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Identification of Rhizobial Symbionts Associated with Lupinus Spp

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Identification of Rhizobial Symbionts Associated with Lupinus Spp IDENTIFICATION OF RHIZOBIAL SYMBIONTS ASSOCIATED WITH LUPINUS SPP. Dilshan Beligala A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2015 Committee: Vipaporn Phuntumart, Advisor Helen Michaels Paul Morris © 2015 Dilshan Beligala All Rights Reserved ii ABSTRACT Vipaporn Phuntumart, Advisor Lupinus, commonly called lupine is a genus of legumes which belongs to the family Fabaceae and its members have the ability to establish a symbiotic association with rhizobia and effectively fix atmospheric nitrogen in root nodules. Although, the main symbiont lineage of most Lupinus spp. is Bradyrhizobium sp., there are evidence of other rhizobial genera nodulating lupines such as Rhizobium, Mesorhizobium, Burkholderia and Microvirga. Furthermore, Burkholderia spp. has been reported to colonize cluster roots of lupine. Accordingly, there is a great diversity among the root nodule bacteria which nodulate lupines in different geographic locations. Therefore, the aim of this work is to identify rhizobial symbionts associated with Lupinus spp. using 16S rRNA, atpD, glnII and dnaK phylogenies. In this study, these marker genes of 17 strains were PCR amplified, sequenced and analyzed using NCBI-BLAST. Sequence alignment was performed using ClustalW2 followed by computing the maximum-likelihood phylogenetic trees using MEGA6 software package. According to the congruence of single gene trees of 16S rRNA, atpD, glnII and dnaK, the strains USDA 3040, 3051, 3709 and SB_J are identified as members that belong to the genus Bradyrhizobium. In addition, L_3d52 strain is identified as Rhizobium sp. whereas USDA 3063 and 3717 are proven to be Mesorhizobium sp. and USDA 3043 and L_OO are identified as Burkholderia sp. whereas USDA 3057a is identified as Microvirga sp. However, further experiments and analysis are needed to confirm the identities of the rest of the studied strains that are not in full agreement among generated phylogenetic trees. iii I would like to dedicate this to my mother Mrs. Renuka Ranaweera, my father Mr. Samarasinghe Beligala, my brother Mr. Lakshan Beligala and my wife Mrs. Gayathri Beligala for being great pillars of support. iv ACKNOWLEDGMENTS As I approach the completion of my Master’s Degree, I realize that I am extremely thankful to the support and assistance of many people. I wish to thank every one of those who have helped me to survive the birthing of this thesis. First and foremost, I would like to express my sincere gratitude to my supervisor, Associate Professor Vipa Phuntumart, for her time and effort spent on continuously leading, advising and encouraging me throughout this research. Her passion for challenges has given me inspiration, her vast knowledge has given me guidance and her enthusiasm in research has given me motivation. I feel lucky to work under the supervision of such a talented advisor. I owe gratitude to my c ommittee members, Associate Professor Paul F . Morris and Associate Professor Helen J. Michaels, whose stimulating vmoti ation and valuable ideas were extremely helpful in completing my research successfully. They have been a constant source of knowledge and support throughout the years. Further, I would like to acknowledge M. Ps atrick Elia, a curator in the United States Department of Agriculture, for providing me with rhizobial strains for the experiment. Also, I am indebted to Mr. Jacob Sublett from Dr. Helen Michaels's lab for providing me with lupine root nodules. In addition, I gratefully acknowledge the support of everyone in the Department of Biological Science, Bowling Green State University, all the academic staff and non -academic staff, especially Ms. DeeDee Wentland and Ms. Denise Holcombe for t heir kindness and support in all administration is sues. I would like to acknowledge everyone in Dr. Paul Morris’s lab who helped me with the necessary equipments and chemicals time to time. My special thanks go to Assistant Professor Dr. Hans Wildschutte for allowing me to use the nanodrop spectrophotometer in his lab when it was needed to continue my experiment. v I would also like to be grateful to all the members in my lab, especially Mr. Alex Howard, for helping me out in the field and Ms. Shannon Miller for helping me with phylogenetic analysis. I feel lucky to have the opportunity to work with such friendly lab mates. I also must mention the valuable support I received from Ms. Menaka Ariyaratna, a PhD candidate in the Department who helped me in numerous ways throughout my research work. With much of happiness I thank my dear friends, especially Ms. Ramadha Dilhani, Mr. Madushanka Sugath and Mr. Dayal Wijayarathne for all the support they have given me throughout until this point. I feel so blessed to have such supportive friends and I would like to acknowledge all of them who offered many words of encouragement throughout the years. Finally, my deepest gratitude goes to my loving parents and my wife Ms. Gayathri Beligala for their support in my studies and for being there with me through thick and thin. I am forever indebted to all that they have done for me. Without their endless support and unconditional love, I wouldn’t have achieved this much. Thank you for being around and for never ending motivations I’ve been getting all this while. vi TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION………………......................