Little cherry disease (LChD), a virus disease of sweet (Prunus avium) and sour cherries (P. cerasus), is caused by members of the Closteroviridae family. Symptoms are especially visible on fruits and leaves. Leaves become red or bronze in late summer and fall. Fruit are small, angular, and pointed. Fruits are unmarketable due to a characteristic bitter flavor. LChD also causes reduction of yield (1). Sweet and sour cherries are the second (after apples) most often grown fruit species in the Czech Republic. Since LChD occurred in Germany (1) and Poland (2) in 2007 and 2008, sweet and sour cherry trees with LChD symptoms were surveyed in orchards in the East Bohemia Region of the Czech Republic. The presence of LChD was determined by reverse transcription (RT)-PCR and woody indicator plants, as recommended by the European and Mediterranean Plant Protection Organization (EPPO). Different parts of plants were taken from trees with suspicious symptoms to observe the dynamics of virus infection during the 2009 growing season. Total RNA was isolated from young leaves, blossoms, fruits, and fully developed leaves with a CONCERT Plant RNA Purification Reagent (Invitrogen, Carlsbad, CA) (3). RT-PCR was performed with a QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany) and oligonucleotides previously described (4). Oligonucleotide LCV3EC (5′-GCTCTAGAGGCACCTTTTATTTTTTATATATGC-3′), complementary to position 16910 to 16934 (GenBankAccession No. Y10237) (with the addition of eight nonviral nucleotides to introduce an XbaI site), was used as a negative-sense primer in RT reactions and PCR. Oligonucleotide LCV16659 (5′-GTTATAGAATTCACTGCAAGTG-3′) was used as a positive-sense primer for PCR amplification. The program used for cDNA synthesis was 50°C for 30 min, followed by denaturation for 10 min at 95°C, 35 cycles of 45 s at 94°C, 45 s at 58°C, and 45 s at 72°C. A final incubation was at 72°C for 5 min (1). The finished PCR products (430 bp) were analyzed on 1% agarose gels (stained with SYBR green). According to the preliminary results, young leaves from buds (67% of samples of selected trees with LChD were positive), blossoms (67% positive), and leaves taken in autumn (67% positive) were optimal for the detection of LChD by RT-PCR. The trial with woody indicator plant species was established in the field. Indicators P. avium cv. Sam and P. avium cvs. Bing, F12/1, and Canindex (4) were inoculated with buds from LChD-infected trees and observed for 2 years. Woody indicators remained symptomless throughout the first year of observation, but the indicators showed red coloration of leaves in late summer of the second year. P. avium cv. Canindex seems to be the best woody indicator for testing of LChD in the climatic conditions of the Czech Republic. To our knowledge, this is the first report of LChD in the Czech Republic. References: (1) W. Jelkmann et al. Acta Hortic. 781:321, 2008. (2) B. Komorowska and M. Cieślińska. Plant Dis. 92:1366, 2008. (3) J. Matoušek et al. Biol.Chem. 388:1, 2007. (4) M. Vitushkina et al. Eur. J. Plant Pathol. 103:803, 1997.
Distribution of Plum pox virus strains in natural sources in the Czech Republic