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

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.