First Report of Citrus leaf blotch virus in Sweet Cherry
First Report of Citrus leaf blotch virus in Sweet Cherry
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DOI：
10.1094/pdis-09-15-0965-pdn
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发表时间：
2016-02
期刊：
影响因子：
4.5
通讯作者：
Jian Wang;D. Zhu;Y. Tan;X. Zong;H. Wei;Qingzhong Liu
中科院分区：
文献类型：
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作者：
Jian Wang;D. Zhu;Y. Tan;X. Zong;H. Wei;Qingzhong Liu
2014年5月，在中国山东省泰安市郊区的果园中发现了5棵有症状的甜樱桃树（Prunus avium L. cv. Red-lamp），其中一棵为严重花叶病，四棵为轻度花叶病。从五棵有症状的树和三棵无症状的树的幼叶样本中提取总RNA。来自严重症状树的总 RNA 用于小 RNA (sRNA) 文库构建。 < 50 nt 的 RNA 从 6% 聚丙烯酰胺中纯化，并在 Illumina HiSeq2000 上测序。使用 CLC Genomics Workbench 7.5（CLCbio，奥胡斯，丹麦）分析原始测序读数。去除宿主sRNA（由保藏于NCBI的甜樱桃RNA-seq序列文库构建的数据库）后，组装了301个重叠群（> 100 nt）（长度分数，0.94；相似性分数，1.0）。 NCBI（非冗余核苷酸数据库）的 BLASTn 搜索显示，重叠群与樱桃病毒 A（CVA，56 个重叠群）、李坏死环斑病毒（PNRSV，13 个重叠群）、小樱桃病毒 1（LChV-1，两个重叠群）和柑橘叶斑病毒（CLBV，三个重叠群）相似。中国已有甜樱桃 CVA、PNRSV 和 LChV-1 感染的报道（Lu et al. 2015；Zong et al. 2014），但没有 CLBV 感染。 RT-PCR 证实了树中的 CVA、PNRSV 和 LChV-1 感染（Lu et al. 2015；Zong et al. 2014）。为了验证CLBV感染，根据确定的序列设计引物（CLBV-F：GCCTACAGTTTAAGTGAGAGGCT和CLBV-R：GTCTAAAAGTTCTTAAAAGACATC），并通过RT-PCR扩增覆盖CLBV运动蛋白（MP）和外壳蛋白（CP）基因的DNA片段。从有严重症状的树、四棵有轻微症状的树中的两棵以及一棵无症状的树中扩增出一个 2.9-kb DNA 片段。来自表现出严重症状的树的扩增子被克隆到pMD18-T（Takara，大连，中国）中。对 6 个克隆进行了测序（Invitrogen，中国北京），结果发现 99.9% 至 100% 相同。一个序列以登录号 KT373979 保藏在 GenBank 中。该序列被映射到 3229 个读数（大约 26 倍覆盖），平均长度为 23.6 nt（长度分数，0.94；相似性分数，1.0）。使用 MEGA6 对 GenBank 和其他 Betaflexiviridae 家族成员中的 CLBV 分离株的 CP 序列进行系统发育分析。 0（Tamura 等人，2013）表明樱桃分离株与柑橘分离株（NC_003877，分离型）和猕猴桃分离株（JN900477）关系最密切。它们与其他分离株形成了一个与 Betaflexiviridae 的其他病毒不同的簇。樱桃分离株的 CP 基因与柑橘和猕猴桃分离株分别具有 87.6% 和 88.3% 的序列同一性，表明它是 CLBV 的一个独特变种。其MP基因具有较高的同一性（柑橘，95.6％；猕猴桃，94.2％）。因此，CLBV可以感染甜樱桃，并且甜樱桃分离物是一个新毒株。据我们所知，这是首次报道甜樱桃树感染 CLBV。
In May 2014, five symptomatic sweet cherry trees (Prunus avium L. cv. Red-lamp), one with severe and four with mild mosaic symptoms, were found in the orchard at the suburb of Tai’an, Shandong Province, China. Total RNA was extracted from young leaf samples of the five symptomatic trees and three symptomless trees. Total RNA from the severely symptomatic tree was used for small RNA (sRNA) library construction. RNAs of< 50 nt were purified from 6% polyacrylamide and sequenced on an Illumina HiSeq2000. Raw sequencing reads were analyzed using a CLC Genomics Workbench 7.5 (CLCbio, Aarhus, Denmark). After removal of the host sRNAs (database constructed by sweet cherry RNA-seq sequence libraries deposited in NCBI), 301 contigs (> 100 nt) were assembled (length fraction, 0.94; similarity fraction, 1.0). BLASTn searches at NCBI (nonredundant nucleotide database) revealed contigs similar to Cherry virus A (CVA, 56 contigs), Prunus necrotic ringspot virus (PNRSV, 13 contigs), Little cherry virus 1 (LChV-1, two contigs) and Citrus leaf blotch virus (CLBV, three contigs). CVA, PNRSV, and LChV-1 infections of sweet cherry have been reported in China (Lu et al. 2015; Zong et al. 2014), but not CLBV. RT-PCR confirmed the CVA, PNRSV, and LChV-1 infections in the tree (Lu et al. 2015; Zong et al. 2014). To verify CLBV infection, primers (CLBV-F: GCCTACAGTTTAAGTGAGAGGCT and CLBV-R: GTCTAAAAGTTCTTAAAAGACATC) were designed according to the determined sequence and used to amplify a DNA fragment covering the movement protein (MP) and coat protein (CP) genes of CLBV by RT-PCR. A 2.9-kb DNA fragment was amplified from the tree with severe symptoms, from two of the four mild symptomatic trees, and from one of the symptomless trees. The amplicon from the tree exhibiting severe symptoms was cloned into pMD18-T (Takara, Dalian, China). Six clones were sequenced (Invitrogen, Beijing, China) and found to be 99.9 to 100% identical. One sequence was deposited in GenBank as Accession No. KT373979. This sequence was mapped to 3229 reads (approximately 26-fold coverage) with an average length of 23.6 nt (length fraction, 0.94; similarity fraction, 1.0). Phylogenetic analysis of the CP sequences of CLBV isolates in GenBank and other Betaflexiviridae family members using MEGA6. 0 (Tamura et al. 2013) showed that the cherry isolate was most closely related to a citrus isolate (NC_003877, type isolate) and a kiwi isolate (JN900477). With other isolates, they formed a cluster distinct from other viruses of Betaflexiviridae. The CP gene of the cherry isolate shares 87.6% and 88.3% sequence identity with the citrus and kiwi isolates, respectively, indicating that it is a distinct variant of CLBV. Its MP gene shares higher identities (citrus, 95.6%; kiwi, 94.2%). Thus, CLBV can infect sweet cherry, and the sweet cherry isolate is a novel strain. To our knowledge, this is the first report of the CLBV infection in sweet cherry trees.