Biological and Molecular Characterization of Two Isolates of Citrus Viroids Recovered from Cuban Plantations

Abstract

Two field sources (PM 26-X and Cl 13-24) were subjected to biological and biochemical analysis for citrus viroids. PM 26-X was recovered from Frost Marsh grapefruit grafted on alemow showing chlorosis, stunting and gum impregnation of the bark, and Cl 13-24 was recovered from a symptomless Clementine tree grafted on Troyer citrange. Biological indexing on Etrog citron, Parson’s Special mandarin and clemeline 11-20 showed symptoms characteristic of citrus viroids. Viroid infection was demonstrated by sPAGE and molecular hybridization analysis of nucleic acid preparations from the inoculated citrons. The nucleic acid extracts were subjected to retrotranscription, PCR amplification, cloning, SSCP analysis and sequencing. In all instances a “master sequence” was identified as the most frequent variant and was compared to other reported sequences of the same viroids.