Conservation of Prasophyllum - understanding orchid mycorrhizal fungi to save threatened orchids from extinction

Abstract

The Orchidaceae are the world's second largest plant family and have a high proportion of threatened species. For some threatened orchids, ex-situ conservation is one of the few options for recovery. However, many orchids are difficult to grow ex situ, an issue complicated by their reliance on orchid mycorrhizal fungi (OMF). Prasophyllum is a large genus of orchids from Australia, with one third of species listed as threatened. However, poor seed germination has consistently affected ex situ conservation efforts. Therefore, the aim of this PhD was to improve ex situ conservation prospects for Prasophyllum by exploring their relationship with OMF, seed viability and propagation methods.

Chapter 1 (Annals of Botany, in press) studied the continental-scale diversity and distribution of Ceratobasidium OMF. As Prasophyllum associate primarily with Ceratobasidium OMF, we reviewed published Australian Ceratobasidium OMF data, unpublished Ceratobasidium OMF sequences, and a continent-wide soil sequencing project. Comparing the distribution of OMF isolated from orchids with soil data showed that Ceratobasidium OMF with cross-continental distributions are common, often have geographic ranges exceeding their host orchids, and can tolerate a wide range of environmental conditions.

Chapter 2 studied the seasonal dynamics of Ceratobasidium OMF in adult orchids and protocorms. Three Prasophyllum species had different patterns of mycorrhizal associations, ranging from generalist to specific in both adults and protocorms. However, there was no evidence of a seasonal turnover of OMF communities, possibly due to moderate climatic conditions or their association with a single OMF genus. The three orchids did not share OMF, suggesting phylogenetic conservatism or habitat may influence OMF associations.

Chapter 3 examined the role of phylogenetic conservatism and habitat in determining mycorrhizal associations in Prasophyllum. We undertook a broad-scale sampling across the Prasophyllum genus, sampling 41 species across nine different sub-generic groups and six habitat types. We showed that both habitat and host orchid taxonomy are correlated with OMF associations in Prasophyllum.

Chapter 4 investigated the effect of pollination on reproductive output in Prasophyllum. We tested the effect of pollination treatment (self, cross, natural, none) on three species using tetrazolium staining and asymbiotic germination. Tetrazolium staining showed naturally-pollinated seed had low to moderate viability, depending on the colour of stain scored as viable. However, asymbiotic percentage germination rates were low, and although high viability from staining was correlated with germination, the results of staining could not predict germination rates. Seed viability was not sufficiently low to explain difficulties with germination in Prasophyllum.

Chapter 5 developed an asymbiotic germination protocol for Prasophyllum. A broad-spectrum experiment identified a new, low macronutrient asymbiotic medium suitable for the germination of P. frenchii, a counterintuitive finding for an orchid found in fertile soils. However, a previously untested commercial medium supported even higher germination rates, and several other media were optimal for supporting seedling development, although transferral to the nursery caused high mortality.

Chapter 6 studied the effect of nutrient media on symbiotic germination of P. frenchii, as propagation with OMF is preferrable for conservation. We observed germination on media high in macronutrients or with an insoluble carbon source, but not on standard oatmeal agar. We trialled three isolates that had similar ITS sequences (>99%), and the optimal medium was different for each, suggesting isolate-level variation in these fungi. The development of effective symbiotic germination has enabled the implementation of ex situ conservation actions for Prasophyllum. Show more