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Orchidace Ae ROOT-ASSOCIATEDORGANISMS OF THE CYPRIPEDIODEAE (ORCHIDACEAE) A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph In partial hilfilment of requirements for the degree of Doctor of Philosophy December, 200 1 O Caria Zelmer. 200 1 National Librery Bibliothèque nationale 1*1 atcando du Canada Acquisions and Acquisitions et BiMiogrsphic Sewkes services biûliographiqubs The author has granted a non- L'auteur a accordé une licence non exc1usive licence aiiowing the exchsive permettant à la National L1hry of Canada to Bibliothèque Pationale du Canada de reproduce, loan, distriibute or seil reproduire, prêter, distribuer ou copies of this thesis in rnicroform, vendre des copies de ceüe thèse sous paper or electronic formats. la forme de microfiche/fdm., de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or othenÿise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Canada ABSTRACT ROOT-ASSOCIATED ORGANISMS OF THE SLIPPER ORCHIDS (CYPRIPEDIODEAE, ORCHIDACEAE) Carla Dale Zelmer Advisor: University of Guelph, 200 1 Dr. R. L Peterson AI1 orchid species are associated with fungi and other organisms during their life cycles. A review of the literature on genera in the Cypripedioideae (Slipper orchids) indicated that few species of Cypriipdium have been studied in terms of root-associated organisms and features of root anatomy that may be related to colonization of roots by these organisms. Likewise, there are few data on the microorganisrns associated with the roots of Pqhiopedilum and Phrugmipedium, two subtropicaVtropical orchid genera in Cypripedioideae. Field samples of roots of five Cyprirpdim species fiom Manitoba and Ontario showed high levels of colonization by mycorrhual fùngi. Microorganisms isolated into stenle culture included several fungal species known to be mycorrhizal with orc hids, demat iaceous fùngi, including PhiaiocephaIaforri>rii, cyanobactetia. aigae and bacteria. Roots of al1 Cypripedium species had an exodennis of suberized ce11 walls t hat may limit the ingress of most microorganisrns into the cortex. Isolates of Epforhia sp., a known mycorrhizal fùngai genus, affected colonization of protocoms of Cypripdi~ircm reghue Merently depending on cuitun substrate. A large expenment to test the effects of various root-associatecl organisms on the growth of nerile-raid C. regime seedlings showed that Epulorhi:a sp. increased shoot phosphorous and the number of roots. One bacterial isolate showed positive growth effects similar to those caused by plant growth promoting rhizobacteria (PGPRs). Many organisms, including mycorrhual fungi, other hngi, cyanobactena, algae and ciliated protozoans were isolated fiom within the multi- layerad epidermis (velamen) of roots collected fiom greenhouse-grown species of Pa~~hioptidi/~~mand I ~hragntijw(Ii11m. M yconhizal tùngi were also localized wit hin the cortex. The diversity of root-associated organisms show in this study should be taken into consideration dunng the commercial propagation of these genera and the re- introduction of seedlings into field sites during conservation programs. When 1 began my Ph. D.. 1 was often askd why 1 had undertaken this task. 1 had the answer then (there were several). but looking back now at the completion of this research, one answer eclipses the others. The best answer was 'for the joumey.' Along this path 1 have had the honour of leaming fiom a multitude ofpeople. 1 hope that those that t have not named here will forgive me for the ornmission. 1 am gratehl for dl of your contributions. Thank you to my Advisor Lany Peterson, who helped me map out the temtory and taught me to find my own way. 1 have leamed fiom you more than you will know. Thank you, Randy Currah. Joe Gerrath and Melissa Farquhar. the members of my advisory cornmittee. You were willing to guide me through some unfamiliar terrain. Thank you to those that panicipated in my research as surnmer students and Research Assistants, including Lara Armstrong, Jen Pnce. Orie Berlasso. Tony Smrnigalski, Chessie Pottier, Karrie Thomas, Kevin Burgess, Greg Vamier, Fengshan Ma and Chris Peterson. You made my load lighter, and your interest in my research quickened the pace. There were others on the trail. I thank the members of the Root Biology and Mycorrhizal Associations lab (Shawna Carneron, Ron Deckert, Ryan Geil, Kevin Stevens, and Trevor Yu) for their companionship, their curiosity and for sharing parts of their joumeys with me. A special thank you to Lewis Melville, a world-class traveller. Thank you to George Barron, Bud Ewacha. Dons Ames. Laune Winn, Shemy Haii. Franliea Ailen, lain Johnston, Allan Anderson, Peter Crozen, Carole Ann Lacroix, Demis Lynn, Al Bickell, Austin Hill, Grace Hill-Rackett, Michael Yee, NSERC, OGS for helping me obtain the resources 1 needed to complete this work: schoiarships, lab space, equipment, books, plant specimens and knowiedge. 1 couldn't have accomplished this without you. Thank you to my family. You are always there for me, and 1 know 1 will always have a place to corne home to. Finaily, thank you to those who never needed to ask me why, and wdked with me much of the way: Kirsten Müller, Dimaris Acosta, Alex Morisson, Yukari Uetake and Tony Smmigalski. 1 continue to be inspired by your joumeys and your discoveries. TABLE OF CONTENTS Acknow ldgcmcnts ......................................................................................................... i List of Tables ................................................................................................................ vi .. List of Figum. Illustrations and Plit~memmem.mmee.mee~~eee~mem~m~eemmmmmeetemeee~me~eeeee~e~~emem~em~~~~~~~o~~~ List OC Appendicts ~~~~~~~~o~~~~~~~~~~~~~~~~~~mmmmmmmee~emmeeeemmmemmemeeemeeeemex Introduction.................................................................................................................... 1 Chapter 1. CypripeJim mots and mycotrhizai associations .a review ................ .... 3 1ntroduction and Clarification of Terminology ......,.......-.................................................. 3 Root Anatomy and Morpho10gy ..,e.....e..mm..e.e..e......t.me.e..........m............~................................5 Gene ral ............................................................................................................................................ 5 Epidcrmis ............................................................................................................................ -.. .......... 6 Hvpodermis ...................................................................................................................................... 7 Corticai parenchym ......................................................................................................................... 8 Endodermis....................................................................................................................................... 9 Periqcle .........................................................................................................................................10 Xylem ............................................................................................................................................. 11 Phlocm ........................................................................................................................................... 11 Pith .............................................................................................................................................. 11 Roots and Cypri'dium Tuonomy ......,..... ......................................................................... 12 CypRpcdium Mycarrhizas and Symbiotic Fungi ......,.................................................... 12 Roots and Cj'pripedium Conservation .................................................................................17 Concluding Rcmarks .......,.................t.m............................t...........................t.....................18 Chqpter 2 :Cypripediuni mycorihi~lrr. unotomy and extent of coloni&on .............. 20 Introduction ......................................................................................................................... 20 Met ho& .......~...~~~~~~~~~~~~~~~H~~~.~~t..~W~~"~~~~~..~.~~L~~~~~~.~~~~~..ee~~~~~.e~~~~~~~~~.~.~~~~~I~~~~~~~~~~~~~t~~~~~~~~.~t.~~.*O 21 Tissue preparation ........................................................................................................................... --7? Clearing and staining for e.dent~fcolonizationassessmenis ..... ......... .. ........................................... 2.1 Scoring of coloniation ...... .... ... .... ................................................................................................ 75 .................................................................................................................................... 28 Concs............................................................................................................................................ 28 Endodermis.................................................................................................................................. 31 Stele
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