Wang M, Dai H
College of Horticulture, Shenyang Agricultural University, 120 Dongling Road, Shenyang 110866, China. National Natural Science Foundation of China (Grant No. 31470678).
Plant Dis. 2015 Jan;99(1):164. doi: 10.1094/PDIS-07-14-0747-PDN.
Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus in the Betaflexiviridae family (1). ACLSV is distributed worldwide in most pome and stone fruit trees of the Rosaceae family, including apple, pear, peach, plum, cherry, apricot, and hawthorn (3). In 2012, a de novo assembly of the fruit transcriptome of a hawthorn (Crataegus pinnatifida) accession maintained in the National Hawthorn Germplasm Repository at Shenyang was conducted using Illumina-based RNA-seq data, and it resulted that a 7,543 nt of the genomic sequence of ACLSV was assembled. To confirm the result of Illumina RNA-Seq analysis, nine pairs of primers were designed according to the assembled sequence of ACLSV to amplify the genomic sequence of ACLSV by RT-PCR with total RNA extracted from hawthorn leaves as template (2). The full-length sequence of the isolate of ACLSV from hawthorn assembled with the sequences of the RT-PCR fragments was also 7,543 nt (GenBank Accession No. KM207212), which shows 99.5% nucleotide identity with the sequence assembled from Illumina RNA-seq data. The isolate of ACLSV from hawthorn was named SY01, which shows about 75% nucleotide identity with the sequences of ACLSV isolated from apple (GenBank Accession No. KJ522693), peach (JN634760), and plum (M58152). The nucleotide sequences of coat protein and RNA polymerase genes of SY01 are about 83 and 88% identical with those of ACLSV isolates in GenBank, respectively. A pair of primers HF/HR (ACCGGCGTCTTTTGCAAACT/TGGGTTCCAGAGTTTGAATGCA), which amplified a 210-bp fragment, was designed according to the sequence of SY01 to detect ACLSV in hawthorns. With RT-PCR, ACLSV was detected in 6 of the 30 accessions of hawthorn, and the nucleotide identity among PCR fragments was 92%. In addition, leaves from six RT-PCR positive plants reacted positively when tested by DAS-ELISA with polyclonal antisera (X-Y Biotechnology, Shanghai, China) raised against ACLSV. These findings, representing the first report of the presence of ACLSV in hawthorn in China, illustrate the need to develop virus-free trees of hawthorn for cultivation and germplasm distribution of this important Rosaceae family plant. References: (1) E. B. Carstens. Arch. Virol. 155:133, 2010. (2) H. Dai et al. PLoS ONE 8(9):e72910, 2013. (3) A. T. Katsiani et al. Plant Pathol. 63:63, 2014.
苹果褪绿叶斑病毒（ACLSV）是柔线病毒属的模式种，属于乙型弯曲病毒科（1）。ACLSV在全球范围内分布于蔷薇科的大多数仁果类和核果类果树，包括苹果、梨、桃、李、樱桃、杏和山楂（3）。2012年，利用基于Illumina的RNA测序数据对保存在沈阳国家山楂种质资源库中的一个山楂（Crataegus pinnatifida）种质的果实转录组进行了从头组装，结果组装出了一条7543 nt的ACLSV基因组序列。为了确认Illumina RNA测序分析的结果，根据ACLSV的组装序列设计了9对引物，以从山楂叶片中提取的总RNA为模板，通过RT-PCR扩增ACLSV的基因组序列（2）。用RT-PCR片段的序列组装得到的山楂ACLSV分离株的全长序列也为7543 nt（GenBank登录号：KM207212），与从Illumina RNA测序数据组装得到的序列具有99.5%的核苷酸同一性。从山楂中分离得到的ACLSV分离株被命名为SY01，与从苹果（GenBank登录号：KJ522693）、桃（JN634760）和李（M58152）中分离得到的ACLSV序列具有约75%的核苷酸同一性。SY01的外壳蛋白基因和RNA聚合酶基因的核苷酸序列分别与GenBank中ACLSV分离株的序列约83%和88%相同。根据SY01的序列设计了一对引物HF/HR（ACCGGCGTCTTTTGCAAACT/TGGGTTCCAGAGTTTGAATGCA），用于扩增一个210 bp的片段，以检测山楂中的ACLSV。通过RT-PCR，在30个山楂种质中的6个中检测到了ACLSV，PCR片段之间的核苷酸同一性为92%。此外，6株RT-PCR阳性植株的叶片在用针对ACLSV的多克隆抗血清（中国上海X-Y生物技术公司）进行DAS-ELISA检测时呈阳性反应。这些发现代表了中国山楂中存在ACLSV的首次报道，说明了为这种重要的蔷薇科植物的栽培和种质分发培育无病毒山楂树的必要性。参考文献：（1）E. B. Carstens。《病毒学档案》155:133，2010年。（2）H. Dai等人。《公共科学图书馆·综合》8(9):e72910，2013年。（3）A. T. Katsiani等人。《植物病理学》63:63，2014年。
