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

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.Phytoplasmas are responsible for plant diseases that damage annual crops as well as perennial cultures such as fruit trees and grapevine (24). These pathogens multiply within the phloem cells of the host plant and are transmitted from plant to plant by phloem-feeding insects (51). As of today, the many diseases induced by phytoplasmas cannot be cured, and the control of disease spread consists of implementing prophylactic measures, such as quarantine, destruction of infected plant material, and pesticide treatment against the insect vectors. Implementing control of phytoplasma-induced diseases requires the taxonomic characterization of the agent, the determination of its plant host range, and the identification of its insect vector(s) (25). All of these studies necessitate the development of methods for diagnosis that rely on the molecular detection of phytoplasma DNA (8,19). Most phytoplasmas have been classified according to 16S rRNA gene phylogeny and restriction fragment length polymorphism profiles into 14 to 20 groups (24, 49), and 20 "Candidatus Phytoplasma" species have been described (16, 46). Differentiation of phytoplasmas occupying distinct biological niches but displaying less than 3% divergence in 16S rRNA genes must be achieved by comparing genetic loci other than 16S rRNA genes (27). Whereas important progress has been made regarding phytoplasma classification and ecology, phytoplasma phytopathogenicity and mechanisms of phytoplasma transmission by insects are still poorly understood and will benefit from the knowledge and the comparative analysis of phytoplasma genomes.However, isolating phytoplasma DNA is still hampered by the...

The Flavescence doré e phytoplasma (FD-P), a non-cultivable, plant-pathogenic bacterium of the class Mollicutes, is the causal agent of a quarantine disease affecting vineyards of southern Europe, mainly in southern France and northern Italy. To investigate FD-P diversity and phytoplasma genetic determinants governing the FD-P life cycle, a genome project has been initiated. A physical map of the chromosome of FD-P strain FD92, purified from infected broad beans, was constructed by performing restriction digests of the chromosome and resolving the fragments by PFGE. Single and double digestions of the chromosome with the enzymes SalI, BssHII, MluI and EagI were performed and used to map 13 restriction sites on the FD-P chromosome. The size of the chromosome was calculated to be 671 kbp. Southern blot analyses using cloned phytoplasma probes were carried out to assist in the arrangement of contiguous restriction fragments and to map eight genetic loci, including the two rRNA operons, the tuf, uvrB-degV and secY-map (FD9) genes, the FD2 marker and two orphan sequences (FDDH29 and FDSH05) isolated through subtractive suppression hybridization.