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

Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases
dried on 3MM blotting paper (Czosnek et al., 1988). Dot-blotted samples were CVMV,
CVMVMxd, RL1, RL2, RL5, K1, K2, K3, IGl, MB8, KN, MB9, IG2, MB7, MB2, IG3,
MB6, and ISOPOT. Two positive samples, i.e. TYLCV-EG and Bean golden mosaic
virus (BGMV), obtained from Maxwell Laboratory, University of Wisconsin-Madison,
were used as positive controls, while samples taken from a healthy tomato plant raised in
our screen house, at Kawanda Agricultural Research Institute, was used for a negative
control.
Out of 75 samples, which had clear leaf curl symptoms and were tested with DNA
hybridisation, 15 were tested as dot-blots in TE buffer, and 60 were squash-blots. Each
sample was tested against both general and specific probes. For the 15 dot-blots,
Dellaporta DNA extraction had to be done (Dellaporta et al., 1983). These samples were
then hybridised using a mixed probe of radioactive BGMV and TYLCV-EG probes.
Membranes were exposed to two charged X-ray films for different durations, i.e. 6 hours
exposure at 4 °C, overnight at -80 °C, or two days exposure respectively (Nahkla et al.,
1993).
Preparation of P³²-labelled Probes
To prepare P³²-labelled probes, the PCR product of Tomato yellow leaf curl virus DNA,
provided by Dr. Medhat Nahkla of Maxwell Laboratory, was thawed on ice and TE (Tris-
EDTA) buffer added (Nahkla et al., 1993). It was then denaturated by heating in a water
bath at 100 ºC for 2 min, and chilled rapidly on ice. Labelling buffer (5X), a mixture of
dNTPs (dTTP, dATP, dGTP), a P³²-labelled dCTP, Klenow DNA polymerase I, and
Nuclease-Free BSA were added together to a final volume of 50 µl.
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