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 manufacturer's 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.

Mixed infection of black currant (Ribes nigrum L.) plants with Blackcurrant reversion associated virus and rhabdovirus-like particles with symptoms of black currant reversion disease.

Black currant plants of cvs. Black Smith and Karlstejnský dlouhohrozen showing symptoms of severe Russian (R) form of black currant reversion disease (BCRD) were found in 1999-2000 in the Czech Republic. Five selected plants of both cultivars originating from two distant loci were tested by polymerase chain reaction (PCR) for presence of the Blackcurrant reversion associated virus (BRAV), the causal agent of BCRD. In all plants, virus-specific 215 nt cDNA fragments proving the presence of BRAV were obtained. Moreover, in two of those five black currant plants, rhabdovirus-like particles were found in ultrathin sections by electron microscopical examinations. The particles measured 200-347 nm by 64-90 nm. They occurred mostly within nuclei of parenchyma cells of vascular bundles as single particles, rafts of particles, but also in aggregates. They were found also in the perinuclear space and occasionally directly in the cytoplasm. Clusters of particles either within the nucleus or in the perinuclear space were membrane-bound. We bring evidence on the occurrence of the severe (R) form of BCRD and the first evidence of BRAV in the Czech Republic.

Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

Occurrence of nepoviruses in Rubus species in the Czech Republic.

The occurrence of arabis mosaic virus (AMV), raspberry ringspot virus (RRV), tomato black ring virus (TBRV), strawberry latent ringspot virus (SLRV) and cherry leaf roll virus (CLRV) in cultivated and wild plants of raspberry and blackberry has been studied in the Czech Republic in 1993-1996. Five hundred and seventy samples were collected at 51 localities and assayed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The results represent the first evidence on the occurrence of AMV, RRV, TBRV and SLRV in cultivated Rubus species in the Czech Republic. Isolates AMV M20 and TBRV ML15 which were successfully transmitted by mechanical inoculation and characterized by reactions of differential host plants and by electron microscopy are the first isolates from Rubus from this territory. CLRV was not detected in either cultivated or wild Rubus species.