedlings of Gentiana zollingeri (Gentianaceae).

Gentiana zollingeri (Gentianaceae) is an initial mycoheterotrophic plant that depends on a specific group of arbuscular mycorrhizal (AM) fungi for carbon source during underground growth after seed germination. In this study, a mycorrhizal fungus dominant in mycoheterotrophic seedlings of G. zollingeri was successfully isolated from a soil core collected from a point close to a flowering G. zollingeri. The AM fungal isolate was identified as conspecific or closely related to Dominikia aurea (Glomeraceae) by spore morphology and molecular phylogeny. Basic Local Alignment Search Tool (BLAST) searches against the MaarjAM database showed that the nuclear small subunit ribosomal DNA sequences of the isolate matched the AM fungal sequences obtained from a wide range of plants in various ecosystems, including several mycoheterotrophs. Thus, it is suggested that the AM fungal isolate is one of the cheating susceptible AM fungi. Furthermore, the sequences corresponded to those of a group of AM fungi dominantly detected in Japanese temperate forests. Accordingly, there is a possibility that mycoheterotrophic plants, including seedlings of G. zollingeri, may target AM fungi with a wide host range and ubiquitous distribution.

Clonal spore populations in sporocarps of arbuscular mycorrhizal fungi.

Some arbuscular mycorrhizal (AM) fungal species known to form sporocarps (i.e., aggregations of spores) are polyphyletic in two orders, Glomerales and Diversisporales. Spore clusters (sporocarp-like structures) often formed in pot cultures or in vitro conditions are supposed to be clonal populations, while sporocarps in natural habitats with a fungal peridium are morphologically similar to those of epigeous sexual (zygosporic) sporocarps of Endogone species. Thus, in this study, we explored the genetics of sporocarpic spores of two AM fungi with a view to possibilities of clonal or sexual reproduction during sporocarps formation. To examine these possibilities, we investigated single-nucleotide polymorphisms (SNPs) in reduced genomic libraries of spores isolated from sporocarps molecularly identified as Rhizophagus irregularis and Diversispora epigaea. In addition, partial sequences of the MAT locus HD2 gene of R. irregularis were phylogenetically analyzed to determine the nuclear status of the spores. We found that most SNPs were shared among the spores isolated from each sporocarp in both species. Furthermore, all HD2 sequences from spores isolated from three R. irregularis sporocarps were identical. These results indicate that those sporocarps comprise clonal spores. Therefore, sporocarps with clonal spores may have different functions than sexual reproduction, such as massive spore production or spore dispersal via mycophagy.

Orchid mycorrhizal fungi and ascomycetous fungi in epiphytic Vanda falcata roots occupy different niches during growth and development.

Epiphytic orchids are commonly found in exposed environments, which plausibly lead to different root fungal community structures from terrestrial orchids. Until recently, few studies have been conducted to show the fungal community structure during the growth of a photosynthetic and epiphytic orchid in its natural growing site. In this study, the Vanda falcata (commonly known as Neofinetia falcata), one of Japan's ornamental orchids, was used to characterize the fungal community structure at different developmental stages. Amplicon sequencing analysis showed that all development stages contain a similar fungal community: Ascomycota dominate half of the community while one-third of the community belongs to Basidiomycota. Rhizoctonia-like fungi, a polyphyletic basidiomycetous fungal group forming mycorrhizas in many orchids, exist even in a smaller portion (around one-quarter) compared to other Basidiomycota members. While ascomycetous fungi exhibit pathogenicity, two Ceratobasidium strains isolated from young and adult plants could initiate seed germination in vitro. It was also found that the colonization of mycorrhizal fungi was concentrated in a part of the root where it directly attaches to the phorophyte bark, while ascomycetous fungi were distributed in the velamen but never colonized cortical cells. Additionally, the root parts attached to the bark have denser exodermal passage cells, and these cells were only colonized by mycorrhizal fungi that further penetrated into the cortical area. Therefore, we confirmed a process that physical regulation of fungal entry to partition the ascomycetes and mycorrhizal fungi results in the balanced mycorrhizal symbiosis in this orchid.

Mycoheterotrophic seedling growth of Gentiana zollingeri, a photosynthetic Gentianaceae plant species, in symbioses with arbuscular mycorrhizal fungi.

