Plum Pox Virus Genome Research Articles

Effect of storage temperature on viral RNA accumulation in Plum pox virus-infected Red Lyon plum fruit from the Central Valley of Chile

Plum pox virus (PPV), the causative agent of Sharka disease, causes serious economic losses to the stone fruit industry. As PPV is not transmitted by seed, fruit from countries with Sharka present are considered low-risk for spreading the disease. However, there have been cases raising concerns about the safety of exporting fruit from countries with Sharka disease. Due to this, the generation of new scientific evidence becomes important to address these concerns. This study aimed to compare the relative accumulation of PPV viral titer between freshly harvested infected fruit with fruit subjected to cold storage, simulating transit conditions to export markets. During two consecutive seasons, the fruit was collected from a PPV-infected ’Red Lyon’ plum orchard, and divided into three treatments (T): freshly harvested fruit (T1); fruit exposed to cold (0 °C) for seven days (T2), and fruit exposed to cold (0 °C) for 15 days (T3). Semi-quantitative RT-PCR was used to estimate the viral titer; band density values obtained for products of amplification of a PPV genome fragment were normalized with the density values obtained from the constitutive gene nad5 and plotted relative to treatment T1, which served as a control for this purpose. The results showed a significant difference (p < 0.05) between T1 and other treatments. For the first season, the viral RNA reduction was of 50% in T2 and 61% in T3, compared to T1. For the second season, the corresponding RNA viral reductions were 45% in T2 and 56% in T3, compared to T1.

Innovative RNAi Strategies and Tactics to Tackle Plum Pox Virus (PPV) Genome in Prunus domestica-Plum.

We developed an innovative RNAi concept based on two gene constructs built from the capsid gene (CP) cistron of the Plum pox virus (PPV) genome. First, designated as amiCPRNA, a potential molecule interfering with PPV genome translation and the second one is the ami-siCPRNA to target viral genome translation and PPV RNA replication. Following the previous engineering of these constructs in an experimental herbaceous host, they were introduced into Prunus domestica (plum tree) genome. Previously propagated onto a susceptible rootstock, these clones were graft-inoculated with PPV. After four dormancy cycles, and consistent with our experience of PPV infection, some clones showed a common phenomenon of silencing that can differ between the detailed plant phenotypes. Three different phenotypes were developed by the amisiCPRNA clones. First, the high resistance character shown by the amisiCPRNA plum-7 that was similar to the resistance expressed by HoneySweet plum. Secondly, a recovery reaction was developed by the two other amisiCPRNA plum-3 and plum-4 that differed from the rest, characterized as susceptible clones, among these were the amiCPRNA plums. Having assessed the behavior of these plums versus the herbaceous host accumulating the similar form of RNAi: ami-, si-, and ami-siRNA, challenging assays in perennials consistently reflect the natural context of viral genome targeting.

Plum pox virusgenome expression in genetically engineered RNAi plants

An important approach to controlling sharka disease, caused by Plum pox virus (PPV), is the development of PPV-resistant plants using small interfering RNA (siRNA) technology. In order to evaluate siRNA-induced gene silencing, we studied the PPV genome sequence virus genome translation and replication. While little is known about interference of the PPV genome in plums, the present manuscript provides evidence that interfering RNA acts with the dicer plant-enzyme complex to induce its degradation into siRNAs.

Identification of possible evolutionary pathways of Plum pox virus and predicting amino acid residues of importance to host adaptation

Plum pox virus (PPV) displays significant genetic diversity that has resulted in the identification of several different strains, some that differ in their biological properties and in their geographic distribution. Some strains are limited in their distribution to a single country, while isolates of PPV strain D are found in every country where the virus has been detected. Many questions remain regarding the evolutionary history of PPV: its genetic (strain) diversification, its origin, its dispersal and the distribution of isolates and/or strains. To explore the evolution of PPV, inferred ancestral sequences were reconstructed at various phylogenetic nodes using PAML. Evidence was obtained that indicates that the strains PPV D and PPV M were likely the earliest strains of PPV to evolve. Specifically considered also was the point at which adaptation occurred that allowed infection of Prunus avium and/or Prunus cerasus, sweet cherry and sour cherry, respectively. Eight amino acid sites were identified that appear to have undergone positive selection during this adaptation process. These amino acid sites cluster into two areas, P1 and VPg. These areas of the genome are known to be important for functions such as the control of viral replication rate. In combination with other data, this information sheds light on functionally significant points along the PPV genome and perhaps even characteristics of ancient PPV populations.

Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance

The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.

Biotechnological strategies and tools for Plum pox virus resistance: trans-, intra-, cis-genesis, and beyond.

Plum pox virus (PPV) is the etiological agent of sharka, the most devastating and economically important viral disease affecting Prunus species. It is widespread in most stone fruits producing countries even though eradication and quarantine programs are in place. The development of resistant cultivars and rootstocks remains the most ecologically and economically suitable approach to achieve long-term control of sharka disease. However, the few PPV resistance genetic resources found in Prunus germplasm along with some intrinsic biological features of stone fruit trees pose limits for efficient and fast breeding programs. This review focuses on an array of biotechnological strategies and tools, which have been used, or may be exploited to confer PPV resistance. A considerable number of scientific studies clearly indicate that robust and predictable resistance can be achieved by transforming plant species with constructs encoding intron-spliced hairpin RNAs homologous to conserved regions of the PPV genome. In addition, we discuss how recent advances in our understanding of PPV biology can be profitably exploited to develop viral interference strategies. In particular, genetic manipulation of host genes by which PPV accomplishes its infection cycle already permits the creation of intragenic resistant plants. Finally, we review the emerging genome editing technologies based on ZFN, TALEN and CRISPR/Cas9 engineered nucleases and how the knockout of host susceptibility genes will open up next generation of PPV resistant plants.

The efficiency of RNA interference for conferring stable resistance to plum pox virus

Plum transformed with an intron hairpin RNA CP (ihpRNA-CP) was resistant to plum pox virus (PPV) infection through the specific process of RNA silencing involving both small interfering-RNA (siRNA) and a methylated virus transgene. Silencing specifically targeted the PPV genome and led to the degradation of viral RNA in the model plant species Nicotiana benthamiana and the natural Prunus domestica host. Plums inoculated with the five major PPV strains, three widespread PPV strains (D, M, and Rec), and the atypical EA strain did not allow systemic spread of PPV in greenhouse-grown transgenic ihRNA-CP plum over multiple cycles of vegetative growth and cold-induced dormancy. PPV ihRNA-CP N. benthamiana displayed an immunity reaction and also allowed for the testing of PPV-C, a strain that was unable to infect P. domestica. This stable resistance demonstrated in plum based on the accumulation of siRNA can prevent PPV infection and can also act as a “curative” when PPV is inoculated through graft inoculation, through a recovery reaction. Regardless PPV strain variability based on geography, host species, epidemiology and serotypes of the CP protein and substitutions of nucleotides at the NH2-terminus of CP of the major five PPV strains tested, we show that the use of a PPV-CP intron hairpin (ihp) RNA is an effective strategy to specifically target the PPV genome. We provide methods and tools that demonstrate a reliable path towards developing PPV resistance suitable for protecting stone fruit orchards.

Plum pox virus and sharka: a model potyvirus and a major disease

Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. PPV diversity is structured into at least eight monophyletic strains. First discovered in Bulgaria, PPV is nowadays present in most of continental Europe (with an endemic status in many central and southern European countries) and has progressively spread to many countries on other continents. Typical of potyviruses, the PPV genome is a positive-sense single-stranded RNA (ssRNA), with a protein linked to its 5' end and a 3'-terminal poly A tail. It is encapsidated by a single type of capsid protein (CP) in flexuous rod particles and is translated into a large polyprotein which is proteolytically processed in at least 10 final products: P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIapro, NIb and CP. In addition, P3N-PIPO is predicted to be produced by a translational frameshift. PPV causes sharka, the most damaging viral disease of stone fruit trees. It also infects wild and ornamental Prunus trees and has a large experimental host range in herbaceous species. PPV spreads over long distances by uncontrolled movement of plant material, and many species of aphid transmit the virus locally in a nonpersistent manner. A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic plum, 'HoneySweet', transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.

