CHARACTERIZATION OF TWO BEGOMOVIRUSES ISOLATED FROM Sida santaremensis Monteiro AND Sida acuta Burm. f By HAMED ADNAN AL-AQEEL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003 Copyright 2003 by Hamed Adnan Al-Aqeel This dedicated to my family my father Dr. Adnan, my mother Fareda and my wife Hanin. TABLE OF CONTENTS page LIST OF TABLES............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii ABSTRACT....................................................................................................................... ix CHAPTER 1 HISTORY AND LITERATURE REVIEW .................................................................1 Geminivirus History .....................................................................................................1 Taxonomy and Nucleotide Functions...........................................................................3 Begomoviruses .............................................................................................................5 The Genus Sida.............................................................................................................6 Viruses Infecting Sida spp............................................................................................7 Begomoviruses Infecting Sida spp. in Florida............................................................10 2 CHARACTERIZATION OF A NEW BEGOMOVIRUS ISOLATED FROM Sida santaremensis Monteiro in Florida.............................................................................12 Materials and Methods ...............................................................................................13 Virus Source ........................................................................................................13 Begomovirus Detection.......................................................................................13 Cloning and Sequencing......................................................................................14 Molecular Characterization of the Virus .............................................................15 Biological characterization..................................................................................15 Biolistic inoculation .....................................................................................16 Whitefly inoculation.....................................................................................16 Detection of SiGMoV in Test Plants...................................................................17 Results.........................................................................................................................18 Phylogenetic Analysis .........................................................................................18 Nucleotide and Amino Acid Sequence Analysis.................................................19 Biological Characterization ........................................................................................19 Discussion...................................................................................................................27 iv 3 AN EPIDEMIC IN TOMATO CAUSED BY VARIANTS OF Sida golden mosaic virus ............................................................................................................................29 Materials and Methods ...............................................................................................29 Sample Source.....................................................................................................29 PCR Analysis and Restriction Analysis ..............................................................29 Cloning ................................................................................................................30 Gap and Blast Analysis .......................................................................................30 Phylogenetic Analysis .........................................................................................30 Results.........................................................................................................................31 Partial Sequence Analysis from Tomato and S. acuta ........................................31 Phylogenetic Analysis .........................................................................................33 Discussion...................................................................................................................44 LIST OF REFERENCES...................................................................................................46 BIOGRAPHICAL SKETCH .............................................................................................51 v LIST OF TABLES Table page 2-1 Comparison of the nucleotide sequence identity of the DNA-A of Sida golden mottle virus...............................................................................................................21 2-2 Comparison of the nucleotide sequence identity of the DNA-B of Sida golden mottle virus...............................................................................................................21 2-3 Comparison of the open reading frame nucleotide and common region sequences identity of the DNA-A of Sida golden mottle virus .................................................22 2-4 Comparison of the open reading frame and common region nucleotide sequences identity of the DNA-B of Sida golden mottle virus .................................................22 2-5 Comparison of the open reading frame amino acid sequences similirity of the DNA-A of Sida golden mottle virus.........................................................................23 2-6 Comparison of the open reading frame amino acid sequences similirity of the DNA-B of Sida golden mottle virus.........................................................................23 2-7 Host range study of SiGMoV...................................................................................24 3-1 The nucleotides identity of partial sequences of SiGMV DNA-A...........................38 3-2 The nucleotides identity of partial sequences of SiGMV DNA-B...........................38 3-3 The Common region nucleotides identity of SiGMV DNA-A sequences isolated from tomato and S. acuta .........................................................................................39 3-4 The Common region nucleotides identity of SiGMV sequences isolated from tomato and S. acuta ..................................................................................................39 3-6 The nucleotide identity of partial sequences DNA-B sequences isolated from tomato and S. acuta ..................................................................................................41 vi LIST OF FIGURES Figure page 2-1 Sida santaremensis infected with Sida golden mottle virus showing typical ........20 2-2 Phylogenic tree of complete nucleotide of a component of selected begomoviruses with SiGMoV................................................................................25 2-3 Phylogenic tree of complete nucleotide of B component of selected begomoviruses with SiGMoV................................................................................26 3-1 Partial sequence of DNA-A (S3-C7A) amplified from Sida acuta collected from Citra Field, Florida........................................................................................33 3-2 Partial sequence of DNA-A (T3-C8A) amplified from tomato plant collected from Citra Field, Florida........................................................................................34 3-3 Partial sequence of DNA-A (T5-C2A) amplified from tomato plant collected from Citra Field, Florida........................................................................................34 3-4 Partial sequence of DNA-A (T10-C8A) amplified from tomato plant collected from Citra Field, Florida........................................................................................35 3-5 Partial sequence of DNA-A (T10-C10A) amplified from tomato plant collected from Citra Field, Florida........................................................................................35 3-6 Partial sequence of DNA-A (T12-C6A) amplified from tomato plant collected from Citra Field, Florida........................................................................................36 3-7 Partial sequence of DNA-B (S3-C4B) amplified from Sida acuta collected from Citra Field, Florida.................................................................................................36 3-8 Partial sequence of DNA-B (T12-C3B) amplified from tomato plant collected from Citra Field, Florida........................................................................................37 3-9 Partial sequence of DNA-B (T12-C5B) amplified from tomato plant collected from Citra Field, Florida........................................................................................37 3-10 Partial sequence of DNA-B (T12-C7B) amplified from tomato plant collected from Citra Field, Florida........................................................................................37 vii 3-11 Partial sequence of DNA-B (T12-C9B) amplified from tomato plant collected from Citra Field,