Hidetaka Umata

Evidence for novel and specialized mycorrhizal parasitism: the orchid Gastrodia confusa gains carbon from saprotrophic Mycena

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 13C but not greatly in 15N, while M. uniflora was highly enriched in both 13C and 15N. The 13C and 15N 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.

Methane uptake and nitrous oxide emission in Japanese forest soils and their relationship to soil and vegetation types

Abstract 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 < 0.05). The mean CH4 uptake rates (µg CH4-C m−2 h−1) were as follows: Black soil (95 ± 39, mean ± standard deviation [SD]) > Brown forest soil (60 ± 27) ≥ other soils (20 ± 24). N2O emission rates differed significantly among vegetation types (P < 0.05). The mean N2O emission rates (µg N2O-N m−2 h−1) were as follows: Japanese cedar (4.0 ± 2.3) ≥ Japanese cypress (2.6 ± 3.4) > hardwoods (0.8 ± 2.2) = other conifers (0.7 ± 1.4). The CH4 uptake rates in Japanese temperate forests were relatively higher than those in Europe and the USA (11–43 µg CH4-C m−2 h−1), and the N2O emission rates in Japan were lower than those reported for temperate forests (0.23–252 µg N2O-N m−2 h−1). Using land area data of vegetation cover and soil distribution, the amount of annual CH4 uptake and N2O emission in the Japanese forest land was estimated to be 124 Gg CH4-C year−1 with 39% uncertainty and 3.3 Gg N2O-N year−1 with 76% uncertainty, respectively.

Seed germination of Galeola altissima, an achlorophyilous orchid, with aphyllophorales fungi

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.

A new biological function of Shiitake mushroom,Lentinula edodes, in a myco-heterotrophic orchid,Erythrorchis ochobiensis

The biological function ofLentinula edodes in a myco-heterotrophic orchid,Erythrorchis ochobiensis was examined, using one local variant each from Japan (JPN), Papua New Guinea (PNG) and New Zealand (NZ). All variants induced seed germination: PNG and NZ isolates were effective at 25°C and JPN isolate showed the highest germination rate at 30°C. Germinated seeds developed into plants and formed normal endomycorrhizas. Hence, it is concluded thatL. edodes has a perfect symbiotic potential withE. ochobiensis, though it has not been observed in the root of the orchid in the field.

Formation of endomycorrhizas by an achlorophyllous orchid, Erythrorchis ochobiensis, and Auricularia polytricha

To test the mycorrhizal function of heterobasidiomycetous fungi on achlorophyllous orchids and to examine the symbiotic fungal range of a myco-heterotrophic orchid,Erythrorchis ochobiensis, synthetic cultures of the orchid seed were carried out withAuricularia polytricha isolates from Japan and Mexico. After three and a half mo of incubation, 57.0–70.7% of seeds germinated but none of them showed further growth. When cultured on peat moss at 25°C, the germination rate was 8.7% in the presence of Mexican isolate and 18.0% in the presence of Japanese isolate. Some germinated seeds developed into protocorms, and several seeds incubated with the Mexican isolate developed into plantlets after 5 mo. Pelotons were observed in the cells of protocorms and roots. The results indicated that some heterobasidiomycetous fungi could form endomycorrhizas with a myco-heterotrophic orchid. The results also showed that the symbiont ofE. ochobiensis extends, at least experimentally, to Heterobasidiomycetes. The variances of germination rate and seedling growth were suggested to be affected by the difference of isolates and culture conditions.

Germination and growth of Erythrorchis ochobiensis (Orchidaceae) accelerated by monokaryons and dikaryons of Lenzites betulinus and Trametes hirsuta

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.

In vitro germination of Erythrorchis ochobiensis (Orchidaceae) in the presence of Lyophyllum shimeji, an ectomycorrhizal fungus

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.

How do fungal partners affect the evolution and habitat preferences of mycoheterotrophic plants? A case study in Gastrodia

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.