Campestris Pathovar Research Articles

Structures of the O4 and O18 antigens of Stenotrophomonas maltophilia: a case of enantiomeric repeating units

The O-specific side-chain polymers of lipopolysaccharides from the reference strains for Stenotrophomonas maltophilia serogroups O4 and O18 are both xylosylated rhamnans. In the O4 polymer, both sugar components are the d isomers, whereas the O18 polymer contains only the l isomers. By means of NMR spectroscopy, methylation analysis and Smith degradation, the repeating unit of the O4 polymer was identified as a doubly-branched pentasaccharide of the structure shown below. The O18 polymer is based on the enantiomeric pentasaccharide, but the xylosyl substituent at the 4-position is apparently absent from some units. The polymers closely resemble the O antigens found in Xanthomonas campestris pathovars.

PCR-mediated Detection of Xanthomonas oryzae pv. oryzae by Amplification of the 16S–23S rDNA Spacer Region Sequence

A detection method specific for Xanthomonas oryzae pv. oryzae, the pathogen responsible for bacterial blight of rice, was based on the polymerase chain reaction (PCR) and designed by amplifying the 16S–23S rDNA spacer region from this bacterium. The nucleotide sequence of the spacer region between the 16S and 23S rDNA, consisting of approximately 580-bp, from X. oryzae pv. oryzae, X. campestris pv. alfalfae, X. campestris pv. campestris, X. campestris pv. cannabis, X. campestris pv. citri, X. campestris pv. cucurbitae, X. campestris pv. pisi, X. campestris pv. pruni and X. campestris pv. vitians, was determined. The determined sequences had more than 95% identity. Therefore, a pair of primers, XOR-F (5′-GCATGACGTCATCGTCCTGT-3′) and XOR-R2 (5′-CTCGGAGCTATATGCCGTGC-3′) was designed and found to specifically amplify a 470-bp fragment from all strains of X. oryzae pv. oryzae isolated from diverse regions in Japan. No PCR product was amplified from X. campestris pathovars alfalfae, campestris, cannabis, carotae, cucurbitae, dieffenbachiae, glycines, pisi, pruni, vitians or zantedeschiae, except for pathovars citri, incanae and zinniae. The method could also detect the pathogen in infected rice leaves within 3 hr, at a detection limit of 4×101 cfu/ml.

188 Effect of Streptomycin Walnut Blight Sprays on Nut Drop

Walnut Blight caused by the bacteria Xanthomonas campestris pathovar juglandis is a very destructive disease for California walnut production. Streptomycin is an effective disease control material; however, Streptomycin sprays can result in significant nut drop 3 to 5 weeks after spray application. We investigated the basis for walnut drop following applications of Streptomycin (Agrimycin) for walnut blight control. Flowers and developing nuts were collected from four treatments, plus an unsprayed control. 200 ppm Streptomycim was applied at 1) budbreak; 2) pre, full, and post-bloom; 3) postbloom; 4) budbreak and postbloom; 5) untreated control. Samples were collected regularly beginning at the first budbreak spray and extending through the period of nut drop. Samples were fixed and prepared for histological examination. In treatments with a high incidence of nut drop, the embryo failed to develop. Examination of the stigma and style in flowers from these treatments showed inhibited pollen tube growth. Results indicate that Streptomycin inhibits pollen tube growth, which precludes fertilization. This pattern of development and timing of nut drop following Streptomycin application at full bloom is similar in all ways to unpollinated walnut flowers. Nut growth and development appear normal for 3 to 5 weeks; then nuts abort. If Streptomycin became available for walnut blight control, sprays timed to coincide with pistillate bloom and pistillate flower receptivity should be avoided.

Novel genes involved in the regulation of pathogenicity factor production within the rpf gene cluster of Xanthomonas campestris The EMBL accession number for the sequence reported in this paper is AJ245997.

