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

Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases
3.2.2 Identification of Viral Diseases
3.2.2.1 Serological Identification of Tomato Viruses
Double Antigen Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA), as
described by Clark and Adams (1977), was selected because it is efficient and cheap, and
after all Nono-Womdim et al. (1996) used it successfully to identify tomato viruses in
Tanzania. Therefore, we used DAS-ELISA to confirm that symptoms seen in the field
were actually due to virus causal organisms. The technique was also used to identify
those viruses infecting collected tomato leafy symptom-bearing samples. Mottling and
mosaic symptom-bearing tomato leaf samples, which were collected, dried and preserved
over calcium chloride, were then packed in self-sealing polythene bags, with five grams
per bag. Dry samples were then crushed to powder. A total of 10 ml of extraction buffer
(0.03 M phosphate buffer, pH 7.4) was added to each bag. The mixture was ground with
a pestle to extract sap, which was used for ELISA tests (Bar-Joseph and Hull, 1974).
Using DAS-ELISA, mottling and mosaic symptoms-bearing tomato leaf sample extracts
were tested for eight viruses commonly encountered in Africa (Brunt et al., 1990; Nono-
Womdim et al., 1996) and for which antisera could be obtained commercially. These
were ToMV, TSWV, PVMV, PVY, PVX, CMV, AMV and ChiVMV. Polyclonal
antisera and conjugates for these viruses were obtained from SANOFI, France.
Based on standard dilution recommendations, polystyrene microtiter plates were coated
with immunoglobulins (IgG) of the eight viruses at the following manufacturer’s given
concentrations; 30µl/15ml (ToMV), 300µl/15ml (PVMV), 30µl/15ml (TSWV),
60µl/15ml (PVY), 150µl/15ml (PVX), 150µl/15ml (CMV), 30µl/15ml (AMV) and
150µl/15ml (ChiVMV) in sodium carbonate coating buffer (0.2M, pH 9.6), and incubated
for 3 hours at 37 ºC to achieve maximum detection of target viruses. One hundred
microlitres of coating buffer and immunoglobulins solution was put in each well. Plates
were then washed three times with 0.01 M phosphate-buffered saline (PBS-Tween 20)
buffer, and dried over tissue paper (Nono-Womdim and Atibalentja, 1993). Plate wells
were filled with 0.10 ml of tomato sample extract ground in PBS pH 7.4 (1:2, W/V) and
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