The molecular epidemiology of bois noir grapevine yellows caused by 'Candidatus Phytoplasma solani' in the Republic of Macedonia

Bois noir (BN), which is induced by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), is an important grapevine yellows disease that causes severe damage in viticultural regions throughout the Euro-Mediterranean basin. An epidemiological survey to determine potential insect vectors and the primary reservoir plants of BN phytoplasma in Macedonian vineyards was undertaken between 2012 and 2013 in the southeastern part of the country. A study on the species diversity from the suborder Auchenorrhycha revealed the prevalence of the principal vector of ‘Ca. P. solani’, which is the planthopper Hyalesthes obsoletus. Reptalus panzeri, which is the second-most documented BN vector, was not recorded in Macedonian vineyards. Three leafhopper species, namely Psammotettix alienus, Artianus manderstjernii and Euscelis incisus, were also widespread in the BN-affected vineyards, but only H. obsoletus tested positive for ‘Ca. P. solani’. Molecular characterizations were performed by the sequencing and/or RFLP typing of tuf, vmp1 and stamp genes, and the results were used to gain detailed insight into the molecular diversity of the ‘Ca. P. solani’ strains associated with grapevines, tentative reservoir plants (Urtica dioica and Convolvulus arvensis) and the H. obsoletus associated with these plants. Among the 91 ‘Ca. P. solani’ strains detected in diverse plant and insect hosts, three tuf, five vmp1 and 11 distinct stamp genotypes were identified. Twelve comprehensive genotypes of ‘Ca. P. solani’ were detected according to the tuf/vmp1/stamp genotyping. The highest diversity of genotypes was detected among the strains from H. obsoletus individuals associated with U. dioica, of which the most frequent genotype was tuf-ab/V18/M1 (43 %). The tuf-b/V2-TA/STOL comprehensive genotype was found in 33 % of naturally infected grapevines. Two ‘Ca. P. solani’ genotypes were associated with U. dioica, namely (i) tuf-ab/V18/M1 (60 %) and tuf-a/V3/M4 (40 %), and only one genotype (tuf-b/V2-TA/Rqg50) was associated with C. arvensis.