FIRST REPORT OF PLUM POX VIRUS STRAIN PPV-T IN ALBANIA

Sharka, caused by Plum pox virus (PPV) is considered the most detrimental disease of plum. Although PPV is widespread in all plum growing areas from Romania and causes serious yield losses, limited information about the occurrence of PPV strains are available in some regions like Moldavia plum area. To supplement this information, fifty PPV isolates collected from five different commercial plum orchards from Moldavia were investigated. PPV detection was made by DAS-ELISA and by IC-RT-PCR. PPV strains were serologically determined by TAS-ELISA using PPV-D and PPV-M specific monoclonal antibodies. Molecular strain typing was done by RT-PCR targeting three genomic regions corresponding to (Cter)CP, (Cter)NIb/(Nter)CP and CI. RFLP analysis at the C-ter of CP cistron was used to distinguish the two major strains, D and M based on a RsaI polymorphism located in this genomic section. All PCR products targeting (Cter)CP and one PCR product spanning the (Cter)NIb/(Nter)CP were sequenced. All PPV isolates typed as PPV-M by serological analysis and by molecular differentiation in the genomic region corresponding to (C-ter)CP proved to be PPV recombinant (PPV-Rec) when the molecular analysis were performed in the region corresponding to NIb/CP. The sequencing results confirmed a high similarity with different sequences of PPV-Rec previously reported. Overall results provided that in Moldavia the predominant strain is PPV-D, followed with a much lower frequency by PPV-Rec which shares the CP gene with M strain. The mixed infections (PPV-D+PPV-Rec), were sporadically recorded in this plum growing area.

Serological and Molecular Typing of Plum pox Virus Isolates in the Transylvania, Romania

This study was conducted to determine the presence of plum pox virus (PPV) (family Potyviridae, genus Potyvirus) in different regions of Moldavia. The disease mainly affects apricot, plum, and peach. The genus Potyvirus was first detected in Bulgaria in 1917; since then, it has spread to most of eastern and central Europe and the Mediterranean basin. In Romania, the PPV is widespread in plum orchards, very limited information about the variability of isolated is known. We collected and investigated twenty PPV samples, who were molecular characterized by targeting (Cter)CP, (Ctre)Nib – (Nter)CP and CI genomic regions with specific PCR markers. RFLP protocol we used distinguished between the two major strains, D and M, based on Rsa I polymorphic loci in the (Cter) CP region. Material and Method: All PPV isolates were collected from field experimental plots of Moldavia areas (Bacau and Vaslui county) based on typical symptoms on leaves. For RNA extraction was used Oiagen One Step Kit, and molecular characterization was confirmed by RT-PCR using pair of primer P1/P2. RFLP method was used to distinguish the D and M strains based on RsaI polymorphism located in this genomic section. Results and Discussions: PPV-infection was detected in all samples. The P1/P2 primers revealed and confirmed the presence of the virus by amplifying the expected 243-bp fragment from the CP gene. Using specific primers, fifteen isolates were identified as PPV-D, five as PPV-M and one revealed the presence of PPV-rec. RFLP analysis confirmed these results by indicating the presences of the Rsa I polymorphism in PPV-D strain.

Plum pox virus (PPV) is the most dangerous viral pathogen of stone fruits causing serious yield losses. Although PPV is widespread in Romanian plum orchards, very limited information about the variability of isolates is known. In this study we collected an d investigated twenty PPV isolates from two experimental plots from Fruit Research and Development Station Bistrita . PPV strains were serologically determined by TAS- ELISA using PPV-D and PPV-M specific monoclonal antibodies. Molecular strain typing was performed by I C- RT-PCR targeting three genomic regions (Cter)CP, (Cter)NIb - (Nter)CP and CI. RFLP analysis was used t o distinguish the two major strains, D and M based on a Rsa I polymorphism located in (Cter) CP. PCR products spanning (Cter)CP and (Cter)NIb - (Nter)CP regions were sequenced. All PPV isolates typed as PPV-M by serological analysis and also by molecular differe ntiation in (Cter)CP region proved to be recombinan ts between PPV-D and PPV-M when the molecular analysis were performed in (Cter) NIb - (Nter)CP region. The sequencing results revealed a high homology with di fferent sequences of PPV recombinant (PPV-Rec) previously reported. All these recombinant isolates share the same recombination breakpoint and have c onserved the DAG motif, which is considered essential for po tyvirus aphid transmission. This genetic similarity confirms that PPV-Rec may represent an ancestral group with a common evolutionary origin. This is the first rep ort of naturally infected plum trees with recombinant PPV strain (PPV-Rec) in Romania and predicted a widespread in the other areas where plum is growing.

