Mycorrhizal associations and phylogenetic relationships of South-east Queensland Bulbophyllum orchids

Throughout their life cycles orchids are reliant on inorganic nutrients provided by mutualistic orchid mycorrhizal fungi (OMF), to which the plants allocate sugars produced in photosynthesis. In this study, fungi were isolated from the roots of the native orchids B. exiguum, B. bracteatum, B. minutissimum, B. elisae and B. shepherdii at 7 sites in south-east Queensland to determine whether they exhibit specificity with regard to their fungal partners. Fungi were identified based on internal transcribed spacer (ITS) gene sequences, and analysis of orchid RuBisCO large subunit (rbcL) gene sequences was also performed to ascertain orchid phylogenetic relationships. Additionally, symbiotic seed germination of B. exiguum was tested using 4 fungal inocula. In all, 90 fungal isolates were obtained. Molecular identification revealed a diversity of putatively mycorrhizal fungi from the OMF genera Tulasnella, Serendipita and Ceratobasidium, and dark septate endophytes (DSEs) from the ascomycete order Helotiales. Significantly, 3 orchid spp. (B. exiguum, B. bracteatum and B. elisae) across 3 sites were found to harbour a single Tulasnella sp. that is likely new to science. This indicated narrow OMF specificity and suggested that these orchids may belong to a common sub-clade within Bulbophyllum, an observation supported by phylogenetic analysis of rbcL genes and by taxonomic reassignments that have been proposed based solely on morphological features. B. exiguum were found to harbour an undescribed Serendipita sp. that warrants investigation as a potential agricultural inoculum. B. shepherdii harboured a Ceratobasidium sp. previously found in Norway, but the plant sampled in this study had been relocated from nearby woodland and thus may not usually associate with this OMF. Isolation of DSE helotialean fungi with highest BLAST matches to ericoid mycorrhizal (ErM) sequences pointed to a possible OMF role for these isolates, however further confirmation is needed to establish whether intracellular nutrient-exchange structures are present. The presence of ErM-like fungi associated with orchids supports recent work suggesting a blurring of functional boundaries between mycorrhizal types. B. exiguum seed germination experiments were impeded by overgrowth of fungal contaminants, which were likely endophytes from seed pod tissue, and by poorly-developed seeds that may have resulted from inbreeding. Future studies with seed germination protocols optimised for very small pods are needed to ascertain whether fungal symbionts can stimulate germination in these Bulbophyllum spp. These results provide evidence for narrow OMF specificity for Tulasnella in some SE Queensland Bulbophyllum spp. and further raise the intriguing possibility of DSE forming mycorrhizas with orchids.