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Isolating and Identifying Fungal Endophytes from Roots of Rare Orchid Cypripedium Arietinum in Grand Sable Dunes, Michigan Kari Farkas-Lasich [email protected]

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Isolating and Identifying Fungal Endophytes from Roots of Rare Orchid Cypripedium Arietinum in Grand Sable Dunes, Michigan Kari Farkas-Lasich Karifarkas@Yahoo.Com Northern Michigan University NMU Commons All NMU Master's Theses Student Works 6-2018 Isolating and Identifying Fungal Endophytes from Roots of Rare Orchid Cypripedium arietinum in Grand Sable Dunes, Michigan Kari Farkas-Lasich [email protected] Follow this and additional works at: https://commons.nmu.edu/theses Part of the Environmental Microbiology and Microbial Ecology Commons, and the Plant Biology Commons Recommended Citation Farkas-Lasich, Kari, "Isolating and Identifying Fungal Endophytes from Roots of Rare Orchid Cypripedium arietinum in Grand Sable Dunes, Michigan" (2018). All NMU Master's Theses. 553. https://commons.nmu.edu/theses/553 This Open Access is brought to you for free and open access by the Student Works at NMU Commons. It has been accepted for inclusion in All NMU Master's Theses by an authorized administrator of NMU Commons. For more information, please contact [email protected],[email protected]. ISOLATING AND IDENTIFYING FUNGAL ENDOPHYTES FROM ROOTS OF RARE ORCHID CYPRIPEDIUM ARIETINUM IN GRAND SABLE DUNES, MICHIGAN By Kari L. Farkas-Lasich THESIS Submitted to Northern Michigan University In partial fulfillment of the requirements For the degree of MASTER OF SCIENCE IN BIOLOGY Office of Graduate Education and Research June 2018 ABSTRACT ISOLATING AND IDENTIFYING FUNGAL ENDOPHYTES FROM ROOTS OF RARE ORCHID CYPRIPEDIUM ARIETINUM IN GRAND SABLE DUNES, MICHIGAN By Kari L. Farkas-Lasich Recently the population of Cypripedium arietinum in Grand Sable Dunes, Michigan was estimated at more than 3.5 million plants, greater than 90% of the world’s known population. There is little research on this species’ root associated fungal endophytes. This new population estimate allows the ethical, destructive sampling required to shed light on root associated symbionts and further successful preservation and restoration of the species. Samples were collected of current year’s roots from 75 C. arietinum orchids at 75 locations within 4 jack pine forests in the Grand Sable Dunes, Pictured Rocks National Lakeshore, Michigan. Fungal endophyte cultures were obtained from roots and subcultured for DNA extraction. DNA was PCR amplified with primers that isolate the highly conserved 5.8s rDNA and the highly variable internal transcribed spacer (ITS) regions on either side of it. DNA was sequenced and sequences were identified using NCBI BLAST. Of the 36 DNA samples 27 were positively identified. Phialocephala fortinii and Leptodontidium orchidicola were the most prevalent identifications. These fungi both have saprotrophic abilities and may play an important role in C. arietinum’s lifecycle. Keywords. Cypripedium arietinum, Phialocephala fortinii, Leptodontidium orchidicola, Rhizoscyphus ericae, Grand Sable Dunes i Copyright by KARI L. FARKAS-LASICH June 2018 ii ACKNOWLEDGMENTS I would like to thank my advisor Dr. Donna Becker and committee members Dr. Robert Belton and Dr. Alan Rebertus for encouragement and support throughout this research. Bruce Leutscher, Chief of Science and Natural Resources at Pictured Rocks National Lakeshore provided great resources to help this project along. Thank you to Jake Rich, Jessica Ford, Steve Caird and Kristin Denryter for braving cold and rainy fall days in the field, to Steve Schaar and Mike Peters for endless helpful lab hours and more, to Adrienne Bozic for the many exciting conversations that led to this project and to the NMU Biology Department faculty, staff and graduate students who are amazing resources with a wealth of knowledge and creative problem solving ideas. Thanks to the Michigan Botanical Club, NMU Excellence in Education Grant and NMU Development Fund for financial support of this project. Finally, I thank my family, especially my husband John Lasich, mother Nancy Bailey and stepfather Doug Bailey for listening, asking questions, and encouraging me every chance they got. This thesis follows the format prescribed by the APA Reference Style Guide of Northern Michigan University’s Olson Library. iii TABLE OF CONTENTS List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii List of Abbreviations ......................................................................................................... ix Introduction ......................................................................................................................... 1 Molecular Identification ..................................................................................... 8 Methods............................................................................................................................. 10 Study Area .......................................................................................................... 10 Root Specimen Collection ............................................................................... 15 Fungal Endophyte Isolation ............................................................................. 18 DNA Extraction ................................................................................................. 20 PCR Amplification ............................................................................................ 21 Electrophoresis .................................................................................................. 22 DNA Sequencing............................................................................................... 23 Identification ...................................................................................................... 23 Results ............................................................................................................................... 24 Fungal Endophyte Isolation ............................................................................. 24 DNA Extraction, PCR Amplification, DNA Sequencing ............................ 32 Identification ...................................................................................................... 34 Discussion ......................................................................................................................... 37 Phialocephala fortinii ....................................................................................... 38 Leptodontidium orchidicola ............................................................................. 40 iv Rhizoscyphus ericae ......................................................................................... 42 The Future of Cypripedium arietinum in GSD ............................................ 42 Conclusion ........................................................................................................................ 44 References ......................................................................................................................... 46 Appendix A ....................................................................................................................... 53 Appendix B ..................................................................................................................... 143 v LIST OF TABLES Table 1.1. Master Mix components: MIDSCI Bullseye Hot Start Taq Polymerase, Primer ITS1-F (Gardes & Bruns, 1993), Primer ITS4 (Innis et al., 1990). 10 reactions at 50µL final volume ...................................................................................................................... 22 Table 1.2. Identification of fungal clone sequences by discontinuous MegaBLAST. Sequences 58 and 59 were positively identified as Phialocephala fortinii, but they did not register as the same sequence as Clone 1 ITS1-F ............................................................. 34 vi LIST OF FIGURES Figure 1.1. Mycelia shown as whitish fibers in the soil surrounding the light colored winter bud (top)................................................................................................................... 3 Figure 1.2. Cypripedium arietinum in Grand Sable Dunes, Pictured Rocks National Lakeshore, Michigan........................................................................................................... 5 Figure 1.3. Range map for Cypripedium arietinum. (https://www.ndstudies.gov/sites/default/file/US-Canada.gif) (Based on Blaney & Mazerolle, 2007 and Brzeskiewicz, 2000) .......................................................................... 6 Figure 1.4. Forward and reverse primer annealing locations on nuclear rDNA. Area between two primer arrows is the target DNA for amplification ....................................... 9 Figure 1.5. Cypripedium arietinum habitat in Grand Sable Dunes................................... 11 Figure 1.6. Jack pine forest nestled in dune-scape within Grand Sable Dunes ................ 13 Figure 1.7. Root sampling sites were located within these 4 jack pine islands within Grand Sable Dunes. Each has a different Cypripedium arietinum density estimated in Bozic’s research (2015) .................................................................................................... 15 Figure 1.8. Sampling site with labeled flags ..................................................................... 16 Figure 1.9. Cypripedium arietinum winter buds, rhizome and roots uncovered for sampling ...........................................................................................................................
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