Biological Role of Xanthomonadin Pigments in Xanthomonas campestris pv. Campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Bacteriocins and temperate phage ofXanthomonas campestris pv. glycines

Sixteen strains ofXanthomonas campestris pathovar (pv.) glycines produced bacteriocins (glycinecins) on agar media. Optimal incubation conditions were for 48 h at 20°C. In addition to strains ofX. campestris pv. glycines, bacteriocins were also inhibitory towardsX. campestris pv. phaseoli andX. campestris pv. vesicatoria. All bacteriocins were susceptible to inactivation by a nonspecific protease and resistant to ribonuclease, but they differed in their sensitivity to trypsin, deoxyribonuclease, and heat treatment. Differential heat and enzyme sensitivities also indicated that some strains ofX. campestris pv. glycines produce more than one bacteriocin. Attempts to induce bacteriocin production in liquid cultures were unsuccessful. However, temperate bacteriophage were released from cultures ofX. campestris pv. glycines strains XP175, B83, 17915, and MINN after addition of mitomycin C or nalidixic acid or after exposure to UV light.Reference to brand or firm names does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Defence-related enzymatic response in cabbage to Xanthomonas campestris pv. campestris

Black rot of cabbage caused by Xanthomonas campestris pv. campestris is one of the most important diseases of crucifers worldwide. Expression of defence-related enzymes in cabbage in response to X. campestris pv. campestris was investigated in the current experiment. Among the defence-related enzymes (phynylalanine ammonia lyase, peroxidase, polyphenol oxidase, superoxide dismutase [SOD] and chitinase) and quantity of phenolic compounds studied in the present investigation, phenylalanine ammonia lyase (PAL), the key enzyme in the phenylpropanoid pathway was the first enzyme suppressed at three days after inoculation in X. campestris pv. campestris-cabbage system. Correlation analysis indicated that PAL and phenolic compounds are the two most important compounds determining the susceptibility of cabbage to X. campestris pv. campestris. Induction of peroxidase isoform-1 (Rf value: 0.059) and SOD isoform-1 (Rf value: 0.179) three days after pathogen inoculation implicated the role of these isozymes in susceptible cabbage – X. campestris pv. campestris interaction. This study demonstrates the susceptibility of cabbage to X. campestris pv. campestris is a result of declination of PAL and phenolic contents at biochemical level as a manifestation of increase in bacterial population at the cellular level within the host tissues.     

Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu^r) strains of the pathogen were isolated. The Cu^r genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu^r genes on pXV10A. A probe containing subcloned Cu^r genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu^r genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu^r plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu^r genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.     

Genomic Relatedness of Xanthomonas campestris Strains Causing Diseases of Citrus

Xanthomonas campestris strains that cause disease in citrus were compared by restriction endonuclease analysis of DNA fragments separated by pulsed-field gel electrophoresis and by DNA reassociation. Strains of X. campestris pv. citrumelo, which cause citrus bacterial spot, were, on average, 88% related to each other by DNA reassociation, although these strains exhibited diverse restriction digest patterns. In contrast, strains of X. campestris pv. citri groups A and B, which cause canker A and canker B, respectively, had relatively homogeneous restriction digest patterns. The groups of strains causing these three different citrus diseases were examined by DNA reassociation and were found to be from 55 to 63% related to one another. Several pathovars of X. campestris, previously shown to cause weakly aggressive symptoms on citrus, ranged from 83 to 90% similar to X. campestris pv. citrumelo by DNA reassociation. The type strain of X. campestris pv. campestris ranged from 30 to 40% similar in DNA reassociation experiments to strains of X. campestris pv. citrumelo and X. campestris pv. citri groups A and B. Whereas DNA reassociation quantified the difference between relatively unrelated groups of bacterial strains, restriction endonuclease analysis distinguished between closely related strains.     

The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment,cell motility,polysaccharide synthesis,virulence, and tolerance to various stresses

Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.     

