Trkulja V, Salapura J Mihić, Kovačić D, Stanković I, Bulajić A, Vučurović A, Krstić B
Department of Plant Protection, University of Banja Luka-Faculty of Agriculture, Bulevar vojvode Petra Bojovića 1A, 78000 Banja Luka, Bosnia and Herzegovina.
Department of Plant Protection, Agricultural Institute of Republic of Srpska, Knjaza Miloša 17, 78000 Banja Luka, Bosnia and Herzegovina.
Plant Dis. 2013 Mar;97(3):430. doi: 10.1094/PDIS-09-12-0893-PDN.
In July 2012, a survey was conducted to determine the presence of tospoviruses in Bosnia and Herzegovina, symptoms resembling those caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) were observed in an onion (Allium cepa) seed crop in the Gornji Karajzovci locality (Region of Banja Luka). Symptoms included chlorotic to necrotic, straw-colored, spindle- and diamond-shaped lesions, variable in size and randomly distributed on the leaves and particularly on the scapes. Later the lesions enlarged and coalesced, causing scape breakage. Affected plants occurred throughout the field and disease incidence was estimated at 20%. Symptomatic plants were sampled and assayed by double-antibody sandwich (DAS)-ELISA test using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) against IYSV and two other tospoviruses, Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV). Commercial positive and negative controls were included in each test. IYSV was detected serologically in 19 of 20 screened samples and none of the samples tested positive for TSWV or INSV. The virus was mechanically transmitted from an ELISA-positive sample (302-12) to five of each Petunia × hybrida and Nicotiana benthamiana using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulfite (1). All inoculated P. × hybrida showed local necrotic spots, while N. benthamiana developed mild mosaic 4 and 10 days post-inoculation, respectively. However, difficulties were encountered in reproducing the disease symptoms on mechanically inoculated onion plants corroborating a previous study (2). Serological findings were verified with reverse transcription (RT)-PCR. Total RNAs from all naturally infected onion plants as well as mechanically infected N. benthamiana plants were extracted with the RNease Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with One-Step RT-PCR Kit (Qiagen) using IYSV-specific primers IYSV56U/IYSV917L (3), designed to amplify an 896-bp fragment of the S RNA which includes whole nucleocapsid (N) gene. Total RNAs from Serbian IYSV isolate from onion (GenBank Accession No. EU586203) and from healthy onion plants were used as positive and negative controls, respectively. An amplicon of the expected size was obtained from each of the plants assayed as well as from positive control, but not from the negative control. The amplified products derived from onion isolate 302-12 was purified (QIAquick PCR Purification Kit, Qiagen), sequenced directly (JX861126), and compared with known IYSV isolates. Sequence analysis of the complete N gene, conducted with MEGA5 software (4), revealed the highest nucleotide identity of 99.5% (100% amino acid identity) with IYSV onion isolate (DQ658242) from Texas. To our knowledge, this is the first report of IYSV in Bosnia and Herzegovina. Onion is an important and traditionally grown vegetable crop in Bosnia and Herzegovina and the presence of IYSV could represent an important constraint to onion and other susceptible host production. The discovery of IYSV on onion should prompt more detailed surveys, thorough inspections and subsequent testing to establish the distribution and incidence of IYSV in Bosnia and Herzegovina. References: (1) A. Kritzman et al. Plant Dis. 85:838, 2001. (2) L. Pozzer et al. Plant Dis. 83:345, 1999. (3) I. Robène-Soustrade et al. Plant Pathol. 55:288, 2006. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.
2012年7月，为确定波斯尼亚和黑塞哥维那是否存在番茄斑萎病毒属病毒，在戈尔尼卡拉伊佐夫齐地区（巴尼亚卢卡区）的一片洋葱（Allium cepa）种子田中观察到了类似由鸢尾黄斑病毒（IYSV；番茄斑萎病毒属，布尼亚病毒科）引起的症状。症状包括从褪绿到坏死的、稻草色的、纺锤形和菱形病斑，大小不一，随机分布在叶片上，尤其是花茎上。随后病斑扩大并融合，导致花茎折断。整个田块都出现了受影响的植株，估计发病率为20%。采集有症状的植株样本，使用针对IYSV以及另外两种番茄斑萎病毒属病毒——番茄斑萎病毒（TSWV）和凤仙坏死斑病毒（INSV）的商业多克隆抗血清（瑞士雷纳赫的Bioreba AG公司），通过双抗体夹心（DAS）-ELISA试验进行检测。每次试验都设置了商业阳性和阴性对照。在20个筛查样本中，有19个通过血清学检测出IYSV，没有样本检测出TSWV或INSV呈阳性。使用含有0.1%亚硫酸钠的冷却0.01 M磷酸盐缓冲液（pH 7），将病毒从一个ELISA阳性样本（302 - 12）机械接种到矮牵牛（Petunia × hybrida）和本氏烟草（Nicotiana benthamiana）各5株上（1）。所有接种的矮牵牛都出现了局部坏死斑，而本氏烟草在接种后第4天和第10天分别出现了轻度花叶症状。然而，在机械接种的洋葱植株上再现病害症状时遇到了困难，这与之前的一项研究结果相符（2）。通过反转录（RT）-PCR验证了血清学检测结果。使用RNease植物小提试剂盒（德国希尔德的Qiagen公司）从所有自然感染的洋葱植株以及机械感染的本氏烟草植株中提取总RNA。使用IYSV特异性引物IYSV56U/IYSV917L（3），通过一步法RT-PCR试剂盒（Qiagen公司）进行RT-PCR，该引物旨在扩增S RNA的一个896 bp片段，其中包括完整的核衣壳（N）基因。分别以来自塞尔维亚洋葱IYSV分离株（GenBank登录号EU586203）和健康洋葱植株的总RNA作为阳性和阴性对照。从每个检测的植株以及阳性对照中都获得了预期大小的扩增子，但阴性对照中没有。对来自洋葱分离株302 - 12的扩增产物进行纯化（Qiagen公司的QIAquick PCR纯化试剂盒），直接测序（JX861126），并与已知的IYSV分离株进行比较。使用MEGA5软件（4）对完整的N基因进行序列分析，结果显示与来自德克萨斯州的IYSV洋葱分离株（DQ658242）的核苷酸同一性最高，为99.5%（氨基酸同一性为100%）。据我们所知，这是IYSV在波斯尼亚和黑塞哥维那的首次报道。洋葱是波斯尼亚和黑塞哥维那一种重要的传统种植蔬菜作物，IYSV的存在可能对洋葱及其他易感寄主作物的生产构成重要限制。在洋葱上发现IYSV应促使进行更详细的调查、全面检查及后续检测，以确定IYSV在波斯尼亚和黑塞哥维那的分布及发病率。参考文献：（1）A. Kritzman等人，《植物病害》85:838，2001年。（2）L. Pozzer等人，《植物病害》83:345，1999年。（3）I. Robène-Soustrade等人，《植物病理学》55:288，2006年。（4）K. Tamura等人，《分子生物学与进化》28:2731，2011年。