RNAI TO SILENCE THE PLUM POX VIRUS GENOME

Sharka is a major disease threatening stone fruit production. Originally reported from the Balkan region of Europe, it is spread throughout the European continent and in other temperate areas of the world: Middle-East, Asia, Japan, North and South America and in parts of the North of Africa. Biotechnology is among the alternative solutions adopted to control Plum pox virus (PPV) infection. Following the success of the 'HoneySweet' plum, small RNA interfering (siRNAs) technologies are the approach to disrupt PPV replication. siRNA technologies via genetic engineering are explored in Prunus domestica, a natural host of PPV, to target the PPV genome. The engineered siRNA in plums was explored by challenging plants with the 4 major strains of PPV that are frequently found in orchards: D (originating from France), EA (from Egypt), M (from Greece) and Rec (from Slovakia). The effects of siRNAs were found to be virus specific because the doublet of 21- and 25-nucleotides was activated and no PPV strains broke the resistance. These studies conducted through more than 3 dormancy cycles pointed out that the resistant phenotype is stable. The intron-hairpin CP RNA construct demonstrated that silencing is an efficient strategy to control PPV infection. These silencing studies were extended through the development of a novel approach to silence the PPV genome: the use of artificial miRNA. Combining the knowledge about the PPV sequence and the results of the computational target predictions, two gene constructs designed as amiCPRNA and amisiCPRNA were engineered in Nicotiana benthamiana model plants. The combined effects of ami-and si-RNAs were found successful to silence the PPV genome because more than 70% of tested clones were resistant. While little is known about the cooperative function of ami- and si-RNAs, our findings show that these RNAi technologies can be successfully applied to inhibit PPV replication in these model host-plants.

Evaluation of the genetic diversity of Plum pox virus in a single plum tree

Genetic diversity of Plum pox virus (PPV) and its distribution within a single perennial woody host (plum, Prunus domestica) has been evaluated. A plum tree was triply infected by chip-budding with PPV-M, PPV-D and PPV-Rec isolates in 2003 and left to develop untreated under open field conditions. In September 2010 leaf and fruit samples were collected from different parts of the tree canopy. A 745-bp NIb-CP fragment of PPV genome, containing the hypervariable region encoding the CP N-terminal end was amplified by RT-PCR from each sample and directly sequenced to determine the dominant sequence. In parallel, the PCR products were cloned and a total of 105 individual clones were sequenced. Sequence analysis revealed that after 7years of infection, only PPV-M was still detectable in the tree and that the two other isolates (PPV-Rec and PPV-D) had been displaced. Despite the fact that the analysis targeted a relatively short portion of the genome, a substantial amount of intra-isolate variability was observed for PPV-M. A total of 51 different haplotypes could be identified from the 105 individual sequences, two of which were largely dominant. However, no clear-cut structuration of the viral population by the tree architecture could be highlighted although the results obtained suggest the possibility of intra-leaf/fruit differentiation of the viral population. Comparison of the consensus sequence with the original source isolate showed no difference, suggesting within-plant stability of this original isolate under open field conditions.

The 3'-proximal part of the Plum pox virus P1 gene determinates the symptom expression in two herbaceous host plants.

Three major strains of the Plum pox virus (PPV) are the most important in Europe: PPV-D, PPV-M, and PPV-Rec. By combining the genomes of two different strains of PPV (PPV-D with PPV-Rec; PPV-D with PPV-M), 20 inter-strain chimeric infectious clones (CICPPV) were constructed. Biological properties of CICPPV were tested by inoculating them on different herbaceous host species susceptible to PPV. Four of the seven species tested, exhibited visible symptoms. In Nicotiana benthamiana all CICPPV induced systemic mosaic and leaf malformation. Pisum sativum showed a broad range of symptom severity (systemic chlorotic and necrotic lesions) but neither qualitative nor quantitative aspects of symptomatology were related to a single PPV genome locus. Nicotiana occidentalis and Nicandra physaloides proved to be suitable for symptom-based differentiation. Depending on the virus strain/chimera, N. occidentalis showed two types of symptoms: mild systemic chlorotic spots or local necrotic lesions/systemic vein necroses. N. physaloides reacted to the PPV infection either symptomless or by local necrotic lesions. Our results demonstrated that the P1/HC-pro region of the PPV genome appears to be the determinant of the symptom manifestation in these host plants. In silico analysis mapped it to the 3'-proximal part of the P1 gene.

