IDENTIFICATION OF PLANT AS A NOVEL AND ALTERNATIVE HOST MODEL FOR BURKHOLDERIA PSEUDOMALLEI LEE YIAN HOON (B Sc, UoM) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgments I would like to express my deepest gratitude to my supervisor, Associate Professor Gan Yunn Hwen, for her constant and continuous supervision, support and encouragement throughout this project My heartfelt appreciation to Associate Professor Chua Kim Lee from the Department of Biochemistry, for providing reagents, bacteria strains and for the use of her laboratory equipment I am grateful to Associate Professor Loh Chiang Shiong from the Department of Biological Sciences for providing the Arabidopsis seeds and for his invaluable advice I am grateful to Dr Yin Zhong Zhao from Temasek Life Sciences Laboratory for providing the rice seeds and his invaluable advice My greatest appreciation to Mr Ouyang Xuezhi from the Electron Microscopy Unit in Temasek Life Sciences Laboratory for his time, assistance and invaluable advice I am grateful to Temasek Polytechnic for the financial support during my studies My greatest thanks to Dr Ong Seng Poon from Temasek Polytechnic, for his support and patience throughout the years I am thankful to Dr Tan Seng Kee for his advice during the early stages of the project My deepest appreciation to Ms Lin Meilin, Phoebe, for helping with the plant tissue cultures A big thank you to all my labmates, Dr Sun Guang Wen, Dr Tan Kai Soo, Chen Yahua, Low Kee Chung, Teh Boon Eng and Isabelle Chen for their daily assistance, valuable discussions and wonderful friendship Lastly, my most sincere gratitude to my family for their understanding during the course of my studies Abstract Burkholderia pseudomallei is a Gram negative soil bacterium and the causative agent for melioidosis The type three secretion system (TTSS) is important in the pathogenesis of B pseudomallei in mammalian hosts B pseudomallei has three TTSS while B thailandensis, a closely related but avirulent species, has two Both bacteria share high homology in the TTSS2 locus with Ralstonia solanacearum, which causes bacterial wilt in various crops and plants In this study, we demonstrated the ability of B pseudomallei and B thailandensis to infect tomato but not rice plants Bacteria were found to multiply intercellularly and localize in the xylem vessels of the vascular bundle Infection with KHW∆TTSS1 or KHW∆TTSS2 mutants shows substantial attenuation in disease, indicating their importance in bacterial pathogenesis in susceptible plants The potential of B pseudomallei as a plant pathogen raises new possibilities of exploiting plant as an alternative host for novel anti-infectives or virulence factor discovery Table of Contents Contents Page Acknowledgements Abstract List of Tables 10 List of Figures 10 Abbreviation List 11 Chapter Burkholderia pseudomallei and Melioidosis 1.1 Melioidosis the disease 15 1.2 Characteristics of