Jaroslava Přibylová

Identification and characterization of a new member of the genus Luteovirus from cherry

The complete genomic sequence of a new virus from cherry trees was determined. Its genome is 5857 nt long and resembles that of members of the genus Luteovirus in its genomic organization and nucleotide sequence. Based on the species demarcation criteria for luteoviruses, the virus represents a new luteovirus species. Furthermore, a 47-nt-long inverted repeat was found at the 3’ end of its genome. The virus has been provisionally named cherry-associated luteovirus (ChALV) and is the fourth member of the family Luteoviridae reported to naturally infect woody plants.

First Report of Blueberry red ringspot virus in Highbush Blueberry in the Czech Republic

A collection of highbush blueberry (Vaccinium corymbosum L.) cultivars planted in the field for propagation in South Bohemia was surveyed in May and July of 2009 for the occurrence of detrimental viruses. A total of 67 plants of 10 cultivars (Berkeley, Burlington, Blue Crop, Bluetta, Darrow, Duke, Gila, Jersey, Late Blue, and Northland), were observed for typical Blueberry red ringspot virus (BRRV) symptoms that appear as reddish ring spots and blotches on stems and fruits, exclusively on the upper surface of the older leaves but not the underside. Samples of leaves were collected and maintained at -20°C until used for DNA extraction, then assayed for BRRV infection using PCR. Controls originated from the same blueberry cultivars in vitro. DNA was extracted from leaf tissue with a NucleoSpin Plant II kit for isolating genomic DNA according to the manufacturers instructions (Macherey-Nagel, Düren, Germany). Primer pair BRRV15/16, which amplified fragments of the reverse transcriptase gene (1), was used in PCR for BRRV detection. The program used for PCR amplification was 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 49°C for 30 s, and 70°C for 45 s, followed by a final extension at 70°C for 5 min. The total PCR volume of 25 μl contained 20 ng of DNA, 200 μmol liter-1 dNTPs, 0.5 μl of each primer BRRV15 and BRRV16 (20 pmol μl-1), 75 mM Tris-HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 2.5 U of Taq Purple DNA polymerase, and stabilizers (Top-Bio Ltd., Prague, Czech Republic). Amplifications were conducted in an MJ Research (Waltham, MA) thermocycler. Aliquots (4 μl) of each PCR product were analyzed by electrophoresis in tris-acetate-EDTA buffer. No BRRV symptoms were observed on the plants in early spring, yet BRRV was detected in one symptom-free bush of cv. Darrow by PCR. In July, typical symptoms developed on that and another cv. Darrow bush that was also positive by PCR. DNA fragments of the expected sizes were amplified from total nucleic acid samples of both infected blueberry bushes using primers BRRV15/16, while no amplification products were detected in plants without symptoms. The amplicons obtained with primers BRRV15/BRRV16 were sequenced and revealed 97.5%-nt identity to the BRRV putative reverse transcriptase gene (GenBank Accession No. AF404509). The 845 nt of the amplicon has been deposited at GenBank under Accession No. HM107773. The disease was likely introduced in infected planting material, since no highbush blueberry plantations exist in the vicinity and V. corymbosum is not native to the Czech Republic. In conclusion, to our knowledge, this is the first report of Blueberry red ringspot virus (genus Soymovirus, family Caulimoviridae) in V. corymbosum L. in the Czech Republic. Symptom observation and PCR testing for BRRV should therefore, be incorporated into the certification scheme for highbush blueberry in the Czech Republic. Reference: (1) J. J. Polashock et al. Plant Dis. 93:727, 2009.

The first detection of Candidatus Phytoplasma trifolii in Rhododendron hybridum

Four Rhododendron hybridum plants (from cvs Moravanka and Don Juan), all exhibited symptoms of shortened axillary shoots, reduced leaves with vein clearing and yellowing, undeveloped flowers, and general stunting in a rhododendron nursery garden in southern Bohemia in 2007. Electron microscopy examination of ultra-thin sections revealed the presence of numerous polymorphic phytoplasma-like bodies in the phloem tissue of leaf midribs and petioles. The phytoplasma etiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal phytoplasma primers. Restriction fragment length polymorphism (RFLP) analysis of amplification products obtained with a R16F2/R16R2 primer pair from all symptomatic plants indicated the presence of phytoplasma from the 16SrVI-A subgroup. A detailed comparison of the amplified sequences and phylogenetic analysis confirmed that the phytoplasma belonged to the subgroup 16SrVI-A (clover proliferation phytoplasma group). This is the first report of the natural occurrence of ‘Candidatus Phytoplasma trifolii’ in plants of Rhododendron hybridum.

Sequence comparison and transmission of Blackcurrant reversion virus isolates in black, red and white currants with black currant reversion disease and full blossom disease symptoms.

