Department of Plant Pests, Institute for Plant Protection and Environment, Zemun, Serbia Occurrence and Distribution of Grapevine Yellows Caused by Stolbur Phytoplasma in Montenegro Sanja anja Radonjic adonjic ´ 1 , Snjezana njez ˇana Hrncic rnc ˇic ´ 1 , Jelena elena Jovic ovic ´ 2 , Tatjana atjana Cvrkovic vrkovic ´ 2 , Oliver liver Krstic rstic ´ 3 , Slobodan lobodan Krnjajic rnjajic ´ 2 and and Ivo vo Tosevski os ˇevski 4 AuthorsÕ addresses: 1 Biotechnical Faculty, Centre for Plant Protection, University of Montenegro, Mihaila Lalic´a 1, 81000 Podgorica, Montenegro; 2 Department of Plant Pests, Institute for Plant Protection and Environment, Banatska 33, 11080 Zemun, Serbia; 3 Jurija Gagarina 184, Belgrade, Serbia; 4 CABI Europe – Switzerland, 1 Rue des Grillons, 2800 Dele´mont, Switzerland (correspondence to Ivo Tosˇevski. E-mail: tosevski_ivo@yahoo.com) Received December 9, 2008; accepted December 23, 2008 Keywords: Bois noir, grapevine yellows, Montenegro, PCR, RFLP, stolbur phytoplasma Abstract In August 2008, grapevine yellows (GY)-like symp- toms were observed in vineyards near Podgorica, Montenegro. A survey of the grape growing area of Montenegro was conducted to identify and determine the distribution of phytoplasmas associated with grapevine. Phytoplasmas were detected in symptomatic vine plants using PCR with universal primers for amplification of the phytoplasma 16S rRNA gene. From the 42 plants sampled, 36 were positive for the presence of phytoplasmas. RFLP profiles of the 16S rDNA of all phytoplasma positive isolates tested indi- cated that they belong to 16SrXII-A phylogenetic group. PCR with stolbur specific primers confirmed the presence of stolbur phytoplasma in grapevine sam- ples from Montenegro. Sequence and RFLP analysis of the tuf gene indicated the presence of the tufAY-a and tufAY-b types of stolbur phytoplasma. To our knowledge, this is the first report of phytoplasma infection of grapevine in Montenegro. Introduction Grapevine yellows (GY) are widespread diseases caused by several different phytoplasmas that are indistinguishable on the basis of symptoms. Identifica- tion of specific phytoplasmas associated with GY is possible using molecular tools, such as PCR, RFLP and⁄or sequencing. In Europe, the most widespread phytoplasmas affecting grapevine are Bois noir (BN) and Flavescence dore´e (FD). FD phytoplasmas, which belong to the 16SrV-C and 16SrV-D subgroups and are transmitted by the Nearctic leafhopper Scaphoideus titanus Ball, are causing severe and rapidly spreading disease of grapevine. FD phytoplasma has been reported from several European countries, including France, Italy, Spain, Serbia, Switzerland and Slovenia (Caudwell, 1957; Daire et al., 1993, Batlle et al., 1997; Duduk et al., 2003; Gugerli et al., 2006; Seljak and Oresˇek, 2007), where it causes serious damage and has a negative effect on grapevine production. The FD phytoplasma is a quarantine pathogen in Montenegro and in the European Community. Bois noir is caused by stolbur phytoplasma which belongs to 16SrXII-A phylogenetic group; it has a wider geographic distribu- tion than FD and is present in surrounding countries to Montenegro: Greece, Croatia, Macedonia, Serbia, Bosnia and Herzegovina, Bulgaria (Davis et al., 1997; S ˇ koric´ et al., 1998; S ˇ eruga et al., 2003; Duduk et al., 2004; Delic´ et al., 2007; Avramov et al., 2008). Montenegro has a long tradition in grape growing, vine production. Wine production has significant domestic economic importance and is an important export for Montenegro. Grapes are grown in the Pod- goricˇko and Primorsko region; however, more than 75% Montenegran vineyards are located near Podgorica in Podgoricˇko region. These cover approximately 4100 hectares of vineyards. At the beginning of August 2008, symptoms of GY were observed on grapevines of the red wine cultivar Vranac in three vineyards near Podgorica. Symptoms were typical of phytoplasma infection, including leaf rolling and discoloration, lack of lignification and drooping canes. No phytoplasma diseases have previ- ously been reported from grapevine in this region of Balkans. To determine if grapevine diseases caused by phytoplasmas are present in Montenegro, a survey was conducted in both grape growing regions. Overall, nine vineyards that showed suspected phytoplasma symp- toms were inspected. Materials and Methods During August and September 2008, a grape growing area of Montenegro was surveyed for the presence of GY diseases. Samples of vines with symptoms of leaf J Phytopathol 157:682–685 (2009) doi: 10.1111/j.1439-0434.2009.01560.x Ó 2009 Blackwell Verlag GmbH