First Report of Grapevine Yellow Speckle Viroid-2 in Grapevine in Greece

Autor: Kriton Kalantidis, Leonidas Lotos, Chrysoula-Lyto Sassalou, Varvara I. Maliogka, C. G. Orfanidou, Konstantina Katsarou, Nikolaos I. Katis, Polyxeni G. Pappi
Rok vydání: 2020
Předmět:
Zdroj: Plant Disease. 104:1879-1879
ISSN: 1943-7692
0191-2917
DOI: 10.1094/pdis-12-19-2540-pdn
Popis: Grapevine yellow speckle viroid-2 is a small, circular, single-stranded RNA molecule that is classified in the genus Apscaviroid (family Pospiviroidae). Grapevine yellow speckle viroid-2 (GYSVd-2) has been identified in many grapevine-producing countries such as India, Australia, China, Thailand (Singhal et al. 2019; Tangkanchanapas et al. 2017), Turkey, and Italy (Gambino et al. 2014). Until now, no information regarding its presence and spread in Greek vineyards was available. For this purpose, a random survey was conducted, and 327 samples of leaves and stems were collected from a large range of cultivars from Crete, Attiki, Peloponnese, Northern Greece, and Dodecanese. Total RNA was extracted according to the protocol of Maliogka et al. (2015), and reverse transcription PCR (RT-PCR) was performed using a new primer pair, GYSVd-2 up (5′-GACCTGCAGAGAAAAGAAGAAGG-3′)/GYSVd-2 do (5′-GCTCGACTAGCGGAGGC-3′). GYSVd-2 was frequently identified in the tested samples (39/327). The full viroid genomes of two isolates from Cretan vines (cv. Soultanina) and two from Peloponnese (cv. Superior) were amplified and sequenced using two different pairs of primers: GYSVd-2up 2 (5′-CCTAAGATGCCTCCGCTAGT-3′)/GYSVd-2do 2 (5′-AGACAGGCCCGCGTTTCG-3′) and GYSVd-2up3 (5′-AGAAGGGCCCAGAGGGGAAT-3′)/GYSVd-2do3 (5′-TCTTTTCTCTGCAGGTCCGC-3′). Sanger sequencing of both amplicons was performed in order to confirm the primer annealing sites. The 363-bp amplified genomes of the Cretan isolates (deposited in ENA under accession nos. LR735992 and LR735993) revealed high identity with GYSVd-2 isolates deposited in GenBank (98.63 to 100% nt identity) and 100% nt identity among them, whereas the isolates from Peloponnese (accession nos. LR735995 and LR735996) revealed 98.9 to 100% nt identity to deposited viroid isolates. At the same time the viroid was detected during a high-throughput sequencing analysis of rRNA depleted total RNA from a grapevine (cv. Mavro Nemeas) from Peloponnese. Sequencing was conducted on an Illumina NovaSeq6000 platform yielding ∼60 million 100-bp paired-end reads. After extraction of the Vitis vinifera genome, the de novo assembly of the reads using SPAdes produced 20,365 contigs. The 1,411 contigs exhibiting a size over 240 nt were screened against the nt/nr databases using BLAST. Among the identified viral sequences, the 363-nt-long genome of GYSVd-2 (LR735994) was retrieved, showing 100% nt identity to the cultivar Superior isolate from Peloponnese as well as to isolates from Croatia, Pakistan, and China (MF979530, KY978405, and FJ490172, respectively). The presence of the viroid in this sample was confirmed with the aforementioned RT-PCRs. To our knowledge, this is the first report of GYSVd-2 in Greece, which is expanding our knowledge on its worldwide distribution. Our initial data indicate that the viroid is dispersed in several regions of Greece, and it shows rather low genetic variability. Additional work is needed to elucidate its spread and impact on Greek grapevines.
Databáze: OpenAIRE
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