Chynoweth Robert, Jimenez Daniel, Liberti Daniele, Bellon-Dona Daniel, Carralero Alejandro, Crespo Ana, Albiach-Marti Maria R R
BASF, Nunhems Spain, Finca Lo Ruiz, La Palma (Cartagena), Murcia, Spain;
ValGenetics, Plant Pathology and Microbiology Laboratory, Paterna, Valencia, Spain;
Plant Dis. 2021 Jul 30. doi: 10.1094/PDIS-12-20-2553-PDN.
During the winter 2018, symptoms of leaf chlorotic spots (Figure 1) followed by symptoms of leaf interveinal chlorosis (Figure 2) and severe chlorosis in basal leaves were observed in cucumber cv Laredo (Cucumis sativus) plants in three separated greenhouses, sited in distinct locations in southern Spain. In all cases, Bemisia tabaci populations were observed on infected plants. The symptomology observed was similar to that caused by whitefly transmitted Cucurbit yellow stunting disorder virus (CYSDV, genus Crinivirus, family Closteroviridae), which is usually found infecting cucumber plants in this geographical area (1). Samples from four different cucumber plants of distinct greenhouses were collected and tested for the presence of CYSDV. Total RNA was extracted from the samples using the NucleoSpin RNA Plant kit (Macherey-Nagel, Germany). Molecular detection of CYSDV was performed using the multiplex and degenerate primer RT-PCR method (2), specific to the region of the highly conserved RNA-dependent RNA polymerase (RdRp) gene of criniviruses, which also detects other criniviruses such as Lettuce infectious yellows virus (LIYV) and Beet pseudo-yellows virus (BPYV). Results indicated that the viral species CYSDV, LIYV and BPYV were not detected in the four cucurbit plant samples. In 2004, an emergent crinivirus (Cucurbit chlorotic yellows virus, CCYV), inducing symptoms similar to those caused by CYSDV, was described infecting cucurbits in Japan (3). Recently, CCYV was detected in 2011 in Greece (4) and in 2014 in Egypt (5) and Saudi Arabia (6). Therefore, the four RNA samples were tested for the presence of the CCYV by a RT-PCR method previously described (7). Specific primers were designed to amplify 336 nt of the capsid protein (CP) gene and 680 nt of the RdRp gene, located on CCYV genomic RNA 1 and RNA 2, respectively. In all cases, clear cDNA bands of both expected sizes were detected for each cucumber sample that were then purified and sequenced via Sanger technology. BLAST analysis of those sequences showed 99% identity with the nucleotide sequence of the CP and RpRd genes from the CCYV isolates from Greece (LT992911, LT992910), China (KY400633.1, KX118632) and Taiwan (JF502222). To our knowledge, this is the first report of CCYV infecting cucurbits in Spain. Probably CCYV has been spread throughout the Mediterranean basin, remaining undetected due to the yellowing symptom similarities between CYSDV and CCYV. Detection of the emergent virus CCYV in Spain represents a new threat for the horticultural area of southern Europe.
2018年冬季，在西班牙南部不同地点的三个独立温室中，拉雷多黄瓜品种（Cucumis sativus）植株上出现了叶片褪绿斑点症状（图1），随后出现叶片脉间褪绿症状（图2）以及基部叶片严重褪绿的情况。在所有病例中，均在受感染植株上观察到烟粉虱种群。观察到的症状与粉虱传播的葫芦科黄化矮缩病毒（CYSDV，隶属毛形病毒属，长线形病毒科）引起的症状相似，该病毒在这一地理区域通常感染黄瓜植株（1）。采集了来自不同温室的四株不同黄瓜植株的样本，并检测是否存在CYSDV。使用德国Macherey-Nagel公司的NucleoSpin RNA Plant试剂盒从样本中提取总RNA。采用多重简并引物RT-PCR方法（2）对CYSDV进行分子检测，该方法针对毛形病毒高度保守的RNA依赖RNA聚合酶（RdRp）基因区域，也可检测其他毛形病毒，如莴苣传染性黄化病毒（LIYV）和甜菜假黄化病毒（BPYV）。结果表明，在四个葫芦科植物样本中未检测到CYSDV、LIYV和BPYV这三种病毒。2004年，一种新出现的毛形病毒（葫芦科褪绿黄化病毒，CCYV）在日本被描述感染葫芦科植物，其引发的症状与CYSDV相似（3）。最近，2011年在希腊（4）以及2014年在埃及（5）和沙特阿拉伯（6）检测到了CCYV。因此采用先前描述的RT-PCR方法（7）对这四个RNA样本进行CCYV检测。设计了特异性引物，分别扩增位于CCYV基因组RNA 1和RNA 2上的衣壳蛋白（CP）基因的336 nt和RdRp基因的680 nt。在所有病例中，每个黄瓜样本均检测到了预期大小的清晰cDNA条带，随后通过桑格技术进行纯化和测序。对这些序列的BLAST分析显示，它们与来自希腊（LT992911、LT992910）、中国（KY400633.（此处原文可能有误，推测为KY400633.1）、KX118632）和台湾（JF502222）的CCYV分离株的CP和RpRd基因的核苷酸序列具有99%的同一性。据我们所知，这是CCYV感染西班牙葫芦科植物的首次报道。CCYV可能已传播至整个地中海盆地，由于CYSDV和CCYV之间的黄化症状相似而一直未被发现。在西班牙检测到这种新出现的病毒CCYV对南欧园艺区构成了新的威胁。
