Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri ) virescence in Michigan, USA, was selected as the reference strain for the novel taxon 'Candidatus Phytoplasma asteris'. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Elm yellows group (16SrV) phytoplasmas, which are associated with devastating diseases in elm, grapevine, blackberry, cherry, peach and several other plant species in America, Europe and Asia, represent one of the most diverse phytoplasma clusters. On the basis of phylogenetic analysis of 16S rDNA sequences, elm yellows group phytoplasmas form a discrete subclade within the phytoplasma clade. Three phylogenetic parameters, namely 16S rRNA, ribosomal protein and secY genes, have been evaluated for their usefulness in differentiating elm yellows group phytoplasmas. RFLP analysis of 16S rRNA sequences differentiated the elm yellows group phytoplasmas into five subgroups. Twelve RFLP subgroups were differentiated on the basis of ribosomal protein and 13 were differentiated using secY gene sequences. Phylogenetic analysis of the ribosomal protein genes and secY gene alone or in combination indicated that the subgroups constitute 12 genetically distinct lineages, each of which appears to have evolved under different ecological constraints such as specific vector or plant hosts. On the basis of unique DNA and biological properties, it is proposed that the elm yellows phytoplasma EY1T represents a novel taxon, ‘Candidatus Phytoplasma ulmi’.

Vineyards of southern France and northernPhytoplasmas are phloem-restricted wall-less bacteria pathogenic to many plant species worldwide (37, 52). Phytoplasmas can be spread both by hemipteran insect vectors (63) and by vegetative multiplication of infected-plant material. Controlling phytoplasma-induced diseases in perennial crops depends on field surveys and implementation of prophylactic sanitary measures requiring sensitive and specific detection of phytoplasmas in plants. Genetically different phytoplasmas can infect the same plant species; therefore, precise identification and typing of phytoplasma strains are necessary to ascertain the causes and origin of new outbreaks and predict the route of disease spread.Vineyards in southern France, northern Italy, and Spain are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen of grapevine (7,8,16,24). The classification of phytoplasmas, which are uncultivable and currently described under the provisional genus "Candidatus Phytoplasma," is mainly based on 16S rRNA gene phylogeny, genomic diversity, and plant and insect host ranges (32,36,59). The FD phytoplasma belongs to the 16SrV taxonomic group (36). Members of this group share high 16S rRNA gene sequence similarity (34, 38), but the group consists of phytoplasmas with an important variety of specific biological niches restricted to woody perennial hosts. "Ca. Phytoplasma ulmi" is responsible for yellows of elm species in North America and Europe (38) and "Ca. Phytoplasma ziziphi" is the agent of jujube witches'-broom and cherry lethal yellows in Asia (34,38). In Europe, other phytoplasmas of group 16SrV are mainly infecting grapevine (23,43), alder (46,51), blackberry (26,50), Spartium, and eucalyptus (44,45). Most of the insect vectors naturally disseminating group 16SrV phytoplasmas have been identified. The elm yellows phytoplasmas are transmitted in North America by Scaphoideus luteolus (Van Duzee) (5) and in Europe by Macropsis mendax (Fieber) (15), whereas FD phytoplasmas are specifically transmitted by Scaphoideus titanus (Ball) (53, 58) and rubus stunt phytoplasma by Macropsis fuscula (Zetterstedt) (26). Phytoplasmas associated with Palatinate grapevine yellows (PGY) and alder yellows (AldY) are both transmitted by the alder leafhopper Oncopsis alni (Schrank) (41, 42) and were classified as members of the group 16SrV on the basis of their high 16S rRNA gene and secY sequence similarity to the corresponding genes of FD phytoplasmas (2, 3).The genomic diversity in this phytoplasma group was recently examined. Sequence and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA genes and the 16S-23S intergenic spacer allowed differentiation of two differ-* Corresponding author. Mailing

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

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