CITRUS TRISTEZA VIRUS ON THE ISLAND OF CRETE: A SURVEY AND DETECTION PROTOCOL APPLICATIONS
M. Shegani, D. Tsikou, A. Velimirovic, H. Afifi, A. Karayanni, A. Gazivoda, K. Manevski, I. Manakos, I.C. Livieratos
Over a period of two years, more than 5,000 citrus trees were tested for the presence of Citrus tristeza virus (CTV) on the island of Crete, resulting in thirty eight positives. Comparisons of the relative transcript levels of CTV p23, coat protein (CP), polymerase (POL) and an intergenic (POL/p33) region using quantitative RT-PCR, revealed consistent differences in abundance for each of these RNAs among flowers, stems, young fruits and leaves of infected orange trees. CTV p23 RNAs accumulated at highest levels, reaching a maximum in the flowers and the lowest levels in the leaves, while POL RNAs consistently accumulated at low levels in all tissues tested. A PCR-amplified dig-labelled CTV p23 DNA probe was applied to stem and leaf prints, and to crude and total RNA leaf extracts, using non isotopic hybridization. This technique, when applied to stem or leaf prints, and particularly to total RNA, unequivocally provided strong signals with minimal backgrounds. Moreover, an antiserum with high sensitivity and specificity of CTV detection as shown by DAS and immuno-print ELISA was produced against bacterially-expressed CTV CP. In the former method, stems and flowers contained higher levels of CTV CP when compared to leaf extracts. Taking into account Cretan geography and the importance of citrus to the island, systematic surveys for CTV eradication, sustainable control measurements and epidemiological studies need to be undertaken. The observations, materials and methods presented here may assist all three tasks at local and national level.