Grapevine rupestris stem pitting-associated virus (GRSPaV) and Vein Necrosis: effect of genetic variability in symptoms expression

Vein necrosis (VN) is a virus-like disease of grapevine that is latent in V. vinifera and in most of rootstock and hybrids, with the only exception of rootstock 110 Richter (V. berlandieri x V. rupestris). Despite its ubiquitous presence in many grape growing regions, its economic impact on production is still largely unknown, because of its latency in V. vinifera. Though the causal agent of VN has not been identified, as the disease is able to be transmitted by grafting, VN is then considered a virus-like disease. Recent studies suggested a correlation between VN and the virus Grapevine rupestris stem pitting-associated virus (GRSPaV). In this research, the presence and molecular variability of GRSPaV in grapevine accessions from Tuscany and California was studied through RT-PCR and sequence analyses. RT-PCR analyses with group-specific primer sets distinguished three molecular groups of GRSPaV variants. All of the three groups were detected both in Tuscan and Californian grapevine accessions. Amplification, cloning and sequencing of two distinct viral genomic regions were carried out in order to obtain a finer molecular characterization of GRSPaV isolates. Depending on the genomic region analyzed, five (ORF5) to six (ORF1) phylogenetic groups of virus variants were observed. Notably, the sixth phylogenetic group identified according to sequence analysis of a 299 nucleotides fragment of ORF1 comprises only GRSPaV variants derived from Tuscan grapevine accessions, that show low nucleotide identity with any other GRSPaV sequence deposited in GenBank. This could represent a new and possibly yet unknown phylogroup of GRSPaV variants, that has named “group 5”. Linking the results of GRSPaV molecular characterization with those of VN biological indexing previously conducted on the accessions object of the study, a strong correlation emerged between VN positive plants and infections by GRSPaV variants belonging to phylogenetic groups 2a and 2b. In order to further assess this correlation, a biological indexing trial was performed for 37 grapevine accessions from Italy (Tuscany and Apulia), USA, Portugal and Japan. Results obtained confirmed the hypothesis that only phylogenetic groups 2a and 2b of the virus are able to induce VN symptoms on indicator host 110 Richter. These findings show evidence of a different role of GRSPaV variants in VN determinism; only phylogenetic groups 2a and 2b appear to be able to induce symptoms of VN. This could suggest the possibility to diagnose VN by RT-PCR assays with group-specific primers, representing a much faster, cheaper and more simple way to monitor the diffusion and incidence of this disease

Characterization of Potato Virus Y Isolates and Assessment of Nanopore Sequencing to Detect and Genotype Potato Viruses

peer-reviewPotato virus Y (PVY) is the most economically important virus infecting cultivated potato (Solanum tuberosum L.). Accurate diagnosis is crucial to regulate the trade of tubers and for the sanitary selection of plant material for propagation. However, high genetic diversity of PVY represents a challenge for the detection and classification of isolates. Here, the diversity of Irish PVY isolates from a germplasm collection and commercial sites was investigated using conventional molecular and serological techniques. Recombinant PVY isolates were prevalent, with PVYNTNa being the predominant genotype. In addition, we evaluated Nanopore sequencing to detect and reconstruct the whole genome sequence of four viruses (PVY, PVX, PVS, PLRV) and five PVY genotypes in a subset of eight potato plants. De novo assembly of Nanopore sequencing reads produced single contigs covering greater than 90% of the viral genome and sharing greater than 99.5% identity to the consensus sequences obtained with Illumina sequencing. Interestingly, single near full genome contigs were obtained for different isolates of PVY co-infecting the same plant. Mapping reads to available reference viral genomes enabled us to generate near complete genome sequences sharing greater than 99.90% identity to the Illumina-derived consensus. This is the first report describing the use of Oxford Nanopore’s MinION to detect and genotype potato viruses. We reconstructed the genome of PVY and other RNA viruses; indicating the technologies potential for virus detection in potato production systems, and for the study of genetic diversity of highly heterogeneous viruses such as PVY

Detection of viruses infecting Lilium spp. by RT-PCR and Real-Time PCR

In order to enhance the Italian lily bulb production, a breeding program was carried out at CRA-VIV in Pescia (PT – Italy) during the last years. Asiatic hybrids (Lilium × elegans Thunb.), lily cultivars and other native species were involved in the program. The obtained lily lines, which presented interesting traits, were preserved in a collection. After years of vegetative propagation, some of the new selections showed symptoms referable to viral infections. Virus diseases represent some of the most dangerous threats of Lilium, so the application of fast and effective diagnostic techniques for early detection is very important. The aim of the present study, in the frame of a phytosanitary survey of the lily collection, is to investigate the presence and incidence of Cucumber mosaic virus (CMV), Lilium symptomless virus (LSV), Lily mottle virus (LMoV) and two tospoviruses (Impatiens necrotic spot virus, INSV, and Tomato spotted wilt virus, TSWV). Among the 60 samples object of this study, infections by LSV and CMV were frequently observed. Also LMoV was detected in a smaller amount of samples. All of the samples were negative to INSV and TSWV

Lily mottle virus isolate 2409 coat protein gene, partial cds.

Genbank accession number: KF15666

Grapevine rupestris stem pitting-associated virus isolate MSAS3-8, 28 kDa coat protein gene, complete sequence

Genbank accession number: HQ39674

Lily mottle virus isolate 207/2 coat protein gene, partial cds.

Genbank accession number: JQ65511

Zantedeschia mild mosaic virus isolate 2082 polyprotein gene, partial cds

Genbank accession number: KF15666

Lily mottle virus isolate 2076 coat protein gene, partial cds.

Genbank accession number: KF15666

Lily mottle virus isolate 117/2 coat protein gene, partial cds.

Genbank accession number: JQ65510

Lily mottle virus isolate 2407 coat protein gene, partial cds.

Genbank accession number: KF15666