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.

By conducting reintroductions of the endangered terrestrial orchid Diuris fragrantissima D.L.Jones & M.A.Clem. we compared planting at the following three stages of the natural perennial growth cycle: as actively growing symbiotic plants in spring and autumn and as dormant tubers in summer. Plants reintroduced in spring and autumn were incorporated into randomised treatments involving soil aeration and addition of a mycorrhizal fungus. The addition of a mycorrhizal fungus and soil aeration together significantly increased survival and flowering of plants reintroduced in spring, whereas they had no significant effect on plants reintroduced in autumn. Addition of a fungus without soil aeration did not improve plant survival or flowering. Reintroducing actively growing plants was more successful than reintroducing dormant tubers, with 32.5% and 29.1% plants (reintroduced in spring and autumn, respectively) and 11.0% of tubers persisting after 4 years. Although survival of reintroduced plants declined at a rate of 16.9% per year for 4 years following reintroduction, survival of remnant plants remained relatively constant, ranging from 80.0 to 93.0%. Tuber size was positively correlated with survival and flowering of reintroduced plants for 2 years following reintroduction. A general trend was observed towards the increased likelihood of re-emergence and flowering of plants that flowered in previous seasons.

Diuris fragrantissima D. L. Jones et M. A. Clem. is a perennial terrestrial orchid endemic to the state of Victoria, Australia. The species is listed as Critically Endangered in Victoria under the criteria of the International Union for the Conservation of Nature and Natural Resources (IUCN), having suf- fered a severe population decline since the 1930s, largely attributable to habitat destruction for agricul- tural and urban development.

Taxa within the Diuris punctata species complex exhibit high levels of variation at both species and population level. Morphometric data collected in situ were used to investigate species boundaries of four Victorian Diuris species within the Diuris punctata species complex. Morphological characters and taxonomic groups identified in the present study were compared to those described under the current taxonomic treatment. Sixty-five multistate and continuous characters, including seven vegetative and 58 floral characters, were measured in situ across the range of each species within Victoria. The importance of flower colour in distinguishing taxa was highlighted but characters used were generally indiscrete. Certain characters used in current taxonomic descriptions, e.g. floral fragrance, were found to be uninformative. D. fragrantissima was confirmed as a separate taxon within the D. punctata group, justifying its recognition as a unique entity for conservation. Clustering of D. daltonii within D. punctata suggests that the recent elevation of the D. punctata var. daltonii to species level is not justified. The in situ measurement of morphological characters made it possible to incorporate sufficient sampling to encompass intra-specific and intra-population variation and is a feasible method to overcome sampling limitations encountered when herbarium specimens and limited destructive sampling are used.