THE DEVELOPMENT OF GENETIC RESISTANCE TO PLUM POX VIRUS IN TRANSGENIC NICOTIANA BENTHAMIANA AND PRUNUS DOMESTICA

Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. It is the causal agent of the most devastating viral disease on many stone-fruit spp. The viral disease, known as Sharka in Europe, was recently found in the United States and Canada. Genetic resistance is considered the most effective means to control PPV, but despite over 60 years of extensive screening for germplasm resistant to PPV, few resistant resources have been identified. Here we report engineering genetic resistance against PPV through the hairpin-mediated RNA silencing (RNAi) approach. Two highly conserved regions of the PPV genome corresponding to portions of viral RNA coding for P1 and CP, respectively, were cloned into a plant transformation vector under the control of the double Cauliflower mosaic virus 35S promoter as inverted repeats spanned by an intron from the peach endo polygalacturonase genomic DNA. These two constructs (pAWp1 and pAWcp) were initially tested in Nicotiana benthamiana. Over 50 transgenic lines transformed with pAWp1, pAWcp and an empty plasmid pAWck (as a control) were generated. The transgenic N. benthamiana plants were mechanically inoculated with PPV. ELISA and RT-PCR analysis revealed that 49.1% of pAWp1-derived lines and 41.2% of pAWcp-derived lines were PPV-negative and 100% of control lines (transformed with pAWck) showed PPV-positive. The pAWp1 plasmid was used to transform plum (Prunus domestica L.). Transgenic plum plants were challenged with PPV by chip-bud inoculation. PPV was detected in all the pAWck-derived plum lines but was not detectable in 50% of pAWp1-derived transgenic lines. Two species of PPV-specific small interfering RNAs, a hallmark of posttranscriptional gene silencing (PTGS), were present in the resistant plum lines, suggesting PTGS is responsible for resistance. The PPV-resistant transgenic plum generated in this study may provide an alternative resistant source for the control of PPV.

Differentiation of Plum pox virus isolates by single-strand conformation polymorphism and low-stringency single specific primer PCR analysis of HC-Pro genome region

Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR were assessed for suitability and reliability in genotyping of Plum pox virus (PPV) isolates. Examined PPV isolates included 16 PPV-D, 12 PPV-M, and 14 PPV-Rec isolates collected in Czech Republic. The analysis was performed on the helper component protease (HC-Pro) region of the PPV genome. SSCP and LSSP-PCR allowed the differentiation of PPV strain, but SSCP was not able to distinguish isolates within the same strain. The individual genotyping of each PPV isolate was obtained by LSSP-PCR. Nevertheless, both SSCP and LSSP-PCR techniques are suitable for preliminary screening of genetic variability of plant RNA viruses.

CONTROVERSIAL CONSIDERATIONS BETWEEN NUCLEOTIDE DIVERSITY OF THE 3' TERMINAL REGION OF PLUM POX VIRUS AND DIFFERENCES IN VIRAL SEROTYPES

Plum pox virus (PPV) is a potyvirus causing economic losses to stone fruit trees. In the present study we have analyzed more than 40 PPV isolates collected from different western, eastern, central European and north African countries To preserve the originality of these isolates, the majority was maintained in GF305 peach (Prunus persica), some in apricot (P. armeniaca), plum (P. domestica, P.insititia), or ornamental (P. tomentosa) trees. Serological tests conducted in 2004, 2005 and 2006 with polyclonal antisera by Agdia (USA) validated the high rate of response with all PPV tested. Two primer pairs were designed to amplify 1200 nucleotides (nt) of the 3‘ region of the PPV genome spanning from the carboxy-terminal part of the Nib cistron to the carboxy- terminus of CP, and 500 nt starting from the conserved EcoRI site of the CP cistron to the polyA tail, respectively. Nucleotide sequences of these isolates were determined either from the amplified fragment or the recombinant plasmid cloned into E. coli. Two regions with variable nucleotides were found, the first 110 nt upstream the cleavage site, and the second in nucleotides encoding the cleavage site of NIa-Pro between the Nib and CP cistrons. Sequence homology among the viral isolates was very high. However, phylogenetic analysis of the sequences coding for CP indicated that diversity resided in the amino-terminal section. This finding was corroborated by the use of monoclonal antibodies to PPV D (Durviz, Spain) and PPV M (Agritest, Italy). We discuss the results of these analyses and, particularly, the significance of these nucleotide diversities.

Identification of Plum pox virus Pathogenicity Determinants in Herbaceous and Woody Hosts

