First Report of Alder Yellows Phytoplasma Associated with Common Alder (Alnus glutinosa) in the Republic of Macedonia

Abstract

Alder yellows phytoplasma (AldYp) is classified as a member of the 16SrV-group of phytoplasmas and is closely related to Flavescence dorée (FD), a quarantined pathogen of economic importance affecting vineyards across Europe. AldYp is associated with common (Alnus glutinosa) and grey alder (A. incana), and has been reported in France, Italy, Germany, Austria, Switzerland, the Baltic region, Serbia, and Montenegro (1,2,4). For Macedonian vineyards, so far, neither infection of grapevine with 16SrV-group of phytoplasmas nor the presence of the main FD phytoplasma vector, Scaphoideus titanus, has been recorded. However, the presence of FD-related phytoplasma was detected in wild Clematis vitalba. In September and October 2013, leaves with petioles from A. glutinosa exhibiting leaf discoloration and yellowing were collected from two sites (41°23′43″ N, 22°54′ E and 41°23′ N, 22°53′ E) in southeast Macedonia near the village of Smolare (Strumica district). Eight samples were collected from each site. Leaves of six asymptomatic alder seedlings collected from the same sites served as a control. Nucleic acids were extracted from fresh leaf midribs and petioles using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Initial phytoplasma identification was carried out by nested PCR assay of the 16S rRNA gene, using universal primers P1/P7 and R16F2n/R16R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania), as previously reported (4). Characterization of detected phytoplasmas was performed by amplifying two genetic loci specific for the members of the 16SrV group phytoplasmas; the ribosomal protein gene operon (rp) using primers rp(V)F1/rpR1 and rp(V)F1A/rp(V)R1A (3), and the non-ribosomal metionine aminopeptidase (map) gene using primer set FD9f5/MAPr1 and FD9f6/MAPr2 (1). The PCR amplicons were sequenced and deposited in NCBI GenBank database under the accession numbers KJ605448 to 52 (map) and KJ605453 to 57 (rp). The obtained sequences were compared with reference sequences of the 16SrV-group phytoplasmas (1,3) using the neighbor-joining method in MEGA5 (5). The presence of phytoplasma was detected in 14 of 16 symptomatic alder samples, while all control plants tested negative. The MseI restriction profiles were identical among all 14 samples and with the reference strains of the 16SrV group phytoplasmas (EY1 - 16SrV-A, FD-C - 16SrV-C, and FD-D - 16SrV-D). The rp-based phylogeny enabled identification of four diverse phytoplasma strains among the AldYp strains from Macedonia. Three strains clustered within the rpV-E subgroup while one belonged to rpV-L subgroup. Phylogenetic analysis of the more variable genetic locus, map, showed the presence of five diverse phytoplasma strains. Four strains belonged to the map-FD2 (FD-D, FD92) cluster, while one grouped within the map-FD1 (FD70) cluster. To our knowledge, this is the first report of 16SrV phytoplasma group occurrence on alder in Macedonia. The significant similarity between AldYp strains and FD sensu stricto indicate the risk of pathogen exchange between the wild ecosystem and the grapevine (1). Alder trees naturally infected with the FDp-related strains could therefore represent a serious risk for FD outbreak in Macedonian vineyards if local S. titanus populations developed. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrković et al. Plant Pathol. 57:773, 2008. (3) M. Martini et al. Int. J. Syst. Evol. Microbiol. 57:2037, 2007. (4) S. Radonjić et al. Plant Dis. 97:686, 2013. (5) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.