Incidence, Distribution and Characteristics of Major Tomato Leaf ...

More documents

Recommendations

Info

Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases M13ReversePrimer Spe1 EcoR1 PCR Product EcoR1 M13 Forward Primer CAG GAA ACA GCT ATG AC....GAT CCA ↓CTA GTA ACG GCC GCC AGT GTG CTG G↓ AA TTC GCC CTT ↓ AAG GGC G ↓ AA TTC TGC......CGT CGT GACTGG GTC CTT TGT CGA TAC TG....CTA GGT GAT CAT TGC CGG CGG TCA CAC GAC C TT AAG CGG GAA TTC CCG C TT AAG ACG..…GCA GCA CTG ACC Spe1 EcoR1 EcoR1 54bp 25bp 11bp PCRproduct 7bp 82bp PM13r PM13f(-20 ) Figure 3.9: Part of a linear map of pCR2.1 TOPO vector and sites of action for restriction enzymes used, Spe 1 and EcoR 1(Invitrogen, 1998), and related restriction sites map (Clark and Russell, 1997) 70
Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases Clones that had clear separation of plasmid DNA and insert DNA bands on the gel were subcultured in LB medium with ampicillin and incubated overnight at 37 ºC (Nakhla et al., 1993). Mature cultures were harvested according to the Wizard Plus SV® Minipreps Spin Column protocol. Using a Promega Kit, pure plasmid DNA with the insert DNA fragment was extracted for sequencing. Of the purified DNA, 20 µl were sent to the University of Wisconsin, Biotechnology Centre for sequencing. For the DNA to qualify for sequencing, its concentration had to be between 200–300 ng/µl (Rojas et al., 1993). Therefore, purified DNA concentration readings for tomato leaf sample isolate IG1 DNA (ToLCV - UG) upper region and tomato sample isolate RL5 DNA (TYLCV-UG) common region were measured using a fluorimeter (Starna, UK). To sequence IG1 DNA (ToLCV-UG) upper viral sense/coat protein and RL5 (TYLCV-UG) common region, primers M13 forward and M13 reverse (Biotechnology Centre primers) were used. Primer TLCV-UG rep1f, which was sequenced during this study, was used to sequence IG1 DNA (ToLCV-UG) common region. 3.2.3 Host Range of Two Major Viruses To further understand identified viruses TYLCV and ChiVMV, alternative host weeds and crops found in the tomato agro-ecosystem, were studied. 3.2.3.1 Tomato Yellow Leaf Curl Viruses Host Range Weed and crop samples bearing leaf curl and/or mottling symptoms were collected from the tomato field environment (Table 3.4 and 3.5). Their sap was used in TAS- ELISA tests (Credi et al., 1989) or blotted onto nylon membrane for DNA hybridisation (Czosnek et al., 1988) in the laboratory. A ToLCV-UG probe, labelled using Gene-A labelling kit from Promega, was used under high stringency hybridisation conditions (65º C). Membranes were exposed to Kodak Bio Max MS film with Bio Max Transcreen LE for 24 hours, whereupon the film was developed to show results. 71
Page 1:
Incidence, Distribution and Charact
Page 4 and 5:
Charles SSEKYEWA Incidence, Distrib
Page 6 and 7:
CONTENTS 1 CHAPTER 1...............
Page 8 and 9:
ACKNOWLEDGEMENT The following perso
Page 10 and 11:
vii 7 mosaic virus (CMV), Alfalfa m
Page 12 and 13:
ix 9 Consequently, our findings con
Page 14 and 15:
SAMENVATTING Tomaat is een economis
Page 16 and 17:
xiii 13 Beide genhomologie percenta
Page 18 and 19:
xv 15 dit onderzoek geven ook aan d
Page 20 and 21:
LIST OF TABLES xvii 17 Table Page 1
Page 22 and 23:
ACRONYMS AND ABBREVIATIONS xix 19 A
Page 24 and 25:
Abbreviation Word in full PCR Polym
Page 26 and 27:
Incidence, distribution and charact
Page 28 and 29:
Page 30 and 31:
Page 32 and 33:
Page 34 and 35:
Page 36 and 37:
Table 2.1: The orthography of some
Page 38 and 39:
Page 40 and 41:
Page 42 and 43:
Page 44 and 45: Incidence, distribution and charact
Page 78 and 79: A B Incidence, distribution and cha
Page 80 and 81: A B Incidence, distribution and cha
Page 82 and 83: A Incidence, distribution and chara
Page 106 and 107: Isolate IG1 Incidence, distribution
Page 112 and 113: 1600 500 700 200 Incidence, distrib
Page 126 and 127: 1200 1200 (A) IG1 (ToLCV-UG) PAL1v
Page 132 and 133: 78 Incidence, distribution and char
Page 134 and 135: 87 87 Incidence, distribution and c
Page 144 and 145:
A A C D Incidence, distribution and
Page 146 and 147:
Opaque and not smeared Translucent
Page 148 and 149:
Page 150 and 151:
Page 152 and 153:
Rainfall (cm) Temp. Cº Mean Whitef
Page 154 and 155:
Page 156 and 157:
Page 158 and 159:
Page 160 and 161:
Page 162 and 163:
Page 164 and 165:
Page 166 and 167:
Page 168 and 169:
Page 170 and 171:
Page 172 and 173:
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
Page 180 and 181:
Page 182 and 183:
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Page 190 and 191:
Page 192 and 193:
Page 194 and 195:
Page 196 and 197:
Page 198 and 199:
8 ANNEXES Incidence, distribution a
Page 200 and 201:
Page 202 and 203:
Table A.1.1 continued Incidence, di
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
Page 212 and 213:
Page 214 and 215:
Page 216 and 217:
Annex 5: Incidence, distribution an
Page 218 and 219:
Annex 6: Incidence, distribution an
Page 220 and 221:
Page 222 and 223:
Page 224 and 225:
Page 226 and 227:
Page 228 and 229:
Page 230 and 231:
Page 232:
Books Incidence, distribution and c
show all

Incidence, Distribution and Characteristics of Major Tomato Leaf ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?