Networks of Communication: Defense-related signal transfer between tree seedlings via mycorrhizal networks and an educational mycorrhizal-focused video game by Julia Amerongen Maddison B.Sc. University of British Columbia, 2011 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2016 © Julia Amerongen Maddison, 2016 i Abstract The majority of terrestrial plants associate with fungi in symbiotic resource- exchange relationships called mycorrhizae. Mycorrhizal networks (MNs) arise when the same fungus is connected to multiple plants, allowing for interplant resource transfer and impacting ecosystem functions. Recent work suggests MNs also transfer defense-related information from pathogen-, herbivore-, or mechanically- damaged plants to unharmed neighbors. I investigated the defense pathways involved in defense-related signal transfer in ectomycorrhizal systems. Paired Douglas-fir seedlings were grown with varying levels of belowground connectivity (soil water only; soil water and MNs; soil water, MNs, and roots), and a defense response was stimulated in donor seedlings by methyl jasmonate. After 24 and 48 hrs, I measured expression of two regulatory genes on the jasmonate and ethylene pathways. Receiver response was unrelated to hormone treatment of donors in either gene, but the jasmonate response of donor and receiver pairs was correlated across treatments. Positive expression of both genes across donors and receivers and pervasive presence of spider mites suggested signal transfer may either have not occurred or been masked by already ongoing defensive responses. Results indicate the complexity of these systems, and further work is needed to better characterize defense signal transfer via ectomycorrhizal networks. Because of the importance of these mycorrhizal systems to ecosystem functioning, it is crucial that resource managers and scientists have a good understanding of mycorrhizal ecology. However, lower student interest in plants and fungi combined with difficulties visualizing belowground processes present ii challenges for teaching and learning mycorrhizal concepts. To address this, I co- created the digital plant-centric action-based game Shroomroot for use in lower level postsecondary settings. I conducted a pre-test/post-test evaluation of Shroomroot in a 2nd year postsecondary Introduction to Soil Science course. Students’ knowledge of mycorrhizal ecology increased after playing Shroomroot, and engagement with mycorrhizal content tended to increase after gameplay. These exploratory results suggest positive potential for action-based plant-oriented digital games in the higher education classrooms. Both studies focus on improving our understanding of mycorrhizae and mycorrhizal networks, ecologically and pedagogically. Greater understanding of mycorrhizae has the potential to improve our multi-faceted relationships with the ecosystems upon which we depend. iii Preface This thesis is an original, unpublished product of the author, Julia Amerongen Maddison (JAM). The projects herin were identified and designed by Dr. Suzanne Simard and JAM. The experimental specifics were designed by JAM and Dr. Suzanne Simard in conjunction with committee members, Dr. Maja Krzic, Dr. Sue Grayston, and Dr. Rik Blok. JAM established and led greenhouse experiments and lab work with the exception of combustion elemental analysis and VHP closed vessel microwave acid digestion for foliar nutrient analysis, performed by the British Columbia Ministry of Environment Analytical Laboratory. JAM co-created Shroomroot with Christopher Adderley and Alex Catamo of Area Denial Games, and wrote educational assessments with suggestions from course instructors. Statistical analyses were conducted by JAM with suggestions from Dr. Suzanne Simard and Dr. Valerie Lemay. Ethical approval for the study in Chapter 2 was obtained from the Behaviour Research Ethics Board (BREB) under the code H14-02382, project name “Educational Efficacy of Root-Mycorrhizae Action-based Mobile Game,” nickname “shroomroot.” iv Table of Contents Abstract ........................................................................................................................................ ii Preface ......................................................................................................................................... iv Table of Contents ...................................................................................................................... v List of Tables ........................................................................................................................... vii List of Figures ............................................................................................................................ ix List of Abbreviations .............................................................................................................. xi Acknowledgements ............................................................................................................... xii Chapter 1: Introduction .......................................................................................................... 1 1.2: Interplant signal transfer via ectomycorrhizal networks ........................................... 3 1.2.1: Mycorrhizae and mycorrhizal networks .................................................................................. 3 1.2.2: Plant defense ....................................................................................................................................... 5 1.2.3: Plant communication ....................................................................................................................... 7 1.2.4: MN-mediated interplant signal transfer .................................................................................. 9 1.2.5: Study species .................................................................................................................................... 13 1.2.6: Summary ............................................................................................................................................ 14 1.3: Shroomroot: an educational, simulation-style, digital game to enhance postsecondary teaching and learning about mycorrhizal networks ............................ 16 1.3.1: The importance of learning about mycorrhizal ecology ................................................ 16 1.3.2: Digital Game-Based Learning .................................................................................................... 18 1.3.3: A case for simple games ............................................................................................................... 22 1.3.4: Shroomroot ....................................................................................................................................... 23 1.4: Thesis objectives ..................................................................................................................... 24 Chapter 2: Investigating defense-related signal transfer between Douglas-fir via ectomycorrhizal networks .......................................................................................... 27 2.1: Introduction .............................................................................................................................. 27 2.2: Methods ...................................................................................................................................... 33 2.2.1: Experimental Setup ........................................................................................................................ 33 2.2.2: Treatment and harvest ................................................................................................................. 35 2.2.3: Harvest ................................................................................................................................................ 37 2.2.4: Gene expression .............................................................................................................................. 38 2.2.5: Morphotyping ................................................................................................................................... 41 2.2.6: Foliar nutrients ................................................................................................................................ 41 2.2.7: Data analysis ..................................................................................................................................... 42 2.3: Results ......................................................................................................................................... 43 2.3.1: Seedling characteristics ............................................................................................................... 43 2.3.2: Mycorrhizal network ..................................................................................................................... 44 2.3.3: Belowground root interactions ................................................................................................. 45 2.3.4: Gene expression .............................................................................................................................. 46 2.4: Discussion .................................................................................................................................