Submitted to: Conference of International Organization of Citrus Virologists
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/11/2001
Publication Date: 2/1/2002
Citation: Garnsey, S. M., Zies, D., Irey, M., Sieburth, P. J., Semancik, J. S., Levy, L., Hilf, M. 2002. Practical Field Detection of Florida Citrus Viroids by RT-PCR. Proceedings of the 15th Conference of the International Organization of Citrus Virologists, 219-229. Interpretive Summary: This paper describes the successful use of the polymerase chain reaction (PCR) to detect viroid pathogens in citrus trees in experimental plots and commercial groves in Florida during warm and cool weather periods. The PCR is a very sensitive technique and this paper demonstrates that PCR can be used to detect citrus viroid pathogens very quickly and accurately even when they are in trees in very low quantities.
Technical Abstract: A practical approach to the detection of the three major citrus viroids found in Florida citrus directly from field collected tissue was developed and tested. Reverse transcriptions were done from total nucleic acid extracts obtained by a SDS-potassium acetate extraction procedure from small amounts of tissue pulverized in Tris buffer. Amplifications were done using previously described primers for citrus exocortis viroid (CEVd), hop stunt viroid (CVd II) and citrus viroid III (CVd III), and the products were analyzed by agarose gel electrophoresis. Effects of cultivar, tissue location and sampling time were investigated. CEVd, CVd II, and CVd III viroids could be detected in tissue from experimentally infected orange, grapefruit, lemon, lime, mandarin and mandarin hybrid cultivars, but not reliably from kumquat. Bark tissue from woody, budwood-sized twigs was the best tissue source, and samples collected in warm weather yielded better results than those collected in winter. Field surveys of several hundred trees in commercial groves and test plots with varying viroid content were conducted. Correlation between RT-PCR results and biological indexing exceeded 90%. The methods developed have been used successfully by different personnel in three laboratories. RT-PCR was especially effective for detection of the CVd II, which has been the most difficult citrus viroid to detect biologically or by sequential PAGE. Several isolates that caused moderate symptoms in Etrog citron were not amplified by our CEVd, CVd II and CVd III primers. One was identified as CVd IV, the first detection of this viroid in Florida. The others were identified as sequence variants of CVd III.