We collected samples from black, red and white currants showing symptoms of blackcurrant reversion disease (BRD) and full blossom disease (FBD), cultivated in the Czech Republic. Blackcurrant reversion virus (BRV) was detected in all symptomatic plants. After amplification, a substantial part of the 3′ non-translated region (3′-NTR) of RNA2 of 15 new isolates of BRV was sequenced and compared with sequences available in the literature and GenBank. We did not find significant sequence diversity among isolates associated with either FBD or BRD. BRV was graft-transmitted from FBD infected red currant to black currant where symptoms of BRD were observed. Further sequence analysis of BRV isolates resulted in a phylogenetic tree with four branches, each consisting of six to nine isolates. No correlation with geographic origin was visible on the tree as isolates from various countries occurred in all four branches. We also found no correlation between the host and the topology of the tree: most of black currant isolates occurred in branches 3 and 4, but also occurred in branches 1 and 2. Only one white currant and one red currant isolate occurred in branches 3 and 4, respectively. The sequence identity of the Czech isolates in this region ranged from 91.9 to 99.8%. The 17 plant species growing within and in the close vicinity of the BRD-infested plantation were tested negative for BRV by RT-PCR as natural hosts of BRV. BRV was successfully transmitted by mechanical inoculation from black currant to Nicotiana occidentalis and N. tabacum cv. Xanthi, the latter being a new host for BRV. The infection was confirmed by PCR and sequencing.

Complete genome sequence of currant latent virus (genus Cheravirus, family Secoviridae)

The complete nucleotide sequences of RNA1 and RNA2 of the Holandský červený strain of currant latent virus (CuLV) were determined using next-generation sequencing. The RNA1 is predicted to encode a polyprotein 2124 amino acid long with RdRp motifs. The RNA2 is predicted to encode a polyprotein 957 amino acid long with homology to the capsid protein of apple latent spherical virus and cherry rasp leaf virus. Phylogenetic analysis confirms that CuLV is a new distinct member of the genus Cheravirus.

Molecular characterization of a novel capillovirus from red currant

The complete nucleotide sequence of a novel virus from red currant, provisionally named currant virus A (CuVA), was determined. The genome is 7925 nucleotides long and has a 3′-poly(A) tail. The genome organization with two overlapping open reading frames is similar to that of capilloviruses, but the CuVA genome is about 600 nucleotides longer than that of the longest known capillovirus, cherry virus A. The RNA is predicted to encode a polyprotein with domains of methyltransferase, 2OG-Fe(II) oxygenase, papain-like protease, RNA helicase, RdRp, and capsid protein. Phylogenetic analysis confirms that CuVA is a new and distinct member of the genus Capillovirus.

Elucidation of the Roles of Blackcurrant reversion virus and Phytoplasma in the Etiology of Full Blossom Disease in Currants

To determine the roles of phytoplasmas and Blackcurrant reversion virus (BRV) in the etiology of full blossom disease (FBD), we conducted graft and dodder transmission experiments. Scions from FBD-affected Ribes rubrum were grafted onto red currants, white currants, and black currants. Red and white cultivars revealed symptoms of FBD, whereas blackcurrant displayed symptoms of BRV. No differences in symptoms were observed between plants infected with BRV only and those infected with BRV and phytoplasma. Aster yellows phytoplasma subgroup 16SrI-C was transferred from FBD-infected red currants to periwinkle, where symptoms of green and yellow petal were observed. Back-transmission of phytoplasma to currant seedlings of red and black currant was not successful. Scions of periwinkle infected with aster yellows phytoplasmas of subgroup 16SrI-C and 16SrI-B, which were bottle-, bark-, and approach-grafted onto seedlings of red and black currant, resulted in positive but symptomless transmission of phytoplasma to red currant. We conclude that FBD symptoms are induced by BRV rather than by phytoplasma, which was originally described as the causal agent of FBD.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species.

Identification and molecular characterization of a novel varicosa-like virus from red clover

During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.

Complete genome sequences of blueberry red ringspot virus (Caulimoviridae) isolates from the Czech Republic and Slovenia

Genomic DNA of blueberry red ringspot virus (genus Soymovirus, family Caulimoviridae) from highbush blueberry plants growing for years in the Czech Republic and Slovenia was sequenced. The circular dsDNA genomes consist of 8,303 and 8,299 nt, respectively, and contain eight open reading frames (ORFs) longer than 100 amino acids. The European isolates are 90% to 99% identical in aa sequences of distinct proteins. In contrast to the American New Jersey isolate, in-frame initiation codons have been found upstream from AUG codons of ORFs I, IV and V in the European isolates. These and other differences resulted in a longer capsid protein, reverse transcriptase, movement protein and protein the encoded by ORF VII and reduced (75.4% to 93.7%) amino acid identity with corresponding proteins of the New Jersey isolate.