Mycorrhizal specificity of Diuris fragrantissima (Orchidaceae) and persistence in a reintroduced population

This study investigated the diversity and specificity of mycorrhizal fungi associated with five Diuris (Orchidaceae) taxa in south-eastern Australia, as part of a reintroduction program for the endangered species Diuris fragrantissima. We compared fungi isolated from D. fragrantissima occurring naturally in the only remaining population with those from artificially cultivated plants and reintroduced plants 18 months after planting in a new field site west of Melbourne. Genetic similarity of nuclear internal transcribed spacer and nuclear large subunit DNA sequences showed that Diuris taxa associate with a narrow taxonomic range of fungi within the cosmopolitan family Tulasnellaceae in the Rhizoctonia alliance. All fungal isolates induced host seed germination and hence were considered mycorrhizal. Fungal isolates from naturally occurring D. fragrantissima plants showed a higher level of genetic similarity than fungi isolated from cultivated plants. This observation suggests that, historically, the species may have associated with a more genetically variable range of Tulasnella fungi. Artificially cultivated D. fragrantissima were propagated aseptically from seed and spontaneously formed mycorrhizal associations within 6 months of transfer to potting media. Wild collected D. fragrantissima plants maintained in cultivation for over 30 years were found to contain mycorrhizal fungi similar to those isolated from naturally occurring plants in 2004-2006. Mycorrhizal associations in artificially cultivated D. fragrantissima were present in 18 randomly sampled plants 18 months after reintroduction. Further, associations formed between several reintroduced plants and a fungus concurrently inoculated into site soil. We propose that future orchid reintroductions may benefit from the concurrent addition of suitable mycorrhizal fungi to site soil. Maintenance of orchid mycorrhizal relationships after reintroduction is essential to improve long-term viability of reintroduced populations.