Mechanical inoculation of Nicotiana tabacum with the PSTVd isolate KF 440-2 from the host plant tomato resulted in the de novo emergence, replication, and accumulation of a new "tobacco variant," designated PSTVd NT. It produces no symptoms in tobacco but, like PSTV KF 440-2, severe ones in tomato. The sequence analysis of PSTVd NT revealed a single nucleotide substitution from C-->U at position 259. Autonomous viroid replication was also induced in tobacco by genomic integration of oligomeric cDNA copies of PSTVd KF 440-2. Although these cDNAs contained the original tomato-specific C259, the circular PSTVd RNA subsequently accumulating in tobacco also exhibited the C259-->U259 substitution. In the secondary structure of PSTVd, nucleotide 259 is part of an internal loop analogous to loop E of eukaryotic 5S rRNA and presumed to be the only bulged extrahelical nucleotide of this loop. The C259 in PSTVd KF 440-2 and in practically all other isolates and the U259 in PSTVd NT of the loop E-like structure might be involved in protein binding and in viroid processing. The new variant PSTVd NT is genetically stable in both tobacco and tomato.

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A viroid was isolated from symptomless Brunfelsia undulata plants using the bidirectional PAGE method for analysis of small circular RNA molecules. The viroid was transmitted to tomato by mechanical inoculation. Infected tomato plants developed symptoms similar to those caused by intermediate strains of potato spindle tuber viroid (PSTVd). Cloning and sequencing revealed that the viroid from Brunfelsia undulata is closely related to the Columnea latent viroid (CLVd). Therefore, the new viroid sequence variant has been named CLVd-B. The Brunfelsia viroid CLVd-B consists of 373 nucleotides, 215 G + C, 158 A + U with a GC content of 57.6%. The most stable rod-like secondary structure of this viroid has 80 G:C, 39 A:U and 11 G:U base pairs with a minimum free energy of -157.2 kcal/mol (-657.1 kJ/mol). The sequence similarity of the right terminal domain of CLVd-B and of tomato apical stunt viroid (TASVd) is higher than the sequence similarity of these domains comparing CLVd and TASVd.

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