LIMITED SEQUENCE RANDOMIZATION: TESTING A STRATEGY TO PRODUCE IMPROVED CITRUS VIROID DWARFING AGENTS

Authors: Owens, Robert; Thompson, Susan; SIEBURTH, P - WINTER HAVEN FL; GARNSEY, S - UNIV OF FL LAKE ALFRED FL

Submitted to: Proceedings of the Conference of the International Organizaion of Citrus Vi
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2002
Publication Date: N/A
Citation: N/A

Interpretive Summary: Viroids are the smallest pathogens yet described; small, circular, single-stranded RNA molecules that encode no proteins. Yet, like the much larger viruses, are able to replicate autonomously in infected host plants. Viroids cause a number of economically important diseases, but, in a crop like citrus, the dwarfing that is often associated with viroid infection can actually be beneficial. We would like to develop improved viroid dwarfing agents suitable for use under subtropical conditions. To develop the technology necessary to isolate stable strains of citrus viroid III having the desired biological properties, a series of trial experiments were carried out in the greenhouse using an indicator host (Etrog citron) rather than a commercially important rootstock-scion combination. The results of these particular experiments will be of greatest interest to other scientists interested in viroid or virus evolution at the molecular level. Similar experiments have just been completed with citrus viroid III, a promising citrus dwarfing agent. The results of a field trial about to get underway in Florida with the assistance of several growers will be of direct interest to the ultimate customers for this research, Florida citrus growers and other industry groups.

Technical Abstract: Viroid infection of citrus growing on trifoliate orange or trifoliate hybrid rootstocks has been shown to increase fruit yield and lower production costs. We would like to adapt citrus viroid III (CVd-III) for use with other rootstocks and, toward that end, have examined the extent of its natural sequence variability. Mutations at positions 44 and 54 of CVd-IIIb were previously shown to greatly reduce symptom expression on the indicator host Etrog citron. Here, we report the isolation of several novel CVd-III variants following inoculation of citron with a "library" of RNA transcripts in which five positions opposing positions 44 through 54 had been randomized. Individual trees tested six months to a year post inoculation contained complex mixtures of sequence variants, some containing multiple spontaneous changes. Distribution of viroids and viruses in woody hosts is often uneven, and thus, buds removed from three infected trees six months post inoculation were used to inoculate uninfected citrons. The resulting infected trees were also examined for possible sequence differences among the viroid progeny. In some trees, progeny sequences were continuing to evolve as much as two years post inoculation. The effects of selected mutations on the secondary structure and biological properties of CVd-III will be described. Sequence randomization followed by in vivo selection provides a promising method to isolate improved viroid dwarfing agents with specific biological properties.