García-Ibarra A, Martínez-Gómez P, Rubio M, Dicenta F, Soler A, Pallás V, Sánchez-Navarro J A
Centro de Edafología y Biología Aplicada del Segura, CEBAS (CSIC), Murcia, Spain.
Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, IMIDA, La Alberca (Murcia), Spain.
Plant Dis. 2010 Feb;94(2):275. doi: 10.1094/PDIS-94-2-0275A.
Representing 2% of world production, 20,000 ha of apricot (Prunus armeniaca L.), are cultivated in Spain, primarily in the southeast. A survey was conducted during the spring of 2008 in orchards in the region of Murcia to assess the incidence of several stone fruit viruses. Leaf and fruit samples from 160 trees from 40 orchards were collected randomly for reverse transcription (RT)-PCR analysis. Total RNA extracted (3) from leaves and fruits was tested by a multiplex one-step RT-PCR protocol with a mix of primers that detect eight distinct viruses (4). Amplicons of 250 bp expected for Plum bark necrosis stem pitting-associated virus (PBNSPaV), corresponding to part of the heat shock 70 protein gene, were obtained from four trees and amplicons of 700 bp expected for Apricot latent virus (ApLV), corresponding to part of the coat protein (CP) gene, were obtained from two trees. In all cases, amplicons were obtained using RNA extracted from leaf and fruit tissues. RT-PCR results were confirmed by uniplex RT-PCR with primers specific for each virus and dot-blot hybridization with virus-specific digoxygenin-labeled RNA probes (2). To further characterize the new viruses, we designed primers to amplify specifically the CP gene of ApLV (5'-CCCGACCATGGCTACAAGC-3' and 5'-TTGCCGTCCCGATTAGGTTG-3') and the minor CP gene of PBNSPaV (5'-GAACAAACTACAGCAGCACC-3' and 5'-CAAGGGTAGGACGGGTAACGC-3'). Amplicons of 1,500 and 950 bp corresponding to the ApLV and PBNSPaV CP genes, respectively, were purified from agarose gels and cloned in the pTZ57R plasmid (Fermentas, Burlington, Ontario, Canada). Blastp analysis of the full-length ApLV CP sequence from one infected tree (GenBank Accession No. GQ919051) revealed 86% amino acid (aa) similarity to the single full-length ApLV CP sequence available (No. AAC16234) and 79 and 66.9% similarity to Peach sooty ringspot virus (No. AAG48314) and Apple stem pitting virus (No. NP604468), respectively. Identity/similarity analysis of the full-length PBNSPaV minor CP genes using the Matrix Global Alignment Tool software, version 2.02 (1), revealed 98.8 to 99.6% aa similarity between the Spanish PBNSPaV isolates (Nos. GQ919047, GQ919048, GQ919049, and GQ919050) and 97.1 to 97.4% with the PBNSPaV isolate from the United States (No. EF546442). None of the six infected trees were associated with any particular field symptoms. Five infected trees were cv. Búlida and one was native cv. Murciana, which was infected with ApLV. All infected trees were located in geographically separated orchards. The incidence of ApLV and PBNSPaV was 1.25 and 2.5%, respectively. The low incidence of both viruses together with the scattered geographic distribution could be due to the recent introduction of virus-contaminated plants, although we cannot exclude that it is a consequence of a low dissemination rate. Even though no symptoms were observed, we cannot discard that the infection could affect fruit production or flowering or even cause a synergistic effect in mixed infection with other stone fruit viruses, a risk especially relevant considering the total area of cultivated apricot. To our knowledge, this is the first report of ApLV and PBNSPaV in Spain. References: (1) J. J. Campanella et al. BMC Bioinformatics 4:29, 2003. (2) M. C. Herranz et al. J. Virol. Methods 124:49, 2005. (3) D. J. Mackenzie et al. Plant Dis. 81:222, 1997. (4) J. A. Sánchez-Navarro et al. Eur. J. Plant Pathol. 111:77, 2005.
西班牙杏（Prunus armeniaca L.）的种植面积为20000公顷，占世界产量的2%，主要分布在东南部地区。2008年春季，在穆尔西亚地区的果园进行了一项调查，以评估几种核果类病毒的发生率。从40个果园的160棵树上随机采集叶片和果实样本，用于逆转录（RT）-PCR分析。用检测8种不同病毒的引物混合物，通过多重一步RT-PCR方案对从叶片和果实中提取的总RNA（3）进行检测。从4棵树上获得了与李树皮坏死茎点陷相关病毒（PBNSPaV）预期的250 bp扩增子，对应于热休克70蛋白基因的一部分；从2棵树上获得了与杏潜隐病毒（ApLV）预期的700 bp扩增子，对应于外壳蛋白（CP）基因的一部分。在所有情况下，使用从叶片和果实组织中提取的RNA获得了扩增子。RT-PCR结果通过用每种病毒特异性引物进行的单重RT-PCR和用病毒特异性地高辛标记RNA探针进行的斑点杂交得到证实（2）。为了进一步鉴定这些新病毒，我们设计引物特异性扩增ApLV的CP基因（5'-CCCGACCATGGCTACAAGC-3'和5'-TTGCCGTCCCGATTAGGTTG-3'）和PBNSPaV的次要CP基因（5'-GAACAAACTACAGCAGCACC-3'和5'-CAAGGGTAGGACGGGTAACGC-3'）。分别对应于ApLV和PBNSPaV CP基因的1500和950 bp扩增子从琼脂糖凝胶中纯化，并克隆到pTZ57R质粒（Fermentas，加拿大安大略省伯灵顿）中。对一棵受感染树的全长ApLV CP序列（GenBank登录号GQ919051）进行Blastp分析，结果显示与现有的单个全长ApLV CP序列（编号AAC16234）的氨基酸（aa）相似性为86%，与桃煤烟环斑病毒（编号AAG48314）和苹果茎痘病毒（编号NP604468）的相似性分别为79%和66.9%。使用Matrix Global Alignment Tool软件版本2.02（1）对全长PBNSPaV次要CP基因进行同一性/相似性分析，结果显示西班牙PBNSPaV分离株（编号GQ919047、GQ919048、GQ第919049和GQ919050）之间的aa相似性为98.8%至99.6%，与来自美国的PBNSPaV分离株（编号EF546442）的相似性为97.1%至97.4%。六棵受感染的树均未出现任何特定的田间症状。五棵受感染的树是“布利达”品种，一棵是当地品种“穆尔西亚纳”，感染了ApLV。所有受感染的树都位于地理上相互隔离的果园中。ApLV和PBNSPaV的发生率分别为1.25%和2.5%。这两种病毒的低发生率以及分散的地理分布可能是由于最近引入了受病毒污染的植物，尽管我们不能排除这是传播率低的结果。尽管未观察到症状，但我们不能排除感染可能影响果实产量或开花，甚至在与其他核果类病毒混合感染时产生协同效应，考虑到杏的总种植面积，这一风险尤为重要。据我们所知，这是西班牙首次报道ApLV和PBNSPaV。参考文献：（1）J. J. Campanella等人，《BMC生物信息学》4:29，2003年。（2）M. C. Herranz等人，《病毒学方法杂志》124:49，2005年。（3）D. J. Mackenzie等人，《植物病害》81:222，1997年。（4）J. A. Sánchez-Navarro等人，《欧洲植物病理学杂志》111:77，2005年。