Plum pox virus (PPV) is the causal agent of sharka disease, which is responsible for severe damage and important economic losses in the stone fruit industry. The disease mainly affects stone-fruit species, mainly apricot, plum and peach . Sharka is originated from Eastern Europe and was described for the first time around 1915 in Bulgari a (Atanasoff, 1932). In this study we collected thi rty PPV isolates from one experimental orchard belonging to the Fruit Research and Development Station Cluj. Molecular strain differentiation was done with the help of RT-PCR technique by analyzing the genomic region (Cter)CP of the virus by using RFLP analysis we were able to distinguish the two major strains, D and M, based on Rsa I polymorphism located in the genomic region (Cter) CP.

A certification program focused on plum was established at Fruit Research & Development Station Bistrita, Romania started in 2008. The objective is to obtain in the first step the prebasic material and, subsequently, to establish plum mother plantations with basic material satisfying the EPPO recommended certification standards. Sixteen plum (Prunus domestica) cultivars (Iulia, Matilda, Geta, Zamfira, Ivan, Dani, Doina, Romaner, Elena, Jubileu 50, Delia, Flora, Renclod d’Althan, Stanley, Anna Spath and Carpatin) were subjected to this work. Selected plum trees from experimental orchards were monitored and tested. One shoot was collected from each candidate and tested before grafting onto virus-free Myrobolan 29C rootstock. Selected clones were also tested on woody indicators GF 305. Propagated material was individually tested one year later. All tests were done according to the EPPO recommendations. DAS-ELISA was used for Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Myrobalan latent ringspot virus (MLRSV). To increase the sensitivity of PPV and PDV detection, IC-RT-PCR was performed. Spot-Real Time RT-PCR was also used to detect PPV. Nested-PCR method was made to detect Candidatus Phytoplasma prunorum. Delia cultivar was found infected by PPV and eliminated from propagation by grafting. This cultivar is subjected to pathogen elimination by using in vitro chemo-thermotherapy. The other fifteen cultivars were found virus-free both before and after propagation. The certification of this material as ‘Prebasic category’ is in progress.

Partial molecular characterization of isolates of Plum pox virus (PPV) from Kazakhstan was described by Spiegel et al. (2004). The isolates were classified as members of the Dideron (D) strain based on: a) RFLP analysis of a 243-bp amplicon in the region of the C terminus of the coat protein (CP) fragment, b) RFLP analysis of an 836-bp amplicon in the P3-6K1 coding region and c) analysis of the CP coding region. The plum isolates were detected in plum trees maintained in the plum selection-orchard of the Pomological Garden, Almaty, Kazakhstan. The apricot isolates were detected in wild apricot seedlings obtained originally from Zailiyski Alatou at the northern edge of the Tien-Shan Mountains, but maintained in the Pomological Garden, Almaty, for about 15 years. Disease symptoms were observed on fruit from one of these infected apricot trees and on the leaves of the infected plum trees. PPV was never detected in any wild apricot plants growing in their natural habitat in Zailiyski Alatou. In this recent study, the virus genomes were sequenced completely and their analysis confirmed their identities as members of the D strain of PPV. Interestingly, in some cases, the genomes of the apricot and plum isolates were very similar, with identities of approximately 99.7% (nucleotide and amino acid sequence). Phylogenetic analyses seem to indicate a close relationship of the Kazakhstan apricot and plum isolates to each other, and to the PPV isolate BIII/2 (GenBank accession no. GU461890), a plum isolate from Slovakia. The Kazakhstan isolates and the Slovakia isolate are approximately 99% identical (nucleotide and amino acid sequence). The deletion event described by Spiegel et al. (2004) in the plum isolate P5R8 (KZPLa1) was confirmed.