We found mycoheterotrophic seedling growth (initial mycoheterotrophy) of Gentiana zollingeri, a spring-flowering photosynthetic species of Gentianaceae family. Small seeds (about 300 µm in length) were buried in a habitat by using seed packets, and development of the subterranean seedlings to form shoots, more than 3 cm in length, was observed in symbiosis with arbuscular mycorrhizal (AM) fungi in the dark (i.e., underground of a field). Hyphal coils and their degenerations were observed in the root cortical cells of the subterranean seedlings as well as those of adult plants. Among the mycobionts identified on the basis of partial small subunit rDNA sequences, it was found that AM fungi of a lineage in Glomeraceae dominantly colonized, and the AM fungi were also dominant in adult individuals of G. zollingeri in three habitats separated one another by 17.2, 34.7, and 49.6 km. Though initial mycoheterotrophy in symbioses with AM fungi has been observed in some pteridophytes, this is the first study to demonstrate this type of symbiosis in a photosynthetic seed plant. The mycoheterotrophy means that an energy distribution occurs through the hyphal bridges of AM fungi among different photosynthetic seed plants, which may be important in constructing plant species diversity in some ecosystems.

Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile.

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (13C and 15 N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in 13C but not in 15 N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 ± 8.2% of their C from fungal sources, using the 13C enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

Gibberellin Promotes Fungal Entry and Colonization during Paris-Type Arbuscular Mycorrhizal Symbiosis in Eustoma grandiflorum.

Arbuscular mycorrhizas (AMs) are divided into two types according to morphology: Arum- and Paris-type AMs. Gibberellins (GAs) mainly inhibit the establishment of Arum-type AM symbiosis in most model plants, whereas the effects of GAs on Paris-type AM symbiosis are unclear. To provide insight into the mechanism underlying this type of symbiosis, the roles of GAs were investigated in Eustoma grandiflorum when used as the host plant for Paris-type AM establishment. Eustoma grandiflorum seedlings were inoculated with the model AM fungus, Rhizophagus irregularis, and the effects of GA and the GA biosynthesis inhibitor uniconazole-P on the symbiosis were quantitatively evaluated. Exogenous GA significantly increased hyphopodium formation at the epidermis, thus leading to the promotion of fungal colonization and arbuscule formation in the root cortex. By contrast, the suppression of GA biosynthesis and signaling attenuated fungal entry to E. grandiflorum roots. Moreover, the exudates from GA-treated roots strongly induced the hyphal branching of R. irregularis. Our results show that GA has an contrasting effect on Paris-type AM symbiosis in E. grandiflorum compared with Arum-type AM symbiosis. This finding could be explained by the differential regulation of the early colonization stage, where fungal hyphae make contact with and penetrate the epidermis.

Communities of arbuscular mycorrhizal fungi in forest ecosystems in Japan's temperate region may be primarily constituted by limited fungal taxa.

We investigated arbuscular mycorrhizal (AM) fungal communities in secondary forests and/or Chamaecyparis obtusa plantations at eight study sites in Japan's temperate region. In the secondary forests, AM plants of the families Lauraceae, Sapindaceae, Rutaceae, Araliaceae, Rosaceae, Magnoliaceae, Cornaceae, Piperaceae, and Anacardiaceae were found. The AM fungal communities were evaluated based on compositions of the AM fungal operational taxonomic units (OTUs), which were clustered at a 97% similarity threshold of the sequences of a partial small subunit of a nuclear ribosomal RNA gene obtained from the plant roots. The compositions of AM fungal OTUs were significantly correlated with the plant family compositions and were significantly differentiated among the study sites and between the study forests. Interestingly, only 19 OTUs remained after selecting for those that had more than 1.0% of the total reads, and these 19 OTUs accounted for 86.3% of the total rarefied reads that were classified into 121 OTUs. Furthermore, three dominant OTUs constituted 48.0% of the total reads, and the most dominant OTU was found at all study sites, except at one. These results indicate that AM fungal communities are primarily constituted by limited AM fungal taxa in the forest ecosystems with diverse plant taxa in Japan's temperate region. The results of basic local algorithm search tool (BLAST) searches against MaarjAM, a database of AM fungal sequences, also revealed that the AM fungi which were the three dominant OTUs are distributed in forest ecosystems on a worldwide scale.

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina, an orchid mainly utilizing saprotrophic rhizoctonia.

The majority of chlorophyllous orchids form mycorrhizal associations with so-called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their 13 C and 15 N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in 13 C but not so greatly in 15 N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, 13 C levels were depleted and those of 15 N were indistinguishable from the co-occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.

