Conservation of a critically endangered orchid Drakaea elastica Lindl. in the context of nutritional requirements and saprophytic competency of the mycorrhizal fungus and its propagation Siti Nurfadilah This thesis is presented for the degree of Master The University of Western Australia School of Plant Biology 2010 ABSTRACT Drakaea elastica is a critically endangered orchid species that is a habitat specialist of nutrient deficient sandy soil and is dependent on a slow growing mycorrhizal fungus for its nutrient supply in its entire life cycle. In the conservation of this species, understanding the biology and ecology of the mycorrhizal fungus, especially the critical aspects determining the growth and the survival of the mycorrhizal fungus, is essential. The aim of this research is to investigate nutritional requirements, factors limiting the growth and survival, and saprophytic competency of the slow growing mycorrhizal fungus of Drakaea elastica in terms of its capacity to utilize a variety of nutrient sources (C, N, and P) relative to other sympatric faster growing orchid mycorrhizal fungi. The assessment of the capacity of Drakaea elastica mycorrhizal fungus in axenic liquid media containing single nutrient sources showed that it utilised a variety of C, N, and P sources for its growth. A wide range of C sources that Drakaea elastica mycorrhizal fungus required for its growth encompassed glucose and mannose (monosaccharide), cellobiose (disaccharide), cellulose, pectin, starch, and xylan (polysaccharides). The mycorrhizal fungus did not grow on the other C substrates, galactose, rhamnose, arabinose (monosaccharide) and tannic acid (polysaccharides). Ammonium, a variety of amino acids (aspartic acid, glutamic acid, glutamine, asparagine, alanine, arginine, and glycine), and protein BSA are essential N sources required for the growth of Drakaea elastica mycorrhizal fungus. Drakaea elastica did not utilize nitrate, histidine, and proline as the N source for its growth. In terms of the requirement of P which is critically needed, Drakaea elastica mycorrhizal fungus utilised all forms of P sources including inorganic P (orthophosphate) and organic P (phytic acid and DNA). This investigation of nutritional requirements of Drakaea elastica is important to understand which nutrient sources required by Drakaea elastica mycorrhizal fungus for its robust growth. These data can also be a basis for an understanding of how Drakaea elastica mycorrhizal fungus utilizes different nutrient sources. Drakaea elastica mycorrhizal fungus utilised the same C, N, and P sources with the sympatric faster growing orchid mycorrhizal fungi suggesting that it has to compete for the same nutrient sources. With its slow growing characteristic, Drakaea elastica mycorrhizal fungus presumably has lower competency to access nutrient sources relative to faster growing fungi. This may explain the specialisation of Drakaea elastica mycorrhizal fungus in nutrient deficient sandy soil to avoid competition with many other fungi. The growth and survival of Drakaea elastica mycorrhizal fungus was limited by the availability of an external source of the organic compounds thiamine and PABA. The ability of Drakaea elastica mycorrhizal fungus to utilize a wide range of C, N, and P sources was determined by the availability of these organic compounds which are important in the major metabolism for growth. Without these organic compounds, Drakaea elastica mycorrhizal fungus grew poorly on most C, N, and P sources. The findings of this study can be incorporated into the biology and ecology of Drakaea elastica mycorrhizal fungus which is important in the conservation of Drakaea elastica. In the propagation of Drakaea elastica, understanding the biology and ecology for the seedlings development and establishment is important to optimise propagation of this critically endangered orchid species. Table of contents Abstract i Table of contents iii Acknowledgements v Declaration of candidate contribution vi Chapter I General introduction 1 1.1. Current status of Drakaea elastica 2 1.2. Distribution of Drakaea elastica 2 1.3. Taxonomy of Drakaea elastica 3 1.4. Biology and ecology of Drakaea elastica 4 1.4.1. Association with a specific pollinator 4 1.4.2. Association with mycorrhizal fungi 5 1.4.3. Habitat specialization of Drakaea elastica 7 1.5. Nutrient sources in orchid habitat 7 1.6. Utilization of nutrient sources by orchid mycorrhizal fungi 11 1.6.1. Media used to asses the capability to utilize nutrient sources 12 1.6.2. The assessment of the capacity to utilize nutrient sources 12 1.7. Saprophytic competency of Drakaea elastica mycorrhizal fungus 14 1.8. Propagation of Drakaea elastica 15 1.9. Project aims 15 Chapter II The capacity of Drakaea elastica mycorrhizal fungus to utilize carbon sources 19 2.1. Introduction 19 2.2. Methods 21 2.3. Results 24 2.4. Discussion 30 Chapter III The capacity of Drakaea elastica mycorrhizal fungus to utilize nitrogen sources 40 3.1. Introduction 40 3.2. Methods 42 3.3. Results 44 3.4. Discussion 48 Chapter IV The capacity of Drakaea elastica mycorrhizal fungus to utilize phosphorus sources 53 4.1. Introduction 53 4.2. Methods 54 4.3. Results 56 4.4. Discussion 61 Chapter V Optimization propagation of Drakaea elastica Lindl. 