B pseudomallei 17 1.3 Diagnosis and Treatment 19 1.4 Animal models for melioidosis 20 1.5 Similarity to plant pathogen Ralstonia solanacearum 21 1.6 Aims and rationale of project 23 Chapter Generation of Type Three Secretion System (TTSS) Mutants 2.1 Introduction 25 2.2 Materials and Methods 28 2.2.1 PCR primers, plasmids and bacteria strains 28 2.2.2 Generation of KHW∆TTSS1 mutant 33 2.2.2.1 Cloning and sub-cloning 33 2.2.2.2 Conjugation 35 2.2.2.3 Selection 36 2.2.2.4 PCR confirmation 36 2.2.3 Generation of KHW∆TTSS2 37 2.2.4 Generation of KHW∆TTSS1/2 37 2.2.5 Generation of Bt∆TTSS2 by conjugation 38 2.2.6 Generation of Bt∆TTSS2 by direct transformation 38 with DNA fragments 2.3 Results 39 2.3.1 39 Generation of KHW∆TTSS1, KHW∆TTSS2, KHW∆TTSS1/2 and Bt∆TTSS2 mutant 2.4 Discussion 44 Chapter Infection of Plants with B pseudomallei and B thailandensis 3.1 Introduction 47 3.2 Materials and Methods 49 3.2.1 Plant materials 49 3.2.1.1 Tomato and Arabidopsis 49 3.2.1.2 Rice 50 3.2.2 Bacterial strains 51 3.2.3 Infection of tomato, rice and Arabidopsis plantlets 51 3.2.4 Multiplication of B thailandensis in tomato 52 plantlets 3.2.5 Transmission Electron Microscopy (TEM) 52 3.3 Results 3.3.1 53 Susceptibility of tomato plantlets to B pseudomallei 53 and B thailandensis infection 3.3.2 Susceptibility of tomato plantlets to other strains of 57 B pseudomallei 3.3.3 Resistance of rice plantlets to B pseudomallei 59 and B thailandensis infection 3.3.4 Resistance of Arabidopsis plantlets to 59 B pseudomallei and B thailandensis infection 3.4 3.3.5 Multiplication of B thailandensis in tomato leaves 59 3.3.6 Localization of bacteria at site of infection 62 Discussion 64 Chapter The role of Type Three Secretion System in plant infection 4.1 Introduction 69 4.2 Materials and Methods 72 4.2.1 Bacterial strains 72 4.2.2 Cell lines 72 4.2.3 BtTTSS3 and BtTTSS2 gene expression after 73 plant infection 4.2.4 Cytotoxicity assay 75 4.2.5 Infection of tomato plantlets with KHW∆TTSS 75 mutants 4.2.6 Growth fitness of KHW∆TTSS mutants in different 75 media 4.2.7 4.3 Statistical analysis 76 Results 76 4.3.1 BtTTSS gene expression after plant infection 76 4.3.2 BpTTSS3 is required in mediating virulence in 79 mammalian cells 4.3.3 Virulence of KHW∆TTSS mutants on tomato 81 plantlets 4.3.4 Growth fitness of wild-type and KHW∆TTSS 83 mutants in different media 4.4 Discussion 85 Chapter Summary and future directions 5.1 Summary 89 5.2 Future directions 90 References 94 Appendices 109 I Accumulative data for the infection of tomato plants with 109 B thailandensis (Daily Disease Score) II Accumulative data for the infection of tomato plants with 110 B pseudomallei (Daily Disease Score) III Accumulative data for the infection of tomato plants with 112 B pseudomallei KHW∆TTSS1 mutant (Daily Disease Score) IV Accumulative data for the infection of tomato plants with 113 B pseudomallei KHW∆TTSS2 mutant (Daily