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Characterization of Resistance in Soybean and Population Diversity Keiddy Esperanza Urrea Romero University of Arkansas, Fayetteville

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Characterization of Resistance in Soybean and Population Diversity Keiddy Esperanza Urrea Romero University of Arkansas, Fayetteville University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 7-2015 Pythium: Characterization of Resistance in Soybean and Population Diversity Keiddy Esperanza Urrea Romero University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Agronomy and Crop Sciences Commons, Plant Biology Commons, and the Plant Pathology Commons Recommended Citation Urrea Romero, Keiddy Esperanza, "Pythium: Characterization of Resistance in Soybean and Population Diversity" (2015). Theses and Dissertations. 1272. http://scholarworks.uark.edu/etd/1272 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Pythium: Characterization of Resistance in Soybean and Population Diversity A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science by Keiddy E. Urrea Romero Universidad Nacional de Colombia Agronomic Engineering, 2003 University of Arkansas Master of Science in Plant Pathology, 2010 July 2015 University of Arkansas This dissertation is approved for recommendation to the Graduate Council. ________________________________ Dr. John C. Rupe Dissertation Director ___________________________________ ___________________________________ Dr. Craig S. Rothrock Dr. Pengyin Chen Committee Member Committee Member ___________________________________ ___________________________________ Dr. Burton H. Bluhm Dr. Brad Murphy Committee Member Committee Member Abstract Pythium spp. are an important group of pathogens causing stand losses in Arkansas soybean production. New inoculation methods and advances in molecular techniques allow a better understanding of cultivar resistance and responses of Pythium communities to cultural practices. The objectives of this research were to i) characterize the resistance of soybean to P. aphanidermatum with two phenotyping assays that evaluated the seed rot phase of the disease; and ii) understand the effect of long term crop rotation on species diversity and iii) to determine the effect of location, temperature and continuous soybean and soybean-rice rotation on Pythium spp. diversity in several locations in Arkansas. For objective one, resistance to seedling disease caused by P. aphanidermatum, was characterized in 84 F2:6 soybean lines derived from a cross of ‘Archer’ and ‘Hutcheson’ cultivars using a seed plate assay and an infested vermiculite assay. The lines were assayed with 5,403 SNP markers and genetic maps and QTL mapping was performed. With both inoculation methods, two quantitative trait loci (QTL) were identified on chromosomes 4 and 7. In objective two, the effect of crop rotation and location on species diversity was determined by using soybean seed to bait Pythium spp. from soil of plots following a ten year rotation. Of the 320 isolates, 12 species identified, P. spinosum, P. irregulare, P. pareocandrum, and P. sylvaticum were the most common. There were significant differences in number of Pythium isolates from the ten rotation systems with the rice (wheat)-soybean (wheat) rotation having the highest recovery of these six Pythium spp. In objective three, soils from a soybean-rice and a soybean- soybean rotation were collected from three locations in 2012. A total of 275 isolates were identified representing 25 species. The most frequently recovered species were P. irregulare, P. pareocandrum, P. sylvaticum, P. corolatum and P. spinosum. Location had a large effect on Pythium population composition and diversity. Distinctive species prevailed in each of the three locations across two temperature and two rotations. Overall, populations of Pythium spp. varied the most among locations, but were not influenced by the previous crop or the isolation temperature. Acknowledgments I would like to thank my major advisor Dr. John Rupe for his direction, support and encouragement during these years and to my dissertation committee members: Dr. Craig Rothrock, Dr. Burton Bluhm, Dr. Pengyin Chen and Dr. Brad Murphy for their guidance, constructive criticism and suggestions. My deepest gratitude to my beloved parents Norberto and Flor and my bothers Edwin and Alexis, for their love and encouragement to pursue my dreams. I want to thank my wonderful husband Ruben Morawicki for all his love, support, care, and encouragement all these years. There are many people in the Plant Pathoplogy department that have been a big part of this project and I want to say thank you to all of them. My gratitude to Dr. Rick Bennett, professors, fellow