First Report of Grapevine Red Globe Virus in Grapevine in Germany

Abstract

Grapevine red globe virus (GRGV) is a member of the genus Maculavirus, family Tymoviridae, found to infect grapevine for the first time in Italy (Sabanadzovic et al. 2000). GRGV has also been reported in Greece, the United States, France, China, and Spain (Cretazzo et al. 2017; Fan et al. 2016). No specific symptoms have been associated with GRGV infection (Martelli et al. 2002). In 2016, grapevine leaves from a Pinot Gris cultivar from the Rhineland-Palatinate area showing leaf mottling and flecking were collected and analyzed by high-throughput sequencing of siRNAs using TrueSeq Illumina technology on a HiSeq 1000 system. De novo assembly of the 5,658,588 reads was performed by Geneious software. The BlastN and BlastX analysis of the 316 contigs obtained revealed the presence of 65 contigs related to GRGV, with a percentage of nucleotide similarity ranging from 85.2 to 100%. Illumina reads were mapped to reference using the Graciano-T101 isolate (GenBank accession no. NC_030693.1), allowing the recovery of 90.1% of the viral sequence scattered along the genome. A partial sequence corresponding to a fragment of the GRGV replicase gene of one of the German isolates has been deposited in GenBank database (accession no. MG779496). Bioinformatic analysis of the remaining contigs showed the presence of Arabis mosaic virus, grapevine rupestris stem pitting associated virus, grapevine yellow speckle viroid-1, and hop stunt viroid. The presence of GRGV in the German vineyards was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) using two different sets of primers, the generic primers for tymoviruses, 5′-CATGCANGTSAGRGGRCCRAA-3′ and 5′-CYCARCAYAARGTVAACGA-3′ (Kominek et al. 2009), and newly designed primers specific for GRGV, GRGV-CF-F1 5′-GAATTCGCTGTCGGCCACTC-3′ and GRGV-CF-R1 5′-AGTGAGAGGAGAGATTCCATC-3′. The expected RT-PCR products, of 453 and 386 bp respectively, were successfully amplified, cloned, and sequenced. BlastN analysis of all amplicons confirmed the presence of the virus. GRGV was also detected by RT-PCR in 14 out of 57 randomly collected samples from symptomatic and asymptomatic grapevines, in different grapevine growing areas from the Rhineland-Palatinate area. To our knowledge, this is the first report of GRGV in Germany. Further studies will be necessary to assess the relevance and potential threat of this virus to the grape production in Germany and to determine whether it should be regulated.