Georgia Southern University Digital Commons@Georgia Southern Electronic Theses and Dissertations Graduate Studies, Jack N. Averitt College of Spring 2009 Molecular Phylogenetic Analysis of Novel Spiroplasma Isolates Haritha Lakshmi Jandhyam Follow this and additional works at: https://digitalcommons.georgiasouthern.edu/etd Recommended Citation Jandhyam, Haritha Lakshmi, "Molecular Phylogenetic Analysis of Novel Spiroplasma Isolates" (2009). Electronic Theses and Dissertations. 721. https://digitalcommons.georgiasouthern.edu/etd/721 This thesis (open access) is brought to you for free and open access by the Graduate Studies, Jack N. Averitt College of at Digital Commons@Georgia Southern. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons@Georgia Southern. For more information, please contact [email protected]. MOLECULAR PHYLOGENETIC ANALYSIS OF NOVEL SPIROPLASMA ISOLATES by HARITHA LAKSHMI JANDHYAM (Under the Direction of Laura B. Regassa) ABSTRACT Spiroplasmas are among the smallest self replicating organisms known. They are helical, motile descendents of Gram-positive bacteria that have evolved to occupy broad host ranges including plants, insects and crustaceans. Spiroplasmas are the causative agent of several economically important agricultural diseases, but most host-microbe interactions appear to be commensal. Given the ubiquitous nature of these microbes, a methodical approach that focused on serologically distinct isolates from a single host family was used as an initial step in understanding spiroplasma diversity and distribution. Tabanid- associated spiroplasmas represent the most thoroughly studied group to date, so this project examined a spiroplasma field isolate collection (>200 isolates) that was obtained from tabanid flies in Costa Rica, Ecuador, Australia, and the United States. The 16S rRNA, 23S rRNA, and rpoB genes and the 16S-23S rRNA spacer region were successfully used to establish evolutionary relationships of the closely related spiroplasma isolates. Phylogenetic analyses and non-genetic character mapping indicated that all study isolates belonged to the Apis clade; arginine hydrolysis was a strong indicator of evolutionary relatedness; surface serology and phylogenetic placement were congruent; and neither host specificity nor geographical ranges were strict. INDEX WORDS: Mollicutes, Spiroplasma, Phylogenetics, Surface serology, Tabanids, Biodiversity, Host specificity, MOLECULAR PHYLOGENETIC ANALYSIS OF NOVEL SPIROPLASMA ISOLATES by HARITHA LAKSHMI JANDHYAM B.V.Sc & AH, Acharya N.G. Ranga Agricultural University, India, 2004 A Thesis Submitted to the Graduate Faculty of Georgia Southern University in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE STATESBORO, GEORGIA 2009 © 2009 HARITHA LAKSHMI JANDHYAM All Rights Reserved iii MOLECULAR PHYLOGENETIC ANALYSIS OF NOVEL SPIROPLASMA ISOLATES by HARITHA LAKSHMI JANDHYAM Major Professor: Laura B. Regassa Committee: Lorenza Beati Oscar J. Pung Electronic Version Approved: May 2009 iv DEDICATION I consider myself very lucky to have Krishnan as my loving, understanding and caring husband. I would like to thank him for his patience and support. I would like to dedicate my research work to my husband Krishnan, to my baby girl and to my parents Ramanaiah and Tulasamma. Thanks to my sisters, brother, aunt and uncle for all their loving care and support. v ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Laura B. Regassa, for giving me the precious opportunity to work with her and for her constant support, guidance and encouragement which allowed me to venture into new areas and grow as a researcher. I would like to thank Dr. Lorenza Beati for her expertise in phylogenetics and for the time she spent on my research work. She is very understanding, patient and overall a great person to work with. I would like to thank Dr. Oscar J. Pung, for spending his valuable time on my research project and for guiding me during the course of my graduate program. I would also like to thank Dr. Frank E. French for generously providing his strain collection and serological analyses, as well as his on-going support and expertise. In addition, I thank Ryan Bates for his technical assistance in lab and Tammie Young for generating the 16S rDNA sequences. Funding and facilities for this research were provided by the Department of Biology, Georgia Southern University (Graduate Student Research Fund, Faculty Research Grant), the National Science Foundation (DEB-0481430, PI: Regassa), and the Southeastern Branch of the American Society for