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在美国爱达荷州红皮甜菜中首次发现甜菜曲顶病毒的辣椒黄矮株系和亚利桑那菠菜曲顶病毒。

First report of Pepper yellow dwarf strain of Beet curly top virus and Spinach curly top Arizona virus in red table beet in Idaho, United States.

作者信息

Ramachandran Vanitharani, Wyatt Nathan, Rivera Santiago Eric, Neher Oliver, Weiland John, Bolton Melvin

机构信息

USDA ARS, Edward T. Schafer Agricultural Research Center, Fargo, North Dakota, United States;

USDA-ARS Northern Plains Area, 57644, Cereal Crops Research Unit, 1616 Albrect Drive, Fargo, ND 58102, Fort Collins, Colorado, United States, 80526-8116;

出版信息

Plant Dis. 2023 Apr 27. doi: 10.1094/PDIS-12-22-2855-PDN.

Abstract

In the fall 2021, red table beet plants (Beta vulgaris L. cv 'Eagle') exhibiting stunted growth with shorter petioles were observed at an incidence of 10 to 15 percent in a production field in Payette County, Idaho, United States. In addition to stunting, beet leaves displayed yellowing and mild curling and crumpling, and the roots exhibited hairy root symptoms (sFig.1). To identify potential causal viruses, total RNA was isolated from the leaf and root tissue using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to high-throughput sequencing (HTS). Two libraries were prepared, one for the leaf sample and another for the root sample using a ribo-minus TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA). HTS was performed with 150 bp paired-end sequencing on a NovaSeq 6000 (Novogene, Sacramento, CA). Following adapter trimming and removal of host transcripts, 5.9 and 16.2 million reads were obtained from the leaf and root samples, respectively. These reads were de novo assembled using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The assembled leaf sample contigs were aligned to the NCBI non-redundant database to identify contigs matching known viruses. A single contig of 2845 nts that shared 96% coverage and 95.6% sequence identity to the pepper yellow dwarf strain of beet curly top virus (BCTV-PeYD, EU921828; Varsani et al., 2014), and 98% coverage and 98.39% identity with an isolate of BCTV-PeYD (KX529650) from Mexico, was identified in the leaf sample (GenBank Accession OP477336). To validate the HTS detection of BCTV-PeYD, total DNA was isolated from the leaf sample and a 454 bp fragment of the C1 gene (replication-associate protein) was PCR amplified and Sanger sequencing of the amplicon revealed 99.7% identity to the HTS assembled BCTV-PeYD sequence. In addition to the PeYD strain of BCTV, the Worland strain of BCTV (BCTV-Wor) was detected as a single 2930 nt contig with 100% coverage and 97.3% identity to the BCTV-Wor isolate CTS14-015 (KX867045) known to infect sugar beet in Idaho. Of note, there are 11 strains of BCTV and among those, the BCTV-Wor strain induces mild symptoms in sugar beet (Strausbaugh et al., 2017), whereas BCTV-PeYD was found only in pepper from New Mexico. Further, two contigs of 2201 nts and 523 nts were assembled generating a nearly complete genome of spinach curly top Arizona virus (SpCTAV) in the leaf sample with 99% coverage and 99.3% identity (GenBank Accession OQ703946) to the reference genome of SpCTAV (HQ443515; Hernandez-Zepeda et al., 2013). To validate the HTS results, total DNA was isolated from the leaf tissue and PCR amplified a 442 bp fragment that overlaps the V1, V2, and V3 ORFs and its sequence revealed 100% identity with the HTS assembled SpCTAV. The roots sample also showed HTS reads corresponding to BCTV-PeYD and SpCTAV. In addition, beet necrotic yellow vein virus (BNYVV) was detected in the root sample with 30% coverage, but no sequence reads matching to BNYVV was detected in the leaf sample. BNYVV is known to infect sugar beet causing rhizomania (Tamada et al., 1973; Schirmer et al., 2005). To further confirm the BNYVV HTS results, total RNA was extracted separately from the root and leaf tissue, and RT-PCR was performed with primers that were designed to amplify portions of BNYVV RNAs (Weiland et al., 2020). RT-PCR analysis generated the appropriate amplicons with expected sequences corresponding to the RNA-1, RNA-2, RNA-3, and RNA-4 of BNYVV as determined by Sanger sequencing implying BNYVV the causal agent of hairy root symptoms. Similar to observations seen for BNYVV infection in conventional sugar beet varieties, no amplification was detected for BNYVV in the RNA extracted from leaf tissue, indicating that the RT-PCR results are consistent with the HTS analysis. This is the first report of BCTV-PeYD and SpCTAV observed naturally infecting red table beet in Idaho suggesting the geographical expansion of these viruses. The co-existence of BCTV-PeYD and SpCTAV with limited host range needs to be investigated to determine the actual cause of the observed foliar symptoms. This report provides the basis for further research to understand the pathogenic nature of these viruses and their potential threat to red table beet and sugar beet production in Idaho.

