A project undertaken at the Royal Botanic Gardens Victoria in collaboration with the Australian National University, supervised by Noushka Reiter
Prasophyllum is a genus of around 140 species of terrestrial orchids, most of which occur in southern and eastern Australia with four species occurring in New Zealand. The ecology and conservation of Prasophyllum is of international significance with 39 species threatened with extinction. All orchids are completely dependent on orchid mycorrhizal fungi (OMF) for germination in the wild and continue to associate with OMF as adults. To adequately conserve threatened Prasophyllum species using ex-situ conservation methods (propagation and translocation back into the wild), a thorough understanding is required of the associated OMF. In particular, information is required on the identity and diversity of the OMF involved in the germination of Prasophyllum in nature and the OMF that associate with adult plants, as well as the optimal time of year at which to isolate OMF from adult plants. Therefore using both culture-dependent and culture-independent sequencing methods we aim to (1) determine the extent of temporal change in OMF communities in Prasophyllum during the course of a six month growing season from leaf emergence to flowering; (2) identify OMF responsible for germination in-situ using seed packets and ex-situ using germination trials; and, (3) identify the time of year when OMF responsible for effective ex-situ cultivation are present and most abundant in the roots of adult plants.
Figure 1. Prasophyhllum spicatum – a threatened orchid from Victoria’s coastal fringe
We studied mycorrhizal associations in adult plants and protocorms of Prasophyllum frenchii, P. lindleyanum and P. sp. aff. validum at four periods in a single growing season in south-east Australia. We buried seed packets to study the mycorrhizal associations of germinating protocorms. Culture dependent and independent sequencing methods were used across the Internal Transcribed Spacer and nuclear Large Sub Unit loci. Germination trials were used to test the mycorrhizal status of Operational Taxonomic Units (OTUs).
Prasophyllum frenchii had highly specific mycorrhizal associations with a single Ceratobasidium OTU, while P. lindleyanum and P. sp. aff. validum had more generalist associations with seven and six OTUs respectively. However, there was no evidence of a significant turnover of OMF communities in Prasophyllum during a growing season. Patterns of OMF associations in protocorms versus adult plants were different among all three species, with protocorms of P. lindleyanum displaying highly specific associations with a single OTU, P. frenchii displaying specific associations with two OTUs, and P. sp. aff_. validum_ displaying highly generalist associations with six OTUs.
Figure 1. Prasophyhllum spicatum – a threatened orchid from Victoria’s coastal fringe
Figure 2. Prasophyllum litorale – known only from one National Park in Victoria’s southwest
Figure 3. Prasophyllum lindleyanum – one of the most widespread of all Leek Orchids
Figure 4. Prasophyllum frenchii root – fungi live in the plants’ cells inside the root
Given the diversity of OTUs found associating with two of the three study species, we recommend that mycorrhizal fungi are sampled from across a population and sequenced before germination trials to ensure the maximum diversity is captured in ex-situ germinations. In addition, where feasible and the watering techniques used in this study are possible, seed packets should be used where feasible to capture OMF in protocorms, alongside isolation from protocorms from seed packets to ensure OMF that are only present in protocorms and not adult plants are isolated for use in conservation programs. This is particularly important because we have demonstrated that OTUs capable of supporting germination in the laboratory do not necessarily associate with germinating protocorms in the wild, and even Prasophyllum species with generalist mycorrhizal associations can have specific mycorrhizal requirements during germination. As there was no temporal turnover in OMF communities, sampling of OMF can be undertaken at any point during the season for Prasophyllum.