First Report of Grapevine leafroll-associated virus 1 Infecting Grapevines (Vitis spp.) in Nigeria
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Grapevine (Vitis spp.) was introduced into Nigeria in the 1970s and is mainly grown as own-rooted vines for fresh fruit consumption in the northern guinea savannah ecological zone of the country. However, there is a lack of information on the repertoire of grapevine viruses occurring in vineyards planted to introduced cultivars in Nigeria. To address this knowledge gap, surveys were conducted during 2016 across 28 vineyards to document the occurrence of grapevine viruses and viroids. A total of 318 leaf tissue samples were collected from different red and white fruited cultivars, dried under CaCl at room temperature, and then shipped under USDA-APHIS-PPQ permit to the Texas A&M AgriLife Research and Extension Center, Weslaco, TX, for diagnosis. Equal amount of leaf tissue was pooled from all samples and distributed evenly into subsamples G1.1.164 and G2.1.165. Total nucleic acid was isolated from each subsample using the MagMAX-96 viral RNA isolation kit (ThermoFisher Scientific), then used for cDNA library construction after a ribosomal RNA depletion step performed with the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Both libraries were sequenced on the Illumina NextSeq 500 platform, generating 39.7 and 24.2 million raw high-throughput sequencing (HTS) reads (76 nt in length) from G1.1.164 and G2.1.165, respectively. Bioinformatic analysis of the HTS reads were performed as previously described (Al Rwahnih et al. 2017), resulting in the identification of 1,767 reads, ranging from 221 to 747 nt, that mapped to the genome of Grapevine leafroll-associated virus 1 (GLRaV-1) from the genus Ampelovirus in the family Closteroviridae. The GLRaV-1-specific reads were present only in subsample G2.1.165 along with viroid-specific reads recovered from both subsamples (data not shown). Total RNA extracts (Spectrum Plant Total RNA Kit, Sigma-Aldrich) prepared from 173 randomly selected individual dried leaf tissue samples were subjected to a two-step RT-PCR using the primer pair GLRaV-1F/GLRaV-1R (Poojari et al. 2016) for the amplification of a 267-bp product from the helicase domain of the replicase gene of GLRaV-1. DNA bands of the expected size were obtained from 48 of these samples (28%). Furthermore, GLRaV-1-specific DNA bands were amplified from two of the positive samples (both from Vitis labrusca cv. Bangalore blue) using eight published primers targeting the HSP70h (540 nt), CP (734 nt), CPd2 (398 nt), and p24 (634 nt) coding regions (Alabi et al. 2011). The obtained gene-specific bands were cloned individually into pCR2.1 TOPO-TA vector (Life Technologies) and two independent clones per amplicon were Sanger sequenced. In pairwise comparisons, the HSP70h (MF565493498), CP (MF565499504), CPd2 (MF565505510), and p24 (MF565511516) sequences from both samples shared 98 to 100% (HSP70h), 99 to 100% (CP), 98 to 100/97 to 100% (CPd2), and 99 to 100/98 to 100% (p24) nt/aa identity levels among themselves. Sequences from both samples also shared 91 to 92/93 to 94% (HSP70h), 91/92 to 93% (CP), 88 to 90/82 to 83% (CPd2), and 88/84 to 86% (p24) nt/aa identity levels with corresponding sequences of the type isolate of GLRaV-1 (AF195822). To our knowledge, this is the first report of GLRaV-1 infecting grapevine in Nigeria. The presence of GLRaV-1 likely resulted from inadvertent introductions of virus-infected cuttings into the country. Studies are ongoing to further elucidate the prevalence of GLRaV-1 in Nigerian vineyards and its occurrence in mixed infections with other graft-transmissible agents of grapevine.
