Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Using the PCR, we amplified the 16s ribosomal DNAs (rDNAs) of an Asian strain and an African strain of the uncultured, gram-negative, walled, phloem-limited bacterium-like organism (BLO) associated with citrus greening disease. We evaded coamplification of chloroplast 16s rDNA by using restriction enzymes; the chloroplast 16s rDNA was sensitive to BclI digestion and resistant to EcoRI digestion, while the 16s rDNA of the BLO was resistant to BclI digestion and sensitive to EcoRI digestion. The 16s rDNA of the African BLO strain was amplified from BclI-digested DNA extracted from infected periwinkle leaf midribs. The Asian strain was isolated from plant extract by using a specific monoclonal antibody coated onto the surface of a PCR tube.The 16s rDNAs of the two BLO strains were cloned and sequenced. Comparisons with sequences of 16s rDNAs obtained from the GenBank data base revealed that the two citrus greening disease BLOs belong to the alpha subdivision of the class Proteobucteriu. Even though their closest relatives are members of the alpha-2 subgroup, these BLOs are distinct from this subgroup as we observed only 87.5% homology between the 16s rDNAs examined. Therefore, the two BLOs which we studied probably are members of a new lineage in the a subdivision of the Proteobucteriu. We propose the trivial name "Iiberobacter" for this new group of bacteria and will wait until additional characteristics have been determined before we propose a formal name.

No abstract

A xylem-limited bacterium resemblingXylella fastidiosa has been shown previously by electron mmcroscopy to be associated with citrus variegated chlorosis (CVC), a new disease of sweet organe tress in Brazil. A bacterium was consistently cultured from plant tissues from CVC twigs of sweet orange trees but not from tissues of healthy trees on several cell-free media known to support the growth ofXylella fastidiosa. Bacterial colonies typical ofX. fastidiosa became visible on PW, CS20, and PD2 agar media after 5 and 7-10 days of incubation, respectively. The cells of the CVC bacterium were rod-shaped, 1.4-3 μm in length, and 0.2-0.4 μm in diameter, with rippled walls. An antiserum against an isolate (8.1.b) of the bacterium gave strong positive reactions to double-antibody-sandwich (DAS), enzyme-linked immunosorbent assay (ELISA) with other cultured isolates from CVC citrus, as well as with several type strains ofX. fastidiosa. This result indicates that the CVC bacterium is a strain ofX. fastidiosa. ELISA was also highly positive with all leaves tested from CVC-affected shoots. Leaves from symptomless tress reacted negatively. Sweet organe seedlings inoculated with a pure culture of the CVC bacterium supported multiplication of the bacterium, which became systemic with 6 months after inoculation and could be reisolated from the inoculated seedlings. Symptoms characteristic of CVC developed 9 months post inoculation.

No abstract

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...

No abstract

16S/23S intergenic spacer regions from the rRNA operons of two strains of "Candidatus Liberobacter asiaticum" and one strain of "Candidatus Liberobacter africanum" were cloned and sequenced. The intergenic spacers of the two "Candidatus L. asiaticum" strains studied are identical and contain the genes for isoleucine tRNA (tRNA"') and alanine tRNA (tRNAMa) separated by 11 nucleotides. The intergenic spacer of the "Candidatus L. africanum" strain contains only one tRNA gene (tRNANa). The level of homology between the intergenic spacers of the two liberobacter species is 79.46%. Ribosomal operons with 16S/23S spacer regions other than those studied might be present in the two "Candidatus Liberobacter" species.Huanglongbing (HLB) (greening) disease is one of the most severe diseases of citrus, occurring in Asian and African countries, in the Arabic Peninsula, and on Madagascar, Reunion, and Mauritius, which are islands in the Indian Ocean (2, 5). The agent of the disease is a bacterium which is restricted to the phloem sieve tubes of infected plants. Infection of plants occurs through two psyllid vectors, Diaphorina citri in Asia and Trioza eiytreae in Africa (3, 10). Today, despite many attempts, the HLB disease bacterium has resisted in vitro cultivation. Recently, we were able to characterize the HLB disease bacterium by cloning its 16s rRNA gene (7), as well as genes of the p operon (13,17). In this work we showed that the HLB disease bacterium belongs to the alpha subdivision of the Proteobacteria, in which it represents a new genus with two species, one associated with HLB disease in Asia and the other associated with HLB disease in Africa. The designations "Candidatus Liberobacter asiaticum" and "Candidatus Liberobacter africanum" were given to the two species according to the rules established for uncultured organisms (12).The genes that we cloned and sequenced for taxonomic characterization have been used to develop detection methods, including DNA-DNA hybridization and PCR detection methods (8, 16). These methods have allowed the detection and identification of the liberobacter species, but they have not permitted differentiation of the various serotypes which occur in each species (4,6). In order to further characterize the two liberobacter species at the molecular level and to find genomic polymorphism, we cloned and sequenced the intergenic 16S/ 23s rRNA spacer region known to be highly variable at both the interspecies level and the intraspecies level (1, 9). DNA was extracted as described by Jagoueix et al. previously (6). The 16S/23S intergenic region of each strain was obtained by PCR amplification with primers 012 and 23S1. Primer 012 was defined on the basis of the 16s ribosomal DNA (rDNA) sequence of "Candidatus L. asiaticum" Poona (7) and is located 240 bp upstream of the 3' OH end of the 16s rDNA. This primer allows differentiation of the three liberobacter strains, since in this region each strain has several characteristic nucleotides. Primer 23S1 was defined at the beginning of the 23s rDNA ...