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 “6A” and “7A” groups based on the particular stretch of adenines at nucleotide positions 39–44/45, 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

Molecular Characteristics and Biological Properties of Bean Yellow Mosaic Virus Isolates from Slovakia

Analysis of the viromes of three symptomatic Fabaceae plants, i.e., red clover (Trifolium pratense L.), pea (Pisum sativum L.), and common bean (Phaseolus vulgaris L.), using high-throughput sequencing revealed complex infections and enabled the acquisition of complete genomes of a potyvirus, bean yellow mosaic virus (BYMV). Based on phylogenetic analysis, the Slovak BYMV isolates belong to two distinct molecular groups, i.e., VI (isolate FA40) and XI (isolates DAT, PS2). Five commercial pea genotypes (Alderman, Ambrosia, Gloriosa, Herkules, Senator) were successfully infected with the BYMV-PS2 inoculum and displayed similar systemic chlorotic mottling symptoms. Relative comparison of optical density values using semi-quantitative DAS-ELISA revealed significant differences among virus titers in one of the infected pea genotypes (Ambrosia) when upper fully developed leaves were tested. Immunoblot analysis of systemically infected Alderman plants showed rather uneven virus accumulation in different plant parts. The lowest virus accumulation was repeatedly detected in the roots, while the highest was in the upper part of the plant stem

Phaseolus vulgaris alphaendornavirus-1 is frequent in bean germplasm in Slovakia and shows low molecular variability

Phaseolus vulgaris alphaendornavirus-1 (PvEV-1, family Endornaviridae) was identified by ribodepleted total RNA high-throughput sequencing in the virome of two bean plants (Phaseouls vulgaris L.) grown in a garden in western Slovakia. Two nearly complete PvEV-1 genomes (ca. 14.06 kb, named PV1 and PV2) were assembled, showing 99.9% nucleotide identity, while their nucleotide identity with the reference PvEV-1 genome (NC_039217) reached 98.4%. Two primer pairs spanning the viral helicase encoding region and sequence upstream of the RNA-dependent RNA polymerase were designed and used to confirm the presence of the virus in the original bean samples by RT-PCR. A subsequent search for PvEV-1 presence in Slovakia was focused on two groups of samples: 1) bean plants grown under open field conditions and sampled during the vegetation period and 2) bean accessions grown from seeds obtained from a Slovak and French bean germplasm collection. Based on RT-PCR results, 4 out of 15 bean samples from open fields and 12 out of 21 bean accessions from the curated germplasm collection tested PvEV-1-positive. Interestingly, sequencing of RT-PCR products revealed that all amplified isolates are identical in the two amplified genomic portion which is also identical to those of the PV1 and PV2 isolates. These results suggest a relatively high incidence of PvEV-1 in bean in Slovakia. This is the first evidence and characterization of PvEV-1 from bean plants in Europe

<i>Clematis vitalba</i> Is a Natural Host of the Novel Ilarvirus, Prunus Virus I

Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases

High-Throughput Sequencing Discloses the Cucumber Mosaic Virus (CMV) Diversity in Slovakia and Reveals New Hosts of CMV from the Papaveraceae Family

Cucumber mosaic virus (CMV; Cucumovirus, Bromoviridae) is an omnipresent virus characterized by a large host range and high genetic variability. Using high-throughput sequencing, we have characterized near complete genomes of 14 Slovak CMV variants from different plant hosts. Of these, three variants originated from the Papaveraceae species (oilseed poppy, common poppy and great celandine), previously poorly described as CMV natural hosts. Based on a BLAST search and phylogenetic analysis, the Slovak CMV isolates can be divided into two genetically different Groups, Ia and II, respectively. The SL50V variant, characterized by a divergent RNA2 sequence, potentially represents a reassortant variant. In four samples (T101, SL50V, CP2, MVU2-21), the presence of satellite CMV RNA was identified along with CMV. Although mechanically transmitted to experimental cucumber plants, the role of satellite RNA in the symptomatology observed could not be established due to a complex infection of original hosts with different viruses