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

Abstract

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.

Blueberry red ringspot virus eliminated from highbush blueberry by shoot tip culture

“…In both Slovenia and the Czech Republic, BRRV occurrence was limited to a few plants putatively generated from infected propagative material. The infection in the Czech Republic was not actively spreading to the neighbouring plants during 3 years of our monitoring of symptoms and PCR testing (Přibylová et al 2010). Similar observations have been made in Japan, where BRRV infections were specifically restricted to distinct cultivars.…”

“…Branchlets of young shoots from BRRVinfected blueberry plants cv. Darrow (Přibylová et al 2010) were collected in January 2010.…”

“…Symptomatic leaves of cv. Darrow infected with BRRV from a field germplasm collection (Přibylová et al 2010) were collected, maintained at −20°C, and served as a positive control in PCR assays.…”

“…To date, the virus has been detected outside of North America on imported plants in Japan (Isogai et al 2009), and in the three European countries: Slovenia, Czech Republic, and Poland (Mavrič Pleško et al 2009;Přibylová et al 2010;Kalinowska et al 2012). The infection in Slovenia was found in one plant of cv.…”

“…These resulted in finding two infected plants of cv. Darrow about 10 years old among 67 tested plants of 10 cultivars at a single location (Přibylová et al 2010). Infected plants developed typical symptoms of red ringspot on the upper surfaces of leaves and faint ringspot on fruits.…”