First report of saffron-associated mastrevirus 1 from saffron in Iran.

IF 4.4 2区 农林科学 Q1 PLANT SCIENCES Plant disease Pub Date : 2024-12-14 DOI:10.1094/PDIS-11-24-2462-PDN
Seyyedeh Atefeh Hosseini, Charlotte Julian, Serge Galzi, Denis Filloux, Philippe Roumagnac
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Abstract

In spring 2022, 40 leaf samples of saffron plants harboring a wide variety of symptoms, including curling, yellowing, mosaic, dwarfing and leaf malformation were collected from three Khorasan provinces in Iran. These samples were processed using the virion-associated nucleic acid-based metagenomics approach (Moubset et al., 2022). Noteworthy, 147 contigs with a size >500 bp distributed among 35 samples with multiple symptomatic patterns (curling, yellowing, mosaic, dwarfing and leaf malformation) shared >90% nucleotide identity with saffron-associated mastrevirus 1 (SaM1) (Martínez-Fajardo et al., 2024). This virus has recently been detected from transcriptomic datasets from saffron (Crocus sativus L.) collected in India. SaM1 was proposed to belong to a new species of the Mastrevirus genus (Geminiviridae family) and was tentatively named Saffron-associated mastrevirus 1 (Martínez-Fajardo et al., 2024). In addition, contigs assigned to saffron potyviruses, including saffron latent virus, turnip mosaic virus and bean yellow mosaic virus were assembled from the 40 saffron samples pinpointing that no conclusion could be made on the causal virus of observed symptoms. Total DNAs of the 40 saffron samples were further extracted using the DNeasy Plant Mini Kit (Qiagen) and were tested for the presence of SaM1 using a primer pair amplifying a 327 bp long fragment located in the coat protein of SaM1 (CSAV_2F 5'-TTTAAGTCAGGGTCTGGAGATG-3'and CSAV_3R 5'-GGCATACTGTAACCTCGTCTTC). Amplification conditions were: an initial denaturation at 95°C for 10 min, 30 cycles at 95°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec, and a final extension step at 72°C for 10 min. This PCR test confirmed that 28/40 samples tested positive for SaM1. Total DNAs from one sample (#69-32) testing positive for SaM1 was further amplified using Phi29 DNA polymerase (TempliPhi, GE Healthcare) by rolling circle amplification (RCA) as previously described (Shepherd et al., 2008). RCA products were used as a template for PCR amplification of the complete genome of SaM1 using abutting primers (CSAV_PST1F 5'-CTGCAGTTGCGGTAAGTCTATGTTGGCTG-3' and CSAV_PST1R 5'- CTGCAGAGACAACGATTCCCAAAATTACTTTGTTCC-3'). Amplification conditions were: an initial denaturation at 95°C for 10 min, 30 cycles at 95°C for 30 sec, 55°C for 1 min, 72°C for 2 min and 30 sec, and a final extension step at 72°C for 10 min. Amplification products of approximately 2.7 Kbp were gel purified, ligated to pGEM-T Easy (Promega) and sequenced by standard Sanger sequencing using a primer walking approach (Azenta, Germany). The arrangement of open reading frames within the 2,724 nt circular DNA of the CSAV_A9PstI clone (accession number PQ392009) is similar to those reported for mastreviruses, including the coat protein (CP), the movement protein (MP), the replication-associated protein (Rep) and the RepA protein. In addition, CSAV_A9PstI sequence shares 95.3% genome-wide identity with SaM1 consensus sequence assembled from transcriptomics data (Martínez-Fajardo et al., 2024). Overall, these results indicate that SaM1 is widely present in saffron in different regions of Northeast Iran. In addition, while all tested plant were infected by several viruses, the etiology of SaM1 remains to be determined precisely. To our knowledge, this is the first report of saffron-associated mastrevirus 1 from saffron in Iran.

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伊朗首次报告藏红花相关乳突病毒 1。
2022 年春,从伊朗的三个呼罗珊省收集了 40 份藏红花植株叶片样本,这些样本带有多种症状,包括卷曲、黄化、马赛克、矮化和叶片畸形。采用基于病毒相关核酸的元基因组学方法对这些样本进行了处理(Moubset 等人,2022 年)。值得注意的是,在 35 个具有多种症状模式(卷曲、黄化、镶嵌、矮化和叶片畸形)的样本中,有 147 个大小大于 500 bp 的等位组与藏红花相关乳突病毒 1(SaM1)(Martínez-Fajardo et al.)最近从印度收集的藏红花(Crocus sativus L.)转录组数据集中检测到了这种病毒。SaM1 被认为属于乳突病毒属(Geminiviridae 科)的一个新种,并被暂时命名为藏红花相关乳突病毒 1(Martínez-Fajardo 等人,2024 年)。此外,还从 40 个藏红花样本中收集到了藏红花潜伏病毒、芜菁花叶病毒和豆黄花叶病毒等藏红花潜伏病毒的等位基因,但无法确定观察到的症状的病原病毒。使用 DNeasy Plant Mini Kit(Qiagen)进一步提取了 40 个番红花样本的总 DNA,并使用一对引物(CSAV_2F 5'-TTTAAGTCAGGTCTGGAGATG-3'and CSAV_3R 5'-GGCATACTGTAACCTCGTCTTC)扩增了位于 SaM1 衣壳蛋白中的 327 bp 长片段,以检测是否存在 SaM1。扩增条件为:95°C 初始变性 10 分钟,95°C 30 秒、55°C 30 秒、72°C 45 秒循环 30 次,最后 72°C 延伸 10 分钟。这项 PCR 检测证实,28/40 个样本的 SaM1 检测结果呈阳性。根据之前的描述(Shepherd 等人,2008 年),使用 Phi29 DNA 聚合酶(TempliPhi,GE Healthcare)通过滚动圈扩增(RCA)进一步扩增了对 SaM1 检测呈阳性的一个样本(#69-32)的总 DNA。以 RCA 产物为模板,使用邻接引物(CSAV_PST1F 5'-CTGCAGTTGCGGTAAGTCTATGTTGGCTG-3' 和 CSAV_PST1R 5'- CTGCAGAGACAACGATTCCCAAAATTACTTTGTTCC-3')对 SaM1 的完整基因组进行 PCR 扩增。扩增条件为:95°C 初始变性 10 分钟,95°C 30 秒、55°C 1 分钟、72°C 2 分钟和 30 秒的 30 个循环,以及 72°C 10 分钟的最后延伸步骤。约 2.7 Kbp 的扩增产物经凝胶纯化后,连接到 pGEM-T Easy(Promega 公司),并使用引物游走法(德国 Azenta 公司)进行标准 Sanger 测序。CSAV_A9PstI 克隆(登录号 PQ392009)的 2,724 nt 环状 DNA 中开放阅读框的排列与已报道的乳突病毒相似,包括衣壳蛋白(CP)、运动蛋白(MP)、复制相关蛋白(Rep)和 RepA 蛋白。此外,CSAV_A9PstI 序列与根据转录组学数据组装的 SaM1 共识序列具有 95.3% 的全基因组一致性(Martínez-Fajardo 等人,2024 年)。总之,这些结果表明,SaM1 广泛存在于伊朗东北部不同地区的藏红花中。此外,虽然所有受测植物都感染了多种病毒,但 SaM1 的病原仍有待精确确定。据我们所知,这是伊朗首次报告藏红花中的藏红花相关乳突病毒 1。
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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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