Direct Nucleotide Sequence of PCR-amplified DNAs of the Closely Related Citrus Viroids IIa and IIb (Cachexia)

Citrus viroids IIa (CVdIIa) and IIb (CVdIIb=cachexia), members of the hop stunt viroid (HSVd) group, can be distinguished by their biological properties in Etrog citron. CVdIIa and CVdIIb isolates from Florida were reverse transcribed and subsequently amplified in vitm from total nucleic acid extracts of infected citrus tissue using a reverse transcription-polymerase chain reaction assay (RT-PCR). The amplified products were gel purified, and subsequently sequenced using five distinct sequencing primers containing different segments of the HSVd prototype sequence. These products were sequenced without cloning or the generation of asymmetric PCR products using the fmol'" sequencing system. CVdIIa is 302 nucleotides in length. CVdIIb (cachexia) was determined to be 299 nucleotides in length and differs from CVdIIa by 3 nucleotide deletions and 2 nucleotide substitutions. CVdIIa and CVdIIb share 3 nucleotide substitutions which differed from the two published Japanese HSVd citrus variant sequences. CVdIIa shares greater than 99% sequence similarity with the Japanese HSVd variants. The HSVd variant associated with the grapefruit dwarfing agent from Israel is similar in size to CVdIIb, however these two viroids have very different biological properties and shares only 96% similarity with the published sequence. Index words: citrus, ~iroids, cachexia, RT-PCR, sequencing. Citrus trees are known to be naturally infected with five distinct viroid groups. These groups are characterized by molecular size, as determined by electrophoretic mobility and nucleotide sequencing, nucleic acid homology, as determined by hybridization with group-specific probes, and biological properties in various hosts, specifically Etrog citron (4,5,6,7, 10). Presently, the best characterized citrus viroid is citrus exocortis viroid (CEVd), the causal agent ofthe exocortis bark shelling disease in some citrus species (9). In citron, exocortis induces severe stunting, leaf epinasty, and veinal necrosis. During evaluation of citrus infected with CEVd in Japan, what was thought to be amild strainof CEVd was found in citron. When mechanically transmitted to curcurbits, this putative mild CEVd variant induced symptoms indistinguishable from hop stunt viroid (HSVd). Sequence analysis of this viroid confirmed it was a vari*Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the products by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. This article reports the results of research only. ant of HSVd (6). To date, nine HSVd variants exist, three of which infect citrus (4, 10). HSVd variants infecting citrus possess physical and biological properties similar to the group I1 citrus viroids (CVdII) and are classified as such (8). These include CVdIIa, the causal agent of a mild exocortis disease of citrus, and CVdIIb the causal agent of the cachexia disease of citrus. Under field conditions, CVdIIa induces a mild bark cracking in trifaliate orange and no reaction in mandarin or tangelo. CVdIIa induces leaf-tip browning, petiole wrinkling, and mid-vein necrosis in citron under controlled greenhouse conditions. CVdIIb causes the cachexia disease in citrus (8). In the field CVdIIb is symptomless in trifoliate orange andinducesgumrning, wood pitting, and bark plugging in mandarin and tangelo. In citron, under greenhouse conditions, CVdIIb is characterized by a latent infection (7). CVdIIa and CVdIIb differ in size by only a few nucleotides (7, l l ) , yet they cause two distinctly different diseases of citrus. Sequencing of CVdIIa and CVdIIb should establish their relatedness to each other, and to the Japanese HSVd citrus variants (lo),