Purification and characterization of Xanthomonas campestris pv. glycines exopolysaccharide

The exopolysaccharides (EPS) of virulent and avirulent strains of Xanthomonas campestris pv. glycines, causal agent of bacterial pustule disease of soybean, and one strain of the soybean non-pathogen X. c. pv. campestris were isolated, purified, and their compositions compared. EPS produced by X. c. pv. glycines in a completely defined medium appears to be identical to the well-characterized EPS produced by X. c. pv. campestris (commonly referred to as xanthan gum). The EPS of all strains was composed of the carbohydrates glucose, mannose and glucuronic acid with acetyl and pyruvyl substituents present. Permethylation analyses indicated EPS preparations had identical hexose substitution patterns. Avirulent strains of X. c. pv. glycines produced as much or more acidic EPS as did virulent strains in vitro. None of the EPS preparations were active as elicitors of the soybean pterocarpanoid phytoalexin glyceollin as determined by a soybean cotyledon bioassay.     

Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from Xanthomonas campestris pv. vesicatoria

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.     

Characterization of Insertions of IS476 and Two Newly Identified Insertion Sequences, IS1478 and IS1479, in Xanthomonas campestris pv. campestris

Thirty-two plasmid insertion mutants were independently isolated from two strains of Xanthomonas campestris pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS3 family member, IS476, and two new insertion sequences (IS), IS1478 and IS1479. While IS1478 does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS1479 demonstrated 73% sequence homology with IS1051 in X. campestris pv. dieffenbachiae, 62% homology with IS52 in Pseudomonas syringae pv. glycinea, and 60% homology with IS5 in Escherichia coli. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS1478 by itself constitutes a new subfamily of the widespread IS5 family, whereas IS1479, along with IS1051, IS52, and IS5, belongs to the IS5 subfamily of the IS5 family. All but one of the IS476 insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS476 insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS1478 and IS1479, on the other hand, were not random, and IS1478 and IS1479 each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS476, IS1478, and IS1479 are present in the genomes of all seven X. campestris pv. campestris strains tested and several X. campestris pathovars.     

Bactericidal Activity of Glycinecin A, a Bacteriocin Derived from Xanthomonas campestris pv. glycines, on Phytopathogenic Xanthomonas campestris pv. vesicatoria Cells

The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group (S. G. Heu et al., Appl. Environ. Microbiol. 67:4105-4110, 2001). In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from X. campestris pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.     

Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.     

Une phytotoxine de Xanthomonas campestris pv. vasculorum

A phytotoxin from Xanthomonas campestris pv. vasculorum I. Purification and partial characterization Xanthomonas campestris pv. vasculorum, the causal agent of sugarcane disease produces, when maintained in pure culture, a host selective phytotoxin. The molecule obtained at a high purity level is a strongly acid polyalcohol.     

Differentiation between Xanthomonas campestris pv. graminis (ISPP List 1980), pv. phleipratensis (ISPP List 1980) emend., pv. poae Egli and Schmidt 1982 and pv. arrhenatheri Egli and Schmidt 1982, by Numerical Analysis of Phenotypic Features and Protein Gel Electrophoregrams

A numerical analysis of 257 phenotypic features of 45 bacterial isolates from grasses, revealed three phenons corresponding to (i) X. campestris pv. graminis (ISPP List 1980), (ii) X. campestris pv. phleipratensis (ISPP List 1980) and (iii) X. campestris pv. poae Egli and Schmidt 1982 and X. campestris pv. arrhenatheri Egli and Schmidt 1982. In each phenon, the strains clustered together regardless of the geographical origin of the isolates orthe year of isolation. Polyacrylamide gel electrophoresis of soluble proteins and host range studies, revealed four groups corresponding to the pathovars mentioned above. The four pathovars constitute definite biological entities that can be differentiated by phenotypic, gel electrophoretic and host range features.     

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta)

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava.     