Ten-year results of the practical immunity investigation of apricot cv. Harlayne are presented. Two-year-old trees of cv. Harlayne were inoculated by chip-budding with six different strains and isolates of Plum pox virus (PPV). PPV inoculated trees grew in the field and were evaluated from 2001 to 2011. No PPV symptoms appeared in the leaves of cv. Harlayne within ten years (2002–2011), and within eight years (2004–2011) in the fruits and stones. None of the six isolates of three different PPV strains was detected in the leaves and fruits by ELISA. Suckers of the rootstock Prunus myrobalana developed around cv. Harlayne trees in 2005–2011 were symptomless and ELISA was negative within seven years. New trees of cv. Harlayne obtained from tested trees by budding on PPV susceptible apricot rootstock MVA-2 in 2007 were PPV free from 2008 through 2011. The presence of PPV was proved by ELISA neither in leaves of cv. Harlayne nor in rootstock MVA-2.

Further characterization of a new recombinant group of Plum pox virus isolates, PPV-T, found in orchards in the Ankara province of Turkey

Prunus virus isolates (PVIs) were found that cross-reacted with plum pox virus (PPV) polyclonal antisera in immunosorbent electron microscopy, immunogold labeling, and Western blot analysis. The positive reactions in Western blot analysis were not affected when bovine serum albumin, fetal calf serum, glycerol, D-glucose, or skimmed (defatted) milk were used as blocking agents. Monoclonal antibodies to PPV reacted with the PVIs in Western blot analysis. The PVIs and some PPV isolates also cross-reacted in immunosorbent electron microscopy and Western blot analysis with apple stem pitting virus polyclonal antisera. The RNAs associated with the PVIs were analyzed to evaluate their homology with PPV RNA. The double-stranded RNA (dsRNA) profile of the PVIs showed that there were 3 to 5 dsRNA species ranging in size from 2.7 to 7.4 x 10 6 Da. Oligonucleotide sequences corresponding to the 3' noncoding region, the 5' noncoding region, and the 3' terminus of the coat protein coding sequence of a non-aphid-transmissible strain of PPV (PPV-NAT) were used as probes in hybridization studies and as primers in reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The PVIs did not react. RT-PCR analysis using PPV-specific primers from the conserved 5' terminus of the coat protein coding sequence of PPV gave negative results with all PVIs. These analyses of the viral genome indicate that the PVIs are not isolates of PPV. Further studies to determine if the PVIs were members of the potyvirus group were negative. No evidence of associated cytoplasmic cylindrical inclusions or reaction with a potyvirus-specific monoclonal antibody was observed. The PVI particles ranged in size from 740 to 791 nm in length, and 10.4 to 14.4 nm in width. The coat protein subunit molecular mass ranged from 48 to 56 kDa, and can be distinguished from PPV by Western blot analysis. The implications of the serological cross-reactions are discussed.

The genomic sequence of an El Amar isolate of plum pox virus (PPV) from Egypt was determined by sequencing overlapping cDNA fragments. This is the first complete sequence of a member of the El Amar (EA) strain of PPV. The genome consists of 9791 nt, excluding a poly(A) tail at the 3' terminus. The complete nt sequence of PPV EA is 79-80%, 80%, 77%, and 77% homologous with isolates of strains D/M, Rec (BOR3), C, and W, respectively. The polyprotein identity ranged from 87-91%. Phylogenetic analysis using the complete genome sequence of PPV EA confirmed its strain status. No significant recombination signals were identified using PhylPro and SimPlot scans of the PPV EA sequence, however an interesting recombination signal was identified in the P1/HC-Pro region of PPV W3174.

The recent observation of the frequent occurrence of natural recombinant Plum pox virus (PPV) isolates has led to the identification of a distinct PPV subgroup, named PPV-Rec. The diversity, origin and geographical spread of the recombinant PPV isolates belonging to this subgroup remain, however, relatively poorly known. In an effort to further our understanding of these isolates, eight PPV isolates from Serbia, the country from which the first such recombinant (PPV-o6) originated, were characterized. Depending on the genomic region targeted by different typing assays, seven of the eight isolates tested presented discrepancies in their typing behavior. Sequence analysis of the (Cter)NIb-(Nter)CP region confirmed the recombinant nature of these seven isolates which all presented an identical recombination breakpoint identical to previously characterized PPV-Rec isolates. Biological indexing and immunoblot analysis provided indications that asymptomatic infection of the GF305 peach indicator and migration of the coat protein as a double-band in immunoblots may represent conserved and discriminating properties of PPV-Rec isolates. The genetic diversity of PPV-Rec isolates from former Yugoslavia (Serbia, Bosnia and Herzegovina) was estimated to be twice as large as that of the PPV-Rec isolates obtained from all other countries to date (Albania, Bulgaria, Czech republic, Germany, Hungary and Slovakia). These last results are consistent with the hypothesis that former Yugoslavia is the center of dispersion of PPV-Rec. Taken together, the results presented here provide further evidence for the wide distribution and temporal genetic stability of these natural PPV recombinant isolates and provide for the first time a possible scenario for their dispersion throughout central and eastern Europe.

