首次报告美国得克萨斯州百合花(Lilium asiatica)自然感染百合斑驳病毒的情况。

IF 4.4 2区 农林科学 Q1 PLANT SCIENCES Plant disease Pub Date : 2024-10-11 DOI:10.1094/PDIS-07-24-1485-PDN
John Oladeji Oladokun, Isaias Hernandez, Aryed N Perez-Baez, Olufemi Joseph Alabi
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One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. 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引用次数: 0

摘要

百合(Lilium asiatica)是一种重要的植物,因其花色繁多、香味浓郁而被广泛种植。2024 年 3 月,在得克萨斯州伊达尔戈县韦斯拉科的一处私人庄园中,12/20 株百合植株上出现了由浅黄色斑驳和马赛克组成的潜在病毒样症状。我们随机抽取了两株出现症状的植物(WTX1 和 WTX2)进行病毒诊断。使用 Agdia 的 Poty ImmunoStrip® (Agdia, Inc.)然而,在接种后 20 至 28 天,将提取物摩擦接种到健康的烟草植物(Nicotiana benthamiana)和木樨(Vigna unguiculata)植物(各 4 株)的系统叶片上会诱发轻微的斑驳症状,这表明样品中存在可机械传播的病原体。在两种草本植物的模拟接种植株(各 1 株)上均未观察到病毒样症状。为了检测是否感染了疑似壶状病毒,使用 Oligo(dT) 引物和 PrimeScript 第一链 cDNA 合成试剂盒(Takara Bio,美国)从 WTX1 和 WTX2 提取 2µg 的总核酸(Dellaporta 等,1983 年)用于合成互补 DNA(cDNA)。用 5 U Taq 聚合酶(罗氏诊断公司,印第安纳波利斯,IN)和两对针对部分圆柱包涵体(CI)基因和壶状病毒辅助成分蛋白酶(HC-Pro)的退化引物(Ha 等人,2008 年),在 12.5µl 常规 PCR 反应体积中以每个 cDNA 的一微升等分量作为模板。从两个样本中扩增了每个目标基因的预期 ~700-bp DNA 产物。扩增子被切除、凝胶洗脱(Zymoclean™ Gel DNA Recovery kit)并分别克隆到 pJET1.2/Blunt 载体(Life Technologies)中。对每个克隆 DNA 插入物的两个转化大肠杆菌细胞中的重组质粒进行了桑格测序。对每个基因特异性核苷酸(nt)序列片段的 BLASTn 分析表明,它们与百合斑驳病病毒(LMoV;Potyvirus 属)的相同度(83%-92% nt)为 93%-98%,查询覆盖率为 93%-98%。在配对比较中,所获得的德克萨斯州百合斑驳病病毒(LMoV)特异分离物序列与CI(GenBank登录号:PQ037260-61)和HC-Pro(PQ037262-63)的序列具有约99%的nt和100%的氨基酸(aa)相同性,与最接近的澳大利亚分离物Bate5(JN127341)的序列具有约91-92%的nt和98-100%的aa相同性。根据这些结果,我们新设计了一对 LMoV 特异引物(LM_v109-F:5'-GGCCAGTAATGTGCACAAGC-3'和 LM_c527-R:5'-TCGCTGTAGCTAGCGACGTAC-3'),以上述通用引物获得的 700-bp 片段内部的部分 CI 基因为靶标。如前所述,该引物对用于通过 RT-PCR 筛选每一株机械接种的试验植株。从 N. benthamiana 和 V. unguiculata 的所有接种植株中都扩增出了预期的 LMoV CI 基因的 438-bp 片段;如上所述,结果通过 Sanger 测序得到了证实。每种实验寄主植物的模拟接种植株对 LMoV 的检测结果均为阴性。LMoV 是一种检疫性害虫,以前在美国(Brierley 和 Smith,1944 年)和世界其他百合生产地区(如荷兰、中国、韩国和巴西)都有报道。据我们所知,这是德克萨斯州首次确诊该病毒,从而扩大了其地理分布范围。为防止 LMoV 通过种植材料进一步传播,在繁殖前对百合基础种群进行检测至关重要。
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First report of lily mottle virus naturally infecting lily (Lilium asiatica) in Texas, USA.

Lily (Lilium asiatica) is an important plant grown for its range of flower colors and heavy scent. In March 2024, potyvirus-like symptoms consisting of light-yellow mottling and mosaic were noticed on 12/20 lily plants in a private property in Weslaco, Hidalgo County, Texas. Two symptomatic plants (WTX1 and WTX2) were sampled randomly for virus diagnosis. The leaf extracts of both samples were negative for the potyvirus group using Agdia's Poty ImmunoStrip® (Agdia, Inc., Elkhart, IN, USA). However, rub-inoculation of the extracts onto healthy Nicotiana benthamiana and Vigna unguiculata plants (n=4, each) induced mild mottle symptoms on the systemic leaves of both herbaceous test plants 20 to 28 days post inoculation, indicating the presence of a mechanically transmissible agent in the samples. No virus-like symptoms were observed on the mock-inoculated plants (n=1, each) of both species. To test for suspected potyvirus infection, 2-µg of total nucleic acid extracts (Dellaporta et al. 1983) from WTX1 and WTX2 were used for complimentary DNA (cDNA) synthesis with Oligo(dT) primer and the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, USA). One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. LMoV is a quarantine pest and was previously reported in the U.S. (Brierley and Smith, 1944) and other lily-producing parts of the world, such as the Netherlands, China, Korea, and Brazil. To our knowledge, this is the first confirmed report of the virus in Texas, thus expanding its geographical range. Testing of lily foundation stocks before propagation is essential to prevent further spread of LMoV via planting material.

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来源期刊
Plant disease
Plant disease 农林科学-植物科学
CiteScore
5.10
自引率
13.30%
发文量
1993
审稿时长
2 months
期刊介绍: Plant Disease is the leading international journal for rapid reporting of research on new, emerging, and established plant diseases. The journal publishes papers that describe basic and applied research focusing on practical aspects of disease diagnosis, development, and management.
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