Andean and Ordinary Strains of Potato Virus S Infecting Potatoes in Southern Kazakhstan

Abstract

Southern Kazakhstan comprises Turkistan, Zhambyl, and Almaty administrative regions; it is the most densely populated territory of the country and one of the biggest potato (Solanum tuberosum L.) producing areas. Around 1.17 million metric tons, 33% of the whole country’s production, are produced here annually. Potato virus S belongs to the genus Carlavirus (Betaflexiviridae); it has a single-stranded positive-sense RNA genome (Mackenzie et al. 1989). Based on biological and molecular properties, two strains of potato virus S (PVS) can be differentiated, ordinary (PVSᴼ) and Andean (PVSᴬ) (Dolby and Jones 1987). PVS was reported in the Eastern region (Khassanov and Vologin 2018); however, there is still no information about strain composition for PVS in southern Kazakhstan. One hundred eighty potato tubers of 47 cultivars grown in the commercial fields of Almaty region were collected randomly in September 2015 and 2016. The tubers were kept in the dark until sprouting and then planted in pots in the greenhouse. Four to 6 weeks after emergence, leaves of the grown-out plants were analyzed by double-antibody sandwich ELISA using commercially available polyclonal antibody (BIOREBA AG, Switzerland). Total RNA was extracted from leaves of all 67 PVS-ELISA positive plants using TRIzol (Sigma-Aldrich, U.S.A.), and cDNA was prepared by reverse transcription (RT) using oligo(dT)₁₈. For PCR amplifications, two pairs of CP-specific primers were used: (i) PVS-S, 5′-TGGCGAACACCGAGCAAATG-3′, and PVS-AS, 5′-ACTGCGCCTGTTGGGAACTCCACAG-3′; and (ii) CP634Fw, 5′-TTTCAATGGAATGCACGCTT-3′, and CP-Rv, 5′-TCATTGGTTTGCTGCATTCC-3′. These primers were designed according to the available genome sequences of isolates Yunnan NN (GenBank accession no. KC430335) and RL5 (GenBank accession no. JX683388) representing, respectively, the strains PVSᴼ and PVSᴬ (Vallejo et al. 2016). The resulting PCR products of the expected sizes, specific to PVSᴬ (252 base pair, bp) and PVSᴼ (169 bp), were detected in 20 and 44 PVS-ELISA-positive samples, respectively. In three samples, both strains were detected. Then the full-length CP gene sequences were amplified from 12 PVSᴼ- and four PVSᴬ-positive samples selected randomly. The resulting PCR products of the expected sizes (890 bp) were sequenced directly using conventional Sanger methodology. BLASTn analysis for all samples revealed 96 to 100% nucleotide identity with CP sequences of several known PVS isolates. Pairwise identity and phylogenetic analyses using MegaX confirmed that the 12 isolates belonged to the PVSᴼ and the other four isolates to the PVSᴬ lineage. The full genomes of two isolates from the potato cultivars Fortune (PVSᴼ) and Ushkonyr (PVSᴬ) were then sequenced using the approach described above with overlapping RT-PCR amplicons obtained with specific primers, and the complete nucleotide sequences were deposited in NCBI GenBank database under accession numbers MK442089 and MN095414, respectively. The nucleotide sequence for the Fortune isolate was found to be 96% identical to Qld1 isolate (MF375506, Australia), and the Ushkonyr was found to be 99% identical to BY isolate (MF033144, China). Nucleotide identity between the sequences of the Fortune and Ushkonyr isolates was only 79%. The data indicate that both PVSᴼ and PVSᴬ strains occur in southern Kazakhstan, and regulatory agencies need to monitor the movement and prevalence of this virus to minimize disease losses. In the Eastern region, PVSᴬ seems to dominate seed potato fields (Khassanov and Vologin 2018), and data generated in this work suggest that PVSᴼ is more prevalent in southern Kazakhstan.