Detection and molecular characterization of phytoplasmas based on 16s rDNA gene region by phylogenetic and in silico RFLP analysis of local grapevine cultivars in Şanlıurfa and Adıyaman

Abstract

During the autumn seasons of 2016-2019, several surveys were carried out in grapevine (Vitis vinifera L.) cultivation areas in Şanlıurfa and Adıyaman provinces of South-eastern part of Turkey, to identify grapevine plants exhibiting phytoplasma-like symptoms including yellowing, small leaf formation, chlorosis, short internodes, severe redness and inward curling and to detect and characterize the causal agent responsible for these symptoms. Purified DNA obtained from collected samples was examined for the highly conserved phytoplasma 16S rDNA gene via nested-PCR with the universal phytoplasma-specific primer sets. DNA amplification via nested-PCR/RFLP analyses with some restriction enzymes confirmed the suspected correlations between the disease symptoms observed and phytoplasma presence in the samples. F2n/R2 amplicons of the phytoplasma strains obtained from the samples showed 99.99% homology with each other and 99% homology with phytoplasma DNA partial sequences belonging to some groups deposited in the GeneBank database. Sequencing and phylogenetic analysis revealed that collected isolates have 99% sequence identity with ‘Candidatus phytoplasma solani’ (16SrXII-A) and ‘Ca.P. asteris’ (16SrI-B). According to our knowledge, the results of this study is the first report of the phylogenetic relationship of phytoplasmas infecting different grapevine cultivars based on 16S rDNA gene. Genetic diversity of genes other than 16S rDNA of the pathogen causing these infections is under study.