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 (Astru c et al., 1996). In this work, we present the molecular characterization of an almond HSVd isolate and report on the problems encountered in the diagn osis of HSVd in almond tissue through dot-blot non-radioactive hybridizatio n procedures. False positives were eliminated through incubation of the mem branes with RNase at high ionic strength after hybridization. Further exper iments which included Northern hybridization, RT-PCR coupled to Southern hy bridization, and cloning and sequencing suggested that spurious hybridizati on signals were due to host RNAs with sequence similarity to HSVd. Genetic characterization of the almond isolate demonstrated the existence of two ne w 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 patho genic (P) domain. Multiple alignments with all the available HSVd sequences and subsequent phylogenetic analyses revealed that the two new almond sequ ence variants were included in the previously described Prunus group of HSV d sequences.