Witches'-broom disease of small-fruited acid lime (WBDL) is a severe disease caused by a mycoplasmalike organism (MLO) in the Sultanate of Oman and the United Arab Emirates. The WBDL MLO was characterized by studying its genome size, the sequences of its 16s ribosomal DNA and the 16s-23s ribosomal DNA spacer region, and hybridization profiles obtained by using WBDL MLO-specific probes. The size of the WBDL MLO genome is 720 kbp. Genomic similarities with the MLOs of sunhemp, sesame, and alfalfa phyllodies were demonstrated, and we found that the WBDL MLO belongs to the sunhemp phyllody phylogenetic subgroup.Witches'-broom disease of small-fruited acid lime [Citrus aurantifolia (L.) Swingle] (WBDL) is a lethal disease which is caused by a mycoplasmalike organism (MLO) and appeared in the Sultanate of Oman in the late 1970s (2, 3, 7) and in the neighboring country the United Arab Emirates in 1989 (4, 7). The WBDL MLO was experimentally transmitted by dodder (Cuscuta campestris Yunker) to periwinkle (Catharantus roseus L.) plants, in which it induces characteristic symptoms different from those reported with other MLOs (7). The WBDL MLO is thought to be naturally spread by the leafhopper Hishimonus phycitis, as many WBDL MLO-infected H. phycitis individuals were collected from lime trees with WBDL in Oman (4). However, experimental transmission of the disease with H. phycitis has not been achieved yet. Widely spread in India, I-€. phycitis was not reported from the Arabic Peninsula (5) before we found it there (4). As lime trees have been grown in Oman for centuries, it is likely that the disease started with the introduction of this new insect vector, probably from India, where it is known to transmit the MLO of eggplant little-leaf disease (1). The MLO agent, however, was probably indigenous to the Sultanate of Oman since the disease has never been described in India or elsewhere.In 1991, monoclonal antibodies and DNA probes specific for the WBDL MLO were produced (7). No serological relationships with any of the other MLOs tested, including the eggplant little-leaf disease MLO from India, were detected, but in this paper we show that DNA extracted from phyllody MLOinfected sunhemp, sesame, and alfalfa plants hybridized with the DNA probes which we tested.In order to determine the phylogenetic position of the WBDL MLO among the mollicutes, we cloned and sequenced its 16s rRNA gene. Indeed, even though MLOs cannot be cultured, their phylogenic positions have been determined from the sequences of their 16s ribosomal DNAs (rDNAs) (10,11,15,19). These organisms have been shown to be true mollicutes, and the name "phytoplasma" has been proposed for them (20). In addition, the sequence of the 16s-23s rDNA spacer region and the genome sue of the WBDL MLO were determined. MATERIALS AND METHODSPlant material. Healthy periwinkle and lime plants were obtained from seeds and were maintained at 25°C during the day and at 20°C at night.Lime and periwinkle plants were infected with the WBDL MLO by graft transmission and were g...

Background: Spiroplama citri, the causal agent of citrus stubborn disease, is a bacterium of the class Mollicutes and is transmitted by phloem-feeding leafhopper vectors. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of S. citri strain GII3-3X is currently being deciphered.

The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the Spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS30, IS481, IS3, and ISNCY) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins. S. citri has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that S. citri possesses a nearly complete pathway for the biosynthesis of a terpenoid.

Significant changes have been made in the systematics of the genus Spiroplasma (class Mollicutes) since it was expanded by revision in 1987 to include 23 groups and eight sub-groups. Since that time, two additional spiroplasmas have been assigned group numbers and species names. More recently, specific epithets have been assigned to nine previously designated groups and three sub-groups. Also, taxonomic descriptions and species names have been published for six previously ungrouped spiroplasmas. These six new organisms are : Spiroplasma alleghenense (strain PLHS-13 (group XXVI), Spiroplasma lineolae (strain TALS-23 (group XXVII), Spiroplasma platyhelix (strain PALS-13 (group XXVlll), Spiroplasma montanense (strain HYOS-13 (group XXXI), Spiroplasma helicoides (strain TABS-23 (group XXXII) and Spiroplasma tabanidicola (strain TAUS-13 (group XXXIII). Also, group XVII, which became vacant when strain DF-IT (Spiroplasma chrysopicola) was transferred to group VIII, has been filled with strain Tab 4c. The discovery of these strains reflects continuing primary search in insect reservoirs, particularly horse flies and deer flies (Diptera :Tabanidae). In the current revision, new group designations for 10 spiroplasma strains, including six recently named organisms, are proposed. Three unnamed but newly grouped spiroplasmas are strain TIUS-I (group XXIX; ATCC 51751) from a typhiid wasp (Hymenoptera : Tiphiidae), strain BIUS-1 (group XXX; ATCC 51750) from floral surfaces of the tickseed sunflower (Bidens sp.) and strain BARC 1901 (group XXXIV; ATCC 700283). Strain BARC 2649 (ATCC 700284) from Tabanus lineola has been proposed as a new sub-group of group VIII. Strains TIUS-1 and BIUS-1 have unusual morphologies, appearing as helices a t only certain stages in culture. In this revision, potentially important intergroup serological relationships observed between strain DW-1 (group II) from a neotropical Drosophila species and certain sub-group representatives of group I spiroplasmas are also reported.

2CRA -Centro di ricerca per la viticoltura, 26 viale XXVIII Aprile 31015, Conegliano (TV), ItalyIn addition to the grapevine flavescence doré e phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were .97.5 %, the proposed threshold to distinguish two 'Candidatus Phytoplasma' taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence doré e (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: 'Candidatus Phytoplasma rubi'.Phytoplasmas are plant-pathogenic bacteria belonging to the class Mollicutes, a group of cell wall-less microorganisms phylogenetically related to Gram-positive bacteria with a low DNA G+C content (Weisburg et al., 1989). Phytoplasmas are still unavailable in culture. They are responsible for hundreds of crop diseases worldwide (Lee et al., 2000) and are transmitted from plant to plant by sap-sucking hemipteran insects (Weintraub & Beanland, 2006). Flavescence dorée (FD) of grapevine, a quarantine disease first reported in the 1950s in south-western France (Caudwell, 1957) is now present in southern Europe (Batlle et al., 1997;Belli et al., 1985;Boudon-Padieu, 2002;Duduk et al., 2003). The causal agent of FD was shown to be a phytoplasma (Caudwell et al., 1971) that is transmitted by a grapevine leafhopper of north American origin, Scaphoideus titanus (Ball.) (Schvester et al., 1961). The FD phytoplasma belongs to the 16SrV taxonomic group (Lee et al., 1998). Members of this group share high 16S rRNA gene sequence similarity (Davis & Dally, 2001), but the group consists of phytoplasmas with an important variety of biological niches restricted to woody perennial hosts. 'Candidatus P. ulmi' in the 16SrV-A subgroup is responsible for yellows of elm species in North America and Europe (Lee et al., 2004) and 'Candidatus P. ziziphi' in the 16SrV-B subgroup is the agent of jujube witches'-broom and cherry lethal yellows in Asia (Jung et al., 2003;Lee et al., 2004). In Europe, other phytoplasmas in the 16SrV group mainly infect grapevines (Maixner et al., 1994), alder (Lederer & Seemüller, 1991; Mäurer et al., 1993), blackberry (de Fluiter & van der Meer, 1953; Mäurer & Seemüller, 1995), species of the genu...