Investigations into Multiple–Herbicide-Resistant Ambrosia artemisiifolia (Common Ragweed) in Ohio and Glyphosate-Resistance Mechanisms Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jason Thomas Parrish Graduate Program in Horticulture and Crop Science The Ohio State University 2015 Dissertation Committee: Dr. Mark M. Loux, Advisor Dr. S. Kent Harrison Dr. James D. Metzger Dr. David M. Mackey Dr. Anne E. Dorrance Copyrighted by Jason Thomas Parrish 2015 Abstract Common ragweed (Ambrosia artemisiifolia) is a weed problem in many places throughout the world. Though it seldom dominates the landscape, common ragweed seems to be able to exploit diverse habitats. Common ragweed is primarily outcrossing and has a high rate of gene polymorphisms, leading to high genetic diversity. This high level of genetic diversity likely plays a major role in the evolution of herbicide-resistant biotypes. Whole-plant bioassays of herbicide dose-response in the greenhouse were used to characterize resistance levels to glyphosate, cloransulam-methyl, and fomesafen herbicides. Additional studies were conducted to provide insight into potential mechanisms that may contribute to the development of resistance to glyphosate in an Ohio ragweed biotype, including 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene sequencing, quantitative PCR of the EPSPS gene, EPSPS enzyme immunoblot and activity/inhibition assays, 31P-nuclear magnetic resonance (NMR) studies of glyphosate- treated tissues, and whole-plant absorption and translocation studies using 14C-labeled glyphosate. A single common ragweed population from Clinton County, Ohio exhibited multiple resistance to herbicides at dosages that exceeded the rate required to kill herbicide-sensitive common ragweed biotypes from 4- to 30-fold for glyphosate, > 1000-fold for cloransulam-methyl, and 14- to > 100-fold for fomesafen. This is the first report of a common ragweed biotype with multiple resistance to herbicides from three site-of-action (SOA) groups. Sequencing data indicated the gene coding for EPSPS has a ii high mutation rate in all studied common ragweed biotypes, but it typically does not code for an altered amino acid sequence in the glyphosate binding area. Additional studies identified alleles of EPSPS coding for proline-to-serine and proline-to-threonine substitutions at amino acid number 106 (based upon the mature maize EPSPS numbering scheme). Previous studies by other authors have found these amino acid substitutions to confer glyphosate resistance in numerous other species. The alleles containing these mutations were not detected in previous studies of Ohio ragweed populations, and it is not known whether these alleles are translated into a functional EPSPS protein. Direct sequence analysis also suggested that there are six-to-eight or more partial- or full-length copies of the EPSPS gene in a typical diploid (2n) common ragweed plant. An immunoblot assay with common ragweed total soluble protein, as well as Palmer amaranth (Amaranthus palmeri) glyphosate-sensitive and EPSPS overexpressing glyphosate-resistant controls, showed a single plant from the glyphosate-resistant biotype with increased EPSPS expression. Quantitative PCR also showed an increased relative EPSPS gene copy number in the same plant. 31P-NMR data showed similar uptake of glyphosate into the leaf cells and no vacuolar sequestration in all common ragweed biotypes, with lower sugar-phosphate (including shikimate-3-phosphate) accumulation relative to glyphosate-susceptible common ragweed plants. Similarly, absorption and translocation of 14C-labeled-glyphosate over 48 hours did not differ between resistant and susceptible biotypes. More research will be required to unequivocally determine the molecular basis of glyphosate resistance in common ragweed, but accumulated evidence supports the hypothesis that multiple mechanisms of glyphosate resistance are possible within a common ragweed population. iii Acknowledgments This work was completed thanks to the many forms of assistance and support from hundreds of people at OSU, Colorado State University, Monsanto Company, Washington University in St. Louis, and the University of Illinois, and the love and encouragement of my friends and family outside of these institutions. You know who you are, and I could not have done this without you. Thank you for the financial support and your patience. You shared your time, knowledge, and experience, your workspaces and equipment, and even your food. We spent many hours talking about plants, the weather, sports, and international foods. Thank you for the long lunches where we shared laughter and frustrations. I enjoyed the drives across the state looking for plants or any other adventure we might encounter. Thank you for helping me when I had questions and last-minute requests. I have learned so much from so many of you. As I complete my dissertation, I am left with many debts and many memories. iv Vita 2002................................................................Firelands High School 2007................................................................B.S. Agriculture, The Ohio State University 2008 to 2014 .................................................Graduate Research/Teaching Associate, Department of Horticulture and Crop Science, The Ohio State University Fields of Study Major Field: Horticulture and Crop Science v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Tables ...................................................................................................................... x List of Figures ................................................................................................................... xii Chapter 1 : Introduction ..................................................................................................... 1 1.1 Common Ragweed ................................................................................................... 1 1.2 Glyphosate Resistance .............................................................................................. 4 1.3 Common Ragweed Resistance to Other Herbicides............................................... 13 1.4 Objectives ............................................................................................................... 15 Chapter 1 References .................................................................................................... 17 Chapter 2 : Characterization of Common Ragweed Resistance to Glyphosate, Cloransulam-Methyl, and Fomesafen Herbicides............................................................. 29 2.1 Materials and Methods: .......................................................................................... 29 2.1.1 Development of a sample population .............................................................. 29 2.1.2 Growing Conditions for Dose-Response ......................................................... 31 vi 2.1.3 Treatments ....................................................................................................... 31 2.1.4 Data Collection and Analysis .......................................................................... 34 2.2 Results and Discussion ........................................................................................... 37 2.2.1 Glyphosate dose-response results .................................................................... 37 2.2.2 Cloransulam-methyl dose-response results ..................................................... 38 2.2.3 Fomesafen dose-response results .................................................................... 39 2.2.4 Discussion........................................................................................................ 40 Chapter 2 References .................................................................................................... 41 Chapter 3 : Common Ragweed Target-Site Glyphosate-Resistance Mechanisms .......... 54 3.1 Materials and Methods ........................................................................................... 54 3.1.1 Plant Materials ................................................................................................. 54 3.1.2 Genomic DNA extraction ................................................................................ 54 3.1.3 RNA extraction and complementary DNA synthesis ....................................... 55 3.1.4 PCR primer design .......................................................................................... 56 3.1.5 Gene sequencing of EPSPS, acetolactate synthase (ALS), and fructan 1-exohydrolase IIa (FEH) .......................................................................................... 58 3.1.6 EPSPS Enzyme Activity.................................................................................. 60 3.1.7 EPSPS Enzyme Quantification ........................................................................ 63 3.1.8 EPSPS relative genomic copy number determination ..................................... 65 3.2 Results and Discussion