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Title: THE USE OF RT-PCR IN THE FLORIDA CITRUS VIROID INDEXING PROGRAM

Author
item SIEBURTH, P - WINTER HAVEN FL
item IREY, M - US SUGAR CROP FLORIDA
item GARNSEY, S - UNIV OF FL LAKE ALFRED FL
item Owens, Robert

Submitted to: Proceedings of the Conference of the International Organizaion of Citrus Vi
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/2/2002
Publication Date: N/A
Citation: N/A

Interpretive Summary: Viroids are the smallest pathogens yet described; small, circular, single-stranded RNA molecules that encode no proteins. Yet, like the much larger viruses, are able to replicate autonomously in infected host plants. Certain viroids cause economic damage to commercial citrus production, and budwood certification programs must test for endemic, graft-transmissible viruses and viroids. In Florida, the largest volume of testing is for citrus tristeza virus and citrus viroids. Here, we describe improved PCR (polymerase chain reaction)-based methodologies for viroid detection using both field trees and greenhouse-grown indicator hosts. Using these methodologies, we identified a new variant of citrus viroid III (CVd-III) and demonstrated for the first time the presence of CVd-IIIa in Florida. Our findings will be of greatest interest to researchers and regulatory officials concerned with virus and viroid diseases of citrus.

Technical Abstract: RT-PCR has been incorporated as an adjunct to the biological indexing program for viroids, especially to test for CVd II, and for all viroids where rapid results are needed. The sensitivity of RT-PCR for testing of composite samples was evaluated with 5, 10, 25, 50, 100 and 200 tree composites. Single positives were detected consistently in 5 and 10 tree composites that have been incorporated into the routine RT-PCR testing for CVd II from citron. When positive samples are found, the sources comprising the composite are re-tested individually. Viroid infected sources found by biological indexing were tested by RT-PCR for CVd II, CVd III and CEVd from both the citron indicators and the corresponding field trees to evaluate the validity of RT-PCR testing. The ability to detect all three viroids from field samples by RT-PCR is important for decreasing testing time and reducing costs. RT-PCR also allows rapid diagnosis of field samples and testing of trees near a citrus canker quarantine zone without exposing our biological test facilities to potential sources of citrus canker inoculum. Agarose gel electrophoresis is adequate for visualization of CVd II RT-PCR products. The sensitivity of detection for CEVd and CVd III has been increased by visualization of RT-PCR products in either acrylamide gels with GelStar stain or Metaphor agarose gels. Improved primers for CEVd have increased our confidence for detecting this viroid by RT-PCR. PCR products from 32 Florida isolates that cause a mild citron reaction were screened with oligonucleotide probes specific for CVd IIIa or CVd IIIb. These viroids were found to occur both separately and as mixtures.