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新西兰无花果树中首次发现无花果病毒B的报告。

First report of Fig virus B in Ficus carica in New Zealand.

作者信息

Veerakone Stella, Kanchiraopally Deepika, Khan Subuhi, Liefting Lia, Thompson Jeremy R

机构信息

New Zealand Ministry for Primary Industries, 91821, Plant health and environment, 231 Morrin Road, Auckland, Auckland, New Zealand, 1140;

New Zealand Ministry for Primary Industries, 91821, Wellington, New Zealand;

出版信息

Plant Dis. 2023 Jul 24. doi: 10.1094/PDIS-05-23-0928-PDN.

Abstract

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

摘要

无花果(Ficus carica)自古以来就被种植,现在在全球范围内都有种植,既因其果实,也作为观赏植物。几种病毒和类病毒与无花果花叶病(FMD)有关,这是一种在全球范围内发生的病害复合体(Preising等人,2021年)。已知无花果花叶病毒(FMV)、无花果叶斑驳相关病毒1(FLMaV-1)、无花果轻度斑驳相关病毒(FMMaV)和无花果杆状DNA病毒1(FBV-1)在新西兰感染无花果(Minafra等人,2012年;Veerakone等人,2015年)。2020年12月,收到了来自奥克兰圣赫利尔斯路边一棵无花果树的叶片样本,该样本显示出枝条枯死并伴有叶片褪绿花叶症状,用于病毒检测。使用KingFisher™ mL纯化系统(赛默飞世尔科技公司,马萨诸塞州沃尔瑟姆)和InviMag植物DNA微量提取试剂盒(德国Invitek Molecular GmbH公司)从有症状的叶片中提取总核酸,并使用Liefting等人(2021年)所述方法在牛津纳米孔技术公司的MinION设备上进行高通量测序。所有序列分析均使用Geneious Prime 2021.1.1(https://www.geneious.com)进行。按照Liefting等人(2021年)所述,对总共355,858条通过质量检查的读数进行了针对NCBI核苷酸数据库的BLASTn搜索。以下病毒产生了匹配结果:FMV、FBV-1、FMMaV和一种无花果长线形病毒。通过物种特异性逆转录聚合酶链反应(RT-PCR)证实了FMV、FBV-1和FMMaV的存在。为了鉴定长线形病毒,将读数映射到在无花果中报道的长线形病毒,包括最近鉴定的暂定种无花果病毒A(FiVA;GenBank登录号MN817232)和无花果病毒B(FiVB;GenBank登录号MN817233)。通过从重叠的RT-PCR中使用根据重叠群序列设计的引物,从映射到FiVB中获得了五个长度从939到2340个核苷酸(nt)的病毒重叠群。随后,使用从重叠群序列设计的引物,通过重叠RT-PCR从新西兰分离株的3'区域扩增出一个6.4 kb的序列(GenBank登录号OQ968551)。该序列与FiVB的核苷酸(nt)同一性为79.5%。使用FiVB特异性RT-PCR和使用引物FiVB-F1(5'-GAGGGAGAGATGTAGATGC-3')和FiVB-R2(5'-TGTCGTCGATATCGTTGTGT-3')进行的桑格测序对从奥克兰地区2016年至2021年期间收集的原始样本以及另外25个有症状和10个无症状的无花果样本进行了检测,这些引物旨在扩增70 kDa热休克蛋白(HSP70)开放阅读框中的一个725 nt片段。从原始样本和三个有症状样本中扩增出了预期大小的产物,并且发现它们的序列相同。BLAST搜索表明,该序列(GenBank登录号ON553403)与FiVB的一个分离株(GenBank登录号MN817233)的核苷酸同一性为82.7%,氨基酸同一性为87.3%。这些额外的阳性样本是从一个小型家庭苗圃收集的,那里的植物通过扦插繁殖并已在当地分发,这表明该病毒很可能在奥克兰地区的传播范围有限。所有三个感染FiVB的样本对FMV也呈阳性。然而,FiVB与FMD症状的关联尚不清楚。FiVB最初是从从日本收集的一棵无花果树渗出的乳胶样本中鉴定出来的(Park等人,2021年),并且是迄今为止世界上关于FiVB的唯一报告。尽管来自阿根廷的一个相同序列,命名为无花果长线形病毒1,已提交到GenBank,但该分离株的来源未知。据我们所知,这是FiVB在新西兰的首次报告。

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