Incidence and molecular characterization of a 16SrI-B phytoplasma strain associated with Vitis vinifera leaf yellowing and reddening in the west of Iran

Abstract

Leaf yellowing and reddening symptoms on Vitis vinifera vines were observed in different grapevine nurseries in the province of Kurdistan in the west of Iran during 2016–2017. Total DNA was extracted from 27 symptomatic and four symptomless plants and tested for the presence of phytoplasma DNA by nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 primer pairs. PCR amplicons of about 1.8 and 1.2 kb in size were obtained from all symptomatic V. vinifera but not from symptomless plants. Restriction fragment length polymorphism (RFLP) profiles of R16F2n/R2 amplicons using AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII and Sau3AI restriction enzymes were identical to each other and to those obtained from 16SrI phytoplasmas, indicating that the phytoplasmas associated with grapevine yellowing and reddening were members of the 16SrI group. A R16F2n/R16R2 16S rDNA sequence of Kurdistan V. vinifera yellowing and reddening strain (KVvY) was submitted to GenBank as accession number MH638316. The Blast analysis of KVvY showed 100% sequence identity with those of the ‘Candidatus Phytoplasma asteris’-related strains. Phylogenetic analysis confirmed that the phytoplasma associated with KVvY clustered within the 16SrI phytoplasma clade closer to the Onion yellows mild strain (OY-M), a 16SrI-B phytoplasma. Restriction analysis using iPhyClassifier confirmed that virtual RFLP patterns of the KVvY were identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession AP006628).