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Pythium, and Fusarium Have Been Shown To ABSTRACT Both Pythium and Fusarium spp. can be readily isolated from corn and soybeans plants where stand establishment is an issue. In the first study, isolations were made from symptomatic corn and soybean seeds and seedlings collected in Ohio from fields with stand establishment problems, and in a second study soil was collected from 88 locations in Ohio using a stratified sampling strategy and baiting procedure. The results of this study provide a comprehensive view of the Pythium species present in Ohio, their pathogenicity, response to fungicide seed treatment, distribution in Ohio, and response to abiotic properties of the soil, as well as the pathogenicity and fungicide sensitivity of F. graminearum to corn and soybean seedlings. The overall goal was to determine which species of Pythium and Fusarium are most commonly associated with seed and seedling disease of corn and soybean, and determine how widespread these pathogens are in the state. In addition, determine which soil properties pH, Ca, Mg, K, P, CEC, OM, percent sand silt and clay, and field capacity are associated with disease caused by Pythium spp., and the formation of Pythium communities. A total of 124 isolates representing 12 different species of Pythium, and 112 isolates of F. graminearum were recovered from diseased corn and soybean seedlings from the first study. The results demonstrated that there is a large diversity of pathogenic Pythium species in the soils of Ohio. These ii included several species that were reported for the first time as pathogens of corn and/or soybeans and also include the description of the new species Pythium delawarii. These investigations also provided the first report of P. attrantheridium as a pathogen of both corn and soybean; the first report of P. dissotocum as a pathogen of soybean; and the first report of P. inflatum as a seed and seedling pathogen of corn and soybean in North America. Of the five commercial seed-treatment fungicides tested, none provided adequate control for all twelve Pythium species tested, and fludioxonil was the only fungicide which provided sufficient inhibition of F. graminearum mycelial growth. However several fludioxonil resistant mutants were identified during the sensitivity experiments. F. graminearum was found to be highly pathogenic to both corn and soybean seedlings in the both the in vitro and soil bioassay pathogenicity test, indicating the importance of F. graminearum as a pathogen of both corn and soybean seedlings. P. irregulare, P. inflatum, P. torulosum, P. ultimum var ultimum, P. ultimum var sporangiiferum and P. dissotocum were the most frequently recovered of the 21 species identified in the second study. Greater disease incidence levels were associated with soils with lower levels of Ca, Mg, CEC, and organic matter. Five Pythium communities were identified in this study using the Jaccard similarity index and Ward’s minimum variance to group locations which have the same Pythium species present. Canonical Discriminant Analysis (CDA) was used to determine if there was a strong association between abiotic components pH, iii Ca, Mg, organic matter, percent clay and field capacity of the soil and the structure of Pythium communities. The results of this study provide a comprehensive view of Pythium species present in Ohio agronomic soils, the distribution in Ohio, and response to abiotic properties of the soil through a large scale sampling strategy that included representative locations from the corn and soybean production region of the state. iv Dedicated to my wife Gloria our son Calvin and my parents Perry and Karen Broders v ACKNOWLEDGMENTS I wish to thank my adviser, Anne Dorrance, for guidance, support, and critiques that made this dissertation possible. Her patience and constant encouragement have made me a better student and professional. I thank Patrick Lipps, whose editorial corrections, have made me a far better writer today than I was when I began. My knowledge of the genus Pythium is largely attributable to Pat and his assistance in familiarizing me with the different keys to the genus and identifying all the morphological structure and the value of pond water. I thank Mike Boehm, Brian McSpadden-Gardener, and Pierce Paul, who along with Anne and Pat formed and excellent student advisory committee. Their input and suggestions have been very valuable in this work. Pierce has provided great assistance in the statistical analysis for all these studies, and has fielded all my SAS related question. I am thankful to Joe Win for all his assistance with the bioinformatics and phylogenetic aspects of this research, to Sue Ann Berry for the endless list of technical assistance, to Matt Wallhead and Grant Austin for all their work with the soil baiting and isolate recovery, and to Robert Mullen for answering all my soil related questions. This work was supported by OARDC Matching Grants Program, SEEDS: The OARDC Graduate Research Enhancement Grant Program, Syngenta Crop Protection Seed Improvement Grant and Pioneer Crop Management Research Awards Program. vi VITA August 11, 1979………………………… Born – Osmond, Nebraska, USA 2004………………………….