First Report of Grapevine Yellow Speckle Viroids and Hop Stunt Viroid in <i>Vitis vinifera</i> in Kazakhstan

Grapevine viroids are the smallest pathogens affecting viticulture. Infected grapevines may show symptoms such as leaf yellowing, mottling, stunted growth, reduced fruit quality, and overall vine decline. Asymptomatic viroid infections in grapevines can still decrease grapevine productivity, thereby posing a hidden economic threat to viticulture (Wolpert et al., 1996; Wu et al., 2023). This study aimed to detect three widely distributed viroid species of the Pospiviroidae family: Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), and Hop stunt viroid (HSVd). The study focused on grapevine (Vitis vinifera) cultivars 'Saperavi' and 'Rkatsiteli' cultivated in Kazakhstan. Three fields in Shirin village and one field in Tyulkubas village (each about 600 ha) in Almaty region were tested for the presence of viroids due to a decline in yield observed despite the negative tests for common viruses (past 3 years). Symptoms typical for viral infections were observed sporadically without a consistent pattern of infection. Total 17 samples from Shirin (July 2023) and 14 from Tyulkubas (June 2024) with visible symptoms were collected randomly. Validated primers were used to identify GYSVd-1 (mF/mR1, 250 bp) (Hajizadeh et al. 2012) GYSVd-2 (P1/P2, 375 bp) (Jiang et al. 2009) and HSVd (78P/83M, 312 bp) (Sano et al. 2001) using conventional RT-PCR. Among the analyzed samples, in Shirin village, 6 samples tested positive for GYSVd-1, 15 for GYSVd-2, and 15 for HSVd, including 2 positive for both HSVd and GYSVd-1. In Tyulkubas village, 2 samples were positive for GYSVd-1 and 12 for HSVd; GYSVd-1 positive samples als were positive for HSVd, including 8 positive for both HSVd and GYSVd-2 and 6 positive for three viroids. Amplicons for each viroid were further sequenced using the Sanger's method on ABI 3500 Genetic Analyser with BigDye v.3.1 kit and using MinION sequencer with the Rapid Barcoding Kit 96 V14 (Oxford Nanopore Technologies) according to the manufacturer's protocol. The obtained reads were mapped to reference sequences from GenBank-GYSVd-1 NC_001920.1, HSVd NC_001351.1, and GYSVd-2 MG780425.1-using Minimap v.2.2.0 with default parameters in Geneious software. Sequencing produced total 2253 reads for GYSVd-1 (average length 159 bp, range 42-296), 2095 for GYSVd-2 (203.6 bp, 49-578), and 1235 for HSVd (218.5 bp, 53-440). Of these, 947 (42.0%) were been mapped to GYSVd-1, 1778 (84.9%) to GYSVd-2, and 678 (54.9%) to HSVd. Genome coverage was 62.3% (GYSVd-1), 98.1% (GYSVd-2), and 73.8% (HSVd), with mean depths of 889X, 1282X, and 659X, respectively. Each viroid resulted a single contig corresponding to the target amplicon. No reads corresponding to other viral or bacterial agents have been identified. NCBI BLASTn search resulted total 141 hits with coverage 59-68% and identity 98.69-99.35% for GYSVd-1; 128 hits with coverage 82-100% and identity 97.59-100% for GYSVd-2; 157 hits with coverage 99-100% and identity 95.49-97.29%; no not-specific matches were identified for three viroids. The assembled viroid sequences obtained using nanopore sequencing have been uploaded into NCBI database (accessions PV341024-PV341026). Viroids GYSVd-1,2 and HSVd have been detected for the first time in Kazakhstan. The results indicate the need for further studies to estimate impact of these viroids on viticulture and lay a basis for their monitoring in the country.