SYMBIOTIC NITROGEN FIXATION AND ESTABLISHMENT OF SIX MONTANA NATIVE LEGUMES SPECIES by Sarah Jean Metcalf A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science in Land Rehabilitation MONTANA STATE UNIVERSITY Bozeman, Montana May 2005 © COPYRIGHT By Sarah Jean Metcalf 2005 All Rights Reserved ii APPROVAL Of a thesis submitted by Sarah Jean Metcalf 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. Jeff Jacobsen Approved for the Department of Land Resources and Environmental Sciences Jon Wraith Approved for the Department of College of Graduate Studies Bruce McLeod iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a master degree at Montana State University-Bozeman, I agree that the Library shall make it available to borrowers under the rules of the Library. 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 part may be granted only by the copyright holder. Sarah Metcalf May 05, 2005 iv ACKNOWLEDGEMENTS First of all, I would like to acknowledge the Natural Resources Conservation Service, the MSU Foundation Seed Program as well as the Department of Land Resources and Environmental Sciences at MSU for funding this project. The following people have helped to make this project a valuable experience for me. Jeff Jacobsen helped me find this project; he has turned my life path in a whole new direction, for which I will always be grateful. Clain Jones, my primary advisor, worked very closely with me and was always around to help me solve problems. Tim McDermott has been a wonderful mentor and provided me with opportunities too numerous to list and Lina Butero-Griffiths, from his lab, who was very patient in teaching protocols. Bill Grey has also given so much of his time and laboratory space to help me with the pathogen aspects of this project. His enthusiasm for research and discovery kept me going numerous times. I also want to thank Mark Majerus and Larry Holzworth; both have given me great advice, encouragement and assistance over the last two years. Dave Wichman and Bernie Schaff were appreciated for their assistance in maintaining my field sites. I would also like to thank Aaron Mugaas, Deirdre Willie, Ann McCauley, Teresa Warne, Ryan Weikel and Cole Germann for their assistance in the laboratory and field. Special thanks to my family, especially my parents who have been very supportive both financially and spiritually throughout this project. Finally, I am thankful for my son Shea who inspired me to obtain this degree though without whom completion would have been much easier. v TABLE OF CONTENTS 1. INTRODUCTION ...........................................................................................................1 Objectives ........................................................................................................................1 Background......................................................................................................................1 Materials and Methods.....................................................................................................1 Factors Effecting Nodulation and Legume Growth.........................................................4 Nodules ............................................................................................................................8 N2 Fixation in the Field....................................................................................................9 2. GREENHOUSE STUDIES: SOIL INOCULATION AND FUNGICIDE EFFECTS ON NODULATION AND BIOMASS.........................................................................12 Introduction....................................................................................................................12 Materials and Methods...................................................................................................12 Trial I: Effects of soil origin and N content on nodulation and biomass.......................12 Materials and Methods Trial 1...................................................................................13 Introduction................................................................................................................13 Seeding.......................................................................................................................15 Data Collection ..........................................................................................................15 Trial I Results..................................................................................................................16 Germination and emergence .....................................................................................16 Biomass.....................................................................................................................16 Nodulation.................................................................................................................19 Pathogenic Observations and Isolations ...................................................................20 Trial II: Effects of soil moisture content on nodulation and biomass..............................22 Materials and Methods..............................................................................................22 Introduction...............................................................................................................22 Seeding......................................................................................................................24 Data Collection .........................................................................................................24 Trial II Results .................................................................................................................24 Biomass.....................................................................................................................24 Nodulation.................................................................................................................25 Trial III: Effects of fungicide coating on nodulation, biomass and plant levels..............29 Materials and Methods..............................................................................................29 Introduction...............................................................................................................29 Seeding......................................................................................................................29 Data Collection .........................................................................................................30 Trial III Results................................................................................................................30 Emergence and Plant Survival..................................................................................30 Nodulation and Biomass...........................................................................................32 Trial IV: Effects of fungicide drench on nodulation and biomass...................................36 vi TABLE OF CONTENTS – Continued Materials and Methods...........................................................................................36 Introduction............................................................................................................36 Seeding...................................................................................................................36 Data Collection ......................................................................................................37 Trail IV Results.............................................................................................................38 Biomass..................................................................................................................38 Root Structure, Nodules and Fungicide.................................................................39 N-content................................................................................................................41 Trial V: Effects of inoculation and fungicide application on nodulation and biomass ..............................................................................................................................42 Materials and Methods...........................................................................................42 Introduction............................................................................................................42 Seeding...................................................................................................................43 Trial V Results ..............................................................................................................44 Introduction............................................................................................................44 A. canadensis Combined Treatments....................................................................44 A. canadensis Nodulation ......................................................................................45