Ludvíková H, Suchá J
Research and Breeding Institute of Pomology Holovousy, Ltd., Holovousy 150801 Hořice, Czech Republic.
Plant Dis. 2011 Sep;95(9):1197. doi: 10.1094/PDIS-12-10-0868.
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.
小叶樱桃病（LChD）是甜樱桃（欧洲甜樱桃）和酸樱桃（欧洲酸樱桃）的一种病毒病，由长线形病毒科成员引起。症状在果实和叶片上尤为明显。夏末和秋季，叶片会变成红色或青铜色。果实小、呈角状且尖端尖锐。由于具有独特的苦味，果实无法进入市场销售。LChD还会导致产量下降（1）。甜樱桃和酸樱桃是捷克共和国种植的第二大水果品种（仅次于苹果）。自2007年德国（1）和2008年波兰（2）出现LChD以来，对捷克共和国东波希米亚地区果园中出现LChD症状的甜樱桃和酸樱桃树进行了调查。按照欧洲和地中海植物保护组织（EPPO）的建议，通过逆转录（RT）-PCR和木本指示植物来确定LChD的存在。从具有可疑症状的树上采集植物的不同部位，以观察2009年生长季节病毒感染的动态情况。使用CONCERT植物RNA纯化试剂（Invitrogen，加利福尼亚州卡尔斯巴德）从幼叶、花朵、果实和完全展开的叶片中分离总RNA（3）。使用QIAGEN一步RT-PCR试剂盒（Qiagen，德国希尔德）和先前描述的寡核苷酸进行RT-PCR（4）。与16910至16934位互补的寡核苷酸LCV3EC（5'-GCTCTAGAGGCACCTTTTATTTTTTATATATGC-3'）（GenBank登录号Y10237）（添加了八个非病毒核苷酸以引入XbaI位点）用作RT反应和PCR中的反义引物。寡核苷酸LCV16659（5'-GTTATAGAATTCACTGCAAGTG-3'）用作PCR扩增的正义引物。用于cDNA合成的程序为50°C 30分钟，然后在95°C变性10分钟，94°C 45秒、58°C 45秒和72°C 45秒进行35个循环。最后在72°C孵育5分钟（1）。将完成的PCR产物（430 bp）在1%琼脂糖凝胶上进行分析（用SYBR green染色）。根据初步结果，芽中的幼叶（所选有LChD症状树木的样本中67%呈阳性）、花朵（67%呈阳性）和秋季采集的叶片（67%呈阳性）最适合通过RT-PCR检测LChD。在田间建立了木本指示植物品种试验。用感染LChD树木的芽接种指示植物欧洲甜樱桃品种Sam和欧洲甜樱桃品种Bing、F12/1和Canindex（4），并观察2年。在观察的第一年，木本指示植物一直没有症状，但在第二年夏末，指示植物的叶片出现红色。在捷克共和国的气候条件下，欧洲甜樱桃品种Canindex似乎是检测LChD的最佳木本指示植物。据我们所知，这是LChD在捷克共和国的首次报道。参考文献：（1）W. Jelkmann等人，《园艺学报》781:321，2008年。（2）B. Komorowska和M. Cieślińska，《植物病害》92:1366，2008年。（3）J. Matoušek等人，《生物化学》388:1，2007年。（4）M. Vitushkina等人，《欧洲植物病理学报》103:803，1997年。
