Detection and characterisation of Plum pox virus (PPV) isolates from Eastern Slovakia revealed the presence of three main viral strains.

Plum pox virus (PPV), the agent responsible for Sharka disease, is the most important viral pathogen of stone fruit trees world-wide, having an endemic status in Slovakia. To increase knowledge of PPV diversity in Slovakia, a set of 11 isolates, originated from the eastern part of the country, was characterised. The isolates were chip-budded from their original Prunus hosts to the susceptible GF305 indicators, exhibiting the symptoms of variable severity. A genomic region encompassing the partial NIb and the hypervariable 5´terminal region of the CP gene was amplified from all 11 isolates in RT-PCR and directly sequenced. The phylogenetic analysis revealed the grouping of the 11 Slovak isolates into 3 distinct clusters, representing the PPV-M (2 isolates), D (7 isolates) and Rec strains (2 isolates). The strain affiliation of isolates was further confirmed by strain-specific RT-PCR, using which the presence of additional mixed infection by minor PPV variants was detected in 2 samples. The results further contribute to the understanding of PPV diversity in Slovakia and confirm the specificity and sensitivity of molecular approaches used for the virus strain determination

Evaluation of the genetic diversity of <em>Plum pox virus</em> 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 7 years 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

Analysis of Hop Stunt Viroid Diversity in Grapevine (Vitis vinifera L.) in Slovakia: Coexistence of Two Particular Genetic Groups

The hop stunt viroid (HSVd) is a widespread subviral pathogen infecting a broad spectrum of plant hosts including grapevine (Vitis vinifera L.). Despite its omnipresence in virtually all grapevine growing areas around the world, molecular data characterizing HSVd populations are missing from Slovakia. Analysis of the complete nucleotide sequences of 19 grapevine variants revealed the existence of two genetic HSVd groups in Slovakia (internally named the &ldquo;6A&rdquo; and &ldquo;7A&rdquo; groups based on the particular stretch of adenines at nucleotide positions 39&ndash;44/45, respectively). Despite their sampling at different times in various unrelated vineyards, the 6A and 7A groups are characterized by low intra-group divergence (~0.3 and 0.2%, respectively). On the other hand, inter-group divergence reached 2.2% due to several mutations, seven of which were found to be group-specific and mainly (except for one) located in the region of the pathogenic domain. Interestingly, in addition to their frequent co-existence within the same geographical location, the mixed infection of the 6A and 7A type sequence variants was also unequivocally and repeatedly proven within single grapevine plants. The RNA secondary structure analysis of representative isolates from each of these two genetic groups indicated a potential compensatory explanation of such mutations. These group-specific sites could be pointing towards the evolutionary selection linked to the necessity of the viroid to retain its structural conformational integrity, crucial for its functional biochemical ability to interact with specific grapevine cellular host factors required for HSVd propagation

Comparative Transcriptome Analysis of Two Cucumber Cultivars with Different Sensitivity to Cucumber Mosaic Virus Infection

Cucumber mosaic virus (CMV), with extremely broad host range including both monocots and dicots around the world, belongs to most important viral crop threats. Either natural or genetically constructed sources of resistance are being intensively investigated; for this purpose, exhaustive knowledge of molecular virus-host interaction during compatible and incompatible infection is required. New technologies and computer-based &ldquo;omics&rdquo; on various levels contribute markedly to this topic. In this work, two cucumber cultivars with different response to CMV challenge were tested, i.e., sensitive cv. Vanda and resistant cv. Heliana. The transcriptomes were prepared from both cultivars at 18 days after CMV or mock inoculation. Subsequently, four independent comparative analyses of obtained data were performed, viz. mock- and CMV-inoculated samples within each cultivar, samples from mock-inoculated cultivars to each other and samples from virus-inoculated cultivars to each other. A detailed picture of CMV-influenced genes, as well as constitutive differences in cultivar-specific gene expression was obtained. The compatible CMV infection of cv. Vanda caused downregulation of genes involved in photosynthesis, and induction of genes connected with protein production and modification, as well as components of signaling pathways. CMV challenge caused practically no change in the transcription profile of the cv. Heliana. The main differences between constitutive transcription activity of the two cultivars relied in the expression of genes responsible for methylation, phosphorylation, cell wall organization and carbohydrate metabolism (prevailing in cv. Heliana), or chromosome condensation and glucan biosynthesis (prevailing in cv. Vanda). Involvement of several genes in the resistant cucumber phenotype was predicted; this can be after biological confirmation potentially applied in breeding programs for virus-resistant crops