Disease Score) V Accumulative data for the infection of tomato plants with 114 B pseudomallei KHW∆TTSS3 mutant (Daily Disease Score) VI Accumulative data for the infection of tomato plants with 115 B pseudomallei KHW∆TTSS1/2 mutant (Daily Disease Score) VII Accumulative data for the infection of tomato plants with 117 various strains of B pseudomallei (Daily Disease Score) List of Tables No 2.1 Title All primers used and their annealing temperatures The Page 29 restriction enzyme sites are indicated in the sequence 2.2 All plasmids used and constructed 30 2.3 Escherichia coli, B thailandensis and B pseudomallei 32 strains used 4.1 Primers for real-time PCR of BtTTSS genes (BTH_IIxxxx 74 refers to the gene accession number) List of Figures No Title Page 2.1 Cloning procedure for Bp/BtTTSS mutants generation 41 2.2 Confirmation of KHW∆TTSS mutants by PCR 42 amplification of selected genes 3.1 Symptoms in tomato plantlets after B thailandensis 55 infection 3.2 Virulence of B pseudomallei and B thailandensis on 56 tomato plantlets 3.3 Infection of tomato plantlets with different B pseudomallei 58 isolates 3.4 B thailandensis multiplication in tomato leaves 61 3.5 Representative transmission electron micrographs of 63 B pseudomallei and B thailandensis in tomato leaves 10 Starkey M and Rahme LG (2009) Modeling Pseudomonas aeruginosa pathogenesis in plant hosts Nat Protoc 4(2):117-124 Staskawicz BJ, Mudgett MB, Dangl JL and Galan JE (2001) Common and contrasting themes of plant and animal diseases Science 292(5525):22852289 Stevens MP, Friebel A, Taylor LA, Wood MW, Brown PJ, Hardt WD and Galyov EE (2003) A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity J Bacteriol 185(16):4992-4996 Stevens MP, Haque A, Atkins T, Hill J, Wood MW, Easton A, Nelson M, Underwood-Fowler C, Titball RW, Bancroft GJ and Galyov EE (2004) Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis Microbiology 150(Pt 8):2669-2676 Stevens MP, Wood MW, Taylor LA, Monaghan P, Hawes P, Jones P.W, Wallis TS and Galyov EE (2002) An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen Mol Microbiol 46(3):649-59 Sun GW, Lu J, Pervaiz S, Cao WP and Gan YH (2005) Caspase-1 dependent macrophage death induced by Burkholderia pseudomallei Cell Microbiol 7(10):1447-1458 Suparak S, Kespichayawattana W, Haque A, Easton A, Damnin S, Lertmemongkolchai G, Bancroft GJ and Korbsrisate S (2005) 105 Multinucleated giant cell formation and apoptosis in infected host cells is mediated by Burkholderia pseudomallei type III secretion protein BipB J Bacteriol 187(18):6556-6560 Tang X, Xiao Y and Zhou JM (2006) Regulation of the type III secretion system in phytopathogenic bacteria Mol Plant Microbe Interact 19(11):1159-1166 Tans-Kersten J, Huang H and Allen C (2001) Ralstonia solanacearum needs motility for invasive virulence on tomato J Bacteriol 183(12):3597-3605 Teplitski M, Robinson JB and Bauer WD (2000) Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria Mol Plant Microbe Interact 13(6):637-648 Thibault FM, Hernandez E, Vidal DR, Girardet M and Cavallo JD (2004) Antibtiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents J Antimicrob Chemother 54(6):1134-8 Thongdee M, Gallagher LA, Schell M, Dharakul T, Songsivilai S and Manoil C (2008) Targeted mutagenesis of Burkholderia thailandensis and Burkholderia pseudomallei through natural transformation of PCR fragments Appl Environ Microbiol 74(10):2985-2989 Titball RW, Russell P, Cuccui J, Easton A, Haque A, Atkins T, Sarkar-Tyson M, Harley V, Wren B and Bancroft GJ (2008) Burkholderia pseudomallei: animal models of infection Trans R Soc Trop Med Hyg 102(S1):S111-S116 106 Trakulsomboon S, Pitt TL and Dance DA (1994) Molecular typing of Pseudomonas pseudomallei from imported primates in Britain Vet Rec 135(3):65-6 Troisfontaines P and Cornelis GR (2005) Type III secretion: more systems than you think Physiology 20:326-339 Uehlinger S, Schwager S, Bernier, SP, Riedel K, Nyuyen DT, Sokol PA and Eberl L (2009) Identification of specific and universal virulence factors in Burkholderia cenocepacia strains by using multiple infection hosts Infect Immun 77(9):4102-4110 Van Gijsegem F (1997) In planta regulation of phytopathogenic bacteria virulence genes: relevance of plant-derived signals European J Plant Pathol 103(4):291-301 Warawa J and Woods DE (2002) Melioidosis vaccines Expert Rev Vaccines 1(4):477-482 Warawa J and Woods DE (2005) Type III secretion system cluster is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model FEMS Microbiol Lett 242(1):101-108 White NJ (2003) Melioidosis Lancet 361(9370):1715-22 Whitmore A and Krishnaswami CS (1912) An account of the discovery of a hitherto undescribed infective disease occurring among the population of Rangoon Indian Med Gaz 47:262-267 107 Wiersinga WJ, van der Poll T, White NJ, Day NP and Peacock SJ (2006) Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei Nat Rev Microbiol 4(4): 272-82 Winstanley C, Hales BA and Hart CA (1999) Evidence for the presence in Burkholderia pseudomallei of a type III secretion system-associated gene cluster J Med Microbiol 48(7):649-656 Wolfgang MC, Kulasekara BR, Liang X, Boyd D, Wu K, Yang Q, Miyada CG and Lory S (2003) Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa Proc Natl Acad Sci U S A 100(14):8484-8489 Wuthiekanun V and Peacock SJ (2006) Management of melioidosis Expert Rev Anti Infect Ther 4(3):445-55 Wuthiekanun V, Smith MD, Dance DA and White NJ (1995a) The isolation of Pseuodomonas pseuodmallei from soil in Northeastern Thailand Trans R Soc Trop Med Hyg 89(1):41-43 Wuthiekanun V, Smith MD and White NJ (1995b) Survival of Burkholderia pseudomallei in the absence of nutrients Trans R Soc Trop Med Hyg 89(5): 491 108 Appendix I Accumulative data for the infection of tomato plants with B thailandensis (Daily Disease Score) Collation of Disease Score