First Report of Pepper vein yellows virus Infecting Pepper (Capsicum spp.) in the United States
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2015 The American Phytopathological Society. A routine inspection of a field planted with hot and sweet pepper (Capsicum spp.) in South Texas was undertaken during late summer of 2014. Plants showing symptoms consisting primarily of interveinal yellowing and vein clearing were observed and disease incidence was estimated at about 75% based on visual symptoms. To explore the virome of the field, symptomatic leaf tissue samples from 12 plants selected randomly across the field were pooled, flash-frozen, and used for isolation of total RNA using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturers instructions. The complementary DNA library constructed from the total RNA extract using the ScriptSeq Complete (Plant Leaf) Kit (Epicentre, Illumina, Inc.) was subjected to next generation sequencing (NGS) at the University of Southern California sequencing facility using the Illumina NextSEquation 500 platform. The CLC Genomics workbench software (CLC Bio, Qiagen) was then used for quality trimming and de novo assembly following which the contigs were analyzed using NCBIs BLASTX program (http://www.ncbi.nlm.nih.gov/blast) against the viral RefSeq database. The analysis generated 23.5 million Illumina reads (76 nucleotides [nt] in length) of which more than 5,328 reads could be mapped to Pepper vein yellows virus (PeVYV; genus Polerovirus; family Luteoviridae), also known as Pepper yellow leaf curl virus and Pepper yellows virus. To confirm the NGS results, fresh leaf tissue samples were collected individually from 18 symptomatic plants from the same field and total RNA isolated from each plant was screened by the reverse transcription (RT)-PCR. The reaction used two sets of PeVYV-specific primers pairs capable of amplifying 870 bp of the open reading frame 1 (ORF1) encoding the P0/P1 proteins and 580 bp of ORF2 encoding the RNA-dependent RNA polymerase (RdRP) and intergenic noncoding region (NCR) (Dombrovsky et al. 2013). DNA fragments of the expected sizes were obtained for both regions from all sampled plants. To further confirm the results, gene-specific amplicons from two of the 18 RT-PCR positive samples were cloned into the pCR2.1 TOPO-TA vector (Life Technologies) and two independent clones per amplicon sequenced. In pairwise comparisons, the P0/P1-specific nt sequences (KP995744 to KP995747) shared 99.1 to 99.7% identity among themselves, 93.1 to 93.6% with corresponding sequences of a PeVYV isolate from Japan (AB594828) and 91 to 91.5% with corresponding sequences of the PeVYV isolate Is from Israel (HM439608). Similarly, the RdRP/NCR-specific nt sequences (KP995748 to KP995751) shared 99.4 to 100% identity among themselves, 93.6 to 93.7% with corresponding sequences of an isolate from Japan (AB594828) and 94.4 to 94.8% with the corresponding sequences of isolate Is (HM439608). PeVYV has been reported in Japan (Murakami et al. 2011), Spain (Villanueva et al. 2013), Sudan (Alfaro-Fernandez et al. 2014), and six additional countries (Knierim et al. 2013). To the best of our knowledge, this is the first report of PeVYV in the United States, thus adding the nation to the areas of occurrence of the virus, and highlighting further its potential as a worldwide threat to pepper and related crops.