……………………………. 1 CHAPTER II. AIMS ………………………….…………………………………………… 10 CHAPTER III. MATERIALS AND METHODS …..……………………………………… 11 3.1 Isolation of rhizobia from root nodules of Lupinus spp. and soybean………….. 11 3.1.1 Surface sterilization………………….......……………………………. 11 3.2 Molecular characterization ……………….……………………………………. 12 3.2.1 DNA isolation ……………………….......……………………………. 12 3.2.2 PCR of gene candidates……………………………….…..………….. 13 3.2.3 DNA sequencing and analysis………………………………………… 16 3.2.4 Phylogenetic analysis ………………………………………………… 16 3.2.4.1 Phylogeny of single gene trees …..…..….……...………….. 16 3.2.4.2 Phylogeny of concatenated gene sequences …....………….. 20 CHAPTER IV. RESULTS …………………….......................……………………………. 21 4.1 DNA Quantification and Quality Analysis ....………………………………….. 21 4.2 Gel electrophoresis of PCR products……………………………………………. 22 4.3 Phylogenetic analysis of root nodule symbionts based on 16S rRNA, glnII, atpD and dnaK genes………………………………………………………..…………………. 29 4.3.1 Phylogeny of the 16S rRNA gene.....………………….…..………….. 29 4.3.2 Phylogeny of the dnaK gene.………………………….…..………….. 32 4.3.3 Phylogeny of the glnII gene..………………………….…..………….. 33 4.3.4 Phylogeny of the atpD gene...………………………….…..………….. 36 4.3.5 Phylogeny of the concatenated dataset….…………….…..………….. 37 vii 4.3.5.1 Concatenation of 16S rRNA, atpD, glnII and dnaK sequences……………………………………..….……...………….. 37 4.3.5.2 Concatenation of 16S rRNA, atpD and glnII sequences...….. 40 4.3.5.3 Concatenation of 16S rRNA, dnaK and glnII sequences...….. 41 4.3.5.4 Concatenation of 16S rRNA and dnaK sequences……....….. 44 CHAPTER V. DISCUSSION…..………………………………………………………….. 46 CHAPTER VI. CONCLUSIONS………………………………………………………….. 51 REFERENCES……………………………………………………………………………… 52 viii LIST OF FIGURES Figure Page 1 Agarose gel electrophoresis of extracted genomic DNA of representative strains… 22 2 Agarose gel electrophoresis of PCR products of 16S rRNA, atpD and glnII gene regions………………………………………………………………………………. 24 3 Agarose gel electrophoresis of PCR products of 16S rRNA and atpD gene regions.. 25 4 Agarose gel electrophoresis of PCR products of glnII gene regions……………..… 26 5 Agarose gel electrophoresis of PCR products of dnaK gene regions with Bradyrhizobium specific primers……………………………………………………………………… 27 6 Agarose gel electrophoresis of temperature gradient PCR products of L_OO dnaK gene regions with Burkholderia specific primers………………………………………… 28 7 Agarose gel electrophoresis of temperature gradient PCR products of L_3d52 dnaK gene regions with Rhizobium specific primers…………………………………………… 29 8 Maximum-likelihood phylogenetic tree of rhizobial strains isolated from lupine and soybean root nodules and representative strains of named genera, based on 16S rRNA gene sequences (1485 bp)…………………………………………………………… 31 9 Maximum-likelihood phylogenetic tree of rhizobial strains isolated from lupine and soybean root nodules and representative strains of named genera, based on dnaK gene sequences………………………………………………………………………….... 32 10 Maximum-likelihood phylogenetic tree of rhizobial strains isolated from lupine and soybean root nodules and representative strains of named genera, based on glnII gene sequences…………………………………………………………………………… 34 ix 11 Maximum-likelihood phylogenetic tree of rhizobial strains isolated from lupine and soybean root nodules and representative strains of named genera, based on atpD gene sequences…………………………………………………………………………… 36 12 Maximum-likelihood phylogenetic tree of rhizobial strains isolated from lupine and soybean root nodules and representative strains of named genera, computed from the concatenation of 16S rRNA, atpD, glnII and dnaK gene sequences………………… 38 13 Maximum-likelihood phylogenetic tree of USDA 3042 and 3717, computed from the concatenation of 16S rRNA, atpD and glnII gene sequences………………………. 40 14 Maximum-likelihood phylogenetic tree of L_3d52 and SB_5, computed from the concatenation of 16S rRNA, dnaK and glnII gene sequences………………………. 42 15 Maximum-likelihood phylogenetic tree of L_OO, USDA 3043 and USDA 3057a, computed from the concatenation of 16S rRNA and dnaK gene sequences………… 44 x LIST OF TABLES Table
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