摘要

2021年秋季,在美国爱达荷州佩耶特县的一个生产田中,观察到红色食用甜菜植株(Beta vulgaris L. cv 'Eagle')生长发育迟缓,叶柄较短,发病率为10%至15%。除了生长发育迟缓外,甜菜叶片还出现黄化、轻度卷曲和皱缩,根部呈现毛状根症状(补充图1)。为了鉴定潜在的致病病毒,使用RNeasy植物微型试剂盒(Qiagen,加利福尼亚州瓦伦西亚)从叶片和根部组织中分离总RNA,并进行高通量测序(HTS)。使用去核糖体TruSeq链特异性总RNA文库制备试剂盒(Illumina,加利福尼亚州圣地亚哥)制备了两个文库,一个用于叶片样本,另一个用于根部样本。在NovaSeq 6000(加利福尼亚州萨克拉门托的Novogene公司)上进行150 bp双端测序的高通量测序。经过接头修剪和去除宿主转录本后,分别从叶片和根部样本中获得了590万和1620万条 reads。使用SPAdes组装器(Bankevitch等人,2012年;Prjibelski等人,2020年)对这些reads进行了从头组装。将组装好的叶片样本重叠群与NCBI非冗余数据库进行比对,以鉴定与已知病毒匹配的重叠群。在叶片样本中鉴定出一个2845 nt的单一重叠群,与甜菜曲顶病毒的辣椒黄矮株系(BCTV-PeYD,EU921828;Varsani等人,2014年)共享96%的覆盖率和95.6%的序列同一性,与来自墨西哥的BCTV-PeYD分离株(KX529650)具有98%的覆盖率和98.39%的同一性(GenBank登录号OP477336)。为了验证HTS对BCTV-PeYD的检测,从叶片样本中分离总DNA,对C1基因(复制相关蛋白)的454 bp片段进行PCR扩增,并对扩增产物进行Sanger测序,结果显示与HTS组装的BCTV-PeYD序列具有99.7%的同一性。除了BCTV的PeYD株系外,还检测到BCTV的沃兰株系(BCTV-Wor),它是一个2930 nt的单一重叠群,与已知感染爱达荷州甜菜的BCTV-Wor分离株CTS14-015(KX867045)具有100%的覆盖率和97.3%的同一性。值得注意的是,BCTV有11个株系,其中BCTV-Wor株系在甜菜中引起轻微症状(Strausbaugh等人,2017年),而BCTV-PeYD仅在新墨西哥州的辣椒中发现。此外,在叶片样本中组装了两个分别为2201 nt和523 nt的重叠群,生成了菠菜曲顶亚利桑那病毒(SpCTAV)的近乎完整的基因组,与SpCTAV的参考基因组(HQ443515;Hernandez-Zepeda等人,2013年)具有99%的覆盖率和99.3%的同一性(GenBank登录号OQ703946)。为了验证HTS结果,从叶片组织中分离总DNA,对与V1、V2和V3 ORF重叠的442 bp片段进行PCR扩增,其序列显示与HTS组装的SpCTAV具有100%的同一性。根部样本也显示出与BCTV-PeYD和SpCTAV对应的HTS reads。此外,在根部样本中检测到甜菜坏死黄脉病毒(BNYVV),覆盖率为30%,但在叶片样本中未检测到与BNYVV匹配的序列 reads。已知BNYVV感染甜菜会导致根瘤病(Tamada等人,1973年;Schirmer等人,2005年)。为了进一步证实BNYVV的HTS结果,分别从根部和叶片组织中提取总RNA,并使用设计用于扩增BNYVV RNA部分的引物进行RT-PCR(Weiland等人,2020年)。RT-PCR分析产生了适当的扩增产物,其预期序列与通过Sanger测序确定的BNYVV的RNA-1、RNA-2、RNA-3和RNA-4相对应,这意味着BNYVV是毛状根症状的病原体。与在传统甜菜品种中观察到的BNYVV感染情况类似,从叶片组织中提取的RNA中未检测到BNYVV的扩增,这表明RT-PCR结果与HTS分析一致。这是首次报道在爱达荷州自然感染红色食用甜菜的BCTV-PeYD和SpCTAV病毒,表明这些病毒的地理范围在扩大。需要对宿主范围有限的BCTV-PeYD和SpCTAV的共存情况进行研究,以确定观察到的叶片症状的实际原因。本报告为进一步研究这些病毒的致病性质及其对爱达荷州红色食用甜菜和甜菜生产的潜在威胁提供了基础。

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