First Report of Grapevine Geminivirus A in Vitis in New Zealand

Abstract

Grapevine geminivirus A (GGVA), a member of the family Geminiviridae, was originally described from the United States on Vitis vinifera, cultivars Black Beet and Nagano Purple, imported from Korea (Al Rwahnih et al. 2017). Since its initial characterization, GGVA has also been reported from the United States, China, and Korea in diverse Vitis spp. (Al Rwahnih et al. 2017; Fan et al. 2017; Jo et al. 2016). The transmission of GGVA by means of a vector and the economic impact of this virus are still unknown. In 2016, a small-scale, untargeted virus survey was done to assess the efficacy of high-throughput sequencing of small RNA (sRNA). Leaf samples from 20 vines were collected from the New Zealand Winegrowers’ germplasm collection, Lincoln, New Zealand. During the survey, GGVA was identified in an asymptomatic sample of Vitis vinifera, an interspecific cross Seibel 7052 (VID 280). sRNA was extracted using a mirVana miRNA Isolation Kit (Thermo Fisher Scientific). A library was prepared using the TruSeq Small RNA Sample Prep Kit (Illumina) and sequenced with 50-bp single-end reads (small RNA-Seq) on an Illumina HiSeq2000 at the Beijing Genomics Institute (Hong Kong), and a total of 32.6 million reads was obtained. When the reads were mapped to the reference sequence of GGVA (GenBank accession no. KX618694), 163,611 reads were assembled with a mean coverage level of 1,183× across the genome, yielding a near complete 2,902-nt GGVA genome sequence. Mapping was done using Bowtie2 (version 2.2.4) (Langmead and Salzberg 2012). Three viruses (grapevine leafroll-associated virus 2, grapevine rupestris stem pitting associated virus, and grapevine asteroid mosaic associated virus) and one viroid (hop stunt viroid) were also detected in this sample using the YABI Virus Surveillance and Diagnosis (VSD) toolkit (Barrero et al. 2017). The grapevine was resampled in July 2018, and the full genome was amplified by PCR using two abutting primers, 23-1F/2R (Fan et al. 2017). BLAST analysis of the sequence obtained (2,905 nt; GenBank accession no. MK690474) showed 99% nt similarity to the full genome of GGVA isolate Tamar (GenBank accession no. KX618694). The virus was detected in only one vine out of 20 (from 12 different cultivars) sequenced by sRNA-Seq from the same germplasm collection. Twenty additional grapevine samples from the South Island tested negative for GGVA by a species-specific PCR using the primers V1-F1/1R (Fan et al. 2017). To the best of our knowledge, this is the first report of GGVA in New Zealand.