NUTRIENT AND DROUGHT EFFECTS ON BIOMASS ALLOCATION, PHYTOCHEMISTRY, AND ECTOMYCORRHIZAE OF BIRCH DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nathan Michael Kleczewski M.S. * * * * * The Ohio State University 2008 Dissertation Committee Associate Professor Pierluigi Bonello, Adviser Professor Daniel Herms Approved by: Associate Professor Landon Rhodes Professor Ralph Boerner ____________________________ Adviser Plant Pathology Graduate Program i ii ABSTRACT Trees acclimate to environmental variation by altering their growth patterns, internal chemistry, and mycorrhizal associations. Growth, chemical synthesis, and mycorrhizae simultaneously compete for limiting resources, such as nitrogen and water, and their interactions can significantly influence tree defenses against biotic and abiotic stress. The Optimal Allocation, Growth-Differentiation Balance, and Treseder and Allen’s models have been proposed, among others, to explain and predict patterns of tree and mycorrhizal responses to soil nutrient availability. To date, these models have been tested in isolation. This dissertation describes how these three models can be integrated into a single model that can explain patterns of tree acclimation over a nutrient availability gradient, the Mycorrhiza-Defense Allocation Synthesis (MyDAS). The MyDAS model was derived from greenhouse-based experiments, but was then tested and partly validated under conditions that more closely mimic field conditions. Testing showed that the initial patterns of acclimation to nutrient availability can have strong impacts on the mechanisms used by trees to respond to drought. An important corollary of this research is that fertilization and mycorrhizal inoculation, which are routinely used as a means to improve tree establishment and health in managed settings, can be irrelevant or even counterproductive to enhancing tree tolerance to biotic and abiotic stress. iii Aside from MyDAS, key outcomes of this research were: (1) lignin deposition likely plays a role in regulating mycorrhizal colonization; (2) acclimation to nutrient availability can significantly affect the nature of tree responses to drought; (3) initial soil type can influence tree growth more than fertilization and mycorrhizal inoculation on plant growth and health; and (4) commercial mycorrhizal products may not benefit trees in managed environments, even when these products are shown to be viable. I believe that these outcomes will open up new areas of investigation on the ecology of woody plants, from natural environments to urban landscape settings. Ultimately, a better understanding of acclimation processes will allow the development of more rational practices to alleviate biotic and abiotic stress in managed tree systems. iv Dedicated to my grandparents Julia, Antone, and Chester. I will see you again. v ACKNOWLEDGEMENTS I would like to express my gratitude to my advisor Enrico Bonello, who in addition to helping me to develop as a scientist and writer, also served as a second father to me while at The Ohio State University. I would also like to thank Dan Herms, who also played a large role in my development as a person and scientist, and always found a way to find something positive out of what I interpreted as a disaster. In addition, I wish to thank my committee members, Dr. Landon Rhodes and Dr. Ralph Boerner who provided insightful comments on my work and progress and were always willing to help me in my efforts. Recognition is due to the many members of the Herms and Bonello labs, particularly Duan Wang and Bryant Chambers, who went above and beyond to assist me with setting up and conducting the many experiments presented in this dissertation. In addition, David Snodgrass and Jim Vent from the Department of Horticultural Science were instrumental in setting up the greenhouse and Cravo studies. I would like to thank Dr. Mike Boehm and the Department of Plant Pathology at the Ohio State University for providing me with the tools necessary to be a well-rounded teacher and researcher. Lastly, I would like to send a special thanks to my friends Chris Wallis and Justin Whitehill, whose support, good humor, and tolerance of my banter made my time at Ohio State a fun and memorable experience. Certainly nothing I have ever done would be possible without the unconditional love and support of my parents and family. vi VITA Nathan Michael Kleczewski November 23rd 1980………………………………. Born-Sheboygan Wisconsin 2004……………………………………………….. B.S. Ecological and Organismal Biology. The University of Wisconsin- Oshkosh. Oshkosh, WI 2007. ………………………………………………. M.S. Plant Pathology, The Ohio State University Columbus, OH 2004-2008………………………………………….. Graduate Associate The Ohio State University Columbus, OH FIELDS OF STUDY Major Field: Plant pathology vii TABLE OF CONTENTS Abstract…………………………………..