The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pathovar campestris (Xcc) is subject to co-ordinate regulation by a cluster of genes called rpf (for regulation of pathogenicity factors). These genes are located within a 21.9 kb region of the chromosome isolated as the cosmid clone pIJ3020. The genes in the left-hand section of this region of the chromosome have previously been characterized. This paper reports on the genes in the right-hand section and on the phenotypes of mutants with transposon insertions in these genes. Sequence analysis identified eight genes or ORFs with the gene order rpfD-orf1-orf2-orf3-orf4-recJ-rpf E-greA. RecJ and GreA have established functions in recombination and transcriptional elongation, respectively. rpfD encoded a protein with some amino acid sequence relatedness to a hypothetical protein from Caulobacter crescentus and an autolysin response regulator in Bacillus subtilis. The predicted protein products of orf1, 2 and 3 were related to each other and had substantial amino acid sequence relatedness to hypothetical proteins from C. crescentus. Transposon insertions in orf1, 2 and 3 had no effect on the synthesis of extracellular enzymes or EPS. The predicted proteins RpfE and Orf4 showed the highest amino acid sequence relatedness to hypothetical proteins from Bordetella pertussis and Klebsiella pneumoniae, respectively. Transposon insertions in rpfE led to reduced levels of some extracellular enzymes (endoglucanase and protease) and increased levels of others (polygalacturonate lyase). Transposon insertions in orf4 had no effect on polygalacturonate lyase but led to reduced levels of protease and endoglucanase. Levels of EPS were reduced in both rpfE and orf4 mutants. These alterations in the levels of extracellular enzymes, which were relatively modest (between two- and threefold), did not affect the pathogenicity of Xcc on turnip. It is proposed that the gene designation should be rpfI for orf4.

Identification of a lexA gene in, and construction of a lexA mutant of, Xanthomonas campestris pv. citri.

The lexA gene of Xanthomonas campestris pathovar citri (X.c. pv. citri) was cloned and sequenced. The 639-bp open reading frame encodes a protein of 213 amino acids that shares substantial sequence homology with the products of previously characterized lexA genes, sharing 46% identity with the LexA protein of Escherichia coli. Amino acids required for autocatalytic cleavage of LexA are conserved in the X.c. pv. citri protein, whereas domains thought to mediate DNA binding differ markedly from those of LexA proteins from E. coli and other bacteria. The X.c. pv. citri LexA protein was overexpressed in E. coli, and SDS-polyacrylamide gel electrophoresis revealed a molecular size of 23 kDa for the purified protein. A lexA mutant of X.c. pv. citri was constructed by gene replacement, and the basal level of recA expression in this mutant was shown to be similar to that for wild-type cells exposed to a DNA-damaging agent. These results indicate that LexA functions as a repressor of recA expression in X.c. pv. citri.

Construction and Characterization of a recA Mutant of Xanthomonas campestris pv. citri

The recA gene of Xanthomonas campestris pathovar citri (X. c. pv. citri) was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame of 1,032 bp that encodes a 344-amino acid protein and which shows a high degree of homology to recA genes from other bacteria. The cloned recA gene of X. c. pv. citri restored to a recA deletion mutant of Escherichia coli the ability to resist killing induced by methylmethane sulfonate or ultraviolet radiation, indicating that the cloned gene was functional in E. coli. A recA mutant of X. c. pv. citri was constructed by gene replacement and was shown both not to possess detectable DNA recombination activity and to be markedly more sensitive to DNA-damaging agents than is wild-type X. c. pv. citri. Transformation of recA mutants of E. coli and X. c. pv. citri with a plasmid containing X. c. pv. citri recA conferred the ability to produce a 37-kDa protein that reacted with antiserum to E. coli RecA protein. Two additional open reading frames were also identified in the recA region of the X. c. pv. citri genome: One located immediately upstream from recA that encodes a 213-amino acid protein with a high degree of similarity to the LexA protein of E. coli, and another located immediately downstream of recA that encodes a 153-amino acid protein with sequence similarity to the RecX protein of Pseudomonas aeruginosa.

Induction of the viable but non-culturable condition in Xanthomonas campestris pv. campestris in liquid microcosms and sterile soil

Xanthomonas campestris pathovar campestris was examined for the ability to enter the viable but non-culturable (VBNC) state. When nutrients were removed from a bacterial culture, the culturability dropped to undetectable levels within 6 weeks while the concentration of viable cells remained above 10(5) cells ml(-1). Addition of cupric sulfate facilitated this process. When bacteria were placed in autoclaved soil, culturability dropped by at least four logs within 5 days of the experiment while the concentration of viable cells remained at least two logs higher. These results indicate that the plant pathogen X. campestris can enter the VBNC condition in sterile soil and in liquid cultures and that cupric sulfate decreases the time for entry into the VBNC condition.

Induction of the viable but non-culturable condition in Xanthomonas campestris pv. campestris in liquid microcosms and sterile soil

Xanthomonas campestris pathovar campestris was examined for the ability to enter the viable but non-culturable (VBNC) state. When nutrients were removed from a bacterial culture, the culturability dropped to undetectable levels within 6 weeks while the concentration of viable cells remained above 10 5 cells ml −1. Addition of cupric sulfate facilitated this process. When bacteria were placed in autoclaved soil, culturability dropped by at least four logs within 5 days of the experiment while the concentration of viable cells remained at least two logs higher. These results indicate that the plant pathogen X. campestris can enter the VBNC condition in sterile soil and in liquid cultures and that cupric sulfate decreases the time for entry into the VBNC condition.

