Mitra Arunabha, Jarugula Sridhar, Hoheisel Gwen, Rayapati Naidu
Washington State University, Plant Pathology, Prosser, Washington, United States;
Washington State University, Plant Pathology, 24106 N. Bunn Rd, PROSSER, Washington, United States, 99350;
Plant Dis. 2021 Feb 16. doi: 10.1094/PDIS-01-21-0183-PDN.
Since 2015, several blueberry plants () of cvs. Draper and Top Shelf in an organic farm in eastern Washington State showed reduced growth with deformed leaves displaying chlorotic spots, rings, and red blotches and producing small and poorly ripened berries. The symptomatic plants showed gradual decline within 2 to 3 years post-planting. In ELISA using antibodies (Agdia, Inc., USA) to , , , , , , and [TRSV]), leaf samples from six symptomatic plants tested positive only to TRSV (). Subsequently, total RNA was isolated from leaves of a symptomatic plant using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, USA). High quality RNA was subjected to high-throughput sequencing (HTS) on the Illumina© NovaSeq™ platform (Huntsman Cancer Institute, UT, USA). An average of ~28 million 150-base pair (bp) paired-end reads obtained were subjected to quality filtering followed by de novo assembly using CLC Genomics Workbench (v12.0) and BLASTn analysis (http://www.ncbi.nlm.nih.gov/blast). Two contigs of 2,778 bp (average coverage: 11,031.7) and 3,589 bp (average coverage: 11,882) showed, respectively, a maximum of 97.3 and 97.6% nucleotide (nt) identity with TRSV RNA1 of a South Korean isolate (KJ556849). Another contig of 3,615 bp (average coverage: 7072.1) showed a maximum of 92.8% nt identity with TRSV RNA2 of an isolate from Iowa (MT563079). The HTS data revealed no other viral sequences reported from blueberry plants (Martin and Tzanetakis 2018). To further confirm the presence of TRSV, extracts of leaf samples from seven symptomatic and ten asymptomatic plants collected randomly from cvs. Draper and Top Shelf were tested by RT-PCR using primers specific to a region of the helicase gene of TRSV RNA1 (Forward: GACTACTGAGCAACATTGCAACTTCC, Reverse: GTCCCCTAACAGCATTGACTACC) and the coat protein gene of TRSV RNA2 (Forward: GCTGATTGGCAGTGTATTGTTAC, Reverse: GTGTTCGCATCTGGTTTCAAATTGG). An approximately 360 bp fragment specific to RNA1 and ~640 bp fragment specific to RNA2 were amplified only from symptomatic samples. Sanger sequence analysis of amplicons specific to RNA1 and RNA2 showed 98.1% and 96.8% nt identity with corresponding sequences of TRSV isolates from South Korea (KJ556849) and Iowa (MT563079), respectively. These results confirmed the presence of TRSV in symptomatic blueberry plants. The complete sequence of RNA1 (7,512 nt, MW495243) and RNA2 (3,925 nt, MW495244) genome segments of the blueberry isolate determined in this study showed 95.9 and 93.2% nt sequence identity, respectively, with corresponding TRSV sequences from South Korea (KJ556849) and Iowa (MT563079). Based on previous reports (Converse and Ramsdell 1982, Martin et al. 2012, Martin and Tzanetakis, 2018), this study represents the first report of TRSV infecting highbush blueberry in Washington State. Since the State has emerged as the national leader in blueberry production, the results will strengthen plant health certification standards to provide virus-tested propagative materials for domestic growers and export to the European Union.
自2015年以来，华盛顿州东部一个有机农场中，多个品种为德雷珀（Draper）和顶级货架（Top Shelf）的蓝莓植株生长减缓，叶片变形，出现褪绿斑点、环斑和红色斑块，结出的浆果小且成熟度差。这些有症状的植株在种植后2至3年内逐渐衰退。在使用针对番茄环纹斑点病毒（TRSV）、番茄斑萎病毒（TSWV）、烟草脆裂病毒（TRV）、烟草环斑病毒（TRSV）、蓝莓潜隐病毒（BLV）、蓝莓叶焦病毒（BLFV）和草莓潜隐环斑病毒（SLRSV）的抗体（美国Agdia公司）进行的酶联免疫吸附测定（ELISA）中，来自6株有症状植株的叶片样本仅对TRSV检测呈阳性。随后，使用Spectrum™植物总RNA提取试剂盒（美国Sigma - Aldrich公司）从一株有症状植株的叶片中分离总RNA。高质量的RNA在Illumina© NovaSeq™平台（美国犹他州亨茨曼癌症研究所）上进行高通量测序（HTS）。获得的平均约280万个150碱基对（bp）的双端读数经过质量过滤，然后使用CLC基因组学工作台（v12.0）进行从头组装，并进行BLASTn分析（http://www.ncbi.nlm.nih.gov/blast）。两个长度分别为2778 bp（平均覆盖度：11031.7）和3589 bp（平均覆盖度：11882）的重叠群，与一株韩国分离株（KJ556849）的TRSV RNA1的核苷酸（nt）同一性最高分别为97.3%和97.6%。另一个长度为3615 bp（平均覆盖度：7072.1）的重叠群，与一株来自爱荷华州的分离株（MT563079）的TRSV RNA2的nt同一性最高为92.8%。高通量测序数据未揭示蓝莓植株中报道的其他病毒序列（Martin和Tzanetakis，2018年）。为进一步确认TRSV的存在，从德雷珀和顶级货架品种中随机采集的7株有症状和10株无症状植株的叶片样本提取物，使用针对TRSV RNA1解旋酶基因区域的特异性引物（正向：GACTACTGAGCAACATTGCAACTTCC，反向：GTCCCCTAACAGCATTGACTACC）和TRSV RNA2外壳蛋白基因的特异性引物（正向：GCTGATTGGCAGTGTATTGTTAC，反向：GTGTTCGCATCTGGTTTCAAATTGG）进行逆转录聚合酶链反应（RT - PCR）检测。仅从有症状样本中扩增出了一个约360 bp的RNA1特异性片段和约640 bp的RNA2特异性片段。对RNA1和RNA2特异性扩增子的桑格测序分析显示，与来自韩国（KJ556849）和爱荷华州（MT563079）的TRSV分离株的相应序列分别具有98.1%和96.8%的nt同一性。这些结果证实了有症状蓝莓植株中存在TRSV。本研究中测定的蓝莓分离株的RNA1（7512 nt，MW495243）和RNA2（3925 nt，MW495244）基因组片段的完整序列，与来自韩国（KJ556849）和爱荷华州（MT563079）的相应TRSV序列的nt序列同一性分别为95.9%和93.2%。根据先前的报道（Converse和Ramsdell，1982年；Martin等人，2012年；Martin和Tzanetakis，2018年），本研究是TRSV感染华盛顿州高丛蓝莓的首次报道。由于该州已成为蓝莓生产的全国领先者，这些结果将加强植物健康认证标准，为国内种植者提供经过病毒检测的繁殖材料，并出口到欧盟。
