Propagation and biology of Arachnorchis (Orchidacae) and their mycorrhizal fungi

Raleigh, R 2005, Propagation and biology of Arachnorchis (Orchidacae) and their mycorrhizal fungi, Doctor of Philosophy (PhD), Biotechnology and Environmental Biology, RMIT University.

Document type: Thesis
Collection: Theses

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Title Propagation and biology of Arachnorchis (Orchidacae) and their mycorrhizal fungi
Author(s) Raleigh, R
Year 2005
Abstract Terrestrial orchids make up one of the most threatened groups of plants in Australia and the genus Arachnorchis is listed as the fourth most threatened. The process of propagation and re-introduction of terrestrial orchid plants to the wild has proven difficult, and so far, nearly impossible for some species. This may be partly because terrestrial orchids form complex relationships with mycorrhizal fungi and in genera like Arachnorchis the dependency on the fungus appears acute.

Arachnorchis has long been considered by amateur growers of terrestrial orchids as one of the most difficult groups to propagate and maintain in cultivation. This lack of knowledge on how to grow Arachnorchis species hinders attempts made by conservation authorities to supplement threatened wild populations in order to achieve a more sustainable future for those species. Natural pollination was absent, but artificial pollination achieved 100% capsule production. Individuals were self-fertile, although seed viability was greater for cross-pollinated samples. This study attempted to track the fate of as many Arachnorchis species as possible from germination through to deflasking and re-emergence, and so destructive and potentially destructive measurements at earlier stages were avoided. This thesis examines germination and subsequent growth of up to eight species of Arachnorchis, but concentrated on A. phaeoclavia, A. tentaculata, A. fulva, A. robinsonii and A. venusta. Two of these are common species: A. pha eoclavia and A. tentaculata, and three carried a threatened classification of "rare" or " endangered": A. fulva, A. robinsonii and A. venusta.

This study monitored the fate of individuals of the endangered A. fulva in the field and showed that large reproductive plants re-emerged and flowered each year, whereas smaller individuals might be absent in one or more years and were less likely to flower. Germination of all species concentrated on using symbiotic culture (using mycorrhizal fungi), since germination is known to be more rapid, resulting in healthier, more robust seedlings than when plants are grown asymbiotically. Tests using A. fulva and A. venusta, two threatened species, showed similar viability to A. tentaculata and A. phaeoclavia, more common species. Germination was maximised by examining the viability of seeds before and after treatment with surface-sterilising solutions required for aseptic culture. The highest levels of germination, with limited contamination, were achieved using 0.5% available chlorine for 3 minutes.

The most effective fungal isolates (>65% germination) were obtained from common species like A. phaeoclavia and A. tentaculata, but there was no correlation between germination and time of year or life stage of the orchid. Collar collection was shown to be non-fatal to robust orchid plants, with large reproductive individuals (at the time of collar collection) re-emerging in the next year and producing a flower bud. Collar collection from small, weedy individuals could be fatal to the plant and isolation of an effective fungus was unlikely. Cross-inoculating seeds with fungi isolated from a different orchid species was not recommended, since the symbiosis failed in all experiments, as late as Stage 4 protocorm development.

A range of substrates was used to produce strong seedlings capable of surviving the transfer to nursery conditions with minimal loss. More than 81% of seedlings survived deflasking from non-agar substrates, while the best result from agar was 55%. Some substrates reduced the time involved from seed to plants in the field to as little as 4 months, but aftercare became critical. Sucrose promoted tuberisation, but led to tuber deaths during dormancy. Potting mixes were tested in the nursery and a free-draining loam mix based on a mix used by the Australasian Native Orchid Society was the best medium for deflasking of seedlings. Watering during dormancy should be avoided. The choice of propagule for re-introduction was examined and the best survival to re-emergence was obtained by planting out actively growing seedlings in autumn.

Identification of cultures using classical morphology grouped cultures as belonging to the form-genera Epulorhiza and Moniliopsis and suggested that most cultures contained more than one fungus. Identification of the most useful fungal cultures was attempted using molecular techniques such as sequencing the ITS region and mitochondrial DNA. One effective culture, CALAPHAER18 SHTX (cultured from a single monilioid cell) was identified as Serendipita vermifera (Oberwinkler) Roberts. All other cultures tested were mixtures of fungi. The use of specific primers designed to amplify a sequence present in the identified isolate (CALAPHAER18 SHTX) showed that nine mixed cultures also contained a fungus most closely related to Serendipita vermifera. Specific primers also showed that Rhizoctonia solani was not present in any of the 10 isolates from Arachnorchis plants. The molecular work showed that, although the sequenced endophytes from Arachnorchis were all most closely related to Serendipita vermifera, three dist inct groups of fungi were present and these associated with separate species of Arachnorchis.

Future work with Arachnorchis species will require the isolation of single fungus cultures and further examination of the development of the orchid plant. In particular, the process of tuberisation and growth in vitro on various non-agar substrates should be investigated further.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Biotechnology and Environmental Biology
Keyword(s) Arachnorchis
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