Genetically distinct strains of the plant bacterium Xylella fastidiosa (Xf) are responsible for a variety of plant diseases, accounting for severe economic damage throughout the world. Using as a reference the genome of Xf 9a5c strain, associated with citrus variegated chlorosis (CVC), we developed a microarray-based comparison involving 12 Xf isolates, providing a thorough assessment of the variation in genomic composition across the group. Our results demonstrate that Xf displays one of the largest flexible gene pools characterized to date, with several horizontally acquired elements, such as prophages, plasmids, and genomic islands (GIs), which contribute up to 18% of the final genome. Transcriptome analysis of bacteria grown under different conditions shows that most of these elements are transcriptionally active, and their expression can be influenced in a coordinated manner by environmental stimuli. Finally, evaluation of the genetic composition of these laterally transferred elements identified differences that may help to explain the adaptability of Xf strains to infect such a wide range of plant species

Phylogenetic relationships of 17 species of Xanthomonas were assessed based on analysis of 16S-23S rDNA intergenic spacer (ITS) sequences; a higher level of resolution was obtained than that revealed by 16S rDNA analysis. ITS sequences varied in size from 492 to 578 nt within the genus and the similarity among sequences ranged from 63 to 99%. Major differences were found for the hyacinthi group, which included Xanthomonas albilineans, Xanthomonas hyacinthi, Xanthomonas sacchari, Xanthomonas theicola and Xanthomonas translucens. A common ITS structure with tRNA(Ala) and tRNA(Ile) embedded within the sequence was found for all ITS sequences of Xanthomonas species and for Stenotrophomonas maltophilia. These tRNAs were highly conserved and divided the ITS sequence into three regions (ITS1, ITS2 and ITS3). ITS1 and ITS2 sequences of Xanthomonas species showed mean similarities of 87.1 and 86.8%, respectively, and differences consisted of substitution and addition/deletion of 1-5 nt. ITS2 showed remarkable variation in sequence length: most species had an ITS2 of 19-20 nt, whereas a long insertion of 51-56 nt was found in Xanthomonas codiaei, X. hyacinthi, Xanthomonas melonis, X. theicola and X. translucens. For ITS3 the most striking alteration was seen in X. hyacinthi, which showed a large deletion of 44 nt. The ITS phylogenetic tree grouped Xanthomonas species into six major clusters. Cluster I included Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas bromi, Xanthomonas campestris, X. campestris pv. gardneri, Xanthomonas cassavae, X. codiaei, Xanthomonas cucurbitae, Xanthomonas fragariae, Xanthomonas hortorum, X. melonis, Xanthomonas oryzae, Xanthomonas pisi, Xanthomonas vasicola and Xanthomonas vesicatoria. The species X. albilineans, X. sacchari, X. hyacinthi, X. theicola and X. translucens represented distinct clusters (II-VI). Topology of the 16S-23S rDNA ITS phylogenetic tree was very similar to that of the 16S rDNA tree reported previously, but more clusters were discriminated because of the higher level of diversity among the ITS sequences (16.2%) compared with the 16S rDNA sequences (1.8%).

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The phylogenetic relationships of Xylella fastidiosa strains isolated from different hosts, including citrus trees, coffee, grapevine, plum and pear, were inferred by sequence analysis of the 16S rDNA and 16S-23S intergenic spacer region. A high level of similarity (97 1-100 %) was found in the 16S rDNA of the Xylella fastidiosa strains. The 16S-23S region showed a higher level of variation, with similarity values ranging from 79 8 to 100 %. Two tRNAs (tRNA Ala and tRNA Ile ) were encountered within the spacer sequence. The phylogenetic trees, constructed using the neighbour-joining method, showed that the citrus, coffee, peach and plum strains were closely related and separate from grapevine strains. The pear strain remained isolated from all the other Xylella strains in both analyses and produced values of less than 20 % in DNA-DNA hybridization experiments with a citrus strain. These results show that this strain does not belong to the Xylella fastidiosa genomic species.

The present study reports the expression of proteins of Xanthomonas axonopodis pv. citri in response to different growth conditions. The bacterium was cultured in the basal medium MM1 and in the presence of leaf extracts from a susceptible host plant (sweet orange) as well as a resistant (ponkan) and a nonhost plant (passion fruit). The protein profiles were analyzed by two-dimensional gel electrophoresis (2-DE). Twelve differential spots (induced, up- and down-regulated and repressed) were observed in the protein profiles of the bacterium cultivated in citrus extract (susceptible host) when compared to that of MM1. The 2-DE profile of the bacterium cultured in the complex medium nutrient yeast glycerol was also obtained and the comparison with that of MM1 revealed 36 differential spots. Five proteins from the different treatments were successfully N-terminally sequenced and the putative functions were assigned by homology searches in databases. Two constitutively expressed proteins, B4 and B5, were identified as pseudouridine synthase and elongation factor P, respectively. The large subunit of ribulose 1,5-biphosphate carboxylase/oxygenase and a sulfate binding protein were found as specifically up-regulated in the presence of citrus extracts. Finally, the heat shock protein G was found exclusively in the complex medium and repressed in all other media.

The genetic diversity of the Stemphylium solani isolates from cotton was assessed by Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromes (REP)-PCR fingerprinting. Twenty eight monosporic isolates of S. solani from cotton were used along with five isolates from tomato and one isolate of Alternaria macrospora from cotton for comparison. The dendrogram obtained revealed clear differences between the cotton and tomato isolates as well as between the tomato isolates from different geographic regions. The genetic relationships among S. solani isolates were also analyzed by sequencing the internal transcribed spacer (ITS) region of four isolates representing the three ERIC and REP groups. The tomato isolate from the State of São Paulo showed a distinct ITS sequence from that of the cotton isolates and tomato isolate from the State of Goiás, giving evidence that it belongs to a different genotype of S. solani. This is the first report of the entire sequence of the ITS1-5.8S-ITS2 regions of S. solani.

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins during treatment of Xanthomonas axonopodis pv. passiflorae in media containing leaf extract of the compatible (passion fruit) and incompatible (tomato) hosts. The results showed that at different times of treatment (5, 25 and 45 h) the global expression of proteins was almost identical in cells grown in minimal medium (MM) and in medium containing leaf extract of the incompatible host (MMT). The protein patterns of cells grown in medium containing passiflorae (MMP) leaf extract and MM were also compared enabling the detection of 17 differential spots. Most of the proteins were induced at earlier times of incubation (5 h) and maintained until 45 h in MMP. By using another carrier ampholyte range, seven additional proteins were identified in MMP treated cells. Five proteins, including one constitutive, two induced and two up-regulated in MMP were microsequenced. All sequences were found in the genome of xanthomonads sharing high level of identity (88-100%). Fructose biphosphate aldolase was expressed in all media employed. A putative membrane-related protein and a hypothetical protein were novel proteins induced specifically by the passiflorae extract. An inorganic pyrophosphatase and a hypothetical protein that showed similarity to the yciF gene of Salmonella thyphimurium were up-regulated in MMP.

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.