Microbiology. vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................................................................................. vi LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix CHAPTER 1 INTRODUCTION ...........................................................................................11 2 MATERIALS AND METHODS.....................................................................23 3 RESULTS ........................................................................................................30 4 DISCUSSION..................................................................................................60 REFERENCES ..................................................................................................................64 APPENDICES A Partial characterization of strain GSU5358 from Australia ............................70 B Spiroplasma isolates/species with identical DNA sequences ………………71 vii LIST OF TABLES Page Table 1: Spiroplasma species and/or representative strains by serogroup ........................22 Table 2: Apis Clade Spiroplasmas from Tabanidae: Diptera of Australia, Costa Rica, Ecuador and U.S.A .……………………………………………………………………..26 Table 3: Conditions for PCR amplification of Spiroplasma genes/region ...……………27 Table 4: New accession numbers for DNA sequences generated from Spiroplasma isolates/species and deposited as part of this study...……………………………………28 Table 5: Genbank accession numbers for sequences from described Mollicutes species used in the phylogenetic analyses……………………………………………….29 Table 6: Maximum parsimony heuristic search values for study genes…………………59 viii LIST OF FIGURES Page Figure 1: Majority Rule Maximum Parsimony tree based on 16S rRNA gene sequence .21 Figure 2A: Bayesian phylogenetic tree derived from partial 16S rDNA sequence...........36 Figure 2B: Maximum Parsimony phylogenetic tree derived from partial 16S rDNA.......37 Figure 2C: Maximum Likelihood phylogenetic tree derived from partial 16S rDNA ......38 Figure 3A: Bayesian phylogenetic tree derived from partial 16S-23S rDNA sequence ...39 Figure 3B: Maximum Parsimony phylogenetic tree derived from partial 16S-23S .........40 Figure 3C: Maximum Likelihood phylogenetic tree derived from partial 16S-23S .........41 Figure 4A: Bayesian phylogenetic tree derived from partial 23S rDNA sequence...........42 Figure 4B: Maximum Parsimony phylogenetic tree derived from partial 23S rDNA.......43 Figure 4C: Maximum Likelihood phylogenetic tree derived from partial 23S rDNA ......44 Figure 5A: Bayesian phylogenetic tree derived from partial rpoB sequence analysis ......45 Figure 5B: Maximum Parsimony phylogenetic tree derived from partial rpoB sequence 46 Figure 5C: Maximum Likelihood phylogenetic tree derived from partial rpoB sequence47 Figure 6A: Bayesian phylogenetic trees derived from concatenated 16S & 23S rRNA ...48 Figure 6B: Maximum Parsimony phylogenetic tree derived from concatenated 16S.......49 Figure 6C: Maximum Likehood phylogenetic tree derived from concatenated 16S .......50 Figure 7A: Bayesian phylogenetic tree derived from concatenated 16S rRNA, ITS ........51 Figure 7B: Maximum Parsimony phylogenetic trees derived from concatenated 16S .....52 Figure 7C: Maximum Likelihood phylogenetic tree derived from concatenated 16S.......53 Figure 8A: Bayesian phylogenetic tree derived from concatenated 16S, 23S rRNA, ......54 Figure 8B: Maximum Parsimony phylogenetic tree derived from concatenated 16S, .....55 ix Figure 8C: Maximum Likelihood phylogenetic tree derived from concatenated 16S, .....56 Figure 9: Optimal doubling time values overlayed on Bayesian phylogenetic tree ..........57 Figure 10: Spiroplasma host information overlayed on Bayesian phylogenetic tree ........58 x CHAPTER 1 INTRODUCTION Spiroplasmas (Procaryotae: Mollicutes: Entomoplasmatales: Spiroplasmataceae) are helical and motile Gram positive prokaryotes that are associated with insects and plants. The genus Spiroplasma was discovered recently and has drawn much attention due to its reductive evolution; characterized by small genome sizes (780 – 2,200 kb), low GC content (24% – 31%), and the lack of a cell wall (Carle et al. 1995, Williamson et al. 1997). Spiroplasmas may be one of the most abundant groups of microbes found on earth, due to their close association with a wide variety of organisms, ticks, plants and crustaceans, including tabanid flies (Hackett and Clark 1989, Hackett et al. 1992, Tully et al. 1987; Williamson et al. 1989; Williamson et al. 1998; Wang et al. 2004, 2005; Nunan et al. 2004). Although insect and plant hosts play a major role in maintenance and dispersal of these spiroplasmas, plant surfaces appear to