The Mycoheterotrophic Symbiosis Between Orchids and Mycorrhizal Fungi Possesses Major Components Shared with Mutualistic Plant-Mycorrhizal Symbioses.

Achlorophylous and early developmental stages of chorolophylous orchids are highly dependent on carbon and other nutrients provided by mycorrhizal fungi, in a nutritional mode termed mycoheterotrophy. Previous findings have implied that some common properties at least partially underlie the mycorrhizal symbioses of mycoheterotrophic orchids and that of autotrophic arbuscular mycorrhizal (AM) plants; however, information about the molecular mechanisms of the relationship between orchids and their mycorrhizal fungi is limited. In this study, we characterized the molecular basis of an orchid-mycorrhizal (OM) symbiosis by analyzing the transcriptome of Bletilla striata at an early developmental stage associated with the mycorrhizal fungus Tulasnella sp. The essential components required for the establishment of mutual symbioses with AM fungi or rhizobia in most terrestrial plants were identified from the B. striata gene set. A cross-species gene complementation analysis showed one of the component genes, calcium and calmodulin-dependent protein kinase gene CCaMK in B. striata, retains functional characteristics of that in AM plants. The expression analysis revealed the activation of homologs of AM-related genes during the OM symbiosis. Our results suggest that orchids possess, at least partly, the molecular mechanisms common to AM plants.

Arbuscular mycorrhizal fungal communities under gradients of grazing in Mongolian grasslands of different aridity.

Communities of arbuscular mycorrhizal (AM) fungi in Mongolian grassland were characterized under gradients of grazing intensity at three study sites of different aridity: mountain forest steppe at Hustai National Park (Hustai), and desert steppe at Mandalgovi and Bulgan. Grazing intensity was classified into three categories: lightly grazed (LG), moderately grazed (MG), and heavily grazed (HG). With regard to floristic composition, grazing decreased the shoot biomass of Poaceae species, especially Stipa spp. Distinctness of the AM fungal communities was observed among the three study sites, but most of the AM fungal operational taxonomic units (OTUs) that comprised over 1.0% of the total reads were ubiquitous. This result indicates that the AM fungal communities may be derived from similar AM fungal floras in correspondence with environmental factors. The composition of AM fungal communities differed significantly among the grazing intensities at all study sites. The relative abundance of the most dominant AM fungal OTU of the LG plots decreased with an increase in grazing intensity at all study sites. The mean proportions of the most dominant AM fungal OTUs also decreased with increased grazing intensity at Hustai. Dominance by a single AM fungal taxon may be a typical ecological feature of the AM fungal symbiosis, and grazing disturbs AM fungal community structure.

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

BACKGROUND: In 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. RESULTS: We 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. CONCLUSIONS: We 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.

Comparison of green and albino individuals of the partially mycoheterotrophic orchid Epipactis helleborine on molecular identities of mycorrhizal fungi, nutritional modes and gene expression in mycorrhizal roots.

Some green orchids obtain carbon from their mycorrhizal fungi, as well as from photosynthesis. These partially mycoheterotrophic orchids sometimes produce fully achlorophyllous, leaf-bearing (albino) variants. Comparing green and albino individuals of these orchids will help to uncover the molecular mechanisms associated with mycoheterotrophy. We compared green and albino Epipactis helleborine by molecular barcoding of mycorrhizal fungi, nutrient sources based on 15 N and 13 C abundances and gene expression in their mycorrhizae by RNA-seq and cDNA de novo assembly. Molecular identification of mycorrhizal fungi showed that green and albino E. helleborine harboured similar mycobionts, mainly Wilcoxina. Stable isotope analyses indicated that albino E. helleborine plants were fully mycoheterotrophic, whereas green individuals were partially mycoheterotrophic. Gene expression analyses showed that genes involved in antioxidant metabolism were upregulated in the albino variants, which indicates that these plants experience greater oxidative stress than the green variants, possibly due to a more frequent lysis of intracellular pelotons. It was also found that some genes involved in the transport of some metabolites, including carbon sources from plant to fungus, are higher in albino than in green variants. This result may indicate a bidirectional carbon flow even in the mycoheterotrophic symbiosis. The genes related to mycorrhizal symbiosis in autotrophic orchids and arbuscular mycorrhizal plants were also upregulated in the albino variants, indicating the existence of common molecular mechanisms among the different mycorrhizal types.

