First report of Grapevine pararetrovirus infecting grapevine in Greece

In recent years, many viruses that infect grapevine (Vitis vinifera), have been identified in Greece (Katsarou et al. , 2023; Panailidou et al. , 2023). Further investigation to determine the presence of other known and unknown viruses in commercial vineyards has led to the detection of Grapevine pararetrovirus (GPRV). GPRV was discovered in 2022 in Russia (Republic of Dagestan) in samples showing symptoms of viral disease (Shvets et al. , 2022). GPRV is a pararetrovirus (family Caulimoviridae) whose genome is circular double-stranded DNA ranging from 7. 2 to 8. 5 Kb in size (Bhat et al. , 2016). During the 2022 growing season, leaf samples from 55 plants were obtained from a commercial vineyard located in the prefecture of Messinia, a significant grapevine-growing region in Greece. Samples from each grower were pooled and sequenced by high-throughput sequencing (HTS) on an Illumina Sequencer, after having extracted total RNA using a Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, USA). One pooled sample of leaves from four plants, collected in the municipality of Trifillia, had a total of 93, 694, 500 reads, with 35, 320 mapping to the genome of GPRV (average depth 579. 1, coverage 98%). To confirm infection by GPRV, each of the four samples (prior to pooling) was tested by RT-PCR using the specific primers GPRV₂669F (5′-TAGGAGACTACTCTGACGACG-3′) and GPRV₃086R (5′-ACTGGAACTCTTCTTCTCTGGT-3′) (Shvets et al. , 2022), which amplified a 418 nt fragment within ORF5 of the virus genome. PCR was conducted in 40 cycles with the following conditions: 95°C for 30 seconds, 54°C for 30 seconds and 72°C for 30 seconds. The expected DNA fragment was amplified from two of the four samples tested. The PCR products were sequenced bidirectionally by Sanger sequencing (Genewiz, Germany) and the sequence of the amplified fragments was 99% identical to the HTS contig. Two sequences were submitted to the GenBank (Accession Nos. PP584599 and PP584600). They had 91. 4% nucleotide identity with GPRV isolate D1454w (OP886324. 1) from Russia. Leaf samples had a mild yellowing but as another virus (Grapevine fleck virus) and viroid (Grapevine yellow speckle viroid-1) were also detected, no conclusion can be made on possible symptoms. To the best of our knowledge, this is the first report of GPRV from Greece. This shows that the distribution of GPRV may be wider than previously thought and highlights the necessity for further surveillance to determine the true distribution. The authors would like to acknowledge Anastasia Nikoloudi for technical support. Samples were collected through a grant for the University of Crete (Category C) entitled 'The effect of changes in rural communities may affect the plant-pathogen interactions' KA10894 – Special account for Research Funds of University of Crete. This work was also supported by the European Union's Horizon 2020 research and innovation program under the Marie Sktodowska-Curie grant agreement No. 872969, project 'Novel approaches for plant health monitoring (PANTHEON) '.