At present isolates of Hop stunt viroid (HSVd) are divided into five groups : three major groups (plum-type, hop-type and citrus-type) each containing isolates from only a limited number of isolation hosts and two minor groups that were presumed to derive from recombination events between members of the main groups. In this work we present the characterization of 16 new sequence variants of HSVd obtained from four Mediterranean countries (Cyprus, Greece, Morocco and Turkey) where this viroid had not previously been described. Molecular variability comparisons considering the totality of the sequence variants characterized so far revealed that most of the variability is found in the pathogenic and variable domains of the viroid molecule whereas both the terminal right (T R ) and left (T L ) domains are regions of low or no variability, respectively, suggesting the existence of constraints limiting the heterogeneity of the sequence variants. Phylogenetic analyses revealed that sequence variants belonging to the two minor recombinant subgroups are more frequent than previously thought. When the cruciform structure alternative to the typical rodlike conformation was considered it was observed that the upper part of this structure (hairpin I) was strictly conserved whereas in the lower part a reduced variability was found. The existence of a covariation in this lower part was notable. Interestingly, a hammerhead-like sequence was found within the T R domain of HSVd and it was strictly conserved in all the sequence variants. The evolutionary implications of the presence of this motif on the HSVd are discussed.

Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5' untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of the CP, the amino acid residues in this region, which were previously thought to be functionally important in the replication cycle of the virus, were strictly conserved. No clear correlation with the type of symptom or host specificity could be observed. The validity of this grouping was confirmed when other isolates recently characterized by other authors were included in these analyses.

The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

No abstract

Grapevine phloem-limited isometric virui (GPLIV) is the name proposed for a non mechanically-transmissible virus found in Italian and Tunisian grapevines. In density gradient ccntrifugaiion purified virus preparations sedimented as two components: T, made up of empty protein shells, and B, cotnposed of intact nuclcoprotein particles. B particles had a buoyant density of 1.45 g/cm^ at equilibrium in CsCl and contained 35 % RNA consisting of a single mokcule with an apparent size of 7A kb. The coat protein consisted of a single species with a mol.wt of 28,000 daltons. Purified virus preparations did not infect herbaceous hosts by manual inoculation. A specific antiserum with a titre of 1 : 64 raised in rabbits, was used for identification of GPLIV in field-grown Tunisian grapevines and in leafroll-affected Italian vines before and after heat treatment. Although heat treatment eliminated the virus from the majority of the plants, leafrol! symptoms persisted in several GPLIVfree vines, indicating that there is no clear-cut relationship between GPLIV and this disease.