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Physiological Characteristics of Fungi Associated With PHYSIOLOGICAL CHARACTERISTICS OF FUNGI ASSOCIATED WITH ANTARCTIC ENVIRONMENTS by Priyanka Sushil Kudalkar A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Land Resources and Environmental Sciences MONTANA STATE UNIVERSITY Bozeman, Montana April 2016 ©COPYRIGHT by Priyanka Sushil Kudalkar 2016 All Rights Reserved ii DEDICATION To my dearest Manjiri, Mumma and Pappa, for always believing in me and letting me follow my dreams. iii ACKNOWLEDGEMENTS First and foremost, I would like to thank my academic advisor Dr. John Priscu, for accepting me as a graduate student and guiding me through research and thesis work. Without his valuable guidance, feedback, and unending patience, this project would not have been possible. I would also like to convey my deepest thanks to my committee members, Dr. Cathy Cripps and Dr. Gary Strobel for serving on my committee and for providing me with their valuable inputs. They both have been instrumental in amplifying my interest in Science since my undergraduate research and have always strived to make research a wonderful learning experience for all their students. I am grateful to Dr. Lillian Lin and Christopher Barbour from the Statistical Consulting and Research Service for providing statistical advice and R code. Thanks to Edward Barge for generously helping me with the initial DNA sequencing of my samples. I would like to thank my labmates for all their help, friendship and advice. My sincere appreciation to Dr. Cathy Cripps and Trista Vick- Majors for providing valuable feedback during earlier iterations of this thesis. I would also like to thank all my friends from India and Bozeman who have played such an important role in making sure that I was not alone during this process. Last but certainly not least, I would like to thank my family for their unconditional love and support. I would not have come this far without all of you. Thanks to my dearest cousins, Gurudatta, Amruta and Aditya for the endless laughs during stressful times. Thank you, Neeraj Gurdasani for always being such a positive influence. My research was funded by the NSF- PLR grants to J. Priscu. I greatly acknowledge the teaching assistantship I received from Department of Ecology at Montana State University. iv TABLE OF CONTENTS 1. INTRODUCTION .......................................................................................................... 1 Background: The McMurdo Dry Valleys, Antarctica .................................................... 1 Ice-covered Lakes ........................................................................................................... 1 Subglacial Lakes ............................................................................................................. 3 Overview of Antarctic Fungi .......................................................................................... 4 Diversity of Fungi in Icy Ecosystems ............................................................................ 6 Role of Fungi in Icy Ecosystems .................................................................................... 7 2. MATERIAL AND METHODS ...................................................................................... 9 Study Sites ...................................................................................................................... 9 Sample Collection and Processing ............................................................................... 10 Fungal Culturing ........................................................................................................... 11 Microscopic Observations ............................................................................................ 12 DNA Extraction ............................................................................................................ 12 PCR Amplification of the ITS Region ......................................................................... 13 Visualization, Purification and Quantification of PCR Products ................................. 14 DNA Sequencing .......................................................................................................... 14 Temperature Experiments ............................................................................................ 15 Test Organisms to Profile Bioactivity: Fungi and Bacteria ......................................... 16 Bioactivity Profiling Test Organisms ........................................................................... 16 Dual Culture Assay ............................................................................................... 17 Test for Volatile Antimicrobials ........................................................................... 18 Measuring Substrate Utilization Using BIOLOG Plate Assay .................................... 20 Statistical Analyses ....................................................................................................... 22 3. HYPOTHESES ............................................................................................................. 24 4. RESULTS ..................................................................................................................... 26 Isolation, Culturing, and Taxonomic Identification ..................................................... 26 Morphological Description Using Microscopic Observations ..................................... 28 Temperature Experiments ............................................................................................ 32 Biological Effects of Fungal Isolates on Test Organisms ............................................ 36 Dual Culture Assay ............................................................................................... 36 Test for Volatile Organic Compounds .................................................................. 37 Substrate Profiling ........................................................................................................ 38 v TABLE OF CONTENTS - CONTINUED 5. DISCUSSION ............................................................................................................... 47 Diversity of Fungal Isolates from AntarcticLake Ice and Subglacial Sediments ............................................................................................. 47 Growth Temperature Preference of Fungal Isolates ..................................................... 50 Biological Inhibition of Fungi by Antarctic Fungal Isolates ........................................ 52 Carbon Substrate Utilization Using Biolog Assay ....................................................... 53 Conclusions .................................................................................................................. 54 REFERENCES CITED ..................................................................................................... 57 APPENDICES .................................................................................................................. 68 APPENDIX A: Growth of Fungal Isolates .................................................................. 69 APPENDIX B: Carbon Guilds Present in Biolog Plates ............................................. 71 APPENDIX C: Heat Maps Of Fungal Isolates ............................................................ 77 vi LIST OF TABLES Table Page 1. Summary of Fungal Sequences from BLAST Search of GenBank . .26 2. Q10 Values of Isolates . 34 3. Effects of Bioactive Compounds on Pathogens. 36 4. ANOVA Showing Transformed Response. 38 5. ANOVA for Interactions Between Temperature and Isolate, and Temperature and Group . .39 vii LIST OF FIGURES Figure Page 1. Conceptual Model Depicting the Dynamic Equilibrium of Sediment Inclusions in Antarctic Lake Ice.. .................................................... 2 2. Conceptual Model Depicting the Location of Sediments in Subglacial Lake Whillans. ............................................................................... 4 3. Maps showing the Locations of Lake Bonney, Lake Chad in Taylor Valley (TV), and Subglacial Lake Whillans in West Antarctica. ............................................................................................ 10 4. Bioactivity Test: Petri Plate Assay ..................................................................... 18 5. Bioactivity Test: Gas Assay ............................................................................... 19 6. Petri Plates Supporting The Growth of Mycelial Colonies ................................ 26 7. Phylogenetic Tree showing the Relationship of T. ellipsoideum to Reference Taxa .................................................................... 29 8. Phylogenetic Tree showing the Relationship of L. hoffmannii to Reference Taxa....................................................................... 30 9. Effect of Temperature on Growth Rates of Mucor sp. ....................................... 33 10. Effect of Temperature on Growth Rates of Lecythophora hoffmannii. .................................................................................. 34 11. Effect of Temperature on Growth Rates of Unidentified Ascomycota. .................................................................................. 34 12. Effect of Temperature on Growth Rates of Tetracladium ellipsoideum................................................................................. 35 13. Carbon Substrate Utilization by all Four Isolates at 4°C.. ............................... 41
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