……………………………………………...iii Dedication………………………………...……………………………………………....v Acknowledgements……………………...………………………………………………vi Vita……………………………………………………………………………………....vii List of figures…………………………………………………………………………....xii List of tables…..………………………………………………………………………..xiv Chapters: 1. Introduction General considerations…...…………………………………………………………... 1 The impacts of drought on plant health and productivity…...………………………. .2 The impacts of nutrients on plant health and productivity…………………………... 4 Effects of nitrogen on plant stress tolerance ……………………………...............6 Plant responses to stress ………………………………………………………………7 Relationships between growth variables ………………………………………...........7 Growth patterns and the environment ……………………………………………….12 Plant chemistry, metabolism, and phenotypic plasticity …………………………….16 Primary metabolites ……………………………………………………..............17 Carbohydrates ……………………………………………………………….17 Carbohydrates and drought tolerance …………………………………...19 viii Amino acids …………………………………………………………………20 Amino acids and drought tolerance ……………………………………..20 Secondary metabolites …………………………………………………………..21 Phenolics: A basic overview of their metabolism …………………...............21 Roles of phenolics in plant stress…………………………………………….24 Protection from visible and UV radiation ………………….....................24 Protection against herbivores and pathogens ……………………………26 Relationships between primary and secondary metabolism as influenced by stress ……………………………………………………………………………………27 The Growth Differentiation Balance Hypothesis …………………...............28 Mycorrhizal associations ……………………………………………………………29 Arbuscular-Mycorrhizal fungi …………………………………………..............31 Ectomycorrhizal fungi …………………………………………………………..32 Process of colonization and symbiosis ………………………………………….34 Benefits of mycorrhizae …………………………………………………………37 Regulation of mycorrhizae ………………………………………………………38 Aims and objectives of this project ………………………………………………….42 2. Integrating models of phenotypic plasticity, plant defense, and mycorrhizal regulation. A case study with paper birch (Betula papyrifera) Introduction ………………………………………………………………………….44 Materials and methods ………………………………………………………………48 Plant material ……………………………………………………………………48 Experimental design ……………………………………………………………..49 Harvesting and sample preparation ……………………………………...............52 Growth and biomass analysis ……………………………………………………53 Chemical analysis: Sugars and starch …………………………………...............53 Chemical analysis: Phenolics ……………………………………………………54 Identification of EM fungi ………………………………………………………54 Statistical analysis ……………………………………………………………….55 Results ……………………………………………………………………………….56 Effects of soil type, fertilization, and drought on plant growth patterns ……………………………………………………………………………............56 Effects of soil type, fertilization, and drought on plant chemistry ………………61 Effects of soil type, fertilization, and drought on EM …………………………..65 ix Discussion …………………………………………………………………………...67 3. A test of the MyDAS model with river birch (Betula nigra) Introduction ………………………………………………………………………….76 Materials and methods ………………………………………………………………78 Plant material ……………………………………………………………………78 Experimental details ……………………………………………………………..79 Compound identification ………………………………………………..............83 Estimation of EM colonization and identification of EM fungi ………...............84 Statistical analysis ……………………………………………………………….84 Results ……………………………………………………………………………….85 Growth, water potential, and EM colonization ………………………………….85 Phytochemistry ………………………………………………………………….94 Total and individual amino acids ………………………………………………108 Discussion ………………………………………………………………………….117 Relationships between plant growth and drought tolerance …………...............119 Effects of fungicide …………………………………………………………….121 4. The impacts of urban tree management practices on growth, physiology, herbivore, and ectomycorrhizae of paper birch (Betula papyrifera) Introduction ..………………………………………………………………………123 Materials and methods ……...……………………………………………………...125 Experiment 1: Effects of EM inoculation on growth of paper birch under controlled conditions ………….………………………………………………..125 Plant material and growing conditions …………..……………………..….125 Treatments ………………………………………………………………….126 Experiment 2: Effects of EM inoculation and mineral fertilization on growth, chemistry, and defoliation of paper birch growing in field-simulated urban soils …………………………………………………………………………………..127 Plant material ………………………………………………………………127 Treatments …………………………………………………………………128 Measurements of growth …………………………………………..............130 Assessment of EM colonization ……………………………………………130 x Assessment of