for B thailandensis Day Day Day Plant Plant Plant 1 4 5 44 2 5 87 1 3 1 1 2 45 2 5 88 2 3 5 1 4 46 2 5 89 2 3 1 3 4 47 2 5 90 2 5 1 3 4 48 2 5 91 2 3 2 49 2 5 92 1 5 2 50 2 3 93 1 3 2 2 51 1 4 94 2 3 2 3 52 1 5 95 2 5 10 2 4 53 2 5 96 3 5 11 2 4 54 1 2 5 97 2 5 12 2 55 3 5 98 1 3 13 2 56 2 5 99 2 5 14 2 57 1 2 5 100 2 3 15 2 4 58 3 5 101 1 3 16 1 4 59 1 5 102 2 3 17 2 3 60 1 5 103 2 3 18 1 4 61 1 5 104 2 3 19 1 4 62 1 5 105 3 20 2 5 63 5 106 3 21 1 64 5 107 3 22 2 65 2 5 108 1 3 23 2 5 66 1 5 109 2 3 24 1 67 2 5 110 2 3 25 2 4 68 1 5 111 2 3 26 1 69 1 5 112 2 3 27 3 4 70 1 3 5 113 2 5 28 1 5 71 1 3 5 114 2 3 29 1 5 72 1 3 5 115 1 3 30 2 5 73 2 3 116 3 5 31 2 5 74 2 3 117 2 5 32 1 3 75 2 3 118 1 5 33 2 76 1 3 119 2 5 34 2 3 5 77 1 4 5 120 2 5 35 2 5 78 2 3 121 2 5 36 2 5 79 2 3 122 2 5 37 2 4 80 2 3 123 2 5 38 2 5 81 2 4 5 124 1 5 39 2 5 82 2 3 5 125 1 5 40 2 5 83 2 3 126 1 5 41 1 2 4 84 1 3 127 2 5 42 2 5 85 2 3 128 3 5 43 2 5 86 2 3 129 1 5 1.7 1.9 2.5 3.6 4.3 4.9 Ave 109 Appendix II Accumulative data for the infection of tomato plants with B pseudomallei (Daily Disease Score) Collation of Disease Score for KHW Day Day Day Plant Plant Plant 1 2 2 51 2 5 101 2 4 5 1 1 52 1 5 102 1 3 1 1 53 3 4 103 1 5 1 2 54 3 104 1 3 5 1 2 3 55 1 3 105 1 3 5 2 4 56 1 3 5 106 1 2 5 1 1 2 57 1 3 107 1 3 5 2 58 1 3 5 108 1 3 5 2 3 59 1 3 5 109 1 3 5 10 2 60 4 5 110 2 5 11 2 3 61 4 5 111 1 3 5 12 2 3 62 3 5 112 1 3 5 13 2 2 63 1 5 113 2 3 14 1 2 64 1 3 5 114 2 4 15 2 3 65 1 3 115 2 3 16 2 2 66 2 3 5 116 2 4 17 1 2 67 1 5 117 1 5 18 2 68 2 5 118 1 3 4 19 1 4 69 3 5 119 1 3 5 20 2 5 70 4 5 120 1 3 5 21 2 5 71 4 5 121 2 3 5 22 2 5 72 3 5 122 2 3 5 23 2 73 2 3 123 2 3 24 1 2 74 1 5 124 2 5 25 1 2 75 2 5 125 1 3 26 1 2 4 76 2 5 126 2 5 27 1 5 77 2 4 127 2 3 28 1 3 78 1 5 128 2 3 29 2 5 79 1 4 5 129 1 3 4 30 2 5 80 2 5 130 1 3 5 31 2 5 81 2 3 131 1 3 5 32 2 5 82 1 5 132 1 3 33 1 3 5 83 3 133 1 3 5 34 3 5 84 1 5 134 2 3 5 35 1 3 5 85 4 5 135 1 36 1 3 4 86 4 5 136 2 5 37 1 3 87 1 5 137 1 5 38 2 3 5 88 4 5 138 1 4 39 1 3 5 89 1 4 139 1 5 40 1 90 1 3 140 1 5 41 3 5 91 4 5 141 1 5 42 2 3 5 92 2 3 5 142 2 5 110 43 3 5 93 2 143 2 44 2 3 94 1 144 1 3 5 45 2 5 95 1 2 145 2 5 46 2 5 96 1 3 146 1 5 47 1 3 97 1 3 147 1 2 5 48 1 5 98 1 3 148 1 3 5 49 2 5 99 1 5 149 1 3 5 50 1 5 100 1 3 150 2 3 151 1 2 5 152 2 4 Ave 1.6 1.8 2.4 3.1 4.2 4.6 sd 0.5 0.6 0.6 0.7 0.8 0.7 111 Appendix III Accumulative data for the infection of tomato plants with B pseudomallei KHW∆TTSS1 mutant (Daily Disease Score) Collation of Disease Score KHW∆TTSS1 Day Plant Day 1 2 2 1 2 2 1 2 2 1 1 1 1 1 1 1 Plant Day 35 1 1 36 1 1 2 37 1 2 38 1 1 1 39 1 2 40 1 2 1 1 41 1 1 1 1 42 1 Plant 69 1 1 2 70 1 1 1 71 1 1 1 2 72 1 1 2 73 1 1 2 2 74 1 1 2 1 2 75 1 1 1 1 2 76 1 1 1 1 2 2 43 1 2 3 77 1 1 2 10 2 2 3 44 1 1 1 78 1 1 2 11 1 1 2 45 1 1 2 79 1 1 1 12 2 2 3 46 1 1 2 80 1 1 2 13 1 2 47 1 1 2 81 1 1 14 1 2 2 48 1 1 2 82 1 1 15 1 2 3 49 1 1 2 83 1 1 2 16 1 