Analysis of the population structure of three phenotypically different PSTVd isolates

Phenotypically dissimilar greenhouse isolates from a Polish collection of potato spindle tuber viroid (PSTVd) were analysed. Partially purified PSTVd genomic RNAs from severe, intermediate and mild isolates was reverse transcribed and the resulting cDNAs enzymatically amplified. Abutting-primer PCR (Ab-P PCR) technology was used to obtain, in a single step, infectious full-length PSTVd cDNA monomers and these were sequenced. The mild isolate was found to be composed of a unique molecular variant (M), closely related to previously described PSTVd mild isolates. In the intermediate isolate, three variants, i2, i3 and i4, were detected. The severe isolate was found to be a mixture containing at least four molecular variants: s23, s27, i4 and i2. Infection of test plants with plasmids carrying monomeric cDNAs corresponding to each of the cloned variants confirmed that they are infectious. In addition, variant M produced mild symptoms, variants i2, i3, i4 intermediate symptoms and variants s23 and s27 severe symptoms. Therefore, the disease symptoms produced by a mixture are determined by the severe variants, masking the presence of milder ones. All the variants detected (except i2 which is identical to previously described PSTVd-DI) represent novel PSTVd sequences with point mutations located in the V and/or P domains. In particular, variants s23 and i4 represent shorter (358 nucleotides) versions of the PSTVd genome.