Distribution of Petrosavia sakuraii (Petrosaviaceae), a rare mycoheterotrophic plant, may be determined by the abundance of its mycobionts.

Petrosavia sakuraii (Petrosaviaceae) is a rare, mycoheterotrophic plant species that has a specific symbiotic interaction with a narrow clade of arbuscular mycorrhizal (AM) fungi. In the present study, we tested the hypothesis that the distribution and abundance of mycobionts in two P. sakuraii habitats, Nagiso and Sengenyama (central Honshu, Japan), determine the distribution pattern of this rare plant. Nagiso is a thriving habitat with hundreds of P. sakuraii individuals per 100 m(2), whereas Sengenyama is a sparsely populated habitat with fewer than 10 individuals per 100 m(2). AM fungal communities associated with tree roots were compared at 20-cm distances from P. sakuraii shoots between the two habitats by molecular identification of AM fungal partial sequences of the small subunit ribosomal RNA gene. The percentage of AM fungal sequences showing over 99 % identity with those of the dominant P. sakuraii mycobionts was high (54.9 %) in Nagiso, but low (13.2 %) in Sengenyama. Accordingly, the abundance of P. sakuraii seems to reflect the proportion of potential mycobionts. It is likely that P. sakuraii mycobionts are not rare in Japanese warm temperate forests since 11.2 % of AM fungal sequences previously obtained from a deciduous broad-leaved forest devoid of P. sakuraii in Mizuho, central Honshu, Japan, were >99 % identical to those of the dominant P. sakuraii mycobionts. Thus, results suggest that the abundant mycobionts may be required for sufficient propagation of P. sakuraii, and this quantitative trait of AM fungal communities required for P. sakuraii may explain the rarity of this plant.

Significant difference in mycorrhizal specificity between an autotrophic and its sister mycoheterotrophic plant species of Petrosaviaceae.

Petrosaviaceae is a monocotyledonous plant family that comprises two genera: the autotrophic Japonolirion and the mycoheterotrophic Petrosavia. Accordingly, this plant family provides an excellent system to examine specificity differences in mycobionts between autotrophic and closely related mycoheterotrophic plant species. We investigated mycobionts of Japonolirion osense, the sole species of the monotypic genus, from all known habitats of this species by molecular identification and detected 22 arbuscular mycorrhizal (AM) fungal phylotypes in Archaesporales, Diversisporales, and Glomerales. In contrast, only one AM fungal phylotype in Glomerales was predominantly detected from the mycoheterotrophic Petrosavia sakuraii in a previous study. The high mycobiont diversity in J. osense and in an outgroup plant, Miscanthus sinensis (Poaceae), indicates that fungal specificity increased during the evolution of mycohetrotrophy in Petrosaviaceae. Furthermore, some AM fungal sequences of J. osense showed >99% sequence similarity to the dominant fungal phylotype of P. sakuraii, and one of them was nested within a clade of P. sakuraii mycobionts. These results indicate that fungal partners are not necessarily shifted, but rather selected for in the course of the evolution of mycoheterotrophy. We also confirmed the Paris-type mycorrhiza in J. osense.

Mycoheterotrophic growth of Cephalanthera falcata (Orchidaceae) in tripartite symbioses with Thelephoraceae fungi and Quercus serrata (Fagaceae) in pot culture condition.

Mixotrophy, obtaining carbon by mycoheterotrophy and photosynthesis, has been suggested in Cephalanthera species (Orchidaceae) by analyses on stable isotopes of carbon. In this study, we examined the growth of Cephalanthera falcata in pot cultured tripartite symbioses with Thelephoraceae fungi and Quercus serrata. Mycorrhizal fungi were isolated from roots of C. falcata in natural habitats. Two fungal isolates identified as Thelephoraceae were cultured and inoculated to fine roots of non-mycorrhizal seedlings of Q. serrata (Fagaceae). After the ectomycorrhizal formation, non-mycorrhizal seedlings of C. falcata were co-planted. The pots with tripartite symbioses were cultured in greenhouse for 30 months, and growth of C. falcata seedlings was examined. Fresh weight of C. falcata seedlings was significantly increased by the tripartite symbioses even in those with no shoot, thus providing further evidence for the mycoheterotrophic nature of this orchid. The achievement of seedling culture in tripartite symbioses would be valuable for conserving many forest orchids and for conducting experiments to understand their physiology and ecology.

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.