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The rare achlorophyllous orchid Cyrtosia septentrionalis (Rchb.f.) Garay is thought to switch mycobiont from a Physisporinus species (‘TK-10’) to an Armillaria species as it develops. We conducted morphological, chemical, and molecular analyses of the root system of three adult plants growing sympatrically in Japan to assess reliance on different mycorrhizae. The root systems were found to comprise several long primary roots that radiate outwards from the emergent stem and many shorter lateral ones. Long roots branched most vigorously where they encountered Armillaria gallica in the soil, and the resulting lateral roots contained the highest density of intra-radical rhizomorphs. One plant was associated with ‘TK-10’ and the other two with A. gallica, but only the apical portion of long roots and a few lateral roots were colonized. Mycobionts isolated from the base of long roots proved incapable of germinating C. septentrionalis seeds. Whereas glucose was the dominant monosaccharide in non-mycorrhizal roots, galactose was in mycorrhizal ones. Lateral roots are specialized in mycorrhization and their velamen, and mucilage may play a key role in preventing desiccation. We hypothesize that C. septentrionalis can associate with either fungus from germination to adulthood, but that detection of A. gallica in the soil can prompt replacement of ‘TK-10’.

Plants produce compounds to resist fungal attack. However, most orchids require fungal colonisation for germination, and fully mycoheterotrophic (MH) orchids are dependent upon fungi for their entire nutritional needs. To elucidate the manner in which MH orchids interact with and accommodate a mycorrhizal partner, we examined the effect of compounds involved in pathogenic resistance on seed germination in Cyrtosia septentrionalis, an MH orchid for which a temperature of 30 °C and an atmosphere comprising reduced O2 (5%) and elevated CO2 (8%) have been shown to be optimal for axenic germination. Seeds were either co-cultured with a Physisporinus mycobiont in sealed containers or monocultured on nutrient-free water agar (WA) supplemented with various compounds under micro-aerophilic conditions (6–12% O2, 5–8% CO2) at 19, 23 or 30 °C. Co-cultured seeds germinated and underwent onward development at all temperatures. In contrast, monocultured seeds failed to germinate on WA alone except at 30 °C, but germination was initiated, as indicated by seed coat rupture, at 19 °C when K+, kinetin and NAA were added. Ca2+ and H2O2 increased germination initiation, whereas salicylic acid and gibberellic acid inhibited it. Asymbiotic initiation also occurred at 30 °C in sealed vessels with a more-or-less regular atmosphere when K+ and kinetin were added. We hypothesise that germination in C. septentrionalis is triggered by the micro-aerophilic conditions established as a result of its mycobiont’s wood-rotting activity, with certain compounds secreted as part of the early defence response having a potentiating effect. However, subsequent mycobiont colonisation is essential for full germination and onward development to ensue.

The climbing orchid Erythrorchis altissima is the largest mycoheterotroph in the world. Although previous in vitro work suggests that E. altissima has a unique symbiosis with wood-decaying fungi, little is known about how this giant orchid meets its carbon and nutrient demands exclusively via mycorrhizal fungi. In this study, the mycorrhizal fungi of E. altissima were molecularly identified using root samples from 26 individuals. Furthermore, in vitro symbiotic germination with five fungi and stable isotope compositions in five E. altissima at one site were examined. In total, 37 fungal operational taxonomic units (OTUs) belonging to nine orders in Basidiomycota were identified from the orchid roots. Most of the fungal OTUs were wood-decaying fungi, but underground roots had ectomycorrhizal Russula. Two fungal isolates from mycorrhizal roots induced seed germination and subsequent seedling development in vitro. Measurement of carbon and nitrogen stable isotope abundances revealed that E. altissima is a full mycoheterotroph whose carbon originates mainly from wood-decaying fungi. All of the results show that E. altissima is associated with a wide range of wood- and soil-inhabiting fungi, the majority of which are wood-decaying taxa. This generalist association enables E. altissima to access a large carbon pool in woody debris and has been key to the evolution of such a large mycoheterotroph.

We found Gastrodia fontinalis T. P. Lin in a bamboo forest from Takeshima Island, which is the northernmost island of the Ryukyu Islands in Japan. This species is apparently rare and was previously considered to be an endemic Taiwanese species. Because there are a few minor differences between the original description and our specimens collected in Takeshima Island, here we report Gastrodia fontinalis from Takeshima Island as the first record outside of Taiwan, with a description of the specimens from Takeshima Island.

Nervilia nipponica is a tuberous terrestrial orchid that has a highly restricted distribution within common secondary evergreen forest communities in central and western Japan. Such a limited occurrence could be attributable to a requirement for a specific mycorrhizal fungus. As part of a broader examination of this hypothesis, we sought to elucidate the mycorrhizal associations of N. nipponica. Seventy-five samples of mycorrhizae from forty individuals were collected at ten populations throughout the orchid's range in Japan. The identity of mycorrhizal fungi was investigated by sequencing two genetic markers (nrDNA ITS and nrDNA 28S LSU) and their relationships were assessed via phylogenetic analyses. The most frequently encountered mycorrhizal fungi consisted of four closely related Agaricomycetes that infected an average of 78.7 % of individuals per population. All four formed a discrete, monophyletic clade with low sequence homology to other fungi registered in GenBank, indicating that they belong to a novel, unnamed family. Two additional fungal groups, belonging to Ceratobasidiaceae and "Group B" Sebacinales, were found in 22.0 and 21.5 % of individuals per population, respectively. The orchid probably uses these two groups opportunistically, because they were found at lower densities and always in combination with the unidentified Agaricomycete. These findings suggest that a group of novel Agaricomycete fungi constitutes the dominant mycobiont of N. nipponica.

