Komorowska B, Cieślińska M
Department of Plant Protection, Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland.
Plant Dis. 2008 Sep;92(9):1366. doi: 10.1094/PDIS-92-9-1366A.
Little cherry disease (LChD) is a serious viral disease of sweet (Prunus avium) and sour (P. cerasus) cherry trees. Infection of sensitive cultivars results in small, angular, and pointed fruits with reduced sweetness. In late summer, leaves show a characteristic red coloration or bronzing of the surfaces. One Ampelovirus species, Little cherry virus 2 (LChV-2) (2), and one unassigned species in the Closteroviridae, Little cherry virus 1 (LChV-1) (3), have been associated with LChD. Twenty-seven sour and sweet cherry trees of six varieties from orchards located in several regions of Poland were tested for LChV-1 and LChV-2. Leaf samples were taken either from trees showing fruit symptoms or from asymptomatic trees during the summer of the 2006 growing season. RNA was isolated from the leaves with an RNeasy Kit (Qiagen, Hilden, CA), and reverse transcription (RT)-PCR was performed using primer pairs LCV1U/LCV1L and LCV2UP2/LCV2LO2, which are specific for a 419-bp fragment of the LChV-1 3' nontranslated region and a 438-bp fragment of the LChV-2 methyltransferase gene, respectively (1). The primer pair L2CPF (5'-GTTCGAAAGTGTTTCTTGAT-3') and L2CPR (5'-GCAACAGAAAAACATATGACTCA-3') was designed from existing LChV-2 sequences (GenBank Accession Nos. AF416335 and NC_005065) to amplify the entire LChV-2 coat protein (CP) gene (nucleotides 13,007 to 14,134). The amplified cDNA fragments of LChV-2 genome were ligated to the bacterial vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA), which was used to transform Escherichia coli TOP10 competent cells following the manufacturer's protocol. Both strands of three clones for each amplified LChV-2 genome fragment were sequenced with an automated nucleotide sequencer at the Institute of Biochemistry and Biophysics in Warsaw. RT-PCR results showed that 6 of 27 trees were infected, with LChV-1 detected in five sweet cherry trees and LChV-2 singly infecting one sweet cherry tree cv Elton (isolate C4/14). The nucleotide sequence of the 438-bp methyltransferase gene fragment of isolate C4/14 showed 86, 85, and 84% identity to GenBank Accession Nos. AF333237, AF531505, and AJ430056, respectively, all previously reported LChV-2 sequences from cherry trees. Sequence analysis of the 1,088-bp coat protein gene showed 89 to 91% and 92 to 93% nucleotide and amino acid identity, respectively, with the aforementioned three LChV-2 isolates. The tree infected with LChV-2 was indexed by graft transmission to the woody indicator, Prunus avium cv. Canindex, which showed reddening of the leaves characteristic of LChD 3 months after inoculation. Since cherry production in Poland is 230,000 t per year, the disease may have a significant economic impact because the affected fruits are unsuitable either for consumption or sale. To our knowledge, this is the first report of LChV-2 in Poland. References: (1) M. E. Rott and W. Jelkmann. Phytopathology 91:261, 2001. (2) M. E. Rott and W. Jelkmann. Arch. Virol. 150:107, 2005. (3) M. Vitushkina et al. Eur. J. Plant Pathol. 103:803, 1997.
小樱桃病（LChD）是甜樱桃（欧洲甜樱桃）和酸樱桃（欧洲酸樱桃）树的一种严重病毒性疾病。敏感品种感染后会结出小的、有棱角且尖端尖锐的果实，甜度降低。夏末时，叶片表面会呈现出特征性的红色或青铜色。一种葡萄病毒属病毒，小樱桃病毒2（LChV - 2）（2），以及线形病毒科中一个未分类的病毒，小樱桃病毒1（LChV - 1）（3），都与小樱桃病有关。对来自波兰几个地区果园的6个品种的27株酸樱桃树和甜樱桃树进行了LChV - 1和LChV - 2检测。在2006年生长季的夏季，从表现出果实症状的树上或无症状的树上采集叶片样本。使用RNeasy试剂盒（Qiagen公司，德国希尔德）从叶片中提取RNA，并使用引物对LCV1U/LCV1L和LCV2UP2/LCV2LO2进行逆转录（RT）-PCR，这两对引物分别对LChV - 1 3'非翻译区的419 bp片段和LChV - 2甲基转移酶基因的438 bp片段具有特异性（1）。根据现有的LChV - 2序列（GenBank登录号AF416335和NC_005065）设计引物对L2CPF（5'-GTTCGAAAGTGTTTCTTGAT-3'）和L2CPR（5'-GCAACAGAAAAACATATGACTCA-3'），以扩增整个LChV - 2外壳蛋白（CP）基因（核苷酸13007至14134）。将扩增得到的LChV - 2基因组cDNA片段连接到细菌载体pCR2.1-TOPO（Invitrogen公司，美国加利福尼亚州卡尔斯巴德），按照制造商的方案用于转化大肠杆菌TOP10感受态细胞。在华沙的生物化学与生物物理研究所，使用自动核苷酸测序仪对每个扩增的LChV - 2基因组片段的三个克隆的两条链进行测序。RT-PCR结果显示，27株树中有6株被感染，在5株甜樱桃树中检测到LChV - 1，LChV - 2单独感染了一株甜樱桃品种埃尔顿（分离株C4/14）。分离株C4/14的438 bp甲基转移酶基因片段的核苷酸序列与GenBank登录号AF333237、AF531505和AJ430056的序列分别具有86%、85%和84%的同一性，这些都是先前报道的来自樱桃树的LChV - 2序列。对1088 bp外壳蛋白基因的序列分析显示，与上述三个LChV - 2分离株的核苷酸同一性分别为89%至91%，氨基酸同一性分别为92%至93%。通过嫁接将感染LChV - 2的树接种到木本指示植物欧洲甜樱桃品种Canindex上，接种3个月后，该指示植物的叶片出现了小樱桃病特有的发红现象。由于波兰的樱桃年产量为23万吨，这种疾病可能会产生重大经济影响，因为受影响的果实既不适合食用也不适合销售。据我们所知，这是LChV - 2在波兰的首次报道。参考文献：（1）M. E. Rott和W. Jelkmann。植物病理学91:261，2001。（2）M. E. Rott和W. Jelkmann。病毒学档案150:107，2005。（3）M. Vitushkina等人。欧洲植物病理学杂志103:803，1997。
