Cho I S, Chung B N, Cho J D, Choi G S, Lim H S
Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 441-440, Korea.
Department of Applied Biology, Chungnam National University, Daejeon 305-764, Korea.
Plant Dis. 2012 Jul;96(7):1074. doi: 10.1094/PDIS-03-12-0227-PDN.
Blueberry red ringspot virus (BRRSV) of the Soymovirus genus in the family Caulimovididae causes red ringspot diseases in highbush blueberry (Vaccinium corymbosum L.) on leaves, stems, and fruits. The virus has been identified in the United States, Japan, Czech Republic, Slovenia, and Poland (1). In July 2010, highbush blueberry with red ringspots on leaves and circular blotches on ripening fruits was found in one plant of cv. Duke in Pyeongtaek, Korea. The symptoms were similar to red ringspot disease caused by BRRSV (3), although stems did not show any characteristic symptoms. Red ringspots on the upper surface of leaves were the most visible symptom and became more prominent as leaves matured in August through October. Leaves of the symptomatic plant were collected and tested for BRRSV infection by PCR, and were also embedded for electron microscopy. DNA was extracted from leaves using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Primer pairs BR1512F/BR2377R (5'-ACAGGACGATTAGAAGATGG-3'/5'-CCTTTAGGGCAATATTTCTG-3', amplifying a fragment of the coat protein region with an expected size of 865 bp) and BR2961F/BR3726R (5'-ACCGATACATCACAGTTCAC-3'/5'-TGGTTGTGATAAGATGATTCC-3', amplifying a fragment of the reverse transcriptase region with an expected size of 766 bp) were used to amplify the indicated region of BRRV in PCR. Primers were designed on the basis of the BRRSV isolate from New Jersey (GenBank Accession No. AF404509). DNA fragments of the expected sizes were obtained from the symptomatic plant, while no amplification products were obtained from highbush blueberry without symptoms. The PCR products were cloned into pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of obtained fragments revealed 91 to 98% nucleotide sequence identity with the coat protein gene (GenBank Accession No. JQ706341) and 96 to 98% nucleotide sequence identity with the reverse transcriptase gene (GenBank Accession No. JQ706340) of known BRRV isolates. Electron microscopy of thin sections revealed particles approximately 50 nm diameter within electron-dense inclusion bodies, characteristic of BRRSV (2) To our knowledge, this is the first report of BRRSV infection of highbush blueberry in Korea. Highbush blueberries are usually propagated by cutting, so BRRSV suspicious plants should be tested with PCR before they are propagated. References: (1) E. Kalinowska et al. Virus Genes. DOI 10.1007/s11262-011-0679-4, 2011. (2) K. S. Kim et al. Phytopathology 71:673, 1981. (3) M. Isogai et al. J. Gen. Plant Pathol. 75:140, 2009.
花椰菜花叶病毒科大豆花叶病毒属的蓝莓红环斑病毒（BRRSV）可在高丛蓝莓（Vaccinium corymbosum L.）的叶片、茎和果实上引发红环斑病。该病毒已在美国、日本、捷克共和国、斯洛文尼亚和波兰被发现（1）。2010年7月，在韩国平泽一株‘公爵’品种的高丛蓝莓植株上，发现叶片出现红环斑，成熟果实上有圆形斑点。这些症状与BRRSV引起的红环斑病相似（3），不过茎部未表现出任何特征性症状。叶片上表面的红环斑是最明显的症状，随着8月至10月叶片成熟，症状愈发显著。采集有症状植株的叶片，通过PCR检测是否感染BRRSV，并进行包埋用于电子显微镜观察。按照制造商的说明，使用DNeasy植物微量提取试剂盒（Qiagen公司，加利福尼亚州瓦伦西亚）从叶片中提取DNA。引物对BR1512F/BR2377R（5'-ACAGGACGATTAGAAGATGG-3'/5'-CCTTTAGGGCAATATTTCTG-3'，扩增衣壳蛋白区域的一个片段，预期大小为865 bp）和BR2961F/BR3726R（5'-ACCGATACATCACAGTTCAC-3'/5'-TGGTTGTGATAAGATGATTCC-3'，扩增逆转录酶区域的一个片段，预期大小为766 bp）用于在PCR中扩增BRRV的指定区域。引物是根据来自新泽西州的BRRSV分离株（GenBank登录号AF404509）设计的。从有症状的植株中获得了预期大小的DNA片段，而从无症状的高丛蓝莓中未获得扩增产物。将PCR产物克隆到pGEM-T简易载体（Promega公司，威斯康星州麦迪逊）中并进行测序。对获得片段的BLAST分析显示，与已知BRRV分离株的衣壳蛋白基因（GenBank登录号JQ706341）的核苷酸序列同一性为91%至98%，与逆转录酶基因（GenBank登录号JQ706340）的核苷酸序列同一性为96%至98%。超薄切片的电子显微镜观察显示，在电子致密的包涵体内有直径约50 nm的颗粒，这是BRRSV的特征（2）。据我们所知，这是韩国高丛蓝莓感染BRRSV的首次报道。高丛蓝莓通常通过扦插繁殖，因此在繁殖BRRSV疑似植株之前，应通过PCR进行检测。参考文献：（1）E. Kalinowska等人。《病毒基因》。DOI 10.1007/s11262-011-0679-4, 2011。（2）K. S. Kim等人。《植物病理学》71:673, 1981。（3）M. Isogai等人。《植物病理学报》75:140, 2009。