Plum pox virus (PPV) is a member of the genus Potyvirus that is able to infect a large variety of plant species, including trees of the genus Prunus, its natural host. When some PPV isolates are propagated for an extended time in herbaceous plants, their ability to infect trees is reduced. The molecular basis of this change in host infectivity is poorly understood. We report the construction of hybrid viruses from cDNA clones of two D-strain isolates of PPV, PPV-D and PPV-R, which differ in their host range. PPV-D can infect GF305 peach seedlings efficiently, however, it is unable to infect Nicotiana clevelandii plants. Conversely, PPV-R infects N. clevelandii, but not GF305 peach seedlings. The analyses of the hybrid viruses showed that, although determinants of PPV pathogenicity are extensively spread throughout the PPV genome, the 3' terminal region of the PPV-R genome, including the 3' noncoding region and the coding regions for the coat protein (CP), NIb, and part of NIa protein, is sufficient to confer infectivity of N. clevelandii in a PPV-D background. Our data demonstrate a high concentration of amino acid substitutions in the CP and a host-specific effect of a deletion at the N terminus of this protein in PPV pathogenicity in peach and N. clevelandii infectivity experiments. These results suggest that relevant host specificity determinants are located in the N-terminal region of the CP. The analyses of the PPV-R and PPV-D chimeras also showed that key host-specific pathogenicity determinants lie in the 5' terminal third of the PPV genome, a region that spans proteins P1, HCPro, and P3. The selection of mutations in only a few specific residues in proteins P1, P3, and 6K1 after partial adaptation of a chimeric virus (BD-GFP) to N. clevelandii further suggests a relevant role for these proteins in host adaptation.

Engineering resistance to Plum pox virus (PPV) through the expression of PPV-specific hairpin RNAs in transgenic plants

Plum pox virus (PPV) causes serious diseases in stone-fruit trees. PPV is widespread in Europe and was recently detected in peach orchards in Canada and the United States. In an effort to engineer resistance to PPV in Prunus species, we produced transgenic plants expressing PPV-specific hairpin RNAs under the control of a constitutive promoter. Two highly conserved regions of the PPV genome were introduced into plants as inverted repeats separated by an intron sequence. In initial resistance assays, 22 Nicotiana benthamiana transgenic lines were inoculated with a Canadian isolate of PPV and tested for the presence of PPV between 4 and 5 weeks post inoculation, using the enzyme-linked immunosorbent assay (ELISA) diagnostic method. The majority of the transgenic lines displayed increased resistance to PPV, with only one transgenic line being completely susceptible to the virus. In the second round of resistance assays, a more sensitive detection method, reverse transcription – polymerase chain reaction, was used to test for the presence of PPV at various times post inoculation. The virus was readily detected in inoculated control plants, but not in six selected transgenic lines, suggesting that the latter are highly resistant to PPV. PPV-specific small interfering RNAs were observed in the six selected transgenic lines, confirming that posttranscriptional gene silencing is the mechanism of resistance in these lines. Our results also indicate that the expression of PPV-specific hairpin RNAs is an effective method of inducing resistance to the virus.

Biological Behavior and Partial Molecular Characterization of Six Chilean Isolates of Plum pox virus.

Plum pox virus (PPV) strain D was first detected in Chile in 1992 infecting Prunus trees including peaches, nectarines, apricots, and plums. Since then, quarantine efforts have included periodic surveys in the central zone of the country, the main region for stone fruit production. This work describes the characterization of six PPV isolates from this area of Chile, using biological and molecular approaches. PPV isolates were introduced into Prunus tomentosa and Nicotiana benthamiana hosts by grafting and mechanical inoculation, respectively. Symptoms were evaluated by following the appearance of circular necrotic spots and mosaic in leaves of P. tomentosa and mosaic and some leaf deformation in N. benthamiana. Molecular analysis was carried out using reverse transcription-polymerase chain reaction, allowing the cloning and sequencing of 1.34-kb fragments corresponding to the 3' region of the replicase gene, the complete coat protein (CP) gene, and the 3' nontranslated region of the PPV genome. Evolutionary distance analysis of these nucleotide sequences and their deduced coat protein amino acid sequences grouped the six Chilean isolates among strain D isolates, with closest genetic distances to those of Central Germany and Poland. Representative sources of these isolates suggest that strain D could be the only type of PPV currently present in Chile.

Molecular variability of the P3-6K1 genomic region among geographically and biologically distinct isolates of Plum pox virus.

To evaluate molecular variability in the 5' region of the Plum pox virus (PPV) genome, a fragment spanning the C-terminal part of P3, the complete 6K1 and start of the CI gene in 12 Slovak and French PPV isolates was amplified in IC-RT-PCR and sequenced. Computer analysis of obtained and previously published sequences showed rather substantial differences among isolates of PPV-M and D subgroups (percentage of nucleotide identity ranged from 85.65 to 87.68%). There was a relatively low percentage of heterogeneity among isolates within the same subgroup. Amino acid multiple sequence alignment showed a total of 14 conserved subgroup-specific amino acid positions among PPV-M and D isolates in the examined region. The geographical origin of the isolates was not correlated with their phylogenetic clustering. No reasonable correlation could be established between amino acid substitutions and host species and/or biological properties of isolates. The present IC-RT-PCR detection method targeting a 5' region of the PPV genome, followed by simple RFLP subgroup determination, could enhance conventional CP-based techniques, especially with peculiar isolates. Using this approach, the Slovak isolate BOR-3 was found to be a natural recombinant between a D and an M PPV-type.

