Analysis of the type IV fimbrial-subunit gene fimA of Xanthomonas hyacinthi: application in PCR-mediated detection of yellow disease in Hyacinths

A sensitive and specific detection method was developed for Xanthomonas hyacinthi; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene fimA from strain S148. The fimA gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of fimA was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as Xanthomonas campestris pv. vesicatoria, Neisseria gonorrhoeae, and Moraxella bovis. In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp DNA fragment in all X. hyacinthi isolates. This PCR was shown to be pathovar specific, as assessed by testing 71 Xanthomonas pathovars and bacterial isolates belonging to other genera, such as Erwinia and Pseudomonas. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in X. hyacinthi. Only two Xanthomonas translucens pathovars cross-reacted weakly in PCR. Primers amplifying a subsequence of the fimA gene of X. campestris pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikstr?m, J. van Doorn, K. Haahtela, E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280-1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes. Under laboratory conditions, approximately 1,000 CFU of X. hyacinthi per ml could be detected. In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml. The results indicated that infected hyacinths with early symptoms could be successfully screened for X. hyacinthi with PCR.     

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.     

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.     

Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.     

Pathovar-Specific Antigens of Xanthomonas campestris pv. begoniae and X. campestris pv. pelargonii Detected with Monoclonal Antibodies

Two monoclonal antibodies specific for lipopolysaccharide antigens of Xanthomonas campestris pv. begoniae and pv. pelargonii reacted with all of their respective pathovar strains and not with 130 strains of other xanthomonads or 89 nonxanthomonads tested. These results, as well as previous results, indicate that pathovar-specific monoclonal antibodies were readily generated to strains of X. campestris pathovars that generally infect single hosts.     

Phenotypic Switching Affecting Chemotaxis, Xanthan Production, and Virulence in Xanthomonas campestris

The chemotaxis towards sucrose and yeast extract of nine strains of Xanthomonas campestris representing pathovars campestris, armoraciae, translucens, vesicatoria, and pelargonii was analyzed by using swarm plates. Unexpectedly, each of these strains formed small or reduced swarms typical of nonmotile or nonchemotactic bacteria. With time, however, chemotactic cells appeared on the swarm plates as blebs of bacteria. These cells were strongly chemotactic and were concomitantly deficient in exopolysaccharide production. The switch from the wild type (exopolysaccharide producing and nonchemotactic) to the swarmer type (exopolysaccharide deficient and chemotactic) appeared irreversible ex planta in bacteriological medium. However, in radish leaves swarmer-type strains of X. campestris pv. campestris were able to revert to the wild type. Swarmer-type derivatives of two X. campestris pv. campestris wild-type isolates showed reduced virulence and growth in the host plants cauliflower and radish. However, exocellular complementation of X. campestris pv. campestris Hrp (nonpathogenic) mutant was achieved by coinoculation with a swarmer-type strain.     

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.     

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.     

Bacterial Streak Disease of Maize (Zea mays L.) in South Africa

Bacterial streak disease of maize is currently causing some concern among breeders in South Africa. The causal organism of this previously undescribed disease was successfully isolated and its pathogenicity established using KoCH's postulates. Standard physiological and biochemical tests used to identify phytopathogenic bacteria indicated that the bacterium is a Xanthomonas campestris pathovar. Comparisons between this organism and other recognized X. campestris pathovars of the Poaceae indicated that apart from some minor differences the maize streak pathogen is physiologically similar to X. campestris pv. holcicola. However, in repeated reciprocal inoculation experiments all attempts to induce disease symptoms in sorghum with the maize streak pathogen were unsuccessful. Conversely, X. campestris pv. holcicola did produce symptoms in maize leaves. In all the maize cultivars tested the symptoms produced by the maize streak pathogen were, however, always considerably more severe than those caused by X. campestris pv. holcicola. Notwithstanding its physiological similarity to X. campestris pv. holicola it would appear that on the grounds of host specificity the maize streak pathogen warrants new pathovar status. The name X. campestris pv. zeae is proposed.     

Fingerprinting of Xanthomonas campestris pv. pelargonii and Related Pathovars Using Random-Primed PCR

Polymerase chain reaction (PCR) amplification of total DNA was evaluated as a method to distinguish Xanthomonas campestris pv. pelargonii from other pathovars within this species. Two sets of highly conserved enterobacterial consensus sequences were used as targets for PCR amplification: (a) enterobacterial repetitive intergenic consensus [ERIC] and (b) repetitive extragenic palindromic [REP] sequences. Nucleic acid was extracted from a total of 37 isolates of bacteria: 19 isolates ofX campestris pv. pelargonii and 18 isolates representing 10 other pathovars of X. campestris. After PCR amplification using the ERIC primer pair the DNA fingerprints of X. campestris pv, pelargonii contained two major DNA products (estimated size 500 and 740 pp) that were conserved among all 19 isolates. With the REP primer pair, the fingerprints were more complex and major DNA products ranging from -690 to 1650 bp were detected. Using information from both ERIC- and REP-primed Imgerprints, the X. campestris pv. pelargonii fingerprints were distinguishable from the fingerprints of the other pathovars examined: pvs. citrumelo. citri, beganiae, vittans B and C. phaseoli. campestris, manihotis, juglandis, carotae and pruni.     

