Vrandečić K, Jurković D, Ćosić J, Stanković I, Vučurović A, Bulajić A, Krstić B
Faculty of Agriculture, Kralja Petra Svačića 1d, 31000 Osijek, Croatia.
Institute of Plant Protection, Department of Phytopathology, University of Belgrade-Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia. This research was supported by grants of SEE-ERA. NET PLUS and III 43001 of the Ministry of Education, Science and Technological Development, Republic of Serbia.
Plant Dis. 2013 Jul;97(7):1002. doi: 10.1094/PDIS-12-12-1142-PDN.
Lavandin (Lavandula × intermedia Emeric ex Loiseleur) is cultivated on a large scale in some South European countries for the extraction of essential oils or as an ornamental plant for gardens and landscapes. In May of 2012, virus-like symptoms including bright yellow calico mosaic, leaf distortion, and growth reduction were observed on 15% of lavandin plants in a commercial nursery in Banovo Brdo locality, Baranja County, Republic of Croatia. Leaves from 15 symptomatic lavandin plants were collected and examined by double-antibody sandwich (DAS)-ELISA using commercial antisera (Bioreba AG, Reinach, Switzerland) against two viruses known to infect Lavandula spp.: Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) (2,3). Commercial positive and negative controls and extracts from healthy lavandin leaves were included in each ELISA. Only AMV was detected serologically in all 15 tested samples. Five plants each of Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana were mechanically inoculated with sap from an ELISA-positive sample (70-12) using 0.01 M phosphate buffer (pH 7). Local chlorotic spots accompanied by systemic mosaic on both Chenopodium species and bright yellow mosaic on N. benthamiana were observed 6 and 12 days post-inoculation, respectively. Test plants were assayed by DAS-ELISA and all inoculated plants of each species tested positive for AMV. The presence of AMV in all symptomatic lavandin plants was further confirmed by reverse transcription (RT)-PCR assay. Total nucleic acid was extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using AMV specific primer pair CP AMV1 (5'-TCCATCATGAGTTCTTCAC-3') and CP AMV2 (5'-AGGACTTCATACCTTGACC-3') (1). Total RNAs obtained from the Serbian AMV isolate from alfalfa (GenBank Accession No. FJ527748) and healthy L. × intermedia plant served as the positive and negative control, respectively. The 751-bp amplicons, covering the partial coat protein (CP) gene and 3'-UTR, were obtained from all 15 samples that were serologically positive to AMV as well as from positive control. No amplification product was observed when extract from healthy L. × intermedia plant was used as template in the RT-PCR assay. The RT-PCR product derived from isolate 70-12 was directly sequenced in both directions using the same primer pair as in RT-PCR and deposited in GenBank (JX996119). Multiple sequence alignment of the CP open reading frame was performed by MEGA5 software (4) and revealed that the isolate 70-12 showed the highest nucleotide identity of 99.4% (99.5% amino acid identity) with Serbian AMV isolate from tobacco (FJ527749). To our knowledge, this is the first report of AMV on L. × intermedia in Croatia. Because lavandin is an aromatic plant traditionally and widely grown in Croatia, the presence of AMV could be a limiting factor for its successful production. References: (1) M. M. Finetti-Sialer et al. J. Plant Pathol. 79:115, 1997. (2) T. Kobylko et al. Plant Dis. 92:978, 2008. (3) L. Martínez-Priego et al. Plant Dis. 88:908, 2004. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.
杂薰衣草（Lavandula × intermedia Emeric ex Loiseleur）在一些南欧国家被大规模种植，用于提取精油或作为园林景观观赏植物。2012年5月，在克罗地亚共和国巴拉尼亚县巴诺沃布尔多地区的一个商业苗圃中，15%的杂薰衣草植株出现了类似病毒的症状，包括亮黄色花布状花叶、叶片扭曲和生长减缓。采集了15株有症状的杂薰衣草植株的叶片，使用针对两种已知感染薰衣草属植物的病毒的商业抗血清（瑞士雷纳赫的Bioreba AG公司），通过双抗体夹心（DAS）-ELISA进行检测。每次ELISA检测都包含商业阳性和阴性对照以及健康杂薰衣草叶片的提取物。在所有15个测试样本中，仅血清学检测到苜蓿花叶病毒（AMV）。使用0.01 M磷酸盐缓冲液（pH 7），将来自ELISA阳性样本（70 - 12）的汁液机械接种到藜麦、苋色藜和本氏烟草各5株植物上。接种后6天和12天，分别在两种藜属植物上观察到局部褪绿斑点并伴有系统花叶，在本氏烟草上观察到亮黄色花叶。通过DAS-ELISA对测试植物进行检测，每个物种的所有接种植物对AMV检测均呈阳性。通过逆转录（RT）-PCR检测进一步证实了所有有症状的杂薰衣草植株中都存在AMV。使用RNeasy植物微量提取试剂盒（德国希尔德的Qiagen公司）提取总核酸。使用AMV特异性引物对CP AMV1（5'-TCCATCATGAGTTCTTCAC-3'）和CP AMV2（5'-AGGACTTCATACCTTGACC-3'）（1），通过一步RT-PCR试剂盒（Qiagen公司）进行RT-PCR。从塞尔维亚苜蓿AMV分离株（GenBank登录号FJ527748）和健康的杂薰衣草植株中获得的总RNA分别作为阳性和阴性对照。从所有15个血清学检测对AMV呈阳性的样本以及阳性对照中均获得了覆盖部分外壳蛋白（CP）基因和3'-UTR的751-bp扩增产物。当使用健康杂薰衣草植株的提取物作为RT-PCR检测的模板时，未观察到扩增产物。使用与RT-PCR相同的引物对，对分离株70 - 12的RT-PCR产物进行双向直接测序，并提交至GenBank（JX996119）。使用MEGA5软件（4）对CP开放阅读框进行多序列比对，结果显示分离株70 - 12与塞尔维亚烟草AMV分离株（FJ527749）的核苷酸同一性最高，为99.4%（氨基酸同一性为99.5%）。据我们所知，这是克罗地亚首次报道杂薰衣草上存在AMV。由于杂薰衣草是克罗地亚传统上广泛种植的芳香植物，AMV的存在可能成为其成功生产的限制因素。参考文献：（1）M. M. Finetti-Sialer等人，《植物病理学杂志》79:115，1997年。（2）T. Kobylko等人，《植物病害》92:978，2008年。（3）L. Martínez-Priego等人，《植物病害》88:908，2004年。（4）K. Tamura等人，《分子生物学与进化》28:2731，2011年。
