View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ResearchArchive at Victoria University of Wellington The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation By Jonathan Frericks A thesis submitted to the Victoria University of Wellington in fulfilment of the requirement for the degree of Masters in Ecology and Biodiversity Te Whare Wānanga o te Ūpoko o te Ika a Māui November 2014 II Abstract Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari- Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae- zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL- trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian- Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes. III Acknowledgements I would like to thank Carlos Lehnebach for his incredible support, time, and enthusiasm for this project. I have really appreciated your supervision and look forward to further collaborations. Thanks to Andrew Munkacsi for sharing his lab and his knowledge. I would also like to thank Peter Ritchie who was always helpful and supportive. Thank you to Aaron Hart for pushing me on, even when things were not going well. Thank you to Kevin Matthews, Yangna Sun and Yibo Luo (Chinese Academy of Sciences), Kuga Yukari, Mike Lusk (New Zealand Native Orchid Group), and Jeremy Rolfe (Department of Conservation), without whom I would not have had so many samples. I am thankful for the support of the Otari-Wilton’s Bush Trust and Phil Parnell. Thanks to Finn Michalak and Rewi Elliot for allowing me to put my experiment in the middle of their garden beds. Thanks to Joe Zuccarello for answering my random questions and Lawrence Zettler for answering the less-random ones. I would also like to thank Shirley Pledger whose skilful statistics took me on a magical journey. I am extremely grateful to the Australian Orchid Foundation for enabling me to learn techniques first-hand from the kind people at Kings Park and Botanic Garden: Belinda Davis, Wei Han, and Kingsley Dixon. Thank you to: the Museum of New Zealand Te Papa Tongarewa for the Te Papa MSc Scholarship in Plant Molecular Systematics; the Wellington Botanical Society for the Student Grant and the Arnold and Ruth Dench Botanical Award; and the San Diego Orchid Society for helping fund this project. Thank you to the Royal Society of New Zealand Marsden Fund and the Victoria University Centre for Biodiscovery for enabling me to present my work, meet my orchid heroes, and gain global context for my work during the 5th International Orchid Conservation Congress (IOCC) at La Réunion. Finally, thank you to my family and friends for all you do in the other half of my life – now you get to see where I’ve been all this time! IV Table of Contents Abstract ......................................................................................... III Acknowledgements .....................................................................................IV Table of Contents ........................................................................................ V List of Tables................................................................................... VII List of Figures ................................................................................. VII Chapter 1: Introduction ................................................................................1 Orchids .............................................................................1 New Zealand orchids .......................................................2 Fungal interaction.............................................................3 Specificity .........................................................................4 Orchid mycorrhizae ..........................................................5 Mycorrhizae isolation and seed germination ...................6 Conservation issues of terrestrial orchids........................8 The aims of this thesis research ......................................9 References......................................................................................10 Chapter 2: Diversity of fungal endophytes in New Zealand orchids and their effect on seed germination .........................................15 Introduction .....................................................................................15 Methods ........................................................................................18 Results ........................................................................................24 Discussion.......................................................................................38 References......................................................................................46 Chapter 3: In situ germination of two terrestrial orchids: Microtis unifolia and Thelymitra longifolia, within an urban bush reserve in Wellington ............................................................52 Introduction .....................................................................................52 Methods ........................................................................................56 Results ........................................................................................60 Discussion.......................................................................................65 References......................................................................................70 Chapter 4: Phylogenetic affinities and symbiotic germination of Spiranthes novae-zelandiae Hook.f., a nationally vulnerable wetland orchid .......................................................73 Introduction .....................................................................................73 Methods ........................................................................................76 Results ........................................................................................81 Discussion.......................................................................................88 References......................................................................................93 V Table of Contents continued… Chapter 5: General discussion and recommendations .........................98 Endophyte diversity ........................................................98 Germination ....................................................................98 Seed baiting .................................................................100 Ex situ orchid ...............................................................101 Conservation