students, and support personnel. In Dr. Rupe’s laboratory, without the help of Adele Steger, Bob Holland, Sandy Goeke, and Kartlyn Jones this project would not have be possible. Also, I would like to thank John Guerber, Scot Winters, Dr. Chunda Fenn, Dr. Joannis Tzanetakis and his laboratory, and Dr. Jim Correll and his laboratory. In the Crop Soil and Environmental Science department, I would like to thanks Dr. Pengyin Chen and Dr. Esten Mason and theirs laboratory, especially, Liliana Florez, Ailan Zeng, Cindy Lopez, Nythia, Topher Addison, and Andrea Acuña for their help with the genotyping analysis. Dr. Edward Gbur for his invaluable help whit the statistical analysis. Dr. Martin Chilvers and Alejandro Rojas at Michigan State University, for their help with the molecular identification of the Pythium isolates. Lastly, I would like to thank my entire family in Colombia and my friends: Camila, Natalia, Juliana, Andrea, Catalina, Valeria, Cristina, Sergio and Claudia that have been supporting me here and from the distance all these years. Dedication I dedicate this dissertation to my parents Flor Maria Romero and Norberto Urrea Bejarano, to my brothers Edwin and Alexis Urrea Romero, and my husband Ruben Morawicki for all their love and support during these years. Tables of Contents Chapter I: Introduction 1 Seedling diseases in Soybean 1 Importance of Pythium spp. 1 Effect of crop rotation on Pythium spp. 4 Environmental factors affecting Pythium spp. 6 Management of Pythium spp. 7 Fungicide seed treatments 7 Soybean resistance to Pythium spp. 8 Research justification and objectives 11 References 13 Chapter II: Characterization seed rot the resistance in soybean to Pythium 19 aphanidermatum Abstract 19 Introduction 20 Materials and Methods 24 Isolate 24 Plant material 24 Phenotypic data 25 Genotyping data 27 Data analysis 28 Results 29 Phenotypic data 30 Quantitative trait loci mapping 30 Discussion 31 Conclusion 36 References 37 Chapter III: Effect of crop rotation on Pythium spp. population composition in 61 Arkansas soybean fields Abstract 61 Introduction 62 Materials and Methods 66 Sample collection 66 Baiting and isolation 67 Molecular identification 67 Pathogenicity tests 68 Statistical analysis 69 Results 69 Isolation and identification 69 Crop rotation 70 Pathogenicity tests 70 Discussion 71 Conclusion 74 References 75 Chapter IV: Effect of crop rotation, location and isolation temperature on 88 Pythium spp. population composition in Arkansas Abstract 88 Introduction 89 Materials and Methods 93 Soil collection 93 Soil sampling 94 Isolation 94 Molecular identification 95 Experimental design and statistical analysis 95 Results 96 Pythium species isolation and identification 96 Pythium species by location 96 Pythium species diversity 97 Discussion 97 Conclusion 100 References 101 Overall conclusion 113 Appendix 114 List of Tables Chapter II Table 1. Analysis of variance of seed plate and infested vermiculite assay with 41 Pythium aphanidermatum of 84 F2:6 lines and the parents, Archer and Hutcheson. Table 2. Percent of seed germination for the 84 F2:6 lines and parents evaluated for the 42 resistance to Pythium aphanidermatum with the seed plate assay. Table 3. Percent of plant stands for the 84 F2:6 lines and parents evaluated for the 45 resistance to Pythium aphanidermatum with the infested vermiculite assay. Table 4. Summary of single nucleotide polymorphism (SNP) markers used to screen 49 the 84 F2:6 lines derived from ‘Archer’ x ‘Hutcheson’ cross. Table 5. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 56 84 F2:6 lines derived from ‘Archer’ X ‘Hutcheson cross that were mapped using Single Marker Analysis (SMA) using seed plate assay. Table 6. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 57 84 F2:6 lines derived from ‘Archer’ X ‘Hutcheson’ cross that were mapped using Single Marker Analysis (SMA) using the infested vermiculite assay. Table 7. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 58 84 F2:6 lines derived from an ‘Archer’ X ‘Hutcheson cross that were mapped using Composite interval mapping (CIM) using three phenotyping methods. Chapter III Table 1. Frequency of isolation of Oomycetes recovered across 10 crop rotation 79 system. Table 2. Isolates of five Pythium spp. recovered with soybean, corn and rice as the last 81 crops across the 10 rotation system. Table 3. Analysis the variance of estimated dead seeds on corn, soybean, rice and 82 wheat Table 4. Dead seeds (logit transformed) of soybean tested with five Pythium species 83 on soybean. Table 5. Dead seeds (logit transformed) of soybean tested for forty nine Pythium 84 species isolates. Table 6. Dead seeds (logit transformed) of corn for five for Pythium species. 85 Table 7.
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