PPV-D Isolates Research Articles

Almond Can Be Infected by Plum Pox Virus-D Isolate Penn4 and Is a Transmission-Competent Host.

Although it is currently eradicated from the United States, Plum pox virus (PPV) poses an ongoing threat to U.S. stone fruit production. Although almond (Prunus dulcis) is known to be largely resistant to PPV, there is conflicting evidence about its potential to serve as an asymptomatic reservoir host for the virus and thus serve as a potential route of entry. Here, we demonstrate that both Tuono and Texas Mission cultivars can be infected by the U.S. isolate PPV Dideron (D) Penn4 and that Tuono is a transmission-competent host, capable of serving as a source of inoculum for aphid transmission of the virus. These findings have important implications for efforts to keep PPV out of the United States and highlight the need for additional research to test the susceptibility of almond to other PPV-D isolates.

Molecular Analysis of New Crimean Isolates of the Plum Pox Virus

Molecular analysis of new isolates of the plum pox virus (PPV) found in the gene pool collections of peach, apricot, and plum from the Nikitsky Botanical Garden (National Science Center, Russian Academy of Sciences), as well as on wild cherry plum trees in the surrounding area, has been carried out. Using the enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction, 19 new PPV isolates have been detected. Most of them belonged to the strain D; single isolates of the strains Rec and M were also found. The 3'-terminal sequences of genome of the new isolates were sequenced, including the coat protein (CP) gene and adjacent sequences of the NIb gene and the 3'-nontranslated region. Phylogenetic analysis of these sequences revealed the distribution of PPV-D isolates into several clusters or clades. An average divergence rate between sequences of Crimean PPV-D isolates was 2.0%. A relatively high level of genetic variation indicates a long-standing introduction of the ancestors of the current PPV-D isolates to Nikitsky Botanical Garden (NBG). Isolate H23, identified in the apricot collection, belonged to strain M. This is the first detection of PPV-M in Crimea. Isolate AL-Ch (strain Rec) was found on wild cherry plum. In the CP of isolate K87 (strain D), the K59T mutation was detected, which prevents its recognition by PPV-D-specific 4DG5 monoclonal antibodies. In CP of AL-Ch and previously characterized isolates K28 and Kisl-1pl (strain Rec), amino acid substitutions were detected at position 68, which did not affect the recognition of these isolates by AL monoclonal antibodies. Apparently, position 68 is outside the PPV-M/Rec-specific epitope. The results of the work show that PPV most likely entered the NBG with contaminated drupaceous fruit material as a result of several independent introductions from different regions of the world and spread over plantations by aphids.

Update on Distribution and Genetic Variability of Plum pox virus Strains in Bulgaria

Field surveys for Plum pox virus (PPV) infection were conducted in stone fruit orchards all over Bulgaria. In total, 1168 out of 3020 leaf samples from cultivated Prunus spp. and wildly growing P. cerasifera trees reacted positive for PPV in DASI-ELISA with the universal monoclonal antibody (MAb) 5B. Further ELISA analyses showed that 987 and 127 isolates belonged to PPV-M and PPV-D serotypes, respectively. The plum and P. cerasifera showed 82.0% and 50.5% levels of infection, respectively followed by the peach (40.0%) and the apricot (32.0%). Five hundred fifty one PPV isolates were further typed by IC-RT-PCR with PPV-Rec, -M and -D-specific primers, targeting (Cter)NIb-(Nter) CP genome region, as 125 isolates were sequenced. The results revealed the presence of PPV-Rec, PPV-M and PPV-D and mixed infections of these strains. PPV-Rec was the most prevalent strain (49.0%), followed by PPV-M (40.1%), while PPV-D was the less spread strain (8.2%). PPV-Rec was the most common strain in plums, including the eight “old-aged” trees from the region of the first Sharka discovery. PPV-M was the most prevalent strain in peach and apricot. Phylogenetic analyses on (Cter)NIb-(Nter)CP of the isolates were performed. PPV-Rec isolates formed a homogeneous group, while PPV-M isolates split into PPV-Ma and PPV-Mb subgroups. Five separated clades were formed by the analyzed PPV-D isolates. Nucleotide sequences of the partial CP coding region of the analyzed isolates revealed a slightly higher intra-strain genetic variability in PPV-Rec and PPV-M isolates, while that of PPV-D strain isolates was higher from the reported for these strains.

