Zindović J, Lanzoni C, Autonell C Rubies, Ratti C
Department of Plant Protection, University of Montenegro, Biotechnical Faculty, Mihajla Lalića 1, 81000 Podgorica, Montenegro.
DipSA, Patologia Vegetale, Università di Bologna, Viale G. Fanin, 40 - 40127 Bologna, Italy.
Plant Dis. 2013 Sep;97(9):1259. doi: 10.1094/PDIS-12-12-1147-PDN.
In September and October 2011, samples were collected from mature peach trees (17 years old) exhibiting symptoms of chlorotic rings and spots, vein clearing, mosaic, necrosis, leaf distortion, stunting, and rosette formation in a major commercial orchard (80 ha) near Podgorica, Montenegro. Samples were collected from nine different peach varieties (cvs. Adriana, Caldesi, Gloria, Maria Marta, May Crest, Morsiani, Rita Star, Spring Belle, and Spring Crest). Samples (n = 58) were tested using DAS-ELISA for the presence of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV). Commercial positive and negative controls were included in each ELISA (antisera and controls supplied by BIOREBA AG, Reinach, Switzerland). Only one symptomatic sample from cv. Gloria tested positive for PDV (sample reference: 399/11), a further 11 samples (cvs. Rita Star [six], May Crest [four] and Spring Crest [one]) were positive for PNRSV. Samples were also tested for Plum pox virus (PPV) by real-time RT-PCR (1). The PDV positive sample (399/11) showing mosaic was in mixed infection with PPV, as were 6 of the 11 PNRSV samples, including sample 373/11 with yellow mottling and leaf distortion symptoms. On single-infected PNRSV, sample 368/11 chlorotic line patterns and leaf deformations were observed. To confirm the presence of PDV and PNRSV, positive samples were also tested by RT-PCR. Total RNA was extracted using RNeasy Plant Mini kit (Qiagen, Hilden, Germany). RT-PCR was performed with primer pairs PDV2F/PDV1R (3) and MG1/MG2 (2) specific for PDV and PNRSV, respectively. Amplicons of the expected size, 173 bp for PDV and 675 bp for PNRSV, were obtained from corresponding ELISA-positive samples. Amplified products from three samples (PDV 399/11 and PNRSV 368/11 and 373/11) were cloned into pGEM-T Easy Vector (Promega, Madison, WI) then sent for sequence analysis (MWG-Biotech AG, Edersberg, Germany). Sequence data was compared to sequences published in GenBank. Analysis of sequence obtained from isolate 399/11 (cv. Gloria) corresponded to partial CP gene of PDV, with a high degree of similarity to isolates reported from other parts of the world ranging from 94.2 to 95.9%, showing highest similarity with isolate Ch 137 (L28145). Sequence analyses of CP gene from PNRSV isolates 368/11 (JX569825) and 373/11 (JX569826) proved to be 89.3 to 99.7% identical with corresponding sequences of isolates previously described. In particular, the Montenegrin PNRSV isolates were most closely related to Chilean NctCl.augl isolate from nectarine (EF565253). To demonstrate that the virus was infectious, seedlings of peach cv. GF305 were side grafted with bud-woods from PDV (sample 399/11) and PNRSV-positive samples (samples 368/11 and 373/11) and a healthy control sample. Grafted seedlings were kept in a greenhouse with a under 16-h light regime at 22 to 24°C and observed for symptom development. No symptoms were observed in grafted plants with the healthy control. All plants inoculated with virus-positive samples exhibited stunted vegetation and mild mottle with no difference in symptoms between the two viruses. Indicator plants of peach cv. GF305 inoculated with PPV dual-infected samples (399/11 and 373/11) were subsequently shown to be positive for PPV by real-time RT-PCR. Subsequent DAS-ELISA test on samples from experimentally inoculated trees using specific antisera as described above confirmed PDV and PNRSV infections as expected. These viruses have recently been reported from sour cherry (Prunus cerasus L.) in Serbia (4), ~600 km to the northeast. However, to our knowledge, this is the first report on the occurrence of PDV and PNRSV in Montenegro. References: (1) N. Capote et al. Int. Microbiol. 12:1, 2009. (2) M. Glasa et al. Ann. Appl. Biol. 140:279, 2002. (3) D. R. Parakh et al. Acta Hortic. 386:421, 1996. (4) S. Radičević et al. Genetika 44:285, 2012.