2 2 50 1 1 2 84 1 1 17 1 2 2 51 1 1 2 85 1 1 2 18 1 2 3 52 1 1 2 86 1 1 1 19 1 2 2 53 1 1 1 87 1 1 1 20 2 2 54 1 1 1 88 1 1 2 21 1 2 2 55 1 1 1 89 1 1 1 22 1 1 2 56 1 1 2 90 1 1 1 23 1 1 3 57 1 2 2 91 1 1 24 1 1 2 58 1 1 1 92 1 1 25 1 2 2 59 1 1 1 93 1 2 26 1 2 2 60 2 2 2 94 1 1 1 27 1 2 2 61 1 1 1 95 1 1 2 28 1 2 2 62 1 1 2 96 1 1 29 1 2 3 63 1 2 97 1 1 30 1 1 64 1 1 1 98 1 1 31 1 1 3 65 1 1 99 1 1 1 32 1 1 66 1 1 1 100 1 1 1 33 1 2 3 67 1 1 1 101 1 1 34 1 2 68 1 1 Ave 1.1 1.5 1.7 1.8 2.2 2.8 sd 0.3 0.5 0.5 0.4 0.6 0.8 112 Appendix IV Accumulative data for the infection of tomato plants with B pseudomallei KHW∆TTSS2 mutant (Daily Disease Score) Collation of Disease Score KHW∆TTSS2 Day Plant Day 1 2 2 1 2 3 1 2 2 1 1 1 1 1 1 1 1 Plant Day 35 1 1 2 36 1 2 2 37 1 1 2 38 1 2 39 1 1 40 1 1 1 41 1 1 1 42 1 Plant 69 1 2 70 1 2 71 1 1 1 2 72 1 1 1 1 73 1 2 2 1 74 1 2 2 1 2 75 1 2 2 1 2 76 1 1 1 2 2 43 1 1 2 77 1 1 1 10 1 1 3 44 1 1 1 78 1 2 2 11 1 1 1 45 1 1 2 79 1 2 12 1 1 1 46 1 2 2 80 1 1 13 1 1 2 47 1 1 1 81 1 1 2 14 1 1 1 48 1 1 82 1 2 2 15 1 1 1 49 1 1 2 83 1 2 2 16 1 1 1 50 1 1 2 84 1 2 3 17 1 1 51 1 1 1 85 1 1 18 1 1 1 52 1 2 3 86 1 1 2 19 1 1 1 53 1 1 1 87 1 1 2 20 1 1 2 54 1 1 2 88 1 2 21 1 1 55 1 2 2 89 1 1 2 22 1 1 1 56 1 1 1 90 1 2 2 23 1 2 57 1 1 91 1 1 2 24 1 1 1 58 1 2 92 1 1 2 25 1 1 3 59 1 1 2 93 1 1 26 1 1 2 60 1 2 2 94 1 1 2 27 1 1 2 61 1 1 95 1 2 28 1 1 1 62 1 1 2 96 1 1 1 29 1 1 1 63 1 2 2 97 1 1 30 1 1 1 64 1 1 1 98 1 1 1 31 1 2 3 65 1 1 1 99 1 1 1 32 1 1 3 66 1 2 3 100 1 1 2 33 1 1 2 67 1 2 101 1 2 2 34 1 2 2 68 1 2 Ave 1 1.1 1.2 1.5 1.8 2.4 sd 0 0.4 0.4 0.6 0.8 0.9 113 Appendix V Accumulative data for the infection of tomato plants with B pseudomallei KHW∆TTSS3 mutant (Daily Disease Score) Collation of Disease Score KHW∆TTSS3 Day Day Day Plant Plant Plant 1 2 4 35 2 69 1 2 1 2 3 36 1 2 3 70 1 2 3 1 2 3 37 2 2 71 1 2 2 1 2 3 38 1 2 72 1 5 1 3 5 39 2 2 73 1 1 2 1 2 4 40 2 3 74 1 1 1 3 41 1 1 2 75 1 1 1 2 3 42 1 2 4 76 1 1 1 1 43 1 1 77 1 2 10 1 1 3 44 1 1 78 1 2 11 1 2 2 45 1 1 2 79 1 2 12 1 1 2 46 1 1 2 80 1 2 2 13 1 1 2 47 1 2 81 1 2 2 14 1 2 3 48 1 3 5 82 1 1 2 15 1 1 3 49 1 1 4 83 1 2 2 16 1 1 2 50 1 1 2 84 1 1 3 17 1 2 51 1 2 3 85 1 1 3 18 1 2 52 1 2 4 86 1 19 1 2 3 53 1 1 3 87 1 1 20 1 1 54 1 88 1 1 21 1 2 3 55 1 1 2 89 1 1 22 1 1 3 56 1 1 2 90 1 1 23 1 2 3 57 1 2 3 91 1 1 24 1 1 2 58 1 2 3 92 1 1 25 1 2 2 59 1 1 4 93 1 2 26 1 1 2 60 1 1 94 1 2 2 27 1 1 2 61 1 1 3 95 1 1 28 1 2 2 62 1 1 96 1 1 29 1 1 3 63 1 1 2 97 1 1 2 30 1 1 64 1 1 2 98 1 3 31 1 2 65 1 1 3 99 1 2 2 32 1 2 66 1 2 100 1 1 33 2 2 67 1 2 2 Ave 1.0 1.4 1.6 2.2 2.7 3.2 34 1 2 68 1 1 0.2 0.5 0.6 0.6 0.8 0.9 sd 114 Appendix VI Accumulative data for the infection of tomato plants with B pseudomallei KHW∆TTSS1/2 mutant (Daily Disease Score) Collation of Disease Score KHW∆TTSS1/2 Day Day Day Plant Plant Plant 1 1 4 45 1 2 89 1 1 2 1 1 2 46 1 1 2 90 1 1 2 1 1 3 47 1 1 91 1 1 2 1 1 2 48 1 1 1 92 1 1 