Taxonomic and functional characterisation of fungi from the Sebacina vermifera complex from common and rare orchids in the genus Caladenia.

The terrestrial orchid genus Caladenia contains many species which are threatened with extinction. They have highly specific associations with Sebacina vermifera and closely related fungi, and conservation of these terrestrial orchids, in part, relies on symbiotic propagation to produce plants for reintroduction and ex situ conservation collections. However, little is known of the diversity of mycorrhizal fungi associating with natural populations. Here, restriction fragment polymorphism analysis, internal transcribed spacer and nuclear large subunit sequencing and symbiotic seed germination were used to investigate the taxonomic and functional diversity of fungal isolates from single populations of six endangered Caladenia species and one common species across the same biogeographic range. Fifty-nine fungal isolates were collected for investigation including ten isolates from the six endangered species Caladenia audasii, Caladenia amoena, Caladenia sp. aff. fragrantissima (Central Victoria), Caladenia sp. aff. patersonii, Caladenia rosella and Caladenia orientalis and 49 isolates from six populations of the common species Caladenia tentaculata. While the common species associated with three distinct S. vermifera-like taxa, the six endangered species were restricted to one of these fungal taxa. No direct relationship between the taxonomic identity of the fungi and their ability to stimulate seed germination was observed; however, the majority of the fungi isolated from the Caladenia species were capable of germinating seed in vitro, indicating their mycorrhizal status and potential for symbiotic propagation in conservation programmes.

Functional and genetic diversity of mycorrhizal fungi from single plants of Caladenia formosa (Orchidaceae).

BACKGROUND AND AIMS: Mycorrhizal associations are essential to the plant kingdom. The largest flowering plant family, the Orchidaceae, relies on mycorrhizal fungi for germination, growth and survival. Evidence suggests varying degrees of fungal-host specificity based on a single fungal isolate from a single plant. This paper shows for the first time the diversity of endophytes colonizing in a single plant over consecutive years and the functional significance of this diversity. METHODS: Stem-collars of Caladenia formosa were collected in different seasons and years. Mycorrhizal fungi isolated were tested for their efficacy to induce leafing and genetically determined using ITS-RFLP and sequencing. RESULTS: Multiple mycorrhizal fungi were repeatedly isolated from a single collar that displayed varying effectiveness in germination percentages and adult leaf length. Additional factors contributed to the isolation of effective mycorrhizal fungi; fungal collection season, year of collection and individual isolates. Surface sterilization only improved the number of isolated mycorrhizal fungi. Dual inoculation did not increase germination. All 59 mycorrhizal fungi effective in germinating seed belonged to one clearly defined ITS (internal transcribed spacer) clade and clustered close to Sebacina vermifera (79-89 % homology). Isolates resulting in the greatest germination were not necessarily those resulting in the greatest survival and growth 1 year after germination. CONCLUSION: Single orchid plants contained multiple mycorrhizal fungal strains of one species that had diverse functional differences. These results suggest that our current knowledge of fungal-host specificity may be incomplete due to experimental and analytical limitations. It also suggests that the long-term effectiveness of a mycorrhizal fungus or fungi could only be found by germination and longer-term growth tests rather than genetically.