2011年9月和10月，在黑山共和国波德戈里察附近一个主要商业果园（约80公顷）中，从表现出褪绿环斑、斑点、叶脉变清、花叶、坏死、叶片变形、生长受阻和莲座状等症状的成熟桃树（约17年树龄）上采集样本。样本取自9个不同的桃品种（品种：阿德里亚娜、卡尔德西、格洛丽亚、玛丽亚·玛尔塔、五月冠、莫尔西亚尼、丽塔之星、春美人、春冠）。使用双抗夹心酶联免疫吸附测定法（DAS - ELISA）对58个样本检测李属矮缩病毒（PDV）和李坏死环斑病毒（PNRSV）的存在情况。每次酶联免疫吸附测定中均包含商业阳性和阴性对照（抗血清和对照由瑞士赖纳赫的BIOREBA AG公司提供）。仅来自格洛丽亚品种的1个有症状样本检测出PDV呈阳性（样本编号：399/11），另有11个样本（丽塔之星品种[6个]、五月冠品种[4个]和春冠品种[1个]）检测出PNRSV呈阳性。还通过实时逆转录 - 聚合酶链反应（real - time RT - PCR）对样本检测李痘病毒（PPV）（1）。表现出花叶症状的PDV阳性样本（399/11）与PPV混合感染，11个PNRSV样本中的6个也是如此，包括具有黄色斑驳和叶片变形症状的373/11样本。在单一感染PNRSV的情况下，观察到368/11样本有褪绿线条图案和叶片变形。为确认PDV和PNRSV的存在，对阳性样本也进行了逆转录 - 聚合酶链反应（RT - PCR）检测。使用RNeasy植物小提试剂盒（德国希尔德的Qiagen公司）提取总RNA。分别用对PDV和PNRSV特异的引物对PDV2F/PDV1R（3）和MG1/MG2（2）进行RT - PCR。从相应的酶联免疫吸附测定阳性样本中获得了预期大小的扩增子，PDV为173 bp，PNRSV为675 bp。将3个样本（PDV 399/11和PNRSV 368/11及373/11）的扩增产物克隆到pGEM - T Easy载体（美国威斯康星州麦迪逊的Promega公司）中，然后送去进行序列分析（德国埃德斯贝格的MWG - Biotech AG公司）。将序列数据与GenBank中公布的序列进行比较。对从399/11分离株（格洛丽亚品种）获得的序列分析对应于PDV的部分外壳蛋白（CP）基因，与世界其他地区报道的分离株具有高度相似性，范围从94.2%到95.9%，与Ch 137分离株（L28145）相似度最高。对PNRSV分离株368/11（JX569825）和373/11（JX569826）的CP基因序列分析表明，与先前描述的分离株的相应序列有89.3%到99.7%的同一性。特别是，黑山的PNRSV分离株与来自油桃的智利NctCl.augl分离株（EF565253）关系最为密切。为证明该病毒具有传染性，用来自PDV（样本399/11）和PNRSV阳性样本（样本368/11和373/11）以及一个健康对照样本的芽木对桃品种GF305的幼苗进行侧接。将嫁接后的幼苗置于温度为22至24°C、光照时间为16小时以下的温室中，并观察症状发展情况。接种健康对照的嫁接植物未观察到症状。所有接种病毒阳性样本的植物均表现出生长受阻和轻度斑驳，两种病毒之间症状无差异。用PPV双重感染样本（399/11和373/11）接种的桃品种GF305指示植物随后通过实时RT - PCR检测显示PPV呈阳性。随后使用上述特异性抗血清对实验接种树的样本进行的DAS - ELISA检测如预期那样证实了PDV和PNRSV感染。最近在东北约600公里处的塞尔维亚酸樱桃（Prunus cerasus L.）中报道了这些病毒（4）。然而，据我们所知，这是黑山关于PDV和PNRSV发生情况的首次报道。参考文献：（1）N. Capote等人，《国际微生物学》12:1，2009年。（2）M. Glasa等人，《应用生物学年鉴》140:279，2002年。（3）D. R. Parakh等人，《园艺学报》386:421，1996年。（4）S. Radičević等人，《遗传学》44:285，2012年。
