Clematis vitalba Is a Natural Host of the Novel Ilarvirus, Prunus Virus I

Abstract

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.

Keywords: Bromoviridae; Clematis; PrVI; alternative host; high-throughput sequencing.

Figure 1

Leaves from a symptomatic Clematis vitalba plant showing bright yellow line pattern and ringspot symptoms collected in July 2018 at Budakeszi (close vicinity to Budapest, Hungary) (photo: Pal Salamon).

Figure 2

Validation of the small RNA HTS. (a) Primers were designed based on the Ilarvirus-genus-specific contig sequences able to amplify most of the each of three genomic RNAs. (b) Agarose gel electrophoresis of the RT-PCR productsobtainedwith the primers shown in (a). M is the GeneRuler 100 bp Plus DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA).

Figure 3

C. vitalba plants from Slovakia showing mosaic like symptoms hosting (a) PL622 and (b) PlCv5 PrVI isolates.

Figure 4

Biotest of the Croatian Cle-1 isolate. (a) Symptoms in experimental plants after inoculation with the Croatian archival Cle isolates of PrVI: local chlorotic mottling 8 dpi in Chenopodium quinoa with Cle-1 isolate 680 (top left). The same plant 30 dpi with systemic mottling (top right). Systemic yellow net in Nicotiana glutinosa top leaves infected with Cle-1 isolate 847 16 dpi (bottom left) and N. megalosiphon with Cle-2 710 with less intense netting 27 dpi (red arrow). (b) RT-PCR confirmation of the PrVI infection in the sampled test plants using diagnostic primers: PrVI_CP-F and PrVI_CP-R amplifying 351 bp of the coat protein encoded on RNA3. M is the GeneRuler 100 bp Plus DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA).

Figure 5

Phylogenetic analysis of the nucleic acid sequences of RNA1 of PrVI and other ilarviruses belonging to group 1. Viruses are referred by the GenBank accession numbers of the reference genomes, following by the full name of the virus. In case of PrVI only the abbreviated PrVI is displayed followed by the host name and the origin of the country (SLO—Slovenia, SK—Slovakia, GR—Greece, HR—Croatia, RU—Russia and HU—Hungary). The red sign indicates a new virus with a reference of which sequence was not included in the phylogenetical analysis in the original description of the virus [14].

Figure 6

Phylogenetic analysis of the nucleic acid sequences of RNA2 of PrVI and other ilarviruses belonging to group 1. Viruses are referred by the GenBank accession numbers of the reference genomes, following by the full name of the virus. In case of PrVI only the abbreviated PrVI is displayed followed by the host name and the origin of the country (SLO—Slovenia, SK—Slovakia, GR—Greece, HR—Croatia, RU—Russia and HU—Hungary). The red sign indicates a new virus with a reference of which sequence was not included in the phylogenetical analysis in the original description of the virus [14].

Figure 7

Phylogenetic analysis of the nucleic acid sequences of RNA3 of PrVI and other ilarviruses belonging to group 1. Viruses are referred to by the GenBank accession numbers of the reference genomes, following by the full name of the virus. In case of PrVI only the abbreviated PrVI is displayed followed by the host name and the origin of the country (SLO—Slovenia, SK—Slovakia, GR—Greece, HR—Croatia, RU—Russia and HU—Hungary). The red sign indicates a new virus with a reference of which sequence was not included in the phylogenetical analysis in the original description of the virus [14].

Figure 8

Clematis mini survey. (a) Symptoms of the sampled leaves of C. vitalba, indicating the geographical origin and the year and month of sampling. (b) Result of the RT-PCR test of the sampled C. vitalba plants using diagnostic primers: PrVI_CP-F and PrVI_CP-R [14] amplifying 351 bp part of the coat protein encoded on RNA3. M is the GeneRuler 100 bp Plus DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA). Sequences of the amplified viral RNA3 segments were directly sequenced (GenBank acc. numbers are CleSZD–OR485309 and CleGD–OR485310).