Identification of the promoter region of the Xanthomonas campestris pv. citri recA gene responsible for induction by DNA-damaging agents

The abundance of the RecA protein and of recA transcripts was markedly increased on exposure of Xanthomonas campestris pathovar citri to various DNA-damaging agents, including mitomycin C. The promoter sequence responsible for mediating the sensitivity of recA expression to DNA damage was investigated by subcloning a 426-bp restriction fragment of the 5′ untranslated and coding region of the gene into a promoterless vector containing the luxAB genes of Vibrio fischeri. Xanthomonas campestris pv. citri cells transformed with this vector responded to DNA-damaging agents with a marked increase in luciferase activity. Deletion of nucleotides from the 5′ end of the recA fragment inserted into the reporter plasmid revealed that the 58 bp upstream of the transcription initiation site are sufficient to mediate induction of recA expression by mitomycin C.

Characterization of transposon-generated protease mutant of Xanthomonas campestris pathovar glycine 8ra. Enzyme activity, cloning, and mapping of flanking DNA.

Protease negative mutant of Xanthomonas campestris pathovar glycine 8ra (prt-mutant) was constructed by mutagenesis employing artificial transposon Omegon-Km. Transposon delivery was conducted through diparental conjugation using X. campestris pathovar glycine 8ra as recipient and Escherichia coli S17-1 carrying pJFF 3500 plasmid as the donor. The frequency of transconjugation was found 1.9 x 10(-7) per recipient. Enzyme analysis indicated the presence of mutant with lower protease activity than that of the wild-type. Genetic analysis employing pulsed-field gel electrophoresis (PFGE) of the genomic DNA digested with AseI or SpeI restriction endonuclease could significantly differentiate X. campestris pathovar glycine 8ra prt from the wild-type parent. The 9.85 kb pLR omega 6 plasmid was constructed from the genomic DNA of the prt mutant after being digested with KpnI restriction endonuclease and ligated with T4 DNA ligase.

The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase.

Xanthomonas campestris pv campestris (Xcc) is a plant pathogenic bacterium that controls the production of pathogenicity factors in part by a cluster of genes designated rpf (regulation of pathogenicity factors). Sequence analysis of one of these genes (rpfA) revealed an open reading frame with amino acid sequence similarity to aconitases from other bacteria. Aconitase activity was lower in cellular extracts of an rpfA::Tn5 mutant than in those from the wild type. A zymogram of aconitase activity after native gel electrophoresis showed the presence of two distinct aconitases in Xcc; the major aconitase was absent in the rpfA::Tn5 mutant. This mutant also had reduced levels of extracellular enzymes and extracellular polysaccharide (EPS). Supplying rpfA in trans to the rpfA::Tn5 mutant restored both the major aconitase activity and the synthesis of these pathogenicity factors. The transcription of the genes for two extracellular enzymes (prtA, encoding a serine protease, and engXCA, encoding endoglucanase) was reduced in the rpfA mutant background. Because some eukaryotic aconitases are also involved in iron regulation, we explored a possible connection between rpfA and iron metabolism. Intracellular iron levels in the mutants were lower than in the wild type as assessed by sensitivity to the iron-activated antibiotic, streptonigrin. Wild-type bacteria grown in iron-deficient conditions had a similar sensitivity to streptonigrin as the aconitase mutant. Overall, these results suggest that a prokaryotic aconitase can also act as a regulator of gene expression and that the regulation is possibly related to changes in intracellular iron levels.

Pathogenicity, Non‐Host Hypersensitivity and Host Defence Non‐Permissibility Regulatory Gene hrpX is Highly Conserved in Xanthomonas Pathovars

Abstract Xanthomonas campestris pv. campestris and X. oryzae pv, oryzae contain the 1428 base pair hrpX gene whose product is involved in the regulation oi hrp genes required for pathogericity, non‐host hypersensitivity and non‐permissibility of compatible host defence responses. Previous Southern blot hybridization studies have suggested that hrpX is conserved in several X. campestris pathovars and X. oryzae. strains. We have confirmed and extended these findings using hrpX gene amplification by polymerase chain reaction, coupled with Southern blot hybridization analyses. Sixteen distinct pathovars of X. campestris and 12 strains of X. oryzae pv, oryzae were shown to contain homologs of hrpX which were not apparent in heterologous bacteria such as Agrobacterium tumefaciens, A. rhizogenes, Erwinia carolovora ssp. carotovora, Pseudomonas syringae pv, glycinea. P. syringae pv, labaci, and Escherichia coli. The hrpX gene is therefore highly conserved among Xanthomonas species and its gene product strongly resembles positive regulatory proteins of the AraC protein family,

A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule.

Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris (Xcc). The phenotype of mutants in one of the genes, rpfF, can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipoplysaccharide and lipid A from Xcc, and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.

Detection of Xanthomonas campestris pv. pelargonii in geranium and greenhouse nutrient solution by serological and PCR techniques

Specificity of a new monoclonal antibody, 2H5, to Xanthomonas campestris pv. pelargonii, causal agent of geranium bacterial blight, was determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence tests on 14 strains of X. c. pelargonii, 12 strains of other X. campestris pathovars, 3 strains of other Xanthomonas spp., 3 strains of other plant pathogens, and 43 saprophytic bacteria isolated from geranium. X. c. pelargonii was detected in tissue from symptomatic and asymptomatic geraniums sampled from commercial growers, and artificially inoculated plants, by monoclonal antibody-based tests. The intensity of response in ELISA was only moderately correlated (r = 0.56) with symptom severity, while symptom severity was not correlated (r = 0.16) with the number of fluorescing cells in immunofluorescence. The bimodal frequency distribution of ELISA and immunofluorescence results served to validate arbitrarily chosen positive/negative threshold values. Most positive ELISA and immunofluorescence test results were confirmed by the polymerase chain reaction (PCR) using published primers (Manulis et al., 1994. Appl. Environ. Microbiol 60, 4094-4099). In contrast to plant tissue, the bacterium was detected in greenhouse nutrient solution with greater sensitivity by immunofluorescence and PCR than by ELISA. Sensitivity of detection was enhanced 100-fold by concentration of the bacteria by centrifugation.

A phagemid shuttle vector based on filamentous phage φLf and theE. coli vector pOK12 for use inXanthomonas campestris

AnE. coli-Xanthomonas shuttle vector, p2GP (3.3 kb), was constructed by fusing pOK12 with a DNA fragment carrying gene II andori from filamentous phage φLf. p2GP retains the advantages afforded by pOK12 and can replicate in differentX. campestris pathovars. Superinfection ofX. c. pv.campestris cells harboring p2GP or its derivatives with φLf causes the release of ssDNA-containing recombinant phage particles which are sources of single-stranded template for sequencing the inserts.

Differentiation of three species of Xanthomonas and Stenotrophomonas maltophilia using cellular fatty acid analyses

Five hundred eighty-eight strains, representing Xanthomonas albilineans, X. fragariae, ten pathovars of X. campestris, and Stenotrophomonas maltophilia from ornamentals, were subjected to fatty acid methyl ester (FAME) analyses. Quantitative variance among FAME profiles enabled identification of the four species with 100% accuracy. Dendrogram cluster analysis placed strains of X. albilineans remotely from those of the other two Xanthomonas species and S. maltophilia. Whereas some profiles of pathovars of X. campestris were distinct, strains within X. albilineans, X. fragariae, and S. maltophilia were homogeneous by their conserved FAME ratios. Pathovars of X. campestris that had conserved profiles were fittonia, hederae, malvacearum, pelargonii, and zinniae. FAME profiles of X. campestris pathovars begoniae, dieffenbachiae, fici, maculifoliigardeniae, and poinsettiicola were, however, quantitatively diverse. These pathovars did not form discrete subgroups, and intercalated randomly with one another on the dendrogram. Certain species or pathovars of X. campestris which have homogeneous FAME profiles can easily be identified with fatty acid analysis; however, pathovars of X. campestris with heterogeneous profiles are not readily identified by fatty acid analysis.

