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Project: 314 / 2017
Title: Oh brother, where art thou? Distribution patterns and characterization of Tulasnella spp. associated with two related Australian orchid species, Diuris fragrantissima and Diuris punctata.

Applicants: Dr Eleonora Egidi and Dr Ashley Franks
Institution:. Department of Physiology Anatomy and Microbiology, School Life Sciences, La Trobe University Bundoora. Victoria 

Orchid reintroduction requires the presence of suitable mycorrhizal fungi to ensure long-term persistence and survival in situ. Tulasnella spp. are the main fungal symbionts of Australian Diuris orchids, including the highly endangered Victorian orchid Diuris fragrantissima and the not-endangered related species Diuris punctata.

Our recent studies have shown a limited occurrence of the fungus in the D. fragrantissima native site, suggesting that Diuris mycorrhizae abundance may be limited by site-specific environmental features. However, the scarce information available on Diuris-associated Tulasnella spp. biology and ecology hampers our ability to draw significant conclusions on the factors constraining the mycorrhizal fungus in natural conditions and, ultimately, the persistence of reintroduced orchids in situ.

In this project, we propose two main objectives: firstly, to improve our understanding of Tulasnella spp. biology, ecology and taxonomy by investigating morphology, genetic diversity and growth characteristics of fungi isolated from highly endangered D. fragrantissima and not-endangered D. punctata root systems; secondly, to identify the environmental factors influencing Tulasnella spp. occurrence in situ. Disentangling the factors which control Diuris-associated mycorrhizal fungi distribution will help to identify the characteristics of microsites that provide favourable abiotic and biotic conditions for seeds reintroduction. Ultimately, this information can be used by those managing the recovery of this critically endangered species to tailor more specific reintroduction programs.


Research outcomes

We investigated morphology, phylogenetic variability, and growth of several fungal isolates associated with a rare and a widespread orchid belonging to the same genus (D. punctata and D. fragrantissima). Morphological examinations yielded the observation of many vegetative and asexual characters, however no sexual spores. Sequence analysis of fungal barcoding and multi-locus markers identified clonal species within the isolates, and determined narrow monophyly among fungi from both orchid species. Finally, growth studies led to the development of a lysate–protein concentration method for assessing fungal growth. This method was later used to reliably determine increased growth in the fungal culture isolated from the common orchid species.

The in situ sampling targeted a site in the Mornington Peninsula rich in orchid species, including the D. fragrantissima-sister orchid D. punctata. Following DNA extraction and NGS DNA sequencing, the fungal community associated with bulk and orchid rhizosphere soil was characterised. However, no Tulasnella species were retrieved with the molecular method implemented in this study. As the suitability of the NGS approach to identify mycorrhizal fungi in soil has been tested and demonstrated in our previous studies (Egidi et al. 2018), the lack of retrieval of Tulasnella sp. sequences may be due to the seasonality of the occurrence of mycorrhizal fungi in soil and provides to option in the future to test seasonal suitability for reintroduction.

Completed Honours research thesis:
K. Aedo: “Characterising Tulasnella sp. mycorrhizal fungus isolates from two Australian Diuris species: fungal roles in endangered orchid conservation”, La Trobe University