Exploring mycorrhizal associations of two terrestrial orchids Pterostylis agathicola and Danhatchia australis

Abstract

Orchid mycorrhizal connections are as essential for orchids as breath is for us. These mycorrhizal connections are seen in both chlorophyllous and achlorophyllous orchids. The fungi and the orchid form a connection, which may or may not be specific to the orchid. Some orchids in Aotearoa follow the distribution patterns of an ecosystem dominated by a specific tree. Are the orchid mycorrhizae the factor in an ecosystem that specifically makes these orchids germinate and thrive only there? I tested this on chlorophyllous and achlorophyllous orchids in New Zealand because one has a lesser dependence on mycorrhizal fungus than the other throughout its lifetime. Pterostylis agathicola follows the distribution of Agathis australis, while Danhatchia australis closely follows the distribution of Beilschmiedia tarairi and Rhopalostylis sapida. I examined the co-occurrence of the orchids and co-locating trees, and the fungal communities of the orchids. In the field, I examined the spatial locations of these orchids in relation to that of their co-occurring tree species, and other features of their environments. I also cultured fungi from pelotons present in the orchid roots on agar media. Once I had achieved pure fungal cultures, these were characterized using Sanger sequencing. I selected some of these fungal cultures and used them in an attempt to germinate the orchid seeds. In addition to Sanger sequencing, I performed metagenomic analysis of the internal fungal microbiome within roots of both the orchids. This analysis returned a wider range of the fungal community present in the roots of each species than did the culturing. I only observed Pterostylis agathicola growing specifically around Agathis australis – dominated ecosystems. The closest tree to several Danhatchia australis was not always B. tarairi or R. sapida, although these were still in the vicinity. This indicates that further evidence is necessary to confirm which tree species D. australis might be associating with. The most dominant species from the culturing and the most abundant sequences (reads) in my metagenomic analyses for P. agathicola was from the Ceratobasidiaceae family. The Sanger sequences of cultured fungi from D. australis were of different families than seen for other similar orchids. Seed germination cultures using Ceratobasidiaceae for both the orchids produced protocorm-like structures. The fungal communities of Pterostylis agathicola was as expected but that of Danhatchia australis indicates there is more to be learnt to uncover their connection to the surrounding ecosystems.