To determine the means and variations in CH4 uptake and N2O emission in the dominant soil and vegetation types to enable estimation of annual gases fluxes in the forest land of Japan, we measured monthly fluxes of both gases using a closed-chamber technique at 26 sites throughout Japan over 2 years. No clear seasonal changes in CH4 uptake rates were observed at most sites. N2O emission was mostly low throughout the year, but was higher in summer at most sites. The annual mean rates of CH4 uptake and N2O emission (all sites combined) were 66 (2.9–175) µg CH4-C m−2 h−1 and 1.88 (0.17–12.5) µg N2O-N m−2 h−1, respectively. Annual changes in these fluxes over the 2 years were small. Significant differences in CH4 uptake were found among soil types (P Brown forest soil (60 ± 27) ≥ other soils (20 ± 24). N2O emission rates differed significantly among vegetation types (P

Premise of the study Since mycoheterotrophic plants (MHPs) completely depend on their mycorrhizal fungi for carbon, selection of fungal partners has an important role in the speciation of MHPs. However, the causes and mechanisms of mycobiont changes during speciation are not clear. We tested fungal partner shifts and changes in mycorrhizal specificity during speciation of three closely related MHPs-Gastrodia confusa (Gc), G. pubilabiata (Gp), and G. nipponica (Gn) (Orchidaceae)-and correlations between these changes and the vegetation types where each species grows. Methods We investigated the diversity of mycobionts of the three species by sequencing nrDNA ITS, and the sequence data were subjected to test changes in fungal specificity and fungal partner shifts among the three species. Furthermore, we conducted multivariate analysis to test for differences in mycobiont communities of vegetation types where each species grows. Key results Two saprobic Basidiomycota, Marasmiaceae and Mycenaceae, were dominant fungal partners of the three species, and Gn was simultaneously associated with the ectomycorrhizal Russulaceae and Sebacinaceae. Although mycobiont composition differed among the three species, they also sometimes shared identical fungal species. Multivariate analysis revealed that mycobiont communities of the three species in bamboo thickets differed significantly from those in other vegetation types. Conclusions Fungal partner shifts are not necessarily associated with the evolution of MHPs, and fungal specificity of Gc and Gp was significantly higher than that of Gn, implying that the specificity fluctuates during speciation. Further, Gc exclusively inhabits bamboo thickets, which suggests that adaptation to particular fungi specific to bamboo thickets triggered speciation of this species.

Four sib-monokaryons and two reconstituted dikaryons of two basidiomycetes,Lenzites betulinus andTrametes hiruta, accelerated the seed germination ofErythrorchis ochobiensis, an achlorophyllous orchid. All isolates ofL. betulinus and three isolates ofT. hirsuta induced the development of plants from germinated seeds. Although three monokaryotic isolates ofT. hirsuta failed to induce the development of plants, the reconstituted dikaryons induced the development.

Seed germination test ofGaleola altissima was carried out with five aphyllophorales fungi:Erythromyces crocicreas, Ganoderma australe, Loweporus tephroporus, Microporus affinus andPhellinus sp.. All five species were effective for seed germination of the orchid.Erythromyces crocicreas, which has hitherto been regarded as the only endomycorrhizal fungus of the orchid, was confirmed to be effective for further development of the orchid.

Some physiological properties of six strains of Hymenochaete crocicreas Berk. et Br., including the symbiotic germination of Galeola altissima Reich. fil., were investigated. H. crocicreas distributed in the tropical Asia is one of white pocket rot fungi, and G. altissima is a chlorophyll free orchid of which habitat is the south of Tanegashima Island of Japan, Formosa, Malaysia, and India.In the temperature-growth test, 6 isolates of H. crocicreas are divided into two groups due to their growth at high temperature, namely one group grows well at 35°C, but another is reduced their growth. In Bavendamm reaction, all isolates show positive reaction which suggests the presence of polyphenol oxidases. In the germination test, the symbiotic germination takes place with 5 isolates at 23°C and 30°C, and protocorms are larger at 30°C than 23°C except one isolate. Some variation in the ratio of germination and the size of protocorms arise from fungal partners.

We investigated the physiological ecology of the Asian non-photosynthetic orchid Gastrodia confusa . We revealed its mycorrhizal partners by using molecular identification and identified its ultimate nutritional source by analysing carbon and nitrogen natural stable isotope abundances. Molecular identification using internal transcribed spacer and large subunit nrDNA sequences showed that G. confusa associates with several species of litter- and wood-decomposer Mycena fungi. The carbon and nitrogen isotope signatures of G. confusa were analysed together with photosynthetic plant reference samples and samples of the ectomycorrhizal epiparasite Monotropa uniflora . We found that G. confusa was highly enriched in 13 C but not greatly in 15 N, while M. uniflora was highly enriched in both 13 C and 15 N. The 13 C and 15 N signatures of G. confusa were the closest to those of the fruit bodies of saprotrophic fungi. Our results demonstrate for the first time using molecular and mass-spectrometric approaches that myco-heterotrophic plants gain carbon through parasitism of wood or litter decaying fungi. Furthermore, we demonstrate that, several otherwise free-living non-mycorrhizal, Mycena can be mycorrhizal partners of orchids.

In vitro germination of a myco-heterotrophic orchid,Erythrorchis ochobiensis, was tested in the presence of ectomycorrhizal fungi,Lyophyllum shimeji andTricholoma fulvocastaneum. Lyophyllum shimeji stimulated the germination after incubation for 1.5 mo. Although most germinated seeds did not grow further after 3 mo, several seeds developed into small protocorms but showed amorphous profiles. Fungal mycelia were observed in the germinated seeds and protocorms, but pelotons were not detected. Since the seeds did not germinate axenically, it may be suggested that the fungus has the ability to stimulate germination.