In July of 2008, during the Canadian Plum pox virus (PPV) eradication survey, three Prunus spp. trees (B-5, B-6, and C-1) in a home owner's yard in Grimsby, ON, Canada were found to be infected with PPV by triple antibody sandwich (TAS)-ELISA using the 5B generic monoclonal detecting antibody (1) and reverse transcription (RT)-PCR using the well-validated universal primer set P1/P2. All three trees were grafted on an unknown plum rootstock cultivar. Tree B-5 contained grafts of unknown peach (P. persica) and plum (P. domestica) cultivars, tree B-6 contained the same peach graft as tree B-5 and an unknown apricot (P. armeniaca) cultivar, and tree C-1 was grafted with the same plum cultivar as tree B-5. Strain typing was done by TAS-ELISA using strain-specific monoclonal antibodies for D, M, C, and EA strains. Positive results were obtained with the M-specific test. Strain typing by RT-PCR also was done using primers specific for D, M, W, and recombinant (Rec) strains. Positive results were obtained with the M and Rec primers (4). The 605-bp fragment generated by the PPV Rec primers, which spans the recombination site, was cloned and sequenced. The nucleotide sequences obtained from B-5, B-6, and C-1 are 99% identical to each other and approximately 98 and 99% identical to the PPV Rec isolates BOR-3 and J4c, respectively. Correspondingly, percentage identities are approximately 90% for PPV M, 84% for PPV isolate D-Fan (a typical Canadian D isolate), and 69% for PPV W. The deduced amino acid sequence of B-5, B-6, and C-1 are 98 to 99% identical to each other, 99% identical to the PPV Rec isolates BOR-3 and J4c, 92% identical to isolates of PPV M, and only 84% identical to the typical Canadian D isolate PPV Fan. The P3-6K1 genomic region was amplified using primers that generate a 836-bp fragment (2). This region was 97 to 98% identical to the PPV Rec isolates BOR-3 and J4c, 96 to 97% identical to isolates of PPV D, but only 86% identical to isolates of PPV M. The data above confirm that the PPV isolates B-5, B-6, and C-1 belong to the strain PPV Rec (3). To our knowledge, this is the first report of PPV Rec in North America, and together with PPV D and W, it represents the third PPV strain found on this continent. An intensive survey of all Prunus spp. within a 1.5-km radius area surrounding the home owner's property failed to reveal any additional PPV-positive plants. The three positive plants were removed.

Oligonucleotide microarray-based detection and genotyping of Plum pox virus

Properties of the sour cherry isolate of plum pox virus (PPV) were investigated by reverse transcription-polymerase chain reaction (RT-PCR), restriction fragment length polymorphism (RFLP), molecular hybridization, nucleotide sequencing, ultrathin sectioning of infected tissue, and graft transmission to different cherry rootstocks. Analysis of RT-PCR-amplified cDNA product from infected tissue with primers for the 3' noncoding region (3'-NCR) of the PPV genome and molecular hybridization of the amplified product with a labeled PPV cRNA probe verified that the potyvirus infecting sour cherry trees (Prunus cerasus) in Moldova is an isolate of PPV. RFLP analysis of RT-PCR products from infected tissue with specific primers for the 3'-terminal region of the PPV coat protein (CP) gene revealed that the sour cherry isolate of PPV is a unique strain of PPV and a prototype of a new group that contains neither the RsaI nor the AluI restriction site. These results were confirmed by nucleotide sequencing analysis. Nucleotide sequencing of the 3'-NCR and the region coding for the 3'-terminal fragment of the PPV CP gene showed about 93% identity to that of other PPV isolates. RT-PCR assays of tissue extracts from three sour cherry cultivars demonstrated that sour cherry PPV was distributed systemically in sour cherry trees and infected leaf, bark, root, flower, fruit, and seed tissues. The virus was successfully transmitted by chip bud grafting to rootstocks of P. avium (sweet cherry) and P. mahaleb.