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Ten years after the original reports demonstrating the feasibility of the detection of plant viruses and viroids by PCR-based techniques, a large body of literature describes the development of PCR primer pairs and of very sensitive assays for essentially all groups of viral pathogens. The tests developed show, in general, a sensitivity in great excess (up to several thousand-fold) of that reported for serological assays. Yet, the assays reported have so far and to a very large extent been confined to laboratory scale set-ups. Transition from this situation to the real-life, large scale application of these techniques will require further validation as well as development of automated or semi-automated pre- and post-PCR steps. Past and current achievements as well as future perspectives are discussed.

Hop stunt viroid (HSVd) is able to infect a number of herbaceous and woody hosts, such as grapevine, Citrus or Prunus plants. Previous phylogenetic analyses have suggested the existence of three major groups of HSVd isolates (plum-type, hop-type and citrus-type). The fact that these groups often contain isolates from only a limited number of isolation hosts prompted the suggestion that group-discriminating sequence variations could, in fact, represent host-specific sequence determinants which may facilitate or be required for replication in a given host. In an effort to further understand the relationships between HSVd and its different hosts, HSVd variants from eight naturally infected Prunus sources, including apricot, peach and Japanese plum have been cloned and sequenced. In total, ten molecular variants of HSVd have been identified, nine of which have not been described before. A detailed phylogenetic analysis of the existing HSVd sequences, including the new ones from Prunus determined in this work, points towards a redefinition of the grouping of variants of this viroid, since two new groups were identified, one of them composed of sequences described here. A bias for the presence of certain sequences and/or structures in certain hosts was observed, although no conclusive host-determinants were found. Surprisingly, our analysis revealed that a number of HSVd isolates probably derived from recombination events and that the previous hop-type group itself is likely to be the result of a recombination between members of the plum-type and citrus-type groups.