Phytohormone Signaling of the Resistance to Plum pox virus (PPV, Sharka Disease) Induced by Almond (Prunus dulcis (Miller) Webb) Grafting to Peach (P. persica L. Batsch).

Plum pox virus (PPV, sharka) is a limiting factor for peach production, and no natural sources of resistance have been described. Recent studies, however, have demonstrated that grafting the almond cultivar “Garrigues” onto the “GF305” peach infected with Dideron-type (PPV-D) isolates progressively reduces disease symptoms and virus accumulation. Furthermore, grafting “Garrigues” onto “GF305” prior to PPV-D inoculation has been found to completely prevent virus infection, showing that resistance is constitutive and not induced by the virus. To unravel the phytohormone signaling of this mechanism, we analyzed the following phytohormones belonging to the principal hormone classes: the growth-related phytohormones cytokinin trans-zeatin (tZ) and the gibberellins GA3 and GA4; and the stress-related phytohormones ethylene acid precursor 1-aminocyclopropane-1-carboxylic acid (ACC), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA). PPV inoculation produced a significant increase in GA3 and ABA in peach, and these imbalances were related to the presence of chlorosis symptoms. However, grafting “Garrigues” almond onto the PPV-inoculated “GF305” peach produced the opposite effect, reducing GA3 and ABA contents in parallel to the elimination of symptoms. Our results showed the significant implication of SA in this induced resistance in peach with an additional effect on tZ and JA concentrations. This SA-induced resistance based in the decrease in symptoms seems to be different from Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR), which are based in other reactions producing necrosis. Further studies are necessary, however, to validate these results against PPV-D isolates in the more aggressive Marcus-type (PPV-M) isolates.

Spread and competitiveness ofPlum Pox Virus:Rec and -D strains in experimental plum orchard

Sharka disease caused by Plum pox virus (PPV) is endemic in Bulgaria. The speed and mode of spread of PPV-Rec and PPV-D isolates, molecularly characterised in advance and introduced in an experimental plum orchard to serve as permanent sources of infection was studied from 2008 to 2015. The trees were visually inspected for symptoms development and tested by DASI-ELISA twice each year. The positive samples were serologically (-M and -D specific MAbs) and molecularly (IC-RT-PCR) strain-typed, and sequenced in (Cter)NIb-(Nter)CP coding region. Artificially inoculated trees were found infected in the spring of 2009. After 7 years 43 trees (11.2%) became naturally infected. The majority of the trees were infected by PPV-Rec (65%), while only one tree (2.5%) by PPV-D. Nevertheless PPV-M was not introduced in the orchard, this strain was found in single and mixed infections with PPV-Rec strain. None of the isolates in naturally infected trees was identical to PPV-Rec and PPV-D inoculums, while several of them were a 100% identical to the respective PPV-Rec, PPV-D or PPV-M isolates from neighbourhood-infected trees. After the initial random pattern of PPV spread, aggregation of the new infections around the trees infected in previous years was observed. The presence of only Phorodon humuli and Hyalopterus pruni complex, known as PPV vectors and their poor abundance (5.0% from all caught aphids), together with some specific features of the landscape influenced the slow natural development of PPV in the experimental orchard.

