Sequence diversity among avocado sunblotch viroids isolated from single avocado trees

In previous studies we developed an avocado sunblotch viroid (ASBVd) specif ic reverse transcription-polymerase chain reaction (RT-PCR) protocol and te sted all avocado germplasm accessions in the U.S. National Germplasm Reposi tory (NGR) for ASBVd. It was found that 19% of the accessions were infected with the viroid. To characterize the population of sequence variants, sing le trees of seven accessions with a history of infection were selected. RT- PCR products were cloned and sequenced from these trees and a total of 60 s equence variants were identified from 122 clones. Most of the variants were found to be unique to the individual tree from which they were isolated. T he fraction of unique variants was 81% in 'Lima Late', 81% in 'Aycock Red', 67% in 'Hawaii', and 60% in 'Young Special'. Analysis of the sequence data suggests that variants evolved within a tree rather than arising from mult iple infection events, supporting the quasispecies concept for ASBVd. Chang es in the right terminal loop (RTL), previously associated with leaf varieg ation and bleaching, were found in all variants isolated from bleached tiss ue; however, 16 clones isolated in nonsymptomatic tissue also had changes i n the RTL. The addition of a U between bases 115 and 118 was found in 21 of the 23 clones isolated from the bleached tissue but not found in the 99 cl ones isolated from green tissue, These data suggest that the insertion of a U between bases 115 and 118 is responsible for the bleaching condition.