Pepper Tree (Schinus terebenthifolius Radii), a New Host Plant for Xanthomonas campestris pv. mangiferaeindicae

Apigmented bacterial colonies were obtained in Reunion Island from angular leaf lesions on Pepper tree (Schinus terebenthifolius Radii), a member of Anacardiaceae. All isolates were identified as Xanthomonas campestris, using physiological and biochemical tests. These strains were reinoculated to Pepper tree leaves, and Koch postulates were verified. Furthermore, they were inoculated to mango leaves and produced lesions identical to those induced by Xanthomonas campestris pv. mangiferaeindicae, the causal agent of bacterial black spot of mangoes. Apigmented and pigmented strains of X. c. pv. mangiferaeindicae from Mango and Ambarella were pathogenic to Pepper tree. Strains isolated from Pepper tree were compared to X. c. pv. mangiferaeindicae, by means of phenotypic features (utilization of 147 carbon sources) and using a serological assay. A high homology among the strains was observed. Thus, It is concluded that strains isolated from Pepper tree belong to pv. mangiferaeindicae, and that Pepper tree is a host species for X. c. pv. mangiferaeindicae.     

Similarity between Copper Resistance Genes from Xanthomonas campestris and Pseudomonas syringae

Plasmid-borne copper resistance genes from copper-resistant strains of Xanthomonas campestris pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of X. campestris pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from X. campestris pv. vesicatoria. Furthermore, a region of the copper resistance genes from X. campestris pv. vesicatoria 07882 hybridized with copA, the first gene of the copper resistance operon (cop) of Pseudomonas syringae pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of X. campestris pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.     

Biological Control of Black Rot (Xanthomonas campestris pv. campestris) of Cabbage in Tanzania with Bacillus strains

We evaluated the biocontrol efficacy of strains of Bacillus from Tanzania against the black rot pathogen, Xanthomonas campestris pv. campestris, in cabbage and the influence of the method of application under field conditions. The incidence and severity of black rot in the foliage, stems and heads of the highly susceptible cultivar, Copenhagen Market, were significantly reduced, especially when antagonists were applied through the roots as compared to application through the seeds or foliage (cotyledons). Promising antagonists included strains of B. cereus, B. lentimorbus and B. pumilus.     

Cross-reactive Antigens between Xanthomonas campestris pv. citri Pathotypes and Citrus Species

Cross-reactive antigens were detected in crude and semi-purified preparations from acetone powder of Citrus aurantifolia and Citrus sinensis leaves with antisera to Xanthomonas campestris pv. citri pathotypes A and C by DAS-ELISA. Antiserum to X. campestris pv. citri pathotype C revealed an antigenic disparity between C. aurantifolia (susceptible host to pathotype C) and C. sinensis (resistant host to pathotype C) whereas antiserum to X, campestris pv. citri pathotype A did not reveal any antigenic disparity between these hosts, both susceptible to pathotype A. The occurrence of “key” cross-reactive antigens in Citrus species and X. campestris pv. citri and their possible involvement in such interaction are discussed.     

Inhibition of Seed Germination and Root Development Caused by Xanthomonas campestris pv. vesicatoria in Pepper and Tomato

Inoculation of pepper seeds with the leaf pathogen Xanthomonas campestris pv. vesicatoria inhibited pepper germination. The inhibitory effect, which was stronger in non-sterilized light textured soils, decreased with time, and after 20, days or more, there was no difference between inoculated and non-inoculated seeds. Inhibitory substance(s) within the cytoplasmatic fraction of pathogen cells inhibited the germination of non-host tomato seeds. No relationship between pathogenicity to pepper leaves and inhibition of pepper seed germination was detected. The inhibitory substance(s) was found in two out of four X. campestris pv. vesicatoria strains. Heat-killed bacteria suppressed growth of pepper but not tomato seedlings. It is, therefore, suggested that the inhibition of seed germination and the decrease in root development are different modes of X. campestris pv. vesicatoria pathogenesis toward pepper plants.