Jarugula Sridhar, Wagstaff Camille, Mitra Arunabha, Crowder David, Gang David, Rayapati Naidu
Washington State University, Plant Pathology, PROSSER, Washington, United States;
Washington State University, 6760, Pullman, Washington, United States;
Plant Dis. 2023 Feb 21. doi: 10.1094/PDIS-12-22-2981-PDN.
In 2021 and 2022, virus-like symptoms were observed in several cultivars of industrial hemp (Cannabis sativa) in two fields in central Washington, USA. Affected plants had a range of symptoms at different developmental stages, with young plants having severe stunting with shortened internodes and reduced flower mass. Young leaves of infected plants also showed light green to total yellowing, and twirling with twisting margins (Fig. S1). Infections of older plants caused less foliar symptoms that consisted of mosaic, mottling, and mild chlorosis on a few branches with tacoing of older leaves. To assess if symptomatic hemp plants were infected with Beet curly top virus (BCTV) as reported earlier (Giladi et al., 2020; Chiginsky et al., 2021), symptomatic leaves were collected from 38 plants, and the extracted total nucleic acids tested by PCR to amplify a 496-base pair (bp) fragment specific to BCTV coat protein (CP) using primers BCTV2-F 5'-GTGGATCAATTTCCAG-ACAATTATC-3' and BCTV2-R 5'-CCCATAAGAGCCATATCA-AACTTC-3' (Strausbaugh et al. 2008). BCTV was found in 37 of the 38 plants. To further assess the virome of symptomatic hemp plants, total RNA was extracted from symptomatic leaves of four plants using Spectrum total RNA isolation kits (Sigma-Aldrich, St. Louis, MO) and subjected to high-throughput sequencing on an Illumina Novaseq platform in paired-end mode (University of Utah, Salt Lake City, UT). The raw reads (33 to 40 million per sample) were trimmed based on quality and ambiguity and resulting paired-end reads of ≈142 bp length were assembled de novo into a pool of contigs (CLC Genomics Workbench 21, Qiagen Inc.). Virus sequences were identified through BLASTn analysis in GenBank (https://www.ncbi.nlm.nih.gov/blast). One contig of 2,929 nucleotides (nt) obtained from one sample (accession no. OQ068391) showed 99.3% identity with BCTV-Wor strain reported from sugar beet in Idaho (accession no. KX867055 Strausbaugh et al., 2017). Another contig of 1,715 nt from a second sample (accession no. OQ068392) shared 97.3% identity with BCTV-CO strain (accession no. KX867022). Two contig sequences of 2,876 nt (accession no. OQ068388) and 1,399 nt (accession no. OQ068389) obtained from the 3rd and 4th samples showed 97.2% and 98.3% identity, respectively, with Citrus yellow vein-associated virus (CYVaV, accession no. MT893740.1) reported in industrial hemp from Colorado (Chiginsky et al., 2021). Contigs of 256 nt sequence (accession no. OQ068390) obtained from the 3rd and 4th samples also showed 99-100% identity with Hop Latent viroid (HLVd) sequences in GenBank (accessions OK143457 and X07397). These results indicated single infections of BCTV strains and co-infection of CYVaV and HLVd in individual plants. To confirm theagents, symptomatic leaves were collected from 28 randomly selected hemp plants and tested by PCR/RT-PCR using primers specific to BCTV (Strausbaugh et al., 2008), CYVaV (Kwon et al., 2021) and HLVd (Matoušek et al., 2001). Amplicons specific to BCTV (496 bp), CYVaV (658 bp) and HLVd (256 bp) were detected in 28, 25, and 2 samples, respectively. BCTV CP sequences obtained by Sanger sequencing from seven samples showed 100% sequence identity with BCTV-CO and BCTV-Wor strains in six and one samples, respectively. Similarly, sequences of CYVaV- and HLVd-specific amplicons showed 100% identity with corresponding sequences in GenBank. To the best of our knowledge, this is the first report of two strains of BCTV (BCTV-CO and BCTV-Wor), CYVaV, and HLVd infecting industrial hemp in Washington state.
