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Maize redness (MR), induced by stolbur phytoplasma ('Candidatus Phytoplasma solani', subgroup 16SrXII-A), is characterized by midrib, leaf, and stalk reddening and abnormal ear development. MR has been reported from Serbia, Romania, and Bulgaria for 50 years, and recent epiphytotics reduced yields by 40 to 90% in South Banat District, Serbia. Potential vectors including leafhoppers and planthoppers in the order Hemiptera, suborder Auchenorrhyncha, were surveyed in MR-affected and low-MR-incidence fields, and 33 different species were identified. Only Reptalus panzeri populations displayed characteristics of a major MR vector. More R. panzeri individuals were present in MR-affected versus low-MR fields, higher populations were observed in maize plots than in field border areas, and peak population levels preceded the appearance of MR in late July. Stolbur phytoplasma was detected in 17% of R. panzeri adults using nested polymerase chain reaction but not in any other insects tested. Higher populations of R. panzeri nymphs were found on maize, Johnsongrass (Sorghum halepense), and wheat (Triticum aestivum) roots. Stolbur phytoplasma was detected in roots of these three plant species, as well as in R. panzeri L3 and L5 nymphs. When stolbur phytoplasma-infected R. panzeri L3 nymphs were introduced into insect-free mesh cages containing healthy maize and wheat plants, 89 and 7%, respectively, became infected. These results suggest that the MR disease cycle in South Banat involves mid-July transmission of stolbur phytoplasma to maize by infected adult R. panzeri. The adult R. panzeri lay eggs on infected maize roots, and nymphs living on these roots acquire the phytoplasma from infected maize. The nymphs overwinter on the roots of wheat planted into maize fields in the autumn, allowing emergence of phytoplasma-infected vectors the following July. [ABSTRACT FROM AUTHOR]

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.

Alder yellows phytoplasmas (AldYp) of the 16SrV-group associated with common alder (Alnus glutinosa) and grey alder (A. incana) are closely related to the grapevine yellows (GY)-associated quarantine phytoplasma Flavescence dorée (FDp). AldYp have been reported in several countries where epidemic appearance of FDp has been confirmed (France, Italy, and Serbia) (1,2). To date, the presence of 16SrV-group of phytoplasmas has not been reported in Montenegro; however, the main vector of FD phytoplasma, Scaphoideus titanus, has been identified in Montenegrin vineyards since 2008. During a survey in September 2011, in the northern part of Montenegro, 12 symptomatic alder trees showing symptoms of leaf discoloration, ranging from yellow to light green, were sampled. Six samples, each comprising several symptomatic leaves, were collected from A. glutinosa at streamside in woodlands near the town Kolašin and other six samples from A. incana close to the river Lim near the town of Bijelo Polje. Leaves of six young A. glutinosa seedlings were used as controls. Total DNA was extracted from fresh leaf midribs and petioles using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Nested PCR assay was conducted on 16S rRNA gene using phytoplasma generic primers P1/P7 and F2n/R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania) (3). Confirmation of identification and characterization of phytoplasma positive samples was performed by amplifying the non-ribosomal metionine aminopeptidase (map) gene using FD9f5/MAPr1 and FD9f6/MAPr2 primer set (1), specific for the members of the 16SrV group phytoplasmas. Amplification products were sequenced and deposited in GenBank (KC188998 through 9001). Comparison of the map gene sequences was performed by phylogenetic analysis along with 20 reference sequences of the 16SrV-group members (1), using the neighbor-joining method in MEGA5 software (4). 16S rRNA gene amplification revealed the presence of phytoplasmas in 11 out of 12 symptomatic samples, while Mse I restriction analysis and comparison with reference strains (AldYp and FDp from Serbia) enabled affiliation of detected phytoplasmas to the 16SrV-group. None of the controls were positive for any phytoplasma. Phylogenetic analysis of the Montenegrin AldYp map gene sequences revealed presence of four different strains clustering within the previously defined clusters of the 16SrV-group members (1). Three different strains associated with symptomatic A. glutinosa were identified and they clustered either within the FD1, FD2, or PGY-C cluster, while a single detected strain from A. incana proved to be identical with PGY-A isolate of AldY phytoplasma infecting grapevine in Germany (AM384892). To our knowledge, this is the first report of the association of 16SrV-group phytoplasmas with common and grey alder in Montenegro, as well as the first report of FD-related phytoplasmas in Montenegro. Since alder trees are considered as a possible natural reservoir of the FD phytoplasmas (1), the finding of alders naturally infected with strains related to the FDp (FD1 and FD2 clusters) indicate a possible threat of economic importance to the grape production in Montenegro, which should be addressed in further research. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrkovic et al. Plant Pathol. 57:773, 2008. (3) I-M. Lee et al. Int. J. Syst. Evol. Bacteriol. 48:1153, 1998. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.