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.

This study investigated the relationships between ericoid mycorrhizal endophytes of the Ericaceae (Northern Hemisphere) and the Epacridaceae (Australia). Over 200 fungi were isolated from the roots of two species of Epacridaceae from Victoria, Australia. The isolates were divided into 12 groups by morphology on quarterstrength potato dextrose agar. All were slow-growing and most were dematiaceous, but groups varied from white through pink to dark olive. The ITS1-5.8S-ITS2 ribosomal DNA was amplified and sequenced from eight isolates, forming typical ericoid mycorrhizal morphology in Epacris impressa and one nonmycorrhizal isolate. Sequences were compared, by using similarities and maximum-parsimony analysis, with those of Hymenoscyphus ericae (Leotiales) and Oidiodendron species (Hyphomycetes), the most common endophytes of the Ericaceae. Maximum-parsimony analysis produced four clusters : (1) all Oidiodendron species (at least 90% similarity) ; (2) all five Victorian dark grey-olive isolates (at least 96% similarity) ; (3) one Victorian isolate and Cistella grevillei (88% similarity) ; (4) two light-coloured Victorian isolates and H. ericae (81% similarity). This suggests that these isolates from the Epacridaceae do not belong to the same species as those forming ericoid mycorrhiza in the Ericaceae.

Six developmental stages (leafing, budding, flowering, fruiting, senescence and dormancy) were chosen in the threatened terrestrial orchid Caladenia formosa G.W.Carr to optimise isolation of effective fungi. Loose (undigested) pelotons were observed by scanning electron microscopy in the old tuber and collar, suggesting a role in infection of new tissue. In collars collected at early life stages (leafing, budding, flowering), pelotons had loosely coiled hyphae that were uniformly fine (1-2 µm diameter), with or without monilioid cells. In collars collected from older life stages (fruiting, senescence), pelotons had increasing proportions (up to 94%) of clumped fine hyphae. Coarser hyphae (4-6 µm diameter) were also present in the fruiting stage in one year. Only fungi isolated from single pelotons in collars of early life stages (leafing, budding, flowering) had fine hyphae with monilioid cells and induced seed germination (to green leaf production). Sectioned protocorms had pelotons of fine, loosely coiled hyphae with monilioid cells, as in field-collected material from early life stages. This suggests that the most effective fungi for conservation of this orchid are likely to be isolated from pelotons of loose fine hyphae with monilioid cells from leafing to flowering stages. B T 0 3 0 9 9 I s o l a t i o n o f m y c o r r h i z a l f u n g i i n C a l a d e n i a f o r m o s a T . T . H u y n h e t a l .

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.

The first axenic synthesis of morphologically typical ericoid mycorrhizas of the Epacridaceae has been achieved in micropropagated Epacris impressa Labill. with eight fungi isolated from roots of two epacrid species, E. impressa and Astroloma pinifolium (R.Br.) Benth. Mycorrhizal synthesis has also been achieved between E. impressa and both Hymenoscyphus ericae (Read) Korf and Kernan and Oidiodendron griseum Robak, recognized endophytes of Ericaceae, suggesting that the endophytes of the Epacridaceae and Ericaceae are capable of cross-infection. Infection rate of epidermal cells on hair roots varied from 3-77 % infection and the density of hyphal coils varied widely. This synthesis makes possible studies of the roles of these endophytes in the Epacridaceae and comparison with their roles in the Ericaceae.