VIEW ARTICLE

Techniques

Enzymatic cDNA Amplification of Citrus Exocortis and Cachexia Viroids from Infected Citrus Hosts. X. Yang, National Germplasm Resources Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 011A, Beltsville, MD 20705, Present address: Institute of Microbiology, The Chinese Academy of Sciences, Beijing, China.; A. Hadidi(2), and S. M. Garnsey(3). (2)National Germplasm Resources Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 011A, Beltsville, MD 20705; and (3)U.S. Horticultural Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 2110 Camden Road, Orlando, FL 32803. Phytopathology 82:279-285. Accepted for publication 16 September 1991. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1992. DOI: 10.1094/Phyto-82-279.

Reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for the detection and identification of citrus exocortis viroid (CEV), citrus cachexia viroid (CCaV), and citrus viroid IIa (CVIIa) from nucleic acid extracts of infected sweet orange or Etrog citron. DNA primers (19-24 nucleotides in length) specific for CEV or hop stunt viroid (HSV) sequence were used for cDNA synthesis and specific amplifications of CEV and the HSV-related CCaV (or CVIIa), respectively. The size of the major RT-PCR product from CEV-infected tissue was the same as full length CEV (371 bp) and hybridized with a SP6-generated CEV cRNA probe. The size of the major RT-PCR product from CCaV or CVIIa-infected tissue was approximately 297 bp and 302–303 bp, respectively, and hybridized with a SP6-generated HSV cRNA probe. These products were absent from amplified extracts of uninfected tissue. The RT-PCR assay is more sensitive than existing detection methods and provides information about viroid detection from sweet orange or Etrog citron without requiring large samples or molecular hybridization.