2021年和2022年，在美国华盛顿州中部的两块田地中，几个工业大麻（Cannabis sativa）品种出现了类似病毒的症状。受影响的植株在不同发育阶段有一系列症状，幼株严重矮化，节间缩短，花量减少。受感染植株的幼叶也表现出从浅绿色到完全变黄，并伴有叶缘卷曲和扭曲（图S1）。 older plants感染导致较少的叶片症状，包括花叶、斑驳，以及少数枝条上的轻度褪绿，老叶呈玉米卷状。为了评估有症状的大麻植株是否如先前报道（Giladi等人，2020年；Chiginsky等人，2021年）那样感染了甜菜曲顶病毒（BCTV），从38株植株上采集了有症状的叶片，提取总核酸后通过PCR检测，使用引物BCTV2-F 5'-GTGGATCAATTTCCAG-ACAATTATC-3'和BCTV2-R 5'-CCCATAAGAGCCATATCA-AACTTC-3'（Strausbaugh等人，2008年）扩增BCTV外壳蛋白（CP）特异的496个碱基对（bp）片段。在38株植株中的37株中发现了BCTV。为了进一步评估有症状的大麻植株的病毒组，使用Spectrum总RNA分离试剂盒（Sigma-Aldrich，密苏里州圣路易斯）从4株植株的有症状叶片中提取总RNA，并在Illumina Novaseq平台上以双端模式进行高通量测序（犹他大学，盐湖城，犹他州）。原始读数（每个样本3300万至4000万）根据质量和模糊度进行修剪，得到长度约为142 bp的双端读数，将其从头组装成重叠群池（CLC Genomics Workbench 21，Qiagen公司）。通过GenBank（https://www.ncbi.nlm.nih.gov/blast）中的BLASTn分析鉴定病毒序列。从一个样本获得的一个2929个核苷酸（nt）的重叠群（登录号OQ068391）与爱达荷州甜菜中报道的BCTV-Wor株（登录号KX867055 Strausbaugh等人，2017年）显示出99.3%的同一性。来自第二个样本的另一个1715 nt的重叠群（登录号OQ068392）与BCTV-CO株（登录号KX867022）有97.3%的同一性。从第3个和第4个样本获得的两个重叠群序列，长度分别为2876 nt（登录号OQ068388）和1399 nt（登录号OQ068389），分别与科罗拉多州工业大麻中报道的柑橘黄脉相关病毒（CYVaV，登录号MT893740.1）有97.2%和98.3%的同一性。从第3个和第4个样本获得的256 nt序列的重叠群（登录号OQ068390）与GenBank中的蛇麻草潜隐类病毒（HLVd）序列（登录号OK143457和X07397）也显示出99 - 100%的同一性。这些结果表明，个别植株中存在BCTV株的单一感染以及CYVaV和HLVd的共同感染。为了确认病原体，从28株随机选择的大麻植株上采集有症状的叶片，使用针对BCTV（Strausbaugh等人，2008年）、CYVaV（Kwon等人，2021年）和HLVd（Matoušek等人，2001年）的引物进行PCR/RT-PCR检测。分别在28株、25株和2株中检测到了BCTV特异的扩增子（496 bp）、CYVaV特异的扩增子（658 bp）和HLVd特异的扩增子（256 bp）。通过Sanger测序从7个样本获得的BCTV CP序列，在6个样本中与BCTV-CO株显示出100%的序列同一性，在1个样本中与BCTV-Wor株显示出100%的序列同一性。同样，CYVaV和HLVd特异扩增子的序列与GenBank中的相应序列显示出100%的同一性。据我们所知，这是首次报道两种BCTV株（BCTV-CO和BCTV-Wor）、CYVaV和HLVd在华盛顿州感染工业大麻。