2 1 1 2 49 1 1 2 93 1 1 3 1 1 50 1 1 2 94 1 1 3 1 1 2 51 1 1 3 95 1 1 3 1 1 52 1 1 2 96 1 3 1 2 3 53 1 1 97 1 2 10 1 1 2 54 1 1 3 98 1 1 2 11 1 1 3 55 1 2 3 99 1 2 12 1 1 3 56 1 1 1 100 1 2 2 13 1 1 57 1 2 2 101 1 2 14 1 2 58 1 1 2 102 1 2 2 15 1 1 3 59 1 1 103 1 2 3 16 1 1 3 60 1 1 3 104 1 1 2 17 1 1 2 61 1 2 4 105 1 1 2 18 1 1 3 62 1 1 4 106 1 1 3 19 1 1 1 63 1 107 1 1 3 20 1 1 1 64 1 1 3 108 1 1 2 21 1 2 65 1 1 109 1 1 22 1 1 66 1 2 110 1 2 3 23 1 1 67 1 111 1 1 1 24 1 2 68 2 112 1 1 2 25 1 2 69 1 1 113 1 1 2 26 1 1 70 1 1 3 114 1 1 2 27 1 71 1 1 115 1 2 28 1 1 72 1 1 116 1 1 29 1 1 73 1 1 117 1 1 30 1 1 74 2 118 1 1 2 31 1 1 75 1 2 119 1 1 2 32 1 1 76 1 2 120 1 2 33 1 2 4 77 1 1 121 1 2 2 34 1 2 4 78 1 1 122 1 2 3 35 1 1 79 1 1 123 1 1 2 36 1 2 80 1 1 2 124 1 1 2 37 1 2 5 81 1 2 4 125 1 1 2 38 1 5 82 1 4 126 1 2 3 39 1 1 83 1 1 4 127 1 2 2 40 1 5 84 1 1 128 1 1 2 41 1 1 2 85 1 1 2 129 1 2 2 115 42 1 2 86 1 2 3 130 1 1 2 43 1 1 3 87 1 1 2 131 1 1 2 44 1 1 2 88 1 2 132 1 1 133 1 2 Ave 1.0 1.1 1.3 2.0 3.1 3.8 sd 0.1 0.3 0.5 0.6 0.9 1.1 116 Appendix VII Accumulative data for the infection of tomato plants with various strains of B pseudomallei (Daily Disease Score) K9 10 11 12 13 Ave sd 77/96 10 11 12 13 14 Ave sd Day 1 1 1 1 1 1 1 Day 1 1 1 1 1 1 1 1 Day 1 1 1 1 1 1.15 0.38 Day 2 1 1 2 1 1 1.21 0.43 Day 2 2 1 1 2 2 1.62 0.51 Day 2 2 2 2 2 1.79 0.43 Day 2 2 2 2 2.23 0.44 Day 2 2 2 2 2 2.14 0.36 Day 3 3 2 3 2.92 0.64 Day 3 3 3 3 3 3.07 0.47 Day 4 4 3 4 3.69 0.63 Day 4 3 3 4 4 3.71 0.61 Day 5 5 5 5 4.54 0.78 Day 5 4 4 5 5 5 4.50 0.65 117 109/96 10 11 12 13 Ave sd 561 10 11 12 13 14 Ave sd Day 1 1 1 1 1 1 1 Day 1 1 1 1 1 1 1 1 Day 1 1 1 1 1.23 0.44 Day 1 1 1 1 1 1.14 0.36 Day 2 2 2 2 2 1.77 0.44 Day 2 2 1 2 2 1.64 0.50 Day 2 2 3 2 2.31 0.48 Day 2 2 2 2 2 2 2.07 0.27 Day 3 3 3 4 3 3.15 0.55 Day 2 3 2 3 3 3 2.64 0.50 Day 3 3 5 3.69 0.75 Day 3 4 3 3 4 3.43 0.51 Day 4 4 5 5 4.54 0.52 Day 4 5 4 4 5 5 4.50 0.52 118 612 10 11 12 13 14 Ave sd Day 1 1 1 1 1 1 1 1 Day 1 1 1 1 1 1.14 0.36 Day 2 1 2 2 2 1.64 0.50 Day 3 2 2 2 2 2.29 0.47 Day 3 2 3 3 2.71 0.73 Day 4 3 3 4 4 3.50 0.52 Day 4 4 3 5 5 4.29 0.73 490 10 11 12 13 14 Ave sd Day 1 1 1 1 1 1 1 1 Day 2 1 1 1 1 1 1.21 0.43 Day 2 1 1 2 2 1.57 0.51 Day 3 2 2 2 3 2 2.29 0.47 Day 3 2 2 2 4 3 2.71 0.73 Day 4 3 3 3 3.50 0.65 Day 5 4 4 4 5 5 4.57 0.51 119 ... Pseudomonas syringae and R solanacearum have been elucidated using Arabidopsis as a plant model (Quirino and Bent, 2003) 3.2 Materials and Methods 3.2.1 Plant Materials 3.2.1.1 Tomato and Arabidopsis... and can be isolated from rice paddy fields in endemic areas such as Thailand Dharakul and Songsivilai first raised the question of a relationship between B pseudomallei and plants (Dharakul and. .. Rice and Arabidopsis plantlets were also infected with B pseudomallei and B thailandensis to evaluate their potential as plant models for 48 disease Rice is chosen for evaluation as B pseudomallei