Recombination analysis of 51 Plum pox virus (PPV) isolates, including 10 genomes of PPV-M Istanbul

Plum pox virus strain M (PPV-M) isolates were previously detected in different districts of Turkey, and they grouped together with European M isolates in phylogenetic trees. However, PPV-M isolates from Istanbul formed a separate group adjacent to European PPV-M isolates. Here, we have obtained full genomes of 10 PPV-M isolates from Istanbul derived from apricot and peach trees. The Recombination Detection Program version 4.16 (RDP4) was used for recombination analysis of 51 genomes of PPV, including the 10 full genomes of PPV-M Istanbul and six Turkish PPV-D isolates. Genome sequences were selected from GenBank to represent global PPV variation. Recombination events, likely parental isolates of recombinants and recombination break points were analysed using nine independent recombination detection methods (RDP, Bootscan, Geneconv, Maxchi, Chimaera, Siscan, 3seq, Lard and Phlopro) implemented in the RDP4 program with default settings. The program identified 19 recombination events. Of the total events, 13 recombination events included Turkish PPV-M and PPV-D isolates. Seven of the recombination detection methods showed that isolates from the PPV-Rec strain (AF357541, GU461889, EU117116, GU474956 and JX013532) included in the 51 genomes are likely to contain recombinant fragments from the one of the Turkish PPV-D isolates and a PPV-M isolate from Istanbul. Recombination events among the 10 PPV-M isolates were also detected at the intrastrain level. This is possible because all PPV-M Istanbul isolates were collected from a narrow district in Istanbul. Genome nucleotide comparisons revealed that PPV-M isolates from Istanbul had identity with European PPV-M, PPV-T, PPV-An, PPV-Rec, PPV-D, PPV-EA, PPV-CR, PPV-W and PPV-C of 97, 94, 92, 89, 88, 076, 74, 74 and 73%, respectively. The average intragroup diversity was found to be 0.014 for the Turkish PPV-M group (10 isolates) and 0.019 for the European PPV-M isolates (four isolates).

Development of an on-site plum pox virus detection kit based on immunochromatography

Sharka disease, caused by plum pox virus (PPV), is the most serious viral disease of stone fruit trees. Among the eight known strains of the virus, PPV-D is the most important due to its recent global spread. Although enzyme-linked immunosorbent assay (ELISA) is the most common approach for diagnosing sharka, it involves time-consuming steps and requires expensive equipment and trained technicians. In this study, an on-site PPV detection kit based on immunochromatography was developed using polyclonal antibodies against the coat protein (CP) of a PPV-D isolate. The immunochromatographic (IC) assay kit was as sensitive as a commercial ELISA system for detecting Japanese PPV-D isolates. Moreover, it was easy to use (a one-step procedure), and results could be obtained on-site within 15 min without special laboratory equipment. The IC assay kit detected the virus from every aerial part of symptomatic Japanese apricot trees. In a detailed study of viral localization in leaves, the most suitable plant parts for use in the IC assay were symptomatic mesophyll tissues and the region from the petiole to the main vein. A positive reaction was also observed using the CP of other major (PPV-M and PPV-Rec) and minor (PPV-EA, PPV-W, and PPV-T) strains.

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.

First report of plum pox virus infecting Japanese apricot (Prunus mume Sieb. et Zucc.) in Japan

For the first time, plum pox virus (PPV) has been detected in commercial Japanese apricot (Prunus mume) trees in Tokyo, Japan. These trees had ringspot or mottle on leaves, color breaking of petals and, occasionally, mild ringspots and malformation on fruits. The virus was identified based on the morphology of virus particles, serology, and RT-PCR. The amplified nucleotide fragment shared 100% identity with a partial coat protein gene of PPV-D isolates.

