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

Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases
incubated at 4ºC overnight. After rinsing off excess antigens, 0.10 ml of conjugated IgGs
were added per well and incubated at 37ºC for 3 hours. Plates were again washed with
PBS-Tween 20 and dried before adding 0.10 ml substrate p-nitrophenyl phosphate
(1mg/ml) in 0.1 M diethanolamine substrate buffer at pH 9.8 (Clark and Adams, 1977).
Microtiter plates, which had the substrate added, were incubated for 1-2 hours at room
temperature in a dark place to reduce background reaction. Background reaction refers to
exposure of loaded microtiter plates to excessive light causing very fast reactions, which
cause yellowing of empty wells, hence masking positive sample well reaction. Positive
samples were recognized from wells that turned yellow, while healthy control remained
colourless (Nono-Womdim et al., 1996). Plates were also read with a MULTISKAN MS,
Primary EIA 1.5-0 plate reader with a 405 nm absorbency filter. A well reading was
considered positive if its absorbency value was three times that of a healthy sample well.
Blanks should have absorbency values of less than 0.1 (Nono-Womdim personal
communication). Data generated on number of samples testing positive or negative were
analysed for percentages and frequencies with MSTATC statistical programme.
3.2.2.2 Molecular Identification of Viruses
Molecular techniques were used to identify those viruses, which caused leaf curl in the
field, but could not be detected in serological tests. Geminiviruses are some of the
possible examples of such viruses (Anderson and Morales, 2005). One of the
geminiviruses causing leaf curl is Tomato yellow leaf curl virus (TYLCV).
Tomato yellow leaf curl is one of the most devastating viral diseases of tomato caused by
a number of virus species (Moriones and Navas-Castillo, 2000). These viruses could not
be tested through DAS-ELISA with polyclonal antisera. Therefore, instead of using
DAS-ELISA, molecular techniques like DNA hybridisation (Czosnek, et al., 1988) and
polymerase chain reaction (PCR) (Nakhla and Maxwell, 1998; Navot et al., 1992) were
used. For PCR, TYLCV-specific and general primers were used as shown in Table 3.3,
whereas for DNA hybridisation analysis samples were either dot-blotted or squash-
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