A Natural Population of Recombinant Plum Pox Virus is Viable and Competitive under Field Conditions

The isolate BOR-3, collected in Slovakia in 1996, was recently identified as a natural recombinant between an M and D type of Plum pox virus (PPV). Biological assays demonstrated its capacity to be aphid- and graft-transmitted to various Prunus spp. hosts. A study was carried out to determine the further presence of PPV recombinants in two epidemiologically distinct areas – Slovakia and France. Tools based on PPV-M and D subgroup typing, targeting P3–6K1, CI and CP regions of the PPV genome were used for recombinant identification. Closely related recombinant variants were detected in different Prunus spp. during a survey conducted in Slovakia in 2001, but not within a set of selected PPV isolates from France collected between 1985 and 2001. Sequence analysis of the (Cter)NIb–(Nter)CP region of 10 recombinant isolates from Slovakia showed their high homology, reaching more than 98%. All the recombinant isolates shared the same recombination breakpoint situated in the C terminus of the NIb gene. Our study demonstrates that the PPV recombinants are viable and competitive with conventional PPV-M and D isolates. The present work indicates that the occurrence of recombinants within PPV isolates might be more common than previously assumed.

Evidence of a Naturally Occurring Recombinant Isolate of Plum pox virus from Slovakia.

Sharka, caused by the Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees worldwide. Two main groups of PPV isolates have been identified, PPV-M and PPV-D (1). Natural variability of Slovak PPV isolates in Prunus hosts has been recently evaluated (3). The PPV isolate BOR-3 was isolated in the summer of 1996 from a 5-year-old apricot tree, cv. VS 123/9, in an orchard in western Slovakia. The host apricot tree was propagated from seed; hence infection via aphid transmission from the immediate surroundings is highly probable. In order to retain its original properties, the isolate was transmitted by chip budding from a diseased tree to seedlings of Prunus persica, cv. GF 305. In contrast to other PPV isolates collected from the same location, infection of GF 305 with BOR-3 was either symptomless or produced only weak symptoms. On the other hand, sap-infected young seedlings of P. insititia × P. domestica St. Julien No. 2 presented leaf chlorotic spots and rings similar to those produced by other tested Slovak PPV isolates. Serological typing with group-specific MAbs (1) and restriction fragment length polymorphism of RT-PCR products amplified from the C terminus of the capsid protein (CP) gene demonstrated that BOR-3 is a member of PPV-M group (3). However, nucleotide (nt) sequence analysis of the P3-6K1 genomic region (GenBank accession AF357541) has shown a strong similarity with PPV-D group isolates (reaching 97%). To obtain more information, a 1846-nt long 3 genomic region encompassing the C-terminus of NIb, CP and 3 noncoding region (NCR) was subsequently sequenced (GenBank accession AY028309). The sequence comparison revealed that the BOR-3 isolate was the most identical to the PPV isolate ð6 (GenBank accession S57404) isolated more than 10 years ago in former Yugoslavia, which is about 550 km from the Slovak focus. The ð6 isolate is known to be the only PPV RNA recombinant at present (2). Nucleotide sequence identity in 3'NIb+CP+NCR region between BOR-3 and ð6 isolates reached >98%, contrary to 96% identity with PPV-M isolates (PS, SK68) and 88% identity with PPV-D isolates (Dideron, Rank). Further, the potential recombination site in the BOR-3 genome has been located in the C-terminus of NIb gene near nt position 8450 (numbered according to PPV-PS isolate, AJ243957). This site corresponds to the location of the recombination crossover identified in the PPV-ð6 isolate (2). Despite geographical and temporal distinctiveness of the two PPV isolates, an identical origin of ð6 and BOR-3 cannot be excluded. The hypothesis that these PPV isolates are the product of two independent recombination events at a recombination hot spot in the PPV genome situated near the C terminus of NIb gene also should be considered. Based on these data, the PPV BOR-3 isolate is a product of a natural homologous RNA recombination event between an M and D isolate. Evidence of homologous RNA recombination in this isolate significantly enriches current knowledge on the occurrence of recombination among potyviruses and emphasizes the role of RNA recombination in viral evolution and adaptation. This report indicates that occurrence of recombinants within PPV isolates infecting perennial crops might not be as rare as previously suggested (4).