Mycorrhizal Symbiosis in Forest-Grown American Ginseng (Panax quinquefolius), and the Effect of Mycorrhizal Colonization on Root Ginsenoside Concentrations A thesis presented to the faculty of the Voinovich School of Leadership and Public Affairs In partial fulfillment of the requirements for the degree Master of Science Tanner R. Filyaw August 2017 © 2017 Tanner R. Filyaw. All Rights Reserved. 2 This thesis titled Mycorrhizal Symbiosis in Forest-Grown American Ginseng (Panax quinquefolius), and the Effect of Mycorrhizal Colonization on Root Ginsenoside Concentrations by TANNER R. FILYAW has been approved for the Program of Environmental Studies and the Voinovich School of Leadership and Public Affairs by Sarah C. Davis Associate Professor of Environmental and Plant Biology Mark Weinberg Dean, Voinovich School of Leadership and Public Affairs 3 ABSTRACT FILYAW, TANNER R., M.S., August 2017, Environmental Studies Mycorrhizal Symbiosis in Forest-Grown American Ginseng (Panax quinquefolius), and the Effect of Mycorrhizal Colonization on Root Ginsenoside Concentrations Director of Thesis: Sarah C. Davis American ginseng (Panax quinquefolius L.) is a valuable medicinal plant that has been harvested from the forests of eastern North America for over 300 years. Arbuscular Mycorrhizal Fungi (AMF) are symbiotic soil organisms that colonize plant roots and assist in the uptake of water and nutrients. The role of AMF in the production of American ginseng has become a topic of increasing interest, but forest-based research on this subject is limited. This study quantified AMF colonization in forest-grown ginseng roots, resolved the relationship between AMF colonization and root ginsenoside concentrations, and identified species of AMF present in forest production sites. Roots from four production sites were measured for AMF colonization, and ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd were quantified by High Performance Liquid Chromatography (HPLC). AMF spores were extracted from soil samples by wet-sieving, and identified morphologically. Results indicate that AMF colonization varied significantly between sites (p < 0.05), but no significant differences in ginsenoside concentrations were resolved between sites (p = 0.104), nor did concentrations vary with the extent of AMF colonization (p = 0.08). Significant inverse relationships between AMF colonization and Rg1 (p < 0.01) were detected, and there was a positive correlation between AMF colonization and Re (p = 0.007). Rhizophagus intraradices was the only species of AMF 4 identified in production sites. Observed differences in total percent colonization between large and small-scale sites suggests that AMF may be influenced by management interventions in WS production. Furthermore, results may also highlight the underlying importance of growing site, with the highest ginsenoside concentrations and root masses observed in sites with differing management characteristics, and differing levels of AMF colonization. 5 DEDICATION This work is dedicated to all of my family and friends who encouraged and supported me throughout the development of this project. 6 ACKNOWLEDGMENTS I would like to acknowledge my academic advisor and committee chair, Dr. Sarah C. Davis, for all of the financial and intellectual support provided during the course of this research project, as well as my other committee members, Dr. Arthur Trese and Dr. James Dyer for supporting the implementation of this research. I would also like to thank Dr. Shiyong Wu, Dr. Jeff Wiseman, and Dr. Haotong Chen for providing essential laboratory equipment and technical expertise. Finally, I would like to thank Dr. Joseph Morton and the International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi for providing technical expertise and analytical services. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3! Dedication ........................................................................................................................... 5! Acknowledgments ............................................................................................................... 6! List of Tables ...................................................................................................................... 9! List of Figures ................................................................................................................... 10! Chapter 1: Introduction ..................................................................................................... 11! Chapter 2: Literature Review ............................................................................................ 14! American Ginseng ........................................................................................................ 14! Arbuscular Mycorrhizal Fungi ...................................................................................... 16! Ginsenosides ................................................................................................................. 20! Chapter 3: Methodology ................................................................................................... 22! Objectives ..................................................................................................................... 22! Study Sites .................................................................................................................... 22! Sampling Site Habitat Descriptions .............................................................................. 25! Site A - Large-Scale Wild-Simulated Operation, Clarion County, Pennsylvania .... 25! Site B - Large-Scale Wild-Simulated Operation, Garrett County, Maryland ........... 26! Site C – Small-Scale Wild-Simulated Operation, Muskingum County, Ohio .......... 27! Site D - Small-Scale Wild-Simulated Operation Meigs County, Ohio .................... 29! Root and Soil Sampling ................................................................................................ 30! Root Clearing and Staining ........................................................................................... 31! Measuring Mycorrhizal Colonization ........................................................................... 32! Ginsenoside Sample Preparation and Analysis ............................................................ 32! Mean Infectivity Percentage ......................................................................................... 34! AMF Species Identification .......................................................................................... 34! Statistical Analysis ........................................................................................................ 35! Chapter 4: Results ............................................................................................................. 36! Mycorrhizal Colonization ............................................................................................. 36! Ginsenoside Analysis .................................................................................................... 43! 8 AMF Species Identification and Mean Infectivity Potential ........................................ 50! Chapter 5: Discussion ....................................................................................................... 51! Limitations and Next Steps ........................................................................................... 61! Conclusion .................................................................................................................... 62! References ......................................................................................................................... 64! Appendix A: Developmental Stages of American Ginseng ............................................. 74! Appendix B: Mycorrhizal Structures ................................................................................ 80! 9 LIST OF TABLES Page Table 1: Sampling site habitat characteristics and management practices .....................23 Table 2: Sampling site composition of forest canopy and understory vegetation ..........24 Table 3: Mean annual temperature (°C) and total precipitation (cm) from 2010-2015 all sites ............................................................................................................................25 Table 4: One-way ANOVA test for significance of Total Percent Colonization (TPC) and Percent Arbuscule Content (PAC) between sites .....................................................36 Table 5: One-way ANOVA test for significance of total root mass (ln) and fibrous root mass (ln) between sites ...........................................................................................39 Table 6: Mean values for total percent colonization (TPC), percent arbuscule content (PAC), total root mass, and fibrous root mass ....................................................42 Table 7: Mean values for percent of root mass represented by ginsenosides ................43 Table 8: Welch’s one-way ANOVA test for significant differences in concentrations of ginsenosides Rg1, Re, and Rb2 between sites ............................................................45 Table 9: One-way ANOVA test for significant differences in concentrations of ginsenosides Rb1, Rc, and Rd between sites.......................................................45 Table 10: Two-way ANOVA test for