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A Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases B C D E F Figure 3. 6: Some miscellaneous symptoms observed on tomato plants in the surveyed area. Some of these (A, E, F) seem to be due to the plant’s reaction to pesticides 58
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 59
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Incidence, Distribution and Charact
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Charles SSEKYEWA Incidence, Distrib
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CONTENTS 1 CHAPTER 1...............
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ACKNOWLEDGEMENT The following perso
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vii 7 mosaic virus (CMV), Alfalfa m
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ix 9 Consequently, our findings con
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SAMENVATTING Tomaat is een economis
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xiii 13 Beide genhomologie percenta
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xv 15 dit onderzoek geven ook aan d
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LIST OF TABLES xvii 17 Table Page 1
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ACRONYMS AND ABBREVIATIONS xix 19 A
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Abbreviation Word in full PCR Polym
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Incidence, distribution and charact
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78 Incidence, distribution and char
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87 87 Incidence, distribution and c
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A A C D Incidence, distribution and
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Opaque and not smeared Translucent
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Rainfall (cm) Temp. Cº Mean Whitef
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8 ANNEXES Incidence, distribution a
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Table A.1.1 continued Incidence, di
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Annex 5: Incidence, distribution an
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Books Incidence, distribution and c
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