ENZYMATIC CDNA AMPLIFICATION OF CITRUS-EXOCORTIS AND CACHEXIA VIROIDS FROM INFECTED CITRUS HOSTS

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.