Cassandra B. Mclean

Functional and genetic diversity of mycorrhizal fungi from single plants of Caladenia formosa (Orchidaceae).

BACKGROUND AND AIMS Mycorrhizal associations are essential to the plant kingdom. The largest flowering plant family, the Orchidaceae, relies on mycorrhizal fungi for germination, growth and survival. Evidence suggests varying degrees of fungal-host specificity based on a single fungal isolate from a single plant. This paper shows for the first time the diversity of endophytes colonizing in a single plant over consecutive years and the functional significance of this diversity. METHODS Stem-collars of Caladenia formosa were collected in different seasons and years. Mycorrhizal fungi isolated were tested for their efficacy to induce leafing and genetically determined using ITS-RFLP and sequencing. RESULTS Multiple mycorrhizal fungi were repeatedly isolated from a single collar that displayed varying effectiveness in germination percentages and adult leaf length. Additional factors contributed to the isolation of effective mycorrhizal fungi; fungal collection season, year of collection and individual isolates. Surface sterilization only improved the number of isolated mycorrhizal fungi. Dual inoculation did not increase germination. All 59 mycorrhizal fungi effective in germinating seed belonged to one clearly defined ITS (internal transcribed spacer) clade and clustered close to Sebacina vermifera (79-89 % homology). Isolates resulting in the greatest germination were not necessarily those resulting in the greatest survival and growth 1 year after germination. CONCLUSION Single orchid plants contained multiple mycorrhizal fungal strains of one species that had diverse functional differences. These results suggest that our current knowledge of fungal-host specificity may be incomplete due to experimental and analytical limitations. It also suggests that the long-term effectiveness of a mycorrhizal fungus or fungi could only be found by germination and longer-term growth tests rather than genetically.

Effect of developmental stage and peloton morphology on success in isolation of mycorrhizal fungi in Caladenia formosa (Orchidaceae)

Six developmental stages (leafing, budding, flowering, fruiting, senescence and dormancy) were chosen in the threatened terrestrial orchid Caladenia formosa G.W.Carr to optimise isolation of effective fungi. Loose (undigested) pelotons were observed by scanning electron microscopy in the old tuber and collar, suggesting a role in infection of new tissue. In collars collected at early life stages (leafing, budding, flowering), pelotons had loosely coiled hyphae that were uniformly fine (1–2 μm diameter), with or without monilioid cells. In collars collected from older life stages (fruiting, senescence), pelotons had increasing proportions (up to 94%) of clumped fine hyphae. Coarser hyphae (4–6 μm diameter) were also present in the fruiting stage in one year. Only fungi isolated from single pelotons in collars of early life stages (leafing, budding, flowering) had fine hyphae with monilioid cells and induced seed germination (to green leaf production). Sectioned protocorms had pelotons of fine, loosely coiled hyphae with monilioid cells, as in field-collected material from early life stages. This suggests that the most effective fungi for conservation of this orchid are likely to be isolated from pelotons of loose fine hyphae with monilioid cells from leafing to flowering stages.

Taxonomic and functional characterisation of fungi from the Sebacina vermifera complex from common and rare orchids in the genus Caladenia

The terrestrial orchid genus Caladenia contains many species which are threatened with extinction. They have highly specific associations with Sebacina vermifera and closely related fungi, and conservation of these terrestrial orchids, in part, relies on symbiotic propagation to produce plants for reintroduction and ex situ conservation collections. However, little is known of the diversity of mycorrhizal fungi associating with natural populations. Here, restriction fragment polymorphism analysis, internal transcribed spacer and nuclear large subunit sequencing and symbiotic seed germination were used to investigate the taxonomic and functional diversity of fungal isolates from single populations of six endangered Caladenia species and one common species across the same biogeographic range. Fifty-nine fungal isolates were collected for investigation including ten isolates from the six endangered species Caladenia audasii, Caladenia amoena, Caladenia sp. aff. fragrantissima (Central Victoria), Caladenia sp. aff. patersonii, Caladenia rosella and Caladenia orientalis and 49 isolates from six populations of the common species Caladenia tentaculata. While the common species associated with three distinct S. vermifera-like taxa, the six endangered species were restricted to one of these fungal taxa. No direct relationship between the taxonomic identity of the fungi and their ability to stimulate seed germination was observed; however, the majority of the fungi isolated from the Caladenia species were capable of germinating seed in vitro, indicating their mycorrhizal status and potential for symbiotic propagation in conservation programmes.

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.

Planting conditions improve translocation success of the endangered terrestrial orchid Diuris fragrantissima (Orchidaceae)

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.

The effect of soil microflora on the survival of cuttings of Epacris impressa

A study was conducted to test the effect of inoculation with soil containing mycorrhizae propagules and other soil micro organisms on survival of stem cuttings of Epacris impressa. With non-sterile soil present, the cuttings were mycorrhizal, more survived and the survivors were healthier, with more flowers and roots, although root area was not increased. Soil inoculum thus increased the success of propagation of Epacris impressa for conservation purposes and in commercial horticulture.

In situ morphometric study of the Diuris punctata species complex (Orchidaceae), with implications for conservation

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.