Bois noir (BN) is an economically important grapevine yellows disease induced by the stolbur phytoplasma and principally vectored by the cixiid Hyalesthes obsoletus. This study addresses the involvement of other planthoppers and/or leafhoppers in BN epidemics in the South Banat district of northeastern Serbia, by performing transmission experiments and multilocus typing of stolbur phytoplasma isolates to determine the vector-related characteristics of the disease. Transmission trials were conducted with adults of two cixiid congeners, Reptalus panzeri and R. quinquecostatus, which were found to harbour stolbur phytoplasma in the vineyards under study. A molecular characterization of stolbur phytoplasma isolates was performed by sequence analysis and/or RFLP typing of the two housekeeping genes tuf and secY and the two membrane proteins stamp and vmp1. Transmission trials with naturally infected R. panzeri adults from either the BN-infected vineyards or maize redness (MR)-affected maize fields revealed a high stolbur phytoplasma transmission efficiency to grapevines. In contrast, experiments conducted with stolbur-positive R. quinquecostatus originating from BN-infected vineyards, provided no evidence for a vector role of this species. Seven stolbur phytoplasma genotypes, all of which were tuf-b types, were detected among the grapevine-and insect-associated field samples according to the tuf/secY/vmp1/stamp typing. STOLg was the genotype most frequently found in naturally infected grapevine (42%), as well as R. panzeri originating from the vineyards (85%) and maize fields (98%). The same genotype was found in all experimental plants inoculated by R. panzeri, confirming its vectorship of the disease.

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.

A survey was conducted over several years in Italy and the Balkans in order to gain an understanding of the relationship between the Flavescence dorée (FD) phytoplasma isolates found in clematis and grapevine. A total of 399 clematis and 107 grapevine samples were analyzed. The results showed that 36% of the Clematis vitalba plant samples were infected by phytoplasmas which, in grapevine, are associated with FD, a quarantine disease in Europe. Infected clematis plants were also found in areas where FD phytoplasma had never previously been reported to infect grapevine, such as Macedonia, Croatia and some areas of Italy and Serbia. Molecular data from three phytoplasma genomic fragments showed the presence of different FD phytoplasma isolates, all belonging to the 16SrV-C subgroup, including the Italian FD-C isolate, the isolate found in Serbia, an isolate similar to the French FD2000 and a new isolate typical of central Italy. A few clematis plants were infected with single nucleotide polymorphism, insertion or deletion mutants of the FD-C isolate. Of all the potential Hemipteran vector species surveyed in Italy and Serbia, only 18 of 527 Dictyophara europaea individuals tested proved to be infected with the FD phytoplasma. Preliminary transmission experiments showed that this species is able to transmit the FD phytoplasma from clematis to grapevine. The presence of FD-infected clematis and of D. europaea could, therefore, constitute a risk for FD epidemics in the European viticultural regions.

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

No abstract

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.

Plant feeding insects and the plants they feed upon represent an ecological association that is thought to be a key factor for the diversification of many plant feeding insects, through differential adaptation to different plant selective pressures. While a number of studies have investigated diversification of plant feeding insects above the species level, relatively less attention has been given to patterns of diversification within species, particularly those that also require plants for oviposition and subsequent larval development. In the case of plant feeding insects that also require plant tissues for the completion of their reproductive cycle through larval development, the divergent selective pressure not only acts on adults, but on the full life history of the insect. Here we focus attention on Rhinusa antirrhini (Curculionidae), a species of weevil broadly distributed across Europe that both feeds on, and oviposits and develops within, species of the plant genus Linaria (Plantaginaceae). Using a combination of mtDNA (COII) and nuclear DNA (EF1-alpha) sequencing and copulation experiments we assess evidence for host associated genetic differentiation within R. antirrhini. We find substantial genetic variation within this species that is best explained by ecological specialisation on different host plant taxa. This genetic differentiation is most pronounced in the mtDNA marker, with patterns of genetic variation at the nuclear marker suggesting incomplete lineage sorting and/or gene flow between different host plant forms of R. antirrhini, whose origin is estimated to date to the mid-Pliocene (3.77 Mya; 2.91-4.80 Mya).