EFFECTS OF NATIVE ECTOMYCORRHIZAL FUNGI ON ASPEN SEEDLINGS IN GREENHOUSE STUDIES: INOCULATION METHODS, FERTILIZER REGIMES, AND PLANT UPTAKE OF SELECTED ELEMENTS IN SMELTER-IMPACTED SOILS by Christopher Paul Mahony A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science In Plant Science Montana State University Bozeman, Montana State University April 2005 © COPYRIGHT by Christopher Paul Mahony 2005 All Rights Reserved ii APPROVAL Of the thesis submitted by Christopher Paul Mahony This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies Dr. Cathy L. Cripps Approved for the Department of Plant Sciences and Plant Pathology Dr. John E. Sherwood Approved For the College of Graduate Studies Dr. Bruce R. McLeod iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this thesis is allowed. If I have indicated my intention to copyright this thesis by including a copyright notice page, copying is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation from or reproduction of this thesis in whole or in parts may be granted only by the copyright holder. Christopher P. Mahony 4/1/2005 iv ACKNOWLEDGMENTS I would like to acknowledge and thank the following individuals: Dr. Cathy Cripps – Advisor Dr. Dennis Neuman – Committee member Dr. Richard Stout – Committee member Dr. Cathy Zabinski – Committee member Paul and Regina – Parents Friends near and far Bridger Bowl Trout These individuals provided support, encouragement, and held me steady in the direction of my LIFE. To them, I am forever indebted. v TABLE OF CONTENTS 1. LITERATURE REVIEW ...………………………………...….…………….……...…1 Introduction....................................................................................................................1 Soils ......................................................................................................................3 Chemical ..................................................................................................................5 Physical....................................................................................................................6 Biological.................................................................................................................6 Plants ......................................................................................................................8 Mycorrhizal Fungi .......................................................................................................9 Survival and Fitness...............................................................................................11 Increased Nutrient Uptake .....................................................................................11 Drought Tolerance .................................................................................................12 Protection From Pathogens....................................................................................12 Heavy Metals .........................................................................................................13 Mechanisms ...........................................................................................................13 Objectives .................................................................................................................18 2. PRELIMINARY EXPERIMENTS...............................................................................19 Introduction..................................................................................................................19 Research Goals.............................................................................................................27 Material and Methods ..................................................................................................27 Site Description......................................................................................................27 Collection of Fungi For Use as Potential Inoculum...............................................28 Source of Plant Material ........................................................................................30 Evaluating Sources of Inoculum............................................................................32 Development and Evaluation of Inoculum Types for Potential Use with Aspen .................................................................................33 Liquid Inoculum.....................................................................................................34 Soil Inoculum.........................................................................................................34 Mycelial Plugs and Spore Inoculum......................................................................35 Evaluation of Techniques For Inoculation of Aspen With Mycorrhizal Fungi.........................................................................35 Preliminary Assessment of Mycorrhizal Formation..............................................37 Identification and Confirmation of Mycorrhizal Types on Roots .........................37 Results..........................................................................................................................38 Growth and Survival of Ectomycorrhizal Fungi in Culture...................................38 Evaluation of Aspen Seed and Rooted Cuttings as Source of Plant Material.........................................................43 vi TABLE OF CONTENTS CONTINUED Evaluation of Methods for Inoculating Aspen with Mycorrhizal Fungi................43 Conditions for Mycorrhization ..............................................................................45 Discussion...................................................................................................................45 3. FERTILIZER AND INOCULATION EFFECTS ON ASPEN SEEDLING MYCORRHIZATION AND GROWTH.............................49 Introduction..................................................................................................................49 Ectomycorrhizal Fungi in Native Habitats ...............................................................51 Difficulties of Mycorrhizal Studies ..........................................................................58 Research Goals..........................................................................................................59 Material and Methods ...................................................................................................60 Native Ectomycorrhizal Fungi..................................................................................60 Inoculation Procedures..............................................................................................61 Fertilizer Treatments.................................................................................................61 Fertilizer Application Rates ......................................................................................62 Water Regime ...........................................................................................................62 Experimental Design.................................................................................................62 Experiment 1: No Fertilizer ..................................................................................62 Experiment 2: Fertilizer/Inoculation Method .......................................................62 Experiment 3: Fertilizer Type...............................................................................64 Seedling Harvesting...............................................................................................64 Data Analysis.........................................................................................................65 Experiment 1..........................................................................................................65 Experiment 2 ........................................................................................................65 Experiment 3 ........................................................................................................66 Results..........................................................................................................................67 Experiment 1: No Fertilizer ..................................................................................67 Percent of Aspen Seedling with Mycorrhizae ....................................................70 Height..................................................................................................................70 Root Collar Diameter..........................................................................................71 Aboveground Biomass........................................................................................71 Belowground Biomass........................................................................................72 Total Biomass .....................................................................................................72