PCR-AMPLIFICATION OF TOMATO YELLOW LEAF CURL VIRUS (TYLCV) DNA FROM SQUASHES OF PLANTS AND WHITEFLY VECTORS - APPLICATION TO THE STUDY OF TYLCV ACQUISITION AND TRANSMISSION

DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitte d by the whitefly Bemisia tabaci, was amplified from squashes of infec ted tomato plants and of viruliferous vectors using the polymerase cha in reaction (PCR). Samples of infected tissues as small as 1 mm(2) wer e squashed onto a nylon membrane. A 1 x 2 mm strip containing the squa sh was introduced into a 25 mu l PCR reaction mix. The reaction produc ts were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from indivi dual viruliferous whiteflies. The same squash could be used several ti mes to amplify different virus DNA fragments with various sets of prim ers. Thus plant and insect squashes can be used as templates for the a mplification of geminiviral DNA with no need to prepare tissue extract s or purify nucleic acids. The squash-PCR procedure was applied to stu dy whitefly transmission of TYLCV. Tomato plants were inoculated by pl acing a single viruliferous insect in the center of a young leaflet. I n some plants TYLCV DNA was detected at the site of inoculation as ear ly as 5 min after the beginning of the access feeding and in all plant s after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the ac cess feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min.