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Characterization and Identification of Pythium On CHARACTERIZATION AND IDENTIFICATION OF PYTHIUM ON SOYBEAN IN NORTH DAKOTA A Dissertation Submitted to the Graduate Faculty Of the North Dakota State University Of Agriculture and Applied Science By Kimberly Korthauer Zitnick-Anderson In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY Major Department: Plant Pathology November 2013 Fargo, North Dakota North Dakota State University Graduate School Title Identification and characterization of Pythium spp. on Glycine max (soybean) in North Dakota By Kimberly Korthauer Zitnick-Anderson The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Dr. Berlin Nelson Chair Dr. Steven Meinhardt Dr. Jay Goos Dr. Laura Aldrich-Wolfe Approved: Dr. Jack Rasmussen 11/08/2013 Date Department Chair ABSTRACT The Oomycete Pythium comprises one of the most important groups of seedling pathogens affecting soybean, causing both pre- and post-emergence damping off. Numerous species of Pythium have been identified and found to be pathogenic on a wide range of hosts. Recent research on Pythium sp. infecting soybean has been limited to regions other than the Northern Great Plains and has not included North Dakota. In addition, little research has been conducted on the pathogenicity of various Pythium species on soybean or associations between Pythium communities and soil properties. Therefore, the objectives of this research were to isolate and identify the Pythium sp. infecting soybean in North Dakota, test their pathogenicity and assess if any associations between Pythium sp. and soil properties exist. Identification of the Pythium sp. was achieved using molecular techniques and morphological features. A total of 26 described Pythium sp. and several unknown species were recovered from soybean roots collected from 138 fields between 2011 and 2012. The majority of Pythium species (P. attrantheridium, P. debaryanum, P. diclinum, P. dissotocum, P. heterothallicum, P. hypogynum, P. inflatum, P. intermedium, P. irregulare, P. kashmirense, P. lutarium, P. minus, P. oopapillum, P. perplexum, P. terrestris, P. viniferum, P. violae,and an unknown Pythium sp.) caused pre-emergence damping off on soybean seedlings with less than 50% emergence and survival. In contrast, P. orthogonon, P. nunn, and P. rostratifingens had approximately 80% or greater emergence and survival of soybean seedlings. The negative and positive controls had 100% and 0% emergence and survival of soybean seedlings. Associations between soil properties and three Pythium groups were performed using logistic regression analysis. Logistic regression analysis determined that the presence of group one characterized by P. ultimum was correlated with zinc levels. Group two was characterized by P. kashmirense and an unknown Pythium sp. and was iii correlated with cation exchange capacity (CEC) values. Group three was characterized by P. irregulare and P. heterothallicum and was correlated with calcium carbonate exchange and CEC. iv ACKNOWLEDGEMENTS I would like to express my gratitude and appreciation to my advisor Dr. Berlin Nelson, for all the time and patience he has shown me through the entire process of getting my PhD. I would also like to thank Dr. Anne-Marie Fortuna, Dr. Luis del Rio-Mendoza, and Dr. Jack Norland, Kendra Rodel, Tracy Christianson, and Susilo for their help with sample preparations, analysis, and general guidance and assistance. In addition I would like to express my gratitude towards the members of my committee, Dr. Steven Mienhardt, Dr. Laura Aldrich-Wolfe, and Dr. Jay Goos. Thank you everyone for the encouragement and honesty. I want to say thanks to Matt Anderson, my family, and friends. Your support and willingness to listen to me talk about my research regardless of how little you understood was greatly appreciated. I could not have asked for better support system and companionship. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES .........................................................................................................................x LIST OF APPENDIX TABLES ................................................................................................... xii LIST OF APPENDIX FIGURES................................................................................................. xiii CHAPTER I: LITERATURE REVIEW ..........................................................................................1 Taxonomy and Phylogeny ........................................................................................................... 1 Taxonomy ................................................................................................................................ 1 Morphology.......................................................................................................................... 2 Molecular ............................................................................................................................. 6 Phylogeny ................................................................................................................................ 8 Life Cycle .................................................................................................................................... 9 Ecology ...................................................................................................................................... 10 Host Range ................................................................................................................................ 11 Environmental Factors .............................................................................................................. 11 Soil texture ............................................................................................................................. 11 Soil organic matter................................................................................................................. 13 Soil metals ............................................................................................................................. 13 Soil salinity ............................................................................................................................ 14 Soil pH ................................................................................................................................... 14 Methods of Control ................................................................................................................... 15 vi CHAPTER II: CHARACTERIZATION AND IDENTIFICATION OF PYTHIUM ON GLYCINE MAX (SOYBEAN) IN NORTH DAKOTA .................................................................18 Introduction ............................................................................................................................... 18 Materials and Methods .............................................................................................................. 19 Collection of plants ................................................................................................................ 19 Isolation and identification of isolates ................................................................................... 20 Pathogenicity trials ................................................................................................................ 22 Results ....................................................................................................................................... 26 Isolation and identification .................................................................................................... 26 Pathogenicity trials ................................................................................................................ 31 Discussion ................................................................................................................................. 35 Conclusion ................................................................................................................................. 40 CHAPTER III: ASSOCIATIONS BETWEEN SOIL PROPERTIES AND PRESENCE OR ABSENCE OF PYTHIUM SPP. IN SOYBEAN FIELDS OF NORTH DAKOTA ...............41 Introduction ............................................................................................................................... 41 Materials and Methods .............................................................................................................. 43 Collection of plants and soils ................................................................................................. 43 Species diversity, evenness, and Spearman correlation ........................................................ 44 Cluster analysis ...................................................................................................................... 45 Logistic regression ................................................................................................................. 46 Results ......................................................................................................................................
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