65 5.1. Introduction 65 5.2. Methods 65 5.3. Results 70 5.4. Discussion 75 Chapter VI General Discussion 78 6.1. Introduction 78 6.2. Nutritional requireents of Drakaea elastica mycorrhizal fungus 79 6.3. Factors limiting the growth and survival of Drakaea elastica mycorrhizal fungus 81 6.4. Saprophytic competency of Drakaea elastica mycorrhizal fungus 83 6.5. Propagation of Drakaea elastica 84 6.6. Conclusion 85 References 86 Appendix 1 100 Appendix 2 102 ACKNOWLEDGEMENTS I am very grateful to my supervisors, Prof. Kingsley W. Dixon, Dr. Nigel D. Swarts, and Dr. Nigel J. Merritt for their excellent guidance, support, encouragement, and advice. I also wish to thank the orchid conservation group of Kings Park Botanic Gardens and Parks Authority, Dr. Ryan Phillips, Dr. Belinda Newman, and Myles Menz for their helps during my study in Perth. I am grateful to Dr. Eric Bunn, Keran Keys, and Beorn Harris that provided assistance and advice in the laboratory. I also would like to thank friends at Kings Park: Liann Smithson, Emma Dalziell, Clare White, Alison Ritchie, Adam Cross, Martha, Wolfgang Lewandrowski, Akshay Menon, Brinn, and other students doing their projects at Kings Park for the friendship during my study in Perth. Nedlanders: Wara, Nisa, mbak Yanti, Ing, Dewi, Heru Wibowo, Emielda Yusiharini, and other Indonesian students in Perth, Laily, Ristin, Tari, etc. thank you so much for the support.Also to ADS 9M friends; Umam, Zahid, Asep, Anto, Irwan, Mufid, Lora, Erna, Ekha, with you I learn how to be brave to reach dreams. I would like to thank The Government of Australia that awarded the scholarship that made it possible for me to study orhid conservation in Australia. My sincerest thanks for my family for their endless support and prayers for me. CHAPTER I General Introduction The Orchidaceae is the largest plant family, comprising approximately 30,000 species classified into five subfamilies and containing 870 genera (Dressler, 1993). Orchids are cosmopolitan, occupying a diverse array of substrates and being found across continents in various climates and altitudes (Arditti, 1992). Despite the great diversity of orchids, many are under threat of extinction (IUCN, 2001). This is thought to be related to the unique biological characteristics of orchids, particularly their high dependency on the other organisms (mycorrhizal fungi and pollinators) to complete their life cycle. External threatening processes also place severe pressure on orchid habitat. Conservation programs are needed to prevent orchids from becoming extinct and the knowledge of the biology and ecology of orchids is important for the success of orchid conservation programs (Swarts and Dixon, 2009; Brundrett, 2007). This thesis aims to increase the understanding of the biology and ecology of Drakaea elastica Lindl., a critically endangered terrestrial orchid endemic to Western Australia that is specialized on a nutrient deficient sandy soil habitat (Hopper and Brown, 2007). In its entire life cycle, Drakaea elastica relies heavily on a mycorrhizal fungus which is typically slow growing (in culture, relative to other terrestrial orchid mycorrhizal fungi) as the nutrient supplier (Ramsay et al, 1986). Thus, understanding factors determining the growth and the survival of the mycorrhizal fungus associate is substantial in the conservation programs of Drakaea elastica. The focal point of this study is to investigate the biological and ecological aspects influencing the fitness and the survival of Drakaea elastica mycorrhizal fungus, including its nutritional requirement, factors limiting its growth and survival, and its saprophytic competency in terms of its capacity to access a variety of nutrients (carbon, nitrogen, and phosphorus sources) relative to faster growing fungi from sympatric orchids. Furthermore, this study also investigated the biological and ecological requirements for seed germination and seedling development of Drakaea elastica for the optimization of propagation of this critically endangered orchid. 1 1.1. Current Status of Drakaea elastica Drakaea is a terrestrial genus containing 10 other taxa endemic to the South-west Australian Floristic Region. Five Drakaea species are threatened with extinction and are legally protected