Analysis of Grapevine rupestris stem pitting-associated virus in Slovakia Reveals Differences in Intra-Host Population Diversity and Naturally Occurring Recombination Events

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a worldwide-distributed pathogen in grapevines with a high genetic variability. Our study revealed differences in the complexity of GRSPaV population in a single host. A single-variant GRSPaV infection was detected from the SK30 grapevine plant. On the contrary, SK704 grapevine was infected by three different GRSPaV variants. Variant-specific RT-PCR detection protocols have been developed in this work to study distribution of the three different variants in the same plant during the season. This study showed their randomized distribution in the infected SK704 grapevine plant. Comparative analysis of fulllength genome sequences of four Slovak GRSPaV isolates determined in this work and 14 database sequences showed that population of the virus cluster into four major phylogenetic lineages. Moreover, our analyses suggest that genetic recombination along with point mutations could play a significant role in shaping evolutionary history of GRSPaV and contributed to its extant genetic diversification

Sequence variability, recombination analysis, and specific detection of the W strain of Plum pox virus

Plum pox virus (PPV), a member of the genus Potyvirus, is the causal agent of Sharka, the most detrimental disease of stone-fruit trees worldwide. PPV isolates are grouped into seven distinct strains. The minor PPV-W strain was established recently for the divergent W3174 isolate found in Canada. Here, the partial or complete genomic sequences of four PPV-W isolates from Latvia have been determined. The completely sequenced isolates LV-141pl and LV-145bt share 93.1 and 92.1% nucleotide identity, respectively, with isolate W3174, with two regions of higher (>20%) divergence in the P1/HC-Pro and NIa (VPg) regions. Further analyses demonstrated that these two regions correspond to two independent recombination events in the W3174 genome, one involving PPV-M (approximate genome positions 692 to 1424) and the other PPV-D (nucleotides 5672 to 5789). The LV-141pl and LV-145bt isolates appear to be representatives of the “ancestral” PPV-W strain, not affected by recombination. The PPV-W intrastrain variability is substantially higher than that of all other PPV strains, with potential implications for the serological detection of PPV-W isolates. A PPV-W-specific primer pair has been developed, allowing the specific reverse-transcription polymerase chain reaction detection of all five presently available W isolates. The characterization of these new PPV-W isolates sheds light on PPV-W evolutionary history, further supports the hypothesis of its East-European origin, and opens the way for the biological and epidemiological characterization of this poorly known PPV strain

A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites.

Grapevine Pinot gris virus (GPGV) is a widely distributed grapevine pathogen that has been associated to the grapevine leaf mottling and deformation disease. With the aim of better understanding the disease epidemiology and providing efficient control strategies a specific and quantitative duplex TaqMan real-time RT-PCR assay has been developed. This method has allowed reliable quantitation of the GPGV titer ranging from 30 up to 3 x 108 transcript copies, with a detection limit of 70 viral copies in plant material. The assay targets a grapevine internal control that reduces the occurrence of false negative results, thus increasing the diagnostic sensitivity of the technique. Viral isolates both associated and non-associated to symptoms from Greece, Slovakia and Spain have been successfully detected. The method has also been applied to the absolute quantitation of GPGV in its putative transmission vector Colomerus vitis. Moreover, the viral titer present in single mites has been determined. In addition, in the current study a new polymorphism in the GPGV genome responsible for a shorter movement protein has been found. A phylogenetic study based on this genomic region has shown a high variability among Spanish isolates and points to a different evolutionary origin of this new polymorphism. The methodology here developed opens new possibilities for basic and epidemiological studies as well as for the establishment of efficient control strategies

Molecular and Biological Characterisation of Turnip mosaic virus Isolates Infecting Poppy (Papaver somniferum and P. rhoeas) in Slovakia

In recent years, the accumulated molecular data of Turnip mosaic virus (TuMV) isolates from various hosts originating from different parts of the world considerably helped to understand the genetic complexity and evolutionary history of the virus. In this work, four complete TuMV genomes (HC9, PK1, MS04, MS15) were characterised from naturally infected cultivated and wild-growing Papaver spp., hosts from which only very scarce data were available previously. Phylogenetic analyses showed the affiliation of Slovak Papaver isolates to the world-B and basal-B groups. The PK1 isolate showed a novel intra-lineage recombination pattern, further confirming the important role of recombination in the shaping of TuMV genetic diversity. Biological assays indicated that the intensity of symptoms in experimentally inoculated oilseed poppy are correlated to TuMV accumulation level in leaves. This is the first report of TuMV in poppy plants in Slovakia