Since first being recorded in 1917–18 in Bulgaria, sharka (plum pox) disease has progressively spread via infected plant material to be present in most Prunus‐growing nations today. The disease has serious agronomic and political consequences because it causes enormous economic losses. In countries in which sharka is endemic, a high percentage of apricot and European plum production is unmarketable because of the disease. To these figures should be added the costs of sanitary controls, surveys and eradication programmes against sharka virus. Estimated costs associated with sharka management worldwide in the last 30 years exceed 10 000 million euros. However, improvements in knowledge of the disease and in techniques used to identify the disease are significantly aiding disease control and management.

Prunus spp. are affected by a large number of viruses, causing significant economic losses through either direct or indirect damage, which results in reduced yield and fruit quality. Among these viruses, members of the genus Ilarvirus (isometric labile ringspot viruses) occupy a significant position due to their distribution worldwide. Although symptoms caused by these types of viruses were reported early in the last century, their molecular characterization was not achieved until the 1990s, much later than for other agronomically relevant viruses. This was mainly due to the characteristic liability of virus particles in tissue extracts. In addition, ilarviruses, together with Alfalfa mosaic virus, are unique among plant viruses in that they require a few molecules of the coat protein in the inoculum in order to be infectious, a phenomenon known as genome activation. Another factor that has made the study of this group of viruses difficult is that infectious clones have been obtained only for the type member of the genus, Tobacco streak virus. Four ilarviruses, Prunus necrotic ringspot virus, Prune dwarf virus, Apple mosaic virus, and American plum line pattern virus, are pathogens of the main cultivated fruit trees. As stated in the 9th Report of the International Committee on Taxonomy of Viruses, virions of this genus are "unpromising subjects for the raising of good antisera." With the advent of molecular approaches for their detection and characterization, it has been possible to get a more precise view of their prevalence and genome organization. This review updates our knowledge on the incidence, genome organization and expression, genetic diversity, modes of transmission, and diagnosis, as well as control of this peculiar group of viruses affecting fruit trees.

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.

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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.

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