Incidence, Distribution and Characteristics of Major Tomato Leaf ...
Incidence, Distribution and Characteristics of Major Tomato Leaf ...
Incidence, Distribution and Characteristics of Major Tomato Leaf ...
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<strong>Incidence</strong>, distribution <strong>and</strong> characteristics <strong>of</strong> major tomato leaf curl <strong>and</strong> mosaic virus diseases<br />
Clones that had clear separation <strong>of</strong> plasmid DNA <strong>and</strong> insert DNA b<strong>and</strong>s on the gel were<br />
subcultured in LB medium with ampicillin <strong>and</strong> incubated overnight at 37 ºC (Nakhla et<br />
al., 1993). Mature cultures were harvested according to the Wizard Plus SV® Minipreps<br />
Spin Column protocol. Using a Promega Kit, pure plasmid DNA with the insert DNA<br />
fragment was extracted for sequencing. Of the purified DNA, 20 µl were sent to the<br />
University <strong>of</strong> Wisconsin, Biotechnology Centre for sequencing. For the DNA to qualify<br />
for sequencing, its concentration had to be between 200–300 ng/µl (Rojas et al., 1993).<br />
Therefore, purified DNA concentration readings for tomato leaf sample isolate IG1 DNA<br />
(ToLCV - UG) upper region <strong>and</strong> tomato sample isolate RL5 DNA (TYLCV-UG)<br />
common region were measured using a fluorimeter (Starna, UK). To sequence IG1 DNA<br />
(ToLCV-UG) upper viral sense/coat protein <strong>and</strong> RL5 (TYLCV-UG) common region,<br />
primers M13 forward <strong>and</strong> M13 reverse (Biotechnology Centre primers) were used.<br />
Primer TLCV-UG rep1f, which was sequenced during this study, was used to sequence<br />
IG1 DNA (ToLCV-UG) common region.<br />
3.2.3 Host Range <strong>of</strong> Two <strong>Major</strong> Viruses<br />
To further underst<strong>and</strong> identified viruses TYLCV <strong>and</strong> ChiVMV, alternative host weeds<br />
<strong>and</strong> crops found in the tomato agro-ecosystem, were studied.<br />
3.2.3.1 <strong>Tomato</strong> Yellow <strong>Leaf</strong> Curl Viruses Host Range<br />
Weed <strong>and</strong> crop samples bearing leaf curl <strong>and</strong>/or mottling symptoms were collected from<br />
the tomato field environment (Table 3.4 <strong>and</strong> 3.5). Their sap was used in TAS- ELISA<br />
tests (Credi et al., 1989) or blotted onto nylon membrane for DNA hybridisation<br />
(Czosnek et al., 1988) in the laboratory. A ToLCV-UG probe, labelled using Gene-A<br />
labelling kit from Promega, was used under high stringency hybridisation conditions (65º<br />
C). Membranes were exposed to Kodak Bio Max MS film with Bio Max Transcreen LE<br />
for 24 hours, whereupon the film was developed to show results.<br />
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