Characterization of Xanthomonas campestris pv. pruni strains from different hosts by pathogenicity tests and analysis of whole‐cell fatty acids and whole‐cell proteins

AbstractStrains of Xanthomonas campestris pv. pruni obtained from Prunus armeniaca. P. domestica, P. persica and P. salicina in different geographical areas were compared for pathogenicity, fatty acid and wholecell protein analysis. Four strains, one per each host plant, were inoculated at the same time, on the foliage of P. armeniaca, P. avium, P. persica and P. salicina cultivars. Mean content of fatty acids of X.c. pv. pruni strains were also compared with those of many strains of X.c. pv. campestris, pv. graminis, pv. hyacinthii, pv. pelargonii and pv. vasculorum. Strains showed a remarkable homogeneity in fatty acids content and whole‐cell protein profiles and principal component and cluster analysis did not reveal any grouping according to original host or geographical origin. However, X.c. pv. pruni strains can be grouped apart from the other X. campestris pathovars. There appears to be no pathogenic specialization among the strains tested, however, they varied in aggressiveness to host plants and host plant in susceptibility. The most of the strains were able to cross‐infect species other that from where they were originally isolated, although, P. avium did not show any symptom of disease. P. persica cv. Sentry and P. salicina cv. Globe Sun, recently licensed as resistant to X.c. pv. pruni. were infected, although to a lesser extent, by some strains.

Recognition and Detection in Seed of the Xanthomonas Pathogens That Cause Cereal Leaf Streak Using rDNA Spacer Sequences and Polymerase Chain Reaction

Two short DNA sequences, situated in the spacer segment between the 16S and 23S rRNA genes and flanking an alanine-tRNA gene, varied among four xanthomonads. Polymerase chain reaction (PCR) primers designed to exploit this variability amplified a 139-bp fragment from strains of five leaf streak pathogens of cereals, Xanthomonas campestris pvs. cerealis, hordei, secalis, translucens, and undulosa, and five X. campestris pathovars pathogenic on forage grasses, X. campestris pvs. arrhenatheri, graminis, phlei, phleipratensis, and poae. These primers did not amplify DNA from 52 other bacteria, including other xanthomonads. PCR specificity was largely determined by one primer site, which is located in a 57-bp sequence present only in the rDNA spacer of DNA homology group I strains. Amplification of extracts of 2 seed lots known to be contaminated with a translucent leaf streak pathogen produced the specific 139-bp PCR fragment ; 4 of 27 additional seed lots with unknown levels of leaf streak pathogens were positive for this amplified fragment. The assays proved to be fast and relatively sensitive (2 x 10 3 CFU/g of seed), indicating the technique might be useful for detecting pathogens in seed.

Variation for Black Rot Resistance in Tronchuda Regenerated from Epicotyl Segments

Tronchuda (Brassica oleracea var. tronchuda Bailey syn. costata) regenerants with resistance to Xanthomonas campestris pathovar campestris Pammel (Dawson) were produced by culturing epicotyl segments of 3-week-old seedlings on Murashige and Skoog medium supplemented with 2 mg BAP and NAA at 0.1 mg·liter–1. Cultures were placed in darkness (1, 2, or 3 weeks) or in a 16-hour·day–1 light regime. Seedlings of all four cultivars were more susceptible (mean disease severity rating of 3.8 to 4.0; where 1 = trace and 4 = more than 2 cm2 diseased leaf tissue) than the respective regenerants. There were significant differences in disease severity of regenerants among and within the four cultivars. Dark incubation of cultured explants generated plants with higher disease resistance than evident with control plants. One week of dark incubation resulted in increased resistance in regenerants of `Penca de Chaves', `Portuguesa', and `Vilinda' (2.1, 2.3, and 2.7 mean disease severity, respectively), whereas 2 weeks of dark incubation increased resistance in regenerants of `Ana Maria' and `Vilinda' (2.1 and 2.7 mean disease severity, respectively). The genotype×plant treatment interaction was significant. Chemical names used: 6-benzylaminopurine (BAP); a-naphthaleneacetic acid (NAA).

Testing for Pseudomonas syringae pv. atrofaciens and Xanthomonas campestris pathovars on cereals in Italy

During recent years, recurrent attacks of basal glume rot/leaf blight attributed to Pseudomonas syringae pv. atrofaciens have been observed on cultivars of durum wheat grown in the Po valley (IT). The main aim of this study was to test for this pathogen and Xanthomonas campestris pathovars in commercial seed lots of cereals produced in different regions of Italy, as well as in symptomatic plants collected in the field. None of the analyses led to the detection of xanthomonads. In testing for P.s. atrofaciens, representative bacterial isolates were selected and characterized; this was done by combining conventional identification tests and computerized densitometric analysis of electrophoretic patterns of cell proteins obtained using the SDS‐PAGE technique. P.s. atrofaciens was detected in seed samples (barley, wheat and durum wheat) and symptomatic plants (durum wheat) grown in northern and central Italy. The possible pathogenic role of other unrelated fluorescent pseudomonads in this interaction was also examined. Difficulties in discriminating pvs syringae and atrofaciens of P. syringae are discussed and the uncertain taxonomic relationship between these two pathovars is emphasized.