Nutritional Similarity between Leaf-Associated Nonpathogenic Bacteria and the Pathogen Is Not Predictive of Efficacy in Biological Control of Bacterial Spot of Tomato

It has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogen Pseudomonas syringae pv. tomato. This relationship was investigated further in this study by using the pathogen Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage. The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogen X. campestris pv. vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log₁₀ of the number of CFU per leaflet) in growth chamber assays. Nutritional similarity between the nonpathogenic bacteria and X. campestris pv. vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates. In contrast to studies with P. syringae pv. tomato, nutritional similarity between the nonpathogenic bacteria and the pathogen X. campestris pv. vesicatoria was not correlated with reductions in disease severity. Nutritional similarity was also not correlated with reductions in pathogen population size. Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size of X. campestris pv. vesicatoria is not related to disease severity and that X. campestris pv. vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that of P. syringae pv. tomato.     

A multilocus sequence analysis of Xanthomonas campestris reveals a complex structure within crucifer-attacking pathovars of this species

Previous classification of Xanthomonas campestris has defined six pathovars (aberrans, armoraciae, barbareae, campestris, incanae, and raphani) that cause diseases on cruciferous plants. However, pathogenicity assays with a range of strains and different hosts identifies only three types of symptom: black rot, leaf spot and bacterial blight. These findings raise the question of the genetic relatedness between strains assigned to different pathovars or symptom phenotypes. Here we have addressed this issue by multilocus sequence analysis of 42 strains. The X. campestris species was polymorphic at the 8 loci analysed and had a high genetic diversity; 23 sequence types were identified of which 16 were unique. All strains that induce black rot (pathovars aberrans and campestris) were genetically close but split in two groups. Only three clonal complexes were found, all within pathovar campestris. The assignment of the genome-sequenced strain 756C to pathovar raphani suggested from disease symptoms was confirmed, although this group of strains was particularly polymorphic. Strains belonging to pathovars barbareae and incanae were closely related, but distinct from pathovar campestris. There is evidence of genetic exchanges of housekeeping genes within this species as deduced from a clear incongruence between individual gene phylogenies and from network structures from SplitsTree analysis. Overall this study showed that the high genetic diversity derived equally from recombination and point mutation accumulation. However, X. campestris remains a species with a clonal evolution driven by a differential adaptation to cruciferous hosts.     

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.     

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.     

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.     

Copper Resistance Gene Homologs in Pathogenic and Saprophytic Bacterial Species from Tomato

Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. In Southern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid and chromosomal DNA in X. compestris pv. vesicatoria, P. putida, and the yellow Pseudomonas sp. Homology was detected only in the chromosomal DNA of P. cichorii and P. fluorescens. Homology with cop was also detected in chromosomal DNA from copper-sensitive strains of P. cichorii, P. fluorescens, and P. syringae pv. tomato, suggesting that the cop homolog may be indigenous to certain Pseudomonas species and have some function other than copper resistance. No homology was detected in DNA from a copper-sensitive X. campestris pv. vesicatoria strain. Copper-inducible protein products were detected in each copper-resistant bacterium by immunoblot analysis with antibodies raised to the CopB protein from the cop operon. The role of the homologous DNA in copper resistance was confirmed for the X. campestris pv. vesicatoria strain by cloning and transferring the cop homolog to a copper-sensitive strain of X. campestris pv. vesicatoria. The possibility and implications of copper resistance gene exchange between different species and genera of pathogenic and saprophytic bacteria on tomato plants are discussed.     

Genetic organization of the lexA,recA and recX genes in Xanthomonas campestris

A gene cluster containing lexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported lexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different X. campestris pathovar. The abundance of RecA protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas lexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.     

Xanthan gum production and rheological behavior using different strains of Xanthomonas sp.

The proposal of the present study was to select and carry out the molecular characterization of strains of Xanthomonas sp. in order to correlate with gum production and determine possible genetic alterations during the study. The gums produced were also evaluated rheologically. Ten strains of Xanthomonas were used in the screening and the best ones in terms of productivity were Xanthomonas campestris pv. mangiferaeindicae 1230 (8.93 g/L), X. campestris pv. campestris 254 (9.49 g/L) and X. campestris pv. campestris 1078 (9.67 g/L). The gum produced by X. campestris pv. mangiferaeindicae presented the best apparent viscosity. The results for the profiles of the bands produced by RAPD showed considerable genetic variability amongst the evaluated strains, making not possible to neither group the strains according to pathovar or species, nor correlate the band profile with the productivity obtained. According to the RAPD analysis, no detectable mutations occurred in these bacteria during the study.     

Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates

In this study, Pseudomonas syringe pathovars isolated from olive, tomato and bean were identified by species-specific PCR and their genetic diversity was assessed by repetitive extragenic palindromic (REP)-PCR. Reverse universal primers for REP-PCR were designed by using the bases of A, T, G or C at the positions of 1, 4 and 11 to identify additional polymorphism in the banding patterns. Binding of the primers to different annealing sites in the genome revealed additional fingerprint patterns in eight isolates of P. savastanoi pv. savastanoi and two isolates of P. syringae pv. tomato. The use of four different bases in the primer sequences did not affect the PCR reproducibility and was very efficient in revealing intra-pathovar diversity, particularly in P. savastanoi pv. savastanoi. At the pathovar level, the primer BOX1AR yielded shared fragments, in addition to five bands that discriminated among the pathovars P. syringae pv. phaseolicola, P. savastanoi pv. savastanoi and P. syringae pv. tomato. REP-PCR with a modified primer containing C produced identical bands among the isolates in a pathovar but separated three pathovars more distinctly than four other primers. Although REP- and BOX-PCRs have been successfully used in the molecular identification of Pseudomonas isolates from Turkish flora, a PCR based on inter-enterobacterial repetitive intergenic concensus (ERIC) sequences failed to produce clear banding patterns in this study.