Multilocus Genotyping Reveals New Molecular Markers for Differentiating Distinct Genetic Lineages among " Phytoplasma Solani" Strains Associated with Grapevine Bois Noir.

Grapevine Bois noir (BN) is associated with infection by " Phytoplasma solani" (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of -type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring , -, --, and --, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene , which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu ( gene) and hemolysin III-like protein ( gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of -type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.