………….. B.S. Biological Sciences, University of Nebraska-Lincoln 2004-present………………………………Graduate Research Associate, The Ohio State University PUBLICATIONS Research Publications 1. Broders, K. D., Lipps P. E., Paul P. A., and Dorrance A. E. 2007. Characterization of Pythium spp. associated with corn and soybean seed and seedling disease in Ohio. Plant Disease 91: 727-735. 2. Broders, K. D., Lipps, P. E., Paul, P. A., and Dorrance, A. E. 2007. Evaluation of Fusarium graminearum associated with corn and soybean seed and seedling disease in Ohio. Plant Disease 91:1155-1160. FIELDS OF STUDY Major Field: Plant Pathology vii TABLE OF CONTENTS Page Abstract……………………………………………………………………………………ii Dedication…………...…………………………………………………………….……....v Acknowledgments………………………………………………………………………..vi Vita..…………………………………………………………………………….…...…..vii List of Tables………………………………………………………………………..…...x List of Figures………………………………………………………………………...….xii Chapter Page 1. Introduction………………………………………………………………… 1 2. Characterization of Pythium spp. associated with corn and soybean seed and seedling disease Ohio………………………………………………… 17 Objectives………………………………………………………….. 20 Materials and Methods…………………………………………… 20 Results…………………………………………………………....... 27 Discussion………………………………………………………….. 39 List of references…………………………………………………… 48 3. Evaluation of Fusarium graminearum associated with corn and soybean seed and seedling disease in Ohio………….……………………………….. 53 Objectives…………………………………………………………... 56 Materials and Methods……………………………………………... 56 Results……………………………………………………………… 62 Discussion………………………………………………………….. 71 List of references……………………………………………........... 75 viii Chapter Page 4. Pythium delawarii: A new species isolated from soybean in Ohio……….… 79 Material and Methods…………………………………………....... 81 Taxonomy.……………………………………………………....... 84 Discussion………………………………………………………….. 90 List of references…………………………………………………… 95 5. Association of soil chemical and physical properties with Pythium species diversity, community composition, and disease incidence……….. 98 Objectives………………………………………………………….. 103 Material and Methods…………………………………………....... 103 Results…………………………………………………………....... 111 Discussion………………………………………………………….. 131 List of references…………………………………………………… 141 Appendices Appendix A: Single Strand Conformational Polymorphism (SSCP) Protocol……………………………………………… 147 Appendix B: Disease incidence, species diversity, and soil property values for the 88 locations sampled in the corn and soybean production region of Ohio…………………………………….. 162 Appendix C: The Pythium species presence/absence data matrix for 88 locations in the corn and soybean production region of Ohio. ………………………………………………. 168 Bibliography …………………………………………………………………… 176 ix LIST OF TABLES Table Page 2.1 F-values and P statistics for effects of Pythium species, fungicide concentration, and experiment on the mycelial growth in plate assays…………………………………………………………………… 34 2.2 Growth of the control of twelve species of Pythium evaluated on media amended with four different fungicide seed treatment active ingredients……………………………………………………………… 35 2.3 Growth of control for nine isolates of P. dissotocum in a comparison of four fungicide seed treatments………………………………………. 36 2.4 Growth of control for 11 isolates of P. sylvaticum in a comparison of four fungicide seed treatments………………………………………… 37 5.1 The GPS coordinates for all 88 locations sampled, and the corresponding region and subregion for each location…………….. 104-105 5.2 Means of soil chemical and physical properties from 88 locations in Ohio. Locations were classifed into classes based on soil texture, measured as the percent sand, silt, and clay, using the hydrometer method. Each location was classified based on the soil texture, according to the USDA guide for soil textural classification…………. 116 5.3 Principal component loadings of soil properties that significantly contributed to differences in the 88 locations sampled from the corn and soybean production region of Ohio………………………………... 117 5.4 Correlation coefficients among soil properties, species diversity and disease incidence from 88 locations in the corn and soybean production region of Ohio……………………………………………… 120 5.5 Principal component loadings of soil properties that significantly contributed to differences in 49 locations sampled in the clay and clay loam soil classes………………………………………………………. 122 x Table Page 5.6 Classification
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