Molecular characterization of an almond isolate of hop stunt viroid (HSVd) and conditions for eliminating spurious hybridization in its diagnosis in almond samples

Hop stunt viroid (HSVd) has a wide range of hosts including herbaceous and woody plants. Recently, HSVd was demonstrated to infect almond trees (Astruc et al., 1996). In this work, we present the molecular characterization of an almond HSVd isolate and report on the problems encountered in the diagnosis of HSVd in almond tissue through dot-blot non-radioactive hybridization procedures. False positives were eliminated through incubation of the membranes with RNase at high ionic strength after hybridization. Further experiments which included Northern hybridization, RT-PCR coupled to Southern hybridization, and cloning and sequencing suggested that spurious hybridization signals were due to host RNAs with sequence similarity to HSVd. Genetic characterization of the almond isolate demonstrated the existence of two new HSVd sequence variants named HSVd.alm1 and HSVd.alm2. The changes of HSVd.alm1 and HSVd.alm2 were located at residues variable among HSVd sequences, in loops on the left part of the rod-like molecule that includes the pathogenic (P) domain. Multiple alignments with all the available HSVd sequences and subsequent phylogenetic analyses revealed that the two new almond sequence variants were included in the previously described Prunus group of HSVd sequences.