Identification and Preliminary Characterization of a Viroid-like RNA in Atalantia citroides

Abstract

Atalantia citroides propagated on rough lemon rootstock was graft-inoculated in the scion with an artificial mixture of Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III) and Citrus viroid IV (CVd-IV). Three years later, bioassays in Etrog citron and slot-blot hybridization with viroid specific probes showed that all the inoculated viroids were detected in the rootstock but none in the scion. However, sequential polyacrylamide gel electrophoresis (sPAGE) showed the presence in the A. citroides scion of a viroid-like RNA with an electrophoretic mobility between HSVd and CVd-III. Further analysis by denaturing PAGE of purified preparations thereof revealed two bands with the mobility expected for the circular and linear forms of viroids. Infectivity of the viroid-like RNA was demonstrated by graft transmission to Etrog citron as well as by slash inoculation with purified preparations. Sequencing of three partial-length cDNA clones of this new RNA species, obtained by retrotranscription and PCR amplification (RT-PCR) approach that does not require prior sequence knowledge showed that the sequences corresponded to the central conserved region characteristic of members of the genus Apscaviroid. Complete molecular characterization is in progress.