First Report of Grapevine Asteroid Mosaic-Associated Virus Infecting Grapevines (Vitis vinifera) in Italy.

Abstract

Asteroid mosaic, a disease of the grapevine fleck complex characterized by starlike spots on the foliage of several cultivars of Vitis vinifera and clearing of primary and secondary veins on V. rupestris, was originally described in California (Hewitt 1954). An isometric virus, named grapevine asteroid mosaic-associated virus (GAMaV), was partially characterized from original Californian sources (Abou Ghanem-Sabanadzovic et al. 2003) and reported in Canada (Xiao and Meng 2016), Uruguay (Jo et al. 2015), and more recently in France (Candresse et al. 2017). In the summer of 2016, 48 grapevine berry samples (24 V. vinifera ‘Cannonau’ and 24 V. vinifera ‘Cabernet Sauvignon’), were collected between veraison and harvest in Alghero (northwest Sardinian Island, 40.650638, 8.241534) and their gene expression investigated by RNAseq. For each sample, total RNA was extracted with the RNeasy Plant mini kit (Qiagen), reverse transcribed, and cDNA libraries were prepared using the TrueSeq RNA kit (Illumina) according to the manufacturer’s instructions. Sequencing was performed on an Illumina Miseq system and produced an average of 40,842,896 paired-end reads (2 × 101 nt). The obtained reads were screened against the virus genome database (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral/) and aligned to the large portions of the GAMaV genome for four samples of cultivar Cannonau. The presence of the virus was confirmed by reverse transcription polymerase chain reaction using primers GAMaV-F3 (5′-ATCTCGCAGCCCAACCTCTG-3′) and GAMaV-R3 (5′-CATGATGACGGCTTGGGTCTT-3′) (Candresse et al. 2017). Detected polymorphisms (GATK pipeline) indicated the coexistence of two distinct virus variants. De novo assembly of the produced reads (by the usage of the software Spades) followed by manual curation of the obtained scaffolds led to the reconstruction of the partial genome of such variants (two scaffolds of 6,204 nt) that were deposited to the NCBI nucleotide database with the accession numbers MH061348 and MH061349. Both sequences covered 92% of the reference genome NC_031692.1 (Vargas-Asencio et al. 2017) with a percentage of identity of 92%, and the reciprocal homology proved to be 97%. Such observations demonstrate the presence of GAMaV in Italy. Noteworthy, although the vineyard was composed of both Cannonau and Cabernet Sauvignon vines, only four samples from the cultivar Cannonau showed the presence of the virus. Although we do not provide conclusive data on a different susceptibility of the used grapevine genotypes, we suggest that clone selection may have played a primary role. No detectable symptoms were observed in any of the infected vines, thus supporting the hypothesis that GAMaV may be largely latent or semilatent as previously reported (Martelli 2014). Analysis of the RNAseq experiments showed that the four infected plants were also coinfected by grapevine fleck virus, grapevine rupestris stem pitting-associated virus, grapevine leafroll-associated virus 2, grapevine yellow speckle viroid 1, and grapevine Syrah virus 1.