Discovery and characterization of Plum pox virus isolates in Montenegro

In 2006, twenty extensive and intensive plum orchards, surrounding Nik s i C in Montenegro, were screened for the presence of symptoms of Plum pox virus (PPV) infection. Mild to severe symptoms were found in 15 orchards, usually only on some trees. In total 19 samples were collected and 17 proved to be positive by DAS-ELISA (Bioreba). Of these positives, 16 were selected for molecular identification. PPV isolates have been assigned to two major (PPV-D and PPV-M) and two minor (PPV-EA, PPVC) groups. Recently, a fifth group (PPV-REC) has been established, which corresponds to naturally-occurring recombinants between PPV-M and PPV-D isolates. All PPV-REC isolates share the same recombination breakpoint on the NIb polymerase gene and therefore conventional CP gene-based molecular and serological methods are not able to discriminate between PPV-M and PPV-REC isolates. Subgroup characterization was performed using two different PCR systems: one positioned downstream of the recombinant crossover (using the PPV-M and -D specific primer pairs P4/P3-M and P4/P3-D respectively; Dallot et al. , in press); and the other upstream of the recombination breakpoint (using the primer pairs CIf/CI-M (PPV-M specific) and CIf/CI-D (PPV-D specific); Glasa et al. , 2002). The results showed the presence of PPV-D type isolates in 4 samples. These samples were also negative for PPV-M when tested by TAS-ELISA (Agritest). The remaining 12 samples were PPV-M positive by TAS-ELISA. Of these, seven proved to be of PPV-REC type by RT-PCR. However, the isolate type could not be determined for the remaining five samples using the previously described method. Therefore they were further analyzed using primer pairs mM5/mM3, mD5/mD3 and mD5/ mM3 ( P ubr et al. , 2004), situated around the recombination breakpoint. All samples gave a positive result only with primer pair mD/mM3 and were therefore also identified as the PPV-REC type. Although plum production is important in Montenegro, data about PPV infection is scarce. Sharka symptoms were observed by Miju s kovi C (2002), but until now the presence of PPV was never confirmed. As a result, this is the first report confirming PPV as being present in Montenegro.

Genetic variability of Plum pox virus isolates in the Czech Republic

Plum pox virus (PPV), the causal agent of Sharka disease, is an important pathogen of stone fruit trees. In this study, 24 new Czech PPV isolates from five different orchards were collected and characterized, molecularly. PPV-D isolates were identified in all orchards studied; whereas PPV-Rec isolates were identified in only two of them. A phylogenetic analysis on (Cter) NIb-(Nter) CP was performed. Three Czech PPV-D isolates BOH11CZ, BOH12CZ, and BOH13CZ diverged into a significantly separated cluster. PPV-Rec isolates formed a fairly homogenous group. However, the Bohutice and the Lipov PPV-Rec isolates clustered in two significantly separated branches.

Plum Pox Virus Mixed Infection Detected on Apricot in Pakistan.

Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees. First reported from Bulgaria in 1917, the virus is now widespread in Europe, the Mediterranean Basin, and Asia Minor and is sporadically present in North and South America. On the basis of molecular and serological properties, six PPV subgroups are recognized, from which PPV-D, PPV-M, and PPV-Rec are the most common (1,2). Several apricot trees (Prunus armeniaca) showing mild, pale green rings and diffuse chlorotic spots on leaves were found in a small orchard in the Baltistan District in northern Pakistan at approximately 2,400 m above sea level. Dried leaf samples from one symptomatic tree randomly selected from the orchard were positive for PPV using double-antibody sandwich enzyme-linked immunosorbent assay with antisera prepared in the laboratory, immunoblot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid protein (CP) gene using standard procedures (1). To check the subgroup affiliation and evaluate the molecular variability, the 562-bp variable region spanning the C-terminus of NIb and the N-terminus of the CP was amplified, the RT-PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI), and positive clones were analyzed by restriction and sequence analyses. Interestingly, sequence analysis of four clones revealed mixed infection, i.e., the presence of two different PPV isolates in the apricot sample. One isolate belonged to PPV-D (GenBank Accession No. DQ422147) and the other belonged to the PPV-Rec subgroup (GenBank Accession No. DQ422148). Multiple alignment of the sequenced genome portion of the Pakistan PPV-D isolate indicated 96 to 99% nt identity with various PPV-D isolates without unique, clear-cut differences. Similarly, the PPV-Rec isolate had 98 to 99% identity with European PPV-Rec isolates and retained the cross-over at nucleotide position 8450 in the 3' terminus of NIb. This sequence had the amino acid signature at the N-terminus of the CP typical of the PPV-Rec subgroup (2). Moreover, no particular clustering of the Pakistan isolates within PPV-D and PPV-Rec could be observed after phylogenetic analysis. The DAG motif, essential for aphid transmission, was present in both sequences. To our knowledge, this is the first indication of PPV occurrence in Pakistan and first identification of the PPV-Rec isolate outside Europe. Together with previous reports on the PPV presence in China and Kazakhstan (3,4), this report indicates the need for more detailed epidemiological studies focusing the PPV spread and its molecular diversity in Asia.

Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction

ABSTRACT The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3' minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected.

Sequence analysis of isolates of the Canadian Plum pox virus, and comparisons to isolates from Europe and the United States

The complete genome sequences of seven isolates of Canadian Plum pox virus (PPV) obtained from infected peach, plum, and flowering almond have been determined. All sequences were most similar to PPV-D strain. Comparison of the sequences of nucleotides and amino acids of the seven isolates indicate that Canadian isolates fall into two main subgroups, I and II, with subgroup I isolates being most closely related to European PPV-D isolates. All isolates of subgroup I (two) were from plum, whereas for isolates of subgroup II, four were obtained from peach and one isolate from flowering almond. Comparisons of complete PPV-D isolates from Canada, Europe, and the United States show that these fall into distinct groupings based on geographic origin. Alignments of Canadian, European, and United States PPV-D isolates revealed that there were only 13 positions in the PPV polyprotein where a variant amino acid exists in more than one isolate. A diagnostic RsaI site, often used to assign strain-D status to an isolate, was found to be absent in several Canadian PPV-D isolates, suggesting that caution should be exerted when employing this restriction site polymorphism for strain identification

Partial molecular characterization of plum pox virus isolates occurring in Canada

Plum pox virus (PPV), recently detected for the first time in Canada, induces serious disease in stone fruits. Serological analyses along with reverse transcription – polymerase chain reaction and subsequent analyses with restriction enzymes indicate that Canadian isolates correspond to the Dideron strain of PPV (PPV-D). Sequence analysis of the 3'-terminal 1.47 kilobases of the genomes of 11 Canadian PPV isolates obtained from peach, nectarine, and plum from different orchards confirmed that all isolates so far analyzed correspond to PPV-D. In addition, these sequencing studies show that: (1) all Canadian PPV-D isolates retain the DAG tripeptide sequence (aspartate–alanine– glycine) known to be required for transmission by aphids, (2) none of the Canadian isolates are identical in nucleotide or amino-acid sequence to any other sequenced PPV isolate, (3) no two Canadian isolates contain identical nucleotide sequences, and (4) the greatest degree of sequence heterogeneity exists in the N-terminal region of PPV coat protein. Sequence comparisons suggest that Canadian isolates fall into two main subgroups, the most prominent subgroup being, by far, the least represented in the sequence databases. The two Canadian subgroups may represent isolates that were independently introduced into Canada

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).

3'-Terminal sequence of the plum pox virus PS and o6 isolates: evidence for RNA recombination within the potyvirus group

The sequence of the 3'-terminal 1768 nucleotides of the PS and ŏ6 isolates of plum pox virus (PPV) has been determined and compared with that of the equivalent regions of other PPV isolates sequenced previously. The sequenced region is part of the PPV open reading frame encoding the last 186 amino acids of the NIb protein and the coat protein (CP, 330 amino acids), followed by a non-coding region of 220 nucleotides and a poly(A) tail. PPV-PS and PPV(-)ŏ6, just like PPV-El Amar, show rather high levels of nucleotide diversity in the sequence encoding the C-terminal region of the NIb protein (19.4 to 31%) and the N terminus of CP (22.8 to 41.1%) when compared with PPV-Rankovic, PPV-D and PPV-NAT, whereas the level of diversity in the rest of the CP sequence and the 3' non-coding region is low (8 to 10.8% and 5.5 to 7.7%, respectively). However, the first 429 sequenced nucleotides of PPV(-)ŏ6 are very similar to those of the PPV-Rankovic, PPV-D and PPV-NAT isolates, whereas the rest of the sequence clearly resembles PPV-PS. Thus, PPV(-)ŏ6 seems to be the result of a natural recombination event between two wild strains of PPV. To our knowledge this is the first evidence of homologous RNA recombination (a process which could play an important role in the evolution of RNA viruses) within the potyvirus group.