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

Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases
blotted onto nylon membranes to test for TYLCV DNA. For each sample squashed, a
portion was dried over anhydrous calcium chloride at 4ºC, in petridishes (Green, 1991)
and stored in the refrigerator.
Consequently, every yellow leaf curl symptom-bearing sample that did not test positive
through DAS-ELISA or had very clear TYLCV associated symptoms and was straight
away suspected to be caused by tomato yellow leaf curl viruses (sensu lato), was
subjected to molecular tests (Table 3.2). Of 800 samples tested in serology, 102 tomato
leaf samples were negative to any of the eight viruses. Tomato samples bearing leaf curl,
mosaic and mottling, but not testing positive through DAS-ELISA included: sample MP4
with mosaic symptoms, MP11 with white mosaic symptoms, IG9 with green mosaic
symptoms, IG8 with green mosaic symptoms, MB6 with bronzing, mild mosaic and curl
symptoms, and sample MPz with severe mosaic symptoms. Some samples were tested
more than once depending on their previous reactions.
3.2.2.2.1 Virus Characterization Using DNA Hybridisation
DNA hybridisation was used to test for the presence of TYLCV. It involved preparation
of P³²-labeled probes and hybridisation of squash-blotted or dot-blotted nylon membranes
(Czosnek et al., 1988).
3.2.2.2.1.1 Sample Preparation for DNA Hybridisation
To prepare dot-blotted nylon membranes, preserved dry as well as fresh leaf curl and
yellow mottling symptom bearing tomato leaf samples (Figure 3.3) were squashed in TE
buffer (Tris-EDTA), with Kontes blue pestles in eppendorf tubes and placed on ice
(Nahkla et al., 1993). Squashed samples were centrifuged for 5 min, at 5,000 rpm. Pellets
formed were discarded and supernatant used for dot-blotting (Nahkla et al., 1993).
QIAGEN Nylon Plus membranes from QIAGEN Inc., 28159 Av. Stanford Santa Clarita
CA 91355, were used. For each sample, a dot-blot spot of 5 µl of supernatant was blotted
twice on separate spots on the membrane to minimize errors. Blotted membranes were
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