Multiple gene analyses identify distinct “bois noir” phytoplasma genotypes in the Republic of Macedonia

Abstract

“Bois noir” (BN) is a grapevine yellows disease, associated with phytoplasma strains related to ‘Candidatus Phytoplasma solani’, that causes severe losses to viticulture in the Euro-Mediterranean basin. Due to the complex ecological cycle of its etiological agent, BN epidemiology is only partially known, and no effective control strategies have been developed. Numerous studies have focused on molecular characterization of BN phytoplasma strains, to identify molecular markers useful to accurately describe their genetic diversity, geographic distribution and host range. In the present study, a multiple gene analysess were carried out on 16S rRNA, tuf, vmp1, and stamp genes to study the genetic variability among 18 BN phytoplasma strains detected in diverse regions of the Republic of Macedonia. Restriction fragment length polymorphism (RFLP) assays showed the presence of one 16S rRNA (16SrXII-A), two tuf (tuf-type a, tuf-type b), five vmp1 (V2-TA, V3, V4, V14, V18), and three stamp (S1, S2, S3) gene patterns among the examined strains. Based on the collective RFLP patterns, seven genotypes (Mac1 to Mac7) were described as evidence for genetic heterogeneity, and highlighting their prevalence and distribution in the investigated regions. Phylogenetic analyses on vmp1 and stamp genes underlined the affiliation of Macedonian BN phytoplasma strains to clusters associated with distinct ecologies.