Flavescence doré e (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and-III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster

Elm yellows phytoplasmas (EY) belonging to the 16SrV-A subgroup were recently proposed as a new candidate species 'Candidatus Phytoplasma ulmi'. These pathogens infect elm trees, causing leaf yellowing and premature drying. In this study, 25 isolates originating from localities in northeast, east and southwest Serbia were characterized by means of RFLP analysis and DNA sequencing of four genomic loci: 16S rRNA, ribosomal protein rpl22-rps3, secY and map. In total, five different genotypes were identified based on collective sequencing of all four genes. Four of these genotypes showed significant nucleotide changes compared with the EY1 T reference strain. Phylogeny based on parsimony analyses of ribosomal protein, secY and map genetic loci indicated a single monophyletic origin of EY1 T and the new 'Ca. Phytoplasma ulmi' strains. Unlike phylogenetic clustering, DNA sequence comparison of EY1 T and the novel strains revealed mutations in oligonucleotide signature sequences for all three genes (16S, rpl22-rps3 and secY) used for the characterization and assignment of 16SrV-A phytoplasmas to the 'Ca. Phytoplasma ulmi' species in the original description. Based on their high degree of genetic variability, the Serbian strains were assigned to four different subtypes of 'Ca. Phytoplasma ulmi' (EY-S1, EY-S2, EY-S3 and EY-S4). New diagnostic enzymes for practical use in 'Ca. Phytoplasma ulmi' identification are proposed for the 16S rRNA, ribosomal protein and secY genes. The implications of genetic variability within signature sequences for taxonomy and identification of 'Ca. Phytoplasma' species, as well as the importance of geographic variability and number of strains characterized for species description, are discussed.

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 L(3) and L(5) nymphs. When stolbur phytoplasma-infected R. panzeri L(3) 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.

Maize redness (MR), a disease causing midrib, leaf and stalk reddening and abnormal ear development in maize, has been reported from Serbia, Romania and Bulgaria for 50 years. Recent epiphytotics reduced yields by 40%-90% in southern Banat, Serbia. MR was recently associated with the presence of the stolbur phytoplasma, although the epidemiology of the disease remained unknown. Diseased fields in southern Banat were surveyed for potential vectors of the phytoplasma during 2005 and 2006, and high populations of Reptalus panzeri were found. In affected fields, 20% of the R. panzeri individuals and 85% of symptomatic maize plants carried the stolbur phytoplasma. When stolbur phytoplasma-infected R. panzeri were introduced into insect-free mesh cages containing healthy maize plants, midrib and leaf reddening developed on 48% of plants and stolbur phytoplasma was detected in 90% of the symptomatic plants. No symptoms or phytoplasma-positive plants were found in cages without insects. These data indicate that MR symptoms are associated with the stolbur phytoplasma. Reptalus panzeri is both abundant in affected fields and can transmit the stolbur phytoplasma, indicating the insect is likely to be a major vector of MR.

Herbal products such as essential oils may play a promising role in the treatment of infections caused by gastrointestinal nematodes (GINs). The aim of this study was to evaluate the in vitro potential of 11 essential oils (EOs) and one binary combination of isolated EO compounds, as well as the in vivo anthelmintic efficacy of two EO formulations. Four GIN genera were identified in the coproculture examination: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The in vitro egg hatch test (EHT) was performed at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) for each EO, whereas in the in vivo faecal egg count reduction test (FECRT), each EO sample was diluted in sunflower oil and orally administrated at a dose of 100 mg/kg to the different group of animals. In the EHT, the EOs of Origanum vulgare, Foeniculum vulgare, Satureja montana, Satureja hortensis and two types of Thymus vulgaris were the most effective. The dominant compounds of these EOs were carvacrol, thymol, anethol, p-cymene and γ-terpinene, indicating their importance for the anthelmintic activity. In the FECRT, both T. vulgaris EO type 1 and linalool:estragole combination show an anthelmintic potential with a mean effect on FECR of approximately 25%. The results suggest the possible role of tested EOs as anthelmintic agents in sheep farms, although further in vivo tests are needed.

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In August 2008, grapevine yellows (GY)-like symptoms 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 indicated that they belong to 16SrXII-A phylogenetic group. PCR with stolbur specific primers confirmed the presence of stolbur phytoplasma in grapevine samples 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.

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